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Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability
发现具有高亲和性、选择性和口服生物利用度的 3,5-二甲基-4-磺酰基-1H-吡咯髓系细胞白血病 1 抑制剂
- Peng-Ju ZhuPeng-Ju ZhuState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Peng-Ju Zhu
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- Ze-Zhou Yu 余泽洲Ze-Zhou YuState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Ze-Zhou Yu
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- Yi-Fei Lv 吕一飞Yi-Fei LvState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Yi-Fei Lv
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- Jing-Long Zhao 赵京龙Jing-Long ZhaoState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Jing-Long Zhao
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- Yuan-Yuan Tong 童媛媛Yuan-Yuan Tong 童媛媛State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
中国药科大学天然药物国家重点实验室、江 苏药物设计与优化重点实验室，南京，210009Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
中国药科大学药学院药物化学系，南京 210009More by Yuan-Yuan Tong 汤媛媛的更多作品 - ,
- Qi-Dong You* 尤启东*Qi-Dong You 游启东*Email: youqd@163.comState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
中国药科大学天然药物国家重点实验室、江 苏药物设计与优化重点实验室，南京，210009Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
中国药科大学药学院药物化学系，南京 210009More by Qi-Dong You 尤启东的更多作品 - , and 和
- Zheng-Yu Jiang* 蒋正宇*Zheng-Yu Jiang*Email: jiangzhengyucpu@163.comState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Zheng-Yu Jiang
Copyright © 2021 American Chemical Society
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版权所有 © 2021 美国化学学会
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Abstract 摘要
Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer therapy. Herein, we describe the rational design, synthesis, and structure–activity relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1 inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition (52%@30 μM) led to the discovery of the most potent compound 40 with high affinity (Kd = 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds). Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-1-dependent manner. 40 exhibited favorable physicochemical properties and pharmacokinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively, these findings implicate that compound 40 is a promising antitumor agent that deserves further preclinical evaluations.
髓系细胞白血病 1（Mcl-1）蛋白是细胞凋亡的一个关键负调控因子，开发 Mcl-1 抑制剂一直是一种极具吸引力的癌症治疗策略。在此，我们介绍了作为 Mcl-1 抑制剂的 3,5-二甲基-4-磺酰基-1H-吡咯基化合物的合理设计、合成和结构-活性关系研究。通过对具有主要 Mcl-1 抑制作用（52%@30 μM）的命中化合物 11 的逐步优化，我们发现了最有效的化合物 40，该化合物具有高亲和力（K d = 0.23 nM）和优于其他 Bcl-2 家族蛋白的选择性（>40,000 倍）。机理研究表明，40 能以 Mcl-1 依赖性方式激活细胞凋亡信号通路。40 具有良好的理化特性和药代动力学特征（F% = 41.3%）。此外，口服 40 的耐受性良好，能有效抑制 MV4-11 异种移植模型中的肿瘤生长（T/C = 37.3%）。总之，这些发现表明化合物 40 是一种很有前景的抗肿瘤药物，值得进一步进行临床前评估。
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Note Added After ASAP Publication
ASAP 出版后添加的注释
The version of this paper that was published ASAP August 3, 2021, contained an error in the structure in the TOC and abstract graphic. The corrected version was reposted August 12, 2021.
本文于 2021 年 8 月 3 日在 ASAP 上发布的版本中，TOC 和摘要图的结构存在错误。更正后的版本已于 2021 年 8 月 12 日重新发布。
The evasion of apoptosis is one of the main reasons that results in cancer. (1) B-cell lymphoma-2 (Bcl-2) family proteins, composed of pro-apoptotic proteins (Bax and Bak), anti-apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1), and BH3-only proteins (Bid, Noxa, and Puma), (2) have long been identified as pivotal apoptosis regulators. (3) In apoptosis signaling, dimerization of pro-apoptotic members Bax and Bak can form oligomers to depolarize the outer mitochondrial membrane (OMM), (4) which can release mitochondrial proteins to induce apoptosis. (5) Mcl-1 was first found by analyzing gene expression of the ML-1 human myeloid leukemia cell line. (6) Like other anti-apoptotic members, Mcl-1 contains a binding groove comprising Bcl-2 homology (BH) 1-3 domains. (7) Such a groove can bind to the BH3 domains of pro-apoptotic members, (8) which disturbs the formation of Bax/Bak homo/heterodimers and further inhibits apoptosis. (9,10) Mcl-1 is important for the survival and development of normal cells, such as embryogenesis, (11) maintenance of hepatocytes, (12) B and T-cells, (13) and neuron development. (14) However, overexpression of Mcl-1 has been widely observed in cancers, such as chronic myeloid leukemia (CML), (15) multiple myeloma (MM), (16) non-small cell lung cancer (NSCLC), (17) and colon cancer. (18) There is also evidence showing that Mcl-1 is closely associated with cancer relapse, (19) immortalization, (20) and drug resistance. (21) Therefore, targeting Mcl-1 is an attractive strategy for cancer therapy. In 2016, venetoclax (Bcl-2 selective inhibitor) reported by AbbVie (22) was approved by the FDA for the treatment of 17p gene deficiency chronic lymphocytic leukemia (CLL), and targeting Bcl-2 family proteins proves to be a practical strategy for cancer therapy.
逃避细胞凋亡是导致癌症的主要原因之一。(1) B细胞淋巴瘤-2（Bcl-2）家族蛋白由促凋亡蛋白（Bax和Bak）、抗凋亡蛋白（Bcl-2、Bcl-X L 、Bcl-w、Mcl-1和A1）和纯BH3蛋白（Bid、Noxa和Puma）组成，(2) 早已被确认为关键的凋亡调节因子。(3）在细胞凋亡信号传导过程中，促凋亡成员 Bax 和 Bak 的二聚体可形成寡聚体，使线粒体外膜（OMM）去极化，（4）从而释放线粒体蛋白，诱导细胞凋亡。(5) Mcl-1 最早是通过分析 ML-1 人类髓性白血病细胞系的基因表达发现的。(6) 与其他抗凋亡成员一样，Mcl-1 包含一个由 Bcl-2 同源（BH）1-3 结构域组成的结合槽。(7) 这种沟槽可与促凋亡成员的 BH3 结构域结合，(8) 从而干扰 Bax/Bak 同源/异源二聚体的形成，进一步抑制细胞凋亡。(9,10）Mcl-1 对正常细胞的生存和发育非常重要，如胚胎发育、（11）肝细胞的维持、（12）B 细胞和 T 细胞、（13）神经元的发育。(14）然而，在慢性髓性白血病（CML）、（15）多发性骨髓瘤（MM）、（16）非小细胞肺癌（NSCLC）、（17）和结肠癌等癌症中已广泛观察到 Mcl-1 的过表达。(18）还有证据表明，Mcl-1 与癌症复发、（19）永生化（20）和耐药性密切相关。(21）因此，靶向 Mcl-1 是一种有吸引力的癌症治疗策略。2016 年，艾伯维公司（AbbVie）报道的 Venetoclax（Bcl-2 选择性抑制剂）（22）被 FDA 批准用于治疗 17p 基因缺失的慢性淋巴细胞白血病（CLL），靶向 Bcl-2 家族蛋白被证明是一种切实可行的癌症治疗策略。
逃避细胞凋亡是导致癌症的主要原因之一。(1) B细胞淋巴瘤-2（Bcl-2）家族蛋白由促凋亡蛋白（Bax和Bak）、抗凋亡蛋白（Bcl-2、Bcl-X L 、Bcl-w、Mcl-1和A1）和纯BH3蛋白（Bid、Noxa和Puma）组成，(2) 早已被确认为关键的凋亡调节因子。(3）在细胞凋亡信号传导过程中，促凋亡成员 Bax 和 Bak 的二聚体可形成寡聚体，使线粒体外膜（OMM）去极化，（4）从而释放线粒体蛋白，诱导细胞凋亡。(5) Mcl-1 最早是通过分析 ML-1 人类髓性白血病细胞系的基因表达发现的。(6) 与其他抗凋亡成员一样，Mcl-1 包含一个由 Bcl-2 同源（BH）1-3 结构域组成的结合槽。(7) 这种沟槽可与促凋亡成员的 BH3 结构域结合，(8) 从而干扰 Bax/Bak 同源/异源二聚体的形成，进一步抑制细胞凋亡。(9,10）Mcl-1 对正常细胞的生存和发育非常重要，如胚胎发育、（11）肝细胞的维持、（12）B 细胞和 T 细胞、（13）神经元的发育。(14）然而，在慢性髓性白血病（CML）、（15）多发性骨髓瘤（MM）、（16）非小细胞肺癌（NSCLC）、（17）和结肠癌等癌症中已广泛观察到 Mcl-1 的过表达。(18）还有证据表明，Mcl-1 与癌症复发、（19）永生化（20）和耐药性密切相关。(21）因此，靶向 Mcl-1 是一种有吸引力的癌症治疗策略。2016 年，艾伯维公司（AbbVie）报道的 Venetoclax（Bcl-2 选择性抑制剂）（22）被 FDA 批准用于治疗 17p 基因缺失的慢性淋巴细胞白血病（CLL），靶向 Bcl-2 家族蛋白被证明是一种切实可行的癌症治疗策略。
Mcl-1 has been identified as the largest member with 350 amino acid residues among anti-apoptotic proteins. (14) Of note, Mcl-1 also differs from other anti-apoptotic members in that it possesses four PEST domains composed of proline (P), glutamate (E), serine (S), and threonine (T) residues. (23) Such domains are targets of E3 ubiquitin-ligases, which promote the degradation of Mcl-1. (24) The binding groove of Mcl-1 is composed of P1–P4 pockets. In the binding mode of Mcl-1 and Bim, four conserved hydrophobic residues h1–h4 (Ile58, Leu62, Ile65, and Phe69) of the Bim BH3 domain protrude into four hydrophobic pockets (P1–P4) of the Mcl-1 (Figure 1) binding groove, respectively. (25) Additionally, there is a salt-bridge interaction between Asp67 of the Bim BH3 domain and Arg263 of Mcl-1. (26) Small molecules that mimic the hot spots of BH3-only proteins can occupy the BH3 binding groove of Mcl-1, which relieves pro-apoptotic proteins to induce tumor cell apoptosis. (27) Therefore, developing BH3 mimetic Mcl-1 inhibitors is a promising strategy for cancer therapy. (28−30)
在抗凋亡蛋白中，Mcl-1 是最大的成员，有 350 个氨基酸残基。(14)值得注意的是，Mcl-1 与其他抗凋亡成员的不同之处还在于，它拥有四个由脯氨酸（P）、谷氨酸（E）、丝氨酸（S）和苏氨酸（T）残基组成的 PEST 结构域。(23）这些结构域是 E3 泛素连接酶的靶标，可促进 Mcl-1 的降解。(24) Mcl-1 的结合槽由 P1-P4 口袋组成。在 Mcl-1 和 Bim 的结合模式中，Bim BH3 结构域的四个保守疏水残基 h1-h4（Ile58、Leu62、Ile65 和 Phe69）分别突入 Mcl-1（图 1）结合沟的四个疏水口袋（P1-P4）。(25）此外，Bim BH3 结构域的 Asp67 与 Mcl-1 的 Arg263 之间存在盐桥相互作用。(26）模仿纯 BH3 蛋白热点的小分子可以占据 Mcl-1 的 BH3 结合沟，从而解除促凋亡蛋白的作用，诱导肿瘤细胞凋亡。(27）因此，开发 BH3 拟态 Mcl-1 抑制剂是一种很有前景的癌症治疗策略。(28-30)
在抗凋亡蛋白中，Mcl-1 是最大的成员，有 350 个氨基酸残基。(14)值得注意的是，Mcl-1 与其他抗凋亡成员的不同之处还在于，它拥有四个由脯氨酸（P）、谷氨酸（E）、丝氨酸（S）和苏氨酸（T）残基组成的 PEST 结构域。(23）这些结构域是 E3 泛素连接酶的靶标，可促进 Mcl-1 的降解。(24) Mcl-1 的结合槽由 P1-P4 口袋组成。在 Mcl-1 和 Bim 的结合模式中，Bim BH3 结构域的四个保守疏水残基 h1-h4（Ile58、Leu62、Ile65 和 Phe69）分别突入 Mcl-1（图 1）结合沟的四个疏水口袋（P1-P4）。(25）此外，Bim BH3 结构域的 Asp67 与 Mcl-1 的 Arg263 之间存在盐桥相互作用。(26）模仿纯 BH3 蛋白热点的小分子可以占据 Mcl-1 的 BH3 结合沟，从而解除促凋亡蛋白的作用，诱导肿瘤细胞凋亡。(27）因此，开发 BH3 拟态 Mcl-1 抑制剂是一种很有前景的癌症治疗策略。(28-30)
Figure 1 图 1
Figure 1. Binding mode of Mcl-1 and Bim BH3 (PDB 2NL9).
图 1.Mcl-1 与 Bim BH3 的结合模式（PDB 2NL9）。
The large and shallow binding groove makes Mcl-1 intrinsically difficult to be targeted. (31) Several groups have developed peptide Mcl-1 inhibitors. (32−35) However, during the last decade, medicinal chemists have contributed more to developing Mcl-1 small molecule inhibitors. (36−38) As shown in Figure 2, Zhang’s team has reported a series of phenalene-based Mcl-1 inhibitors, (39−41) of which the most potent 1 showed a Ki of 5 nM. (42) Indole-based Mcl-1 inhibitors have also been investigated in depth. In 2008, Abbott’s work pioneered the discovery of indole Mcl-1 inhibitors. (43,44) In 2013, Fesik’s team discovered an indole-based Mcl-1 inhibitor 2 by using the fragment merging method. (45) Further modifications (46−48) led to VU661013 (3) (49) with an ideal in vivo antitumor efficacy. (50) In 2015, AbbVie identified A1210477 (4) as an indole-based Mcl-1 inhibitor, (51,52) which is widely used as an in vitro chemical tool. (27,53) Based on A1210477, some other Mcl-1 inhibitors with an indole core were developed. (54,55) Recently, the macrocyclization strategy has been widely used in the discovery of Mcl-1 inhibitors. (56) In 2018, AstraZeneca reported the macrocyclic clinical candidate AZD5991 (5) (57) (NCT03218683) with excellent antitumor efficacy. (58,59) In 2018, Amgen (60) discovered the first orally efficacious Mcl-1 inhibitor AMG176 (6) with a macrocyclic structure. AMG176 has also been promoted into the clinical trial (NCT02675452). Apart from that, compounds with other skeletons have also been identified as potent Mcl-1 inhibitors. (61−66) Geneste’s group developed thieno[2,3-d]pyrimidine-based Mcl-1 inhibitors S63845 (7) (67) and MIK665 (8) (68) with excellent in vivo potency. To date, the phase I clinical trial of MIK665 has been completed (NCT02979366).
Mcl-1 的结合沟槽大而浅，因此很难成为靶向药物。(31）一些研究小组开发了多肽 Mcl-1 抑制剂。(32-35）然而，在过去十年中，药物化学家在开发 Mcl-1 小分子抑制剂方面做出了更多贡献。(36-38）如图 2 所示，Zhang 团队报告了一系列苯亚甲基 Mcl-1 抑制剂，（39-41）其中最有效的 1 种抑制剂的 K 值为 5 nM。2008 年，Abbott 的研究率先发现了吲哚类 Mcl-1 抑制剂。(43,44）2013 年，Fesik 团队利用片段合并法发现了一种基于吲哚的 Mcl-1 抑制剂 2。(45) 进一步修饰（46-48）后，VU661013 (3) (49)具有理想的体内抗肿瘤疗效。(50）2015 年，艾伯维公司发现 A1210477（4）是一种基于吲哚的 Mcl-1 抑制剂，（51,52）被广泛用作体外化学工具。(27,53）在 A1210477 的基础上，又开发出了其他一些以吲哚为核心的 Mcl-1 抑制剂。(54,55）最近，大环化策略被广泛应用于 Mcl-1 抑制剂的发现。(56）2018年，阿斯利康公司报道了大环化临床候选药物AZD5991（5）（57）（NCT03218683），具有极佳的抗肿瘤疗效。(58,59）2018年，安进公司（60）发现了首个具有口服疗效的大环结构Mcl-1抑制剂AMG176（6）。AMG176 也已被推进临床试验（NCT02675452）。除此之外，具有其他骨架的化合物也被确认为强效的 Mcl-1 抑制剂。(61-66）Geneste 小组开发的基于噻吩并[2,3-d]嘧啶的 Mcl-1 抑制剂 S63845 (7) (67) 和 MIK665 (8) (68)具有极佳的体内效力。迄今为止，MIK665 的 I 期临床试验已经完成（NCT02979366）。
Mcl-1 的结合沟槽大而浅，因此很难成为靶向药物。(31）一些研究小组开发了多肽 Mcl-1 抑制剂。(32-35）然而，在过去十年中，药物化学家在开发 Mcl-1 小分子抑制剂方面做出了更多贡献。(36-38）如图 2 所示，Zhang 团队报告了一系列苯亚甲基 Mcl-1 抑制剂，（39-41）其中最有效的 1 种抑制剂的 K 值为 5 nM。2008 年，Abbott 的研究率先发现了吲哚类 Mcl-1 抑制剂。(43,44）2013 年，Fesik 团队利用片段合并法发现了一种基于吲哚的 Mcl-1 抑制剂 2。(45) 进一步修饰（46-48）后，VU661013 (3) (49)具有理想的体内抗肿瘤疗效。(50）2015 年，艾伯维公司发现 A1210477（4）是一种基于吲哚的 Mcl-1 抑制剂，（51,52）被广泛用作体外化学工具。(27,53）在 A1210477 的基础上，又开发出了其他一些以吲哚为核心的 Mcl-1 抑制剂。(54,55）最近，大环化策略被广泛应用于 Mcl-1 抑制剂的发现。(56）2018年，阿斯利康公司报道了大环化临床候选药物AZD5991（5）（57）（NCT03218683），具有极佳的抗肿瘤疗效。(58,59）2018年，安进公司（60）发现了首个具有口服疗效的大环结构Mcl-1抑制剂AMG176（6）。AMG176 也已被推进临床试验（NCT02675452）。除此之外，具有其他骨架的化合物也被确认为强效的 Mcl-1 抑制剂。(61-66）Geneste 小组开发的基于噻吩并[2,3-d]嘧啶的 Mcl-1 抑制剂 S63845 (7) (67) 和 MIK665 (8) (68)具有极佳的体内效力。迄今为止，MIK665 的 I 期临床试验已经完成（NCT02979366）。
Figure 2 图 2
Figure 2. Representative Mcl-1 inhibitors.
图 2.具有代表性的 Mcl-1 抑制剂。
However, most Mcl-1 inhibitors suffer druggability issues like poor membrane permeability, (47) low oral bioavailability, (38) and big molecular weight. Such problems hinder potent Mcl-1 inhibitors from coming into clinical trials and further from being approved for cancer therapy. As pan Bcl-2 inhibition leads to undesired side effects, (69) selectivity against Mcl-1 is another issue that deserves consideration. Herein, we describe the design, synthesis, and structure–activity relationships (SARs) of a series of pyrrole-based Mcl-1 inhibitors. Replacing the indole core of 2 with a pyrrole ring yielded hit compound 11 with primary Mcl-1 inhibitory potency (52%@30 μM). Further optimization of 11 led to the discovery of the most potent compound 40 (Mcl-1, Ki = 20 nM). 40 possessed high Mcl-1 binding affinity (Kd = 0.23 nM) and remarkable selectivity over other Bcl-2 family members (Bcl-2, Bcl2A1, Bcl-xL, and Bcl-w, Kd > 10,000 nM). In addition, 40 exhibited ideal cellular activity and oral bioavailability (F% = 41.3%). In balb/c nude mice loaded with MV4-11 xenografts, 40 showed desired antitumor effects through oral administration (T/C = 37.3%).
然而，大多数 Mcl-1 抑制剂都存在可药性问题，如膜渗透性差、（47）口服生物利用度低、（38）分子量大。这些问题阻碍了强效 Mcl-1 抑制剂进入临床试验，更阻碍了它们被批准用于癌症治疗。由于泛用 Bcl-2 抑制会导致不良副作用，（69）针对 Mcl-1 的选择性是另一个值得考虑的问题。在此，我们介绍了一系列基于吡咯的 Mcl-1 抑制剂的设计、合成和结构-活性关系 (SAR)。用吡咯环取代 2 的吲哚核心，得到了具有主要 Mcl-1 抑制效力（52%@30 μM）的热门化合物 11。进一步优化 11 后，发现了最有效的化合物 40（Mcl-1，K = 20 nM）。40 具有很高的 Mcl-1 结合亲和力（K d = 0.23 nM），并对其他 Bcl-2 家族成员（Bcl-2、Bcl2A1、Bcl-xL 和 Bcl-w，K d > 10,000 nM）具有显著的选择性。此外，40 表现出理想的细胞活性和口服生物利用度（F% = 41.3%）。在装有 MV4-11 异种移植物的 balb/c 裸鼠体内，40 通过口服显示出理想的抗肿瘤效果（T/C = 37.3%）。
然而，大多数 Mcl-1 抑制剂都存在可药性问题，如膜渗透性差、（47）口服生物利用度低、（38）分子量大。这些问题阻碍了强效 Mcl-1 抑制剂进入临床试验，更阻碍了它们被批准用于癌症治疗。由于泛用 Bcl-2 抑制会导致不良副作用，（69）针对 Mcl-1 的选择性是另一个值得考虑的问题。在此，我们介绍了一系列基于吡咯的 Mcl-1 抑制剂的设计、合成和结构-活性关系 (SAR)。用吡咯环取代 2 的吲哚核心，得到了具有主要 Mcl-1 抑制效力（52%@30 μM）的热门化合物 11。进一步优化 11 后，发现了最有效的化合物 40（Mcl-1，K = 20 nM）。40 具有很高的 Mcl-1 结合亲和力（K d = 0.23 nM），并对其他 Bcl-2 家族成员（Bcl-2、Bcl2A1、Bcl-xL 和 Bcl-w，K d > 10,000 nM）具有显著的选择性。此外，40 表现出理想的细胞活性和口服生物利用度（F% = 41.3%）。在装有 MV4-11 异种移植物的 balb/c 裸鼠体内，40 通过口服显示出理想的抗肿瘤效果（T/C = 37.3%）。
Results and Discussion 结果与讨论
Discovery of Initial Hit 11 as an Mcl-1 Inhibitor
发现作为 Mcl-1 抑制剂的首发药物 11
In 2013, through the fragment-based drug design, Fesik’s team (45) reported a series of indole-based Mcl-1 inhibitors, of which the most potent compound 2 showed nanomolar Mcl-1 inhibitory potency (Ki = 55 nM) and proper selectivity over Bcl-2 (Ki = 870 nM) and Bcl-xL (Ki > 15,000 nM). The deep P2 pocket and polar Arg263 differ the binding groove of Mcl-1 from that of other Bcl-2 family members. (45,70) As shown in Figure 3A, the complex of 2/Mcl-1 (PDB 4HW2) shows that the 4-atom linker of 2 protrudes into the P2 pocket and the indole-2-carboxyl acid group forms key polar interaction with Arg263. In their SAR study, tethering a 4-atom linker to the NH of the indole core (9b) retained the Mcl-1 inhibitory activity of the corresponding 3-substituted indole compound (9a) (Figure 3B). Compound 10 was reported to bind to Mcl-1 with high affinity. (51) The binding mode of 10 (PDB 6B4U) demonstrates that the morpholine ring is solvent-exposed and the phenyl ring is kept out of the P2 pocket due to the rigid indole core. Combining these findings, in our design, the pyrrole-2-carboxylic acid group was kept for maintaining the key polar interaction with Agr263. The 3,5-dimethyl-4-chlorine phenyl-linked 4-atom linker has been fully reported to be suitable for P2 pocket binding. (47,49) Consequently, this moiety was kept and attached to the NH group of the pyrrole core to occupy the P2 pocket. For locating the solvent-exposed phenyl ring of 10 in the P2 pocket, a flexible methylene linker was introduced between the 4-methyl phenyl group and the pyrrole core. Compound 11 was then designed and prepared with a moderate Mcl-1 inhibition ratio (52%@30 μM) determined by a fluorescence polarization (FP)-based assay (Figure 3C).
2013年，Fesik团队（45）通过基于片段的药物设计，报道了一系列基于吲哚的Mcl-1抑制剂，其中最有效的化合物2显示出纳摩尔级的Mcl-1抑制效力（K = 55 nM），并对Bcl-2（K = 870 nM）和Bcl-xL（K > 15,000 nM）具有适当的选择性。深 P2 袋和极性 Arg263 使 Mcl-1 的结合槽与其他 Bcl-2 家族成员的结合槽不同。(45,70) 如图 3A 所示，2/Mcl-1 的复合物（PDB 4HW2）显示，2 的 4 原子连接体突入 P2 口袋，吲哚-2-羧酸基团与 Arg263 形成关键的极性相互作用。在他们的 SAR 研究中，将 4 原子连接体拴在吲哚核心的 NH 上（9b）保留了相应的 3 取代吲哚化合物（9a）的 Mcl-1 抑制活性（图 3B）。据报道，化合物 10 与 Mcl-1 具有高亲和力。(51) 10 的结合模式（PDB 6B4U）表明吗啉环是溶剂外露的，而苯基环由于吲哚核心的刚性而被挡在 P2 口袋之外。结合这些发现，我们在设计中保留了吡咯-2-羧酸基团，以保持与 Agr263 的关键极性相互作用。据充分报道，3,5-二甲基-4-氯苯基连接的 4 原子连接体适用于 P2 口袋结合。(47,49) 因此，我们保留了这一分子，并将其连接到吡咯核心的 NH 基团上，以占据 P2 口袋。为了将 10 的暴露于溶剂的苯基环置于 P2 口袋中，在 4-甲基苯基和吡咯核心之间引入了柔性亚甲基连接体。随后设计并制备了化合物 11，通过基于荧光偏振（FP）的检测方法测定，其对 Mcl-1 的抑制率适中（52%@30 μM）（图 3C）。
2013年，Fesik团队（45）通过基于片段的药物设计，报道了一系列基于吲哚的Mcl-1抑制剂，其中最有效的化合物2显示出纳摩尔级的Mcl-1抑制效力（K = 55 nM），并对Bcl-2（K = 870 nM）和Bcl-xL（K > 15,000 nM）具有适当的选择性。深 P2 袋和极性 Arg263 使 Mcl-1 的结合槽与其他 Bcl-2 家族成员的结合槽不同。(45,70) 如图 3A 所示，2/Mcl-1 的复合物（PDB 4HW2）显示，2 的 4 原子连接体突入 P2 口袋，吲哚-2-羧酸基团与 Arg263 形成关键的极性相互作用。在他们的 SAR 研究中，将 4 原子连接体拴在吲哚核心的 NH 上（9b）保留了相应的 3 取代吲哚化合物（9a）的 Mcl-1 抑制活性（图 3B）。据报道，化合物 10 与 Mcl-1 具有高亲和力。(51) 10 的结合模式（PDB 6B4U）表明吗啉环是溶剂外露的，而苯基环由于吲哚核心的刚性而被挡在 P2 口袋之外。结合这些发现，我们在设计中保留了吡咯-2-羧酸基团，以保持与 Agr263 的关键极性相互作用。据充分报道，3,5-二甲基-4-氯苯基连接的 4 原子连接体适用于 P2 口袋结合。(47,49) 因此，我们保留了这一分子，并将其连接到吡咯核心的 NH 基团上，以占据 P2 口袋。为了将 10 的暴露于溶剂的苯基环置于 P2 口袋中，在 4-甲基苯基和吡咯核心之间引入了柔性亚甲基连接体。随后设计并制备了化合物 11，通过基于荧光偏振（FP）的检测方法测定，其对 Mcl-1 的抑制率适中（52%@30 μM）（图 3C）。
Figure 3 图 3
Optimizations of the Linker between the 4-Methyl Phenyl Group and the Pyrrole Core
优化 4-甲基苯基和吡咯核心之间的连接物
According to our design, the linker between the 4-methyl phenyl group and the pyrrole core was vital for locating the phenyl group into the P2 pocket. To explore a preferable linker for potency, 12–15 with various linkers were prepared. The FP assay was used to evaluate the Mcl-1 inhibitory activities of our compounds. AZD5991 and A1210477 were selected as positive controls. As shown in Table 1, the introduction of the rigid carbonyl linker (12) and the sulfone linker (13) improved Mcl-1 inhibitory activity. However, two-atom linkers (14, 15) showed abolished potency. The primary SAR results indicated that the conformation and length of the sulfone linker were the most favorable. As a result, compound 13 with a sulfone linker was selected for further modifications.
根据我们的设计，4-甲基苯基和吡咯核心之间的连接体对于将苯基定位到 P2 口袋中至关重要。为了探索更合适的连接体以提高药效，我们制备了 12-15 种具有不同连接体的化合物。FP 试验用于评估我们化合物的 Mcl-1 抑制活性。AZD5991 和 A1210477 被选为阳性对照。如表 1 所示，刚性羰基连接体（12）和砜连接体（13）的引入提高了 Mcl-1 抑制活性。然而，双原子连接体（14、15）的效力被削弱。主要的 SAR 结果表明，砜连接体的构象和长度是最有利的。因此，带有砜连接体的化合物 13 被选中作进一步修饰。
根据我们的设计，4-甲基苯基和吡咯核心之间的连接体对于将苯基定位到 P2 口袋中至关重要。为了探索更合适的连接体以提高药效，我们制备了 12-15 种具有不同连接体的化合物。FP 试验用于评估我们化合物的 Mcl-1 抑制活性。AZD5991 和 A1210477 被选为阳性对照。如表 1 所示，刚性羰基连接体（12）和砜连接体（13）的引入提高了 Mcl-1 抑制活性。然而，双原子连接体（14、15）的效力被削弱。主要的 SAR 结果表明，砜连接体的构象和长度是最有利的。因此，带有砜连接体的化合物 13 被选中作进一步修饰。
Table 1. Effects of Linkers on Mcl-1 Inhibition
表 1.连接体对 Mcl-1 抑制作用的影响
表 1.连接体对 Mcl-1 抑制作用的影响
Then, we investigated the binding mode of 13 with Mcl-1 by molecular docking. As shown in Figure 4, as expected, the pyrrole-2-carboxyl group formed a hydrogen bond with Arg263. The 3,5-dimethyl-4-chlorine phenyl-linked 4-atom linker protruded into the P2 pocket. The docking study of 13 also gave the reason for the advantage of the sulfone linker for potency: the sulfone linker located the phenyl ring into the P2 pocket. Consequently, the phenyl ring could form additional hydrophobic interactions with Ala227 and Met231.
然后，我们通过分子对接研究了 13 与 Mcl-1 的结合模式。如图 4 所示，正如预期的那样，吡咯-2-羧基与 Arg263 形成了氢键。3,5-二甲基-4-氯苯基连接的 4 个原子连接体突出到 P2 口袋中。对 13 的对接研究也揭示了砜基连接体在药效方面具有优势的原因：砜基连接体将苯环置于 P2 口袋中。因此，苯基环可以与 Ala227 和 Met231 形成额外的疏水相互作用。
然后，我们通过分子对接研究了 13 与 Mcl-1 的结合模式。如图 4 所示，正如预期的那样，吡咯-2-羧基与 Arg263 形成了氢键。3,5-二甲基-4-氯苯基连接的 4 个原子连接体突出到 P2 口袋中。对 13 的对接研究也揭示了砜基连接体在药效方面具有优势的原因：砜基连接体将苯环置于 P2 口袋中。因此，苯基环可以与 Ala227 和 Met231 形成额外的疏水相互作用。
Figure 4 图 4
Figure 4. Proposed binding mode of compound 13 with Mcl-1 using molecular docking. The docking site was derived from the position of the small-molecular ligand cocrystallized in the binding site of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. The hydrogen bonds are represented by green dashed lines, and the π–σ and π–alkyl interactions are represented by pink dashed lines. The carbon atoms of small molecules and Mcl-1 residues are colored cyan and green, respectively.
图 4.利用分子对接法推测的化合物 13 与 Mcl-1 的结合模式。对接位点是根据小分子配体在 Mcl-1 结合位点（PDB 4HW2）中的共晶体位置推导出来的。配体用棍棒表示。氢键用绿色虚线表示，π-σ 和 π-烷基相互作用用粉色虚线表示。小分子和 Mcl-1 残基的碳原子分别用青色和绿色表示。
Reinspection of the binding mode of 13 gave an idea that substitution at the 5-position of the pyrrole core could form more interactions in the P2 pocket (Figure 4). 3,5-Dimethyl pyrrole-based compound 16 was then prepared. As shown in Table 2, 16 showed better performance compared to 13. However, 3,5-diethyl pyrrole-based compound 17 was not preferred. Only replacement of the 5-methyl group of 16 with an ethyl group (18) impaired potency. In contrast, replacement of the 3-methyl group of 16 with an ethyl group (19) retained comparable activity with 16. Both removal of 5-methyl substitution at the pyrrole core (20) and further extension at the 5-position (21) were unfavorable. These results demonstrated that the appropriate size of 5-substitution was important for Mcl-1 inhibition and the methyl substituent was optimal. Further extension at the 3-position of 19 (22) retained potency. To further confirm the advantage of the sulfone linker for activity, analogues 23 with a flexible sulfide linker and 24 with a 2-atom length linker were prepared. As shown in Table 2, the sulfide linker (23) showed a minor decreased potency compared to the sulfone linker (16). Introduction of a 2-atom linker (24) remarkably impaired Mcl-1 inhibition.
对 13 的结合模式进行重新检查后发现，在吡咯核心的 5 位进行取代可以在 P2 口袋中形成更多的相互作用（图 4）。于是制备了基于 3,5-二甲基吡咯的化合物 16。如表 2 所示，与 13 相比，16 表现出更好的性能。然而，3,5-二乙基吡咯基化合物 17 并不理想。只有将 16 的 5-甲基基团替换为乙基基团（18）才会降低药效。相反，用乙基取代 16 的 3-甲基（19），其活性与 16 相当。去除吡咯核心的 5-甲基取代基（20）和进一步扩展 5-位（21）都是不利的。这些结果表明，5-取代基的适当大小对抑制 Mcl-1 非常重要，而甲基取代基是最佳选择。19 的 3 位进一步延伸（22）保留了药效。为了进一步证实砜连接体在活性方面的优势，我们制备了具有柔性硫化物连接体的类似物 23 和具有 2 个原子长度连接体的类似物 24。如表 2 所示，与砜连接物（16）相比，硫化物连接物（23）的药效略有下降。引入 2 个原子的连接体（24）明显削弱了对 Mcl-1 的抑制作用。
对 13 的结合模式进行重新检查后发现，在吡咯核心的 5 位进行取代可以在 P2 口袋中形成更多的相互作用（图 4）。于是制备了基于 3,5-二甲基吡咯的化合物 16。如表 2 所示，与 13 相比，16 表现出更好的性能。然而，3,5-二乙基吡咯基化合物 17 并不理想。只有将 16 的 5-甲基基团替换为乙基基团（18）才会降低药效。相反，用乙基取代 16 的 3-甲基（19），其活性与 16 相当。去除吡咯核心的 5-甲基取代基（20）和进一步扩展 5-位（21）都是不利的。这些结果表明，5-取代基的适当大小对抑制 Mcl-1 非常重要，而甲基取代基是最佳选择。19 的 3 位进一步延伸（22）保留了药效。为了进一步证实砜连接体在活性方面的优势，我们制备了具有柔性硫化物连接体的类似物 23 和具有 2 个原子长度连接体的类似物 24。如表 2 所示，与砜连接物（16）相比，硫化物连接物（23）的药效略有下降。引入 2 个原子的连接体（24）明显削弱了对 Mcl-1 的抑制作用。
Table 2. Effects of Pyrrole Substituents on Mcl-1 Inhibition
表 2.吡咯取代基对 Mcl-1 抑制作用的影响
表 2.吡咯取代基对 Mcl-1 抑制作用的影响
Then, the binding mode of 16 was elucidated by the docking study. As shown in Figure 5, The 5-methyl of the pyrrole core inserted into a narrow cavity and formed additional hydrophobic interactions with Phe228, Met231, and Phe270 in the P2 pocket, which led to an improvement in activity.
然后，通过对接研究阐明了 16 的结合模式。如图 5 所示，吡咯核心的 5-甲基插入到一个狭窄的空腔中，并与 P2 口袋中的 Phe228、Met231 和 Phe270 形成额外的疏水相互作用，从而提高了活性。
然后，通过对接研究阐明了 16 的结合模式。如图 5 所示，吡咯核心的 5-甲基插入到一个狭窄的空腔中，并与 P2 口袋中的 Phe228、Met231 和 Phe270 形成额外的疏水相互作用，从而提高了活性。
Figure 5 图 5
Figure 5. Proposed binding mode of 16 with Mcl-1 using molecular docking. The docking site was derived from the position of the small-molecular ligand cocrystallized in the binding site of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. The hydrogen bonds are represented by green dashed lines, and the π–σ and π–alkyl interactions are represented by pink dashed lines. The carbon atoms of small molecules and Mcl-1 residues are colored cyan and green, respectively.
图 5.利用分子对接法推测的 16 与 Mcl-1 的结合模式。对接位点是根据小分子配体在 Mcl-1 结合位点（PDB 4HW2）中的共晶体位置推导出来的。配体用棍棒表示。氢键用绿色虚线表示，π-σ 和 π-烷基相互作用用粉色虚线表示。小分子和 Mcl-1 残基的碳原子分别用青色和绿色表示。
Effects of Ring Transformation at the Sulfone Linker on Mcl-1 Inhibition
砜连接体上的环转变对 Mcl-1 抑制作用的影响
Then, we evaluated the effects of various rings at the sulfone linker on Mcl-1 inhibitory activity. Replacing the phenyl ring with more polar pyridyl rings (25, 26) exhibited a more than 10-fold decreased potency. The thienyl ring (27) showed improved activity, while the bicyclic naphthyl ring (28) showed weaker activity compared to 16. The cycloalkane analogs are inactive (29, 30), which suggested that the π-system of the rings attached to the sulfone linker was necessary for Mcl-1 inhibition (Table 3).
然后，我们评估了砜连接环上的各种环对 Mcl-1 抑制活性的影响。用极性更强的吡啶环取代苯基环（25、26）后，其效力降低了 10 倍以上。噻吩环（27）的活性有所提高，而双环萘环（28）的活性则比 16 弱。环烷类似物没有活性（29、30），这表明连接到砜连接体上的环的π-系统是抑制 Mcl-1 的必要条件（表 3）。
然后，我们评估了砜连接环上的各种环对 Mcl-1 抑制活性的影响。用极性更强的吡啶环取代苯基环（25、26）后，其效力降低了 10 倍以上。噻吩环（27）的活性有所提高，而双环萘环（28）的活性则比 16 弱。环烷类似物没有活性（29、30），这表明连接到砜连接体上的环的π-系统是抑制 Mcl-1 的必要条件（表 3）。
Table 3. SAR Study Focused on Rings on the Sulfone Linker
表 3.以砜基连接体上的环为重点的 SAR 研究
表 3.以砜基连接体上的环为重点的 SAR 研究
In addition to Mcl-1 inhibition activity, we also predicted drug-like properties of all compounds by ADMET 10.0 software for avoiding potential druggability issues (Supporting Information, Table S1). Most compounds possessed proper predicated solubility, absorption property, and CYP inhibition profiles. Thus, the detailed SAR study of the substituted phenyl ring was carried out to enhance the activity.
除了 Mcl-1 抑制活性外，我们还通过 ADMET 10.0 软件预测了所有化合物的类药物特性，以避免潜在的可药性问题（佐证资料，表 S1）。大多数化合物都具有适当的预测溶解度、吸收特性和 CYP 抑制特征。因此，我们对取代的苯基环进行了详细的 SAR 研究，以提高其活性。
除了 Mcl-1 抑制活性外，我们还通过 ADMET 10.0 软件预测了所有化合物的类药物特性，以避免潜在的可药性问题（佐证资料，表 S1）。大多数化合物都具有适当的预测溶解度、吸收特性和 CYP 抑制特征。因此，我们对取代的苯基环进行了详细的 SAR 研究，以提高其活性。
Modifications on the Substituents of the Phenyl Ring
对苯环取代基的修改
First, we compared the substituent position effects of the phenyl ring. A library of compounds (31–43) with different substitution patterns on the phenyl ring was prepared. As shown in Table 4, 31 with a non-substituted phenyl ring possessed similar potency to 4-methyl-substituted 16. Substitutions at the 2-position and 3-position (32–37 and 39) yielded similar or decreased potency compared to 31 except for 38 with 3-Br substitution exhibiting improved inhibitory activity. Potency was improved by substitutions at the 4-position, as exemplified by compounds 40–43. The most potent compounds, 40 with 4-acetyl substitution and 43 with 4-OH substitution, showed about 10-fold improved potency.
首先，我们比较了苯基环上取代基位置的影响。我们制备了一个苯环上具有不同取代模式的化合物库（31-43）。如表 4 所示，未取代苯环的 31 与 4-甲基取代的 16 具有相似的效力。2 位和 3 位取代（32-37 和 39）产生的效力与 31 相似或有所降低，只有 3-Br 取代的 38 的抑制活性有所提高。在 4 位进行取代后，化合物 40-43 的效力有所提高。效力最强的化合物是 4-乙酰取代的 40 和 4-OH 取代的 43，其效力提高了约 10 倍。
首先，我们比较了苯基环上取代基位置的影响。我们制备了一个苯环上具有不同取代模式的化合物库（31-43）。如表 4 所示，未取代苯环的 31 与 4-甲基取代的 16 具有相似的效力。2 位和 3 位取代（32-37 和 39）产生的效力与 31 相似或有所降低，只有 3-Br 取代的 38 的抑制活性有所提高。在 4 位进行取代后，化合物 40-43 的效力有所提高。效力最强的化合物是 4-乙酰取代的 40 和 4-OH 取代的 43，其效力提高了约 10 倍。
Table 4. SAR Study Focused on Substitution on the Phenyl Ring
表 4.以苯环取代为重点的 SAR 研究
表 4.以苯环取代为重点的 SAR 研究
| cpd | R | Ki ± SE (μM) | cpd | R | Ki ± SE (μM) |
|---|---|---|---|---|---|
| 16 | 4-CH3 | 0.17 ± 0.04 | 45 | 4-isopropyl 4-异丙基 | 0.17 ± 0.03 |
| 31 | H | 0.19 ± 0.03 | 46 | 4-tert-butyl 4-叔丁基 | 0.34 ± 0.09 |
| 32 | 2-COCH3 | 0.50 ± 0.13 | 47 | 4-OEt | 0.14 ± 0.03 |
| 33 | 2-OCH3 | 0.43 ± 0.12 | 48 | 4-Ph 4 相 | 0.13 ± 0.03 |
| 34 | 2-Br 2 卧室 | >10 | 49 | 4-COPh | 0.38 ± 0.05 |
| 35 | 2-OH | 0.15 ± 0.02 | 50 | 4-OPh | 0.20 ± 0.03 |
| 36 | 3-COCH3 | 0.55 ± 0.16 | 51 | 4-F | 0.26 ± 0.10 |
| 37 | 3-OCH3 | 0.20 ± 0.05 | 52 | 4-Cl | 0.18 ± 0.03 |
| 38 | 3-Br 3 卧室 | 0.060 ± 0.013 | 53 | 4-NO2 4-无 {{0} | 0.42 ± 0.08 |
| 39 | 3-OH | 0.15 ± 0.04 | 54 | 4-COOH | 0.42 ± 0.07 |
| 40 | 4-COCH3 | 0.020 ± 0.001 | 55 | 4-NH2 4-NH {{0} | 0.34 ± 0.06 |
| 41 | 4-OCH3 | 0.070 ± 0.010 | 56 | 2,6-diMe 2,6-二甲基甲酰胺 | >10 |
| 42 | 4-Br | 0.040 ± 0.005 | 57 | 3,5-diMe 3,5-二甲基甲酰胺 | >10 |
| 43 | 4-OH | 0.018 ± 0.002 | AZD5991 | <0.001 <0.001 | |
| 44 | 4-ethyl 4-乙基 | 0.070 ± 0.020 0.070 ± 0.020 | A1210477 | 0.006 ± 0.001 0.006 ± 0.001 |
Compounds with various groups at the 4-position were then prepared (44–55). Generally, as shown in Table 4, the phenyl ring can tolerate a variety of substituents at the 4-position. 44 with 4-ethyl substitution exhibited a minor increased potency over 31. Meanwhile, disubstitution on the phenyl ring led to compounds 56–57 with abolished activity.
然后制备出了在 4 位上带有各种基团的化合物（44-55）。一般来说，如表 4 所示，苯基环可以容忍 4 位上的各种取代基。44 与 31 相比，4-乙基取代物的效力略有提高。同时，苯基环上的二取代导致 56-57 号化合物的活性降低。
然后制备出了在 4 位上带有各种基团的化合物（44-55）。一般来说，如表 4 所示，苯基环可以容忍 4 位上的各种取代基。44 与 31 相比，4-乙基取代物的效力略有提高。同时，苯基环上的二取代导致 56-57 号化合物的活性降低。
The most potent compounds of this set, 40 and 43, possessed suitable predicated ADME properties, except the poor predicated membrane permeability of 43 (Supporting Information, Table S1).
这组化合物中最强效的 40 和 43 具有合适的预示 ADME 特性，但 43 的预示膜渗透性较差（佐证资料，表 S1）。
这组化合物中最强效的 40 和 43 具有合适的预示 ADME 特性，但 43 的预示膜渗透性较差（佐证资料，表 S1）。
The binding modes of 40 and 43 were investigated by docking. As depicted in Figure 6, compared with 16, 40 and 43 possessed similar binding modes. In addition, the 4-acetyl group of 40 formed a hydrogen bond with Lys234 and the 4-OH of 43 formed a hydrogen bond with Ala227. The hydrogen bonds may contribute to the 10-fold improved Ki of both compounds.
通过对接研究了 40 和 43 的结合模式。如图 6 所示，与 16 相比，40 和 43 具有相似的结合模式。此外，40 的 4-乙酰基与 Lys234 形成了氢键，43 的 4-OH 与 Ala227 形成了氢键。氢键可能是这两种化合物的 K 值提高 10 倍的原因。
通过对接研究了 40 和 43 的结合模式。如图 6 所示，与 16 相比，40 和 43 具有相似的结合模式。此外，40 的 4-乙酰基与 Lys234 形成了氢键，43 的 4-OH 与 Ala227 形成了氢键。氢键可能是这两种化合物的 K 值提高 10 倍的原因。
Figure 6 图 6
Figure 6. Purposed binding modes of 40 (A) and 43 (B) with Mcl-1 using molecular docking. The docking site was derived from the position of the small-molecular ligand cocrystallized in the binding site of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. Hydrogen bonds are represented by green dashed lines, and π–σ and π–alkyl interactions are represented by pink dashed lines. The carbon atoms of small molecules and Mcl-1 residues are colored purple and green, respectively.
图 6.利用分子对接法推测的 40（A）和 43（B）与 Mcl-1 的结合模式。对接位点来自 Mcl-1 结合位点（PDB 4HW2）中共晶体化的小分子配体的位置。配体用棍棒表示。氢键用绿色虚线表示，π-σ 和 π-烷基相互作用用粉色虚线表示。小分子和 Mcl-1 残基的碳原子分别用紫色和绿色表示。
In Vitro Selectivity Evaluation
体外选择性评估
Representative compounds 27, 40, 41, 42, 43, and 44 were selected for testing their selectivity over other Bcl-2 family members (Bcl-2, Bcl-xL, and Bfl-1) by an FP-based assay or time-resolved fluorescence resonance energy transfer (TR-FRET) based assay. The results implicated that all compounds showed promising selectivity as no significant inhibition against other Bcl-2 family members was observed at a concentration of 10 μM (Table 5). 40 was selected for the binding selectivity assay by DiscoverX’s Bcl-2scan technology. (71) As shown in Figure 7, 40 possessed high Mcl-1 binding affinity with a Kd of 0.23 nM, whereas no binding was observed against Bcl-2, Bcl-xL, Bcl-w, and Bcl2A1 (Kd ≥ 10,000 nM). 40 exhibited hugely improved selectivity (>40,000 folds) over Bcl-2 compared to compound 2 (Mcl-1, Ki = 55 nM; Bcl-2, Ki = 870 nM). The remarkably improved selectivity of 40 could be illustrated by the overlay of 40 and 2 (Figure 8A). The sulfone linker constrained the angle between the phenyl group and the pyrrole core. As a result, the 4-(phenylsulfonyl)-1H-pyrrole core of 40 occupied the entire upper part of the P2 pocket. Also, the proper angle forced the 4-atom linker of 40 to insert into the lower part of Mcl-1’s P2 pocket deeper than that of 2. The P2 pocket of Bcl-2 was narrower and shallower than that of Mcl-1 (Figure 8B). Consequently, the P2 pocket of Bcl-2 could hardly accommodate 40, which led to the high selectivity of 40.
通过基于 FP 的检测或基于时间分辨荧光共振能量转移（TR-FRET）的检测，选出了具有代表性的化合物 27、40、41、42、43 和 44，以测试它们对其他 Bcl-2 家族成员（Bcl-2、Bcl-xL 和 Bfl-1）的选择性。结果表明，所有化合物都显示出良好的选择性，因为在 10 μM 的浓度下，没有观察到对其他 Bcl-2 家族成员的明显抑制作用（表 5）。通过 DiscoverX 的 Bcl-2scan 技术，40 被选中进行结合选择性检测。(71）如图 7 所示，40 与 Mcl-1 的结合亲和力很高，K d 为 0.23 nM，而与 Bcl-2、Bcl-xL、Bcl-w 和 Bcl2A1 的结合亲和力不高（K d ≥ 10,000 nM）。与化合物 2 相比，40 对 Bcl-2 的选择性大大提高（>40,000 倍）（Mcl-1，K = 55 nM；Bcl-2，K = 870 nM）。40 和 2 的重叠图（图 8A）可以说明 40 的选择性显著提高。砜连接物限制了苯基和吡咯核心之间的角度。因此，40 的 4-（苯磺酰基）-1H-吡咯核心占据了 P2 口袋的整个上部。此外，由于角度适当，40 的 4 原子连接体插入 Mcl-1 P2 袋下部的深度比 2 的深。Bcl-2 的 P2 袋比 Mcl-1 的窄而浅（图 8B）。因此，Bcl-2 的 P2 袋很难容纳 40，这就导致了 40 的高选择性。
通过基于 FP 的检测或基于时间分辨荧光共振能量转移（TR-FRET）的检测，选出了具有代表性的化合物 27、40、41、42、43 和 44，以测试它们对其他 Bcl-2 家族成员（Bcl-2、Bcl-xL 和 Bfl-1）的选择性。结果表明，所有化合物都显示出良好的选择性，因为在 10 μM 的浓度下，没有观察到对其他 Bcl-2 家族成员的明显抑制作用（表 5）。通过 DiscoverX 的 Bcl-2scan 技术，40 被选中进行结合选择性检测。(71）如图 7 所示，40 与 Mcl-1 的结合亲和力很高，K d 为 0.23 nM，而与 Bcl-2、Bcl-xL、Bcl-w 和 Bcl2A1 的结合亲和力不高（K d ≥ 10,000 nM）。与化合物 2 相比，40 对 Bcl-2 的选择性大大提高（>40,000 倍）（Mcl-1，K = 55 nM；Bcl-2，K = 870 nM）。40 和 2 的重叠图（图 8A）可以说明 40 的选择性显著提高。砜连接物限制了苯基和吡咯核心之间的角度。因此，40 的 4-（苯磺酰基）-1H-吡咯核心占据了 P2 口袋的整个上部。此外，由于角度适当，40 的 4 原子连接体插入 Mcl-1 P2 袋下部的深度比 2 的深。Bcl-2 的 P2 袋比 Mcl-1 的窄而浅（图 8B）。因此，Bcl-2 的 P2 袋很难容纳 40，这就导致了 40 的高选择性。
Figure 7 图 7
Figure 7. Binding affinities of 40 to Mcl-1 and other Bcl-2 family members.
图 7.40 与 Mcl-1 及其他 Bcl-2 家族成员的结合亲和力。
Figure 8 图 8
Figure 8. Bcl-2 could not accommodate 40 due to the shallow and narrow P2 pocket. (A) Overlay of compound 2 (green) and 40 (purple). (B) Comparison of P2 pockets of Mcl-1 and Bcl-2.
图 8.由于 P2 袋又浅又窄，Bcl-2 无法容纳 40。(A）化合物 2（绿色）和 40（紫色）的叠加。(B）Mcl-1 和 Bcl-2 P2 袋的比较。
Table 5. In Vitro Selectivity Evaluation
表 5.体外选择性评估
表 5.体外选择性评估
| cpd | Mcl-1 (Ki ± SE, μM) | Bcl-2a | Bcl-xLb | Bfl-1a |
|---|---|---|---|---|
| 27 | 0.080 ± 0.020 | 7.54 | 37 | NA |
| 40 | 0.020 ± 0.001 | 10.00 | NA | 1.57 |
| 41 | 0.070 ± 0.010 | 14.16 | 27 | 1.82 |
| 42 | 0.040 ± 0.005 | 11.64 | 13 | 4.14 |
| 43 | 0.018 ± 0.002 | NA | NA | NA |
| 44 | 0.070 ± 0.020 0.070 ± 0.020 | 12.14 | 36 | 8.77 |
a
a FP, inhibition@10 μM (%).
FP, inhibition@10 μM (%).
a FP, inhibition@10 μM (%).
b
b TR-FRET, inhibition@10 μM (%).
TR-FRET, inhibition@10 μM (%).
b TR-FRET, inhibition@10 μM (%).
In Vitro Anticancer Effects
体外抗癌效果
After confirming the Mcl-1-targeting ability in vitro, 27, 40, 41, 42, 43, and 44 were then tested for their antiproliferative activities against the tumor cell lines sensitive to Mcl-1 inhibitors (H929, MV4-11, SK-BR-3, and NCI-H23) (53,67,72) and the insensitive K562 cell line (49,53) through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colourimetric (MTT) assay. A1210477 and AZD5991 were selected as positive controls. As shown in Table 6, all compounds showed submicromolar activities against the MM cell line H929. 27, 40, 41, 42, and 44 showed better in vitro antitumor efficacy compared to the known Mcl-1 inhibitor A1210477. In spite of the high Mcl-1 inhibitory activity, 43 did not perform that potency against cancer cell lines in vitro. The result may attribute to its poor permeability resulted by 4-OH. In the acute myeloid leukemia (AML) cell line MV4-11, all compounds showed decreased activities while 40 retained the submicromolar potency. Decreased potencies were observed in breast cancer cell line SK-BR-3 and NSCLC cell line NCI-H23 of all compounds, which was in line with other Mcl-1 inhibitors’ performance in solid cancer cell lines. (67,68)40, 41, 42, and 44 showed ideal selectivity against CML cell line K562 (IC50 > 30 μM).
在体外确认了 Mcl-1 靶向能力后，27、40、41、42、43 和 44 对 Mcl-1 抑制剂敏感的肿瘤细胞系（H929、MV4-11、SK-BR-3 和 NCI-H23）（53、67、72）和不敏感的 K562 细胞系（49、53）进行了抗增殖活性测试、MV4-11、SK-BR-3 和 NCI-H23）（53,67,72）和对 Mcl-1 抑制剂不敏感的 K562 细胞系（49,53）的抗增殖活性进行了 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑比色法（MTT）试验。A1210477 和 AZD5991 被选为阳性对照。如表 6 所示，所有化合物对 MM 细胞株 H929 都显示出亚摩尔活性。与已知的 Mcl-1 抑制剂 A1210477 相比，27、40、41、42 和 44 显示出更好的体外抗肿瘤功效。尽管 43 具有很高的 Mcl-1 抑制活性，但其体外抗癌细胞株的效力并不明显。这可能是因为 4-OH 导致其渗透性较差。在急性髓性白血病（AML）细胞系 MV4-11 中，所有化合物的活性都有所下降，而 40 仍保持亚摩尔效力。在乳腺癌细胞株 SK-BR-3 和 NSCLC 细胞株 NCI-H23 中，所有化合物的效力都有所下降，这与其他 Mcl-1 抑制剂在实体癌细胞株中的表现一致。(67,68)40、41、42 和 44 对 CML 细胞株 K562 表现出理想的选择性（IC 50 > 30 μM）。
在体外确认了 Mcl-1 靶向能力后，27、40、41、42、43 和 44 对 Mcl-1 抑制剂敏感的肿瘤细胞系（H929、MV4-11、SK-BR-3 和 NCI-H23）（53、67、72）和不敏感的 K562 细胞系（49、53）进行了抗增殖活性测试、MV4-11、SK-BR-3 和 NCI-H23）（53,67,72）和对 Mcl-1 抑制剂不敏感的 K562 细胞系（49,53）的抗增殖活性进行了 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑比色法（MTT）试验。A1210477 和 AZD5991 被选为阳性对照。如表 6 所示，所有化合物对 MM 细胞株 H929 都显示出亚摩尔活性。与已知的 Mcl-1 抑制剂 A1210477 相比，27、40、41、42 和 44 显示出更好的体外抗肿瘤功效。尽管 43 具有很高的 Mcl-1 抑制活性，但其体外抗癌细胞株的效力并不明显。这可能是因为 4-OH 导致其渗透性较差。在急性髓性白血病（AML）细胞系 MV4-11 中，所有化合物的活性都有所下降，而 40 仍保持亚摩尔效力。在乳腺癌细胞株 SK-BR-3 和 NSCLC 细胞株 NCI-H23 中，所有化合物的效力都有所下降，这与其他 Mcl-1 抑制剂在实体癌细胞株中的表现一致。(67,68)40、41、42 和 44 对 CML 细胞株 K562 表现出理想的选择性（IC 50 > 30 μM）。
| cpd | H929 | MV4-11 | SK-BR-3 | NCI-H23 | K562 |
|---|---|---|---|---|---|
| 27 | 0.70 ± 0.12 | 2.30 ± 0.45 | 5.13 ± 0.66 | 3.10 ± 1.73 | 11.3 ± 1.76 |
| 40 | 0.36 ± 0.09 | 0.70 ± 0.07 | 2.84 ± 0.66 | 3.02 ± 1.35 | >30 |
| 41 | 0.39 ± 0.09 | 1.56 ± 0.24 | 6.27 ± 1.89 | 9.72 ± 2.33 | >30 |
| 42 | 0.48 ± 0.17 | 1.70 ± 0.63 | 5.60 ± 1.21 | 3.84 ± 1.24 | >30 |
| 43 | 1.39 ± 0.31 | 2.02 ± 0.28 | 7.02 ± 1.07 | 2.16 ± 0.75 | 14.8 ± 3.07 14.8 ± 3.07 |
| 44 | 0.49 ± 0.05 | 2.59 ± 0.31 | 3.92 ± 1.65 | 5.41 ± 1.26 | >30 |
| A1210477 | 0.90 ± 0.29 | 1.55 ± 0.47 | 4.31 ± 1.29 4.31 ± 1.29 | 13.5 ± 2.81 | >30 |
| AZD5991 | 0.036 ± 0.008 0.036 ± 0.008 | 0.024 ± 0.006 0.024 ± 0.006 | >30 | 0.19 ± 0.04 0.19 ± 0.04 | 29.6 ± 5.3 |
a
a 所示数值为平均 IC 50 ± SD（μM）（n = 3）。± SD（μM，n = 3）。
Values shown are the mean IC50 ± SD (μM, n = 3).
a 所示数值为平均 IC 50 ± SD（μM）（n = 3）。± SD（μM，n = 3）。
40 Effectively Induced Apoptosis in an Mcl-1-Dependent Manner
40 以 Mcl-1 依赖性方式有效诱导细胞凋亡
As 40 showed ideal target affinity and antiproliferative activities, it was selected for further biological evaluation. A coimmunoprecipitation (Co-ip) experiment was performed to evaluate the on-target Mcl-1 inhibition of 40 in living cells. H929 cells were treated with increasing concentrations of 40 or positive control AZD5991 (0.05 μM), respectively. The Mcl-1-Bak complex was monitored by western blot (WB) analysis. The results (Figure 9A) demonstrated that 40 could displace Bak protein from Mcl-1 in a concentration-dependent manner. To verify if compound 40 could induce apoptosis in cells, an annexin-V and propidium iodide (PI) double staining assay was applied in H929 cells at concentrations of 1 and 5 μM. As shown in Figure 9B–G, 40 and positive control A1210477 significantly induced apoptosis in a concentration-dependent manner. Apoptosis ratio analysis demonstrated that 40 was more potent in inducing apoptosis than A1210477. To further prove that 40 could activate apoptosis signaling, effects of 40 on the poly ADP-ribose polymerase (PARP) cleavage and caspase activation were evaluated in H929 cells and also in K562 cells to assess off-target effects. Both cell lines were incubated with 40 and A1210477 at concentrations of 0, 0.1, 0.5, 1, and 5 μM. The amount of cleaved PARP (cPARP) was evaluated by the WB assay. As shown in Figure 9H,I, 40 and A1210477 remarkably upregulated PARP cleavage in H929 cells in a concentration-dependent manner. However, the same treatment in K562 cells did not induce comparable PARP cleavage. In caspase activation assay, K562 cells and H929 cells were exposed to 40 and A1210477 at indicated concentrations for 4 h and the caspase activation folds were determined by caspase assay kits. As shown in Figure 9J,K, 40 concentration-dependently activated caspase 3/7 in H929 cells, which is more potent than A1210477. However, caspase activation was not observed in K562 cells after the same treatment of 40 and A1210477. Collectively, these results showed that 40 activated the apoptosis in an Mcl-1-dependent manner.
由于 40 表现出理想的靶向亲和力和抗增殖活性，因此被选中进行进一步的生物学评估。为了评估 40 在活细胞中对 Mcl-1 的靶向抑制作用，我们进行了共免疫沉淀（Co-ip）实验。分别用浓度递增的 40 或阳性对照 AZD5991（0.05 μM）处理 H929 细胞。通过免疫印迹（WB）分析监测 Mcl-1-Bak 复合物。结果（图 9A）表明，40 能以浓度依赖的方式将 Bak 蛋白从 Mcl-1 中置换出来。为了验证化合物 40 能否诱导细胞凋亡，在浓度为 1 μM 和 5 μM 的 H929 细胞中进行了附件素-V 和碘化丙啶（PI）双重染色试验。如图 9B-G 所示，40 和阳性对照 A1210477 以浓度依赖性方式显著诱导细胞凋亡。凋亡比率分析表明，40 比 A1210477 更能诱导细胞凋亡。为了进一步证明 40 能激活细胞凋亡信号，我们在 H929 细胞和 K562 细胞中评估了 40 对多聚 ADP 核糖聚合酶（PARP）裂解和 caspase 激活的影响，以评估其脱靶效应。这两种细胞株均与浓度为 0、0.1、0.5、1 和 5 μM 的 40 和 A1210477 一起培养。裂解 PARP（cPARP）的量通过 WB 检测法进行评估。如图 9H,I 所示，40 和 A1210477 显著上调了 H929 细胞中 PARP 的裂解，且呈浓度依赖性。然而，同样的处理在 K562 细胞中并没有诱导类似的 PARP 分裂。在caspase活化试验中，K562细胞和H929细胞暴露于40和A1210477（指定浓度）4小时，用caspase检测试剂盒测定caspase活化倍数。如图 9J、K 所示，40 浓度依赖性地激活了 H929 细胞中的 caspase 3/7，其作用比 A1210477 更强。然而，在 K562 细胞中，同样处理 40 和 A1210477 后，未观察到 caspase 激活。总之，这些结果表明 40 能以 Mcl-1 依赖性方式激活细胞凋亡。
由于 40 表现出理想的靶向亲和力和抗增殖活性，因此被选中进行进一步的生物学评估。为了评估 40 在活细胞中对 Mcl-1 的靶向抑制作用，我们进行了共免疫沉淀（Co-ip）实验。分别用浓度递增的 40 或阳性对照 AZD5991（0.05 μM）处理 H929 细胞。通过免疫印迹（WB）分析监测 Mcl-1-Bak 复合物。结果（图 9A）表明，40 能以浓度依赖的方式将 Bak 蛋白从 Mcl-1 中置换出来。为了验证化合物 40 能否诱导细胞凋亡，在浓度为 1 μM 和 5 μM 的 H929 细胞中进行了附件素-V 和碘化丙啶（PI）双重染色试验。如图 9B-G 所示，40 和阳性对照 A1210477 以浓度依赖性方式显著诱导细胞凋亡。凋亡比率分析表明，40 比 A1210477 更能诱导细胞凋亡。为了进一步证明 40 能激活细胞凋亡信号，我们在 H929 细胞和 K562 细胞中评估了 40 对多聚 ADP 核糖聚合酶（PARP）裂解和 caspase 激活的影响，以评估其脱靶效应。这两种细胞株均与浓度为 0、0.1、0.5、1 和 5 μM 的 40 和 A1210477 一起培养。裂解 PARP（cPARP）的量通过 WB 检测法进行评估。如图 9H,I 所示，40 和 A1210477 显著上调了 H929 细胞中 PARP 的裂解，且呈浓度依赖性。然而，同样的处理在 K562 细胞中并没有诱导类似的 PARP 分裂。在caspase活化试验中，K562细胞和H929细胞暴露于40和A1210477（指定浓度）4小时，用caspase检测试剂盒测定caspase活化倍数。如图 9J、K 所示，40 浓度依赖性地激活了 H929 细胞中的 caspase 3/7，其作用比 A1210477 更强。然而，在 K562 细胞中，同样处理 40 和 A1210477 后，未观察到 caspase 激活。总之，这些结果表明 40 能以 Mcl-1 依赖性方式激活细胞凋亡。
Figure 9 图 9
Figure 9. 40 induced apoptosis in an Mcl-1-dependent manner. (A) Co-ip experiment of 40 and AZD5991 in H929 cells. (B–G) Flow cytometry assay of 40 and A1210477 in H929 cells at concentrations of 1 μM and 5 μM. (H,I) WB assay of 40 and A1210477 in H929 cells and K562 cells at concentrations of 0, 0.1, 0.5, 1, and 5 μM. (J,K) Caspase activation abilities of 40 and A1210477 in H929 cells and K562 cells at concentrations of 0, 0.1, 0.5, 1, and 5 μM. The values shown are the means ± SEM (n = 3 independent observations), ns = no significant, ***p < 0.001, ****p < 0.0001 one-way ANOVA with Tukey–Kramer posttest.
图 9.40 以 Mcl-1 依赖性方式诱导细胞凋亡。(A）40 和 AZD5991 在 H929 细胞中的 Co-ip 实验。(B-G）40 和 A1210477 在 1 μM 和 5 μM 浓度下对 H929 细胞的流式细胞术检测。(H,I) 40 和 A1210477 在浓度为 0、0.1、0.5、1 和 5 μM 的 H929 细胞和 K562 细胞中的 WB 检测。(J,K）浓度为 0、0.1、0.5、1 和 5 μM 的 40 和 A1210477 在 H929 细胞和 K562 细胞中的 Caspase 活化能力。所示数值为平均值 ± SEM（n = 3 个独立观察值），ns = 无显著性，***p < 0.001，****p < 0.0001 单因素方差分析，Tukey-Kramer 后验。
Drug-Like Properties Evaluation of 40
40 种药物的类药物特性评估
After confirming that 40 possessed desired cellular activity, drug-like properties evaluations were carried out. As shown in Table 7, 40 showed favorable water solubility and permeability in the parallel artificial membrane permeability assay (PAMPA). CYP inhibition determination showed that 40 had no pronounced inhibition against five major isozymes at a concentration of 10 μM (Table 7). In further evaluations, 40 exhibited acceptable stability in simulated intestinal fluid (SIF), simulated gastric fluid (SGF), mouse and human plasma, and rat and human liver microsomes (RLM and HLM) (Table 8). In the Caco-2 cell model, 40 showed moderate permeability and no significant efflux was observed. In addition, 40 displayed no hERG channel inhibition (Table 8 and Supporting Information, Figure S1). In vivo pharmacokinetic (PK) profiles of 40 were evaluated in SD rats. Intravenous (iv) administration of 40 at a dose of 3 mg/kg showed an acceptable half-life of 2.3 h. Of note, 40 showed favorable bioavailability (F% = 41.3%) by oral (po) administration (Figure 10). Taken together, 40 exhibited good druglike properties.
在确认 40 具有所需的细胞活性后，进行了类药物特性评估。如表 7 所示，在平行人工膜渗透性试验（PAMPA）中，40 表现出良好的水溶性和渗透性。CYP 抑制测定显示，在 10 μM 浓度下，40 对五种主要同工酶没有明显的抑制作用（表 7）。在进一步的评估中，40 在模拟肠液（SIF）、模拟胃液（SGF）、小鼠和人血浆以及大鼠和人肝微粒体（RLM 和 HLM）中表现出可接受的稳定性（表 8）。在 Caco-2 细胞模型中，40 表现出中等渗透性，未观察到明显的外流。此外，40 对 hERG 通道没有抑制作用（表 8 和证明资料，图 S1）。在 SD 大鼠体内评估了 40 的体内药代动力学（PK）特征。以 3 毫克/千克的剂量静脉注射（iv）40，半衰期为 2.3 小时，可以接受。值得注意的是，口服（po）40 表现出良好的生物利用度（F% = 41.3%）（图 10）。综上所述，40 表现出良好的类药物特性。
在确认 40 具有所需的细胞活性后，进行了类药物特性评估。如表 7 所示，在平行人工膜渗透性试验（PAMPA）中，40 表现出良好的水溶性和渗透性。CYP 抑制测定显示，在 10 μM 浓度下，40 对五种主要同工酶没有明显的抑制作用（表 7）。在进一步的评估中，40 在模拟肠液（SIF）、模拟胃液（SGF）、小鼠和人血浆以及大鼠和人肝微粒体（RLM 和 HLM）中表现出可接受的稳定性（表 8）。在 Caco-2 细胞模型中，40 表现出中等渗透性，未观察到明显的外流。此外，40 对 hERG 通道没有抑制作用（表 8 和证明资料，图 S1）。在 SD 大鼠体内评估了 40 的体内药代动力学（PK）特征。以 3 毫克/千克的剂量静脉注射（iv）40，半衰期为 2.3 小时，可以接受。值得注意的是，口服（po）40 表现出良好的生物利用度（F% = 41.3%）（图 10）。综上所述，40 表现出良好的类药物特性。
Figure 10 图 10
Figure 10. In vivo PK parameters of 40. The values shown are the means. Bars represent SEM.
图 10.40 的体内 PK 参数。所示数值为平均值。条形图代表 SEM。
| parameters | 40 |
|---|---|
| PAMPA permeability PAMPA 渗透率 | 52.6 ± 1.10 52.6 ± 1.10 |
| solubility (μg/mL) 溶解度（微克/毫升） | 32.3 |
| CYP450 isozymes: 1A2, 2C9, 2C19, 2D6, 3A4 CYP450 同工酶：1A2、2C9、2C19、2D6、3A4 | Inhibition rates (%): 19.30, 26.04, 2.85, 9.13, 29.36 抑制率（%）：19.30, 26.04, 2.85, 9.13, 29.36 |
a
a 两次运行的平均值 ± 标准偏差 (SD)。PAMPA 渗透率值是在 pH 值为 7.4（10 –6 cm/s ）时测定的。
Average of two runs ± standard deviation (SD). PAMPA permeability values were determined at pH 7.4 (10–6 cm/s).
a 两次运行的平均值 ± 标准偏差 (SD)。PAMPA 渗透率值是在 pH 值为 7.4（10 –6 cm/s ）时测定的。
b
b 浓度为 10 μM 时对 CYP450 酶的抑制率（%）。7-乙氧基香豆素、磺胺苯环唑、奥美拉唑、异丙嗪和氟康唑分别是 CYP1A2、CYP2C9、CYP2C19、CYP2D6 和 CYP3A4 的阳性对照药
Inhibitory rates (%) against the CYP450 enzymes at a concentration of 10 μM. 7-ethoxycoumarin, sulfaphenazole, omeprazole, promethazine, and fluconazole were positive controls for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively
b 浓度为 10 μM 时对 CYP450 酶的抑制率（%）。7-乙氧基香豆素、磺胺苯环唑、奥美拉唑、异丙嗪和氟康唑分别是 CYP1A2、CYP2C9、CYP2C19、CYP2D6 和 CYP3A4 的阳性对照药
| parameters | 40 |
|---|---|
| SIF | 85.19 |
| SGF | 84.50 |
| mouse plasma 小鼠血浆 | 100.00 |
| human plasma 人血浆 | 96.50 |
| RLM T1/2 (hr) RLM T 1/2 （小时） | 0.75 |
| RLM Clint (μL/min/mg) RLM Cl int (微升/分钟/毫克） | 31.40 |
| RLM 30 min remaining (%) RLM 30 分钟剩余量（%） | 58.11 |
| HLM T1/2 (hr) HLM T 1/2 （小时） | 0.83 |
| HLM Clint (μL/min/mg) HLM Cl int （微升/分钟/毫克） | 67.50 |
| HLM 30 min remaining (%) HLM 30 分钟剩余时间 (%) | 64.00 |
| Caco-2 permeability (nm/s) (A → B) Caco-2 渗透率（纳米/秒）（A → B） | 36.2 |
| efflux ratio 流出比 | 0.1 |
| hERG (IC50, μM) hERG（IC 50 ，μM） | > 30 > 30 |
a
a 培养 6 小时后的剩余量（%）。
Remaining (%) after 6 h of incubation.
a 培养 6 小时后的剩余量（%）。
b
b 培养 2 小时后的剩余量（%）。
Remaining (%) after 2 h of incubation.
b 培养 2 小时后的剩余量（%）。
In Vivo Antitumor Efficacy of 40
40 种抗肿瘤药物的体内抗肿瘤功效
Then, in vivo antitumor efficacy evaluation of 40 was carried out on balb/c nude female mice loaded with MV4-11 xenograft. The clinical candidate AZD5991 was selected as a positive control. After po administration of 40 (n = 6, 60 mpk) and intraperitoneal (ip) administration of 40 (n = 6, 20 mpk) and AZD5991 (n = 6, 10 mpk) every two days for 14 days, as shown in Figure 11A and Figure S2, po administration of 40 showed desired in vivo tumor growth inhibition activity (T/C = 37.30%) and ip administration of 40 shared equal antitumor activity (T/C = 5.52%) with positive control AZD5991 (T/C = 5.18%). Moreover, the administration of 40 did not induce significant changes in body weight and organ weight (Figure 11B,C). Hematoxylin–eosin (H&E) staining revealed that 40 could induce necrosis of MV4-11 tumor tissues but showed no effect on hearts, livers, or kidneys (Figure 11D). To ascertain the apoptosis pathway activation of 40in vivo, the WB assay was used to evaluate PARP cleavage in tumor biopsies of each group. As shown in Figure 11E, compared with the vehicle group, administration of 40 and AZD5991 could activate cleavage of PARP. Consistent with antitumor efficacy and WB results, increased TUNEL staining was observed in isolated tumors of treated groups (Figure 11F and Supporting Information, Figure S3). These results demonstrated that 40 was well tolerated and orally efficacious in the MV4-11 xenograft model.
然后，在装有 MV4-11 xenograft 的 balb/c 裸露雌性小鼠身上对 40 进行了体内抗肿瘤疗效评估。临床候选药物 AZD5991 被选为阳性对照。如图 11A 和图 S2 所示，腹腔注射 40（n = 6，60 mpk）和腹腔注射 40（n = 6，20 mpk）和 AZD5991（n = 6，10 mpk）14 天后，40 表现出理想的体内肿瘤生长抑制活性（T/C = 37.30%），ip给药40与阳性对照AZD5991（T/C = 5.18%）具有相同的抗肿瘤活性（T/C = 5.52%）。此外，服用 40 不会引起体重和器官重量的显著变化（图 11B,C）。血红素-伊红（H&E）染色显示，40能诱导MV4-11肿瘤组织坏死，但对心脏、肝脏或肾脏没有影响（图11D）。为了确定 40 在体内激活凋亡途径的情况，我们用 WB 检测法评估了各组肿瘤活检组织中 PARP 的裂解情况。如图 11E 所示，与给药组相比，服用 40 和 AZD5991 可激活 PARP 的裂解。与抗肿瘤疗效和 WB 结果一致的是，在治疗组的离体肿瘤中观察到 TUNEL 染色增加（图 11F 和证明资料，图 S3）。这些结果表明，40 在 MV4-11 异种移植模型中具有良好的耐受性和口服疗效。
然后，在装有 MV4-11 xenograft 的 balb/c 裸露雌性小鼠身上对 40 进行了体内抗肿瘤疗效评估。临床候选药物 AZD5991 被选为阳性对照。如图 11A 和图 S2 所示，腹腔注射 40（n = 6，60 mpk）和腹腔注射 40（n = 6，20 mpk）和 AZD5991（n = 6，10 mpk）14 天后，40 表现出理想的体内肿瘤生长抑制活性（T/C = 37.30%），ip给药40与阳性对照AZD5991（T/C = 5.18%）具有相同的抗肿瘤活性（T/C = 5.52%）。此外，服用 40 不会引起体重和器官重量的显著变化（图 11B,C）。血红素-伊红（H&E）染色显示，40能诱导MV4-11肿瘤组织坏死，但对心脏、肝脏或肾脏没有影响（图11D）。为了确定 40 在体内激活凋亡途径的情况，我们用 WB 检测法评估了各组肿瘤活检组织中 PARP 的裂解情况。如图 11E 所示，与给药组相比，服用 40 和 AZD5991 可激活 PARP 的裂解。与抗肿瘤疗效和 WB 结果一致的是，在治疗组的离体肿瘤中观察到 TUNEL 染色增加（图 11F 和证明资料，图 S3）。这些结果表明，40 在 MV4-11 异种移植模型中具有良好的耐受性和口服疗效。
Figure 11 图 11
Figure 11. Antitumor efficacy of compound 40 in nude mice loaded with MV4-11 xenografts. (A) Tumor volume change. The values shown are the means ± SEM **p < 0.01, and ****p < 0.0001, one-way ANOVA with the Tukey–Kramer posttest. (B) Body weight change. (C) Organ weight change. The values shown are the means. Bars represent SEM. (D) Representative HE staining images of tumor tissues and organ tissues. Scale bar 200 μM. (E) WB assay of tumor tissues. (n = 2 for each group). (F) Representative images of tunel assay of tumor tissues. Scale bar 200 μM.
图 11.化合物 40 在装有 MV4-11 异种移植物的裸鼠中的抗肿瘤效果。(A）肿瘤体积变化。所示数值为平均值 ± SEM **p < 0.01，****p < 0.0001，单因素方差分析，Tukey-Kramer 后验。(B) 体重变化。(C) 器官重量变化。所示数值为平均值。条代表 SEM。(D) 肿瘤组织和器官组织的代表性 HE 染色图像。标尺条 200 μM。(E）肿瘤组织的 WB 检测。(每组 n = 2）。(F）肿瘤组织 Tunel 检测的代表性图像。缩放条 200 μM。
Chemistry 化学
Target compounds 11 and 12 were synthesized according to Scheme 1. In the presence of K2CO3, the reaction of commercially available 4-chloro-3,5-dimethylphenol (58) with 1,3-dibromopropane by nucleophilic substitution yielded intermediate 59. With AlCl3 as the Lewis acid, the Friedel–Crafts acylation reaction of starting material methyl 1H-pyrrole-2-carboxylate 60 and 4-methylbenzoyl chloride in DCM gave intermediate 61. Carbonyl reduction of 61 by triethylsilane in THF under nitrogen yielded intermediate 62. In the presence of Cs2CO3, 61 and 62 reacted with 59 to obtain 64 and 63 in DMF. Final hydrolysis of 63 and 64 by NaOH in THF/MeOH gave target compounds 11 and 12.
目标化合物 11 和 12 是根据方案 1 合成的。在 K 2 的存在下CO 3 的条件下的条件下，市售的 4-氯-3,5-二甲基苯酚（58）与 1,3-二溴丙烷发生亲核取代反应，生成中间体 59。以 AlCl 3 作为路易斯酸，1H-吡咯-2-羧酸甲酯 60 和 4-甲基苯甲酰氯在 DCM 中进行弗里德尔-卡夫斯酰化反应，得到中间体 61。在氮气条件下，在 THF 中用三乙基硅烷对 61 进行羰基还原，得到中间体 62。在 Cs 2 的存在下CO 3 61 和 62 与 59 反应，在 DMF 中得到 64 和 63。最后，63 和 64 在 THF/MeOH 中被 NaOH 水解，得到目标化合物 11 和 12。
目标化合物 11 和 12 是根据方案 1 合成的。在 K 2 的存在下CO 3 的条件下的条件下，市售的 4-氯-3,5-二甲基苯酚（58）与 1,3-二溴丙烷发生亲核取代反应，生成中间体 59。以 AlCl 3 作为路易斯酸，1H-吡咯-2-羧酸甲酯 60 和 4-甲基苯甲酰氯在 DCM 中进行弗里德尔-卡夫斯酰化反应，得到中间体 61。在氮气条件下，在 THF 中用三乙基硅烷对 61 进行羰基还原，得到中间体 62。在 Cs 2 的存在下CO 3 61 和 62 与 59 反应，在 DMF 中得到 64 和 63。最后，63 和 64 在 THF/MeOH 中被 NaOH 水解，得到目标化合物 11 和 12。
Scheme 1 方案 1
Scheme 1. Synthesis of Target Compounds 11 and 12a
方案 1.目标化合物 11 和 12 的合成 a
方案 1.目标化合物 11 和 12 的合成 a
aReagents and conditions: (a) 1,3-dibromopropane, K2CO3, CH3CN, 90 °C, 3 h, 75%. (b) 4-methylbenzoyl chloride, AlCl3, DCM, N2, 0 °C–r.t. 2 h, 68%. (c) triethylsilane, THF, N2, r.t. 2 h, 65%. (d) 59, Cs2CO3, DMF, 60 °C, 1 h, 84–89%. (e) NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 78–95%.
a 试剂和条件： (a) 1,3-二溴丙烷，K 2 CO 3 CH 3 CNCN，90 °C，3 小时，75%。(b) 4-甲基苯甲酰氯，AlCl 3 ，DCM，N 2 。(b) 4-甲基苯甲酰氯，AlCl {{4} ，DCM，N 2 。，0℃-r.t. 2 小时，68%。(c) 三乙基硅烷，四氢呋喃，N 2 ，2 小时，68%。2 小时，65%。(d) 59，Cs 2 CO 3 ，DMF，60℃，1 小时，84-89%。(e) NaOH（2 M），THF/MeOH = 1:1，50℃，过夜，78-95%。
Target compound 13 and analogue 20 were synthesized according to Scheme 2 from commercially available 65a-b. With Ag2CO3 as the catalyst, in the presence of methyl isocyanate, silver-catalyzed isocyanide-alkyne cycloaddition of 65a-b yielded 66a-b in 1,4-dioxane. (73) Subsequently, in the presence of NaH, the electrophilic substitution reaction of 66a-b and 59 gave 67a-b. Methyl ester groups of 67a-b were hydrolyzed by NaOH (2 M) in MeOH/THF to yield target compounds 13 and 20.
目标化合物 13 和类似物 20 是根据方案 2 从市售 65a-b 合成的。以 Ag 2 CO 3 为催化剂，在异氰酸甲酯存在的情况下，银催化CO 3 作为催化剂，在异氰酸甲酯存在下，银催化 65a-b 的异氰酸-炔环加成反应在 1,4- 二氧六环中生成了 66a-b。(73) 随后，在 NaH 的存在下，66a-b 和 59 发生亲电取代反应，生成 67a-b。67a-b 的甲酯基团在 MeOH/THF 中被 NaOH（2 M）水解，得到目标化合物 13 和 20。
目标化合物 13 和类似物 20 是根据方案 2 从市售 65a-b 合成的。以 Ag 2 CO 3 为催化剂，在异氰酸甲酯存在的情况下，银催化CO 3 作为催化剂，在异氰酸甲酯存在下，银催化 65a-b 的异氰酸-炔环加成反应在 1,4- 二氧六环中生成了 66a-b。(73) 随后，在 NaH 的存在下，66a-b 和 59 发生亲电取代反应，生成 67a-b。67a-b 的甲酯基团在 MeOH/THF 中被 NaOH（2 M）水解，得到目标化合物 13 和 20。
Scheme 2 方案 2
Scheme 2. Synthesis of Target Compound 13 and 20a
方案 2.目标化合物 13 和 20 的合成 a
方案 2.目标化合物 13 和 20 的合成 a
aReagents and conditions: (a) methyl isocyanate, Ag2CO3, 1,4-dioxane, 80 °C, 30 min, 18–25%. (b) 59, NaH, DMF, 0 °C–r.t. 0.5 h, 35–42%. (c) NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 62–72%.
a 试剂和条件： (a) 异氰酸甲酯、Ag 2 CO 3 ，1,4-二氧六环，80℃，30 分钟，18-25%。(b) 59，NaH，DMF，0℃-r.t. 0.5 小时，35-42%。(c) NaOH（2 M），THF/MeOH = 1:1，50℃，过夜，62-72%。
Target compounds 14 and 15 were synthesized according to Scheme 3. In the presence of Cs2CO3, starting material ethyl 4-nitro-1H-pyrrole-2-carboxylate 68 reacted with 59 to give 69. The nitro group of 69 was reduced to amino group by tin(II) dichloride dehydrate (SnCl2·2H2O) in EtOH and subsequent sulfonylation with TosCl gave intermediate 70. Hydrolysis of 70 by NaOH (2 M) in MeOH/THF gave target compound 14. Nitro reduction of 69 by SnCl2 and subsequent tert-butyloxycarbonyl (Boc) protection of the amino group gave compound 71. In the presence of NaH, the electrophilic substitution reaction of 71 and 4-methylbenzyl bromide yielded 72. Subsequent deprotection of the Boc group by CF3COOH gave 73 and hydrolysis of 73 yielded target compound 15.
目标化合物 14 和 15 是根据方案 3 合成的。在 Cs 2 的存在下CO 3 的条件下起始原料 4-硝基-1H-吡咯-2-羧酸乙酯 68 与 59 反应生成 69。在 EtOH 中，69 的硝基被脱水二氯化锡（SnCl 2 -2H 2 O）还原成氨基，然后用 TosCl 进行磺化反应，得到中间体 70。在 MeOH/THF 中用 NaOH（2 M）水解 70，得到目标化合物 14。用 SnCl 2 对 69 进行硝基还原，然后对氨基进行叔丁氧羰基（Boc）保护，得到化合物 71。在 NaH 存在下，71 与 4-甲基溴化苄发生亲电取代反应，得到 72。随后用 CF 3 COOH 对 Boc 基团进行脱保护，得到 73。COOH 对 Boc 基团进行脱保护，得到 73，73 的水解反应得到目标化合物 15。
目标化合物 14 和 15 是根据方案 3 合成的。在 Cs 2 的存在下CO 3 的条件下起始原料 4-硝基-1H-吡咯-2-羧酸乙酯 68 与 59 反应生成 69。在 EtOH 中，69 的硝基被脱水二氯化锡（SnCl 2 -2H 2 O）还原成氨基，然后用 TosCl 进行磺化反应，得到中间体 70。在 MeOH/THF 中用 NaOH（2 M）水解 70，得到目标化合物 14。用 SnCl 2 对 69 进行硝基还原，然后对氨基进行叔丁氧羰基（Boc）保护，得到化合物 71。在 NaH 存在下，71 与 4-甲基溴化苄发生亲电取代反应，得到 72。随后用 CF 3 COOH 对 Boc 基团进行脱保护，得到 73。COOH 对 Boc 基团进行脱保护，得到 73，73 的水解反应得到目标化合物 15。
Scheme 3 方案 3
Scheme 3. Synthesis of Target Compound 14 and 15a
方案 3.目标化合物 14 和 15 的合成 a
方案 3.目标化合物 14 和 15 的合成 a
aReagents and conditions: (a) 59, Cs2CO3, DMF, 60 °C, 1 h, 75%. (b) SnCl2·2H2O, N2, EtOH, 60 °C, 4 h. (c) Pyridine, TosCl, r.t. 1 h, 38%. (d) NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 43–64%. (e) (Boc)2O, EtOH, r.t. 1 h, 46%. (f) 4-Methylbenzyl bromide, NaH, r.t. 0.5 h, 73%. (g) CF3COOH, r.t. 1.5 h, 95%.
a 试剂和条件： (a) 59, Cs 2 CO 3 ，DMF，60 °C，1 小时，75%。(b) SnCl 2 -2H 2 -2H 2 O, N 2 (c) 吡啶，TosCl，反应 1 小时，38%。(d) NaOH（2 M），THF/MeOH = 1:1，50℃，过夜，43-64%。(e) (Boc) 2 O, EtOH, r t.O，EtOH，回流 1 小时，46%。(f) 4-甲基溴化苄，NaH，回流 0.5 小时，73%。(g) CF 3 COOH, r.t. 1.5 h, 95%。
Target compounds 16, 23–26, 29–31, 33 and 34, 37 and 38, 41 and 42, 44–47, 51–53, and 55–57 were synthesized according to Scheme 4. Commercially available mercaptans 74a–v were first activated by SO2Cl2 in DCM and then reacted with ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate to give intermediates 75a–v. 75a–v further coupled with 59 to yield 76a–v. Hydrolysis of 76a by NaOH (2 M) in THF/EtOH yielded target compound 23. Oxidation of thio bonds of 76a–v by 3-chloroperbenzoic acid (m-CPBA) yielded compounds 77a–v. Hydrolysis of 77a-v by NaOH (2 M) in EtOH/THF yielded target compounds 16, 24–26, 29–31, 33–34, 37 and 38, 41 and 42, 44–47, 51–53, and 56–57. Reduction of 53 by Pd/C under hydrogen in EtOAc yielded target compound 55.
根据方案 4 合成了目标化合物 16、23-26、29-31、33 和 34、37 和 38、41 和 42、44-47、51-53 和 55-57。市售硫醇 74a-v 首先在二甲基二硅氧烷中被 SO 2 活化。Cl 2 在二氯甲烷中活化，然后与 3,5-二甲基-1H-吡咯-2-羧酸乙酯反应，得到中间产物 75a-v。3-氯过苯甲酸 (m-CPBA) 氧化 76a-v 的硫键，得到 77a-v 化合物。NaOH（2 M）在 EtOH/THF 中水解 77a-v 得到目标化合物 16、24-26、29-31、33-34、37 和 38、41 和 42、44-47、51-53 和 56-57。在氢气作用下，Pd/C 在 EtOAc 中还原 53，得到目标化合物 55。
根据方案 4 合成了目标化合物 16、23-26、29-31、33 和 34、37 和 38、41 和 42、44-47、51-53 和 55-57。市售硫醇 74a-v 首先在二甲基二硅氧烷中被 SO 2 活化。Cl 2 在二氯甲烷中活化，然后与 3,5-二甲基-1H-吡咯-2-羧酸乙酯反应，得到中间产物 75a-v。3-氯过苯甲酸 (m-CPBA) 氧化 76a-v 的硫键，得到 77a-v 化合物。NaOH（2 M）在 EtOH/THF 中水解 77a-v 得到目标化合物 16、24-26、29-31、33-34、37 和 38、41 和 42、44-47、51-53 和 56-57。在氢气作用下，Pd/C 在 EtOAc 中还原 53，得到目标化合物 55。
Scheme 4 方案 4
Scheme 4. Synthesis of Target Compounds 16, 23–26, 29–31, 33 and 34, 37 and 38, 41 and 42, 44–47, 51–53, and 55–57a
方案 4.目标化合物 16、23-26、29-31、33 和 34、37 和 38、41 和 42、44-47、51-53 和 55-57 的合成 a
方案 4.目标化合物 16、23-26、29-31、33 和 34、37 和 38、41 和 42、44-47、51-53 和 55-57 的合成 a
aReagents and conditions: (a) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, SO2Cl2, Et3N, DCM, r.t. 45 min, 56–85%; (b) 59, Cs2CO3, DMF, 50 °C, 1 h, 78–85%; (c) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 63–85%. (d) m-CPBA, DCM, r.t. 0.5 h, 55–70%; (e) Pd/C, H2, EA, 50 °C, 3 h, 95%.
a 试剂和条件： (a) 3,5-二甲基-1H-吡咯-2-羧酸乙酯，SO 2 Cl 2 N, DCM, r. t.N, DCM, r.t. 45 min, 56-85%; (b) 59, Cs 2 CO 3 ，DMF，50 °C，1 小时，78-85%；（c）NaOH（2 M），C 2 H 5 H 5 OH/THF=1:1，50℃，过夜，63-85%。(d) m-CPBA, DCM, r.t. 0.5 h, 55-70%; (e) Pd/C, H 2 H 2 OH/THF = 1:1, 50 °C, 过夜，63-85%。，EA，50℃，3 小时，95%。
Target compounds 17–19 and 21–22 were synthesized according to Scheme 5. Starting material diethyl malonate 78 was first converted to diethyl oximinomalonate (79) through a reported method, (74) and then in the presence of Zn, 79 reacted with diethyl oxalate 80a to give intermediate 81a. The reaction of 79 and 80b or 80c yielded 81b-c or 81d-e. Further reaction with p-toluenethiol, nucleophilic substitution with 59, oxidation by m-CPBA, and hydrolysis were as depicted in Scheme 4, which gave target compounds 17–19 and 21 and 22.
目标化合物 17-19 和 21-22 是根据方案 5 合成的。起始原料丙二酸二乙酯 78 首先通过已报道的方法（74）转化为草酰丙二酸二乙酯（79），然后在锌存在下，79 与草酸二乙酯 80a 反应得到中间体 81a。79 与 80b 或 80c 反应生成 81b-c 或 81d-e。如方案 4 所示，进一步与对甲苯硫酚反应，与 59 发生亲核取代反应，被 m-CPBA 氧化，水解，得到目标化合物 17-19、21 和 22。
目标化合物 17-19 和 21-22 是根据方案 5 合成的。起始原料丙二酸二乙酯 78 首先通过已报道的方法（74）转化为草酰丙二酸二乙酯（79），然后在锌存在下，79 与草酸二乙酯 80a 反应得到中间体 81a。79 与 80b 或 80c 反应生成 81b-c 或 81d-e。如方案 4 所示，进一步与对甲苯硫酚反应，与 59 发生亲核取代反应，被 m-CPBA 氧化，水解，得到目标化合物 17-19、21 和 22。
Scheme 5 方案 5
Scheme 5. Synthesis of Target Compounds 17–19 and 21 and 22a
方案 5.目标化合物 17-19 和 21 及 22 的合成 a
方案 5.目标化合物 17-19 和 21 及 22 的合成 a
aReagents and conditions: (a) AcOH, NaNO2, 0 °C–r.t. overnight; (b) AcONa, AcOH, Zn, r.t.–80 °C, 3 h, 36–43%. (c) p-Toluenethiol, SO2Cl2, Et3N, DCM, r.t. 45 min, 45–62%; (d) 59, Cs2CO3, DMF, 50 °C, 1 h, 57–72%; (e) m-CPBA, DCM, r.t. 0.5 h, 45–56%; (f) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 74–86%.
a 试剂和条件：(a) AcOH，NaNO {{1} ，0℃-r.t 过夜；(b) AcONa，AcOH，Zn，r.t.-80℃，3 小时，36-43%。0℃-r.t.过夜；(b) AcONa，AcOH，Zn，r.t.-80℃，3 小时，36-43%。(c) 对甲苯硫酚，SO 2 Cl 2 ，Et 3 N, DCM, r.t. 45 分钟，45-62%； (d) 59, Cs 2 CO 3 CO 3 DMF, 50 °C, 1 h, 57-72%; (e) m-CPBA, DCM, r.t. 0.5 h, 45-56%; (f) NaOH (2 M), C 2 H 5 .H 5 OH/THF = 1:1，50 °C，过夜，74-86%。
Target compounds 27 and 28 were synthesized according to Scheme 6. With AlCl3 as the Lewis acid, Friedel–Crafts acylation of the starting material ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate and 2-thiophenesulfonyl chloride (85a) or 1-naphthalenesulfonyl chloride (85b) in PhCl gave intermediates 86a-b. Further nucleophilic substitution with 59 and hydrolysis of 87a-b were the same as depicted in Scheme 4, which yielded target compounds 27 and 28.
目标化合物 27 和 28 是根据方案 6 合成的。以 AlCl 3 为路易斯酸，起始原料 3,5-二甲基-1H-吡咯-2-甲酸乙酯和 2-噻吩磺酰氯（85a）或 1-萘磺酰氯（85b）在 PhCl 中进行弗里德尔-卡夫酰化反应，得到中间体 86a-b。进一步与 59 发生亲核取代反应以及 87a-b 的水解过程与方案 4 中描述的相同，从而得到目标化合物 27 和 28。
目标化合物 27 和 28 是根据方案 6 合成的。以 AlCl 3 为路易斯酸，起始原料 3,5-二甲基-1H-吡咯-2-甲酸乙酯和 2-噻吩磺酰氯（85a）或 1-萘磺酰氯（85b）在 PhCl 中进行弗里德尔-卡夫酰化反应，得到中间体 86a-b。进一步与 59 发生亲核取代反应以及 87a-b 的水解过程与方案 4 中描述的相同，从而得到目标化合物 27 和 28。
Scheme 6 方案 6
Scheme 6. Synthesis of Target Compounds 27 and 28a
方案 6.目标化合物 27 和 28 的合成 a
方案 6.目标化合物 27 和 28 的合成 a
aReagents and conditions: (a) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, N2, AlCl3, PhCl, 90 °C, 2 h, 59–68%; (b) 59, Cs2CO3, DMF, 50 °C, 1 h, 63–75%; (c) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 54–63%.
a 试剂和条件： (a) 3,5-二甲基-1H-吡咯-2-羧酸乙酯，N 2 ，氯化铝 {{2}N {{1} ，AlCl 3 ，PhClPhCl, 90 °C, 2 h, 59-68%; (b) 59, Cs 2 .CO 3 ，DMF，50 °C，1 小时，63-75%；（c）NaOH（2 M），C 2 H 5 H 5 OH/THF = 1:1，50 °C，过夜，54-63%。
The target compounds 32, 35, 36, 39, 40, 43, 48–50, and 54 were synthesized according to Scheme 7. With CuSO4·5H2O as a catalyst, aryl iodides 88a–j reacted with ethanedithiol to give aryl thiols 89a–j in the presence of Cs2CO3 in DMSO under nitrogen. Further coupling with 59, sulfide oxidation, and hydrolysis were the same as depicted in Scheme 4, which yielded target compounds 32, 36, 40, 48–50, and 54 and intermediates 93h–j. Deprotection of the benzyl group of 93h–j by Pd/C and hydrogen in EtOAc yielded target compounds 35, 39, and 43.
目标化合物 32、35、36、39、40、43、48-50 和 54 是根据方案 7 合成的。用 CuSO 4 -5H 2 O 作为催化剂，在 Cs 2 CO 3 的存在下，芳基碘化物 88a-j 与乙二硫醇反应生成芳基硫醇 89a-j。CO 3 在二甲基亚砜（DMSO）中，在氮气条件下反应生成芳基硫醇 89a-j。与 59 的进一步偶联、硫化物氧化和水解过程与方案 4 中描述的相同，得到了目标化合物 32、36、40、48-50 和 54 以及中间体 93h-j。在 EtOAc 中用 Pd/C 和氢气对 93h-j 的苄基进行脱保护，得到目标化合物 35、39 和 43。
目标化合物 32、35、36、39、40、43、48-50 和 54 是根据方案 7 合成的。用 CuSO 4 -5H 2 O 作为催化剂，在 Cs 2 CO 3 的存在下，芳基碘化物 88a-j 与乙二硫醇反应生成芳基硫醇 89a-j。CO 3 在二甲基亚砜（DMSO）中，在氮气条件下反应生成芳基硫醇 89a-j。与 59 的进一步偶联、硫化物氧化和水解过程与方案 4 中描述的相同，得到了目标化合物 32、36、40、48-50 和 54 以及中间体 93h-j。在 EtOAc 中用 Pd/C 和氢气对 93h-j 的苄基进行脱保护，得到目标化合物 35、39 和 43。
Scheme 7 方案 7
Scheme 7. Synthesis of Target Compounds 32, 35, 36, 39, 40, 43, 48–50, and 54a
方案 7.目标化合物 32、35、36、39、40、43、48-50 和 54 的合成 a
方案 7.目标化合物 32、35、36、39、40、43、48-50 和 54 的合成 a
aReagents and conditions: (a) ethanedithiol, CuSO4·5H2O, Cs2CO3, DMSO, N2, 90 °C, 4 h, 76–85%; (b) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, SO2Cl2, Et3N, DCM, r.t. 45 min, 54–75%; (c) 59, Cs2CO3, DMF, 50 °C, 1 h, 75–85%; (d) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 42–80%; (e) m-CPBA, DCM, r.t. 0.5 h, 63–84%; (f) Pd/C, H2, EA, 50 °C, 3 h, 63–67%.
a 试剂和条件： (a) 乙二硫醇、CuSO 4 -5H 2 O、Cs 2 CO 3 dmso, n 2 90 °C，4 小时，76%-85%；（b）3,5-二甲基-1H-吡咯-2-羧酸乙酯，SO 2 Cl 2 。Cl 2N, DCM, r. t.N, DCM, r.t. 45 分钟，54-75%；（c）59, Cs 2 CO 3 CO 3 ，DMF，50 °C，1 小时，75-85%；（d）NaOH（2 M），C 2 H 5 H 5 OH/THF = 1:1，50 °C，过夜，42-80%；（e）m-CPBA，DCM，0.5 小时后，63-84%；（f）Pd/C，H 2 ，EA，50℃，3 小时，63-67%。
In this paper, we described the discovery of a series of Mcl-1 inhibitors with 3,5-dimethyl-4-sulfonyl-1H-pyrrole core. The sulfone linker and the 5-methyl group of the pyrrole core furnished inhibitors with improved Mcl-1 inhibition. Further modifications on the phenyl ring yielded 40 with high Mcl-1 binding affinity. The sulfone linker constrained the structure to a proper conformation, which led to the remarkable selectivity over other Bcl-2 family members. The in vitro antitumor assay demonstrated that 40 inhibited the growth of H929 and MV4-11 cells with appropriate potency. 40 was then validated to induce apoptosis in the H929 cell line in an Mcl-1-dependent manner. Further studies showed that 40 also possessed good druglike properties, including appropriate PK profiles and no inhibition toward hERG and CYPs. In MV4-11 xenografts, oral administration of 40 was well tolerated to significantly inhibit tumor growth. We hope our work is useful for developing more potent Mcl-1 inhibitors and promote cancer therapy targeting Mcl-1.
本文介绍了一系列以 3,5-二甲基-4-磺酰基-1H-吡咯为核心的 Mcl-1 抑制剂的发现。砜连接物和吡咯核心的 5-甲基基团使抑制剂具有更好的 Mcl-1 抑制作用。对苯基环的进一步修饰产生了具有高 Mcl-1 结合亲和力的 40。砜连接物将结构限制在一个适当的构象中，从而使其对其他 Bcl-2 家族成员具有显著的选择性。体外抗肿瘤试验表明，40 能以适当的效力抑制 H929 和 MV4-11 细胞的生长。随后又验证了 40 能以 Mcl-1 依赖性方式诱导 H929 细胞系凋亡。进一步的研究表明，40 还具有良好的类药物特性，包括适当的 PK 曲线以及对 hERG 和 CYPs 无抑制作用。在 MV4-11 异种移植中，口服 40 的耐受性良好，能显著抑制肿瘤生长。我们希望我们的工作有助于开发更有效的 Mcl-1 抑制剂，并促进以 Mcl-1 为靶点的癌症治疗。
本文介绍了一系列以 3,5-二甲基-4-磺酰基-1H-吡咯为核心的 Mcl-1 抑制剂的发现。砜连接物和吡咯核心的 5-甲基基团使抑制剂具有更好的 Mcl-1 抑制作用。对苯基环的进一步修饰产生了具有高 Mcl-1 结合亲和力的 40。砜连接物将结构限制在一个适当的构象中，从而使其对其他 Bcl-2 家族成员具有显著的选择性。体外抗肿瘤试验表明，40 能以适当的效力抑制 H929 和 MV4-11 细胞的生长。随后又验证了 40 能以 Mcl-1 依赖性方式诱导 H929 细胞系凋亡。进一步的研究表明，40 还具有良好的类药物特性，包括适当的 PK 曲线以及对 hERG 和 CYPs 无抑制作用。在 MV4-11 异种移植中，口服 40 的耐受性良好，能显著抑制肿瘤生长。我们希望我们的工作有助于开发更有效的 Mcl-1 抑制剂，并促进以 Mcl-1 为靶点的癌症治疗。
General Procedures 一般程序
The synthesis of target compounds is shown in Schemes 1–7. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (GF254) and visualized under UV light. Melting points were determined with a Melt-Temp II apparatus. The 1H NMR and 13C NMR spectra were measured on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane as the internal standard. ESI-mass and high-resolution mass spectra (HRMS) were recorded on a Water Q-Tof micro mass spectrometer. The purity (≥95%) of the compounds was verified by HPLC performed on a Shimadzu C18 (4.6 mm × 150 mm, 3.5 μm) column using a mixture of 90:10 or 80:20 solvent methanol/water with 1‰ TFA at a flow rate of 0.5 mL/min and peak detection at 254 nm.
目标化合物的合成过程见图 1-7。在 0.25 毫米硅胶板（GF254）上用薄层色谱法（TLC）监测反应，并在紫外光下观察。熔点用 Melt-Temp II 仪器测定。 1 H核磁共振和{{1H NMR 和 13 C NMR 光谱是在布鲁克 AV-300 仪器上测量的，使用氚代溶剂，以四甲基硅烷为内标。ESI 质谱和高分辨率质谱（HRMS）在 Water Q-Tof 微型质谱仪上记录。化合物的纯度（≥95%）由高效液相色谱（HPLC）验证，该色谱采用岛津 C18（4.6 mm × 150 mm，3.5 μm）色谱柱，使用 90:10 或 80:20 混合溶剂甲醇/水和 1‰反式脂肪酸，流速为 0.5 mL/min，色谱峰检测波长为 254 nm。
目标化合物的合成过程见图 1-7。在 0.25 毫米硅胶板（GF254）上用薄层色谱法（TLC）监测反应，并在紫外光下观察。熔点用 Melt-Temp II 仪器测定。 1 H核磁共振和{{1H NMR 和 13 C NMR 光谱是在布鲁克 AV-300 仪器上测量的，使用氚代溶剂，以四甲基硅烷为内标。ESI 质谱和高分辨率质谱（HRMS）在 Water Q-Tof 微型质谱仪上记录。化合物的纯度（≥95%）由高效液相色谱（HPLC）验证，该色谱采用岛津 C18（4.6 mm × 150 mm，3.5 μm）色谱柱，使用 90:10 或 80:20 混合溶剂甲醇/水和 1‰反式脂肪酸，流速为 0.5 mL/min，色谱峰检测波长为 254 nm。
5-(3-Bromopropoxy)-2-chloro-1,3-dimethylbenzene (59)
5-(3-溴丙氧基)-2-氯-1,3-二甲基苯 (59)
To a mixture of K2CO3 (8.3 g, 60 mmol, 2 equiv), 1,3-dibromopropane (6 mL, 60 mmol, 2 equiv), and CH3CN (40 mL), 58 (4.7 g, 30 mmol, 1 equiv) was added and the mixture was stirred for 5 h at 90 °C. After cooling to room temperature, the reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3 times). The combined organic layer was then washed with brine and dried over Na2SO4. Removal of solvent in vacuo yielded a crude product. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 500:1 to 300:1 v/v) gave 59. Colorless liqiud, 6.2 g, yield: 75%. 1H NMR (300 MHz, Chloroform-d) δ 6.68 (s, 2H), 4.11 (t, J = 7.1 Hz, 2H), 3.56 (t, J = 7.1 Hz, 2H), 2.32 (s, 6H), 2.19 (p, J = 7.1 Hz, 2H). ESI-MS m/z: 277.0 [M + 1]+.
加入 K 2 的混合物中CO 3 (8.3 克，60 毫摩尔，2 等分）、1,3-二溴丙烷（6 毫升，60 毫摩尔，2 等分）和 CH 3 CN（40 毫升）的混合物中，加入 58（4.7 克，30 毫摩尔，1 等分CN (40 mL)，加入 58 (4.7 g, 30 mmol, 1 equiv)，混合物在 90 °C 下搅拌 5 小时。冷却至室温后，用水（100 mL）稀释反应混合物，并用 EtOAc 进行萃取（3 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.在真空中去除溶剂，得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 500:1 至 300:1 v/v）进一步提纯，得到 59.无色液体，6.2 克，收率：75%。 1 H NMR（300 MHz，氯仿-d）δ 6.68（s，2H），4.11（t，J＝7.1 Hz，2H），3.56（t，J＝7.1 Hz，2H），2.32（s，6H），2.19（p，J＝7.1 Hz，2H）。ESI-MS m/z: 277.0 [M + 1] + ..
加入 K 2 的混合物中CO 3 (8.3 克，60 毫摩尔，2 等分）、1,3-二溴丙烷（6 毫升，60 毫摩尔，2 等分）和 CH 3 CN（40 毫升）的混合物中，加入 58（4.7 克，30 毫摩尔，1 等分CN (40 mL)，加入 58 (4.7 g, 30 mmol, 1 equiv)，混合物在 90 °C 下搅拌 5 小时。冷却至室温后，用水（100 mL）稀释反应混合物，并用 EtOAc 进行萃取（3 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.在真空中去除溶剂，得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 500:1 至 300:1 v/v）进一步提纯，得到 59.无色液体，6.2 克，收率：75%。 1 H NMR（300 MHz，氯仿-d）δ 6.68（s，2H），4.11（t，J＝7.1 Hz，2H），3.56（t，J＝7.1 Hz，2H），2.32（s，6H），2.19（p，J＝7.1 Hz，2H）。ESI-MS m/z: 277.0 [M + 1] + ..
Methyl-4-(4-methylbenzoyl)-1H-pyrrole-2-carboxylate (61)
4-(4-甲基苯甲酰基)-1H-吡咯-2-甲酸甲酯 (61)
At 0 °C, to a solution of 4-methylbenzoyl chloride (5 g, 32 mmol) and DCM (40 mL), AlCl3 (4.4 g, 32 mmol) was added under nitrogen. The mixture was stirred for 10 min, and 60 (2 g, 16 mmol) was added. The reaction was then allowed to stir at room temperature for 2 h under nitrogen. After the completion of the reaction, 100 mL of water was carefully added to the mixture. DCM (80 mL) was added to the mixture and stirred for 15 min. After filtration, the organic layer was isolated and the inorganic layer was extracted with DCM (2 times). The combined organic layer was then washed with brine and dried with Na2SO4. Removal of solvent under reduced pressure yielded the crude product. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 30:1 to 15:1 v/v) gave 61. White powder, 2.6 g, yield: 68%, 1H NMR (300 MHz, Chloroform-d) δ 10.37 (s, 1H), 7.80 (d, J = 7.9 Hz, 2H), 7.60 (s, 1H), 7.40 (s, 1H), 7.31 (d, J = 7.6 Hz, 2H), 3.92 (s, 3H), 2.46 (s, 3H). ESI-MS m/z: 244.1 [M + 1]+.
0 °C 时，向 4-甲基苯甲酰氯（5 克，32 毫摩尔）和 DCM（40 毫升）的溶液中，在氮气下加入 AlCl 3 （4.4 克，32 毫摩尔）。(4.4 克，32 毫摩尔）。混合物搅拌 10 分钟，然后加入 60（2 克，16 毫摩尔）。然后在氮气环境下于室温搅拌 2 小时。反应完成后，小心地向混合物中加入 100 mL 水。向混合物中加入 DCM（80 mL）并搅拌 15 分钟。过滤后，分离出有机层，无机层用二氯甲烷萃取（2 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 30:1 至 15:1 v/v）进一步纯化，得到 61.白色粉末，2.6 克，收率：68%， 1 。H NMR（300 MHz，氯仿-d）δ 10.37（s，1H），7.80（d，J = 7.9 Hz，2H），7.60（s，1H），7.40（s，1H），7.31（d，J = 7.6 Hz，2H），3.92（s，3H），2.46（s，3H）。ESI-MS m/z: 244.1 [M + 1] + ..
0 °C 时，向 4-甲基苯甲酰氯（5 克，32 毫摩尔）和 DCM（40 毫升）的溶液中，在氮气下加入 AlCl 3 （4.4 克，32 毫摩尔）。(4.4 克，32 毫摩尔）。混合物搅拌 10 分钟，然后加入 60（2 克，16 毫摩尔）。然后在氮气环境下于室温搅拌 2 小时。反应完成后，小心地向混合物中加入 100 mL 水。向混合物中加入 DCM（80 mL）并搅拌 15 分钟。过滤后，分离出有机层，无机层用二氯甲烷萃取（2 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 30:1 至 15:1 v/v）进一步纯化，得到 61.白色粉末，2.6 克，收率：68%， 1 。H NMR（300 MHz，氯仿-d）δ 10.37（s，1H），7.80（d，J = 7.9 Hz，2H），7.60（s，1H），7.40（s，1H），7.31（d，J = 7.6 Hz，2H），3.92（s，3H），2.46（s，3H）。ESI-MS m/z: 244.1 [M + 1] + ..
Methyl-4-(4-methylbenzyl)-1H-pyrrole-2-carboxylate (62)
4-(4-甲基苄基)-1H-吡咯-2-甲酸甲酯 (62)
To a solution of 61 (1 g, 4.1 mmol) and THF (30 mL), triethylsilane (0.93 g, 8.2 mmol) was added under nitrogen. The solution was then allowed to stir for 2 h at room temperature under nitrogen. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 80:1 to 40:1 v/v) gave 62, white powder, 0.61 g, yield: 65% 1H NMR (300 MHz, Chloroform-d) δ 9.13 (s, 1H), 7.14 (s, 4H), 6.76 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H), 3.82 (s, 2H), 2.36 (s, 3H). ESI-MS m/z: 247.1 [M + NH4]+.
在氮气环境下，向 61（1 克，4.1 毫摩尔）和 THF（30 毫升）的溶液中加入三乙基硅烷（0.93 克，8.2 毫摩尔）。然后让溶液在室温氮气下搅拌 2 小时。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 80:1 至 40:1 v/v）进一步纯化，得到 62，白色粉末，0.61 克，收率：65% 1 。H NMR（300 MHz，氯仿-d）δ 9.13（s，1H），7.14（s，4H），6.76（d，J = 5.6 Hz，2H），3.86（s，3H），3.82（s，2H），2.36（s，3H）。ESI-MS m/z: 247.1 [M + NH 4 ] + ..
在氮气环境下，向 61（1 克，4.1 毫摩尔）和 THF（30 毫升）的溶液中加入三乙基硅烷（0.93 克，8.2 毫摩尔）。然后让溶液在室温氮气下搅拌 2 小时。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 80:1 至 40:1 v/v）进一步纯化，得到 62，白色粉末，0.61 克，收率：65% 1 。H NMR（300 MHz，氯仿-d）δ 9.13（s，1H），7.14（s，4H），6.76（d，J = 5.6 Hz，2H），3.86（s，3H），3.82（s，2H），2.36（s，3H）。ESI-MS m/z: 247.1 [M + NH 4 ] + ..
Methyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-(4-methylbenzyl)-1H-pyrrole-2-carboxylate (63)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-(4-甲基苄基)-1H-吡咯-2-甲酸甲酯 (63)
To a solution of 62 (1 g, 4.4 mmol) and DMF (20 mL), Cs2CO3 (2.5 g, 8 mmol) was added. The mixture was stirred for 10 min at 60 °C and subsequently, 59 (2.2 g, 8 mmol) was added. The mixture was allowed to stir for another 1 h. After cooling to room temperature, the reaction mixture was then diluted in 60 mL of water. The mixture was extracted with EtOAc (3 times). The combined organic layer was then washed with brine and dried with Na2SO4. Removal of solvent under reduced pressure yielded a crude product. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 80:1 to 30:1 v/v) gave 63, white powder, yield: 89%. 1H NMR (300 MHz, Chloroform-d) δ 7.11 (s, 4H), 6.86 (s, 1H), 6.73 (s, 1H), 6.68 (s, 2H), 4.50 (t, J = 6.6 Hz, 2H), 3.88 (t, J = 5.5 Hz, 2H), 3.85 (s, 3H), 3.76 (s, 2H), 2.42 (s, 6H), 2.38 (s, 3H), 2.27 (p, J = 6.0 Hz, 2H). ESI-MS m/z: 464.1 [M + K]+.
向 62（1 克，4.4 毫摩尔）和 DMF（20 毫升）的溶液中加入 Cs 2 CO 3 (2.5 克，8 毫摩尔）。混合物在 60 °C 下搅拌 10 分钟，随后加入 59（2.2 克，8 毫摩尔）。冷却至室温后，用 60 mL 水稀释反应混合物。混合物用乙酸乙酯萃取（3 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯=80:1 至 30:1 v/v）进一步提纯，得到白色粉末 63，收率：89%。 1 H NMR（300 MHz，氯仿-d）δ 7.11（s，4H），6.86（s，1H），6.73（s，1H），6.68（s，2H），4.50（t，J = 6.6 Hz，2H），3.88（t，J = 5.5 Hz，2H），3.85（s，3H），3.76（s，2H），2.42（s，6H），2.38（s，3H），2.27（p，J = 6.0 Hz，2H）。ESI-MS m/z: 464.1 [M + K] + ..
向 62（1 克，4.4 毫摩尔）和 DMF（20 毫升）的溶液中加入 Cs 2 CO 3 (2.5 克，8 毫摩尔）。混合物在 60 °C 下搅拌 10 分钟，随后加入 59（2.2 克，8 毫摩尔）。冷却至室温后，用 60 mL 水稀释反应混合物。混合物用乙酸乙酯萃取（3 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯=80:1 至 30:1 v/v）进一步提纯，得到白色粉末 63，收率：89%。 1 H NMR（300 MHz，氯仿-d）δ 7.11（s，4H），6.86（s，1H），6.73（s，1H），6.68（s，2H），4.50（t，J = 6.6 Hz，2H），3.88（t，J = 5.5 Hz，2H），3.85（s，3H），3.76（s，2H），2.42（s，6H），2.38（s，3H），2.27（p，J = 6.0 Hz，2H）。ESI-MS m/z: 464.1 [M + K] + ..
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(4-methylbenzyl)-1H-pyrrole-2-carboxylic Acid (11)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-(4-甲基苄基)-1H-吡咯-2-羧酸 (11)
To a solution of 63 (500 mg, 1.2 mmol) in MeOH/THF (5 mL/5 mL) was added 1 mL of NaOH (2 M). The solution was stirred overnight at 50 °C. After completion of the reaction, the mixture was quenched with 1 M hydrochloric acid to pH 2 and then extracted with ethyl acetate (3 times). The organic layer was washed with brine and dried with Na2SO4. Removal of the solvent and recrystallization from ethyl acetate/n-hexane gave title compound 11 as a white solid, yield: 78%; mp: 201–203 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1H), 7.04 (s, 4H), 6.82 (d, J = 1.6 Hz, 1H), 6.77 (s, 2H), 6.64 (d, J = 1.9 Hz, 1H), 4.38 (t, J = 6.6 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H), 3.64 (s, 2H), 2.31 (s, 6H), 2.26 (s, 3H), 2.10 (p, J = 6.3 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.24, 156.94, 138.94, 136.94, 134.99, 129.25, 128.68, 128.06, 125.53, 122.60, 121.89, 118.27, 115.11, 64.97, 45.46, 32.35, 30.87, 20.91. HRMS (ESI): calcd for C24H26ClNO3 [M-H]− 410.1523, found 410.1525. HPLC (90:10 methanol:water with 1‰ TFA): tR = 8.51 min, 98.5%.
向 63（500 毫克，1.2 毫摩尔）在 MeOH/THF （5 毫升/5 毫升）中的溶液中加入 1 毫升 NaOH（2 兆）。该溶液在 50 °C 下搅拌过夜。反应完成后，用 1 M 盐酸淬灭混合物至 pH 2，然后用乙酸乙酯萃取（3 次）。有机层用盐水洗涤，并用 Na 2 SO { 4 干燥。SO {{1} 干燥。｝.除去溶剂，从乙酸乙酯/正己烷中重结晶，得到标题化合物 11，为白色固体，产率：78%；熔点：201-203 °C：201-203 °C. 1 H NMR（300 MHz，DMSO-d 6 ）δ 12.10（s，1H），7.04（s，4H），6.82（d，J = 1.6 Hz，1H），6.77（s，2H），6.64（d，J = 1.9Hz，1H）、4.38（t，J = 6.6Hz，2H）、3.82（t，J = 6.0Hz，2H）、3.64（s，2H）、2.31（s，6H）、2.26（s，3H）、2.10（p，J = 6.3Hz，2H）。 13 C NMR（75 MHz，DMSO-d 6 ）δ 162.24，156.94，138.94，136.94，134.99，129.25，128.68，128.06，125.53，122.60，121.89，118.27，115.11，64.97，45.46，32.35，30.87，20.91。HRMS (ESI)：煅烧为 C 24 H { 26 H 26 ClNO 3 [M-H] {{9}410.1523，发现值 410.1525。HPLC（90：10 甲醇：水，含 1‰反式脂肪酸）：t R = 8.51 分钟，98.5%。
向 63（500 毫克，1.2 毫摩尔）在 MeOH/THF （5 毫升/5 毫升）中的溶液中加入 1 毫升 NaOH（2 兆）。该溶液在 50 °C 下搅拌过夜。反应完成后，用 1 M 盐酸淬灭混合物至 pH 2，然后用乙酸乙酯萃取（3 次）。有机层用盐水洗涤，并用 Na 2 SO { 4 干燥。SO {{1} 干燥。｝.除去溶剂，从乙酸乙酯/正己烷中重结晶，得到标题化合物 11，为白色固体，产率：78%；熔点：201-203 °C：201-203 °C. 1 H NMR（300 MHz，DMSO-d 6 ）δ 12.10（s，1H），7.04（s，4H），6.82（d，J = 1.6 Hz，1H），6.77（s，2H），6.64（d，J = 1.9Hz，1H）、4.38（t，J = 6.6Hz，2H）、3.82（t，J = 6.0Hz，2H）、3.64（s，2H）、2.31（s，6H）、2.26（s，3H）、2.10（p，J = 6.3Hz，2H）。 13 C NMR（75 MHz，DMSO-d 6 ）δ 162.24，156.94，138.94，136.94，134.99，129.25，128.68，128.06，125.53，122.60，121.89，118.27，115.11，64.97，45.46，32.35，30.87，20.91。HRMS (ESI)：煅烧为 C 24 H { 26 H 26 ClNO 3 [M-H] {{9}410.1523，发现值 410.1525。HPLC（90：10 甲醇：水，含 1‰反式脂肪酸）：t R = 8.51 分钟，98.5%。
Methyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-(4-methylbenzoyl)-1H-pyrrole-2-carboxylate (64)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-(4-甲基苯甲酰基)-1H-吡咯-2-甲酸甲酯 (64)
64 was synthesized according to the procedure of 63, white solid, yield 84%; 1H NMR (300 MHz, Chloroform-d) 7.64 (d, J = 7.2 Hz, 2H), 7.47 (s, 1H), 7.38 (s, 1H), 7.14 (d, J = 7.5 Hz, 2H), 6.64 (s, 2H), 4.58 (t, J = 6.4 Hz, 2H), 3.87 (d, J = 7.6 Hz, 5H), 2.42 (s, 3H), 2.36 (s, 6H), 2.33–2.21 (m, 2H). ESI-MS m/z: 462.2 [M + Na]+.
64 按照 63 的步骤合成，白色固体，收率 84%； 1 H NMR（300 MHz，氯仿-dH NMR（300 MHz，氯仿-d）7.64（d，J = 7.2 Hz，2H），7.47（s，1H），7.38（s，1H），7.14（d，J = 7.5 Hz，2H），6.64（s，2H），4.58（t，J = 6.4 Hz，2H），3.87（d，J = 7.6 Hz，5H），2.42（s，3H），2.36（s，6H），2.33-2.21（m，2H）。ESI-MS m/z：462.2 [M + Na] + ..
64 按照 63 的步骤合成，白色固体，收率 84%； 1 H NMR（300 MHz，氯仿-dH NMR（300 MHz，氯仿-d）7.64（d，J = 7.2 Hz，2H），7.47（s，1H），7.38（s，1H），7.14（d，J = 7.5 Hz，2H），6.64（s，2H），4.58（t，J = 6.4 Hz，2H），3.87（d，J = 7.6 Hz，5H），2.42（s，3H），2.36（s，6H），2.33-2.21（m，2H）。ESI-MS m/z：462.2 [M + Na] + ..
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(4-methylbenzoyl)-1H-pyrrole-2-carboxylic Acid (12)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-(4-甲基苯甲酰基)-1H-吡咯-2-羧酸 (12)
12 was synthesized from 64 according to the procedure of 9, white powder, yield: 95%. mp: 174–175 °C, 1H NMR (300 MHz, Chloroform-d) δ 7.68 (d, J = 8.2 Hz, 2H), 7.66 (s, 1H), 7.48 (s, 1H), 7.17 (d, J = 7.8 Hz, 2H), 6.67 (s, 2H), 4.62 (t, J = 5.7 Hz, 2H), 3.92 (t, J = 4.8 Hz, 2H), 2.46 (s, 3H), 2.38 (s, 6H), 2.34 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 188.74, 162.00, 156.85, 142.42, 136.97, 136.48, 134.95, 129.37, 129.10, 125.57, 124.12, 122.39, 119.05, 115.08, 64.85, 46.57, 30.33, 21.53, 20.90. HRMS (ESI): calcd for C24H24ClNO4 [M + Na]+ 448.1292, found 448.1293. HPLC (90:10 methanol:water with 1‰ TFA): tR = 6.97 min, 98.5%.
12 由 64 按照 9 的步骤合成，白色粉末，收率：95%：174-175 °C, 1 H NMR（300 MHz，氯仿-d）δ 7.68（d，J = 8.2 Hz，2H），7.66（s，1H），7.48（s，1H），7.17（d，J = 7.8 Hz，2H），6.67（s，2H），4.62（t，J = 5.7 Hz，2H），3.92（t，J = 4.8 Hz，2H），2.46（s，3H），2.38（s，6H），2.34（s，2H）。 13 C NMR（75 MHz，DMSO-d 6 ）δ 188.74，162.00，156.85，142.42，136.97，136.48，134.95，129.37，129.10，125.57，124.12，122.39，119.05，115.08，64.85，46.57，30.33，21.53，20.90。HRMS (ESI)：煅烧为 C 24 H { 24 H 24 ClNO 4 [M + Na] + 448.1292，发现 448.1293。HPLC（90：10 甲醇：水，含 1‰反式脂肪酸）：t R = 6.97 分钟，98.5%。
12 由 64 按照 9 的步骤合成，白色粉末，收率：95%：174-175 °C, 1 H NMR（300 MHz，氯仿-d）δ 7.68（d，J = 8.2 Hz，2H），7.66（s，1H），7.48（s，1H），7.17（d，J = 7.8 Hz，2H），6.67（s，2H），4.62（t，J = 5.7 Hz，2H），3.92（t，J = 4.8 Hz，2H），2.46（s，3H），2.38（s，6H），2.34（s，2H）。 13 C NMR（75 MHz，DMSO-d 6 ）δ 188.74，162.00，156.85，142.42，136.97，136.48，134.95，129.37，129.10，125.57，124.12，122.39，119.05，115.08，64.85，46.57，30.33，21.53，20.90。HRMS (ESI)：煅烧为 C 24 H { 24 H 24 ClNO 4 [M + Na] + 448.1292，发现 448.1293。HPLC（90：10 甲醇：水，含 1‰反式脂肪酸）：t R = 6.97 分钟，98.5%。
Methyl-4-tosyl-1H-pyrrole-2-carboxylate (66a)
4-对甲苯磺酰基-1H-吡咯-2-羧酸甲酯（66a）
A mixture of 65a (500 mg, 3 mmol), Ag2CO3 (0.08 g, 0.3 mmol), and dioxane (20 mL) was charged with methyl isocyanate (1 equiv). The mixture was heated to 80 °C and stirred for 1 h. Then, the mixture was cooled to room temperature, charged with 60 mL water, and extracted with EtOAc (3 times). The combined organic layer was then washed with brine and dried with Na2SO4. Removal of solvent under reduced pressure yielded a crude product. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 20:1 to 5:1 v/v) gave 66a. Yellow oil, 150 mg, yield: 18%. 1H NMR (300 MHz, DMSO-d6) δ 12.87 (s, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.03 (s, 1H), 3.77 (s, 3H), 2.35 (s, 3H). ESI-MS m/z: 280.1 [M + 1]+.
65a （500 毫克，3 毫摩尔）、Ag 2 和 CO 3 的混合物。CO 3(0.08 克，0.3 毫摩尔）和二噁烷（20 毫升）的混合物中加入异氰酸甲酯（1 等分）。然后，将混合物冷却至室温，加入 60 mL 水，并用 EtOAc 进行萃取（3 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 20:1 至 5:1 v/v）进一步纯化，得到 66a。黄色油状物，150 毫克，收率：18%。 1 H NMR（300 MHz，DMSO-d 6 ）δ 12.87（s，1H），7.80（d，J = 8.3 Hz，2H），7.64（s，1H），7.37（d，J = 8.0 Hz，2H），7.03（s，1H），3.77（s，3H），2.35（s，3H）。ESI-MS m/z: 280.1 [M + 1] + ..
65a （500 毫克，3 毫摩尔）、Ag 2 和 CO 3 的混合物。CO 3(0.08 克，0.3 毫摩尔）和二噁烷（20 毫升）的混合物中加入异氰酸甲酯（1 等分）。然后，将混合物冷却至室温，加入 60 mL 水，并用 EtOAc 进行萃取（3 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 20:1 至 5:1 v/v）进一步纯化，得到 66a。黄色油状物，150 毫克，收率：18%。 1 H NMR（300 MHz，DMSO-d 6 ）δ 12.87（s，1H），7.80（d，J = 8.3 Hz，2H），7.64（s，1H），7.37（d，J = 8.0 Hz，2H），7.03（s，1H），3.77（s，3H），2.35（s，3H）。ESI-MS m/z: 280.1 [M + 1] + ..
Methyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-tosyl-1H-pyrrole-2-carboxylate (67a)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-对甲苯磺酰基-1H-吡咯-2-羧酸甲酯 (67a)
At 0 °C, to a solution of 66a (0.150 g, 0.5 mmol) and distilled DMF (15 mL), NaH (0.04 g, 1 mmol) was added in portions. After the effervescence of the mixture, 59 (0.3 g, 1 mmol) was added and the reaction was stirred at room temperature for 0.5 h. After the consumption of 66a monitored by TLC, 45 mL water was carefully added to the mixture and the mixture was extracted with EtOAc (3 times). The combined organic layer was then washed with brine and dried over Na2SO4. Removal of solvent under reduced pressure yielded a crude product. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 70:1 to 20:1 v/v) gave 67a. Yellow oil, 0.1 mg, yield: 42%. 1H NMR (300 MHz, Chloroform-d) δ 7.75 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 1.5 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 1.5 Hz, 1H), 6.69 (s, 2H), 4.15 (t, J = 7.1 Hz, 2H), 4.06 (t, J = 7.1 Hz, 2H), 3.90 (s, 3H), 2.40 (s, 3H), 2.32 (s, 6H), 2.08 (p, J = 7.1 Hz, 2H). ESI-MS m/z: 476.1 [M + 1]+.
0 °C 时，向 66a（0.150 克，0.5 毫摩尔）和蒸馏 DMF（15 毫升）的溶液中分次加入 NaH（0.04 克，1 毫摩尔）。TLC 监测到 66a 消耗后，小心地向混合物中加入 45 mL 水，然后用 EtOAc（3 次）萃取混合物。然后用盐水洗涤合并的有机层，并用 Na 2 SO { 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 70:1 至 20:1 v/v）进一步纯化，得到 67a。黄色油状物，0.1 毫克，收率：42%。 1 H NMR（300 MHz，氯仿-d）δ 7.75（d，J = 7.6 Hz，2H），7.69（d，J = 1.5 Hz，1H），7.28（d，J = 8.0 Hz，2H），6.95（d，J = 1.5 Hz，1H），6.69（s，2H），4.15（t，J = 7.1 Hz，2H），4.06（t，J = 7.1 Hz，2H），3.90（s，3H），2.40（s，3H），2.32（s，6H），2.08（p，J = 7.1 Hz，2H）。ESI-MS m/z：476.1 [M + 1] + ..
0 °C 时，向 66a（0.150 克，0.5 毫摩尔）和蒸馏 DMF（15 毫升）的溶液中分次加入 NaH（0.04 克，1 毫摩尔）。TLC 监测到 66a 消耗后，小心地向混合物中加入 45 mL 水，然后用 EtOAc（3 次）萃取混合物。然后用盐水洗涤合并的有机层，并用 Na 2 SO { 4 干燥。SO 4 干燥。.减压去除溶剂后得到粗产品。通过硅胶柱色谱法（石油醚/乙酸乙酯 = 70:1 至 20:1 v/v）进一步纯化，得到 67a。黄色油状物，0.1 毫克，收率：42%。 1 H NMR（300 MHz，氯仿-d）δ 7.75（d，J = 7.6 Hz，2H），7.69（d，J = 1.5 Hz，1H），7.28（d，J = 8.0 Hz，2H），6.95（d，J = 1.5 Hz，1H），6.69（s，2H），4.15（t，J = 7.1 Hz，2H），4.06（t，J = 7.1 Hz，2H），3.90（s，3H），2.40（s，3H），2.32（s，6H），2.08（p，J = 7.1 Hz，2H）。ESI-MS m/z：476.1 [M + 1] + ..
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (13)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-3-甲基-4-对甲苯磺酰基-1H-吡咯-2-羧酸 (13)
13 was synthesized from 67a according to the hydrolysis procedure of 11. White powder, yield: 68%; mp: 194–196 °C. 1H NMR (300 MHz, Chloroform-d) δ 12.89 (s, 1H), 7.81 (s, 1H), 7.72 (d, J = 6.8 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.05 (s, 1H), 6.71 (s, 2H), 4.46 (t, J = 5.1 Hz, 2H), 3.83 (t, J = 6.2 Hz, 2H), 2.35 (s, 3H), 2.28 (s, 6H), 2.17–2.08 (m, 2H). HRMS (ESI): calcd for C23H24ClNO5S [M + NH4]+ 479.1407, found 479.1402. HPLC (80:20 methanol:water with 1‰ TFA): tR = 8.14 min, 99.8%.
白色粉末，收率：68%；熔点：194-196°C：194-196 °C. 1 H NMR（300 MHz，氯仿-d）δ 12.89（s，1H），7.81（s，1H），7.72（d，J = 6.8 Hz，2H），7.34（d，J = 7.9 Hz，2H），7.05（s，1H），6.71（s，2H），4.46（t，J = 5.1 Hz，2H），3.83（t，J = 6.2 Hz，2H），2.35（s，3H），2.28（s，6H），2.17-2.08（m，2H）。HRMS (ESI)：煅烧为 C 23 H { 24 H 24 ClNO 5 S [M + NH 4 ] + 479.1407，发现值 479.1402。HPLC（80:20 甲醇:水，含 1‰反式脂肪酸）：t R = 8.14 分钟，99.8%。
白色粉末，收率：68%；熔点：194-196°C：194-196 °C. 1 H NMR（300 MHz，氯仿-d）δ 12.89（s，1H），7.81（s，1H），7.72（d，J = 6.8 Hz，2H），7.34（d，J = 7.9 Hz，2H），7.05（s，1H），6.71（s，2H），4.46（t，J = 5.1 Hz，2H），3.83（t，J = 6.2 Hz，2H），2.35（s，3H），2.28（s，6H），2.17-2.08（m，2H）。HRMS (ESI)：煅烧为 C 23 H { 24 H 24 ClNO 5 S [M + NH 4 ] + 479.1407，发现值 479.1402。HPLC（80:20 甲醇:水，含 1‰反式脂肪酸）：t R = 8.14 分钟，99.8%。
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (20)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-3-甲基-4-对甲苯磺酰基-1H-吡咯-2-羧酸 (20)
Starting from 67b, 20 was synthesized according to the procedure of 11. White powder, yield: 10%; mp: 194–196 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.71 (d, J = 8.2 Hz, 2H), 7.54 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 6.64 (s, 2H), 4.54 (t, J = 6.5 Hz, 2H), 3.83 (t, J = 5.5 Hz, 2H), 2.48 (s, 3H), 2.44 (s, 3H), 2.38 (s, 6H), 2.25 (p, J = 6.1 Hz, 2H). HRMS (ESI): calcd for C24H26ClNO5S [M + Na]+ 498.1118, found 498.1116. HPLC (80:20 methanol:water with 1‰ TFA): tR = 9.08 min, 98.8%.
白色粉末，收率：10%；熔点：194-196°C：194-196 °C. 1 H NMR（300 MHz，氯仿-d）δ 7.71（d，J = 8.2 Hz，2H），7.54（s，1H），7.26（d，J = 8.2 Hz，2H），6.64（s，2H），4.54（t，J = 6.5 Hz，2H），3.83（t，J = 5.5 Hz，2H），2.48（s，3H），2.44（s，3H），2.38（s，6H），2.25（p，J = 6.1 Hz，2H）。HRMS (ESI)：煅烧为 C 24 H { 26 H 26 ClNO 5 S [M + Na] + 498.1118，发现值为 498.1116。HPLC（80：20 甲醇：水，含 1‰反式脂肪酸）：t R = 9.08 分钟，98.8%。
白色粉末，收率：10%；熔点：194-196°C：194-196 °C. 1 H NMR（300 MHz，氯仿-d）δ 7.71（d，J = 8.2 Hz，2H），7.54（s，1H），7.26（d，J = 8.2 Hz，2H），6.64（s，2H），4.54（t，J = 6.5 Hz，2H），3.83（t，J = 5.5 Hz，2H），2.48（s，3H），2.44（s，3H），2.38（s，6H），2.25（p，J = 6.1 Hz，2H）。HRMS (ESI)：煅烧为 C 24 H { 26 H 26 ClNO 5 S [M + Na] + 498.1118，发现值为 498.1116。HPLC（80：20 甲醇：水，含 1‰反式脂肪酸）：t R = 9.08 分钟，98.8%。
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-nitro-1H-pyrrole-2-carboxylate (69)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-硝基-1H-吡咯-2-甲酸乙酯 (69)
69 was synthesized from 68 according to the procedure of 63, yield 75%; 1H NMR (300 MHz, Chloroform-d) δ 7.67 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 6.65 (s, 2H), 4.61 (t, J = 6.8 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.95 (t, J = 5.6 Hz, 2H), 2.38 (s, 6H), 2.31 (p, J = 6.5 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 381.1 [M + H]+.
69 由 68 按照 63 的步骤合成，收率为 75%； 1 H NMR（300 MHz，氯仿-dH NMR（300 MHz，氯仿-d）δ 7.67（d，J = 2.0 Hz，1H），7.49（d，J = 2.0 Hz，1H），6.65（s，2H），4.61（t，J = 6.8 Hz，2H），4.34（q，J = 7.1 Hz，2H），3.95（t，J = 5.6 Hz，2H），2.38（s，6H），2.31（p，J = 6.5 Hz，2H），1.40（t，J = 7.1 Hz，3H）。ESI-MS m/z: 381.1 [M + H] + ..
69 由 68 按照 63 的步骤合成，收率为 75%； 1 H NMR（300 MHz，氯仿-dH NMR（300 MHz，氯仿-d）δ 7.67（d，J = 2.0 Hz，1H），7.49（d，J = 2.0 Hz，1H），6.65（s，2H），4.61（t，J = 6.8 Hz，2H），4.34（q，J = 7.1 Hz，2H），3.95（t，J = 5.6 Hz，2H），2.38（s，6H），2.31（p，J = 6.5 Hz，2H），1.40（t，J = 7.1 Hz，3H）。ESI-MS m/z: 381.1 [M + H] + ..
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-((4-methylphenyl)sulfonamido)-1H-pyrrole-2-carboxylate (70)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-((4-甲基苯基)磺酰胺基)-1H-吡咯-2-甲酸乙酯 (70)
To a solution of 69 (1.5 g, 4.1 mmol) and EtOH (50 mL), SnCl2·2H2O (4.6 g, 20 mmol) was added under nitrogen. The mixture was then allowed to stir at 60 °C for 4 h under nitrogen. Upon the consumption of 69, the solution was cooled to room temperature and quenched with aqueous NaOH (2 M) to pH 9. DCM (70 mL) was added, and the mixture was stirred for 15 min. The organic layer was isolated after the undissolved white solid was filtered. The aqueous phase was then extracted with DCM (2 times). The combined organic layer was then washed with brine and dried with Na2SO4. Removal of solvent under reduced pressure yielded a crude product as a deep brown oil without further purification. 0.6 g (1.7 mmol) of the crude product was dissolved in pyridine (15 mL), and p-toluenesulfonyl chloride (0.65 g, 3.4 mmol) was added. The solution was stirred at room temperature for 1 h. Upon completion of the reaction, the solution was concentrated under reduced pressure, and the crude product 70 was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 60:1 to 20:1 v/v) as yellow oil, 0.63 g, yield: 38%, 1H NMR (300 MHz, Chloroform-d) δ 7.62 (s, 1H), 7.32 (d, J = 7.5 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.92 (s, 1H), 6.68 (s, 2H), 4.29 (q, J = 8.0 Hz, 2H), 4.14 (t, J = 7.1 Hz, 2H), 4.05 (t, J = 7.1 Hz, 2H), 2.34 (s, 3H), 2.33 (s, 6H), 2.08 (p, J = 7.2 Hz, 2H), 1.35 (t, J = 8.0 Hz, 3H). ESI-MS m/z: 505.1 [M + H]+.
向 69（1.5 克，4.1 毫摩尔）和 EtOH（50 毫升）溶液中加入 SnCl 2 -2H 2 O（4.6 克，20 毫摩尔）。然后在氮气环境下将混合物在 60 °C 下搅拌 4 小时。69 消耗后，将溶液冷却至室温，并用 NaOH 水溶液（2 M）淬火至 pH 9。过滤未溶解的白色固体，分离出有机层。然后用二氯甲烷萃取水相（2 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后，得到深棕色油状粗品，无需进一步提纯。将 0.6 克（1.7 毫摩尔）粗产物溶于吡啶（15 毫升）中，然后加入对甲苯磺酰氯（0.65 克，3.4 毫摩尔）。反应完成后，减压浓缩溶液，粗产物 70 经硅胶柱层析（石油醚/乙酸乙酯 = 60:1 至 20:1 v/v）纯化，为黄色油状物，0.63 克，收率：38%， 1 。H NMR（300 MHz，氯仿-d）δ 7.62（s，1H），7.32（d，J = 7.5 Hz，2H），7.15（d，J = 8.0 Hz，2H），6.92（s，1H），6.68（s，2H），4.29（q，J = 8.0Hz，2H），4.14（t，J = 7.1Hz，2H），4.05（t，J = 7.1Hz，2H），2.34（s，3H），2.33（s，6H），2.08（p，J = 7.2Hz，2H），1.35（t，J = 8.0Hz，3H）。ESI-MS m/z: 505.1 [M + H] + ..
向 69（1.5 克，4.1 毫摩尔）和 EtOH（50 毫升）溶液中加入 SnCl 2 -2H 2 O（4.6 克，20 毫摩尔）。然后在氮气环境下将混合物在 60 °C 下搅拌 4 小时。69 消耗后，将溶液冷却至室温，并用 NaOH 水溶液（2 M）淬火至 pH 9。过滤未溶解的白色固体，分离出有机层。然后用二氯甲烷萃取水相（2 次）。然后用盐水洗涤合并的有机层，并用 Na 2 SO 4 干燥。SO 4 干燥。.减压去除溶剂后，得到深棕色油状粗品，无需进一步提纯。将 0.6 克（1.7 毫摩尔）粗产物溶于吡啶（15 毫升）中，然后加入对甲苯磺酰氯（0.65 克，3.4 毫摩尔）。反应完成后，减压浓缩溶液，粗产物 70 经硅胶柱层析（石油醚/乙酸乙酯 = 60:1 至 20:1 v/v）纯化，为黄色油状物，0.63 克，收率：38%， 1 。H NMR（300 MHz，氯仿-d）δ 7.62（s，1H），7.32（d，J = 7.5 Hz，2H），7.15（d，J = 8.0 Hz，2H），6.92（s，1H），6.68（s，2H），4.29（q，J = 8.0Hz，2H），4.14（t，J = 7.1Hz，2H），4.05（t，J = 7.1Hz，2H），2.34（s，3H），2.33（s，6H），2.08（p，J = 7.2Hz，2H），1.35（t，J = 8.0Hz，3H）。ESI-MS m/z: 505.1 [M + H] + ..
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-methylphenyl)sulfonamido)-1H-pyrrole-2-carboxylic Acid (14)
1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-4-((4-甲基苯基)磺酰胺基)-1H-吡咯-2-羧酸 (14)
14 was synthesized from 70 according to the hydrolysis procedure of 9. Pale yellow solid, yield: 64%; mp: 179–181 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.76 (s, 1H), 7.58 (d, J = 7.6 Hz, 2H), 7.29 (d, J = 7.6 Hz, 2H), 6.89 (s, 1H), 6.75 (s, 2H), 6.48 (s, 1H), 4.45–4.29 (m, 2H), 3.78 (s, 2H), 2.31 (s, 9H), 2.03 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 160.64, 156.81, 143.24, 137.14, 136.96, 129.86, 129.73, 127.16, 125.56, 121.39, 119.44, 115.20, 111.92, 67.50, 51.48, 25.60, 21.35, 20.91. HRMS (ESI): calcd for C23H25ClN2O5S [M-H]− 475.1094, found 475.1093. HPLC (90:10 methanol:water with 1‰ TFA): tR = 6.85 min, 98.1%.
淡黄色固体，收率：64%；熔点：179-181 °C：179-181 °C. 1 H NMR（300 MHz，DMSO-d 6 ）δ 9.76（s，1H），7.58（d，J = 7.6 Hz，2H），7.29（d，J = 7.6 Hz，2H），6.89（s，1H），6.75（s，2H），6.48（s，1H），4.45-4.29（m，2H），3.78（s，2H），2.31（s，9H），2.03（s，2H）。 13 C NMR（75 MHz，DMSO-d 6 ）δ 160.64，156.81，143.24，137.14，136.96，129.86，129.73，127.16，125.56，121.39，119.44，115.20，111.92，67.50，51.48，25.60，21.35，20.91。HRMS (ESI)：煅烧为 C 23 H { 25 H 25 ClN 2 O 5 S [M-H] − 475.1094，发现值 475.1093。HPLC（90：10 甲醇：水，含 1‰反式脂肪酸）：t R = 6.85 分钟，98.1%。
淡黄色固体，收率：64%；熔点：179-181 °C：179-181 °C. 1 H NMR（300 MHz，DMSO-d 6 ）δ 9.76（s，1H），7.58（d，J = 7.6 Hz，2H），7.29（d，J = 7.6 Hz，2H），6.89（s，1H），6.75（s，2H），6.48（s，1H），4.45-4.29（m，2H），3.78（s，2H），2.31（s，9H），2.03（s，2H）。 13 C NMR（75 MHz，DMSO-d 6 ）δ 160.64，156.81，143.24，137.14，136.96，129.86，129.73，127.16，125.56，121.39，119.44，115.20，111.92，67.50，51.48，25.60，21.35，20.91。HRMS (ESI)：煅烧为 C 23 H { 25 H 25 ClN 2 O 5 S [M-H] − 475.1094，发现值 475.1093。HPLC（90：10 甲醇：水，含 1‰反式脂肪酸）：t R = 6.85 分钟，98.1%。
Ethyl-4-((tert-butoxycarbonyl)amino)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-pyrrole-2-carboxylate (71)
4-((叔丁氧羰基)氨基)-1-(3-(4-氯-3,5-二甲基苯氧基)丙基)-1H-吡咯-2-甲酸乙酯 (71)
Reduction of 69 was depicted in the synthesis of 70, and the crude product was used in the next step directly. 0.6 g (1.7 mmol) of the crude product was dissolved in EtOH (20 mL), and (Boc)2O (0.65 g, 3.4 mmol) was added. The solution was stirred at room temperature for 1 h. Upon completion of the reaction, the solution was concentrated under reduced pressure and purification by silica gel column chromatography (petroleum ether/ethyl acetate = 80:1 to 20:1 v/v), which gave 71 as a yellow oil, 0.42 g, yield: 46%, 1H NMR (300 MHz, Chloroform-d) δ 7.72 (d, J = 1.5 Hz, 1H), 6.70 (s, 1H), 6.67 (d, J = 1.5 Hz, 1H), 6.61 (s, 2H), 4.34 (t, J = 7.7 Hz, 2H), 4.28 (q, J = 8.0 Hz, 2H), 4.22 (t, J = 7.5 Hz, 2H), 2.31 (s, 6H), 1.98 (p, J = 7.6 Hz, 2H), 1.50 (s, 9H), 1.36 (t, J = 8.1 Hz, 3H). ESI-MS m/z: 451.2 [M + H]+.
Ethyl-4-((tert-butoxycarbonyl)(4-methylbenzyl)amino)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-pyrrole-2-carboxylate (72)
To a solution of 71 (0.4 g, 0.9 mmol) and DMF (15 mL), NaH (0.08 g, 2 mmol) was added in portions. After the effervescence of the mixture, 4-methylbenzyl bromide (0.36 g, 2 mmol) was added and the reaction was stirred at room temperature for 0.5 h. After the consumption of 71 monitored by TLC, 45 mL of water was carefully added to the mixture and then the mixture was extracted with EtOAc (3 times). The combined organic layer was then washed with brine and dried over Na2SO4. The combined organic layer was concentrated under reduced pressure and further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 100:1 to 40:1 v/v) gave 72. Light yellow oil, 0.4 g, yield: 73%. 1H NMR (300 MHz, Chloroform-d) 1H NMR (300 MHz, Chloroform-d) δ 7.26 (d, J = 1.5 Hz, 1H), 7.19 (d, J = 7.6 Hz, 2H), 7.11 (d, J = 7.5 Hz, 2H), 6.85 (d, J = 1.5 Hz, 1H), 6.69 (s, 2H), 5.24 (s, 2H), 4.28 (q, J = 7.5 Hz, 2H), 4.04 (dt, J = 10.0, 7.1 Hz, 4H), 2.33 (s, 3H), 2.32 (s, 6H), 2.11 (p, J = 7.1 Hz, 2H), 1.47 (s, 9H), 1.34 (t, J = 8.0 Hz, 3H). ESI-MS m/z: 555.2 [M + H]+.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-((4-methylbenzyl)amino)-1H-pyrrole-2-carboxylate (73)
72 (0.3 g, 0.56 mmol) was dissolved in CF3COOH (5 mL) and the solution was stirred for 1.5 h at room temperature. Upon the consumption of 72, saturated aqueous NaHCO3 was added carefully in portions to pH 8. The mixture was extracted with EtOAc (3 times). The combined organic layer was then washed with saturated aqueous NaHCO3 and brine and then dried with Na2SO4. Removal of solvent under reduced pressure gave 73 as light purple oil, 0.23 g, Yield: 95%, 1H NMR (300 MHz, Chloroform-d) δ 7.34 (d, J = 1.5 Hz, 1H), 7.23 (d, J = 1.5 Hz, 1H), 7.18 (d, J = 6.6 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 6.68 (s, 2H), 4.52 (s, 2H), 4.26 (q, J = 8.0 Hz, 2H), 4.12–4.02 (m, 4H), 2.34 (s, 3H), 2.32 (s, 6H), 2.10 (p, J = 7.1 Hz, 2H), 1.35 (t, J = 8.0 Hz, 3H). ESI-MS m/z: 455.2 [M + H]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-methylbenzyl)amino)-1H-pyrrole-2-carboxylic Acid (15)
15 was synthesized from 73 according to the hydrolysis procedure of 11. Brown powder, yield: 43%. mp: 157–158 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.20 (d, J = 7.9 Hz, 2H), 7.09 (d, J = 7.9 Hz, 2H), 6.77 (s, 2H), 6.37 (d, J = 2.1 Hz, 1H), 6.28 (d, J = 2.1 Hz, 1H), 4.29 (t, J = 6.5 Hz, 2H), 3.94 (s, 2H), 3.83 (t, J = 6.0 Hz, 2H), 2.31 (s, 6H), 2.27 (s, 3H), 2.06 (p, J = 6.2, 2H). 3C NMR (75 MHz, DMSO) δ 162.20, 156.96, 138.08, 136.94, 135.86, 135.66, 129.09, 127.78, 125.48, 119.55, 115.18, 114.39, 106.25, 65.02, 49.99, 45.05, 30.96, 21.12, 20.92. HRMS (ESI): calcd for C24H27ClN2O3 [M + H]+ 427.1788, found 427.1781. HPLC (90:10 methanol:water with 1‰ TFA): tR = 7.25 min, 98.2%.
Ethyl-3,5-dimethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate (75a)
At 0 °C, to a solution of p-toluenethiol (74a, 1.5 g, 12 mmol), Et3N (5 drops), and distilled DCM (20 mL), SO2Cl2 (1.6 g, 12 mmol) was carefully added in portions. The solution was allowed to stir for 10 min at room temperature. Ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate (1.1 g, 6 mmol) was then added, and the solution was stirred for another 45 min. Upon the consumption of ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, the reaction solution was concentrated under reduced pressure and purification by silica gel column chromatography (petroleum ether/ethyl acetate = 70:1 to 20:1 v/v) gave 75a, white solid, 73%, 1H NMR (300 MHz, Chloroform-d) δ 9.23 (s, 1H), 7.06 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.3 Hz, 2H), 4.39 (q, J = 7.1 Hz, 2H), 2.36 (s, 3H), 2.34 (s, 3H), 2.32 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 288.1 [M-H]−.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate (76a)
76a was synthesized from 75a according to the procedure of 63, colorless oil, yield: 75%; 1H NMR (300 MHz, Chloroform-d) δ 7.03 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 6.65 (s, 2H), 4.54 (t, J = 5.6 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.40 (s, 6H), 2.38 (s, 3H), 2.37 (s, 3H), 2.35 (s, 3H), 2.24 (p, J = 6.2 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 486.2 [M + 1]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylic Acid (23)
To a solution of 76a (380 mg, 0.78 mmol) in EtOH/THF (10 mL/10 mL), NaOH (2 M, 3 mL) was added. The mixture was stirred overnight at 50 °C. After the completion of the reaction, the mixture was then acidified with 1 M HCl and extracted with ethyl acetate. The organic layer was washed with brine and dried with Na2SO4. Removal of solution and further recrystallization from EtOH/n-hexane gave title compound 23. Yellow solid, Yield: 85%; mp: 125–127 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.40 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 6.1 Hz, 2H), 6.63 (s, 2H), 4.49 (t, J = 6.7 Hz, 2H), 3.88 (t, J = 5.3 Hz, 2H), 2.42 (s, 3H), 2.38 (s, 3H), 2.36 (s, 9H), 2.18 (m, J = 12.3, 6.4 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.72 , 156.75 , 141.18 , 136.95 , 135.52 , 134.31 , 132.79 , 130.05 , 125.54 , 125.27 , 119.64 , 114.97 , 107.66 , 64.90 , 43.21 , 30.57 , 20.88 , 20.80 , 12.74 , 10.91. HRMS (ESI): calcd for C25H28ClNO3S [M–H]− 456.1400, found 456.1399. HPLC (80:20 methanol: water with 1‰ TFA): tR = 14.8 min, 98.6%.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-tosyl-1H-pyrrole-2-carboxylate (77a)
At 0 °C, to a solution of 77a (0.8 g, 1 mmol) and distilled DCM (20 mL), 3-chloroperbenzoic acid (m-CPBA, 85%, 0.4 g, 2 mmol) was added. The solution was allowed to stir at room temperature for 0.5 h and then quenched with 20 mL of saturated aqueous NaHCO3. The organic layer was isolated and the aqueous phase was extracted with DCM (2 times). The combined organic layer was concentrated under reduced pressure and further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 20:1 to 4:1 v/v) gave 77a. Light yellow oil, yield: 56%. 1H NMR (300 MHz, Chloroform-d) δ 7.77 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.7 Hz, 2H), 6.70 (s, 2H), 4.32 (q, J = 7.1 Hz, 2H), 4.11 (t, J = 5.8 Hz, 2H), 3.64 (t, J = 6.4 Hz, 2H), 2.44 (s, 3H), 2.39 (s, 12H), 2.37–2.30 (m, 2H), 1.38 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 540.2 [M + Na]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (16)
16 was synthesized from 77a according to the hydrolysis procedure of 23. White powder, 172 mg, Yield: 57%, mp: 183–184 °C 1H NMR (300 MHz, chloroform-d) δ 12.87 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 7.7 Hz, 2H), 6.73 (s, 2H), 4.41 (t, J = 6.8 Hz, 2H), 3.87 (t, J = 4.8 Hz, 2H), 2.54 (s, 3H), 2.36 (s, 6H), 2.28 (s, 6H), 2.03 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.57, 156.70, 143.59, 141.31, 139.07, 136.94, 130.22, 127.93, 126.26, 125.55, 121.07, 119.06, 115.01, 65.16, 42.82, 30.22, 21.36, 20.86, 11.96, 11.02. HRMS (ESI): calcd for C25H28ClNO5S [M + Na]+ 512.1274, found 512.1269. HPLC (80:20 methanol:water with 1‰ TFA): tR = 11.93 min, 99.6%.
Starting from corresponding thiol compounds 74b–v, target compounds 24–26, 29–31, 33–34, 37 and 38, 41 and 42, 44–47, 51–53, and 56–57 were synthesized analogously according to 16.
4-(Benzylsulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (24)
Light yellow solid. Yield: 22%; mp: 144–146 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.32 (s, 3H), 7.12 (d, J = 6.7 Hz, 2H), 6.65 (s, 2H), 4.45 (s, 2H), 4.29 (s, 2H), 3.93 (s, 2H), 2.59 (s, 3H), 2.39 (s, 6H), 2.10 (s, 2H), 2.02 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 162.69, 156.65, 140.35, 136.93, 131.40, 129.91, 129.17, 128.53, 128.37, 125.55, 120.54, 115.81, 115.08, 65.05, 62.79, 42.54, 30.32, 20.86, 11.71, 10.17. HRMS (ESI): calcd for C25H28ClNO5S [M + Na]+ 512.1274, found 512.1266. HPLC (80:20 methanol:water with 1‰ TFA): tR = 9.79 min, 97.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(pyridin-2-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (25)
Pink solid, Yield: 15%; mp: 183–185 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.74 (s, 1H), 8.64 (d, J = 3.7 Hz, 1H), 8.08 (s, 2H), 7.63 (s, 1H), 6.73 (s, 2H), 4.42 (s, 2H), 3.87 (s, 2H), 2.53 (s, 3H), 2.39 (s, 3H), 2.28 (s, 6H), 2.03 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 168.07, 164.81, 164.27, 158.54, 141.08, 138.73, 131.16, 131.03, 129.70, 127.39, 123.16, 120.38, 118.86, 118.56, 116.93, 67.15, 44.69, 32.05, 22.56, 13.62, 12.84. HRMS (ESI): calcd for C23H25ClN2O5S [M + H]+ 477.1251, found 477.1242. HPLC (80:20 methanol:water with 1‰ TFA): tR = 6.49 min, 98.1%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(pyridin-4-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (26)
Purple solid, Yield: 13%; mp: 179–181 °C. 1H NMR (300 MHz, Chloroform-d) δ 8.74 (d, J = 2.7 Hz, 2H), 7.50 (d, J = 5.2 Hz, 2H), 6.62 (s, 2H), 4.52 (t, J = 6.8 Hz, 2H), 3.90 (t, J = 5.3 Hz, 2H), 2.41 (s, 3H), 2.36 (s, 6H), 2.32 (s, 3H), 2.21 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.53, 156.72, 155.92, 151.80, 150.45, 139.61, 137.00, 128.69, 121.17, 119.74, 119.53, 115.04, 65.12, 42.84, 30.33, 20.90, 11.60, 10.58. HRMS (ESI): calcd for C23H25ClN2O5S [M + Na]+ 499.1070, found 499.1094. HPLC (90:10 methanol:water with 1‰ TFA): tR = 6.07 min, 97.0%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(cyclohexylsulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (29)
White solid, yield: 27%; mp: 159–161 °C. 1H NMR (300 MHz, Chloroform-d) δ 6.61 (s, 2H), 4.53 (s, 2H), 3.87 (s, 2H), 3.40 (s, 1H), 2.61 (d, J = 13.0 Hz, 6H), 2.34 (s, 6H), 2.18 (s, 2H), 2.07–1.52 (m, 10H). HRMS (ESI): calcd for C24H32ClNO5S [M + Na]+ 504.1587, found 504.1580. HPLC (80:20 methanol:water with 1‰ TFA): tR = 9.16 min, 96.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(cyclopentylsulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (30)
White solid, yield: 13%; mp: 164–166 °C. 1H NMR (300 MHz, Chloroform-d) δ 6.61 (s, 2H), 4.53 (s, 2H), 3.87 (s, 2H), 3.40 (s, 1H), 2.61 (d, J = 13.0 Hz, 6H), 2.34 (s, 6H), 2.18 (s, 2H), 2.01–1.60 (m, 8H). HRMS (ESI): calcd for C23H30ClNO5S [M + NH4]+ 485.1877, found 485.1877. HPLC (90:10 methanol:water with 1‰ TFA): tR = 9.10 min, 99.4%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(phenylsulfonyl)-1H-pyrrole-2-carboxylic Acid (31)
White solid, yield: 28%; mp: 186–188 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.94–7.86 (m, 2H), 7.61–7.49 (m, 3H), 6.63 (s, 2H), 4.53 (s, 2H), 3.89 (s, 2H), 2.68 (s, 3H), 2.60 (s, 3H), 2.36 (s, 6H), 2.18 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.56, 156.68, 144.04, 139.30, 136.93, 133.21, 129.82, 128.03, 126.20, 125.56, 121.16, 118.67, 114.99, 65.12, 42.87, 30.21, 42.87, 30.21, 20.88, 11.96, 11.04. HRMS (ESI): calcd for C24H26ClNO5S [M + Na]+ 498.1118, found 498.1114. HPLC (80:20 methanol:water with 1‰ TFA): tR = 12.11 min, 99.2%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((2-methoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (33)
White solid, yield: 28%; mp: 198–200 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.76 (s, 1H), 7.95 (d, J = 7.4 Hz, 1H), 7.67–7.55 (m, 1H), 7.13 (d, J = 5.1 Hz, 2H), 6.73 (s, 2H), 4.44 (s, 2H), 3.90 (s, 2H), 3.72 (s, 3H), 2.52 (s, 3H), 2.26 (d, J = 8.3 Hz, 9H), 2.04 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.67, 157.09, 156.64, 140.36, 136.93, 135.46, 130.52, 128.76, 128.43, 125.53, 120.34, 118.45, 115.03, 113.52, 65.08, 56.29, 42.61, 30.27, 20.83, 11.81, 10.93. HRMS (ESI): calcd for C25H28ClNO6S [M + NH4]+ 523.1670, found 523.1667. HPLC (90:10 methanol:water with 1‰ TFA): tR = 7.37 min, 99.5%.
4-((2-Bromophenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (34)
White solid, Yield: 25%; mp: 171–173 °C. 1H NMR (300 MHz, Chloroform-d) δ 8.24 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H), 6.60 (s, 2H), 4.57–4.40 (m, 2H), 3.80 (t, J = 5.1 Hz, 2H), 2.42 (s, 3H), 2.37 (s, 6H), 2.35 (s, 3H), 2.23–2.11 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.58, 156.57, 142.66, 139.86, 136.92, 133.83, 132.36, 128.91, 127.96, 125.55, 120.60, 119.09, 118.48, 114.93, 64.61, 42.44, 30.25, 20.88, 11.49, 10.85. HRMS (ESI): calcd for C24H25BrClNO5S [M + Na]+ 578.0203, found 578.0195. HPLC (90:10 methanol:water with 1‰ TFA): tR = 9.49 min, 97.3%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((3-methoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (37)
White solid, yield: 22%; mp: 159–161 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.55–7.45 (m, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.31–7.16 (m, 2H), 6.77 (s, 2H), 4.42 (s, 2H), 3.90 (d, J = 11.5 Hz, 2H), 3.80 (s, 3H), 2.55 (s, 3H), 2.39 (s, 3H), 2.28 (s, 6H), 2.04 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.53, 159.84, 156.64, 145.30, 139.33, 136.90, 131.15, 128.02, 125.53, 121.16, 118.80, 118.56, 118.25, 114.94, 111.08, 65.12, 55.92, 42.85, 30.17, 20.82, 11.95, 11.01. HRMS (ESI): calcd for C25H28ClNO6S [M + NH4]+ 523.1670, found 523.1663. HPLC (80:20 methanol:water with 1‰ TFA): tR = 8.25 min, 96.1%.
4-((3-Bromophenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (38)
White solid, yield: 28%; mp: 181–183 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.73–7.64 (m, 2H), 7.51–7.37 (m, 2H), 6.77 (s, 2H), 4.43 (s, 2H), 3.89 (s, 2H), 2.37 (s, 3H), 2.31 (s, 6H), 2.12 (s, 3H), 2.06 (d, J = 7.0 Hz, 2H). HRMS (ESI): calcd for C24H25BrClNO5S [M + Na]+ 578.0203, found 578.0195. HPLC (90:10 methanol:water with 1‰ TFA): tR = 9.93 min, 96.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-methoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (41)
White solid, yield: 16%; mp: 180–182 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H), 6.73 (s, 2H), 4.45–4.35 (m, 2H), 3.87 (t, J = 4.3 Hz, 2H), 3.81 (s, 3H), 2.53 (s, 3H), 2.36 (s, 3H), 2.28 (s, 6H), 2.02 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.84, 162.59, 156.72, 138.83, 136.97, 135.89, 128.49, 127.77, 125.56, 120.99, 119.55, 115.06, 114.96, 65.20, 56.11, 42.82, 30.26, 20.88, 11.95, 11.03. HRMS (ESI): calcd for C25H28ClNO6S [M + Na]+ 528.1224, found 528.1218. HPLC (80:20 methanol:water with 1‰ TFA): tR = 9.52 min, 99.6%.
4-((4-Bromophenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (42)
White solid, yield: 17%; mp: 174–176 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.65 (dd, J = 16.7, 8.6 Hz, 4H), 6.63 (s, 2H), 4.32 (t, J = 5.6 Hz, 2H), 3.78 (t, J = 5.0 Hz, 2H), 2.43 (s, 3H), 2.27 (s, 3H), 2.18 (s, 6H), 1.94 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.52, 156.71, 143.29, 139.57, 136.97, 132.96, 128.30, 127.98, 127.18, 125.57, 121.34, 118.10, 115.05, 65.20, 60.21, 42.94, 30.18, 20.90, 11.94, 11.09. HRMS (ESI): calcd for C24H25BrClNO5S [M + Na]+ 578.0203, found 578.0198. HPLC (80:20 methanol:water with 1‰ TFA): tR = 8.64 min, 98.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-ethylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (44)
White solid, yield: 12%; mp: 181–183 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.79 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 6.63 (s, 2H), 4.52 (t, J = 7.0 Hz, 2H), 3.90 (t, J = 5.5 Hz, 2H), 2.81–2.70 (m, 2H), 2.68 (s, 3H), 2.59 (s, 3H), 2.36 (s, 6H), 2.17 (t, J = 6.3 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 162.56, 156.77, 149.58, 141.66, 139.10, 136.96, 129.10, 127.97, 126.36, 125.61, 121.19, 119.14, 115.13, 65.31, 42.87, 30.29, 28.43, 20.84, 15.41, 11.95, 11.07. HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1424. HPLC (80:20 methanol:water with 1‰ TFA): tR = 15.28 min, 98.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-isopropylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (45)
White solid, Yield: 23%; mp: 178–180 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.88 (s, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 6.72 (s, 2H), 4.39 (t, J = 6.8 Hz, 2H), 3.86 (t, J = 5.4 Hz, 2H), 2.92 (dt, J = 13.5, 7.0 Hz, 1H), 2.54 (s, 3H), 2.35 (s, 3H), 2.26 (s, 6H), 2.08–1.97 (m, 2H), 1.16 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C27H32ClNO5S [M + Na]+ 540.1587, found 540.1577. HPLC (80:20 methanol:water with 1‰ TFA): tR = 20.33 min, 99.6%.
4-((4-(tert-Butyl)phenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (46)
White solid, yield: 31%; mp: 187–189 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 6.65 (s, 2H), 4.31 (s, 2H), 3.79 (s, 2H), 2.47 (s, 3H), 2.28 (s, 3H), 2.18 (s, 6H), 1.94 (s, 2H), 1.17 (s, 9H). 13C NMR (75 MHz, DMSO-d6) δ 162.58, 156.74, 156.29, 141.34, 139.17, 136.98, 127.90, 126.73, 126.12, 125.56, 121.15, 119.01, 115.10, 65.24, 42.87, 35.29, 31.17, 30.24, 20.92, 12.03, 11.10. HRMS (ESI): calcd for C28H34ClNO5S [M + Na]+ 554.1744, found 554.1736. HPLC (80:20 methanol:water with 1‰ TFA): tR = 25.54 min, 99.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-ethoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (47)
White solid, yield: 8%; mp: 183–185 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.87 (s, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 6.73 (s, 2H), 4.48–4.34 (m, 2H), 4.08 (q, J = 7.3, 6.7 Hz, 2H), 3.87 (t, J = 5.6 Hz, 2H), 2.54 (s, 3H), 2.36 (s, 3H), 2.28 (s, 6H), 2.02 (s, 2H), 1.33 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 162.60, 162.17, 156.77, 138.78, 136.97, 135.83, 128.47, 127.83, 125.61, 121.09, 119.67, 115.29, 115.12, 65.31, 64.20, 42.82, 30.33, 20.83, 14.81, 11.93, 11.04. HRMS (ESI): calcd for C26H30ClNO6S [M + Na]+ 542.1380, found 542.1373. HPLC (80:20 methanol:water with 1‰ TFA): tR = 12.22 min, 99.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-fluorophenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (51)
White solid, yield: 21%; mp: 158–160 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.76 (d, J = 5.3 Hz, 1H), 7.65 (dd, J = 16.2, 8.5 Hz, 2H), 7.29 (t, J = 8.8 Hz, 1H), 6.63 (s, 2H), 4.32 (t, J = 6.9 Hz, 2H), 3.77 (t, J = 4.9 Hz, 2H), 2.44 (s, 3H), 2.27 (s, 3H), 2.18 (s, 6H), 1.94 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 161.78, 156.80, 143.26, 137.09, 136.94, 129.85, 127.10, 125.52, 122.31, 120.78, 120.50, 115.18, 112.03, 64.87, 45.50, 30.72, 21.34, 20.91. HRMS (ESI): calcd for C24H25ClFNO5S [M + Na]+ 516.1024, found 516.1021. HPLC (80:20 methanol:water with 1‰ TFA): tR = 10.37 min, 95.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-chlorophenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (52)
White solid, Yield: 14%; mp: 165–167 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.93 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 6.73 (s, 2H), 4.42 (t, J = 8.1 Hz, 2H), 3.87 (t, J = 4.7 Hz, 2H), 2.54 (s, 3H), 2.36 (s, 3H), 2.28 (s, 6H), 2.04 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.55, 156.69, 142.87, 139.52, 138.19, 136.94, 129.97, 128.22, 128.02, 125.58, 121.34, 118.18, 115.00, 65.17, 42.94, 30.20, 20.87, 11.95, 11.08. HRMS (ESI): calcd for C24H25Cl2NO5S [M + Na]+ 532.0728, found 532.0726. HPLC (80:20 methanol:water with 1‰ TFA): tR = 15.25 min, 99.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-((4-nitrophenyl)sulfonyl)-1H-pyrrole-2-carboxylic Acid (53)
Yellow solid, yield: 13%; mp: 196–198 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.89 (s, 1H), 8.25 (d, J = 8.7 Hz, 2H), 7.96 (d, J = 8.7 Hz, 2H), 6.63 (s, 2H), 4.33 (t, J = 6.8 Hz, 2H), 3.78 (t, J = 5.2 Hz, 2H), 2.47 (s, 3H), 2.29 (s, 3H), 2.18 (s, 6H), 1.95 (s, 2H). HRMS (ESI): calcd for C24H25ClN2O7S [M - H]− 519.0993, found 519.0990. HPLC (80:20 methanol:water with 1‰ TFA): tR = 10.45 min, 99.5%.
4-((4-Aminophenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (55)
To a solution of 53 (0.2 g, 0.34 mmol) and EtOAc (10 mL), 5% Pd/C (0.05 equiv) was added. The mixture was stirred at 50 °C for 3 h. Upon the consumption of 53, the raction mixture was cooled to room temperature and Pd/C was then filtered. The solvent was evaporated under reduced pressure to give 55 as a gray solid, yield: 15%; mp: 156–158 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.77 (s, 1H), 7.42 (d, J = 8.7 Hz, 2H), 6.75 (s, 2H), 6.58 (d, J = 8.7 Hz, 2H), 6.03 (s, 2H), 4.39 (t, J = 6.9 Hz, 2H), 3.87 (t, J = 5.7 Hz, 2H), 2.51 (s, 3H), 2.35 (s, 3H), 2.28 (s, 6H), 2.01 (dd, J = 8.6, 5.5 Hz, 2H). HRMS (ESI): calcd for C24H27ClN2O5S [M + Na]+ 513.1227, found 513.1225. HPLC (80:20 methanol:water with 1‰ TFA): tR = 5.13 min, 99.2%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((2,6-dimethylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (56)
White solid, yield: 25%; mp: 179–181 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.32 (t, J = 8.5 Hz, 1H), 7.12 (d, J = 7.6 Hz, 2H), 6.61 (s, 2H), 4.54 (t, J = 6.9 Hz, 2H), 3.89 (t, J = 5.4 Hz, 2H), 2.60 (s, 6H), 2.57 (s, 3H), 2.36 (s, 6H), 2.30 (s, 3H), 2.23–2.14 (m, 2H). 13C NMR (75 MHz, DMSO) δ 164.31, 158.51, 141.62, 140.36, 140.07, 139.99, 138.71, 134.35, 133.57, 127.41, 123.09, 122.47, 116.90, 67.00, 44.31, 32.12, 23.61, 22.57, 12.82, 12.64. HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1423. HPLC (80:20 methanol:water with 1‰ TFA): tR = 15.33 min, 99.8%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((3,5-dimethylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (57)
White solid. Yield: 18%; mp: 184–186 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.49 (s, 2H), 7.20 (s, 1H), 6.63 (s, 2H), 4.53 (t, J = 7.0 Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 2.68 (s, 3H), 2.61 (s, 3H), 2.39 (s, 6H), 2.36 (s, 6H), 2.23–2.13 (m, 2H). HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1425. HPLC (80:20 methanol:water with 1‰ TFA): tR = 15.59 min, 96.1%.
Ethyl-3,5-diethyl-1H-pyrrole-2-carboxylate (81a)
78 (8 g; 50 mmol) was first added to an aqueous solution of NaNO2 (10.3 g in 25 mL, 150 mmol). After stirring for 24 h at 0 °C, the solution was allowed to reach room temperature. After extracting the aqueous layer several times with DCM, the combined organic layers were dried over MgSO4 and then evaporated to dryness. The resulting crude product was dissolved in acetic acid (50 mL) and added dropwise to a stirred solution of 80a (4.4 g, 44 mmol) in acetic acid (50 mL). A mixture of Zn (10 g; 156 mmol) and AcONa (12.7 g; 156 mmol) was added alternatively. The mixture was then stirred at 80 °C for 2 h, poured into water (300 mL), and extracted with DCM (3 times) The combined organic layer was concentrated under reduced pressure and purification by silica gel column chromatography (petroleum ether/ethyl acetate = 50:1 to 20:1 v/v) gave 81a, light yellow powder, yield: 43%. 1H NMR (300 MHz, Chloroform-d) δ 8.82 (s, 1H), 5.99–5.89 (m, 1H), 4.35 (q, J = 7.1 Hz, 2H), 2.82 (q, J = 7.5 Hz, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.31 (t, J = 7.6 Hz, 3H), 1.24 (t, J = 7.7 Hz, 3H). ESI-MS m/z: 194.1 [M-1]−.
Ethyl-3,5-diethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate (82a)
81a was synthesized according to the procedure of 75a, yellow powder, yield: 62%. 1H NMR (300 MHz, Chloroform-d) δ 9.30 (s, 1H), 7.04 (d, J = 7.8 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 2.82 (q, J = 7.3 Hz, 2H), 2.75 (q, J = 7.5, 6.0 Hz, 2H), 2.31 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.23 (t, J = 7.6 Hz, 3H), 1.09 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 318.3 [M + 1]+.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-diethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate (83a)
83a was synthesized according to the procedure of 63, colorless oil, yield: 72%. 1H NMR (300 MHz, Chloroform-d) δ 7.01 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 6.66 (s, 2H), 4.60–4.51 (m, 2H), 4.35 (q, J = 7.1 Hz, 2H), 3.96 (t, J = 5.7 Hz, 2H), 2.81 (dq, J = 15.1, 7.4 Hz, 4H), 2.38 (s, 6H), 2.30 (s, 3H), 2.24 (dd, J = 13.0, 6.9 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H), 1.14–1.04 (m, 6H). ESI-MS m/z: 514.2 [M + 1]+.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-diethyl-4-tosyl-1H-pyrrole-2-carboxylate (84a)
84a was synthesized according to the procedure of 77a, colorless oil, yield: 56%. 1H NMR (300 MHz, Chloroform-d) δ 7.75 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 6.65 (s, 2H), 4.56–4.45 (m, 2H), 4.32 (q, J = 7.1 Hz, 2H), 3.95 (t, J = 5.6 Hz, 2H), 3.12 (q, J = 7.4 Hz, 2H), 2.97 (q, J = 7.3 Hz, 2H), 2.44 (s, 3H), 2.39 (s, 6H), 2.19 (p, J = 5.8 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H), 1.26 (t, J = 7.4 Hz, 3H), 1.06 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 568.2 [M + Na]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-diethyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (17)
White powder, yield: 74%; mp: 180–182 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.76 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 6.64 (s, 2H), 4.53 (t, J = 7.1 Hz, 2H), 3.93 (t, J = 5.4 Hz, 2H), 3.15 (q, J = 7.4 Hz, 2H), 3.06 (q, J = 7.3 Hz, 2H), 2.45 (s, 3H), 2.37 (s, 6H), 2.21 (p, J = 5.9 Hz, 2H), 1.27 (t, J = 7.4 Hz, 3H), 1.09 (t, J = 7.3 Hz, 3H). HRMS (ESI): calcd for C27H32ClNO5S [M + Na]+ 540.1587, found 540.1582. HPLC (80:20 methanol:water with 1‰ TFA): tR = 15.84 min, 99.2%.
81b–e were synthesized from the cyclization reaction of 79 and 80b-c according to the procedure of 81a. Starting from the corresponding 81b–e, 18 and 19 and 21 and 22 were synthesized according to the procedure of 17.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-5-ethyl-3-methyl-4-tosyl-1H-pyrrole-2 carboxylic Acid (18)
White powder, yield: 77%; mp: 186–188 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.64 (d, J = 8.9 Hz, 2H), 6.98 (d, J = 8.9 Hz, 2H), 6.63 (s, 2H), 4.29 (t, J = 6.7 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.46 (s, 3H), 2.26 (s, 3H), 2.18 (s, 6H), 1.93 (s, 2H), 1.14 (t, J = 7.1 Hz, 3H). HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1425. HPLC (80:20 methanol:water with 1‰ TFA): tR = 14.70 min, 97.7%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-ethyl-5-methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (19)
White solid, yield: 83%; mp: 178–180 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.84 (s, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 6.73 (s, 2H), 4.42 (t, J = 6.2, 2H), 3.88 (t, J = 5.0 Hz, 2H), 2.87 (q, J = 7.3 Hz, 2H), 2.54 (s, 3H), 2.35 (s, 3H), 2.28 (s, 6H), 2.04 (dt, J = 10.9, 5.4 Hz, 2H), 0.94 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO) δ 162.39, 156.73, 143.61, 141.54, 139.11, 136.96, 134.67, 130.25, 126.29, 125.54, 120.57, 118.28, 115.05, 65.24, 42.92, 30.20, 21.41, 20.90, 18.83, 16.35, 11.17. HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1426. HPLC (80:20 methanol:water with 1‰ TFA): tR = 12.78 min, 98.1%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-5-isobutyl-3-methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (21)
White solid, yield: 79%; mp: 162–164 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.94 (s, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 6.71 (s, 2H), 4.42 (t J = 6.7 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.88 (d, J = 7.3 Hz, 2H), 2.33 (s, 3H), 2.26 (s, 6H), 2.24 (s, 3H), 2.00–1.88 (m, 3H), 0.87 (d, J = 6.4 Hz, 6H). HRMS (ESI): calcd for C28H34ClNO5S [M + NH4]+ 549.2187, found 549.2185. HPLC (90:10 methanol:water with 1‰ TFA): tR = 12.2 min, 99.4%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-isobutyl-5-methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (22)
White solid, yield: 86%; mp: 149–151 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.89 (s, 1H), 7.59 (d, J = 7.8 Hz, 2H), 7.31 (d, J = 7.7 Hz, 2H), 6.70 (s, 2H), 4.51–4.33 (m, 2H), 3.81 (t, J = 5.1 Hz, 2H), 2.82 (d, J = 6.9 Hz, 2H), 2.46 (s, 3H), 2.32 (s, 3H), 2.25 (s, 6H), 2.07–1.96 (m, 2H), 1.83–1.73 (m, 1H), 0.76 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ 166.33, 156.20, 143.26, 141.29, 140.55, 137.19, 136.92, 129.55, 126.56, 126.23, 120.24, 119.11, 114.26, 64.48, 43.10, 33.24, 30.81, 30.38, 22.28, 21.53, 20.96, 11.47. HRMS (ESI): calcd for C28H34ClNO5S [M + NH4]+ 549.2187, found 549.2180. HPLC (80:20 methanol:water with 1‰ TFA): tR = 16.92 min, 99.3%.
Ethyl-3,5-dimethyl-4-(thiophen-2-ylsulfonyl)-1H-pyrrole-2-carboxylate (86a)
To a solution of 85a (2 g, 11 mmol) and PhCl (40 mL), AlCl3 (1.5 g, 11 mmol) was added under nitrogen at 0 °C. The mixture was srirred for 10 min and ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate (1 g, 6 mmol) was added. The mixture was then allowed to stir at 90 °C for 2 h under nitrogen. After completing the reaction monitored by TLC, 100 mL of water was carefully added to the mixture. The mixture was added 40 mL DCM and stirred for 15 min. After filtration, the organic layer was isolated and the aqueous phase was extracted with DCM (2 times). The combined organic layer was then washed with brine and dried with Na2SO4. Removal of solvent under reduced pressure yielded crude product. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate = 40:1 to 10:1 v/v) gave 86a, yellow powder, 2.6 g, yield: 68%. 1H NMR (300 MHz, Chloroform-d) δ 9.42 (s, 1H), 7.68 (dd, J = 3.7, 1.3 Hz, 1H), 7.60 (dd, J = 5.0, 1.3 Hz, 1H), 7.09 (dd, J = 4.9, 3.8 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), 2.64 (s, 3H), 2.58 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 312.0 [M - 1]−.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(thiophen-2-ylsulfonyl)-1H-pyrrole-2-carboxylate (87a)
87a was synthesized according to the procedure of 63, yellow powder, yield: 63%. 1H NMR (300 MHz, Chloroform-d) δ 7.63 (dd, J = 3.7, 1.3 Hz, 1H), 7.57 (dd, J = 5.0, 1.3 Hz, 1H), 7.07 (dd, J = 5.0, 3.8 Hz, 1H), 6.63 (s, 2H), 4.51 (t, J = 7.1 Hz, 2H), 4.32 (q, J = 7.1 Hz, 2H), 3.90 (t, J = 5.6 Hz, 2H), 2.65 (s, 3H), 2.59 (s, 3H), 2.38 (s, 6H), 2.17 (p, J = 6.0 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 510.1 [M + 1]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(thiophen-2-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (27)
Starting from 87a, 27 was synthesized through the hydrolysis procedure of 23. Brown solid, yield: 54%; mp: 178–180 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.95 (s, 1H), 7.65 (s, 1H), 7.15 (s, 1H), 6.73 (s, 2H), 4.42 (s, 2H), 3.87 (s, 2H), 2.54 (s, 3H), 2.44 (s, 3H), 2.28 (s, 6H), 2.03 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.56, 156.72, 146.06, 139.14, 136.98, 133.85, 131.75, 128.43, 127.90, 125.58, 121.24, 119.64, 115.08, 65.19, 42.94, 30.22, 20.90, 11.99, 11.15. HRMS (ESI): calcd for C22H24ClNO5S2 [M + Na]+ 504.0682, found 504.0686. HPLC (80:20 methanol:water with 1‰ TFA): tR = 16.32 min, 97.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-(naphthalen-1-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (28)
Starting from 85b, 28 was synthesized according to the procedure of 27, white solid, yield: 63%; mp: 184–186 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.88 (s, 1H), 8.43–8.33 (m, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 7.2 Hz, 1H), 8.11–8.01 (m, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.60 (dd, J = 6.3, 3.3 Hz, 2H), 6.70 (s, 2H), 4.44 (t, J = 6.7 Hz, 2H), 3.84 (t, J = 5.5 Hz, 2H), 2.61 (s, 3H), 2.27 (s, 6H), 2.24 (s, 3H), 2.10–1.99 (m, 2H). HRMS (ESI): calcd for C28H28ClNO5S [M + Na]+ 548.1274, found 548.1266. HPLC (80:20 methanol:water with 1‰ TFA): tR = 13.96 min, 99.6%.
1-(4-Mercaptophenyl)ethan-1-one (89a)
To a mixture of 4-iodoacetophenone (88a, 5 g, 20 mmol), CuSO4·5H2O (0.25 g, 1 mmol), Cs2CO3 (32.4 g, 100 mmol), and DMSO (70 mL), 1,2-ethanedithiol (3.7 g, 40 mmol) was added under nitrogen at room temperature. The mixture was stirred in the preheated oil bath at 90 °C for 4 h. After being cooled to room temperature, the reaction mixture was carefully quenched by aqueous HCl (1 N) to pH 2. EtOAc (80 mL) was added, and the mixture was stirred for another 10 min. The organic layer was separated and washed with brine, dried over MgSO4, and concentrated under vacuum. The crude product was further purified by column chromatography (petroleum ether/ethyl acetate = 50:1 to 10:1 v/v) to provide 89a as a light yellow powder, yield: 76%. 1H NMR (300 MHz, Chloroform-d) δ 7.93 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 2.60 (s, 3H). ESI-MS m/z: 150.9 [M-1]−.
Ethyl-4-((4-acetylphenyl)thio)-3,5-dimethyl-1H-pyrrole-2-carboxylate (90a)
90a was synthesized according to the procedure of 75a, yellow solid. Yield: 54%. 1H NMR (300 MHz, Chloroform-d) δ 9.60 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 4.41 (q, J = 7.1 Hz, 2H), 2.59 (s, 3H), 2.36 (s, 3H), 2.32 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 316.2 [M–1]−.
Ethyl-4-((4-acetylphenyl)thio)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (91a)
91a was synthesized according to the procedure of 63, yellow oil, yield: 75%. 1H NMR (300 MHz, Chloroform-d) δ 7.79 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.64 (s, 2H), 4.58 (t, J = 6.9 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 3.93 (t, J = 5.6 Hz, 2H), 2.56 (s, 3H), 2.36 (s, 6H), 2.32 (s, 6H), 2.25 (p, J = 6.5 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 536.1 [M + Na]+.
Ethyl-4-((4-acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (92a)
92a was synthesized according to the procedure of 75a, yellow solid, yield: 65%. 1H NMR (300 MHz, Chloroform-d) δ 8.06 (d, J = 7.0 Hz, 2H), 7.96 (d, J = 7.0 Hz, 2H), 6.64 (s, 2H), 4.51 (t, J = 6.8 Hz, 2H), 4.37–4.25 (m, 2H), 3.92 (t, J = 5.0 Hz, 2H), 2.67 (s, 6H), 2.51 (s, 3H), 2.38 (s, 6H), 2.17 (p, J = 5.5 Hz, 2H), 1.38 (t, J = 7.7 Hz, 3H). ESI-MS m/z: 546.2 [M + H]+.
4-((4-Acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (40)
40 was synthesized from 92a according to the hydrolysis procedure of 16, yellow powder, Yield: 42%, mp: 215–216 °C, 1H NMR (300 MHz, DMSO-d6) δ 12.93 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 6.71 (s, 2H), 4.40 (t, J = 6.7 Hz, 2H), 3.85 (t, J = 5.9 Hz, 2H), 2.59 (s, 3H), 2.54 (s, 3H), 2.35 (s, 3H), 2.25 (s, 6H), 2.01 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 197.73, 162.57, 156.74, 150.93, 139.28, 137.99, 137.00, 129.13, 128.67, 125.59, 125.09, 121.06, 120.48, 115.06, 65.13, 42.77, 30.37, 27.29, 20.89, 11.57, 10.56. HRMS (ESI): calcd for C26H28ClNO6S [M + Na]+ 540.1224, found 540.1217. HPLC (90:10 methanol:water with 1‰ TFA): tR = 7.50 min, 99.8%.
Starting from corresponding iodobenzenes 88b–g, 32, 36, 48–50, and 54 were synthesized according to the procedure of 40.
4-((2-Acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (32)
Yellow solid, yield: 9%; mp: 180–182 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.95 (s, 1H), 7.67 (dd, J = 47.1, 19.2 Hz, 4H), 6.77 (s, 2H), 4.44 (s, 2H), 3.92 (s, 2H), 2.45 (s, 6H), 2.25 (d, J = 15.6 Hz, 9H), 2.06 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 202.53, 162.59, 156.77, 141.23, 140.23, 139.75, 137.00, 133.48, 130.35, 128.40, 127.21, 125.59, 121.12, 118.14, 115.14, 65.13, 42.76, 32.18, 30.36, 20.93, 11.83, 11.09. HRMS (ESI): calcd for C26H28ClNO6S [M + NH4]+ 535.1664, found 535.1661. HPLC (90:10 methanol:water with 1‰ TFA): tR = 23.10 min, 97.2%.
4-((3-Acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (36)
Pale yellow solid, yield: 14%; mp: 172–174 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.98 (s, 1H), 8.25 (d, J = 12.4 Hz, 2H), 8.05 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 6.75 (s, 2H), 4.45 (t, J = 6.2 Hz, 2H), 3.90 (t, J = 4.5 Hz, 2H), 2.65 (s, 3H), 2.59 (s, 3H), 2.42 (s, 3H), 2.30 (s, 6H), 2.07 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 197.13, 162.53, 156.68, 144.68, 139.61, 137.88, 136.94, 133.04, 130.75, 130.41, 128.03, 125.57, 124.96, 121.38, 118.11, 114.99, 65.14, 42.96, 30.21, 27.21, 20.87, 11.98, 11.10. HRMS (ESI): calcd for C26H28ClNO6S [M + NH4]+ 535.1664, found 535.1660. HPLC (80:20 methanol:water with 1‰ TFA): tR = 7.76 min, 99.6%.
4-([1,1′-Biphenyl]-4-ylsulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (48)
White solid, Yield: 35%; mp: 158–160 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.93 (s, 1H), 7.93–7.37 (m, 9H), 6.74 (s, 2H), 4.42 (s, 2H), 3.88 (s, 2H), 2.59 (s, 3H), 2.41 (s, 3H), 2.26 (s, 6H), 2.05 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.58, 156.76, 144.82, 142.91, 139.33, 138.88, 136.97, 129.53, 128.98, 128.06, 127.50, 126.92, 125.63, 121.34, 118.86, 115.11, 65.30, 42.93, 30.30, 20.84, 12.02, 11.15. HRMS (ESI): calcd for C30H30ClNO5S [M + H]+ 552.1611, found 552.1603. HPLC (90:10 methanol:water with 1‰ TFA): tR = 17.4 min, 98.2%.
4-((4-Benzoylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (49)
Light yellow solid, yield: 16%; mp: 146–148 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.99 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.85–7.79 (m, 2H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H), 6.63 (s, 2H), 4.55 (t, J = 6.9 Hz, 2H), 3.91 (t, J = 5.4 Hz, 2H), 2.71 (s, 3H), 2.61 (s, 3H), 2.35 (s, 6H), 2.19 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 195.13, 162.54, 156.75, 147.06, 140.99, 139.80, 136.97, 136.63, 133.67, 130.88, 130.22, 129.12, 128.21, 126.47, 125.63, 121.58, 117.99, 115.11, 65.31, 43.01, 30.26, 20.83, 11.99, 11.16. HRMS (ESI): calcd for C31H30ClNO6S [M + NH4]+ 597.1821, found 597.1815. HPLC (90:10 methanol:water with 1‰ TFA): tR = 15.33 min, 99.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-4-((4-phenoxyphenyl)sulfonyl)-1H-pyrrole-2-carboxylic Acid (50)
White solid, yield: 15%; mp: 164–166 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.82 (d, J = 8.6 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H), 7.06 (dd, J = 15.6, 8.3 Hz, 4H), 6.63 (s, 2H), 4.53 (t, J = 6.6 Hz, 2H), 3.91 (t, J = 5.0 Hz, 2H), 2.68 (s, 3H), 2.59 (s, 3H), 2.36 (s, 6H), 2.24–2.12 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.58, 161.19, 156.77, 155.20, 139.07, 138.20, 136.98, 130.80, 128.84, 127.94, 125.63, 125.40, 121.23, 120.56, 119.20, 118.10, 115.12, 65.32, 42.89, 30.31, 20.85, 11.96, 11.09. HRMS (ESI): calcd for C30H30ClNO6S [M + NH4]+ 585.1826, found 585.1819. HPLC (80:20 methanol:water with 1‰ TFA): tR = 22.46 min, 99.2%.
4-((4-Carboxyphenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (54)
Light purple solid, yield: 15%; mp: 183–185 °C. 1H NMR (300 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.3 Hz, 2H), 6.76 (s, 2H), 4.45 (s, 2H), 3.89 (s, 2H), 2.58 (s, 3H), 2.40 (s, 3H), 2.30 (s, 6H), 2.07 (s, 2H). 13C NMR (75 MHz, DMSO) δ 166.48, 162.48, 156.73, 147.55, 139.73, 136.96, 135.00, 130.74, 128.16, 126.50, 125.63, 121.50, 117.99, 115.09, 65.31, 42.98, 30.23, 20.82, 11.90, 11.12. HRMS (ESI): calcd for C25H26ClNO7S [M + NH4]+ 537.1462, found 537.1461. HPLC (80:20 methanol:water with 1‰ TFA): tR = 6.63 min, 97.7%.
4-(Benzyloxy)benzenethiol (89h)
89h was synthesized from 1-(benzyloxy)-4-iodobenzene (88h) according to the procedure of 89a, white solid. Yield: 68%. 1H NMR (300 MHz, Chloroform-d) δ 7.45–7.41 (m, 2H), 7.39–7.34 (m, 3H), 7.33 (d, J = 7.4 Hz, 2H), 6.81 (d, J = 7.5 Hz, 2H), 5.03 (s, 2H), 3.73 (s, 1H). ESI-MS m/z: 215.1 [M-H]−.
Ethyl-4-((4-(benzyloxy)phenyl)thio)-3,5-dimethyl-1H-pyrrole-2-carboxylate (90h)
90h was synthesized from 4-(benzyloxy)benzenethiol according to the procedure of 75a, yellow solid. Yield: 73%. 1H NMR (300 MHz, Chloroform-d) δ 9.27 (s, 1H), 7.51–7.35 (m, 5H), 7.01 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 5.05 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 2.38 (s, 3H), 2.36 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 380.1 [M–H]−.
Ethyl-4-((4-(benzyloxy)phenyl)thio)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (91h)
91h was synthesized according to the procedure of 63, colorless oil, yield: 81%. 1H NMR (300 MHz, Chloroform-d) δ 7.45–7.35 (m, 5H), 6.95 (d, J = 8.9 Hz, 2H), 6.85 (d, J = 8.9 Hz, 2H), 6.64 (s, 2H), 5.03 (s, 2H), 4.55 (t, J = 7.0 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.92 (t, J = 5.6 Hz, 2H), 2.37 (s, 6H), 2.37 (s, 3H), 2.36 (s, 3H), 2.23 (p, J = 6.2 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 578.2 [M + H]+.
Ethyl-4-((4-(benzyloxy)phenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (92h)
92h was synthesized according to the procedure of 77a, white powder, yield: 63%. 1H NMR (300 MHz, Chloroform-d) δ 7.81 (d, J = 8.9 Hz, 2H), 7.44 (d, J = 4.2 Hz, 5H), 7.04 (d, J = 8.9 Hz, 2H), 6.64 (s, 2H), 5.14 (s, 2H), 4.49 (t, J = 7.1 Hz, 2H), 4.31 (q, J = 7.1 Hz, 2H), 3.92 (t, J = 5.5 Hz, 2H), 2.66 (s, 3H), 2.51 (s, 3H), 2.38 (s, 6H), 2.17 (p, J = 6.0 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 610.2 [M + H]+.
4-((4-(Benzyloxy)phenyl)sulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid (93h)
To a solution of 92h (380 mg, 0.62 mmol) in EtOH/THF (10 mL/10 mL) was added NaOH (2 M, 3 mL). The mixture was stirred overnight at 50 °C. After completion of the reaction, the mixture was then acidified with 1 M HCl and extracted with ethyl acetate. The organic layer was washed with brine and dried with Na2SO4. Removal of solution and further recrystallization from EtOH/nhexane gave title compound 93h. White powder, yield: 80%. 1H NMR (300 MHz, Chloroform-d) δ 7.83 (d, J = 8.6 Hz, 2H), 7.46–7.39 (m, 5H), 7.06 (d, J = 8.6 Hz, 2H), 6.64 (s, 2H), 5.15 (s, 2H), 4.53 (t, J = 6.8 Hz, 2H), 3.92 (t, J = 5.2 Hz, 2H) 2.68 (s, 3H), 2.60 (s, 3H), 2.37 (s, 6H), 2.19 (p, J = 6.7 Hz, 2H). ESI-MS m/z: 604.1 [M + Na]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-hydroxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (43)
To a solution of 93h (0.2 g, 0.34 mmol) and EtOAc (10 mL), 5% Pd/C (0.05 equiv) was added. The mixture was stirred at 50 °C for 3 h. Upon the consumption of 91h, the reaction mixture was cooled to room temperature and Pd/C was then filtered. The solvent was evaporated under reduced pressure to give 43 as a gray solid, yield: 96%; mp: 177–179 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.65 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 6.77 (s, 2H), 4.43 (t, J = 6.7 Hz, 2H), 3.89 (t, J = 5.5 Hz, 2H), 2.55 (s, 3H), 2.39 (s, 3H), 2.31 (s, 6H), 2.05 (dt, J = 13.0, 6.6 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.64, 161.74, 156.74, 138.59, 136.98, 134.22, 128.69, 127.70, 125.56, 120.94, 119.94, 116.16, 115.07, 65.23, 42.76, 30.29, 20.89, 11.96, 11.02. HRMS (ESI): calcd for C24H26ClNO6S [M + Na]+ 514.1067, found 514.1061. HPLC (80:20 methanol:water with 1‰ TFA): tR = 6.33 min, 99.2%.
Starting from 1-(benzyloxy)-2-iodobenzene (88j) and 1-(benzyloxy)-3-iodobenzene (88i), 35 and 39 were obtained through the procedure of 43.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((2-hydroxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (35)
White solid, yield: 10%; mp: 175–177 °C. 1H NMR (300 MHz, Chloroform-d) δ 9.44 (s, 1H), 7.44 (d, J = 7.7 Hz, 2H), 7.03 (d, J = 8.2 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.63 (s, 2H), 4.55 (t, J = 7.0 Hz, 2H), 3.90 (t, J = 5.4 Hz, 2H), 2.68 (s, 3H), 2.57 (s, 3H), 2.37 (s, 6H), 2.20 (dt, J = 12.1, 5.9 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.73, 158.73, 156.73, 156.22, 140.24, 139.05, 136.98, 135.05, 129.23, 128.71, 125.55, 120.18, 118.89, 117.80, 115.12, 112.59, 65.17, 42.58, 21.50, 20.91, 11.91, 11.26. HRMS (ESI): calcd for C24H26ClNO6S [M + NH4]+ 509.1513, found 509.1505. HPLC (80:20 methanol:water with 1‰ TFA): tR = 14.95 min, 99.3%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((3-hydroxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (39)
Gray solid, yield: 12%; mp: 173–175 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.91 (s, 1H), 10.18 (s, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.22 (d, J = 9.9 Hz, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.76 (s, 2H), 4.44 (s, 2H), 3.91 (s, 2H), 2.56 (s, 3H), 2.41 (s, 3H), 2.31 (s, 6H), 2.05 (dd, J = 12.3, 6.3 Hz, 2H). HRMS (ESI): calcd for C24H26ClNO6S [M + NH4]+ 509.1513, found 509.1507. HPLC (80:20 methanol:water with 1‰ TFA): tR = 6.47 min, 97.1%.
Protein Purification
GST-tagged proteins Mcl-1 (residues 171–327), Bcl-2 (residues 1–205) and A1/Bfl-1 (residues1–151) were expressed from the pGEX-4 T-1 vector in E. coli BL21 (DE3) cells. Protein expression was induced by 1.0 mM IPTG at 11 °C for 8 h. Cells were lysed by an ultrasonic cracker in 25 mM Tris pH 8.0 buffer containing 300 mM NaCl, 0.1% Tween 20, and 0.1% mercaptoethanol. Proteins were further purified on a GSTtrap column (5 cm) in 50 mM Tris pH 8.0 buffer, with 200 mM NaCl, 10 mM reduced glutathione, and 1 mM DTT. Lastly, 10% SDS-PAGE was used for confirming the molecular weight of the purified proteins.
FP-Based Competition Assay
An FP competition assay was used to evaluate the inhibitory activities of the compounds against Mcl-1, (75) Bcl-2, (68) and Bfl-1. (76) Different concentrations of compounds were prepared by serial dilution in assay buffer (Mcl-1: 20 mM Tris, 150 mM NaCl, 3 mM DTT, pH = 7.5; Bfl-1 and Bcl-2: 55 mM Hepes, 274 mM NaCl, 1.48 mM Na2HPO4, pH = 7.0). FITC-Bak-BH3 was chosen as a probe for Mcl-1 (10 nM final). Fam-Bid was chosen as probes for Bcl-2 and Bfl-1, and the final probe concentrations were 0.66 and 0.33 nM, respectively. The protein final concentrations for Mcl-1, Bfl-1, and Bcl-2 were 20, 13.3, and 20 nM, respectively. The experiments were conducted in 384-well black flat-bottomed polystyrene plates (Corning #3575). For each assay, equal volumes of the diluted compounds (20 μL), proteins (20 μL), and probes (20 μL) were added to the wells in an orderly fashion. Plates were rocked for 30 min at room temperature in the dark, and then the FP values were detected using a SpectraMax multimode microplate reader (Molecular Devices) with excitation and emission wavelengths at 485 and 535 nm, respectively. For each assay, the FP values of blank controls (probes only) were recorded as Pmin; the FP values of negative controls (probes and proteins) were recorded as Pmax, and the FP values of test wells (compounds, probes, and protein) were recorded as Ptest. The inhibition rates of the compounds at each concentration point were calculated using the equation as follows: inhibition rate (%) = [1 – (Ptest – Pmin)/(Pmax – Pmin)] × 100%, and the IC50 values were calculated using Graphpad Prism 6.0 software. Ki was calculated as previously reported. (47,77)
TR-FRET Based Assay
The Bcl-xL inhibitory ratio was assessed by time-resolved fluorescence resonance energy transfer (TR-FRET) technology (48) using a recombinant Bcl-xL and a peptide-ligand substrate. All of the reactions were conducted at room temperature. The 20 μL reaction mixture in buffer contains Bcl-xL, the indicated amount of the inhibitor (10 μM final), ligand, and the reaction dyes. The reaction mixture was incubated for 120 min before reading the TR-FRET signal. For the background, the ligand was replaced with assay buffer. Fluorescence signals for both the donor and acceptor dyes were measured using a Tecan Infinite M1000 plate reader. TR-FRET was recorded as the ratio of the fluorescence of the acceptor and the donor dyes (acceptor/donor).
Molecular Docking
Compounds 13, 16, 40, and 43 were imported to Discovery Studio (DS) 4.0, and the 3D conformation was generated by the “Prepare Ligands” protocol at pH 7.0. Then, the compound was energy minimized in a CHARMm force field for docking. The Mcl-1-ligand complex (PDB ID: 4HW2) downloaded from the Protein Data Bank was chosen for docking studies. Protein residues around the original ligand (radius = 8.0 Å) completely covered the BH3 binding cavity. Libdock was used to dock 13, 16, 40, and 43 into the BH3 binding groove. The top 10 of 100 poses generated were selected for analyzing binding interactions.
BCL2scan Binding Assays
Kd values against Mcl-1, Bcl-2, Bcl-xL, Bcl-w, and Bcl2A1 of 40 were determined by Discovery X’s BCL2scan technology. Compounds interacting with the Bcl-2 family proteins prevented binding of these proteins to an immobilized known peptide ligand and reduced the amount of target protein captured on the solid support. Dissociation constants (Kds) for test compound-Bcl-2 family protein interactions were calculated by measuring the amount of target protein captured on the solid support. Assay conditions were optimized for the measurement of true thermodynamic test compound Kd values.
MTT Assay
Antiproliferative activities of the compounds against different cancer cell lines were determined using MTT assays. Cells were seeded into 96-well plates at 5000 cells per well and incubated for 24 h. Then, the cells were treated with either serial dilutions of the compounds or DMSO for 72 h. MTT solution (5 mg/mL) was added, and the plates were incubated at 37 °C for another 4 h. After removing the solution in the wells of the plates, 150 μL of DMSO was added to dissolve the MTT formazan crystals. The absorbance values (OD values) were determined using the Elx800 absorbance microplate reader (BioTek, Vermont, USA) at 570 nm. Cell viability inhibition rates of the compounds at each concentration point were calculated using the equation as follows: inhibition rate (%) = [1 – (ODtest – ODblank)/(ODcontrol – ODblank)] × 100%. The IC50 values were calculated using Graphpad Prism 6.0 software.
Co-ip Experiment
H929 cells were administered with different concentrations of 40, DMSO or AZD5991 (Biochempartner, BCP23513, China) for 4 h. Next, the cells were shaken in RIPA buffer (Thermo Fisher Scientific, 78501, US) for 45 min on ice. The supernatant was obtained from centrifugation at 12,000 rpm for 20 min. Then, 5 μL of the Mcl-1 antibody (Cell Signaling Technology, D35A5, US) was added into 1 mL of the supernatant. The mixture was shaken at 4 °C overnight. Then, Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003, US) was used to precipitate the Mcl-1 complex after incubation for 4 h. The beads were centrifuged and washed with RIPA buffer four times and subjected to SDS-PAGE following with WB analysis. The primary antibodies used in WB analysis were the Mcl-1 antibody (Cell Signaling Technology, D35A5, US) and Bak antibody (Abcam, Y164, UK).
Apoptosis Assay by Annexin V/Propidium Iodide Staining
H929 cells were seeded in a 6-well plate and incubated with 40 and A1210477 (TOPSCIENCE, T2632, China) of indicated concentrations for 48 h. After incubation, cells were collected and washed with PBS buffer. Cells were then dispersed with 500 μL of binding buffer per tube and then 5 μL of Annexin V-FITC and 5 μL of propidium iodide were added. After incubation for 15 min at 20 °C in the dark, apoptosis was analyzed by flow cytometry in a flow cytometer (Becton-Dickinson , FACS Calibur, US).
WB Analysis
PARP cleavage induction of the compounds was determined by the WB assay. After treatment with serially diluted 40 and A1210477 (TOPSCIENCE, T2632, China) , the H929 cells or K562 cells were washed with PBS and lysed in RIPA lysis buffer. The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected. The protein concentration was determined using the BCA protein assay kit (KeyGen BioTech, KGA902, China). Equal amounts of the total protein extracts were subjected to SDS-PAGE and then transferred onto NC membranes (PALL, 66485, USA). After blocking the nonspecific binding sites with 5% nonfat milk for 1 h, membranes were incubated at 4 °C overnight with the primary antibody anti-cPARP (Abcam, ab32064, UK). After washing with TBST for 10 min three times, the membranes were treated with an IgG-HRP secondary antibody (Abcam, ab6721, UK) at 37 °C for 1–2 h and scanned using the Odyssey infrared imaging system (SYNGENE, G:BOXChemiXR5, UK).
Caspase 3/7 Activation Assay
Caspase 3/7 activation was analyzed according to the previous report. (78) H929 or K562 Cells were dispensed in a 96-well plate and incubated with 40 and A1210477 (TOPSCIENCE, T2632, China) at indicated concentrations for 4 h. After removal of compounds, cells were rinsed by culture medium and 1 μL/mL Caspase 3/7 assay buffer (KeyGen BioTech, KGA S037–50, China) was added. The mixture was incubated at room temperature in the dark for 30 min. Luminescence was measured using a multifunctional microplate reader (Molecular Devices, Spectramax M3, USA).
PAMPA Evaluation
Membrane permeability coefficients were evaluated via double-sink PAMPA on a PAMPA Explorer instrument (pION). The detailed experimental procedures have been reported previously. (79) Briefly, the test compound was diluted with donor buffer at pH 7.4 and placed in the donor side. The test compound was allowed to permeate to the acceptor side through the artificial membrane over 4 h incubation at 25 °C. After incubation, the “sandwich” plate was separated. The donor and acceptor solutions were then measured with a UV plate reader, and the permeability value was calculated by pION software.
CYP450 Inhibition Assay
The CYP450 inhibition activities of 40 on the five major isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were evaluated. The assay was conducted in phosphate buffer (100 mM, pH 7.4). For each assay, equal volumes (25 μL) of diluted microsomes (0.2 mg/mL final), tested compounds or positive control inhibitors (10 μM final, DMSO as the negative control), and specific substrates of the isozymes (10 μM final) were preincubated at 37 °C for 5 min. Then, 25 μL of NADPH (1 mM, final) was added to initiate the reaction. After 20 min of incubation, 100 μL of cold acetonitrile was added to terminate the reaction. The samples were centrifuged, and the supernatants were analyzed by LC–MS/MS to determine the metabolites. The inhibition rate was calculated using the following equation: inhibition rate (%) = [1 – (formation of the metabolite in the presence of tested compound)/(formation of the metabolite of negative control)] × 100%.
HLM and RLM Stability
Compound 40 was preincubated with human liver microsomes (0.2 mg/mL) or rat liver microsomes (0.5 mg/mL) for 5 min at 37 °C in phosphate buffer (100 mM, pH 7.4). Then, 1 mM NADPH was added to initiate the reaction. After incubation for different times (0, 15, 30, 60, and 120 min) at 37 °C, cold acetonitrile was added to precipitate the protein. Then, the samples were centrifuged, and the supernatants were analyzed by LC–MS/MS.
Human and Mouse Plasma Stability
40 was dissolved in DMSO at 10 mM and 4 μL of compound solution was then incubated with 996 μL of prewarmed mouse or human plasma in 37 °C. 5-fold volumes of cold acetonitrile were added to stop the reaction at 0, 0.5, 1, and 2 h. The samples were centrifuged, the supernatant was transferred to a new 96-well plate, and the samples were mixed with purity water (v:v = 1:2). The compound concentration was measured by LC–MS–MS.
Caco-2 Permeability
Caco-2 cells purchased from ATCC were seeded onto polyethylene membranes (PET) in 24-well Corning transwell plates at 1 × 105 cells/cm2, and the medium was refreshed every other day until the 21st day for confluent cell monolayer formation. The standard transport buffer in the study was HBSS at pH 7.4. Before and after the transport studies, the monolayer integrity was evaluated by measuring transepithelial electrical resistance (TEER). The pre-transport and post-transport TEER were required to be no less than 200 Ω·cm2. The standard transport buffer in the study was HBSS at pH 7.4. 40 was diluted with transport buffer from a 10 mM stock solution to a concentration of 2 or 10 μM. The bidirectional permeability of the 40 was investigated by adding the compound solution either in the apical chamber or the basolateral chamber. The efflux ratio was used to identify whether 40 was the Pgp substrate or not. 40 and the reference compound were quantified by LC–MS/MS analysis.
In Vivo Pharmacokinetics Study
All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. The pharmacokinetics study of 40 was conducted in SD rats (200-250 g), which were purchased from Jinan Pengyue Laboratory Animal Breeding Co. Ltd. The rats were randomly divided into two groups (n = 6 for each group). The two groups were administered compound 40 intravenously at 3 mg/kg and orally at 10 mg/kg. Blood samples (150 μL) were collected into heparinized Eppendorf tubes at predetermined time points (0, 0.016, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h) and centrifuged immediately at 4 °C and 8000 rpm for 5 min. The plasma samples were analyzed by LC–MS/MS.
In Vivo Antitumor Efficacy Evaluation
All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. The female balb/c nude mice (7 weeks) were purchased from GemPharmatech Co. Ltd. The mice were maintained in standard conditions (12/12 h light/dark cycle, 22 ± 3 °C, relative humidity of 40%) for 3 days and fed with a standard laboratory rodent diet and water. The mice were then injected subcutaneously with MV4–11 cells (5 × 106) in the right flank. When the average tumor volume reached approximately 200 mm3, the mice were randomized into four groups (n = 6 per group). Administration groups were intraperitoneally or orally treated every 2 days for 14 days. Tumor growth and body weight were monitored at the same interval. The mice were given free access to diet and water during the course of experiments. The tumor volume was calculated using the formula (smaller diameter)2 × (larger diameter)/2, and the RTV was calculated accordingly as RTV = Vt/V0, where Vt is the tumor volume measured at each time point after treatment and V0 is the tumor volume at the beginning of treatment. The drug efficacy was assessed by calculating the T/C values on the final day of the study using the formula T/C = (mean RTVTreated/mean RTVControl) × 100%.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00682.
Molecular formula strings and SAR data (CSV)
Predicated binding modes of 13, 16, 40, and 43 (ZIP)
Predicated drug-like properties, hERG channel inhibition assay, hematoxylin and eosin (H&E) staining, TUNEL analysis, statistical analysis and 1H NMR, 13C NMR, HRMS and HPLC spectra for representative target compounds (PDF)
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- Qi-Dong You - State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China; Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China;
https://orcid.org/0000-0002-8587-0122;
- Zheng-Yu Jiang - State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China; Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China;https://orcid.org/0000-0002-1671-1582;
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
This study was supported by Projects 81930100, 81773581, and 81773639 of the National Natural Science Foundation of China, the Priority Academic Program Development of Jiangsu Higher Education Institutions, CPU2018GY02 of Double First Class Innovation Team of China Pharmaceutical University, the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. SKLNMZZ202003); the “Qing Lan” Project of Jiangsu Province, and the Young Elite Scientists Sponsorship Program by CAST (No.YESS20180146).
| A1/Bfl1 | Bcl-2-related protein A1 |
| Bak | Bcl-2 homologous antagonist-killer protein |
| Bax | Bcl-2-associated X protein |
| Bcl-2 | B-cell lymphoma 2 |
| Bcl-xl | B-cell lymphoma x long isoform |
| Bcl-w | Bcl-2-like-2 |
| CML | chronic myeloid leukemia |
| CYP | cytochrome P450 |
| Co-ip | coimmunoprecipitation |
| FP | fluorescence polarization |
| hERG | human Ether-a-go-go-Related Gene |
| H&E | hematoxylin and eosin |
| HLM | human liver microsome |
| Mcl-1 | myeloid cell leukemia 1 |
| MM | multiple myeloma |
| MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
| PAMPA | parallel artificial membrane permeability assay |
| PARP | poly ADP-ribose polymerase |
| PPIs | protein–protein interactions |
| RLM | rat liver microsome |
| SAR | structure–activity relationships |
| SGF | simulated gastric fluid |
| SIF | simulated intestinal fluid |
| TR-FRET | time-resolved fluorescence resonance energy transfer |
| TUNEL | terminal deoxynucleotidyl transferase dutp nick end labeling |
| WB | western blot |
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- 4Ren, D.; Tu, H.-C.; Kim, H.; Wang, G. X.; Bean, G. R.; Takeuchi, O.; Jeffers, J. R.; Zambetti, G. P.; Hsieh, J. J.-D.; Cheng, E. H.-Y. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 2010, 330, 1390– 1393, DOI: 10.1126/science.1190217Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVyrsbnE&md5=443c28b09c7d260319eb207c56cd7835BID, BIM, and PUMA Are Essential for Activation of the BAX- and BAK-Dependent Cell Death ProgramRen, Decheng; Tu, Ho-Chou; Kim, Hyungjin; Wang, Gary X.; Bean, Gregory R.; Takeuchi, Osamu; Jeffers, John R.; Zambetti, Gerard P.; Hsieh, James J.-D.; Cheng, Emily H.-Y.Science (Washington, DC, United States) (2010), 330 (6009), 1390-1393CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are assocd. with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only mols. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.
- 5Kluck, R. M.; Bossy-Wetzel, E.; Green, D. R.; Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 1997, 275, 1132– 1136, DOI: 10.1126/science.275.5303.1132Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXhtlKjtro%253D&md5=c73d6f594b23584219028345c0c97c1fThe release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosisKluck, Ruth M.; Bossy-Wetzel, Ella; Green, Douglas R.; Newmeyer, Donald D.Science (Washington, D. C.) (1997), 275 (5303), 1132-1136CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphol. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.
- 6Kozopas, K. M.; Yang, T.; Buchan, H. L.; Zhou, P.; Craig, R. W. Mcl-1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to Bcl-2. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 3516– 3520, DOI: 10.1073/pnas.90.8.3516Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXks1CmtL8%253D&md5=b1f43de6c911f095d53c5a3f38df6a63MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2Kozopas, Karen M.; Yang, Tao; Buchan, Heather L.; Zhou, Ping; Craig, Ruth W.Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (8), 3516-20CODEN: PNASA6; ISSN:0027-8424.A gene, MCL1, was isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Expression of MCL1 increases early in the induction, or programming, of differentiation in ML-1 (at 1-3 h), before the appearance of differentiation markers and mature morphol. (at 1-3 days). Further, MCL1 has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(14;18) chromosome translocation. MCL1 and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the assocn. of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-assocd. genes, expression of MCL1 and BCL2 relates to the programming of differentiation and cell viability/death.
- 7Herrant, M.; Jacquel, A.; Marchetti, S.; Belhacène, N.; Colosetti, P.; Luciano, F.; Auberger, P. Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis. Oncogene 2004, 23, 7863– 7873, DOI: 10.1038/sj.onc.1208069Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotl2kur4%253D&md5=91ff0749a52f4244b6480652a2e79033Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosisHerrant, Magali; Jacquel, Arnaud; Marchetti, Sandrine; Belhacene, Nathalie; Colosetti, Pascal; Luciano, Frederic; Auberger, PatrickOncogene (2004), 23 (47), 7863-7873CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)Mcl-1 is an antiapoptotic member of the Bcl-2 family that can promote cell viability. We report here that Mcl-1 is a new substrate for caspases during induction of apoptosis. Mcl-1 cleavage occurs after Asp127 and Asp157 and generates four fragments of 24, 19, 17 and 12 kDa in both intact cells and in vitro, an effect prevented by selective caspase inhibitors. As a consequence, the resulting protein that lacks the first 127 or 157 amino acids contains only the BH1-BH3 domains of Bcl-2 family members. Mutation of Asp127 and Asp157 abolishes the generation of the 24 and 12 kDa fragments and that of the 19 and 17 kDa fragments, resp. Interestingly, when expressed in HeLa cells Mcl-1 wt and Mcl-1 Δ127 showed a markedly different intracellular distribution. Mcl-1 wt colocalized with α-Tubulin near the internal face of the plasma membrane, while Mcl-1 Δ127 co-assocd. with Bim-EL at the mitochondrial level. Coimmunopptn. expts. also demonstrated that Mcl1 Δ127 exhibited increased binding to Bim when compared to Mcl-1 wt. Finally, Mcl-1 wt unlike Mcl-1 Δ127 inhibited Bim-EL-induced caspase activation. Altogether, our findings demonstrate that cleavage of Mcl-1 by caspases modifies its subcellular localization, increases its assocn. with Bim and inhibits its antiapoptotic function.
- 8Adams, J. M.; Cory, S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 2001, 26, 61– 66, DOI: 10.1016/S0968-0004(00)01740-0Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXptV2itQ%253D%253D&md5=6af92fbfe37a6e389302fc45314c30fdLife-or-death decisions by the Bcl-2 protein familyAdams, J. M.; Cory, S.Trends in Biochemical Sciences (2001), 26 (1), 61-66CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)A review, with 51 refs. In response to intracellular damage and certain physiol. cues, cells enter the suicide program termed apoptosis, executed by proteases called caspases. Commitment to apoptosis is typically governed by opposing factions of the Bcl-2 family of cytoplasmic proteins. Initiation of the proteolytic cascade requires assembly of certain caspase precursors on a scaffold protein, and the Bcl-2 family dets. whether this complex can form. Its pro-survival members can act by sequestering the scaffold protein and/or by preventing the release of apoptogenic mols. from organelles such as mitochondria. Pro-apoptotic family members act as sentinels for cellular damage: cytotoxic signals induce their translocation to the organelles where they bind to their pro-survival relatives, promote organelle damage and trigger apoptosis.
- 9Gomez-Bougie, P.; Wuillème-Toumi, S.; Ménoret, E.; Trichet, V.; Robillard, N.; Philippe, M.; Bataille, R.; Amiot, M. Noxa up-regulation and Mcl-1 cleavage are associated to apoptosis induction by bortezomib in multiple myeloma. Cancer Res. 2007, 67, 5418– 5424, DOI: 10.1158/0008-5472.CAN-06-4322Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmtVaitL4%253D&md5=70edbc5d1ddc89ba41bc18db35054430Noxa Up-regulation and Mcl-1 Cleavage Are Associated to Apoptosis Induction by Bortezomib in Multiple MyelomaGomez-Bougie, Patricia; Wuilleme-Toumi, Soraya; Menoret, Emmanuelle; Trichet, Valerie; Robillard, Nelly; Philippe, Moreau; Bataille, Regis; Amiot, MartineCancer Research (2007), 67 (11), 5418-5424CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Targeting the ubiquitin-proteasome pathway has emerged as a potent anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma. Multiple myeloma cell survival is highly dependent on Mcl-1 antiapoptotic mols. In a recent study, proteasome inhibitors induced Mcl-1 accumulation that slowed down their proapoptotic effects. Consequently, we investigated the role of Bcl-2 family members in bortezomib-induced apoptosis. We found that bortezomib induced apoptosis in five of seven human myeloma cell lines (HMCL). Bortezomib-induced apoptosis was assocd. with Mcl-1 cleavage regardless of Mcl-1L accumulation. Furthermore, RNA interference mediated Mcl-1 decrease and sensitized RPMI-8226 HMCL to bortezomib, highlighting the contribution of Mcl-1 in bortezomib-induced apoptosis. Interestingly, an important induction of Noxa was found in all sensitive HMCL both at protein and mRNA level. Concomitant to Mcl-1 cleavage and Noxa induction, we also found caspase-3, caspase-8, and caspase-9 activation. Under bortezomib treatment, Mcl-1L/Noxa complexes were highly increased, Mcl-1/Bak complexes were disrupted, and there was an accumulation of free Noxa. Finally, we obsd. a dissocn. of Mcl-1/Bim complexes that may be due to a displacement of Bim induced by Noxa. Thus, in myeloma cells, the mechanistic basis for bortezomib sensitivity can be explained mainly by the model in which the sensitizer Noxa can displace Bim, a BH3-only activator, from Mcl-1, thus leading to Bax/Bak activation.
- 10Pierson, W.; Cauwe, B.; Policheni, A.; Schlenner, S. M.; Franckaert, D.; Berges, J.; Humblet-Baron, S.; Schönefeldt, S.; Herold, M. J.; Hildeman, D.; Strasser, A.; Bouillet, P.; Lu, L.-F.; Matthys, P.; Freitas, A. A.; Luther, R. J.; Weaver, C. T.; Dooley, J.; Gray, D. H.; Liston, A. Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3+ regulatory T cells. Nat. Immunol. 2013, 14, 959– 965, DOI: 10.1038/ni.2649Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFShs77L&md5=46a3e7ae00ecacc2294ad3381c6e0c66Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3+ regulatory T cellsPierson, Wim; Cauwe, Benedicte; Policheni, Antonia; Schlenner, Susan M.; Franckaert, Dean; Berges, Julien; Humblet-Baron, Stephanie; Schoenefeldt, Susann; Herold, Marco J.; Hildeman, David; Strasser, Andreas; Bouillet, Philippe; Lu, Li-Fan; Matthys, Patrick; Freitas, Antonio A.; Luther, Rita J.; Weaver, Casey T.; Dooley, James; Gray, Daniel H. D.; Liston, AdrianNature Immunology (2013), 14 (9), 959-965CODEN: NIAMCZ; ISSN:1529-2908. (Nature Publishing Group)Foxp3+ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4+ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was crit. for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via crit. survival pathways.
- 11Renjini, A. P.; Titus, S.; Narayan, P.; Murali, M.; Jha, R. K.; Laloraya, M. STAT3 and MCL-1 associate to cause a mesenchymal epithelial transition. J. Cell Sci. 2014, 127, 1738– 1750, DOI: 10.1242/jcs.138214Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXos1Chur4%253D&md5=76aa474af8d7dd8b5b469a1aa633535bSTAT3 and MCL-1 associate to cause a mesenchymal epithelial transitionRenjini, A. P.; Titus, Shiny; Narayan, Prashanth; Murali, Megha; Jha, Rajesh Kumar; Laloraya, MaliniJournal of Cell Science (2014), 127 (8), 1738-1750CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)Embryo implantation is effected by a myriad of signaling cascades acting on the embryo-endometrium axis. Here we show, by using MALDI TOF anal., far-western anal. and colocalization and co-transfection studies, that STAT3 and MCL-1 are interacting partners during embryo implantation. We show in vitro that the interaction between the two endogenous proteins is strongly regulated by estrogen and progesterone. Implantation, pregnancy and embryogenesis are distinct from any other process in the body, with extensive, but controlled, proliferation, cell migration, apoptosis, cell invasion and differentiation. Cellular plasticity is vital during the early stages of development for morphogenesis and organ homeostasis, effecting the epithelial to mesenchymal transition (EMT) and, the reverse process, mesenchymal to epithelial transition (MET). STAT3 functionally assocs. with MCL-1 in the mammalian breast cancer cell line MCF7 that overexpresses STAT3 and MCL-1, which leads to an increased rate of apoptosis and decreased cellular invasion, disrupting the EMT. Assocn. of MCL-1 with STAT3 modulates the normal, anti-apoptotic, activity of MCL-1, resulting in pro-apoptotic effects. Studying the impact of the assocn. of STAT3 with MCL-1 on MET could lead to an enhanced understanding of pregnancy and infertility, and also metastatic tumors.
- 12Hikita, H.; Takehara, T.; Shimizu, S.; Kodama, T.; Li, W.; Miyagi, T.; Hosui, A.; Ishida, H.; Ohkawa, K.; Kanto, T.; Hiramatsu, N.; Yin, X. M.; Hennighausen, L.; Tatsumi, T.; Hayashi, N. Mcl-1 and Bcl-xL cooperatively maintain integrity of hepatocytes in developing and adult murine liver. Hepatology 2009, 50, 1217– 1226, DOI: 10.1002/hep.23126Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlWlu7fP&md5=1344b4717ed1014041c9e59b216a8158Mcl-1 and Bcl-xL cooperatively maintain integrity of hepatocytes in developing and adult murine liverHikita, Hayato; Takehara, Tetsuo; Shimizu, Satoshi; Kodama, Takahiro; Li, Wei; Miyagi, Takuya; Hosui, Atsushi; Ishida, Hisashi; Ohkawa, Kazuyoshi; Kanto, Tatsuya; Hiramatsu, Naoki; Yin, Xiao-Ming; Hennighausen, Lothar; Tatsumi, Tomohide; Hayashi, NorioHepatology (Hoboken, NJ, United States) (2009), 50 (4), 1217-1226CODEN: HPTLD9; ISSN:0270-9139. (John Wiley & Sons, Inc.)Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a crit. apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, esp. in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1-deficient mice by crossing mcl-1flox/flox mice and AlbCre mice and further crossed them with bcl-xflox/flox mice, giving Mcl-1/Bcl-xL-deficient mice. The mcl-1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased no. of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL)-pos. cells in the liver; these phenotypes were very dose to those previously found in hepatocyte-specific Bcl-xL-deficient mice. Although mcl-1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-xflox/+ mcl-1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL-deficient or Mcl-1-deficient mice. In contrast, bcl-xflox/flox mcl-1flox/+ AlbCre, bcl-xflox/+ mcl-1flox/flox AlbCre, and bcl-xflox/flox mcl-1flox/flox AlbCre mice displayed a decreased no. of hepatocytes and a reduced vol. of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl-1 is crit. for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner.
- 13Opferman, J. T.; Letai, A.; Beard, C.; Sorcinelli, M. D.; Ong, C. C.; Korsmeyer, S. J. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 2003, 426, 671– 676, DOI: 10.1038/nature02067Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXps1Cksr4%253D&md5=e34f76ef4a07eeb92b18797ae484dcc5Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1Opferman, Joseph T.; Letai, Anthony; Beard, Caroline; Sorcinelli, Mia D.; Ong, Christy C.; Korsmeyer, Stanley J.Nature (London, United Kingdom) (2003), 426 (6967), 671-676CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Regulated apoptosis is essential for both the development and the subsequent maintenance of the immune system. Interleukins, including IL-2, IL-4, IL-7 and IL-15, heavily influence lymphocyte survival during the vulnerable stages of VDJ rearrangement and later in ensuring cellular homeostasis, but the genes specifically responsible for the development and maintenance of lymphocytes have not been identified. The antiapoptotic protein MCL-1 is an attractive candidate, as it is highly regulated, appears to enhance short-term survival and functions at an apical step in genotoxic deaths. However, Mcl-1 deficiency results in peri-implantation lethality. Here we show that mice conditional for Mcl-1 display a profound redn. in B and T lymphocytes when MCL-1 is removed. Deletion of Mcl-1 during early lymphocyte differentiation increased apoptosis and arrested the development at pro-B-cell and double-neg. T-cell stages. Induced deletion of Mcl-1 in peripheral B- and T-cell populations resulted in their rapid loss. Moreover, IL-7 both induced and required MCL-1 to mediate lymphocyte survival. Thus, MCL-1, which selectively inhibits the proapoptotic protein BIM, is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes.
- 14Thomas, L. W.; Lam, C.; Edwards, S. W. Mcl-1: the molecular regulation of protein function. FEBS Lett. 2010, 584, 2981– 2989, DOI: 10.1016/j.febslet.2010.05.061Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXosVyhu7s%253D&md5=5deee464702c00444349236698dcf9b1Mcl-1; the molecular regulation of protein functionThomas, Luke W.; Lam, Connie; Edwards, Steven W.FEBS Letters (2010), 584 (14), 2981-2989CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. Apoptosis, an essential and basic biol. phenomenon, is regulated in a complex manner by a multitude of factors. Myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, exemplifies a no. of the mechanisms by which a protein's contribution to cell fate may be modified. The N-terminus of Mcl-1 is unique among the Bcl-2 family, in that it is rich in exptl. confirmed and putative regulatory residues and motifs. These include sites for ubiquitination, cleavage, and phosphorylation, which influence the protein's stability, localization, dimerization, and function. Here, the authors review what is known about the regulation of Mcl-1 expression and function, with particular focus on post-translational modifications and how phosphorylation interconnects the complex mol. control of Mcl-1 with the cellular state.
- 15Aichberger, K. J.; Mayerhofer, M.; Krauth, M. T.; Skvara, H.; Florian, S.; Sonneck, K.; Akgul, C.; Derdak, S.; Pickl, W. F.; Wacheck, V.; Selzer, E.; Monia, B. P.; Moriggl, R.; Valent, P.; Sillaber, C. Identification of Mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides. Blood 2005, 105, 3303– 3311, DOI: 10.1182/blood-2004-02-0749Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjtlSlsrg%253D&md5=eb32b6ef8b9b9b54936a9368c8b079cdIdentification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): Evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotidesAichberger, Karl J.; Mayerhofer, Matthias; Krauth, Maria-Theresa; Skvara, Hans; Florian, Stefan; Sonneck, Karoline; Akgul, Cahit; Derdak, Sophia; Pickl, Winfried F.; Wacheck, Volker; Selzer, Edgar; Monia, Brett P.; Moriggl, Richard; Valent, Peter; Sillaber, ChristianBlood (2005), 105 (8), 3303-3311CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)Antiapoptotic members of the bcl-2 family have recently been implicated in the pathogenesis of chronic myeloid leukemia (CML), a hematopoietic neoplasm assocd. with the BCR/ABL oncogene. The authors have examd. expression of MCL-1 in primary CML cells and BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed myeloid cell leukemia-1 (mcl-1) mRNA and the MCL-1 protein in a constitutive manner. The BCR/ABL inhibitor imatinib (=STI571) decreased the expression of MCL-1 in these cells. Correspondingly, BCR/ABL enhanced mcl-1 promoter activity, mcl-1 mRNA expression, and the MCL-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of MCL-1 in Ba/F3 cells was counteracted by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Identical results were obtained for constitutive expression of MCL-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of MCL-1 as a survival-related target in CML cells, mcl-1 siRNA and mcl-1 antisense oligonucleotides (ASOs) were applied. The resulting down-regulation of MCL-1 was found to be assocd. with a substantial decrease in viability of K562 cells. Moreover, the mcl-1 ASO was found to synergize with imatinib in producing growth inhibition in these cells. Together, the authors' data identify MCL-1 as a BCR/ABL-dependent survival factor and interesting target in CML.
- 16Steven, L. G.; Klaus, P.; Jean-Luc, H.; Anderson, K. C. Mcl-1 regulation and its role in multiple myeloma. Cell Cycle 2004, 3, 1259– 1262, DOI: 10.4161/cc.3.10.1196Google ScholarThere is no corresponding record for this reference.
- 17Song, L.; Coppola, D.; Livingston, S.; Cress, W. D.; Haura, E. B. Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells. Cancer Biol. Ther. 2005, 4, 267– 276, DOI: 10.4161/cbt.4.3.1496Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFyrug%253D%253D&md5=b79c35e287895a356d74740c41e03baeMcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cellsSong, Lanxi; Coppola, Domenico; Livingston, Sandy; Cress, Doug; Haura, Eric B.Cancer Biology & Therapy (2005), 4 (3), 267-276CODEN: CBTAAO; ISSN:1538-4047. (Landes Bioscience)Overexpression of anti-apoptotic Bcl-2 family members and deregulation of the pathways that regulate pro-apoptotic family members have been obsd. in non-small cell lung cancers (NSCLC). Previous reports have identified both Bcl-2 and Bcl-xL proteins as survival factors in lung cancer cells since redns. in these proteins can induce apoptosis and sensitize lung cancer cells to apoptosis induced by chemotherapy agents. Myeloid cell leukemia-1 (Mcl-1), another member of the Bcl-2 family, has been found to be a crit. survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human lung cancers. We used NSCLC cell lines to explore how Mcl-1 levels affect lung cancer cell survival and studied tumors from patients to det. expression patterns of Mcl-1. NSCLC cells express abundant Mcl-1 protein and depletion of Mcl-1 levels by antisense Mcl-1 oligonucleotides induces apoptosis in A549 and H1299 lung cancer cells. Redn. in Mcl-1 levels can sensitize lung cancer cells to apoptosis induced by cytotoxic agents as well as by ionizing radiation. Lung cancer cells overexpressing Mcl-1 are less sensitive to apoptosis induced by chemotherapeutic agents, ZD1839 (an inhibitor of EGFR tyrosine kinase) and Bcl-2 or Bcl-xL antisense oligonucleotides. We find that epidermal growth factor (EGF) can enhance Mcl-1 protein levels in an ERK-dependent manner. Signal transduction agents that reduce Mcl-1 levels correlated with their individual ability to induce apoptosis in lung cancer cells. Finally, NSCLC tumors taken directly from patients have elevated levels of Mcl-1 protein compared with normal adjacent lung tissue. Therefore, agents that target Mcl-1 can induce apoptosis and sensitize cells to apoptosis induced by cytotoxic agents. Mcl-1 protein is overexpressed in a subset of human NSCLC and enhanced levels of Mcl-1 may protect lung cancer cells from death induced by a variety of pro-apoptotic stimuli.
- 18Tong, J.; Wang, P.; Tan, S.; Chen, D.; Nikolovska-Coleska, Z.; Zou, F.; Yu, J.; Zhang, L. Mcl-1 degradation is required for targeted therapeutics to eradicate colon cancer cells. Cancer Res. 2017, 77, 2512– 2521, DOI: 10.1158/0008-5472.CAN-16-3242Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvFakurw%253D&md5=40461e21325a846f66d68346ef955e98Mcl-1 Degradation Is Required for Targeted Therapeutics to Eradicate Colon Cancer CellsTong, Jingshan; Wang, Peng; Tan, Shuai; Chen, Dongshi; Nikolovska-Coleska, Zaneta; Zou, Fangdong; Yu, Jian; Zhang, LinCancer Research (2017), 77 (9), 2512-2521CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degrdn. has been assocd. with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degrdn. and anticancer drug responses has not been directly established, esp. in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in detg. effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degrdn. was induced by a variety of multikinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1-KI also suppressed the antiangiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degrdn.-resistant Mcl-1 bound and sequestered PUMA from other prosurvival proteins to maintain cell survival, which was abolished by small-mol. Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degrdn. in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1-targeting agents that can overcome drug resistance.
- 19Pepper, C.; Lin, T. T.; Pratt, G.; Hewamana, S.; Brennan, P.; Hiller, L.; Hills, R.; Ward, R.; Starczynski, J.; Austen, B.; Hooper, L.; Stankovic, T.; Fegan, C. Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers. Blood 2008, 112, 3807– 3817, DOI: 10.1182/blood-2008-05-157131Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlCntrjF&md5=d7a626b0de96a0f82956f3e1da8ea9cfMcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markersPepper, Chris; Lin, Thet Thet; Pratt, Guy; Hewamana, Saman; Brennan, Paul; Hiller, Louise; Hills, Robert; Ward, Rachel; Starczynski, Jane; Austen, Belinda; Hooper, Laura; Stankovic, Tatjana; Fegan, ChrisBlood (2008), 112 (9), 3807-3817CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)Bcl-2 family proteins play a crit. role in the regulation of apoptosis in chronic lymphocytic leukemia (CLL). However, their assocn. with established prognostic markers is unknown. In this study, we analyzed the expression of Bcl-2, Bax, and Mcl-1 in 185 CLL patients and evaluated their relationship with other prognostic markers, in vitro sensitivity to fludarabine, and clin. outcome. Mcl-1 expression was significantly correlated with stage of disease (P < .001), lymphocyte doubling time (P = .01), VH gene mutation status (P < .001), CD38 expression (P < .001), and ZAP-70 expression (P = .003). In addn., Mcl-1 and Mcl-1/Bax ratios showed strong correlations with in vitro resistance to fludarabine (P = .005 and P < .001, resp.). Furthermore, elevated Mcl-1 expression and Mcl-1/Bax ratios were predictive of time to first treatment in the whole cohort (P < .001 and P < .001, resp.) and in stage A patients only (P = .002 and P = .001, resp.). Taken together, our data show that Mcl-1 is a key controller of in vitro drug resistance and is an important regulator of disease progression and outcome in CLL. It therefore represents a promising therapeutic target in this incurable condition. The close correlation between Mcl-1 expression and VH gene mutation status, CD38 expression, and ZAP-70 expression offers a biol. explanation for their assocn. with adverse prognosis.
- 20Zhou, P.; Qian, L.; Bieszczad, C. K.; Noelle, R.; Binder, M.; Levy, N. B.; Craig, R. W. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 1998, 92, 3226– 3239, DOI: 10.1182/blood.V92.9.3226Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntFCqsbg%253D&md5=06d39268f7f856a192ccc6e7be449abeMcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineageZhou, Ping; Qian, Liping; Bieszczad, Christine K.; Noelle, Randolph; Binder, Michael; Levy, Norman B.; Craig, Ruth W.Blood (1998), 92 (9), 3226-3239CODEN: BLOOAW; ISSN:0006-4971. (W. B. Saunders Co.)Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addn., among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell no., but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at "viability gates" along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization.
- 21Touzeau, C.; Maciag, P.; Amiot, M.; Moreau, P. Targeting Bcl-2 for the treatment of multiple myeloma. Leukemia 2018, 32, 1899– 1907, DOI: 10.1038/s41375-018-0223-9Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVCns77E&md5=dfbc63cc6ca0b471ad40bb38d064bbd8Targeting Bcl-2 for the treatment of multiple myelomaTouzeau, Cyrille; Maciag, Paulo; Amiot, Martine; Moreau, PhilippeLeukemia (2018), 32 (9), 1899-1907CODEN: LEUKED; ISSN:0887-6924. (Nature Research)Despite advances in the treatment of multiple myeloma, the disease still remains incurable for the majority of patients. The overexpression of anti-apoptotic proteins (i.e., Bcl-2, Bcl-XL or Mcl-1) is a hallmark of cancer and favors tumor cell survival and resistance to therapy. The oral drug venetoclax is the first-in-class Bcl-2-specific BH3 mimetic. In myeloma, in vitro sensitivity to venetoclax is mainly obsd. in plasma cells harboring the t(11;14) translocation, a mol. subgroup assocd. with high Bcl-2 and low Mcl-1/Bcl-XL expression. In addn. with Bcl-2 members expression profile, functional tests as BH3 profiling or in vitro BH3 mimetic drug testing also predict sensitivity to the drug. Phase 1 clin. trials recently confirmed the efficacy of venetoclax monotherapy in heavily pretreated myeloma patients, mostly in patients with t(11;14). In combination with the proteasome inhibitor bortezomib, venetoclax therapy was found to be feasible and allowed promising response rate in relapsed myeloma patients, independent of t(11;14) status. The present review summarizes the current knowledge, "from bench to bedside", about venetoclax for the treatment of multiple myeloma.
- 22Souers, A. J.; Leverson, J. D.; Boghaert, E. R.; Ackler, S. L.; Catron, N. D.; Chen, J.; Dayton, B. D.; Ding, H.; Enschede, S. H.; Fairbrother, W. J.; Huang, D. C. S.; Hymowitz, S. G.; Jin, S.; Khaw, S. L.; Kovar, P. J.; Lam, L. T.; Lee, J.; Maecker, H. L.; Marsh, K. C.; Mason, K. D.; Mitten, M. J.; Nimmer, P. M.; Oleksijew, A.; Park, C.-H.; Park, C. M.; Phillips, D. C.; Roberts, A. W.; Sampath, D.; Seymour, J. F.; Smith, M. L.; Sullivan, G. M.; Tahir, S. K.; Tse, C.; Wendt, M. D.; Xiao, Y.; Xue, J. C.; Zhang, H.; Humerickhouse, R. A.; Rosenberg, S. H.; Elmore, S. W. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 2013, 19, 202– 208, DOI: 10.1038/nm.3048Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslagtQ%253D%253D&md5=6a24b72303eb2062da653f1f61ac74fbABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing plateletsSouers, Andrew J.; Leverson, Joel D.; Boghaert, Erwin R.; Ackler, Scott L.; Catron, Nathaniel D.; Chen, Jun; Dayton, Brian D.; Ding, Hong; Enschede, Sari H.; Fairbrother, Wayne J.; Huang, David C. S.; Hymowitz, Sarah G.; Jin, Sha; Khaw, Seong Lin; Kovar, Peter J.; Lam, Lloyd T.; Lee, Jackie; Maecker, Heather L.; Marsh, Kennan C.; Mason, Kylie D.; Mitten, Michael J.; Nimmer, Paul M.; Oleksijew, Anatol; Park, Chang H.; Park, Cheol-Min; Phillips, Darren C.; Roberts, Andrew W.; Sampath, Deepak; Seymour, John F.; Smith, Morey L.; Sullivan, Gerard M.; Tahir, Stephen K.; Tse, Chris; Wendt, Michael D.; Xiao, Yu; Xue, John C.; Zhang, Haichao; Humerickhouse, Rod A.; Rosenberg, Saul H.; Elmore, Steven W.Nature Medicine (New York, NY, United States) (2013), 19 (2), 202-208CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-XL), which has shown clin. efficacy in some BCL-2-dependent hematol. cancers. However, concomitant on-target thrombocytopenia caused by BCL-XL inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compd. inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacol. inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematol. cancers.
- 23Quinn, B. A.; Dash, R.; Azab, B.; Sarkar, S.; Das, S. K.; Kumar, S.; Oyesanya, R. A.; Dasgupta, S.; Dent, P.; Grant, S.; Rahmani, M.; Curiel, D. T.; Dmitriev, I.; Hedvat, M.; Wei, J.; Wu, B.; Stebbins, J. L.; Reed, J. C.; Pellecchia, M.; Sarkar, D.; Fisher, P. B. Targeting Mcl-1 for the therapy of cancer. Expert Opin. Invest. Drugs 2011, 20, 1397– 1411, DOI: 10.1517/13543784.2011.609167Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFGgur3N&md5=7e2e0ab1cedb25a32b8e97a935238013Targeting Mcl-1 for the therapy of cancerQuinn, Bridget A.; Dash, Rupesh; Azab, Belal; Sarkar, Siddik; Das, Swadesh K.; Kumar, Sachin; Oyesanya, Regina A.; Dasgupta, Santanu; Dent, Paul; Grant, Steven; Rahmani, Mohamed; Curiel, David T.; Dmitriev, Igor; Hedvat, Michael; Wei, Jun; Wu, Bainan; Stebbins, John L.; Reed, John C.; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B.Expert Opinion on Investigational Drugs (2011), 20 (10), 1397-1411CODEN: EOIDER; ISSN:1354-3784. (Informa Healthcare)A review. Introduction:Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.Areas covered:Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacol. approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.Expert opinion:We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of addnl. signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochem. pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
- 24Akgul, C. Mcl-1 is a potential therapeutic target in multiple types of cancer. Cell. Mol. Life Sci. 2009, 66, 1326– 1336, DOI: 10.1007/s00018-008-8637-6Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksFGhtb4%253D&md5=a6eca2f0ff24caaa199397b3e4c959d5Mcl-1 is a potential therapeutic target in multiple types of cancerAkgul, C.Cellular and Molecular Life Sciences (2009), 66 (8), 1326-1336CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)A review. Resistance to apoptosis is a common challenge in human malignancies contributing to both progress of cancer and resistance to conventional therapeutics. Abnormalities in a variety of cell intrinsic and extrinsic mol. mechanisms cooperatively promote tumor formation. Therapeutic approaches that specifically target components of these mol. mechanisms are getting widespread attention. Mcl-1 is a highly expressed pro-survival protein in human malignancies and its cellular expression is tightly regulated via multiple mechanisms. Mcl-1 differs from other members of the Bcl-2 family in having a very short half-life. So inhibition of its expression and/or neutralization of its anti-apoptotic function will rapidly make Mcl-1-dependent cells more susceptible to apoptosis and provide an opportunity to combat several types of cancers. This review summarizes the current knowledge on the regulation of Mcl-1 expression and discusses the alternative approaches targeting Mcl-1 in human cancer cells whose survivals mainly depend on Mcl-1.
- 25Lessene, G.; Czabotar, P. E.; Colman, P. M. BCL-2 family antagonists for cancer therapy. Nat. Rev. Drug. Discov. 2008, 7, 989– 1000, DOI: 10.1038/nrd2658Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVenu73P&md5=14886161a66c3ed7f754061fa964e1edBCL-2 family antagonists for cancer therapyLessene, Guillaume; Czabotar, Peter E.; Colman, Peter M.Nature Reviews Drug Discovery (2008), 7 (12), 989-1000CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Overexpression of members of the BCL-2 family of pro-survival proteins is commonly assocd. with unfavorable pathogenesis in cancer. The convergence of cytotoxic stress signals on the extended BCL-2 protein family provides the biol. rationale for directly targeting this family to induce apoptotic cell death. Recently, several compds. have been described that inhibit the interaction between BCL-2 family members and their natural ligand, a helical peptide sequence known as the BH3 domain. Here, we review preclin. and clin. data on these compds., and recommend four criteria that define antagonists of the BCL-2 protein family.
- 26Fire, E.; Gullá, S. V.; Grant, R. A.; Keating, A. E. Mcl-1-Bim complexes accommodate surprising point mutations via minor structural changes. Protein Sci. 2010, 19, 507– 519, DOI: 10.1002/pro.329Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisFOhurw%253D&md5=58343d62b4cbfcfb03c53b2b667abadeMcl-1-Bim complexes accommodate surprising point mutations via minor structural changesFire, Emiko; Gulla, Stefano V.; Grant, Robert A.; Keating, Amy E.Protein Science (2010), 19 (3), 507-519CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)Mcl-1 is an antiapoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the α-helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl-2 family function. We report the crystal structure of human Mcl-1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochem. changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl-1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine-to-alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also obsd., with shifting of helix α3 accommodating an isoleucine-to-tyrosine mutation at this same position. We surveyed the variation in available Mcl-1 and Bcl-xL structures and obsd. moderate flexibility that is likely crit. for facilitating interactions of diverse BH3-only proteins with Mcl-1. With the antiapoptotic Bcl-2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl-1.
- 27Gomez-Bougie, P.; Maiga, S.; Tessoulin, B.; Bourcier, J.; Bonnet, A.; Rodriguez, M. S.; Le Gouill, S.; Touzeau, C.; Moreau, P.; Pellat-Deceunynck, C.; Amiot, M. BH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatment. Blood 2018, 132, 2656– 2669, DOI: 10.1182/blood-2018-03-836718Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntlygs70%253D&md5=3c50a02c2e05a402bbfdfd4528dcfabcBH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatmentGomez-Bougie, Patricia; Maiga, Sophie; Tessoulin, Benoit; Bourcier, Jessie; Bonnet, Antoine; Rodriguez, Manuel S.; Le Gouill, Steven; Touzeau, Cyrille; Moreau, Philippe; Pellat-Deceunynck, Catherine; Amiot, MartineBlood (2018), 132 (25), 2656-2669CODEN: BLOOAW; ISSN:1528-0020. (American Society of Hematology)BH3 mimetics are promising drugs for hematol. malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addn., 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Interestingly, this complex was also dissocd. in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clin. evaluation in myeloma at both diagnosis and relapse.
- 28Zhang, Z.; Song, T.; Li, X.; Wu, Z.; Feng, Y.; Xie, F.; Liu, C.; Qin, J.; Chen, H. Novel soluble myeloid cell leukemia sequence 1 (Mcl-1) inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g) developed using a fragment-based approach. Eur. J. Med. Chem. 2013, 59, 141– 149, DOI: 10.1016/j.ejmech.2012.10.050Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1ersbw%253D&md5=c2b6afc34c8b7253cb7f765fc8f68c1cNovel soluble myeloid cell leukemia sequence 1 (Mcl-1) inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g) developed using a fragment-based approachZhang, Zhichao; Song, Ting; Li, Xiangqian; Wu, Zhiyong; Feng, Yingang; Xie, Feibo; Liu, Chengwu; Qin, Jianquan; Chen, HongboEuropean Journal of Medicinal Chemistry (2013), 59 (), 141-149CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Based on a known nanomolar Bcl-2 homol. domain 3 (BH3) mimetic 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b] pyrrole-9-carbonitrile (I, MW: 331), we applied a fragment-based approach to obtain BH3 mimetics with improved affinity and improved soly. in a water-ethanol (9:1) cosolvent. After the deconstruction of I, we obtained fragment cyanoacetamide (II), which was detd. to be a ligand efficiency (LE) hot part. After a rational optimization through fragment evolution beginning with fragment II, a smaller Mcl-1 inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (III, MW: 288) with a 6-fold increase in affinity compared to I was obtained, as predicted by our optimization curve and identified by Mcl-1 protein NMR (NMR).
- 29Zhang, Z.; Liu, C.; Li, X.; Song, T.; Wu, Z.; Liang, X.; Zhao, Y.; Shen, X.; Chen, H. Fragment-based design, synthesis, and biological evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxynicotinamide derivatives as novel Mcl-1 inhibitors. Eur. J. Med. Chem. 2013, 60, 410– 420, DOI: 10.1016/j.ejmech.2012.12.016Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtVemsrk%253D&md5=418bafc7eeab83e01f5399e122b3af7dFragment-based design, synthesis, and biological evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxynicotinamide derivatives as novel Mcl-1 inhibitorsZhang, Zhichao; Liu, Chengwu; Li, Xiangqian; Song, Ting; Wu, Zhiyong; Liang, Xiaomeng; Zhao, Yan; Shen, Xiaoyun; Chen, HongboEuropean Journal of Medicinal Chemistry (2013), 60 (), 410-420CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)We have previously reported a nanomolar inhibitor of antiapoptotic Mcl-1 protein, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1). S1 plays its function by binding to the BH3 groove of Mcl-1. Basing on this spacial structural characteristic, we developed a novel class of Mcl-1 inhibitor using fragment-based drug discovery approach. By dissecting S1, we identified 2-hydroxynicotinonitrile (4) with a 2-hydroxypyridine core as the starting fragment. In the following mol. growth, we used the ligand efficiency evaluation and fit quality score to assess the fragments. A novel potent compd., N-benzyl-5-(4-isopropylthiophenol)-2-hydroxyl nicotinamide (12c), which binds Mcl-1 with an IC50 value of 54 nM was obtained. Compd. 12c demonstrated a better aq. soly. than S1.
- 30Liu, L.; Liu, R.; Yang, X.; Hou, X.; Fang, H. Design, synthesis and biological evaluation of tyrosine derivatives as Mcl-1 inhibitors. Eur. J. Med. Chem. 2020, 191, 112142, DOI: 10.1016/j.ejmech.2020.112142Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjs1OnsLw%253D&md5=e07c3dcd86e299d2bfedb4d2bb91c698Design, synthesis and biological evaluation of tyrosine derivatives as Mcl-1 inhibitorsLiu, Lulu; Liu, Renshuai; Yang, Xinying; Hou, Xuben; Fang, HaoEuropean Journal of Medicinal Chemistry (2020), 191 (), 112142CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)The upregulation of the protein myeloid cell leukemia-1 (Mcl-1) is closely assocd. with various human cancers, which can result in the evasion of apoptosis and a low survival rate. Therefore, developing Mcl-1 inhibitors has become a promising paradigm for cancer therapy. Herein, we designed and synthesized a novel series of tyrosine derivs., among which compds. (I) (R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = OBut, n = 2; R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = Ph, n = 1; R1 = naphthyl, X = Br, R2 = 4-Me-benzyl, n = 1) exhibited very high binding affinity to Mcl-1 with Ki values of 0.18, 0.27 and 0.23μM, resp. Interestingly, compd. I (R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = Ph, n = 1) showed not only potent activity against Mcl-1 but also considerable selectivity over Bcl-2 and Bcl-xL, which was rationalized by mol. docking and fragment-centric topog. mapping (FCTM). It is worth noting that compds. I (R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = OBut, n = 2; R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = Ph, n = 1; R1 = naphthyl, X = Br, R2 = 4-Me-benzyl, n = 1) displayed potent antiproliferative activity against several cancer cell lines and could induce apoptosis of KM3 and HepG2 cells in a dose-dependent manner.
- 31Arkin, M. R.; Wells, J. A. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat. Rev. Drug. Discov. 2004, 3, 301– 317, DOI: 10.1038/nrd1343Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXis1GktrY%253D&md5=3e102c6207be420b4232056b4acdea12Small-molecule inhibitors of protein-protein interactions: progressing towards the dreamArkin, Michelle R.; Wells, James A.Nature Reviews Drug Discovery (2004), 3 (4), 301-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Protein-protein interactions have a key role in most biol. processes, and offer attractive opportunities for therapeutic intervention. Developing small mols. that modulate protein-protein interactions is difficult, owing to issues such as the lack of well-defined binding pockets. Nevertheless, there was important progress in this endeavor in recent years. Here, the authors use illustrative examples to discuss general strategies for addressing the challenges inherent in the discovery and characterization of small-mol. inhibitors of protein-protein interactions.
- 32Horne, W. S.; Boersma, M. D.; Windsor, M. A.; Gellman, S. H. Sequence-based design of alpha/beta-peptide foldamers that mimic BH3 domains. Angew. Chem., Int. Ed. Engl. 2008, 47, 2853– 2856, DOI: 10.1002/anie.200705315Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1c3gvVSgsg%253D%253D&md5=bf421b54bd4b3bf6bee39d70bd681851Sequence-based design of alpha/beta-peptide foldamers that mimic BH3 domainsHorne W Seth; Boersma Melissa D; Windsor Matthew A; Gellman Samuel HAngewandte Chemie (International ed. in English) (2008), 47 (15), 2853-6 ISSN:.There is no expanded citation for this reference.
- 33de Araujo, A. D.; Lim, J.; Wu, K.-C.; Xiang, Y.; Good, A. C.; Skerlj, R.; Fairlie, D. P. Bicyclic helical peptides as dual inhibitors selective for Bcl2A1 and Mcl-1 proteins. J. Med. Chem. 2018, 61, 2962– 2972, DOI: 10.1021/acs.jmedchem.8b00010Google ScholarThere is no corresponding record for this reference.
- 34Muppidi, A.; Doi, K.; Edwardraja, S.; Drake, E. J.; Gulick, A. M.; Wang, H. G.; Lin, Q. Rational design of proteolytically stable, cell-permeable peptide-based selective Mcl-1 inhibitors. J. Am. Chem. Soc. 2012, 134, 14734– 14737, DOI: 10.1021/ja306864vGoogle Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1Ghu7vN&md5=b21e87ed6a2163e51825675eb3391e5dRational Design of Proteolytically Stable, Cell-Permeable Peptide-Based Selective Mcl-1 InhibitorsMuppidi, Avinash; Doi, Kenichiro; Edwardraja, Selvakumar; Drake, Eric J.; Gulick, Andrew M.; Wang, Hong-Gang; Lin, QingJournal of the American Chemical Society (2012), 134 (36), 14734-14737CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Direct chem. modifications provide a simple and effective means to "translate" bioactive helical peptides into potential therapeutics targeting intracellular protein-protein interactions. We previously showed that distance-matching bisaryl cross-linkers can reinforce peptide helixes contg. two cysteines at the i and i+7 positions and confer cell permeability to the cross-linked peptides. Here we report the first crystal structure of a biphenyl-cross-linked Noxa peptide in complex with its target Mcl-1 at 2.0 Å resoln. Guided by this structure, we remodeled the surface of this cross-linked peptide through side-chain substitution and N-methylation and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells.
- 35Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat. Chem. Biol. 2010, 6, 595– 601, DOI: 10.1038/nchembio.391Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXns1Gis7c%253D&md5=4cc80db931dac5360d650ac9c45334eaThe MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizerStewart, Michelle L.; Fire, Emiko; Keating, Amy E.; Walensky, Loren D.Nature Chemical Biology (2010), 6 (8), 595-601CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathol. cell survival remains a formidable but pressing challenge. Such precisely tailored compds. would serve as mol. probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of stabilized alpha-helix of BCL-2 domains (SAHBs), we detd. that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallog. and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature's soln. to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.
- 36Doi, K.; Li, R.; Sung, S. S.; Wu, H.; Liu, Y.; Manieri, W.; Krishnegowda, G.; Awwad, A.; Dewey, A.; Liu, X.; Amin, S.; Cheng, C.; Qin, Y.; Schonbrunn, E.; Daughdrill, G.; Loughran, T. P., Jr.; Sebti, S.; Wang, H. G. Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation. J. Biol. Chem. 2012, 287, 10224– 10235, DOI: 10.1074/jbc.M111.334532Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktlKrsrg%253D&md5=009ebb3b74b44ad905b502e6cb505094Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal DegradationDoi, Kenichiro; Li, Rongshi; Sung, Shen-Shu; Wu, Hongwei; Liu, Yan; Manieri, Wanda; Krishnegowda, Gowdahalli; Awwad, Andy; Dewey, Alden; Liu, Xin; Amin, Shantu; Cheng, Chunwei; Qin, Yong; Schonbrunn, Ernst; Daughdrill, Gary; Loughran, Thomas P., Jr.; Sebti, Said; Wang, Hong-GangJournal of Biological Chemistry (2012), 287 (13), 10224-10235CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small mols. have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compds. that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-XL, and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degrdn. via the proteasome system, which is assocd. with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-XL-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematol. malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degrdn.
- 37Lanning, M. E.; Yu, W.; Yap, J. L.; Chauhan, J.; Chen, L.; Whiting, E.; Pidugu, L. S.; Atkinson, T.; Bailey, H.; Li, W.; Roth, B. M.; Hynicka, L.; Chesko, K.; Toth, E. A.; Shapiro, P.; MacKerell, A. D., Jr.; Wilder, P. T.; Fletcher, S. Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein. Eur. J. Med. Chem. 2016, 113, 273– 292, DOI: 10.1016/j.ejmech.2016.02.006Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktlCitbc%253D&md5=ccf3a93af1ace19229a3e3b562e83190Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoproteinLanning, Maryanna E.; Yu, Wenbo; Yap, Jeremy L.; Chauhan, Jay; Chen, Lijia; Whiting, Ellis; Pidugu, Lakshmi S.; Atkinson, Tyler; Bailey, Hala; Li, Willy; Roth, Braden M.; Hynicka, Lauren; Chesko, Kirsty; Toth, Eric A.; Shapiro, Paul; MacKerell, Alexander D., Jr.; Wilder, Paul T.; Fletcher, StevenEuropean Journal of Medicinal Chemistry (2016), 113 (), 273-292CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-mol. inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 pocket of the protein. Significantly, target mols. were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Mol. modeling using the Site-Identification by Ligand Competitive Satn. (SILCS) approach was used to qual. direct ligand design as well as develop quant. models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions in the p2 pocket dominated affinity of the most favorable binding ligand (3bl: Ki = 31 nM). Compds. were up to 19-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 vs. Bcl-xL. The SILCS-based SAR of the present compds. represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumors and hematol. cancers, including acute myeloid leukemia.
- 38Rescourio, G.; Gonzalez, A. Z.; Jabri, S.; Belmontes, B.; Moody, G.; Whittington, D.; Huang, X.; Caenepeel, S.; Cardozo, M.; Cheng, A. C.; Chow, D.; Dou, H.; Jones, A.; Kelly, R. C.; Li, Y.; Lizarzaburu, M.; Lo, M. C.; Mallari, R.; Meleza, C.; Rew, Y.; Simonovich, S.; Sun, D.; Turcotte, S.; Yan, X.; Wong, S. G.; Yanez, E.; Zancanella, M.; Houze, J.; Medina, J. C.; Hughes, P. E.; Brown, S. P. Discovery and in vivo evaluation of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. J. Med. Chem. 2019, 62, 10258– 10271, DOI: 10.1021/acs.jmedchem.9b01310Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFKjsL3I&md5=8717b6e16a8f3084f0b5a809911c3cc9Discovery and in Vivo Evaluation of Macrocyclic Mcl-1 Inhibitors Featuring an α-Hydroxy Phenylacetic Acid Pharmacophore or BioisostereRescourio, Gwenaella; Gonzalez, Ana Z.; Jabri, Salman; Belmontes, Brian; Moody, Gordon; Whittington, Doug; Huang, Xin; Caenepeel, Sean; Cardozo, Mario; Cheng, Alan C.; Chow, David; Dou, Hannah; Jones, Adrie; Kelly, Ron C.; Li, Yihong; Lizarzaburu, Mike; Lo, Mei-Chu; Mallari, Rommel; Meleza, Cesar; Rew, Yosup; Simonovich, Scott; Sun, Daqing; Turcotte, Simon; Yan, Xuelei; Wong, Simon G.; Yanez, Evelyn; Zancanella, Manuel; Houze, Jonathan; Medina, Julio C.; Hughes, Paul E.; Brown, Sean P.Journal of Medicinal Chemistry (2019), 62 (22), 10258-10271CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Overexpression of the antiapoptotic protein Mcl-1 provides a survival advantage to some cancer cells, making inhibition of this protein an attractive therapeutic target for the treatment of certain types of tumors. Herein, we report our efforts toward the identification of a novel series of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. This work led to the discovery of 1(I), a potent Mcl-1 inhibitor (IC50 = 19 nM in an OPM-2 cell viability assay) with good pharmacokinetic properties and excellent in vivo efficacy in an OPM-2 multiple myeloma xenograft model.
- 39Song, T.; Wang, Z.; Ji, F.; Feng, Y.; Fan, Y.; Chai, G.; Li, X.; Li, Z.; Zhang, Z. Deactivation of Mcl-1 by dual-function small-molecule inhibitors targeting the bcl-2 homology 3 domain and facilitating mcl-1 ubiquitination. Angew. Chem., Int. Ed. Engl. 2016, 55, 14250– 14256, DOI: 10.1002/anie.201606543Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2svmsVyrtA%253D%253D&md5=687a3613109274c1a2fd2519cc1af45fDeactivation of Mcl-1 by Dual-Function Small-Molecule Inhibitors Targeting the Bcl-2 Homology 3 Domain and Facilitating Mcl-1 UbiquitinationSong Ting; Wang Ziqian; Chai Gaobo; Li Zhiqiang; Zhang Zhichao; Ji Fangling; Fan Yudan; Feng Yingang; Li XiangqianAngewandte Chemie (International ed. in English) (2016), 55 (46), 14250-14256 ISSN:.By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. Noxa(BH3) binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, Bim(BH3) binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.
- 40Wang, Z.; He, N.; Guo, Z.; Niu, C.; Song, T.; Guo, Y.; Cao, K.; Wang, A.; Zhu, J.; Zhang, X.; Zhang, Z. Proteolysis targeting chimeras for the selective degradation of Mcl-1/Bcl-2 derived from nonselective target binding ligands. J. Med. Chem. 2019, 62, 8152– 8163, DOI: 10.1021/acs.jmedchem.9b00919Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFemtLvN&md5=f962278d69b69117c501bc7c0c31ebfdProteolysis Targeting Chimeras for the Selective Degradation of Mcl-1/Bcl-2 Derived from Nonselective Target Binding LigandsWang, Ziqian; He, Nianzhe; Guo, Zongwei; Niu, Cuili; Song, Ting; Guo, Yafei; Cao, Keke; Wang, Anhui; Zhu, Junjie; Zhang, Xiaodong; Zhang, ZhichaoJournal of Medicinal Chemistry (2019), 62 (17), 8152-8163CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Proteolysis Targeting Chimera (PROTAC) recruits an E3 ligase to a target protein to induce its ubiquitination and subsequent degrdn. We reported the success in the development of two PROTACs (C3(I) and C5(II)) that potently and selectively induce the degrdn. of Mcl-1 and Bcl-2 (DC50 = 0.7 and 3.0 μM), resp., by introducing the E3 ligase cereblon (CRBN)-binding ligand pomalidomide to Mcl-1/Bcl-2 dual inhibitors S1-6 and Nap-1 with μM-range affinity. I-induced Mcl-1 ubiquitination translated into much more lethality in Mcl-1-dependent H23 cells than the most potent Mcl-1 occupancy-based inhibitor A-1210477 with nM-range affinity. Moreover, structure-activity relationship (SAR) anal. and mol. dynamic (MD) simulations discovered the structural basis for turning nonselective or promiscuous Bcl-2 family ligands into selective PROTACs. I and II exhibited reversible depletion in living cells, which provides a new potent toolkit for gain-of-function studies to probe the dynamic roles of Bcl-2 and Mcl-1 in apoptosis networks.
- 41Wang, Z.; Guo, Z.; Song, T.; Zhang, X.; He, N.; Liu, P.; Wang, P.; Zhang, Z. Proteome-wide identification of on- and off-targets of Bcl-2 inhibitors in native biological systems by using affinity-based probes (afbps). ChemBioChem 2018, 19, 2312– 2320, DOI: 10.1002/cbic.201800380Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFequr%252FI&md5=8ca5bbf1ae20f5ce024f72f431dd9166Proteome-Wide Identification of On- and Off-Targets of Bcl-2 Inhibitors in Native Biological Systems by Using Affinity-Based Probes (AfBPs)Wang, Ziqian; Guo, Zongwei; Song, Ting; Zhang, Xiaodong; He, Nianzhe; Liu, Peng; Wang, Peiran; Zhang, ZhichaoChemBioChem (2018), 19 (21), 2312-2320CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Selective inhibition of proteins of the Bcl-2 family by small-mol. inhibitors is a promising new approach in drug discovery. However, information about how these mols. interact with their cellular targets (on- and off-) is highly limited. We have designed and synthesized photoreactive and "clickable" affinity-based probes (AfBPs)-Nap-2 and Nap-5-by introducing photo-crosslinkers onto Nap-1, a fluorescent deriv. of small-mol. Bcl-2 inhibitor S1-6. The resulting trifunctional probes Nap-2 and Nap-5 can enrich, visualize, and enable the identification of cellular on- and off-targets of Bcl-2 inhibitors both in vitro and in situ. Tubulin was validated as an off-target of Bcl-2 inhibitors (Nap-1 and S1-6) by large-scale cell-based proteome profiling and pull-down/western blotting (PD/WB) with Nap-2 and Nap-5. It was preliminarily illustrated to be a BH3-contg. protein because some well-known Bcl-2 inhibitors can block the labeling of tubulin by Nap-2.
- 42Zhang, Z.; Wu, G.; Xie, F.; Song, T.; Chang, X. 3-Thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) based molecules as potent, dual inhibitors of B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1): structure-based design and structure-activity relationship studies. J. Med. Chem. 2011, 54, 1101– 1105, DOI: 10.1021/jm101181uGoogle Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVeiuw%253D%253D&md5=08ad63a3a7bfb4afd477f63572d2f16c3-Thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) Based Molecules as Potent, Dual Inhibitors of B-Cell Lymphoma 2 (Bcl-2) and Myeloid Cell Leukemia Sequence 1 (Mcl-1): Structure-Based Design and Structure-Activity Relationship StudiesZhang, Zhichao; Wu, Guiye; Xie, Feibo; Song, Ting; Chang, XilongJournal of Medicinal Chemistry (2011), 54 (4), 1101-1105CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)the authors recently described the discovery of a dual inhibitor of Bcl-2 and Mcl-1, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3, S1). Here the authors report a structure-guided design in combination with structure-activity relationship studies to exploit the difference in the p2 binding pocket of Bcl-2 and Mcl-1, from which a novel dual inhibitor 3-(4-aminophenylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (6h) was obtained, which showed significant enhanced IC50 value against Mcl-1 (5 nM), greater Mcl-1/Bak disruption potential, and accordingly a 10-fold increased cytotoxicity over 3.
- 43Bruncko, M.; Song, X.; Tao, Z.; Kunzer, A. R. 7-nonsubstituted Indole Mcl-1 Inhibitors. WO2008130970Al, 2008.Google ScholarThere is no corresponding record for this reference.
- 44Elmore, S. W.; Souers, A. J.; Bruncko, M.; Song, X.; Wang, X.; Hasvold, L. A.; Wang, L.; Kunzer, A. R.; Park, C.; Wendt, M. D.; Tao, Z. 7-sonsubstituted Indole Mcl-1 Inhibitors. WO2008131000A2, 2008.Google ScholarThere is no corresponding record for this reference.
- 45Friberg, A.; Vigil, D.; Zhao, B.; Daniels, R. N.; Burke, J. P.; Garcia-Barrantes, P. M.; Camper, D.; Chauder, B. A.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design. J. Med. Chem. 2013, 56, 15– 30, DOI: 10.1021/jm301448pGoogle Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCjur3J&md5=74e72c3d56cdff85d263bd974319b2dbDiscovery of Potent Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods and Structure-Based DesignFriberg, Anders; Vigil, Dominico; Zhao, Bin; Daniels, R. Nathan; Burke, Jason P.; Garcia-Barrantes, Pedro M.; Camper, DeMarco; Chauder, Brian A.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2013), 56 (1), 15-30CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chem. distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compds. that bind to Mcl-1 with a dissocn. const. of <100 nM with selectivity for Mcl-1 over Bcl-xL and Bcl-2. Structures of merged compds. when complexed to Mcl-1 were obtained by x-ray crystallog. and provide detailed information about the mol. recognition of small-mol. ligands binding Mcl-1. The compds. represent starting points for the discovery of clin. useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.
- 46Burke, J. P.; Bian, Z.; Shaw, S.; Zhao, B.; Goodwin, C. M.; Belmar, J.; Browning, C. F.; Vigil, D.; Friberg, A.; Camper, D. V.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of tricyclic indoles that potently inhibit Mcl-1 using fragment-based methods and structure-based design. J. Med. Chem. 2015, 58, 3794– 3805, DOI: 10.1021/jm501984fGoogle Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtVWkurw%253D&md5=ea5fc51491138dc7a79dd12f813c4bcaDiscovery of Tricyclic Indoles That Potently Inhibit Mcl-1 Using Fragment-Based Methods and Structure-Based DesignBurke, Jason P.; Bian, Zhiguo; Shaw, Subrata; Zhao, Bin; Goodwin, Craig M.; Belmar, Johannes; Browning, Carrie F.; Vigil, Dominico; Friberg, Anders; Camper, DeMarco V.; Rossanese, Olivia W.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2015), 58 (9), 3794-3805CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with various chemotherapies. From an NMR-based screen of a large fragment library, several distinct chem. scaffolds that bind to Mcl-1 were discovered. Here, the authors describe the discovery of potent tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100-fold selectivity over Bcl-2. X-ray structures of these compds. when complexed to Mcl-1 provide detailed information on how these small-mols. bind to the target, which was used to guide compd. optimization.
- 47Pelz, N. F.; Bian, Z.; Zhao, B.; Shaw, S.; Tarr, J. C.; Belmar, J.; Gregg, C.; Camper, D. V.; Goodwin, C. M.; Arnold, A. L.; Sensintaffar, J. L.; Friberg, A.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of 2-indole-acylsulfonamide myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods. J. Med. Chem. 2016, 59, 2054– 2066, DOI: 10.1021/acs.jmedchem.5b01660Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisVygsbs%253D&md5=e667fa5bc866d29ad02d6d52c679f47aDiscovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based MethodsPelz, Nicholas F.; Bian, Zhiguo; Zhao, Bin; Shaw, Subrata; Tarr, James C.; Belmar, Johannes; Gregg, Claire; Camper, DeMarco V.; Goodwin, Craig M.; Arnold, Allison L.; Sensintaffar, John L.; Friberg, Anders; Rossanese, Olivia W.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2016), 59 (5), 2054-2066CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins responsible for the regulation of programmed cell death. Amplification of Mcl-1 is a common genetic aberration in human cancer whose overexpression contributes to the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-apoptotic BH3 contg. proteins that achieve high affinity for the target by utilizing four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1 inhibitors using fragment-based methods and structure-based design. These novel inhibitors exhibit low nanomolar binding affinities to Mcl-1 and >500-fold selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to Mcl-1 provided detailed information on how these small-mols. bind to the target and were used extensively to guide compd. optimization.
- 48Shaw, S.; Bian, Z.; Zhao, B.; Tarr, J. C.; Veerasamy, N.; Jeon, K. O.; Belmar, J.; Arnold, A. L.; Fogarty, S. A.; Perry, E.; Sensintaffar, J. L.; Camper, D. V.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Optimization of potent and selective tricyclic indole diazepinone myeloid cell leukemia-1 inhibitors using structure-based design. J. Med. Chem. 2018, 61, 2410– 2421, DOI: 10.1021/acs.jmedchem.7b01155Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotVyktA%253D%253D&md5=719511e2b52849e2bff5cf1de6d6c2beOptimization of Potent and Selective Tricyclic Indole Diazepinone Myeloid Cell Leukemia-1 Inhibitors Using Structure-Based DesignShaw, Subrata; Bian, Zhiguo; Zhao, Bin; Tarr, James C.; Veerasamy, Nagarathanam; Jeon, Kyu Ok; Belmar, Johannes; Arnold, Allison L.; Fogarty, Stuart A.; Perry, Evan; Sensintaffar, John L.; Camper, DeMarco V.; Rossanese, Olivia W.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2018), 61 (6), 2410-2421CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, has emerged as an attractive target for cancer therapy. Mcl-1 upregulation is often found in many human cancers and is assocd. with high tumor grade, poor survival, and resistance to chemotherapy. Here, we describe a series of potent and selective tricyclic indole diazepinone Mcl-1 inhibitors that were discovered and further optimized using structure-based design. These compds. exhibit picomolar binding affinity and mechanism-based cellular efficacy, including growth inhibition and caspase induction in Mcl-1-sensitive cells. Thus, they represent useful compds. to study the implication of Mcl-1 inhibition in cancer and serve as potentially useful starting points toward the discovery of anti-Mcl-1 therapeutics.
- 49Lee, T.; Christov, P. P.; Shaw, S.; Tarr, J. C.; Zhao, B.; Veerasamy, N.; Jeon, K. O.; Mills, J. J.; Bian, Z.; Sensintaffar, J. L.; Arnold, A. L.; Fogarty, S. A.; Perry, E.; Ramsey, H. E.; Cook, R. S.; Hollingshead, M.; Davis Millin, M.; Lee, K. M.; Koss, B.; Budhraja, A.; Opferman, J. T.; Kim, K.; Arteaga, C. L.; Moore, W. J.; Olejniczak, E. T.; Savona, M. R.; Fesik, S. W. Discovery of potent myeloid cell leukemia-1 (Mcl-1) inhibitors that demonstrate in vivo activity in mouse xenograft models of human cancer. J. Med. Chem. 2019, 62, 3971– 3988, DOI: 10.1021/acs.jmedchem.8b01991Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtlWitb0%253D&md5=a3e80ec8c20cc39a71c78beb85d2ec90Discovery of Potent Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors That Demonstrate in Vivo Activity in Mouse Xenograft Models of Human CancerLee, Taekyu; Christov, Plamen P.; Shaw, Subrata; Tarr, James C.; Zhao, Bin; Veerasamy, Nagarathanam; Jeon, Kyu Ok; Mills, Jonathan J.; Bian, Zhiguo; Sensintaffar, John L.; Arnold, Allison L.; Fogarty, Stuart A.; Perry, Evan; Ramsey, Haley E.; Cook, Rebecca S.; Hollingshead, Melinda; Davis Millin, Myrtle; Lee, Kyung-min; Koss, Brian; Budhraja, Amit; Opferman, Joseph T.; Kim, Kwangho; Arteaga, Carlos L.; Moore, William J.; Olejniczak, Edward T.; Savona, Michael R.; Fesik, Stephen W.Journal of Medicinal Chemistry (2019), 62 (8), 3971-3988CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Overexpression of myeloid cell leukemia-1 (Mcl-1) in cancers correlates with high tumor grade and poor survival. Addnl., Mcl-1 drives intrinsic and acquired resistance to many cancer therapeutics, including B cell lymphoma 2 family inhibitors, proteasome inhibitors, and antitubulins. Therefore, Mcl-1 inhibition could serve as a strategy to target cancers that require Mcl-1 to evade apoptosis. Herein, we describe the use of structure-based design to discover a novel compd. (42) that robustly and specifically inhibits Mcl-1 in cell culture and animal xenograft models. Compd. 42 binds to Mcl-1 with picomolar affinity and inhibited growth of Mcl-1-dependent tumor cell lines in the nanomolar range. Compd. 42 also inhibited the growth of hematol. and triple neg. breast cancer xenografts at well-tolerated doses. These findings highlight the use of structure-based design to identify small mol. Mcl-1 inhibitors and support the use of 42 as a potential treatment strategy to block Mcl-1 activity and induce apoptosis in Mcl-1-dependent cancers.
- 50Ramsey, H. E.; Fischer, M. A.; Lee, T.; Gorska, A. E.; Arrate, M. P.; Fuller, L.; Boyd, K. L.; Strickland, S. A.; Sensintaffar, J.; Hogdal, L. J.; Ayers, G. D.; Olejniczak, E. T.; Fesik, S. W.; Savona, M. R. A novel Mcl-1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia. Cancer. Discov. 2018, 8, 1566– 1581, DOI: 10.1158/2159-8290.CD-18-0140Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFOmsLfN&md5=85fecfd85be4606dfa867193f4cf871dA novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemiaRamsey, Haley E.; Fischer, Melissa A.; Lee, Taekyu; Gorska, Agnieszka E.; Arrate, Maria Pia; Fuller, Londa; Boyd, Kelli L.; Strickland, Stephen A.; Sensintaffar, John; Hogdal, Leah J.; Ayers, Gregory D.; Olejniczak, Edward T.; Fesik, Stephen W.; Savona, Michael R.Cancer Discovery (2018), 8 (12), 1566-1581CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 assocn., leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addn., VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clin. trials.
- 51Bruncko, M.; Wang, L.; Sheppard, G. S.; Phillips, D. C.; Tahir, S. K.; Xue, J.; Erickson, S.; Fidanze, S.; Fry, E.; Hasvold, L.; Jenkins, G. J.; Jin, S.; Judge, R. A.; Kovar, P. J.; Madar, D.; Nimmer, P.; Park, C.; Petros, A. M.; Rosenberg, S. H.; Smith, M. L.; Song, X.; Sun, C.; Tao, Z. F.; Wang, X.; Xiao, Y.; Zhang, H.; Tse, C.; Leverson, J. D.; Elmore, S. W.; Souers, A. J. Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity. J. Med. Chem. 2015, 58, 2180– 2194, DOI: 10.1021/jm501258mGoogle Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXislGjt70%253D&md5=e8c575a04fecaf47e4a9fc5c8348e034Structure-Guided Design of a Series of MCL-1 Inhibitors with High Affinity and SelectivityBruncko, Milan; Wang, Le; Sheppard, George S.; Phillips, Darren C.; Tahir, Stephen K.; Xue, John; Erickson, Scott; Fidanze, Steve; Fry, Elizabeth; Hasvold, Lisa; Jenkins, Gary J.; Jin, Sha; Judge, Russell A.; Kovar, Peter J.; Madar, David; Nimmer, Paul; Park, Chang; Petros, Andrew M.; Rosenberg, Saul H.; Smith, Morey L.; Song, Xiaohong; Sun, Chaohong; Tao, Zhi-Fu; Wang, Xilu; Xiao, Yu; Zhang, Haichao; Tse, Chris; Leverson, Joel D.; Elmore, Steven W.; Souers, Andrew J.Journal of Medicinal Chemistry (2015), 58 (5), 2180-2194CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein the authors report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compds. from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compd. I rapidly induced caspase activation with assocd. loss in cell viability. The small mols. described herein thus comprise effective tools for studying MCL-1 biol.
- 52Leverson, J. D.; Zhang, H.; Chen, J.; Tahir, S. K.; Phillips, D. C.; Xue, J.; Nimmer, P.; Jin, S.; Smith, M.; Xiao, Y.; Kovar, P.; Tanaka, A.; Bruncko, M.; Sheppard, G. S.; Wang, L.; Gierke, S.; Kategaya, L.; Anderson, D. J.; Wong, C.; Eastham-Anderson, J.; Ludlam, M. J. C.; Sampath, D.; Fairbrother, W. J.; Wertz, I.; Rosenberg, S. H.; Tse, C.; Elmore, S. W.; Souers, A. J. Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax). Cell. Death. Dis. 2015, 6, e1590 DOI: 10.1038/cddis.2014.561Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFGrurg%253D&md5=8ceb779fc6868fc0828757364d529e34Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax)Leverson, J. D.; Zhang, H.; Chen, J.; Tahir, S. K.; Phillips, D. C.; Xue, J.; Nimmer, P.; Jin, S.; Smith, M.; Xiao, Y.; Kovar, P.; Tanaka, A.; Bruncko, M.; Sheppard, G. S.; Wang, L.; Gierke, S.; Kategaya, L.; Anderson, D. J.; Wong, C.; Eastham-Anderson, J.; Ludlam, M. J. C.; Sampath, D.; Fairbrother, W. J.; Wertz, I.; Rosenberg, S. H.; Tse, C.; Elmore, S. W.; Souers, A. J.Cell Death & Disease (2015), 6 (1), e1590CODEN: CDDEA4; ISSN:2041-4889. (Nature Publishing Group)The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-mol. BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small mols. potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compd. A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue expts. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-mol. MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these mols. as chem. tools for dissecting the basic biol. of MCL-1 and the promise of small-mol. MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
- 53Xiao, Y.; Nimmer, P.; Sheppard, G. S.; Bruncko, M.; Hessler, P.; Lu, X.; Roberts-Rapp, L.; Pappano, W. N.; Elmore, S. W.; Souers, A. J.; Leverson, J. D.; Phillips, D. C. Mcl-1 is a key determinant of breast cancer cell survival: validation of Mcl-1 dependency utilizing a highly selective small molecule inhibitor. Mol. Cancer Ther. 2015, 14, 1837– 1847, DOI: 10.1158/1535-7163.MCT-14-0928Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1yrs7vL&md5=c6d4077d4e1f1feb92d2fa22b88ab2fbMCL-1 Is a Key Determinant of Breast Cancer Cell Survival: Validation of MCL-1 Dependency Utilizing a Highly Selective Small Molecule InhibitorXiao, Yu; Nimmer, Paul; Sheppard, George S.; Bruncko, Milan; Hessler, Paul; Lu, Xin; Roberts-Rapp, Lisa; Pappano, William N.; Elmore, Steven W.; Souers, Andrew J.; Leverson, Joel D.; Phillips, Darren C.Molecular Cancer Therapeutics (2015), 14 (8), 1837-1847CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Hyperexpression of antiapoptotic BCL-2 family proteins allows cells to survive despite the receipt of signals that would ordinarily induce their deletion, a facet frequently exploited by tumors. Tumors addicted to the BCL-2 family proteins for survival are now being targeted therapeutically. For example, navitoclax, a BCL-2/BCL-XL/BCL-W inhibitor, is currently in phase I/II clin. trials in numerous malignancies. However, the related family member, MCL-1, limits the efficacy of navitoclax and other chemotherapeutic agents. In the present study, we identify breast cancer cell lines that depend upon MCL-1 for survival and subsequently det. the mechanism of apoptosis mediated by the MCL-1 selective inhibitor A-1210477. We demonstrate that apoptosis resulting from a loss in MCL-1 function requires expression of the proapoptotic protein BAK. However, expression of BCL-XL can limit apoptosis resulting from loss in MCL-1 function through sequestration of free BIM. Finally, we demonstrate substantial synergy between navitoclax and MCL-1 siRNA, the direct MCL-1 inhibitor A-1210477, or the indirect MCL-1 inhibitor flavopiridol, highlighting the therapeutic potential for inhibiting BCL-XL and MCL-1 in breast cancer. Mol Cancer Ther; 14(8); 1837-47. cpr2015 AACR.
- 54Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chain. Nat. Chem. Biol. 2016, 12, 931– 936, DOI: 10.1038/nchembio.2174Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKqtb3P&md5=bda5720579a9b5e6027632d2cffba2e1Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chainAkcay, Gizem; Belmonte, Matthew A.; Aquila, Brian; Chuaqui, Claudio; Hird, Alexander W.; Lamb, Michelle L.; Rawlins, Philip B.; Su, Nancy; Tentarelli, Sharon; Grimster, Neil P.; Su, QibinNature Chemical Biology (2016), 12 (11), 931-936CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Targeted covalent inhibition of disease-assocd. proteins has become a powerful methodol. in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here the authors used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to the knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chem. strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochem. and cell-based assays. The authors identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biol. phenomena.
- 55Papatzimas, J. W.; Gorobets, E.; Maity, R.; Muniyat, M. I.; MacCallum, J. L.; Neri, P.; Bahlis, N. J.; Derksen, D. J. From inhibition to degradation: targeting the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1). J. Med. Chem. 2019, 62, 5522– 5540, DOI: 10.1021/acs.jmedchem.9b00455Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVSnsb%252FL&md5=4a28b945723e00cf9a2ef3916a07da42From Inhibition to Degradation: Targeting the Antiapoptotic Protein Myeloid Cell Leukemia 1 (MCL1)Papatzimas, James W.; Gorobets, Evgueni; Maity, Ranjan; Muniyat, Mir Ishruna; MacCallum, Justin L.; Neri, Paola; Bahlis, Nizar J.; Derksen, Darren J.Journal of Medicinal Chemistry (2019), 62 (11), 5522-5540CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Protein-protein interactions (PPIs) have emerged as significant targets for therapeutic development, owing to their crit. nature in diverse biol. processes. An ideal PPI-based target is the protein myeloid cell leukemia 1 (MCL1), a crit. prosurvival factor in cancers such as multiple myeloma where MCL1 levels directly correlate to disease progression. Current strategies for halting the antiapoptotic properties of MCL1 revolve around inhibiting its sequestration of proapoptotic factors. Existing inhibitors disrupt endogenous regulatory proteins; however, this strategy actually increases MCL1 protein levels. Here, the authors show the development of hetero-bifunctional small mols. capable of selectively targeting MCL1 using a proteolysis targeting chimera (PROTAC) methodol. leading to successful degrdn. The authors confirmed the involvement of the E3 ligase CUL4A-DDB1 cereblon ubiquitination pathway, making these PROTACs a first step toward a new class of antiapoptotic B-cell lymphoma 2 family protein degraders.
- 56Johannes, J. W.; Bates, S.; Beigie, C.; Belmonte, M. A.; Breen, J.; Cao, S.; Centrella, P. A.; Clark, M. A.; Cuozzo, J. W.; Dumelin, C. E.; Ferguson, A. D.; Habeshian, S.; Hargreaves, D.; Joubran, C.; Kazmirski, S.; Keefe, A. D.; Lamb, M. L.; Lan, H.; Li, Y.; Ma, H.; Mlynarski, S.; Packer, M. J.; Rawlins, P. B.; Robbins, D. W.; Shen, H.; Sigel, E. A.; Soutter, H. H.; Su, N.; Troast, D. M.; Wang, H.; Wickson, K. F.; Wu, C.; Zhang, Y.; Zhao, Q.; Zheng, X.; Hird, A. W. Structure based design of non-natural peptidic macrocyclic Mcl-1 inhibitors. ACS Med. Chem. Lett. 2017, 8, 239– 244, DOI: 10.1021/acsmedchemlett.6b00464Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFCqtLzP&md5=42f9a8d863aedfe95231e29ffa4673b8Structure based design of non-natural peptidic macrocyclic Mcl-1 inhibitorsJohannes, Jeffrey W.; Bates, Stephanie; Beigie, Carl; Belmonte, Matt; Breen, John; Cao, Shenggen; Centrella, Paolo A.; Clark, Matthew A.; Cuozzo, John W.; Dumelin, Christoph E.; Ferguson, Andrew D.; Habeshian, Sevan; Hargreaves, David; Joubran, Camil; Kazmirski, Steven; Keefe, Anthony D.; Lamb, Michelle L.; Lan, Haiye; Li, Yunxia; Ma, Hao; Mlynarski, Scott; Packer, Martin J.; Rawlins, Philip B.; Robbins, Daniel W.; Shen, Haidong; Sigel, Eric A.; Soutter, Holly H.; Su, Nancy; Troast, Dawn M.; Wang, Haiyun; Wickson, Kate F.; Wu, Chengyan; Zhang, Ying; Zhao, Qiuying; Zheng, Xiaolan; Hird, Alexander W.ACS Medicinal Chemistry Letters (2017), 8 (2), 239-244CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compd. (I), a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compd. I bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approx. 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to addnl. potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compd. (II) is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4-11 cells with an IC50 of 3 μM after 6 h.
- 57Tron, A. E.; Belmonte, M. A.; Adam, A.; Aquila, B. M.; Boise, L. H.; Chiarparin, E.; Cidado, J.; Embrey, K. J.; Gangl, E.; Gibbons, F. D.; Gregory, G. P.; Hargreaves, D.; Hendricks, J. A.; Johannes, J. W.; Johnstone, R. W.; Kazmirski, S. L.; Kettle, J. G.; Lamb, M. L.; Matulis, S. M.; Nooka, A. K.; Packer, M. J.; Peng, B.; Rawlins, P. B.; Robbins, D. W.; Schuller, A. G.; Su, N.; Yang, W.; Ye, Q.; Zheng, X.; Secrist, J. P.; Clark, E. A.; Wilson, D. M.; Fawell, S. E.; Hird, A. W. Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia. Nat. Commun. 2018, 9, 5341, DOI: 10.1038/s41467-018-07551-wGoogle Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFersLjK&md5=10ccf1fda947aca212bede3927eaabfaDiscovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemiaTron, Adriana E.; Belmonte, Matthew A.; Adam, Ammar; Aquila, Brian M.; Boise, Lawrence H.; Chiarparin, Elisabetta; Cidado, Justin; Embrey, Kevin J.; Gangl, Eric; Gibbons, Francis D.; Gregory, Gareth P.; Hargreaves, David; Hendricks, J. Adam; Johannes, Jeffrey W.; Johnstone, Ricky W.; Kazmirski, Steven L.; Kettle, Jason G.; Lamb, Michelle L.; Matulis, Shannon M.; Nooka, Ajay K.; Packer, Martin J.; Peng, Bo; Rawlins, Philip B.; Robbins, Daniel W.; Schuller, Alwin G.; Su, Nancy; Yang, Wenzhan; Ye, Qing; Zheng, Xiaolan; Secrist, J. Paul; Clark, Edwin A.; Wilson, David M.; Fawell, Stephen E.; Hird, Alexander W.Nature Communications (2018), 9 (1), 5341CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic mol. with high selectivity and affinity for Mcl-1 currently in clin. development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clin. trial has been launched for evaluation of AZD5991 in patients with hematol. malignancies (NCT03218683).
- 58Sale, M. J.; Minihane, E.; Monks, N. R.; Gilley, R.; Richards, F. M.; Schifferli, K. P.; Andersen, C. L.; Davies, E. J.; Vicente, M. A.; Ozono, E.; Markovets, A.; Dry, J. R.; Drew, L.; Flemington, V.; Proia, T.; Jodrell, D. I.; Smith, P. D.; Cook, S. J. Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors. Nat. Commun. 2019, 10, 5167, DOI: 10.1038/s41467-019-12409-wGoogle Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mjpt1CqtQ%253D%253D&md5=5486bbf53a2adc4521226cd45a76daefTargeting melanoma's MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitorsSale Matthew J; Minihane Emma; Gilley Rebecca; Ozono Eiko; Cook Simon J; Monks Noel R; Schifferli Kevin P; Richards Frances M; Jodrell Duncan I; Andersen Courtney L; Markovets Aleksandra; Dry Jonathan R; Drew Lisa; Proia Theresa; Davies Emma J; Flemington Vikki; Smith Paul D; Vicente Mario AladrenNature communications (2019), 10 (1), 5167 ISSN:.BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.
- 59Zhang, H.; Nakauchi, Y.; Köhnke, T.; Stafford, M.; Bottomly, D.; Thomas, R.; Wilmot, B.; McWeeney, S. K.; Majeti, R.; Tyner, J. W. Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia. Nat. Cancer. 2020, 1, 826– 839, DOI: 10.1038/s43018-020-0103-xGoogle Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7otFGitA%253D%253D&md5=44f856de2ba9280f69683c24373c39fcIntegrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemiaZhang Haijiao; Tyner Jeffrey W; Nakauchi Yusuke; Kohnke Thomas; Stafford Melissa; Thomas Rozario; Majeti Ravindra; Bottomly Daniel; Wilmot Beth; McWeeney Shannon KNature cancer (2020), 1 (8), 826-839 ISSN:.Deregulation of the BCL2 gene family plays an important role in the pathogenesis of acute myeloid leukemia (AML). The BCL2 inhibitor, venetoclax, has received FDA approval for the treatment of AML. However, upfront and acquired drug resistance ensues due, in part, to the clinical and genetic heterogeneity of AML, highlighting the importance of identifying biomarkers to stratify patients onto the most effective therapies. By integrating clinical characteristics, exome and RNA sequencing, and inhibitor data from primary AML patient samples, we determined that myelomonocytic leukemia, upregulation of BCL2A1 and CLEC7A, as well as mutations of PTPN11 and KRAS conferred resistance to venetoclax and multiple venetoclax combinations. Venetoclax in combination with an MCL1 inhibitor AZD5991 induced synthetic lethality and circumvented venetoclax resistance.
- 60Caenepeel, S.; Brown, S. P.; Belmontes, B.; Moody, G.; Keegan, K. S.; Chui, D.; Whittington, D. A.; Huang, X.; Poppe, L.; Cheng, A. C.; Cardozo, M.; Houze, J.; Li, Y.; Lucas, B.; Paras, N. A.; Wang, X.; Taygerly, J. P.; Vimolratana, M.; Zancanella, M.; Zhu, L.; Cajulis, E.; Osgood, T.; Sun, J.; Damon, L.; Egan, R. K.; Greninger, P.; McClanaghan, J. D.; Gong, J.; Moujalled, D.; Pomilio, G.; Beltran, P.; Benes, C. H.; Roberts, A. W.; Huang, D. C.; Wei, A.; Canon, J.; Coxon, A.; Hughes, P. E. AMG 176, a selective Mcl-1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapies. Cancer. Discov. 2018, 8, 1582– 1597, DOI: 10.1158/2159-8290.CD-18-0387Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFOmsb7E&md5=795a713908c3635bbd5e9fc3d95f3ae0AMG 176, a selective MCL1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapiesCaenepeel, Sean; Brown, Sean P.; Belmontes, Brian; Moody, Gordon; Keegan, Kathleen S.; Chui, Danny; Whittington, Douglas A.; Huang, Xin; Poppe, Leszek; Cheng, Alan C.; Cardozo, Mario; Houze, Jonathan; Li, Yunxiao; Lucas, Brian; Paras, Nick A.; Wang, Xianghong; Taygerly, Joshua P.; Vimolratana, Marc; Zancanella, Manuel; Zhu, Liusheng; Cajulis, Elaina; Osgood, Tao; Sun, Jan; Damon, Leah; Egan, Regina K.; Greninger, Patricia; McClanaghan, Joseph D.; Gong, Jianan; Moujalled, Donia; Pomilio, Giovanna; Beltran, Pedro; Benes, Cyril H.; Roberts, Andrew W.; Huang, David C.; Wei, Andrew; Canon, Jude; Coxon, Angela; Hughes, Paul E.Cancer Discovery (2018), 8 (12), 1582-1597CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges assocd. with inhibition of MCL1 protein-protein interactions, we rigorously applied small-mol. conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematol. cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by redns. in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics.
- 61Lu, X.; Liu, Y. C.; Orvig, C.; Liang, H.; Chen, Z. F. Discovery of a Copper-Based Mcl-1 Inhibitor as an Effective Antitumor Agent. J. Med. Chem. 2020, 63, 9154– 9167, DOI: 10.1021/acs.jmedchem.9b02047Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Sku7zF&md5=cb3ffc5ac05fb8dc509c2d82d730faf7Discovery of a Copper-Based Mcl-1 Inhibitor as an Effective Antitumor AgentLu, Xing; Liu, Yan-Cheng; Orvig, Chris; Liang, Hong; Chen, Zhen-FengJournal of Medicinal Chemistry (2020), 63 (17), 9154-9167CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), which belongs to the Bcl-2 family of prosurvival proteins, is a key regulator of cancer cell survival. To date, few drug-like Mcl-1 inhibitors have been reported. Herein, it is reported the prepn. of 10 copper complexes with 9-substituted β-carboline ligands that act as metal-based Mcl-1 inhibitors. Complex (I) was identified as a potent and selective Mcl-1 inhibitor with strong in vitro antitumor activity. Mechanistic studies demonstrated that I disrupted Mcl-1-Bax/Bak heterodimerization and induced Bax/Bak-dependent apoptosis. In addn., I significantly (P < 0.001) inhibited tumor growth in vivo, induced tumor necrosis, and extended survival time in an NCI-H460 xenograft model. Furthermore, I showed no apparent toxicity in mice. Together, these findings indicate that I is a copper-based Mcl-1 inhibitor with high efficacy and low toxicity that could be developed for the treatment of Mcl-1-related cancers.
- 62Abulwerdi, F.; Liao, C.; Liu, M.; Azmi, A. S.; Aboukameel, A.; Mady, A. S.; Gulappa, T.; Cierpicki, T.; Owens, S.; Zhang, T.; Sun, D.; Stuckey, J. A.; Mohammad, R. M.; Nikolovska-Coleska, Z. A novel small-molecule inhibitor of Mcl-1 blocks pancreatic cancer growth in vitro and in vivo. Mol. Cancer Ther. 2014, 13, 565– 575, DOI: 10.1158/1535-7163.MCT-12-0767Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjslGisL8%253D&md5=8f1686d2e7ddc6e1aa59dcfb902176bbA Novel Small-Molecule Inhibitor of Mcl-1 Blocks Pancreatic Cancer Growth In Vitro and In VivoAbulwerdi, Fardokht; Liao, Chenzhong; Liu, Meilan; Azmi, Asfar S.; Aboukameel, Amro; Mady, Ahmed S. A.; Gulappa, Thippeswamy; Cierpicki, Tomasz; Owens, Scott; Zhang, Tao; Sun, Duxin; Stuckey, Jeanne A.; Mohammad, Ramzi M.; Nikolovska-Coleska, ZanetaMolecular Cancer Therapeutics (2014), 13 (3), 565-575CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Using a high-throughput screening (HTS) approach, we have identified and validated several small-mol. Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chem. modifications of the lead compd. 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochem., biophys., and computational methods and detd. its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunopptn. expts. revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot anal. in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclin. investigations. Mol Cancer Ther; 13(3); 565-75. 2013 AACR.
- 63Lee, T.; Bian, Z.; Zhao, B.; Hogdal, L. J.; Sensintaffar, J. L.; Goodwin, C. M.; Belmar, J.; Shaw, S.; Tarr, J. C.; Veerasamy, N.; Matulis, S. M.; Koss, B.; Fischer, M. A.; Arnold, A. L.; Camper, D. V.; Browning, C. F.; Rossanese, O. W.; Budhraja, A.; Opferman, J.; Boise, L. H.; Savona, M. R.; Letai, A.; Olejniczak, E. T.; Fesik, S. W. Discovery and biological characterization of potent myeloid cell leukemia-1 inhibitors. FEBS Lett. 2017, 591, 240– 251, DOI: 10.1002/1873-3468.12497Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFSlsr%252FP&md5=16ad6c7e0fe49b8eaa20185676284eafDiscovery and biological characterization of potent myeloid cell leukemia-1 inhibitorsLee, Taekyu; Bian, Zhiguo; Zhao, Bin; Hogdal, Leah J.; Sensintaffar, John L.; Goodwin, Craig M.; Belmar, Johannes; Shaw, Subrata; Tarr, James C.; Veerasamy, Nagarathanam; Matulis, Shannon M.; Koss, Brian; Fischer, Melissa A.; Arnold, Allison L.; Camper, DeMarco V.; Browning, Carrie F.; Rossanese, Olivia W.; Budhraja, Amit; Opferman, Joseph; Boise, Lawrence H.; Savona, Michael R.; Letai, Anthony; Olejniczak, Edward T.; Fesik, Stephen W.FEBS Letters (2017), 591 (1), 240-251CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is assocd. with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, the authors describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compds. represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clin. useful Mcl-1 inhibitors.
- 64Lu, X.; Liu, Y. C.; Orvig, C.; Liang, H.; Chen, Z. F. Discovery of β-carboline copper(II) complexes as Mcl-1 inhibitor and in vitro and in vivo activity in cancer models. Eur. J. Med. Chem. 2019, 181, 111567, DOI: 10.1016/j.ejmech.2019.111567Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFKksbnL&md5=54f0a728016fdfeb0752418ad4386209Discovery of β-carboline copper(II) complexes as Mcl-1 inhibitor and in vitro and in vivo activity in cancer modelsLu, Xing; Liu, Yan-Cheng; Orvig, Chris; Liang, Hong; Chen, Zhen-FengEuropean Journal of Medicinal Chemistry (2019), 181 (), 111567CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Mcl-1 is an anti-apoptotic member of Bcl-2 family proteins. The development of inhibitors of Mcl-1 has been challenging. To develop metal-based Mcl-1 inhibitors, twenty two copper(II) complexes 25-46 with 9-substituted β-carboline derivs. were reported. Complexes 38 and 39 showed higher cytotoxicity than the corresponding ligands or cisplatin. The most potent complex 39 presented higher selectivity to Mcl-1 than other Bcl-2 family proteins, and killed cancer cells via Bax/Bak mediated apoptosis. Complex 39 showed an excellent safety profile in mouse model, and significantly inhibited the tumor growth in NCI-H460 tumor bearing model, which is more potent than AZD5991 at the same dosage. Complex 39 prolonged the survival time of the tumor bearing mice. Complex 39 is the first metal-based Mcl-1 inhibitor acting as a potential anticancer agent.
- 65Richard, D. J.; Lena, R.; Bannister, T.; Blake, N.; Pierceall, W. E.; Carlson, N. E.; Keller, C. E.; Koenig, M.; He, Y.; Minond, D.; Mishra, J.; Cameron, M.; Spicer, T.; Hodder, P.; Cardone, M. H. Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker. Bioorg. Med. Chem. 2013, 21, 6642– 6649, DOI: 10.1016/j.bmc.2013.08.017Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtlGrurbJ&md5=a2875b8bc8fce034d17def9920dd31a4Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarkerRichard, David J.; Lena, Ryan; Bannister, Thomas; Blake, Noel; Pierceall, William E.; Carlson, Nicole E.; Keller, Christina Eberhart; Koenig, Marcel; He, Yuanjun; Minond, Dmitriy; Mishra, Jitendra; Cameron, Michael; Spicer, Timothy; Hodder, Peter; Cardone, Michael H.Bioorganic & Medicinal Chemistry (2013), 21 (21), 6642-6649CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Anti-apoptotic Bcl-2 family proteins are important oncol. therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One obsd. mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small mol. inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compds. has been challenging due to difficulties in translating the target selectivity obsd. at the biochem. level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compds. identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compds. at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compds. that genuinely target those proteins. The identification of compd. 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.
- 66Petros, A. M.; Swann, S. L.; Song, D.; Swinger, K.; Park, C.; Zhang, H.; Wendt, M. D.; Kunzer, A. R.; Souers, A. J.; Sun, C. Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein. Bioorg. Med. Chem. Lett. 2014, 24, 1484– 1488, DOI: 10.1016/j.bmcl.2014.02.010Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjtl2htbk%253D&md5=e3e7de08b4f23cfa0578ad228659f9ebFragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 proteinPetros, Andrew M.; Swann, Steven L.; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D.; Kunzer, Aaron R.; Souers, Andrew J.; Sun, ChaohongBioorganic & Medicinal Chemistry Letters (2014), 24 (6), 1484-1488CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is through overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biol. to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chem. optimization efforts.
- 67Kotschy, A.; Szlavik, Z.; Murray, J.; Davidson, J.; Maragno, A. L.; Le Toumelin-Braizat, G.; Chanrion, M.; Kelly, G. L.; Gong, J. N.; Moujalled, D. M.; Bruno, A.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos, S.; Radics, G.; Proszenyak, A.; Balint, B.; Ondi, L.; Blasko, G.; Robertson, A.; Surgenor, A.; Dokurno, P.; Chen, I.; Matassova, N.; Smith, J.; Pedder, C.; Graham, C.; Studeny, A.; Lysiak-Auvity, G.; Girard, A. M.; Grave, F.; Segal, D.; Riffkin, C. D.; Pomilio, G.; Galbraith, L. C.; Aubrey, B. J.; Brennan, M. S.; Herold, M. J.; Chang, C.; Guasconi, G.; Cauquil, N.; Melchiore, F.; Guigal-Stephan, N.; Lockhart, B.; Colland, F.; Hickman, J. A.; Roberts, A. W.; Huang, D. C.; Wei, A. H.; Strasser, A.; Lessene, G.; Geneste, O. The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature 2016, 538, 477– 482, DOI: 10.1038/nature19830Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2srjs1aquw%253D%253D&md5=c435a7621b84063133e41e0cdbad3ac8The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer modelsKotschy Andras; Szlavik Zoltan; Csekei Marton; Paczal Attila; Szabo Zoltan B; Sipos Szabolcs; Radics Gabor; Proszenyak Agnes; Balint Balazs; Ondi Levente; Blasko Gabor; Murray James; Davidson James; Robertson Alan; Surgenor Allan; Dokurno Pawel; Chen Ijen; Matassova Natalia; Smith Julia; Pedder Christopher; Graham Christopher; Maragno Ana Leticia; Le Toumelin-Braizat Gaetane; Chanrion Maia; Bruno Alain; Studeny Aurelie; Lysiak-Auvity Gaelle; Girard Anne-Marie; Grave Fabienne; Guasconi Ghislaine; Cauquil Nicolas; Colland Frederic; Hickman John A; Geneste Olivier; Kelly Gemma L; Gong Jia-Nan; Segal David; Riffkin Chris D; Galbraith Laura C A; Aubrey Brandon J; Brennan Margs S; Herold Marco J; Chang Catherine; Roberts Andrew W; Huang David C S; Strasser Andreas; Lessene Guillaume; Kelly Gemma L; Gong Jia-Nan; Segal David; Riffkin Chris D; Galbraith Laura C A; Aubrey Brandon J; Brennan Margs S; Herold Marco J; Chang Catherine; Roberts Andrew W; Huang David C S; Strasser Andreas; Lessene Guillaume; Moujalled Donia M; Pomilio Giovanna; Wei Andrew H; Aubrey Brandon J; Roberts Andrew W; Melchiore Fabien; Guigal-Stephan Nolwen; Lockhart Brian; Roberts Andrew W; Wei Andrew H; Lessene GuillaumeNature (2016), 538 (7626), 477-482 ISSN:.Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.
- 68Szlavik, Z.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos, S.; Radics, G.; Proszenyak, A.; Balint, B.; Murray, J.; Davidson, J.; Chen, I.; Dokurno, P.; Surgenor, A. E.; Daniels, Z. M.; Hubbard, R. E.; Le Toumelin-Braizat, G.; Claperon, A.; Lysiak-Auvity, G.; Girard, A. M.; Bruno, A.; Chanrion, M.; Colland, F.; Maragno, A. L.; Demarles, D.; Geneste, O.; Kotschy, A. Discovery of S64315, a potent and selective Mcl-1 inhibitor. J. Med. Chem. 2020, 63, 13762– 13795, DOI: 10.1021/acs.jmedchem.0c01234Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Grs7jI&md5=adb0242cb3292fca804f9e6cb0448c67Discovery of S64315, a Potent and Selective Mcl-1 InhibitorSzlavik, Zoltan; Csekei, Marton; Paczal, Attila; Szabo, Zoltan B.; Sipos, Szabolcs; Radics, Gabor; Proszenyak, Agnes; Balint, Balazs; Murray, James; Davidson, James; Chen, Ijen; Dokurno, Pawel; Surgenor, Allan E.; Daniels, Zoe Marie; Hubbard, Roderick E.; Le Toumelin-Braizat, Gaetane; Claperon, Audrey; Lysiak-Auvity, Gaelle; Girard, Anne-Marie; Bruno, Alain; Chanrion, Maia; Colland, Frederic; Maragno, Ana-Leticia; Demarles, Didier; Geneste, Olivier; Kotschy, AndrasJournal of Medicinal Chemistry (2020), 63 (22), 13762-13795CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is assocd. with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clin. candidate S64315, a selective small mol. inhibitor of Mcl-1. Starting from a fragment derived lead compd., we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compds. against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclin. candidate has drug-like properties that have enabled its development and entry into clin. trials.
- 69Zhu, P. J.; Yu, Z. Z.; You, Q. D.; Jiang, Z. Y. Myeloid cell leukemin-1 inhibitors: a growing arsenal for cancer therapy. Drug Discovery Today 2020, 25, 1873– 1882, DOI: 10.1016/j.drudis.2020.07.021Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGmu7jK&md5=544dba0e7990594db20b8e9a8e9e010bMyeloid cell leukemin-1 inhibitors and a growing arsenal for cancer therapyZhu, Peng-Ju; Yu, Ze-Zhou; You, Qi-Dong; Jiang, Zheng-YuDrug Discovery Today (2020), 25 (10), 1873-1882CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)A Review. B-cell lymphoma-2 (Bcl-2) family proteins, comprising proapoptotic proteins (Bax and Bak), antiapoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) and BCL-2 homol. domain 3 (BH3)-only proteins (Bid, Noxa, and Puma), have long been identified as pivotal apoptosis regulators. As an antiapoptotic member, myeloid cell leukemin-1 (Mcl-1) can bind with proapoptotic proteins and inhibit apoptosis. Mcl-1 is frequently overexpressed and closely assocd. with oncogenesis and poor prognosis in several cancers, posing a tremendous obstacle for cancer therapy. Recently, an increasing no. of Mcl-1-selective small-mol. inhibitors have entered preclin. studies and advanced into clin. trials. In this review, we briefly introduce the role of Mcl-1 in apoptosis and highlight the recent development of Mcl-1 small-mol. inhibitors.
- 70Li, X.; Dou, J.; You, Q.; Jiang, Z. Inhibitors of BCL2A1/Bfl-1 protein: Potential stock in cancer therapy. Eur. J. Med. Chem. 2021, 220, 113539, DOI: 10.1016/j.ejmech.2021.113539Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFGlt7zE&md5=cd2472d31fd571818796cbd209ad63e2Inhibitors of BCL2A1/Bfl-1 protein: Potential stock in cancer therapyLi, Xue; Dou, Junwei; You, Qidong; Jiang, ZhengyuEuropean Journal of Medicinal Chemistry (2021), 220 (), 113539CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)The Bcl-2 family members rigorously regulate cell endogenous apoptosis, and targeting anti-apoptotic members is a hot topic in design of anti-cancer drugs. At present, FDA and EMA have approved Bcl-2 inhibitor Venetoclax (ABT-199) for treating chronic lymphocytic leukemia (CLL). However, inhibitors of anti-apoptotic protein BCL2A1/Bfl-1 have not been vigorously developed, and no mol. with ideal activity and selectivity has been found yet. Here we review the biol. function and protein structure of Bfl-1, discuss the therapeutic potential and list the currently reported inhibitory peptides and small mols. This will provide a ref. for Bfl-1 targeting drug discovery in the future.
- 71Chang, J.; Wang, Y.; Shao, L.; Laberge, R. M.; Demaria, M.; Campisi, J.; Janakiraman, K.; Sharpless, N. E.; Ding, S.; Feng, W.; Luo, Y.; Wang, X.; Aykin-Burns, N.; Krager, K.; Ponnappan, U.; Hauer-Jensen, M.; Meng, A.; Zhou, D. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat. Med. 2016, 22, 78– 83, DOI: 10.1038/nm.4010Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVWqtrrM&md5=6b8ae21d47f74e00e0522a1a60529bb6Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in miceChang, Jianhui; Wang, Yingying; Shao, Lijian; Laberge, Remi-Martin; Demaria, Marco; Campisi, Judith; Janakiraman, Krishnamurthy; Sharpless, Norman E.; Ding, Sheng; Feng, Wei; Luo, Yi; Wang, Xiaoyan; Aykin-Burns, Nukhet; Krager, Kimberly; Ponnappan, Usha; Hauer-Jensen, Martin; Meng, Aimin; Zhou, DaohongNature Medicine (New York, NY, United States) (2016), 22 (1), 78-83CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradn. (TBI). Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-assocd. disorders, suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacol. agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of compds. and identified ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent senolytic drug. We show that ABT263 selectively kills SCs in culture in a cell type- and species-independent manner by inducing apoptosis. Oral administration of ABT263 to either sublethally irradiated or normally aged mice effectively depleted SCs, including senescent bone marrow hematopoietic stem cells (HSCs) and senescent muscle stem cells (MuSCs). Notably, this depletion mitigated TBI-induced premature aging of the hematopoietic system and rejuvenated the aged HSCs and MuSCs in normally aged mice. Our results demonstrate that selective clearance of SCs by a pharmacol. agent is beneficial in part through its rejuvenation of aged tissue stem cells. Thus, senolytic drugs may represent a new class of radiation mitigators and anti-aging agents.
- 72Munkhbaatar, E.; Dietzen, M.; Agrawal, D.; Anton, M.; Jesinghaus, M.; Boxberg, M.; Pfarr, N.; Bidola, P.; Uhrig, S.; Höckendorf, U.; Meinhardt, A. L.; Wahida, A.; Heid, I.; Braren, R.; Mishra, R.; Warth, A.; Muley, T.; Poh, P. S. P.; Wang, X.; Fröhling, S.; Steiger, K.; Slotta-Huspenina, J.; van Griensven, M.; Pfeiffer, F.; Lange, S.; Rad, R.; Spella, M.; Stathopoulos, G. T.; Ruland, J.; Bassermann, F.; Weichert, W.; Strasser, A.; Branca, C.; Heikenwalder, M.; Swanton, C.; McGranahan, N.; Jost, P. J. MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically. Nat. Commun. 2020, 11, 4527, DOI: 10.1038/s41467-020-18372-1Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFSitbjE&md5=ca1e26757966980a2bab01fc82448788MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeuticallyMunkhbaatar, Enkhtsetseg; Dietzen, Michelle; Agrawal, Deepti; Anton, Martina; Jesinghaus, Moritz; Boxberg, Melanie; Pfarr, Nicole; Bidola, Pidassa; Uhrig, Sebastian; Hoeckendorf, Ulrike; Meinhardt, Anna-Lena; Wahida, Adam; Heid, Irina; Braren, Rickmer; Mishra, Ritu; Warth, Arne; Muley, Thomas; Poh, Patrina S. P.; Wang, Xin; Froehling, Stefan; Steiger, Katja; Slotta-Huspenina, Julia; van Griensven, Martijn; Pfeiffer, Franz; Lange, Sebastian; Rad, Roland; Spella, Magda; Stathopoulos, Georgios T.; Ruland, Juergen; Bassermann, Florian; Weichert, Wilko; Strasser, Andreas; Branca, Caterina; Heikenwalder, Mathias; Swanton, Charles; McGranahan, Nicholas; Jost, Philipp J.Nature Communications (2020), 11 (1), 4527CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Evasion of programmed cell death represents a crit. form of oncogene addiction in cancer cells. Understanding the mol. mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a mol. basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly assocd. with subclonal gains of MCL-1. In mice, tumor progression is delayed upon pharmacol. or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.
- 73Liu, J.; Fang, Z.; Zhang, Q.; Liu, Q.; Bi, X. Silver-catalyzed isocyanide-alkyne cycloaddition: a general and practical method to oligosubstituted pyrroles. Angew. Chem. Int. Ed. Engl. 2013, 52, 6953– 6957, DOI: 10.1002/anie.201302024Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3snhtlSlsw%253D%253D&md5=b5653c2a15e2fffaeeaa98cff3bf08d6Silver-catalyzed isocyanide-alkyne cycloaddition: a general and practical method to oligosubstituted pyrrolesLiu Jianquan; Fang Zhongxue; Zhang Qian; Liu Qun; Bi XiheAngewandte Chemie (International ed. in English) (2013), 52 (27), 6953-7 ISSN:.There is no expanded citation for this reference.
- 74Kattamuri, P. V.; Yin, J.; Siriwongsup, S.; Kwon, D. H.; Ess, D. H.; Li, Q.; Li, G.; Yousufuddin, M.; Richardson, P. F.; Sutton, S. C.; Kürti, L. Practical singly and doubly electrophilic aminating agents: a new, more sustainable platform for carbon-nitrogen bond formation. J. Am. Chem. Soc. 2017, 139, 11184– 11196, DOI: 10.1021/jacs.7b05279Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCkt7jP&md5=38f07ddb4a79a3a4fecdcc40520c0d7cPractical Singly and Doubly Electrophilic Aminating Agents: A New, More Sustainable Platform for Carbon-Nitrogen Bond FormationKattamuri, Padmanabha V.; Yin, Jun; Siriwongsup, Surached; Kwon, Doo-Hyun; Ess, Daniel H.; Li, Qun; Li, Guigen; Yousufuddin, Muhammed; Richardson, Paul F.; Sutton, Scott C.; Kurti, LaszloJournal of the American Chemical Society (2017), 139 (32), 11184-11196CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Given the importance of amines in a large no. of biol. active natural products, active pharmaceutical ingredients, agrochems., and functional materials, the development of efficient C-N bond-forming methods with wide substrate scope continues to be at the frontier of research in synthetic org. chem. Here, authors present a general and fundamentally new synthetic approach for the direct, transition-metal-free prepn. of sym. and unsym. diaryl-, arylalkyl-, and dialkylamines that relies on the facile single or double addn. of readily available C-nucleophiles to the nitrogen atom of bench-stable electrophilic aminating agents. Practical single and double polarity reversal (i.e., umpolung) of the nitrogen atom is achieved using sterically and electronically tunable ketomalonate-derived imines and oximes. Overall, this novel approach represents an operationally simple, scalable, and environmentally friendly alternative to transition-metal-catalyzed C-N cross-coupling methods that are currently used to access structurally diverse secondary amines.
- 75Cohen, N. A.; Stewart, M. L.; Gavathiotis, E.; Tepper, J. L.; Bruekner, S. R.; Koss, B.; Opferman, J. T.; Walensky, L. D. A competitive stapled peptide screen identifies a selective small molecule that overcomes MCL-1-dependent leukemia cell survival. Chem. Biol. 2012, 19, 1175– 1186, DOI: 10.1016/j.chembiol.2012.07.018Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhtl2rsL7M&md5=5306c2b2c2ff29a70ecfc83674f07423A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-Dependent Leukemia Cell SurvivalCohen, Nicole A.; Stewart, Michelle L.; Gavathiotis, Evripidis; Tepper, Jared L.; Bruekner, Susanne R.; Koss, Brian; Opferman, Joseph T.; Walensky, Loren D.Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (9), 1175-1186CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochem. by an antiapoptotic surface groove that neutralizes the proapoptotic BH3 α helix of death proteins. Antiapoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Although targeting the BCL-2 antiapoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of mols. tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor mol. that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochem. lock-hold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence.
- 76Kump, K. J.; Miao, L.; Mady, A. S. A.; Ansari, N. H.; Shrestha, U. K.; Yang, Y.; Pal, M.; Liao, C.; Perdih, A.; Abulwerdi, F. A.; Chinnaswamy, K.; Meagher, J. L.; Carlson, J. M.; Khanna, M.; Stuckey, J. A.; Nikolovska-Coleska, Z. Discovery and characterization of 2,5-substituted benzoic acid dual inhibitors of the anti-apoptotic Mcl-1 and Bfl-1 proteins. J. Med. Chem. 2020, 63, 2489– 2510, DOI: 10.1021/acs.jmedchem.9b01442Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1emsr4%253D&md5=a0ee24b840cde38f05fcb41e34323aceDiscovery and Characterization of 2,5-Substituted Benzoic Acid Dual Inhibitors of the Anti-apoptotic Mcl-1 and Bfl-1 ProteinsKump, Karson J.; Miao, Lei; Mady, Ahmed S. A.; Ansari, Nurul H.; Shrestha, Uttar K.; Yang, Yuting; Pal, Mohan; Liao, Chenzhong; Perdih, Andrej; Abulwerdi, Fardokht A.; Chinnaswamy, Krishnapriya; Meagher, Jennifer L.; Carlson, Jacob M.; Khanna, May; Stuckey, Jeanne A.; Nikolovska-Coleska, ZanetaJournal of Medicinal Chemistry (2020), 63 (5), 2489-2510CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compd. 24, which binds both Mcl-1 and Bfl-1 with Ki values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.
- 77Bernardo, P. H.; Sivaraman, T.; Wan, K.-F.; Xu, J.; Krishnamoorthy, J.; Song, C. M.; Tian, L.; Chin, J. S. F.; Lim, D. S. W.; Mok, H. Y. K.; Yu, V. C.; Tong, J. C.; Chai, C. L. Structural insights into the design of small molecule inhibitors that selectively antagonize Mcl-1. J. Med. Chem. 2010, 53, 2314– 2318, DOI: 10.1021/jm901469pGoogle Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhvF2ktbg%253D&md5=c033784e92b321c2455a5d8b7594d5f6Structural Insights into the Design of Small Molecule Inhibitors That Selectively Antagonize Mcl-1Bernardo, Paul H.; Sivaraman, Thirunavukkarasu; Wan, Kah-Fei; Xu, Jin; Krishnamoorthy, Janarthanan; Song, Chun Meng; Tian, Liming; Chin, Jasmine S. F.; Lim, Diane S. W.; Mok, Henry Y. K.; Yu, Victor C.; Tong, Joo Chuan; Chai, Christina L. L.Journal of Medicinal Chemistry (2010), 53 (5), 2314-2318CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The screening of a small focused library of rhodanine derivs. as inhibitors of Bcl-2 proteins led to the discovery of two structurally related compds. with different binding profiles against the Bcl-XL and the Mcl-1 proteins. Subsequent NMR studies with mutant proteins and in silico docking studies provide a possible rationale for the obsd. specificity.
- 78Szlavik, Z.; Ondi, L.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Radics, G.; Murray, J.; Davidson, J.; Chen, I.; Davis, B.; Hubbard, R. E.; Pedder, C.; Dokurno, P.; Surgenor, A.; Smith, J.; Robertson, A.; LeToumelin-Braizat, G.; Cauquil, N.; Zarka, M.; Demarles, D.; Perron-Sierra, F.; Claperon, A.; Colland, F.; Geneste, O.; Kotschy, A. Structure-guided discovery of a selective Mcl-1 inhibitor with cellular activity. J. Med. Chem. 2019, 62, 6913– 6924, DOI: 10.1021/acs.jmedchem.9b00134Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1ensrvN&md5=e94a3a9ac96f5693406cafa185484470Structure-Guided Discovery of a Selective Mcl-1 Inhibitor with Cellular ActivitySzlavik, Zoltan; Ondi, Levente; Csekei, Marton; Paczal, Attila; Szabo, Zoltan B.; Radics, Gabor; Murray, James; Davidson, James; Chen, Ijen; Davis, Ben; Hubbard, Roderick E.; Pedder, Christopher; Dokurno, Pawel; Surgenor, Allan; Smith, Julia; Robertson, Alan; LeToumelin-Braizat, Gaetane; Cauquil, Nicolas; Zarka, Marion; Demarles, Didier; Perron-Sierra, Francoise; Claperon, Audrey; Colland, Frederic; Geneste, Olivier; Kotschy, AndrasJournal of Medicinal Chemistry (2019), 62 (15), 6913-6924CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when obsd. in human cancers is assocd. with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small mol. inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using NMR and X-ray-derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compds., and cellular activity was brought on scale by offsetting the neg. charge of the anchoring carboxylate group. The obtained compds. described here exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction, and growth inhibition. These early research efforts illustrate drug discovery optimization from thienopyrimidine hits to a lead compd., the chem. series leading to the identification of our more advanced compds. S63845 and S64315.
- 79Wu, Y.; Jiang, Z.; Li, Z.; Gu, J.; You, Q.; Zhang, X. Click chemistry-based discovery of [3-hydroxy-5-(1h-1,2,3-triazol-4-yl)picolinoyl]glycines as orally active hypoxia-inducing factor prolyl hydroxylase inhibitors with favorable safety profiles for the treatment of anemia. J. Med. Chem. 2018, 61, 5332– 5349, DOI: 10.1021/acs.jmedchem.8b00549Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVKktbzL&md5=11a4b00f8d9a883a387acb553510ba2aClick Chemistry-Based Discovery of [3-Hydroxy-5-(1H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia-Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of AnemiaWu, Yue; Jiang, Zhensheng; Li, Zhihong; Gu, Jing; You, Qidong; Zhang, XiaojinJournal of Medicinal Chemistry (2018), 61 (12), 5332-5349CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)As a gene assocd. with anemia, the erythropoiesis gene is physiol. expressed under hypoxia regulated by †hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study, we applied click chem. to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time, and a series of triazole compds. showed preferable inhibitory activity in fluorescence polarization assays. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the Hb of cisplatin-induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity obsd. in vivo. These results confirm that triazole compd. 15i is a promising candidate for the treatment of renal anemia.
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Abstract 摘要
Figure 1 图 1
Figure 1. Binding mode of Mcl-1 and Bim BH3 (PDB 2NL9).
Figure 2
Figure 2. Representative Mcl-1 inhibitors.
Figure 3
Figure 4
Figure 4. Proposed binding mode of compound 13 with Mcl-1 using molecular docking. The docking site was derived from the position of the small-molecular ligand cocrystallized in the binding site of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. The hydrogen bonds are represented by green dashed lines, and the π–σ and π–alkyl interactions are represented by pink dashed lines. The carbon atoms of small molecules and Mcl-1 residues are colored cyan and green, respectively.
Figure 5
Figure 5. Proposed binding mode of 16 with Mcl-1 using molecular docking. The docking site was derived from the position of the small-molecular ligand cocrystallized in the binding site of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. The hydrogen bonds are represented by green dashed lines, and the π–σ and π–alkyl interactions are represented by pink dashed lines. The carbon atoms of small molecules and Mcl-1 residues are colored cyan and green, respectively.
Figure 6
Figure 6. Purposed binding modes of 40 (A) and 43 (B) with Mcl-1 using molecular docking. The docking site was derived from the position of the small-molecular ligand cocrystallized in the binding site of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. Hydrogen bonds are represented by green dashed lines, and π–σ and π–alkyl interactions are represented by pink dashed lines. The carbon atoms of small molecules and Mcl-1 residues are colored purple and green, respectively.
Figure 7
Figure 7. Binding affinities of 40 to Mcl-1 and other Bcl-2 family members.
Figure 8
Figure 8. Bcl-2 could not accommodate 40 due to the shallow and narrow P2 pocket. (A) Overlay of compound 2 (green) and 40 (purple). (B) Comparison of P2 pockets of Mcl-1 and Bcl-2.
Figure 9
Figure 9. 40 induced apoptosis in an Mcl-1-dependent manner. (A) Co-ip experiment of 40 and AZD5991 in H929 cells. (B–G) Flow cytometry assay of 40 and A1210477 in H929 cells at concentrations of 1 μM and 5 μM. (H,I) WB assay of 40 and A1210477 in H929 cells and K562 cells at concentrations of 0, 0.1, 0.5, 1, and 5 μM. (J,K) Caspase activation abilities of 40 and A1210477 in H929 cells and K562 cells at concentrations of 0, 0.1, 0.5, 1, and 5 μM. The values shown are the means ± SEM (n = 3 independent observations), ns = no significant, ***p < 0.001, ****p < 0.0001 one-way ANOVA with Tukey–Kramer posttest.
Figure 10
Figure 10. In vivo PK parameters of 40. The values shown are the means. Bars represent SEM.
Figure 11
Figure 11. Antitumor efficacy of compound 40 in nude mice loaded with MV4-11 xenografts. (A) Tumor volume change. The values shown are the means ± SEM **p < 0.01, and ****p < 0.0001, one-way ANOVA with the Tukey–Kramer posttest. (B) Body weight change. (C) Organ weight change. The values shown are the means. Bars represent SEM. (D) Representative HE staining images of tumor tissues and organ tissues. Scale bar 200 μM. (E) WB assay of tumor tissues. (n = 2 for each group). (F) Representative images of tunel assay of tumor tissues. Scale bar 200 μM.
Scheme 1
Scheme 1. Synthesis of Target Compounds 11 and 12aaReagents and conditions: (a) 1,3-dibromopropane, K2CO3, CH3CN, 90 °C, 3 h, 75%. (b) 4-methylbenzoyl chloride, AlCl3, DCM, N2, 0 °C–r.t. 2 h, 68%. (c) triethylsilane, THF, N2, r.t. 2 h, 65%. (d) 59, Cs2CO3, DMF, 60 °C, 1 h, 84–89%. (e) NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 78–95%.
Scheme 2
Scheme 2. Synthesis of Target Compound 13 and 20aaReagents and conditions: (a) methyl isocyanate, Ag2CO3, 1,4-dioxane, 80 °C, 30 min, 18–25%. (b) 59, NaH, DMF, 0 °C–r.t. 0.5 h, 35–42%. (c) NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 62–72%.
Scheme 3
Scheme 3. Synthesis of Target Compound 14 and 15aaReagents and conditions: (a) 59, Cs2CO3, DMF, 60 °C, 1 h, 75%. (b) SnCl2·2H2O, N2, EtOH, 60 °C, 4 h. (c) Pyridine, TosCl, r.t. 1 h, 38%. (d) NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 43–64%. (e) (Boc)2O, EtOH, r.t. 1 h, 46%. (f) 4-Methylbenzyl bromide, NaH, r.t. 0.5 h, 73%. (g) CF3COOH, r.t. 1.5 h, 95%.
Scheme 4
Scheme 4. Synthesis of Target Compounds 16, 23–26, 29–31, 33 and 34, 37 and 38, 41 and 42, 44–47, 51–53, and 55–57aaReagents and conditions: (a) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, SO2Cl2, Et3N, DCM, r.t. 45 min, 56–85%; (b) 59, Cs2CO3, DMF, 50 °C, 1 h, 78–85%; (c) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 63–85%. (d) m-CPBA, DCM, r.t. 0.5 h, 55–70%; (e) Pd/C, H2, EA, 50 °C, 3 h, 95%.
Scheme 5
Scheme 5. Synthesis of Target Compounds 17–19 and 21 and 22aaReagents and conditions: (a) AcOH, NaNO2, 0 °C–r.t. overnight; (b) AcONa, AcOH, Zn, r.t.–80 °C, 3 h, 36–43%. (c) p-Toluenethiol, SO2Cl2, Et3N, DCM, r.t. 45 min, 45–62%; (d) 59, Cs2CO3, DMF, 50 °C, 1 h, 57–72%; (e) m-CPBA, DCM, r.t. 0.5 h, 45–56%; (f) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 74–86%.
Scheme 6
Scheme 6. Synthesis of Target Compounds 27 and 28aaReagents and conditions: (a) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, N2, AlCl3, PhCl, 90 °C, 2 h, 59–68%; (b) 59, Cs2CO3, DMF, 50 °C, 1 h, 63–75%; (c) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 54–63%.
Scheme 7
Scheme 7. Synthesis of Target Compounds 32, 35, 36, 39, 40, 43, 48–50, and 54aaReagents and conditions: (a) ethanedithiol, CuSO4·5H2O, Cs2CO3, DMSO, N2, 90 °C, 4 h, 76–85%; (b) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, SO2Cl2, Et3N, DCM, r.t. 45 min, 54–75%; (c) 59, Cs2CO3, DMF, 50 °C, 1 h, 75–85%; (d) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 42–80%; (e) m-CPBA, DCM, r.t. 0.5 h, 63–84%; (f) Pd/C, H2, EA, 50 °C, 3 h, 63–67%.
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- 4Ren, D.; Tu, H.-C.; Kim, H.; Wang, G. X.; Bean, G. R.; Takeuchi, O.; Jeffers, J. R.; Zambetti, G. P.; Hsieh, J. J.-D.; Cheng, E. H.-Y. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 2010, 330, 1390– 1393, DOI: 10.1126/science.11902174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVyrsbnE&md5=443c28b09c7d260319eb207c56cd7835BID, BIM, and PUMA Are Essential for Activation of the BAX- and BAK-Dependent Cell Death ProgramRen, Decheng; Tu, Ho-Chou; Kim, Hyungjin; Wang, Gary X.; Bean, Gregory R.; Takeuchi, Osamu; Jeffers, John R.; Zambetti, Gerard P.; Hsieh, James J.-D.; Cheng, Emily H.-Y.Science (Washington, DC, United States) (2010), 330 (6009), 1390-1393CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are assocd. with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only mols. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.
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- 6Kozopas, K. M.; Yang, T.; Buchan, H. L.; Zhou, P.; Craig, R. W. Mcl-1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to Bcl-2. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 3516– 3520, DOI: 10.1073/pnas.90.8.35166https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXks1CmtL8%253D&md5=b1f43de6c911f095d53c5a3f38df6a63MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2Kozopas, Karen M.; Yang, Tao; Buchan, Heather L.; Zhou, Ping; Craig, Ruth W.Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (8), 3516-20CODEN: PNASA6; ISSN:0027-8424.A gene, MCL1, was isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Expression of MCL1 increases early in the induction, or programming, of differentiation in ML-1 (at 1-3 h), before the appearance of differentiation markers and mature morphol. (at 1-3 days). Further, MCL1 has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(14;18) chromosome translocation. MCL1 and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the assocn. of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-assocd. genes, expression of MCL1 and BCL2 relates to the programming of differentiation and cell viability/death.
- 7Herrant, M.; Jacquel, A.; Marchetti, S.; Belhacène, N.; Colosetti, P.; Luciano, F.; Auberger, P. Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis. Oncogene 2004, 23, 7863– 7873, DOI: 10.1038/sj.onc.12080697https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotl2kur4%253D&md5=91ff0749a52f4244b6480652a2e79033Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosisHerrant, Magali; Jacquel, Arnaud; Marchetti, Sandrine; Belhacene, Nathalie; Colosetti, Pascal; Luciano, Frederic; Auberger, PatrickOncogene (2004), 23 (47), 7863-7873CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)Mcl-1 is an antiapoptotic member of the Bcl-2 family that can promote cell viability. We report here that Mcl-1 is a new substrate for caspases during induction of apoptosis. Mcl-1 cleavage occurs after Asp127 and Asp157 and generates four fragments of 24, 19, 17 and 12 kDa in both intact cells and in vitro, an effect prevented by selective caspase inhibitors. As a consequence, the resulting protein that lacks the first 127 or 157 amino acids contains only the BH1-BH3 domains of Bcl-2 family members. Mutation of Asp127 and Asp157 abolishes the generation of the 24 and 12 kDa fragments and that of the 19 and 17 kDa fragments, resp. Interestingly, when expressed in HeLa cells Mcl-1 wt and Mcl-1 Δ127 showed a markedly different intracellular distribution. Mcl-1 wt colocalized with α-Tubulin near the internal face of the plasma membrane, while Mcl-1 Δ127 co-assocd. with Bim-EL at the mitochondrial level. Coimmunopptn. expts. also demonstrated that Mcl1 Δ127 exhibited increased binding to Bim when compared to Mcl-1 wt. Finally, Mcl-1 wt unlike Mcl-1 Δ127 inhibited Bim-EL-induced caspase activation. Altogether, our findings demonstrate that cleavage of Mcl-1 by caspases modifies its subcellular localization, increases its assocn. with Bim and inhibits its antiapoptotic function.
- 8Adams, J. M.; Cory, S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 2001, 26, 61– 66, DOI: 10.1016/S0968-0004(00)01740-08https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXptV2itQ%253D%253D&md5=6af92fbfe37a6e389302fc45314c30fdLife-or-death decisions by the Bcl-2 protein familyAdams, J. M.; Cory, S.Trends in Biochemical Sciences (2001), 26 (1), 61-66CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)A review, with 51 refs. In response to intracellular damage and certain physiol. cues, cells enter the suicide program termed apoptosis, executed by proteases called caspases. Commitment to apoptosis is typically governed by opposing factions of the Bcl-2 family of cytoplasmic proteins. Initiation of the proteolytic cascade requires assembly of certain caspase precursors on a scaffold protein, and the Bcl-2 family dets. whether this complex can form. Its pro-survival members can act by sequestering the scaffold protein and/or by preventing the release of apoptogenic mols. from organelles such as mitochondria. Pro-apoptotic family members act as sentinels for cellular damage: cytotoxic signals induce their translocation to the organelles where they bind to their pro-survival relatives, promote organelle damage and trigger apoptosis.
- 9Gomez-Bougie, P.; Wuillème-Toumi, S.; Ménoret, E.; Trichet, V.; Robillard, N.; Philippe, M.; Bataille, R.; Amiot, M. Noxa up-regulation and Mcl-1 cleavage are associated to apoptosis induction by bortezomib in multiple myeloma. Cancer Res. 2007, 67, 5418– 5424, DOI: 10.1158/0008-5472.CAN-06-43229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmtVaitL4%253D&md5=70edbc5d1ddc89ba41bc18db35054430Noxa Up-regulation and Mcl-1 Cleavage Are Associated to Apoptosis Induction by Bortezomib in Multiple MyelomaGomez-Bougie, Patricia; Wuilleme-Toumi, Soraya; Menoret, Emmanuelle; Trichet, Valerie; Robillard, Nelly; Philippe, Moreau; Bataille, Regis; Amiot, MartineCancer Research (2007), 67 (11), 5418-5424CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Targeting the ubiquitin-proteasome pathway has emerged as a potent anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma. Multiple myeloma cell survival is highly dependent on Mcl-1 antiapoptotic mols. In a recent study, proteasome inhibitors induced Mcl-1 accumulation that slowed down their proapoptotic effects. Consequently, we investigated the role of Bcl-2 family members in bortezomib-induced apoptosis. We found that bortezomib induced apoptosis in five of seven human myeloma cell lines (HMCL). Bortezomib-induced apoptosis was assocd. with Mcl-1 cleavage regardless of Mcl-1L accumulation. Furthermore, RNA interference mediated Mcl-1 decrease and sensitized RPMI-8226 HMCL to bortezomib, highlighting the contribution of Mcl-1 in bortezomib-induced apoptosis. Interestingly, an important induction of Noxa was found in all sensitive HMCL both at protein and mRNA level. Concomitant to Mcl-1 cleavage and Noxa induction, we also found caspase-3, caspase-8, and caspase-9 activation. Under bortezomib treatment, Mcl-1L/Noxa complexes were highly increased, Mcl-1/Bak complexes were disrupted, and there was an accumulation of free Noxa. Finally, we obsd. a dissocn. of Mcl-1/Bim complexes that may be due to a displacement of Bim induced by Noxa. Thus, in myeloma cells, the mechanistic basis for bortezomib sensitivity can be explained mainly by the model in which the sensitizer Noxa can displace Bim, a BH3-only activator, from Mcl-1, thus leading to Bax/Bak activation.
- 10Pierson, W.; Cauwe, B.; Policheni, A.; Schlenner, S. M.; Franckaert, D.; Berges, J.; Humblet-Baron, S.; Schönefeldt, S.; Herold, M. J.; Hildeman, D.; Strasser, A.; Bouillet, P.; Lu, L.-F.; Matthys, P.; Freitas, A. A.; Luther, R. J.; Weaver, C. T.; Dooley, J.; Gray, D. H.; Liston, A. Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3+ regulatory T cells. Nat. Immunol. 2013, 14, 959– 965, DOI: 10.1038/ni.264910https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFShs77L&md5=46a3e7ae00ecacc2294ad3381c6e0c66Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3+ regulatory T cellsPierson, Wim; Cauwe, Benedicte; Policheni, Antonia; Schlenner, Susan M.; Franckaert, Dean; Berges, Julien; Humblet-Baron, Stephanie; Schoenefeldt, Susann; Herold, Marco J.; Hildeman, David; Strasser, Andreas; Bouillet, Philippe; Lu, Li-Fan; Matthys, Patrick; Freitas, Antonio A.; Luther, Rita J.; Weaver, Casey T.; Dooley, James; Gray, Daniel H. D.; Liston, AdrianNature Immunology (2013), 14 (9), 959-965CODEN: NIAMCZ; ISSN:1529-2908. (Nature Publishing Group)Foxp3+ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4+ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was crit. for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via crit. survival pathways.
- 11Renjini, A. P.; Titus, S.; Narayan, P.; Murali, M.; Jha, R. K.; Laloraya, M. STAT3 and MCL-1 associate to cause a mesenchymal epithelial transition. J. Cell Sci. 2014, 127, 1738– 1750, DOI: 10.1242/jcs.13821411https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXos1Chur4%253D&md5=76aa474af8d7dd8b5b469a1aa633535bSTAT3 and MCL-1 associate to cause a mesenchymal epithelial transitionRenjini, A. P.; Titus, Shiny; Narayan, Prashanth; Murali, Megha; Jha, Rajesh Kumar; Laloraya, MaliniJournal of Cell Science (2014), 127 (8), 1738-1750CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)Embryo implantation is effected by a myriad of signaling cascades acting on the embryo-endometrium axis. Here we show, by using MALDI TOF anal., far-western anal. and colocalization and co-transfection studies, that STAT3 and MCL-1 are interacting partners during embryo implantation. We show in vitro that the interaction between the two endogenous proteins is strongly regulated by estrogen and progesterone. Implantation, pregnancy and embryogenesis are distinct from any other process in the body, with extensive, but controlled, proliferation, cell migration, apoptosis, cell invasion and differentiation. Cellular plasticity is vital during the early stages of development for morphogenesis and organ homeostasis, effecting the epithelial to mesenchymal transition (EMT) and, the reverse process, mesenchymal to epithelial transition (MET). STAT3 functionally assocs. with MCL-1 in the mammalian breast cancer cell line MCF7 that overexpresses STAT3 and MCL-1, which leads to an increased rate of apoptosis and decreased cellular invasion, disrupting the EMT. Assocn. of MCL-1 with STAT3 modulates the normal, anti-apoptotic, activity of MCL-1, resulting in pro-apoptotic effects. Studying the impact of the assocn. of STAT3 with MCL-1 on MET could lead to an enhanced understanding of pregnancy and infertility, and also metastatic tumors.
- 12Hikita, H.; Takehara, T.; Shimizu, S.; Kodama, T.; Li, W.; Miyagi, T.; Hosui, A.; Ishida, H.; Ohkawa, K.; Kanto, T.; Hiramatsu, N.; Yin, X. M.; Hennighausen, L.; Tatsumi, T.; Hayashi, N. Mcl-1 and Bcl-xL cooperatively maintain integrity of hepatocytes in developing and adult murine liver. Hepatology 2009, 50, 1217– 1226, DOI: 10.1002/hep.2312612https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlWlu7fP&md5=1344b4717ed1014041c9e59b216a8158Mcl-1 and Bcl-xL cooperatively maintain integrity of hepatocytes in developing and adult murine liverHikita, Hayato; Takehara, Tetsuo; Shimizu, Satoshi; Kodama, Takahiro; Li, Wei; Miyagi, Takuya; Hosui, Atsushi; Ishida, Hisashi; Ohkawa, Kazuyoshi; Kanto, Tatsuya; Hiramatsu, Naoki; Yin, Xiao-Ming; Hennighausen, Lothar; Tatsumi, Tomohide; Hayashi, NorioHepatology (Hoboken, NJ, United States) (2009), 50 (4), 1217-1226CODEN: HPTLD9; ISSN:0270-9139. (John Wiley & Sons, Inc.)Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a crit. apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, esp. in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1-deficient mice by crossing mcl-1flox/flox mice and AlbCre mice and further crossed them with bcl-xflox/flox mice, giving Mcl-1/Bcl-xL-deficient mice. The mcl-1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased no. of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL)-pos. cells in the liver; these phenotypes were very dose to those previously found in hepatocyte-specific Bcl-xL-deficient mice. Although mcl-1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-xflox/+ mcl-1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL-deficient or Mcl-1-deficient mice. In contrast, bcl-xflox/flox mcl-1flox/+ AlbCre, bcl-xflox/+ mcl-1flox/flox AlbCre, and bcl-xflox/flox mcl-1flox/flox AlbCre mice displayed a decreased no. of hepatocytes and a reduced vol. of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl-1 is crit. for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner.
- 13Opferman, J. T.; Letai, A.; Beard, C.; Sorcinelli, M. D.; Ong, C. C.; Korsmeyer, S. J. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 2003, 426, 671– 676, DOI: 10.1038/nature0206713https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXps1Cksr4%253D&md5=e34f76ef4a07eeb92b18797ae484dcc5Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1Opferman, Joseph T.; Letai, Anthony; Beard, Caroline; Sorcinelli, Mia D.; Ong, Christy C.; Korsmeyer, Stanley J.Nature (London, United Kingdom) (2003), 426 (6967), 671-676CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Regulated apoptosis is essential for both the development and the subsequent maintenance of the immune system. Interleukins, including IL-2, IL-4, IL-7 and IL-15, heavily influence lymphocyte survival during the vulnerable stages of VDJ rearrangement and later in ensuring cellular homeostasis, but the genes specifically responsible for the development and maintenance of lymphocytes have not been identified. The antiapoptotic protein MCL-1 is an attractive candidate, as it is highly regulated, appears to enhance short-term survival and functions at an apical step in genotoxic deaths. However, Mcl-1 deficiency results in peri-implantation lethality. Here we show that mice conditional for Mcl-1 display a profound redn. in B and T lymphocytes when MCL-1 is removed. Deletion of Mcl-1 during early lymphocyte differentiation increased apoptosis and arrested the development at pro-B-cell and double-neg. T-cell stages. Induced deletion of Mcl-1 in peripheral B- and T-cell populations resulted in their rapid loss. Moreover, IL-7 both induced and required MCL-1 to mediate lymphocyte survival. Thus, MCL-1, which selectively inhibits the proapoptotic protein BIM, is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes.
- 14Thomas, L. W.; Lam, C.; Edwards, S. W. Mcl-1: the molecular regulation of protein function. FEBS Lett. 2010, 584, 2981– 2989, DOI: 10.1016/j.febslet.2010.05.06114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXosVyhu7s%253D&md5=5deee464702c00444349236698dcf9b1Mcl-1; the molecular regulation of protein functionThomas, Luke W.; Lam, Connie; Edwards, Steven W.FEBS Letters (2010), 584 (14), 2981-2989CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. Apoptosis, an essential and basic biol. phenomenon, is regulated in a complex manner by a multitude of factors. Myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, exemplifies a no. of the mechanisms by which a protein's contribution to cell fate may be modified. The N-terminus of Mcl-1 is unique among the Bcl-2 family, in that it is rich in exptl. confirmed and putative regulatory residues and motifs. These include sites for ubiquitination, cleavage, and phosphorylation, which influence the protein's stability, localization, dimerization, and function. Here, the authors review what is known about the regulation of Mcl-1 expression and function, with particular focus on post-translational modifications and how phosphorylation interconnects the complex mol. control of Mcl-1 with the cellular state.
- 15Aichberger, K. J.; Mayerhofer, M.; Krauth, M. T.; Skvara, H.; Florian, S.; Sonneck, K.; Akgul, C.; Derdak, S.; Pickl, W. F.; Wacheck, V.; Selzer, E.; Monia, B. P.; Moriggl, R.; Valent, P.; Sillaber, C. Identification of Mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides. Blood 2005, 105, 3303– 3311, DOI: 10.1182/blood-2004-02-074915https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjtlSlsrg%253D&md5=eb32b6ef8b9b9b54936a9368c8b079cdIdentification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): Evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotidesAichberger, Karl J.; Mayerhofer, Matthias; Krauth, Maria-Theresa; Skvara, Hans; Florian, Stefan; Sonneck, Karoline; Akgul, Cahit; Derdak, Sophia; Pickl, Winfried F.; Wacheck, Volker; Selzer, Edgar; Monia, Brett P.; Moriggl, Richard; Valent, Peter; Sillaber, ChristianBlood (2005), 105 (8), 3303-3311CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)Antiapoptotic members of the bcl-2 family have recently been implicated in the pathogenesis of chronic myeloid leukemia (CML), a hematopoietic neoplasm assocd. with the BCR/ABL oncogene. The authors have examd. expression of MCL-1 in primary CML cells and BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed myeloid cell leukemia-1 (mcl-1) mRNA and the MCL-1 protein in a constitutive manner. The BCR/ABL inhibitor imatinib (=STI571) decreased the expression of MCL-1 in these cells. Correspondingly, BCR/ABL enhanced mcl-1 promoter activity, mcl-1 mRNA expression, and the MCL-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of MCL-1 in Ba/F3 cells was counteracted by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Identical results were obtained for constitutive expression of MCL-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of MCL-1 as a survival-related target in CML cells, mcl-1 siRNA and mcl-1 antisense oligonucleotides (ASOs) were applied. The resulting down-regulation of MCL-1 was found to be assocd. with a substantial decrease in viability of K562 cells. Moreover, the mcl-1 ASO was found to synergize with imatinib in producing growth inhibition in these cells. Together, the authors' data identify MCL-1 as a BCR/ABL-dependent survival factor and interesting target in CML.
- 16Steven, L. G.; Klaus, P.; Jean-Luc, H.; Anderson, K. C. Mcl-1 regulation and its role in multiple myeloma. Cell Cycle 2004, 3, 1259– 1262, DOI: 10.4161/cc.3.10.1196There is no corresponding record for this reference.
- 17Song, L.; Coppola, D.; Livingston, S.; Cress, W. D.; Haura, E. B. Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells. Cancer Biol. Ther. 2005, 4, 267– 276, DOI: 10.4161/cbt.4.3.149617https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFyrug%253D%253D&md5=b79c35e287895a356d74740c41e03baeMcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cellsSong, Lanxi; Coppola, Domenico; Livingston, Sandy; Cress, Doug; Haura, Eric B.Cancer Biology & Therapy (2005), 4 (3), 267-276CODEN: CBTAAO; ISSN:1538-4047. (Landes Bioscience)Overexpression of anti-apoptotic Bcl-2 family members and deregulation of the pathways that regulate pro-apoptotic family members have been obsd. in non-small cell lung cancers (NSCLC). Previous reports have identified both Bcl-2 and Bcl-xL proteins as survival factors in lung cancer cells since redns. in these proteins can induce apoptosis and sensitize lung cancer cells to apoptosis induced by chemotherapy agents. Myeloid cell leukemia-1 (Mcl-1), another member of the Bcl-2 family, has been found to be a crit. survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human lung cancers. We used NSCLC cell lines to explore how Mcl-1 levels affect lung cancer cell survival and studied tumors from patients to det. expression patterns of Mcl-1. NSCLC cells express abundant Mcl-1 protein and depletion of Mcl-1 levels by antisense Mcl-1 oligonucleotides induces apoptosis in A549 and H1299 lung cancer cells. Redn. in Mcl-1 levels can sensitize lung cancer cells to apoptosis induced by cytotoxic agents as well as by ionizing radiation. Lung cancer cells overexpressing Mcl-1 are less sensitive to apoptosis induced by chemotherapeutic agents, ZD1839 (an inhibitor of EGFR tyrosine kinase) and Bcl-2 or Bcl-xL antisense oligonucleotides. We find that epidermal growth factor (EGF) can enhance Mcl-1 protein levels in an ERK-dependent manner. Signal transduction agents that reduce Mcl-1 levels correlated with their individual ability to induce apoptosis in lung cancer cells. Finally, NSCLC tumors taken directly from patients have elevated levels of Mcl-1 protein compared with normal adjacent lung tissue. Therefore, agents that target Mcl-1 can induce apoptosis and sensitize cells to apoptosis induced by cytotoxic agents. Mcl-1 protein is overexpressed in a subset of human NSCLC and enhanced levels of Mcl-1 may protect lung cancer cells from death induced by a variety of pro-apoptotic stimuli.
- 18Tong, J.; Wang, P.; Tan, S.; Chen, D.; Nikolovska-Coleska, Z.; Zou, F.; Yu, J.; Zhang, L. Mcl-1 degradation is required for targeted therapeutics to eradicate colon cancer cells. Cancer Res. 2017, 77, 2512– 2521, DOI: 10.1158/0008-5472.CAN-16-324218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvFakurw%253D&md5=40461e21325a846f66d68346ef955e98Mcl-1 Degradation Is Required for Targeted Therapeutics to Eradicate Colon Cancer CellsTong, Jingshan; Wang, Peng; Tan, Shuai; Chen, Dongshi; Nikolovska-Coleska, Zaneta; Zou, Fangdong; Yu, Jian; Zhang, LinCancer Research (2017), 77 (9), 2512-2521CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degrdn. has been assocd. with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degrdn. and anticancer drug responses has not been directly established, esp. in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in detg. effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degrdn. was induced by a variety of multikinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1-KI also suppressed the antiangiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degrdn.-resistant Mcl-1 bound and sequestered PUMA from other prosurvival proteins to maintain cell survival, which was abolished by small-mol. Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degrdn. in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1-targeting agents that can overcome drug resistance.
- 19Pepper, C.; Lin, T. T.; Pratt, G.; Hewamana, S.; Brennan, P.; Hiller, L.; Hills, R.; Ward, R.; Starczynski, J.; Austen, B.; Hooper, L.; Stankovic, T.; Fegan, C. Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers. Blood 2008, 112, 3807– 3817, DOI: 10.1182/blood-2008-05-15713119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlCntrjF&md5=d7a626b0de96a0f82956f3e1da8ea9cfMcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markersPepper, Chris; Lin, Thet Thet; Pratt, Guy; Hewamana, Saman; Brennan, Paul; Hiller, Louise; Hills, Robert; Ward, Rachel; Starczynski, Jane; Austen, Belinda; Hooper, Laura; Stankovic, Tatjana; Fegan, ChrisBlood (2008), 112 (9), 3807-3817CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)Bcl-2 family proteins play a crit. role in the regulation of apoptosis in chronic lymphocytic leukemia (CLL). However, their assocn. with established prognostic markers is unknown. In this study, we analyzed the expression of Bcl-2, Bax, and Mcl-1 in 185 CLL patients and evaluated their relationship with other prognostic markers, in vitro sensitivity to fludarabine, and clin. outcome. Mcl-1 expression was significantly correlated with stage of disease (P < .001), lymphocyte doubling time (P = .01), VH gene mutation status (P < .001), CD38 expression (P < .001), and ZAP-70 expression (P = .003). In addn., Mcl-1 and Mcl-1/Bax ratios showed strong correlations with in vitro resistance to fludarabine (P = .005 and P < .001, resp.). Furthermore, elevated Mcl-1 expression and Mcl-1/Bax ratios were predictive of time to first treatment in the whole cohort (P < .001 and P < .001, resp.) and in stage A patients only (P = .002 and P = .001, resp.). Taken together, our data show that Mcl-1 is a key controller of in vitro drug resistance and is an important regulator of disease progression and outcome in CLL. It therefore represents a promising therapeutic target in this incurable condition. The close correlation between Mcl-1 expression and VH gene mutation status, CD38 expression, and ZAP-70 expression offers a biol. explanation for their assocn. with adverse prognosis.
- 20Zhou, P.; Qian, L.; Bieszczad, C. K.; Noelle, R.; Binder, M.; Levy, N. B.; Craig, R. W. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 1998, 92, 3226– 3239, DOI: 10.1182/blood.V92.9.322620https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntFCqsbg%253D&md5=06d39268f7f856a192ccc6e7be449abeMcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineageZhou, Ping; Qian, Liping; Bieszczad, Christine K.; Noelle, Randolph; Binder, Michael; Levy, Norman B.; Craig, Ruth W.Blood (1998), 92 (9), 3226-3239CODEN: BLOOAW; ISSN:0006-4971. (W. B. Saunders Co.)Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addn., among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell no., but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at "viability gates" along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization.
- 21Touzeau, C.; Maciag, P.; Amiot, M.; Moreau, P. Targeting Bcl-2 for the treatment of multiple myeloma. Leukemia 2018, 32, 1899– 1907, DOI: 10.1038/s41375-018-0223-921https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVCns77E&md5=dfbc63cc6ca0b471ad40bb38d064bbd8Targeting Bcl-2 for the treatment of multiple myelomaTouzeau, Cyrille; Maciag, Paulo; Amiot, Martine; Moreau, PhilippeLeukemia (2018), 32 (9), 1899-1907CODEN: LEUKED; ISSN:0887-6924. (Nature Research)Despite advances in the treatment of multiple myeloma, the disease still remains incurable for the majority of patients. The overexpression of anti-apoptotic proteins (i.e., Bcl-2, Bcl-XL or Mcl-1) is a hallmark of cancer and favors tumor cell survival and resistance to therapy. The oral drug venetoclax is the first-in-class Bcl-2-specific BH3 mimetic. In myeloma, in vitro sensitivity to venetoclax is mainly obsd. in plasma cells harboring the t(11;14) translocation, a mol. subgroup assocd. with high Bcl-2 and low Mcl-1/Bcl-XL expression. In addn. with Bcl-2 members expression profile, functional tests as BH3 profiling or in vitro BH3 mimetic drug testing also predict sensitivity to the drug. Phase 1 clin. trials recently confirmed the efficacy of venetoclax monotherapy in heavily pretreated myeloma patients, mostly in patients with t(11;14). In combination with the proteasome inhibitor bortezomib, venetoclax therapy was found to be feasible and allowed promising response rate in relapsed myeloma patients, independent of t(11;14) status. The present review summarizes the current knowledge, "from bench to bedside", about venetoclax for the treatment of multiple myeloma.
- 22Souers, A. J.; Leverson, J. D.; Boghaert, E. R.; Ackler, S. L.; Catron, N. D.; Chen, J.; Dayton, B. D.; Ding, H.; Enschede, S. H.; Fairbrother, W. J.; Huang, D. C. S.; Hymowitz, S. G.; Jin, S.; Khaw, S. L.; Kovar, P. J.; Lam, L. T.; Lee, J.; Maecker, H. L.; Marsh, K. C.; Mason, K. D.; Mitten, M. J.; Nimmer, P. M.; Oleksijew, A.; Park, C.-H.; Park, C. M.; Phillips, D. C.; Roberts, A. W.; Sampath, D.; Seymour, J. F.; Smith, M. L.; Sullivan, G. M.; Tahir, S. K.; Tse, C.; Wendt, M. D.; Xiao, Y.; Xue, J. C.; Zhang, H.; Humerickhouse, R. A.; Rosenberg, S. H.; Elmore, S. W. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 2013, 19, 202– 208, DOI: 10.1038/nm.304822https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslagtQ%253D%253D&md5=6a24b72303eb2062da653f1f61ac74fbABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing plateletsSouers, Andrew J.; Leverson, Joel D.; Boghaert, Erwin R.; Ackler, Scott L.; Catron, Nathaniel D.; Chen, Jun; Dayton, Brian D.; Ding, Hong; Enschede, Sari H.; Fairbrother, Wayne J.; Huang, David C. S.; Hymowitz, Sarah G.; Jin, Sha; Khaw, Seong Lin; Kovar, Peter J.; Lam, Lloyd T.; Lee, Jackie; Maecker, Heather L.; Marsh, Kennan C.; Mason, Kylie D.; Mitten, Michael J.; Nimmer, Paul M.; Oleksijew, Anatol; Park, Chang H.; Park, Cheol-Min; Phillips, Darren C.; Roberts, Andrew W.; Sampath, Deepak; Seymour, John F.; Smith, Morey L.; Sullivan, Gerard M.; Tahir, Stephen K.; Tse, Chris; Wendt, Michael D.; Xiao, Yu; Xue, John C.; Zhang, Haichao; Humerickhouse, Rod A.; Rosenberg, Saul H.; Elmore, Steven W.Nature Medicine (New York, NY, United States) (2013), 19 (2), 202-208CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-XL), which has shown clin. efficacy in some BCL-2-dependent hematol. cancers. However, concomitant on-target thrombocytopenia caused by BCL-XL inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compd. inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacol. inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematol. cancers.
- 23Quinn, B. A.; Dash, R.; Azab, B.; Sarkar, S.; Das, S. K.; Kumar, S.; Oyesanya, R. A.; Dasgupta, S.; Dent, P.; Grant, S.; Rahmani, M.; Curiel, D. T.; Dmitriev, I.; Hedvat, M.; Wei, J.; Wu, B.; Stebbins, J. L.; Reed, J. C.; Pellecchia, M.; Sarkar, D.; Fisher, P. B. Targeting Mcl-1 for the therapy of cancer. Expert Opin. Invest. Drugs 2011, 20, 1397– 1411, DOI: 10.1517/13543784.2011.60916723https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFGgur3N&md5=7e2e0ab1cedb25a32b8e97a935238013Targeting Mcl-1 for the therapy of cancerQuinn, Bridget A.; Dash, Rupesh; Azab, Belal; Sarkar, Siddik; Das, Swadesh K.; Kumar, Sachin; Oyesanya, Regina A.; Dasgupta, Santanu; Dent, Paul; Grant, Steven; Rahmani, Mohamed; Curiel, David T.; Dmitriev, Igor; Hedvat, Michael; Wei, Jun; Wu, Bainan; Stebbins, John L.; Reed, John C.; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B.Expert Opinion on Investigational Drugs (2011), 20 (10), 1397-1411CODEN: EOIDER; ISSN:1354-3784. (Informa Healthcare)A review. Introduction:Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.Areas covered:Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacol. approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.Expert opinion:We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of addnl. signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochem. pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
- 24Akgul, C. Mcl-1 is a potential therapeutic target in multiple types of cancer. Cell. Mol. Life Sci. 2009, 66, 1326– 1336, DOI: 10.1007/s00018-008-8637-624https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksFGhtb4%253D&md5=a6eca2f0ff24caaa199397b3e4c959d5Mcl-1 is a potential therapeutic target in multiple types of cancerAkgul, C.Cellular and Molecular Life Sciences (2009), 66 (8), 1326-1336CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)A review. Resistance to apoptosis is a common challenge in human malignancies contributing to both progress of cancer and resistance to conventional therapeutics. Abnormalities in a variety of cell intrinsic and extrinsic mol. mechanisms cooperatively promote tumor formation. Therapeutic approaches that specifically target components of these mol. mechanisms are getting widespread attention. Mcl-1 is a highly expressed pro-survival protein in human malignancies and its cellular expression is tightly regulated via multiple mechanisms. Mcl-1 differs from other members of the Bcl-2 family in having a very short half-life. So inhibition of its expression and/or neutralization of its anti-apoptotic function will rapidly make Mcl-1-dependent cells more susceptible to apoptosis and provide an opportunity to combat several types of cancers. This review summarizes the current knowledge on the regulation of Mcl-1 expression and discusses the alternative approaches targeting Mcl-1 in human cancer cells whose survivals mainly depend on Mcl-1.
- 25Lessene, G.; Czabotar, P. E.; Colman, P. M. BCL-2 family antagonists for cancer therapy. Nat. Rev. Drug. Discov. 2008, 7, 989– 1000, DOI: 10.1038/nrd265825https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVenu73P&md5=14886161a66c3ed7f754061fa964e1edBCL-2 family antagonists for cancer therapyLessene, Guillaume; Czabotar, Peter E.; Colman, Peter M.Nature Reviews Drug Discovery (2008), 7 (12), 989-1000CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Overexpression of members of the BCL-2 family of pro-survival proteins is commonly assocd. with unfavorable pathogenesis in cancer. The convergence of cytotoxic stress signals on the extended BCL-2 protein family provides the biol. rationale for directly targeting this family to induce apoptotic cell death. Recently, several compds. have been described that inhibit the interaction between BCL-2 family members and their natural ligand, a helical peptide sequence known as the BH3 domain. Here, we review preclin. and clin. data on these compds., and recommend four criteria that define antagonists of the BCL-2 protein family.
- 26Fire, E.; Gullá, S. V.; Grant, R. A.; Keating, A. E. Mcl-1-Bim complexes accommodate surprising point mutations via minor structural changes. Protein Sci. 2010, 19, 507– 519, DOI: 10.1002/pro.32926https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisFOhurw%253D&md5=58343d62b4cbfcfb03c53b2b667abadeMcl-1-Bim complexes accommodate surprising point mutations via minor structural changesFire, Emiko; Gulla, Stefano V.; Grant, Robert A.; Keating, Amy E.Protein Science (2010), 19 (3), 507-519CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)Mcl-1 is an antiapoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the α-helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl-2 family function. We report the crystal structure of human Mcl-1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochem. changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl-1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine-to-alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also obsd., with shifting of helix α3 accommodating an isoleucine-to-tyrosine mutation at this same position. We surveyed the variation in available Mcl-1 and Bcl-xL structures and obsd. moderate flexibility that is likely crit. for facilitating interactions of diverse BH3-only proteins with Mcl-1. With the antiapoptotic Bcl-2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl-1.
- 27Gomez-Bougie, P.; Maiga, S.; Tessoulin, B.; Bourcier, J.; Bonnet, A.; Rodriguez, M. S.; Le Gouill, S.; Touzeau, C.; Moreau, P.; Pellat-Deceunynck, C.; Amiot, M. BH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatment. Blood 2018, 132, 2656– 2669, DOI: 10.1182/blood-2018-03-83671827https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntlygs70%253D&md5=3c50a02c2e05a402bbfdfd4528dcfabcBH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatmentGomez-Bougie, Patricia; Maiga, Sophie; Tessoulin, Benoit; Bourcier, Jessie; Bonnet, Antoine; Rodriguez, Manuel S.; Le Gouill, Steven; Touzeau, Cyrille; Moreau, Philippe; Pellat-Deceunynck, Catherine; Amiot, MartineBlood (2018), 132 (25), 2656-2669CODEN: BLOOAW; ISSN:1528-0020. (American Society of Hematology)BH3 mimetics are promising drugs for hematol. malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addn., 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Interestingly, this complex was also dissocd. in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clin. evaluation in myeloma at both diagnosis and relapse.
- 28Zhang, Z.; Song, T.; Li, X.; Wu, Z.; Feng, Y.; Xie, F.; Liu, C.; Qin, J.; Chen, H. Novel soluble myeloid cell leukemia sequence 1 (Mcl-1) inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g) developed using a fragment-based approach. Eur. J. Med. Chem. 2013, 59, 141– 149, DOI: 10.1016/j.ejmech.2012.10.05028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1ersbw%253D&md5=c2b6afc34c8b7253cb7f765fc8f68c1cNovel soluble myeloid cell leukemia sequence 1 (Mcl-1) inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g) developed using a fragment-based approachZhang, Zhichao; Song, Ting; Li, Xiangqian; Wu, Zhiyong; Feng, Yingang; Xie, Feibo; Liu, Chengwu; Qin, Jianquan; Chen, HongboEuropean Journal of Medicinal Chemistry (2013), 59 (), 141-149CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Based on a known nanomolar Bcl-2 homol. domain 3 (BH3) mimetic 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b] pyrrole-9-carbonitrile (I, MW: 331), we applied a fragment-based approach to obtain BH3 mimetics with improved affinity and improved soly. in a water-ethanol (9:1) cosolvent. After the deconstruction of I, we obtained fragment cyanoacetamide (II), which was detd. to be a ligand efficiency (LE) hot part. After a rational optimization through fragment evolution beginning with fragment II, a smaller Mcl-1 inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (III, MW: 288) with a 6-fold increase in affinity compared to I was obtained, as predicted by our optimization curve and identified by Mcl-1 protein NMR (NMR).
- 29Zhang, Z.; Liu, C.; Li, X.; Song, T.; Wu, Z.; Liang, X.; Zhao, Y.; Shen, X.; Chen, H. Fragment-based design, synthesis, and biological evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxynicotinamide derivatives as novel Mcl-1 inhibitors. Eur. J. Med. Chem. 2013, 60, 410– 420, DOI: 10.1016/j.ejmech.2012.12.01629https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtVemsrk%253D&md5=418bafc7eeab83e01f5399e122b3af7dFragment-based design, synthesis, and biological evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxynicotinamide derivatives as novel Mcl-1 inhibitorsZhang, Zhichao; Liu, Chengwu; Li, Xiangqian; Song, Ting; Wu, Zhiyong; Liang, Xiaomeng; Zhao, Yan; Shen, Xiaoyun; Chen, HongboEuropean Journal of Medicinal Chemistry (2013), 60 (), 410-420CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)We have previously reported a nanomolar inhibitor of antiapoptotic Mcl-1 protein, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1). S1 plays its function by binding to the BH3 groove of Mcl-1. Basing on this spacial structural characteristic, we developed a novel class of Mcl-1 inhibitor using fragment-based drug discovery approach. By dissecting S1, we identified 2-hydroxynicotinonitrile (4) with a 2-hydroxypyridine core as the starting fragment. In the following mol. growth, we used the ligand efficiency evaluation and fit quality score to assess the fragments. A novel potent compd., N-benzyl-5-(4-isopropylthiophenol)-2-hydroxyl nicotinamide (12c), which binds Mcl-1 with an IC50 value of 54 nM was obtained. Compd. 12c demonstrated a better aq. soly. than S1.
- 30Liu, L.; Liu, R.; Yang, X.; Hou, X.; Fang, H. Design, synthesis and biological evaluation of tyrosine derivatives as Mcl-1 inhibitors. Eur. J. Med. Chem. 2020, 191, 112142, DOI: 10.1016/j.ejmech.2020.11214230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjs1OnsLw%253D&md5=e07c3dcd86e299d2bfedb4d2bb91c698Design, synthesis and biological evaluation of tyrosine derivatives as Mcl-1 inhibitorsLiu, Lulu; Liu, Renshuai; Yang, Xinying; Hou, Xuben; Fang, HaoEuropean Journal of Medicinal Chemistry (2020), 191 (), 112142CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)The upregulation of the protein myeloid cell leukemia-1 (Mcl-1) is closely assocd. with various human cancers, which can result in the evasion of apoptosis and a low survival rate. Therefore, developing Mcl-1 inhibitors has become a promising paradigm for cancer therapy. Herein, we designed and synthesized a novel series of tyrosine derivs., among which compds. (I) (R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = OBut, n = 2; R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = Ph, n = 1; R1 = naphthyl, X = Br, R2 = 4-Me-benzyl, n = 1) exhibited very high binding affinity to Mcl-1 with Ki values of 0.18, 0.27 and 0.23μM, resp. Interestingly, compd. I (R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = Ph, n = 1) showed not only potent activity against Mcl-1 but also considerable selectivity over Bcl-2 and Bcl-xL, which was rationalized by mol. docking and fragment-centric topog. mapping (FCTM). It is worth noting that compds. I (R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = OBut, n = 2; R1 = 3,5-di-Me-4-Cl-Ph, X = H, R2 = Ph, n = 1; R1 = naphthyl, X = Br, R2 = 4-Me-benzyl, n = 1) displayed potent antiproliferative activity against several cancer cell lines and could induce apoptosis of KM3 and HepG2 cells in a dose-dependent manner.
- 31Arkin, M. R.; Wells, J. A. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat. Rev. Drug. Discov. 2004, 3, 301– 317, DOI: 10.1038/nrd134331https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXis1GktrY%253D&md5=3e102c6207be420b4232056b4acdea12Small-molecule inhibitors of protein-protein interactions: progressing towards the dreamArkin, Michelle R.; Wells, James A.Nature Reviews Drug Discovery (2004), 3 (4), 301-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Protein-protein interactions have a key role in most biol. processes, and offer attractive opportunities for therapeutic intervention. Developing small mols. that modulate protein-protein interactions is difficult, owing to issues such as the lack of well-defined binding pockets. Nevertheless, there was important progress in this endeavor in recent years. Here, the authors use illustrative examples to discuss general strategies for addressing the challenges inherent in the discovery and characterization of small-mol. inhibitors of protein-protein interactions.
- 32Horne, W. S.; Boersma, M. D.; Windsor, M. A.; Gellman, S. H. Sequence-based design of alpha/beta-peptide foldamers that mimic BH3 domains. Angew. Chem., Int. Ed. Engl. 2008, 47, 2853– 2856, DOI: 10.1002/anie.20070531532https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1c3gvVSgsg%253D%253D&md5=bf421b54bd4b3bf6bee39d70bd681851Sequence-based design of alpha/beta-peptide foldamers that mimic BH3 domainsHorne W Seth; Boersma Melissa D; Windsor Matthew A; Gellman Samuel HAngewandte Chemie (International ed. in English) (2008), 47 (15), 2853-6 ISSN:.There is no expanded citation for this reference.
- 33de Araujo, A. D.; Lim, J.; Wu, K.-C.; Xiang, Y.; Good, A. C.; Skerlj, R.; Fairlie, D. P. Bicyclic helical peptides as dual inhibitors selective for Bcl2A1 and Mcl-1 proteins. J. Med. Chem. 2018, 61, 2962– 2972, DOI: 10.1021/acs.jmedchem.8b00010There is no corresponding record for this reference.
- 34Muppidi, A.; Doi, K.; Edwardraja, S.; Drake, E. J.; Gulick, A. M.; Wang, H. G.; Lin, Q. Rational design of proteolytically stable, cell-permeable peptide-based selective Mcl-1 inhibitors. J. Am. Chem. Soc. 2012, 134, 14734– 14737, DOI: 10.1021/ja306864v34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1Ghu7vN&md5=b21e87ed6a2163e51825675eb3391e5dRational Design of Proteolytically Stable, Cell-Permeable Peptide-Based Selective Mcl-1 InhibitorsMuppidi, Avinash; Doi, Kenichiro; Edwardraja, Selvakumar; Drake, Eric J.; Gulick, Andrew M.; Wang, Hong-Gang; Lin, QingJournal of the American Chemical Society (2012), 134 (36), 14734-14737CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Direct chem. modifications provide a simple and effective means to "translate" bioactive helical peptides into potential therapeutics targeting intracellular protein-protein interactions. We previously showed that distance-matching bisaryl cross-linkers can reinforce peptide helixes contg. two cysteines at the i and i+7 positions and confer cell permeability to the cross-linked peptides. Here we report the first crystal structure of a biphenyl-cross-linked Noxa peptide in complex with its target Mcl-1 at 2.0 Å resoln. Guided by this structure, we remodeled the surface of this cross-linked peptide through side-chain substitution and N-methylation and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells.
- 35Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat. Chem. Biol. 2010, 6, 595– 601, DOI: 10.1038/nchembio.39135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXns1Gis7c%253D&md5=4cc80db931dac5360d650ac9c45334eaThe MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizerStewart, Michelle L.; Fire, Emiko; Keating, Amy E.; Walensky, Loren D.Nature Chemical Biology (2010), 6 (8), 595-601CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathol. cell survival remains a formidable but pressing challenge. Such precisely tailored compds. would serve as mol. probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of stabilized alpha-helix of BCL-2 domains (SAHBs), we detd. that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallog. and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature's soln. to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.
- 36Doi, K.; Li, R.; Sung, S. S.; Wu, H.; Liu, Y.; Manieri, W.; Krishnegowda, G.; Awwad, A.; Dewey, A.; Liu, X.; Amin, S.; Cheng, C.; Qin, Y.; Schonbrunn, E.; Daughdrill, G.; Loughran, T. P., Jr.; Sebti, S.; Wang, H. G. Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation. J. Biol. Chem. 2012, 287, 10224– 10235, DOI: 10.1074/jbc.M111.33453236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktlKrsrg%253D&md5=009ebb3b74b44ad905b502e6cb505094Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal DegradationDoi, Kenichiro; Li, Rongshi; Sung, Shen-Shu; Wu, Hongwei; Liu, Yan; Manieri, Wanda; Krishnegowda, Gowdahalli; Awwad, Andy; Dewey, Alden; Liu, Xin; Amin, Shantu; Cheng, Chunwei; Qin, Yong; Schonbrunn, Ernst; Daughdrill, Gary; Loughran, Thomas P., Jr.; Sebti, Said; Wang, Hong-GangJournal of Biological Chemistry (2012), 287 (13), 10224-10235CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small mols. have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compds. that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-XL, and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degrdn. via the proteasome system, which is assocd. with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-XL-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematol. malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degrdn.
- 37Lanning, M. E.; Yu, W.; Yap, J. L.; Chauhan, J.; Chen, L.; Whiting, E.; Pidugu, L. S.; Atkinson, T.; Bailey, H.; Li, W.; Roth, B. M.; Hynicka, L.; Chesko, K.; Toth, E. A.; Shapiro, P.; MacKerell, A. D., Jr.; Wilder, P. T.; Fletcher, S. Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein. Eur. J. Med. Chem. 2016, 113, 273– 292, DOI: 10.1016/j.ejmech.2016.02.00637https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktlCitbc%253D&md5=ccf3a93af1ace19229a3e3b562e83190Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoproteinLanning, Maryanna E.; Yu, Wenbo; Yap, Jeremy L.; Chauhan, Jay; Chen, Lijia; Whiting, Ellis; Pidugu, Lakshmi S.; Atkinson, Tyler; Bailey, Hala; Li, Willy; Roth, Braden M.; Hynicka, Lauren; Chesko, Kirsty; Toth, Eric A.; Shapiro, Paul; MacKerell, Alexander D., Jr.; Wilder, Paul T.; Fletcher, StevenEuropean Journal of Medicinal Chemistry (2016), 113 (), 273-292CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-mol. inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 pocket of the protein. Significantly, target mols. were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Mol. modeling using the Site-Identification by Ligand Competitive Satn. (SILCS) approach was used to qual. direct ligand design as well as develop quant. models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions in the p2 pocket dominated affinity of the most favorable binding ligand (3bl: Ki = 31 nM). Compds. were up to 19-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 vs. Bcl-xL. The SILCS-based SAR of the present compds. represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumors and hematol. cancers, including acute myeloid leukemia.
- 38Rescourio, G.; Gonzalez, A. Z.; Jabri, S.; Belmontes, B.; Moody, G.; Whittington, D.; Huang, X.; Caenepeel, S.; Cardozo, M.; Cheng, A. C.; Chow, D.; Dou, H.; Jones, A.; Kelly, R. C.; Li, Y.; Lizarzaburu, M.; Lo, M. C.; Mallari, R.; Meleza, C.; Rew, Y.; Simonovich, S.; Sun, D.; Turcotte, S.; Yan, X.; Wong, S. G.; Yanez, E.; Zancanella, M.; Houze, J.; Medina, J. C.; Hughes, P. E.; Brown, S. P. Discovery and in vivo evaluation of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. J. Med. Chem. 2019, 62, 10258– 10271, DOI: 10.1021/acs.jmedchem.9b0131038https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFKjsL3I&md5=8717b6e16a8f3084f0b5a809911c3cc9Discovery and in Vivo Evaluation of Macrocyclic Mcl-1 Inhibitors Featuring an α-Hydroxy Phenylacetic Acid Pharmacophore or BioisostereRescourio, Gwenaella; Gonzalez, Ana Z.; Jabri, Salman; Belmontes, Brian; Moody, Gordon; Whittington, Doug; Huang, Xin; Caenepeel, Sean; Cardozo, Mario; Cheng, Alan C.; Chow, David; Dou, Hannah; Jones, Adrie; Kelly, Ron C.; Li, Yihong; Lizarzaburu, Mike; Lo, Mei-Chu; Mallari, Rommel; Meleza, Cesar; Rew, Yosup; Simonovich, Scott; Sun, Daqing; Turcotte, Simon; Yan, Xuelei; Wong, Simon G.; Yanez, Evelyn; Zancanella, Manuel; Houze, Jonathan; Medina, Julio C.; Hughes, Paul E.; Brown, Sean P.Journal of Medicinal Chemistry (2019), 62 (22), 10258-10271CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Overexpression of the antiapoptotic protein Mcl-1 provides a survival advantage to some cancer cells, making inhibition of this protein an attractive therapeutic target for the treatment of certain types of tumors. Herein, we report our efforts toward the identification of a novel series of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. This work led to the discovery of 1(I), a potent Mcl-1 inhibitor (IC50 = 19 nM in an OPM-2 cell viability assay) with good pharmacokinetic properties and excellent in vivo efficacy in an OPM-2 multiple myeloma xenograft model.
- 39Song, T.; Wang, Z.; Ji, F.; Feng, Y.; Fan, Y.; Chai, G.; Li, X.; Li, Z.; Zhang, Z. Deactivation of Mcl-1 by dual-function small-molecule inhibitors targeting the bcl-2 homology 3 domain and facilitating mcl-1 ubiquitination. Angew. Chem., Int. Ed. Engl. 2016, 55, 14250– 14256, DOI: 10.1002/anie.20160654339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2svmsVyrtA%253D%253D&md5=687a3613109274c1a2fd2519cc1af45fDeactivation of Mcl-1 by Dual-Function Small-Molecule Inhibitors Targeting the Bcl-2 Homology 3 Domain and Facilitating Mcl-1 UbiquitinationSong Ting; Wang Ziqian; Chai Gaobo; Li Zhiqiang; Zhang Zhichao; Ji Fangling; Fan Yudan; Feng Yingang; Li XiangqianAngewandte Chemie (International ed. in English) (2016), 55 (46), 14250-14256 ISSN:.By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. Noxa(BH3) binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, Bim(BH3) binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.
- 40Wang, Z.; He, N.; Guo, Z.; Niu, C.; Song, T.; Guo, Y.; Cao, K.; Wang, A.; Zhu, J.; Zhang, X.; Zhang, Z. Proteolysis targeting chimeras for the selective degradation of Mcl-1/Bcl-2 derived from nonselective target binding ligands. J. Med. Chem. 2019, 62, 8152– 8163, DOI: 10.1021/acs.jmedchem.9b0091940https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFemtLvN&md5=f962278d69b69117c501bc7c0c31ebfdProteolysis Targeting Chimeras for the Selective Degradation of Mcl-1/Bcl-2 Derived from Nonselective Target Binding LigandsWang, Ziqian; He, Nianzhe; Guo, Zongwei; Niu, Cuili; Song, Ting; Guo, Yafei; Cao, Keke; Wang, Anhui; Zhu, Junjie; Zhang, Xiaodong; Zhang, ZhichaoJournal of Medicinal Chemistry (2019), 62 (17), 8152-8163CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Proteolysis Targeting Chimera (PROTAC) recruits an E3 ligase to a target protein to induce its ubiquitination and subsequent degrdn. We reported the success in the development of two PROTACs (C3(I) and C5(II)) that potently and selectively induce the degrdn. of Mcl-1 and Bcl-2 (DC50 = 0.7 and 3.0 μM), resp., by introducing the E3 ligase cereblon (CRBN)-binding ligand pomalidomide to Mcl-1/Bcl-2 dual inhibitors S1-6 and Nap-1 with μM-range affinity. I-induced Mcl-1 ubiquitination translated into much more lethality in Mcl-1-dependent H23 cells than the most potent Mcl-1 occupancy-based inhibitor A-1210477 with nM-range affinity. Moreover, structure-activity relationship (SAR) anal. and mol. dynamic (MD) simulations discovered the structural basis for turning nonselective or promiscuous Bcl-2 family ligands into selective PROTACs. I and II exhibited reversible depletion in living cells, which provides a new potent toolkit for gain-of-function studies to probe the dynamic roles of Bcl-2 and Mcl-1 in apoptosis networks.
- 41Wang, Z.; Guo, Z.; Song, T.; Zhang, X.; He, N.; Liu, P.; Wang, P.; Zhang, Z. Proteome-wide identification of on- and off-targets of Bcl-2 inhibitors in native biological systems by using affinity-based probes (afbps). ChemBioChem 2018, 19, 2312– 2320, DOI: 10.1002/cbic.20180038041https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFequr%252FI&md5=8ca5bbf1ae20f5ce024f72f431dd9166Proteome-Wide Identification of On- and Off-Targets of Bcl-2 Inhibitors in Native Biological Systems by Using Affinity-Based Probes (AfBPs)Wang, Ziqian; Guo, Zongwei; Song, Ting; Zhang, Xiaodong; He, Nianzhe; Liu, Peng; Wang, Peiran; Zhang, ZhichaoChemBioChem (2018), 19 (21), 2312-2320CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Selective inhibition of proteins of the Bcl-2 family by small-mol. inhibitors is a promising new approach in drug discovery. However, information about how these mols. interact with their cellular targets (on- and off-) is highly limited. We have designed and synthesized photoreactive and "clickable" affinity-based probes (AfBPs)-Nap-2 and Nap-5-by introducing photo-crosslinkers onto Nap-1, a fluorescent deriv. of small-mol. Bcl-2 inhibitor S1-6. The resulting trifunctional probes Nap-2 and Nap-5 can enrich, visualize, and enable the identification of cellular on- and off-targets of Bcl-2 inhibitors both in vitro and in situ. Tubulin was validated as an off-target of Bcl-2 inhibitors (Nap-1 and S1-6) by large-scale cell-based proteome profiling and pull-down/western blotting (PD/WB) with Nap-2 and Nap-5. It was preliminarily illustrated to be a BH3-contg. protein because some well-known Bcl-2 inhibitors can block the labeling of tubulin by Nap-2.
- 42Zhang, Z.; Wu, G.; Xie, F.; Song, T.; Chang, X. 3-Thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) based molecules as potent, dual inhibitors of B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1): structure-based design and structure-activity relationship studies. J. Med. Chem. 2011, 54, 1101– 1105, DOI: 10.1021/jm101181u42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVeiuw%253D%253D&md5=08ad63a3a7bfb4afd477f63572d2f16c3-Thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) Based Molecules as Potent, Dual Inhibitors of B-Cell Lymphoma 2 (Bcl-2) and Myeloid Cell Leukemia Sequence 1 (Mcl-1): Structure-Based Design and Structure-Activity Relationship StudiesZhang, Zhichao; Wu, Guiye; Xie, Feibo; Song, Ting; Chang, XilongJournal of Medicinal Chemistry (2011), 54 (4), 1101-1105CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)the authors recently described the discovery of a dual inhibitor of Bcl-2 and Mcl-1, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3, S1). Here the authors report a structure-guided design in combination with structure-activity relationship studies to exploit the difference in the p2 binding pocket of Bcl-2 and Mcl-1, from which a novel dual inhibitor 3-(4-aminophenylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (6h) was obtained, which showed significant enhanced IC50 value against Mcl-1 (5 nM), greater Mcl-1/Bak disruption potential, and accordingly a 10-fold increased cytotoxicity over 3.
- 43Bruncko, M.; Song, X.; Tao, Z.; Kunzer, A. R. 7-nonsubstituted Indole Mcl-1 Inhibitors. WO2008130970Al, 2008.There is no corresponding record for this reference.
- 44Elmore, S. W.; Souers, A. J.; Bruncko, M.; Song, X.; Wang, X.; Hasvold, L. A.; Wang, L.; Kunzer, A. R.; Park, C.; Wendt, M. D.; Tao, Z. 7-sonsubstituted Indole Mcl-1 Inhibitors. WO2008131000A2, 2008.There is no corresponding record for this reference.
- 45Friberg, A.; Vigil, D.; Zhao, B.; Daniels, R. N.; Burke, J. P.; Garcia-Barrantes, P. M.; Camper, D.; Chauder, B. A.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design. J. Med. Chem. 2013, 56, 15– 30, DOI: 10.1021/jm301448p45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCjur3J&md5=74e72c3d56cdff85d263bd974319b2dbDiscovery of Potent Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods and Structure-Based DesignFriberg, Anders; Vigil, Dominico; Zhao, Bin; Daniels, R. Nathan; Burke, Jason P.; Garcia-Barrantes, Pedro M.; Camper, DeMarco; Chauder, Brian A.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2013), 56 (1), 15-30CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chem. distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compds. that bind to Mcl-1 with a dissocn. const. of <100 nM with selectivity for Mcl-1 over Bcl-xL and Bcl-2. Structures of merged compds. when complexed to Mcl-1 were obtained by x-ray crystallog. and provide detailed information about the mol. recognition of small-mol. ligands binding Mcl-1. The compds. represent starting points for the discovery of clin. useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.
- 46Burke, J. P.; Bian, Z.; Shaw, S.; Zhao, B.; Goodwin, C. M.; Belmar, J.; Browning, C. F.; Vigil, D.; Friberg, A.; Camper, D. V.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of tricyclic indoles that potently inhibit Mcl-1 using fragment-based methods and structure-based design. J. Med. Chem. 2015, 58, 3794– 3805, DOI: 10.1021/jm501984f46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtVWkurw%253D&md5=ea5fc51491138dc7a79dd12f813c4bcaDiscovery of Tricyclic Indoles That Potently Inhibit Mcl-1 Using Fragment-Based Methods and Structure-Based DesignBurke, Jason P.; Bian, Zhiguo; Shaw, Subrata; Zhao, Bin; Goodwin, Craig M.; Belmar, Johannes; Browning, Carrie F.; Vigil, Dominico; Friberg, Anders; Camper, DeMarco V.; Rossanese, Olivia W.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2015), 58 (9), 3794-3805CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with various chemotherapies. From an NMR-based screen of a large fragment library, several distinct chem. scaffolds that bind to Mcl-1 were discovered. Here, the authors describe the discovery of potent tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100-fold selectivity over Bcl-2. X-ray structures of these compds. when complexed to Mcl-1 provide detailed information on how these small-mols. bind to the target, which was used to guide compd. optimization.
- 47Pelz, N. F.; Bian, Z.; Zhao, B.; Shaw, S.; Tarr, J. C.; Belmar, J.; Gregg, C.; Camper, D. V.; Goodwin, C. M.; Arnold, A. L.; Sensintaffar, J. L.; Friberg, A.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of 2-indole-acylsulfonamide myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods. J. Med. Chem. 2016, 59, 2054– 2066, DOI: 10.1021/acs.jmedchem.5b0166047https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisVygsbs%253D&md5=e667fa5bc866d29ad02d6d52c679f47aDiscovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based MethodsPelz, Nicholas F.; Bian, Zhiguo; Zhao, Bin; Shaw, Subrata; Tarr, James C.; Belmar, Johannes; Gregg, Claire; Camper, DeMarco V.; Goodwin, Craig M.; Arnold, Allison L.; Sensintaffar, John L.; Friberg, Anders; Rossanese, Olivia W.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2016), 59 (5), 2054-2066CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins responsible for the regulation of programmed cell death. Amplification of Mcl-1 is a common genetic aberration in human cancer whose overexpression contributes to the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-apoptotic BH3 contg. proteins that achieve high affinity for the target by utilizing four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1 inhibitors using fragment-based methods and structure-based design. These novel inhibitors exhibit low nanomolar binding affinities to Mcl-1 and >500-fold selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to Mcl-1 provided detailed information on how these small-mols. bind to the target and were used extensively to guide compd. optimization.
- 48Shaw, S.; Bian, Z.; Zhao, B.; Tarr, J. C.; Veerasamy, N.; Jeon, K. O.; Belmar, J.; Arnold, A. L.; Fogarty, S. A.; Perry, E.; Sensintaffar, J. L.; Camper, D. V.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Optimization of potent and selective tricyclic indole diazepinone myeloid cell leukemia-1 inhibitors using structure-based design. J. Med. Chem. 2018, 61, 2410– 2421, DOI: 10.1021/acs.jmedchem.7b0115548https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotVyktA%253D%253D&md5=719511e2b52849e2bff5cf1de6d6c2beOptimization of Potent and Selective Tricyclic Indole Diazepinone Myeloid Cell Leukemia-1 Inhibitors Using Structure-Based DesignShaw, Subrata; Bian, Zhiguo; Zhao, Bin; Tarr, James C.; Veerasamy, Nagarathanam; Jeon, Kyu Ok; Belmar, Johannes; Arnold, Allison L.; Fogarty, Stuart A.; Perry, Evan; Sensintaffar, John L.; Camper, DeMarco V.; Rossanese, Olivia W.; Lee, Taekyu; Olejniczak, Edward T.; Fesik, Stephen W.Journal of Medicinal Chemistry (2018), 61 (6), 2410-2421CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, has emerged as an attractive target for cancer therapy. Mcl-1 upregulation is often found in many human cancers and is assocd. with high tumor grade, poor survival, and resistance to chemotherapy. Here, we describe a series of potent and selective tricyclic indole diazepinone Mcl-1 inhibitors that were discovered and further optimized using structure-based design. These compds. exhibit picomolar binding affinity and mechanism-based cellular efficacy, including growth inhibition and caspase induction in Mcl-1-sensitive cells. Thus, they represent useful compds. to study the implication of Mcl-1 inhibition in cancer and serve as potentially useful starting points toward the discovery of anti-Mcl-1 therapeutics.
- 49Lee, T.; Christov, P. P.; Shaw, S.; Tarr, J. C.; Zhao, B.; Veerasamy, N.; Jeon, K. O.; Mills, J. J.; Bian, Z.; Sensintaffar, J. L.; Arnold, A. L.; Fogarty, S. A.; Perry, E.; Ramsey, H. E.; Cook, R. S.; Hollingshead, M.; Davis Millin, M.; Lee, K. M.; Koss, B.; Budhraja, A.; Opferman, J. T.; Kim, K.; Arteaga, C. L.; Moore, W. J.; Olejniczak, E. T.; Savona, M. R.; Fesik, S. W. Discovery of potent myeloid cell leukemia-1 (Mcl-1) inhibitors that demonstrate in vivo activity in mouse xenograft models of human cancer. J. Med. Chem. 2019, 62, 3971– 3988, DOI: 10.1021/acs.jmedchem.8b0199149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtlWitb0%253D&md5=a3e80ec8c20cc39a71c78beb85d2ec90Discovery of Potent Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors That Demonstrate in Vivo Activity in Mouse Xenograft Models of Human CancerLee, Taekyu; Christov, Plamen P.; Shaw, Subrata; Tarr, James C.; Zhao, Bin; Veerasamy, Nagarathanam; Jeon, Kyu Ok; Mills, Jonathan J.; Bian, Zhiguo; Sensintaffar, John L.; Arnold, Allison L.; Fogarty, Stuart A.; Perry, Evan; Ramsey, Haley E.; Cook, Rebecca S.; Hollingshead, Melinda; Davis Millin, Myrtle; Lee, Kyung-min; Koss, Brian; Budhraja, Amit; Opferman, Joseph T.; Kim, Kwangho; Arteaga, Carlos L.; Moore, William J.; Olejniczak, Edward T.; Savona, Michael R.; Fesik, Stephen W.Journal of Medicinal Chemistry (2019), 62 (8), 3971-3988CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Overexpression of myeloid cell leukemia-1 (Mcl-1) in cancers correlates with high tumor grade and poor survival. Addnl., Mcl-1 drives intrinsic and acquired resistance to many cancer therapeutics, including B cell lymphoma 2 family inhibitors, proteasome inhibitors, and antitubulins. Therefore, Mcl-1 inhibition could serve as a strategy to target cancers that require Mcl-1 to evade apoptosis. Herein, we describe the use of structure-based design to discover a novel compd. (42) that robustly and specifically inhibits Mcl-1 in cell culture and animal xenograft models. Compd. 42 binds to Mcl-1 with picomolar affinity and inhibited growth of Mcl-1-dependent tumor cell lines in the nanomolar range. Compd. 42 also inhibited the growth of hematol. and triple neg. breast cancer xenografts at well-tolerated doses. These findings highlight the use of structure-based design to identify small mol. Mcl-1 inhibitors and support the use of 42 as a potential treatment strategy to block Mcl-1 activity and induce apoptosis in Mcl-1-dependent cancers.
- 50Ramsey, H. E.; Fischer, M. A.; Lee, T.; Gorska, A. E.; Arrate, M. P.; Fuller, L.; Boyd, K. L.; Strickland, S. A.; Sensintaffar, J.; Hogdal, L. J.; Ayers, G. D.; Olejniczak, E. T.; Fesik, S. W.; Savona, M. R. A novel Mcl-1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia. Cancer. Discov. 2018, 8, 1566– 1581, DOI: 10.1158/2159-8290.CD-18-014050https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFOmsLfN&md5=85fecfd85be4606dfa867193f4cf871dA novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemiaRamsey, Haley E.; Fischer, Melissa A.; Lee, Taekyu; Gorska, Agnieszka E.; Arrate, Maria Pia; Fuller, Londa; Boyd, Kelli L.; Strickland, Stephen A.; Sensintaffar, John; Hogdal, Leah J.; Ayers, Gregory D.; Olejniczak, Edward T.; Fesik, Stephen W.; Savona, Michael R.Cancer Discovery (2018), 8 (12), 1566-1581CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 assocn., leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addn., VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clin. trials.
- 51Bruncko, M.; Wang, L.; Sheppard, G. S.; Phillips, D. C.; Tahir, S. K.; Xue, J.; Erickson, S.; Fidanze, S.; Fry, E.; Hasvold, L.; Jenkins, G. J.; Jin, S.; Judge, R. A.; Kovar, P. J.; Madar, D.; Nimmer, P.; Park, C.; Petros, A. M.; Rosenberg, S. H.; Smith, M. L.; Song, X.; Sun, C.; Tao, Z. F.; Wang, X.; Xiao, Y.; Zhang, H.; Tse, C.; Leverson, J. D.; Elmore, S. W.; Souers, A. J. Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity. J. Med. Chem. 2015, 58, 2180– 2194, DOI: 10.1021/jm501258m51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXislGjt70%253D&md5=e8c575a04fecaf47e4a9fc5c8348e034Structure-Guided Design of a Series of MCL-1 Inhibitors with High Affinity and SelectivityBruncko, Milan; Wang, Le; Sheppard, George S.; Phillips, Darren C.; Tahir, Stephen K.; Xue, John; Erickson, Scott; Fidanze, Steve; Fry, Elizabeth; Hasvold, Lisa; Jenkins, Gary J.; Jin, Sha; Judge, Russell A.; Kovar, Peter J.; Madar, David; Nimmer, Paul; Park, Chang; Petros, Andrew M.; Rosenberg, Saul H.; Smith, Morey L.; Song, Xiaohong; Sun, Chaohong; Tao, Zhi-Fu; Wang, Xilu; Xiao, Yu; Zhang, Haichao; Tse, Chris; Leverson, Joel D.; Elmore, Steven W.; Souers, Andrew J.Journal of Medicinal Chemistry (2015), 58 (5), 2180-2194CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein the authors report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compds. from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compd. I rapidly induced caspase activation with assocd. loss in cell viability. The small mols. described herein thus comprise effective tools for studying MCL-1 biol.
- 52Leverson, J. D.; Zhang, H.; Chen, J.; Tahir, S. K.; Phillips, D. C.; Xue, J.; Nimmer, P.; Jin, S.; Smith, M.; Xiao, Y.; Kovar, P.; Tanaka, A.; Bruncko, M.; Sheppard, G. S.; Wang, L.; Gierke, S.; Kategaya, L.; Anderson, D. J.; Wong, C.; Eastham-Anderson, J.; Ludlam, M. J. C.; Sampath, D.; Fairbrother, W. J.; Wertz, I.; Rosenberg, S. H.; Tse, C.; Elmore, S. W.; Souers, A. J. Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax). Cell. Death. Dis. 2015, 6, e1590 DOI: 10.1038/cddis.2014.56152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFGrurg%253D&md5=8ceb779fc6868fc0828757364d529e34Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax)Leverson, J. D.; Zhang, H.; Chen, J.; Tahir, S. K.; Phillips, D. C.; Xue, J.; Nimmer, P.; Jin, S.; Smith, M.; Xiao, Y.; Kovar, P.; Tanaka, A.; Bruncko, M.; Sheppard, G. S.; Wang, L.; Gierke, S.; Kategaya, L.; Anderson, D. J.; Wong, C.; Eastham-Anderson, J.; Ludlam, M. J. C.; Sampath, D.; Fairbrother, W. J.; Wertz, I.; Rosenberg, S. H.; Tse, C.; Elmore, S. W.; Souers, A. J.Cell Death & Disease (2015), 6 (1), e1590CODEN: CDDEA4; ISSN:2041-4889. (Nature Publishing Group)The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-mol. BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small mols. potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compd. A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue expts. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-mol. MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these mols. as chem. tools for dissecting the basic biol. of MCL-1 and the promise of small-mol. MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
- 53Xiao, Y.; Nimmer, P.; Sheppard, G. S.; Bruncko, M.; Hessler, P.; Lu, X.; Roberts-Rapp, L.; Pappano, W. N.; Elmore, S. W.; Souers, A. J.; Leverson, J. D.; Phillips, D. C. Mcl-1 is a key determinant of breast cancer cell survival: validation of Mcl-1 dependency utilizing a highly selective small molecule inhibitor. Mol. Cancer Ther. 2015, 14, 1837– 1847, DOI: 10.1158/1535-7163.MCT-14-092853https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1yrs7vL&md5=c6d4077d4e1f1feb92d2fa22b88ab2fbMCL-1 Is a Key Determinant of Breast Cancer Cell Survival: Validation of MCL-1 Dependency Utilizing a Highly Selective Small Molecule InhibitorXiao, Yu; Nimmer, Paul; Sheppard, George S.; Bruncko, Milan; Hessler, Paul; Lu, Xin; Roberts-Rapp, Lisa; Pappano, William N.; Elmore, Steven W.; Souers, Andrew J.; Leverson, Joel D.; Phillips, Darren C.Molecular Cancer Therapeutics (2015), 14 (8), 1837-1847CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Hyperexpression of antiapoptotic BCL-2 family proteins allows cells to survive despite the receipt of signals that would ordinarily induce their deletion, a facet frequently exploited by tumors. Tumors addicted to the BCL-2 family proteins for survival are now being targeted therapeutically. For example, navitoclax, a BCL-2/BCL-XL/BCL-W inhibitor, is currently in phase I/II clin. trials in numerous malignancies. However, the related family member, MCL-1, limits the efficacy of navitoclax and other chemotherapeutic agents. In the present study, we identify breast cancer cell lines that depend upon MCL-1 for survival and subsequently det. the mechanism of apoptosis mediated by the MCL-1 selective inhibitor A-1210477. We demonstrate that apoptosis resulting from a loss in MCL-1 function requires expression of the proapoptotic protein BAK. However, expression of BCL-XL can limit apoptosis resulting from loss in MCL-1 function through sequestration of free BIM. Finally, we demonstrate substantial synergy between navitoclax and MCL-1 siRNA, the direct MCL-1 inhibitor A-1210477, or the indirect MCL-1 inhibitor flavopiridol, highlighting the therapeutic potential for inhibiting BCL-XL and MCL-1 in breast cancer. Mol Cancer Ther; 14(8); 1837-47. cpr2015 AACR.
- 54Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chain. Nat. Chem. Biol. 2016, 12, 931– 936, DOI: 10.1038/nchembio.217454https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKqtb3P&md5=bda5720579a9b5e6027632d2cffba2e1Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chainAkcay, Gizem; Belmonte, Matthew A.; Aquila, Brian; Chuaqui, Claudio; Hird, Alexander W.; Lamb, Michelle L.; Rawlins, Philip B.; Su, Nancy; Tentarelli, Sharon; Grimster, Neil P.; Su, QibinNature Chemical Biology (2016), 12 (11), 931-936CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Targeted covalent inhibition of disease-assocd. proteins has become a powerful methodol. in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here the authors used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to the knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chem. strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochem. and cell-based assays. The authors identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biol. phenomena.
- 55Papatzimas, J. W.; Gorobets, E.; Maity, R.; Muniyat, M. I.; MacCallum, J. L.; Neri, P.; Bahlis, N. J.; Derksen, D. J. From inhibition to degradation: targeting the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1). J. Med. Chem. 2019, 62, 5522– 5540, DOI: 10.1021/acs.jmedchem.9b0045555https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVSnsb%252FL&md5=4a28b945723e00cf9a2ef3916a07da42From Inhibition to Degradation: Targeting the Antiapoptotic Protein Myeloid Cell Leukemia 1 (MCL1)Papatzimas, James W.; Gorobets, Evgueni; Maity, Ranjan; Muniyat, Mir Ishruna; MacCallum, Justin L.; Neri, Paola; Bahlis, Nizar J.; Derksen, Darren J.Journal of Medicinal Chemistry (2019), 62 (11), 5522-5540CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Protein-protein interactions (PPIs) have emerged as significant targets for therapeutic development, owing to their crit. nature in diverse biol. processes. An ideal PPI-based target is the protein myeloid cell leukemia 1 (MCL1), a crit. prosurvival factor in cancers such as multiple myeloma where MCL1 levels directly correlate to disease progression. Current strategies for halting the antiapoptotic properties of MCL1 revolve around inhibiting its sequestration of proapoptotic factors. Existing inhibitors disrupt endogenous regulatory proteins; however, this strategy actually increases MCL1 protein levels. Here, the authors show the development of hetero-bifunctional small mols. capable of selectively targeting MCL1 using a proteolysis targeting chimera (PROTAC) methodol. leading to successful degrdn. The authors confirmed the involvement of the E3 ligase CUL4A-DDB1 cereblon ubiquitination pathway, making these PROTACs a first step toward a new class of antiapoptotic B-cell lymphoma 2 family protein degraders.
- 56Johannes, J. W.; Bates, S.; Beigie, C.; Belmonte, M. A.; Breen, J.; Cao, S.; Centrella, P. A.; Clark, M. A.; Cuozzo, J. W.; Dumelin, C. E.; Ferguson, A. D.; Habeshian, S.; Hargreaves, D.; Joubran, C.; Kazmirski, S.; Keefe, A. D.; Lamb, M. L.; Lan, H.; Li, Y.; Ma, H.; Mlynarski, S.; Packer, M. J.; Rawlins, P. B.; Robbins, D. W.; Shen, H.; Sigel, E. A.; Soutter, H. H.; Su, N.; Troast, D. M.; Wang, H.; Wickson, K. F.; Wu, C.; Zhang, Y.; Zhao, Q.; Zheng, X.; Hird, A. W. Structure based design of non-natural peptidic macrocyclic Mcl-1 inhibitors. ACS Med. Chem. Lett. 2017, 8, 239– 244, DOI: 10.1021/acsmedchemlett.6b0046456https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFCqtLzP&md5=42f9a8d863aedfe95231e29ffa4673b8Structure based design of non-natural peptidic macrocyclic Mcl-1 inhibitorsJohannes, Jeffrey W.; Bates, Stephanie; Beigie, Carl; Belmonte, Matt; Breen, John; Cao, Shenggen; Centrella, Paolo A.; Clark, Matthew A.; Cuozzo, John W.; Dumelin, Christoph E.; Ferguson, Andrew D.; Habeshian, Sevan; Hargreaves, David; Joubran, Camil; Kazmirski, Steven; Keefe, Anthony D.; Lamb, Michelle L.; Lan, Haiye; Li, Yunxia; Ma, Hao; Mlynarski, Scott; Packer, Martin J.; Rawlins, Philip B.; Robbins, Daniel W.; Shen, Haidong; Sigel, Eric A.; Soutter, Holly H.; Su, Nancy; Troast, Dawn M.; Wang, Haiyun; Wickson, Kate F.; Wu, Chengyan; Zhang, Ying; Zhao, Qiuying; Zheng, Xiaolan; Hird, Alexander W.ACS Medicinal Chemistry Letters (2017), 8 (2), 239-244CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compd. (I), a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compd. I bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approx. 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to addnl. potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compd. (II) is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4-11 cells with an IC50 of 3 μM after 6 h.
- 57Tron, A. E.; Belmonte, M. A.; Adam, A.; Aquila, B. M.; Boise, L. H.; Chiarparin, E.; Cidado, J.; Embrey, K. J.; Gangl, E.; Gibbons, F. D.; Gregory, G. P.; Hargreaves, D.; Hendricks, J. A.; Johannes, J. W.; Johnstone, R. W.; Kazmirski, S. L.; Kettle, J. G.; Lamb, M. L.; Matulis, S. M.; Nooka, A. K.; Packer, M. J.; Peng, B.; Rawlins, P. B.; Robbins, D. W.; Schuller, A. G.; Su, N.; Yang, W.; Ye, Q.; Zheng, X.; Secrist, J. P.; Clark, E. A.; Wilson, D. M.; Fawell, S. E.; Hird, A. W. Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia. Nat. Commun. 2018, 9, 5341, DOI: 10.1038/s41467-018-07551-w57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFersLjK&md5=10ccf1fda947aca212bede3927eaabfaDiscovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemiaTron, Adriana E.; Belmonte, Matthew A.; Adam, Ammar; Aquila, Brian M.; Boise, Lawrence H.; Chiarparin, Elisabetta; Cidado, Justin; Embrey, Kevin J.; Gangl, Eric; Gibbons, Francis D.; Gregory, Gareth P.; Hargreaves, David; Hendricks, J. Adam; Johannes, Jeffrey W.; Johnstone, Ricky W.; Kazmirski, Steven L.; Kettle, Jason G.; Lamb, Michelle L.; Matulis, Shannon M.; Nooka, Ajay K.; Packer, Martin J.; Peng, Bo; Rawlins, Philip B.; Robbins, Daniel W.; Schuller, Alwin G.; Su, Nancy; Yang, Wenzhan; Ye, Qing; Zheng, Xiaolan; Secrist, J. Paul; Clark, Edwin A.; Wilson, David M.; Fawell, Stephen E.; Hird, Alexander W.Nature Communications (2018), 9 (1), 5341CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic mol. with high selectivity and affinity for Mcl-1 currently in clin. development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clin. trial has been launched for evaluation of AZD5991 in patients with hematol. malignancies (NCT03218683).
- 58Sale, M. J.; Minihane, E.; Monks, N. R.; Gilley, R.; Richards, F. M.; Schifferli, K. P.; Andersen, C. L.; Davies, E. J.; Vicente, M. A.; Ozono, E.; Markovets, A.; Dry, J. R.; Drew, L.; Flemington, V.; Proia, T.; Jodrell, D. I.; Smith, P. D.; Cook, S. J. Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors. Nat. Commun. 2019, 10, 5167, DOI: 10.1038/s41467-019-12409-w58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mjpt1CqtQ%253D%253D&md5=5486bbf53a2adc4521226cd45a76daefTargeting melanoma's MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitorsSale Matthew J; Minihane Emma; Gilley Rebecca; Ozono Eiko; Cook Simon J; Monks Noel R; Schifferli Kevin P; Richards Frances M; Jodrell Duncan I; Andersen Courtney L; Markovets Aleksandra; Dry Jonathan R; Drew Lisa; Proia Theresa; Davies Emma J; Flemington Vikki; Smith Paul D; Vicente Mario AladrenNature communications (2019), 10 (1), 5167 ISSN:.BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.
- 59Zhang, H.; Nakauchi, Y.; Köhnke, T.; Stafford, M.; Bottomly, D.; Thomas, R.; Wilmot, B.; McWeeney, S. K.; Majeti, R.; Tyner, J. W. Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia. Nat. Cancer. 2020, 1, 826– 839, DOI: 10.1038/s43018-020-0103-x59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7otFGitA%253D%253D&md5=44f856de2ba9280f69683c24373c39fcIntegrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemiaZhang Haijiao; Tyner Jeffrey W; Nakauchi Yusuke; Kohnke Thomas; Stafford Melissa; Thomas Rozario; Majeti Ravindra; Bottomly Daniel; Wilmot Beth; McWeeney Shannon KNature cancer (2020), 1 (8), 826-839 ISSN:.Deregulation of the BCL2 gene family plays an important role in the pathogenesis of acute myeloid leukemia (AML). The BCL2 inhibitor, venetoclax, has received FDA approval for the treatment of AML. However, upfront and acquired drug resistance ensues due, in part, to the clinical and genetic heterogeneity of AML, highlighting the importance of identifying biomarkers to stratify patients onto the most effective therapies. By integrating clinical characteristics, exome and RNA sequencing, and inhibitor data from primary AML patient samples, we determined that myelomonocytic leukemia, upregulation of BCL2A1 and CLEC7A, as well as mutations of PTPN11 and KRAS conferred resistance to venetoclax and multiple venetoclax combinations. Venetoclax in combination with an MCL1 inhibitor AZD5991 induced synthetic lethality and circumvented venetoclax resistance.
- 60Caenepeel, S.; Brown, S. P.; Belmontes, B.; Moody, G.; Keegan, K. S.; Chui, D.; Whittington, D. A.; Huang, X.; Poppe, L.; Cheng, A. C.; Cardozo, M.; Houze, J.; Li, Y.; Lucas, B.; Paras, N. A.; Wang, X.; Taygerly, J. P.; Vimolratana, M.; Zancanella, M.; Zhu, L.; Cajulis, E.; Osgood, T.; Sun, J.; Damon, L.; Egan, R. K.; Greninger, P.; McClanaghan, J. D.; Gong, J.; Moujalled, D.; Pomilio, G.; Beltran, P.; Benes, C. H.; Roberts, A. W.; Huang, D. C.; Wei, A.; Canon, J.; Coxon, A.; Hughes, P. E. AMG 176, a selective Mcl-1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapies. Cancer. Discov. 2018, 8, 1582– 1597, DOI: 10.1158/2159-8290.CD-18-038760https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFOmsb7E&md5=795a713908c3635bbd5e9fc3d95f3ae0AMG 176, a selective MCL1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapiesCaenepeel, Sean; Brown, Sean P.; Belmontes, Brian; Moody, Gordon; Keegan, Kathleen S.; Chui, Danny; Whittington, Douglas A.; Huang, Xin; Poppe, Leszek; Cheng, Alan C.; Cardozo, Mario; Houze, Jonathan; Li, Yunxiao; Lucas, Brian; Paras, Nick A.; Wang, Xianghong; Taygerly, Joshua P.; Vimolratana, Marc; Zancanella, Manuel; Zhu, Liusheng; Cajulis, Elaina; Osgood, Tao; Sun, Jan; Damon, Leah; Egan, Regina K.; Greninger, Patricia; McClanaghan, Joseph D.; Gong, Jianan; Moujalled, Donia; Pomilio, Giovanna; Beltran, Pedro; Benes, Cyril H.; Roberts, Andrew W.; Huang, David C.; Wei, Andrew; Canon, Jude; Coxon, Angela; Hughes, Paul E.Cancer Discovery (2018), 8 (12), 1582-1597CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges assocd. with inhibition of MCL1 protein-protein interactions, we rigorously applied small-mol. conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematol. cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by redns. in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics.
- 61Lu, X.; Liu, Y. C.; Orvig, C.; Liang, H.; Chen, Z. F. Discovery of a Copper-Based Mcl-1 Inhibitor as an Effective Antitumor Agent. J. Med. Chem. 2020, 63, 9154– 9167, DOI: 10.1021/acs.jmedchem.9b0204761https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Sku7zF&md5=cb3ffc5ac05fb8dc509c2d82d730faf7Discovery of a Copper-Based Mcl-1 Inhibitor as an Effective Antitumor AgentLu, Xing; Liu, Yan-Cheng; Orvig, Chris; Liang, Hong; Chen, Zhen-FengJournal of Medicinal Chemistry (2020), 63 (17), 9154-9167CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), which belongs to the Bcl-2 family of prosurvival proteins, is a key regulator of cancer cell survival. To date, few drug-like Mcl-1 inhibitors have been reported. Herein, it is reported the prepn. of 10 copper complexes with 9-substituted β-carboline ligands that act as metal-based Mcl-1 inhibitors. Complex (I) was identified as a potent and selective Mcl-1 inhibitor with strong in vitro antitumor activity. Mechanistic studies demonstrated that I disrupted Mcl-1-Bax/Bak heterodimerization and induced Bax/Bak-dependent apoptosis. In addn., I significantly (P < 0.001) inhibited tumor growth in vivo, induced tumor necrosis, and extended survival time in an NCI-H460 xenograft model. Furthermore, I showed no apparent toxicity in mice. Together, these findings indicate that I is a copper-based Mcl-1 inhibitor with high efficacy and low toxicity that could be developed for the treatment of Mcl-1-related cancers.
- 62Abulwerdi, F.; Liao, C.; Liu, M.; Azmi, A. S.; Aboukameel, A.; Mady, A. S.; Gulappa, T.; Cierpicki, T.; Owens, S.; Zhang, T.; Sun, D.; Stuckey, J. A.; Mohammad, R. M.; Nikolovska-Coleska, Z. A novel small-molecule inhibitor of Mcl-1 blocks pancreatic cancer growth in vitro and in vivo. Mol. Cancer Ther. 2014, 13, 565– 575, DOI: 10.1158/1535-7163.MCT-12-076762https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjslGisL8%253D&md5=8f1686d2e7ddc6e1aa59dcfb902176bbA Novel Small-Molecule Inhibitor of Mcl-1 Blocks Pancreatic Cancer Growth In Vitro and In VivoAbulwerdi, Fardokht; Liao, Chenzhong; Liu, Meilan; Azmi, Asfar S.; Aboukameel, Amro; Mady, Ahmed S. A.; Gulappa, Thippeswamy; Cierpicki, Tomasz; Owens, Scott; Zhang, Tao; Sun, Duxin; Stuckey, Jeanne A.; Mohammad, Ramzi M.; Nikolovska-Coleska, ZanetaMolecular Cancer Therapeutics (2014), 13 (3), 565-575CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Using a high-throughput screening (HTS) approach, we have identified and validated several small-mol. Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chem. modifications of the lead compd. 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochem., biophys., and computational methods and detd. its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunopptn. expts. revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot anal. in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclin. investigations. Mol Cancer Ther; 13(3); 565-75. 2013 AACR.
- 63Lee, T.; Bian, Z.; Zhao, B.; Hogdal, L. J.; Sensintaffar, J. L.; Goodwin, C. M.; Belmar, J.; Shaw, S.; Tarr, J. C.; Veerasamy, N.; Matulis, S. M.; Koss, B.; Fischer, M. A.; Arnold, A. L.; Camper, D. V.; Browning, C. F.; Rossanese, O. W.; Budhraja, A.; Opferman, J.; Boise, L. H.; Savona, M. R.; Letai, A.; Olejniczak, E. T.; Fesik, S. W. Discovery and biological characterization of potent myeloid cell leukemia-1 inhibitors. FEBS Lett. 2017, 591, 240– 251, DOI: 10.1002/1873-3468.1249763https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFSlsr%252FP&md5=16ad6c7e0fe49b8eaa20185676284eafDiscovery and biological characterization of potent myeloid cell leukemia-1 inhibitorsLee, Taekyu; Bian, Zhiguo; Zhao, Bin; Hogdal, Leah J.; Sensintaffar, John L.; Goodwin, Craig M.; Belmar, Johannes; Shaw, Subrata; Tarr, James C.; Veerasamy, Nagarathanam; Matulis, Shannon M.; Koss, Brian; Fischer, Melissa A.; Arnold, Allison L.; Camper, DeMarco V.; Browning, Carrie F.; Rossanese, Olivia W.; Budhraja, Amit; Opferman, Joseph; Boise, Lawrence H.; Savona, Michael R.; Letai, Anthony; Olejniczak, Edward T.; Fesik, Stephen W.FEBS Letters (2017), 591 (1), 240-251CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is assocd. with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, the authors describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compds. represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clin. useful Mcl-1 inhibitors.
- 64Lu, X.; Liu, Y. C.; Orvig, C.; Liang, H.; Chen, Z. F. Discovery of β-carboline copper(II) complexes as Mcl-1 inhibitor and in vitro and in vivo activity in cancer models. Eur. J. Med. Chem. 2019, 181, 111567, DOI: 10.1016/j.ejmech.2019.11156764https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFKksbnL&md5=54f0a728016fdfeb0752418ad4386209Discovery of β-carboline copper(II) complexes as Mcl-1 inhibitor and in vitro and in vivo activity in cancer modelsLu, Xing; Liu, Yan-Cheng; Orvig, Chris; Liang, Hong; Chen, Zhen-FengEuropean Journal of Medicinal Chemistry (2019), 181 (), 111567CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Mcl-1 is an anti-apoptotic member of Bcl-2 family proteins. The development of inhibitors of Mcl-1 has been challenging. To develop metal-based Mcl-1 inhibitors, twenty two copper(II) complexes 25-46 with 9-substituted β-carboline derivs. were reported. Complexes 38 and 39 showed higher cytotoxicity than the corresponding ligands or cisplatin. The most potent complex 39 presented higher selectivity to Mcl-1 than other Bcl-2 family proteins, and killed cancer cells via Bax/Bak mediated apoptosis. Complex 39 showed an excellent safety profile in mouse model, and significantly inhibited the tumor growth in NCI-H460 tumor bearing model, which is more potent than AZD5991 at the same dosage. Complex 39 prolonged the survival time of the tumor bearing mice. Complex 39 is the first metal-based Mcl-1 inhibitor acting as a potential anticancer agent.
- 65Richard, D. J.; Lena, R.; Bannister, T.; Blake, N.; Pierceall, W. E.; Carlson, N. E.; Keller, C. E.; Koenig, M.; He, Y.; Minond, D.; Mishra, J.; Cameron, M.; Spicer, T.; Hodder, P.; Cardone, M. H. Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker. Bioorg. Med. Chem. 2013, 21, 6642– 6649, DOI: 10.1016/j.bmc.2013.08.01765https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtlGrurbJ&md5=a2875b8bc8fce034d17def9920dd31a4Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarkerRichard, David J.; Lena, Ryan; Bannister, Thomas; Blake, Noel; Pierceall, William E.; Carlson, Nicole E.; Keller, Christina Eberhart; Koenig, Marcel; He, Yuanjun; Minond, Dmitriy; Mishra, Jitendra; Cameron, Michael; Spicer, Timothy; Hodder, Peter; Cardone, Michael H.Bioorganic & Medicinal Chemistry (2013), 21 (21), 6642-6649CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Anti-apoptotic Bcl-2 family proteins are important oncol. therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One obsd. mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small mol. inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compds. has been challenging due to difficulties in translating the target selectivity obsd. at the biochem. level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compds. identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compds. at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compds. that genuinely target those proteins. The identification of compd. 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.
- 66Petros, A. M.; Swann, S. L.; Song, D.; Swinger, K.; Park, C.; Zhang, H.; Wendt, M. D.; Kunzer, A. R.; Souers, A. J.; Sun, C. Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein. Bioorg. Med. Chem. Lett. 2014, 24, 1484– 1488, DOI: 10.1016/j.bmcl.2014.02.01066https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjtl2htbk%253D&md5=e3e7de08b4f23cfa0578ad228659f9ebFragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 proteinPetros, Andrew M.; Swann, Steven L.; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D.; Kunzer, Aaron R.; Souers, Andrew J.; Sun, ChaohongBioorganic & Medicinal Chemistry Letters (2014), 24 (6), 1484-1488CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is through overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biol. to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chem. optimization efforts.
- 67Kotschy, A.; Szlavik, Z.; Murray, J.; Davidson, J.; Maragno, A. L.; Le Toumelin-Braizat, G.; Chanrion, M.; Kelly, G. L.; Gong, J. N.; Moujalled, D. M.; Bruno, A.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos, S.; Radics, G.; Proszenyak, A.; Balint, B.; Ondi, L.; Blasko, G.; Robertson, A.; Surgenor, A.; Dokurno, P.; Chen, I.; Matassova, N.; Smith, J.; Pedder, C.; Graham, C.; Studeny, A.; Lysiak-Auvity, G.; Girard, A. M.; Grave, F.; Segal, D.; Riffkin, C. D.; Pomilio, G.; Galbraith, L. C.; Aubrey, B. J.; Brennan, M. S.; Herold, M. J.; Chang, C.; Guasconi, G.; Cauquil, N.; Melchiore, F.; Guigal-Stephan, N.; Lockhart, B.; Colland, F.; Hickman, J. A.; Roberts, A. W.; Huang, D. C.; Wei, A. H.; Strasser, A.; Lessene, G.; Geneste, O. The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature 2016, 538, 477– 482, DOI: 10.1038/nature1983067https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2srjs1aquw%253D%253D&md5=c435a7621b84063133e41e0cdbad3ac8The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer modelsKotschy Andras; Szlavik Zoltan; Csekei Marton; Paczal Attila; Szabo Zoltan B; Sipos Szabolcs; Radics Gabor; Proszenyak Agnes; Balint Balazs; Ondi Levente; Blasko Gabor; Murray James; Davidson James; Robertson Alan; Surgenor Allan; Dokurno Pawel; Chen Ijen; Matassova Natalia; Smith Julia; Pedder Christopher; Graham Christopher; Maragno Ana Leticia; Le Toumelin-Braizat Gaetane; Chanrion Maia; Bruno Alain; Studeny Aurelie; Lysiak-Auvity Gaelle; Girard Anne-Marie; Grave Fabienne; Guasconi Ghislaine; Cauquil Nicolas; Colland Frederic; Hickman John A; Geneste Olivier; Kelly Gemma L; Gong Jia-Nan; Segal David; Riffkin Chris D; Galbraith Laura C A; Aubrey Brandon J; Brennan Margs S; Herold Marco J; Chang Catherine; Roberts Andrew W; Huang David C S; Strasser Andreas; Lessene Guillaume; Kelly Gemma L; Gong Jia-Nan; Segal David; Riffkin Chris D; Galbraith Laura C A; Aubrey Brandon J; Brennan Margs S; Herold Marco J; Chang Catherine; Roberts Andrew W; Huang David C S; Strasser Andreas; Lessene Guillaume; Moujalled Donia M; Pomilio Giovanna; Wei Andrew H; Aubrey Brandon J; Roberts Andrew W; Melchiore Fabien; Guigal-Stephan Nolwen; Lockhart Brian; Roberts Andrew W; Wei Andrew H; Lessene GuillaumeNature (2016), 538 (7626), 477-482 ISSN:.Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.
- 68Szlavik, Z.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos, S.; Radics, G.; Proszenyak, A.; Balint, B.; Murray, J.; Davidson, J.; Chen, I.; Dokurno, P.; Surgenor, A. E.; Daniels, Z. M.; Hubbard, R. E.; Le Toumelin-Braizat, G.; Claperon, A.; Lysiak-Auvity, G.; Girard, A. M.; Bruno, A.; Chanrion, M.; Colland, F.; Maragno, A. L.; Demarles, D.; Geneste, O.; Kotschy, A. Discovery of S64315, a potent and selective Mcl-1 inhibitor. J. Med. Chem. 2020, 63, 13762– 13795, DOI: 10.1021/acs.jmedchem.0c0123468https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Grs7jI&md5=adb0242cb3292fca804f9e6cb0448c67Discovery of S64315, a Potent and Selective Mcl-1 InhibitorSzlavik, Zoltan; Csekei, Marton; Paczal, Attila; Szabo, Zoltan B.; Sipos, Szabolcs; Radics, Gabor; Proszenyak, Agnes; Balint, Balazs; Murray, James; Davidson, James; Chen, Ijen; Dokurno, Pawel; Surgenor, Allan E.; Daniels, Zoe Marie; Hubbard, Roderick E.; Le Toumelin-Braizat, Gaetane; Claperon, Audrey; Lysiak-Auvity, Gaelle; Girard, Anne-Marie; Bruno, Alain; Chanrion, Maia; Colland, Frederic; Maragno, Ana-Leticia; Demarles, Didier; Geneste, Olivier; Kotschy, AndrasJournal of Medicinal Chemistry (2020), 63 (22), 13762-13795CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is assocd. with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clin. candidate S64315, a selective small mol. inhibitor of Mcl-1. Starting from a fragment derived lead compd., we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compds. against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclin. candidate has drug-like properties that have enabled its development and entry into clin. trials.
- 69Zhu, P. J.; Yu, Z. Z.; You, Q. D.; Jiang, Z. Y. Myeloid cell leukemin-1 inhibitors: a growing arsenal for cancer therapy. Drug Discovery Today 2020, 25, 1873– 1882, DOI: 10.1016/j.drudis.2020.07.02169https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGmu7jK&md5=544dba0e7990594db20b8e9a8e9e010bMyeloid cell leukemin-1 inhibitors and a growing arsenal for cancer therapyZhu, Peng-Ju; Yu, Ze-Zhou; You, Qi-Dong; Jiang, Zheng-YuDrug Discovery Today (2020), 25 (10), 1873-1882CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)A Review. B-cell lymphoma-2 (Bcl-2) family proteins, comprising proapoptotic proteins (Bax and Bak), antiapoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) and BCL-2 homol. domain 3 (BH3)-only proteins (Bid, Noxa, and Puma), have long been identified as pivotal apoptosis regulators. As an antiapoptotic member, myeloid cell leukemin-1 (Mcl-1) can bind with proapoptotic proteins and inhibit apoptosis. Mcl-1 is frequently overexpressed and closely assocd. with oncogenesis and poor prognosis in several cancers, posing a tremendous obstacle for cancer therapy. Recently, an increasing no. of Mcl-1-selective small-mol. inhibitors have entered preclin. studies and advanced into clin. trials. In this review, we briefly introduce the role of Mcl-1 in apoptosis and highlight the recent development of Mcl-1 small-mol. inhibitors.
- 70Li, X.; Dou, J.; You, Q.; Jiang, Z. Inhibitors of BCL2A1/Bfl-1 protein: Potential stock in cancer therapy. Eur. J. Med. Chem. 2021, 220, 113539, DOI: 10.1016/j.ejmech.2021.11353970https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFGlt7zE&md5=cd2472d31fd571818796cbd209ad63e2Inhibitors of BCL2A1/Bfl-1 protein: Potential stock in cancer therapyLi, Xue; Dou, Junwei; You, Qidong; Jiang, ZhengyuEuropean Journal of Medicinal Chemistry (2021), 220 (), 113539CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)The Bcl-2 family members rigorously regulate cell endogenous apoptosis, and targeting anti-apoptotic members is a hot topic in design of anti-cancer drugs. At present, FDA and EMA have approved Bcl-2 inhibitor Venetoclax (ABT-199) for treating chronic lymphocytic leukemia (CLL). However, inhibitors of anti-apoptotic protein BCL2A1/Bfl-1 have not been vigorously developed, and no mol. with ideal activity and selectivity has been found yet. Here we review the biol. function and protein structure of Bfl-1, discuss the therapeutic potential and list the currently reported inhibitory peptides and small mols. This will provide a ref. for Bfl-1 targeting drug discovery in the future.
- 71Chang, J.; Wang, Y.; Shao, L.; Laberge, R. M.; Demaria, M.; Campisi, J.; Janakiraman, K.; Sharpless, N. E.; Ding, S.; Feng, W.; Luo, Y.; Wang, X.; Aykin-Burns, N.; Krager, K.; Ponnappan, U.; Hauer-Jensen, M.; Meng, A.; Zhou, D. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat. Med. 2016, 22, 78– 83, DOI: 10.1038/nm.401071https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVWqtrrM&md5=6b8ae21d47f74e00e0522a1a60529bb6Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in miceChang, Jianhui; Wang, Yingying; Shao, Lijian; Laberge, Remi-Martin; Demaria, Marco; Campisi, Judith; Janakiraman, Krishnamurthy; Sharpless, Norman E.; Ding, Sheng; Feng, Wei; Luo, Yi; Wang, Xiaoyan; Aykin-Burns, Nukhet; Krager, Kimberly; Ponnappan, Usha; Hauer-Jensen, Martin; Meng, Aimin; Zhou, DaohongNature Medicine (New York, NY, United States) (2016), 22 (1), 78-83CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradn. (TBI). Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-assocd. disorders, suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacol. agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of compds. and identified ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent senolytic drug. We show that ABT263 selectively kills SCs in culture in a cell type- and species-independent manner by inducing apoptosis. Oral administration of ABT263 to either sublethally irradiated or normally aged mice effectively depleted SCs, including senescent bone marrow hematopoietic stem cells (HSCs) and senescent muscle stem cells (MuSCs). Notably, this depletion mitigated TBI-induced premature aging of the hematopoietic system and rejuvenated the aged HSCs and MuSCs in normally aged mice. Our results demonstrate that selective clearance of SCs by a pharmacol. agent is beneficial in part through its rejuvenation of aged tissue stem cells. Thus, senolytic drugs may represent a new class of radiation mitigators and anti-aging agents.
- 72Munkhbaatar, E.; Dietzen, M.; Agrawal, D.; Anton, M.; Jesinghaus, M.; Boxberg, M.; Pfarr, N.; Bidola, P.; Uhrig, S.; Höckendorf, U.; Meinhardt, A. L.; Wahida, A.; Heid, I.; Braren, R.; Mishra, R.; Warth, A.; Muley, T.; Poh, P. S. P.; Wang, X.; Fröhling, S.; Steiger, K.; Slotta-Huspenina, J.; van Griensven, M.; Pfeiffer, F.; Lange, S.; Rad, R.; Spella, M.; Stathopoulos, G. T.; Ruland, J.; Bassermann, F.; Weichert, W.; Strasser, A.; Branca, C.; Heikenwalder, M.; Swanton, C.; McGranahan, N.; Jost, P. J. MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically. Nat. Commun. 2020, 11, 4527, DOI: 10.1038/s41467-020-18372-172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFSitbjE&md5=ca1e26757966980a2bab01fc82448788MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeuticallyMunkhbaatar, Enkhtsetseg; Dietzen, Michelle; Agrawal, Deepti; Anton, Martina; Jesinghaus, Moritz; Boxberg, Melanie; Pfarr, Nicole; Bidola, Pidassa; Uhrig, Sebastian; Hoeckendorf, Ulrike; Meinhardt, Anna-Lena; Wahida, Adam; Heid, Irina; Braren, Rickmer; Mishra, Ritu; Warth, Arne; Muley, Thomas; Poh, Patrina S. P.; Wang, Xin; Froehling, Stefan; Steiger, Katja; Slotta-Huspenina, Julia; van Griensven, Martijn; Pfeiffer, Franz; Lange, Sebastian; Rad, Roland; Spella, Magda; Stathopoulos, Georgios T.; Ruland, Juergen; Bassermann, Florian; Weichert, Wilko; Strasser, Andreas; Branca, Caterina; Heikenwalder, Mathias; Swanton, Charles; McGranahan, Nicholas; Jost, Philipp J.Nature Communications (2020), 11 (1), 4527CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Evasion of programmed cell death represents a crit. form of oncogene addiction in cancer cells. Understanding the mol. mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a mol. basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly assocd. with subclonal gains of MCL-1. In mice, tumor progression is delayed upon pharmacol. or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.
- 73Liu, J.; Fang, Z.; Zhang, Q.; Liu, Q.; Bi, X. Silver-catalyzed isocyanide-alkyne cycloaddition: a general and practical method to oligosubstituted pyrroles. Angew. Chem. Int. Ed. Engl. 2013, 52, 6953– 6957, DOI: 10.1002/anie.20130202473https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3snhtlSlsw%253D%253D&md5=b5653c2a15e2fffaeeaa98cff3bf08d6Silver-catalyzed isocyanide-alkyne cycloaddition: a general and practical method to oligosubstituted pyrrolesLiu Jianquan; Fang Zhongxue; Zhang Qian; Liu Qun; Bi XiheAngewandte Chemie (International ed. in English) (2013), 52 (27), 6953-7 ISSN:.There is no expanded citation for this reference.
- 74Kattamuri, P. V.; Yin, J.; Siriwongsup, S.; Kwon, D. H.; Ess, D. H.; Li, Q.; Li, G.; Yousufuddin, M.; Richardson, P. F.; Sutton, S. C.; Kürti, L. Practical singly and doubly electrophilic aminating agents: a new, more sustainable platform for carbon-nitrogen bond formation. J. Am. Chem. Soc. 2017, 139, 11184– 11196, DOI: 10.1021/jacs.7b0527974https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCkt7jP&md5=38f07ddb4a79a3a4fecdcc40520c0d7cPractical Singly and Doubly Electrophilic Aminating Agents: A New, More Sustainable Platform for Carbon-Nitrogen Bond FormationKattamuri, Padmanabha V.; Yin, Jun; Siriwongsup, Surached; Kwon, Doo-Hyun; Ess, Daniel H.; Li, Qun; Li, Guigen; Yousufuddin, Muhammed; Richardson, Paul F.; Sutton, Scott C.; Kurti, LaszloJournal of the American Chemical Society (2017), 139 (32), 11184-11196CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Given the importance of amines in a large no. of biol. active natural products, active pharmaceutical ingredients, agrochems., and functional materials, the development of efficient C-N bond-forming methods with wide substrate scope continues to be at the frontier of research in synthetic org. chem. Here, authors present a general and fundamentally new synthetic approach for the direct, transition-metal-free prepn. of sym. and unsym. diaryl-, arylalkyl-, and dialkylamines that relies on the facile single or double addn. of readily available C-nucleophiles to the nitrogen atom of bench-stable electrophilic aminating agents. Practical single and double polarity reversal (i.e., umpolung) of the nitrogen atom is achieved using sterically and electronically tunable ketomalonate-derived imines and oximes. Overall, this novel approach represents an operationally simple, scalable, and environmentally friendly alternative to transition-metal-catalyzed C-N cross-coupling methods that are currently used to access structurally diverse secondary amines.
- 75Cohen, N. A.; Stewart, M. L.; Gavathiotis, E.; Tepper, J. L.; Bruekner, S. R.; Koss, B.; Opferman, J. T.; Walensky, L. D. A competitive stapled peptide screen identifies a selective small molecule that overcomes MCL-1-dependent leukemia cell survival. Chem. Biol. 2012, 19, 1175– 1186, DOI: 10.1016/j.chembiol.2012.07.01875https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhtl2rsL7M&md5=5306c2b2c2ff29a70ecfc83674f07423A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-Dependent Leukemia Cell SurvivalCohen, Nicole A.; Stewart, Michelle L.; Gavathiotis, Evripidis; Tepper, Jared L.; Bruekner, Susanne R.; Koss, Brian; Opferman, Joseph T.; Walensky, Loren D.Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (9), 1175-1186CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochem. by an antiapoptotic surface groove that neutralizes the proapoptotic BH3 α helix of death proteins. Antiapoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Although targeting the BCL-2 antiapoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of mols. tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor mol. that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochem. lock-hold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence.
- 76Kump, K. J.; Miao, L.; Mady, A. S. A.; Ansari, N. H.; Shrestha, U. K.; Yang, Y.; Pal, M.; Liao, C.; Perdih, A.; Abulwerdi, F. A.; Chinnaswamy, K.; Meagher, J. L.; Carlson, J. M.; Khanna, M.; Stuckey, J. A.; Nikolovska-Coleska, Z. Discovery and characterization of 2,5-substituted benzoic acid dual inhibitors of the anti-apoptotic Mcl-1 and Bfl-1 proteins. J. Med. Chem. 2020, 63, 2489– 2510, DOI: 10.1021/acs.jmedchem.9b0144276https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1emsr4%253D&md5=a0ee24b840cde38f05fcb41e34323aceDiscovery and Characterization of 2,5-Substituted Benzoic Acid Dual Inhibitors of the Anti-apoptotic Mcl-1 and Bfl-1 ProteinsKump, Karson J.; Miao, Lei; Mady, Ahmed S. A.; Ansari, Nurul H.; Shrestha, Uttar K.; Yang, Yuting; Pal, Mohan; Liao, Chenzhong; Perdih, Andrej; Abulwerdi, Fardokht A.; Chinnaswamy, Krishnapriya; Meagher, Jennifer L.; Carlson, Jacob M.; Khanna, May; Stuckey, Jeanne A.; Nikolovska-Coleska, ZanetaJournal of Medicinal Chemistry (2020), 63 (5), 2489-2510CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compd. 24, which binds both Mcl-1 and Bfl-1 with Ki values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.
- 77Bernardo, P. H.; Sivaraman, T.; Wan, K.-F.; Xu, J.; Krishnamoorthy, J.; Song, C. M.; Tian, L.; Chin, J. S. F.; Lim, D. S. W.; Mok, H. Y. K.; Yu, V. C.; Tong, J. C.; Chai, C. L. Structural insights into the design of small molecule inhibitors that selectively antagonize Mcl-1. J. Med. Chem. 2010, 53, 2314– 2318, DOI: 10.1021/jm901469p77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhvF2ktbg%253D&md5=c033784e92b321c2455a5d8b7594d5f6Structural Insights into the Design of Small Molecule Inhibitors That Selectively Antagonize Mcl-1Bernardo, Paul H.; Sivaraman, Thirunavukkarasu; Wan, Kah-Fei; Xu, Jin; Krishnamoorthy, Janarthanan; Song, Chun Meng; Tian, Liming; Chin, Jasmine S. F.; Lim, Diane S. W.; Mok, Henry Y. K.; Yu, Victor C.; Tong, Joo Chuan; Chai, Christina L. L.Journal of Medicinal Chemistry (2010), 53 (5), 2314-2318CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The screening of a small focused library of rhodanine derivs. as inhibitors of Bcl-2 proteins led to the discovery of two structurally related compds. with different binding profiles against the Bcl-XL and the Mcl-1 proteins. Subsequent NMR studies with mutant proteins and in silico docking studies provide a possible rationale for the obsd. specificity.
- 78Szlavik, Z.; Ondi, L.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Radics, G.; Murray, J.; Davidson, J.; Chen, I.; Davis, B.; Hubbard, R. E.; Pedder, C.; Dokurno, P.; Surgenor, A.; Smith, J.; Robertson, A.; LeToumelin-Braizat, G.; Cauquil, N.; Zarka, M.; Demarles, D.; Perron-Sierra, F.; Claperon, A.; Colland, F.; Geneste, O.; Kotschy, A. Structure-guided discovery of a selective Mcl-1 inhibitor with cellular activity. J. Med. Chem. 2019, 62, 6913– 6924, DOI: 10.1021/acs.jmedchem.9b0013478https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1ensrvN&md5=e94a3a9ac96f5693406cafa185484470Structure-Guided Discovery of a Selective Mcl-1 Inhibitor with Cellular ActivitySzlavik, Zoltan; Ondi, Levente; Csekei, Marton; Paczal, Attila; Szabo, Zoltan B.; Radics, Gabor; Murray, James; Davidson, James; Chen, Ijen; Davis, Ben; Hubbard, Roderick E.; Pedder, Christopher; Dokurno, Pawel; Surgenor, Allan; Smith, Julia; Robertson, Alan; LeToumelin-Braizat, Gaetane; Cauquil, Nicolas; Zarka, Marion; Demarles, Didier; Perron-Sierra, Francoise; Claperon, Audrey; Colland, Frederic; Geneste, Olivier; Kotschy, AndrasJournal of Medicinal Chemistry (2019), 62 (15), 6913-6924CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when obsd. in human cancers is assocd. with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small mol. inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using NMR and X-ray-derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compds., and cellular activity was brought on scale by offsetting the neg. charge of the anchoring carboxylate group. The obtained compds. described here exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction, and growth inhibition. These early research efforts illustrate drug discovery optimization from thienopyrimidine hits to a lead compd., the chem. series leading to the identification of our more advanced compds. S63845 and S64315.
- 79Wu, Y.; Jiang, Z.; Li, Z.; Gu, J.; You, Q.; Zhang, X. Click chemistry-based discovery of [3-hydroxy-5-(1h-1,2,3-triazol-4-yl)picolinoyl]glycines as orally active hypoxia-inducing factor prolyl hydroxylase inhibitors with favorable safety profiles for the treatment of anemia. J. Med. Chem. 2018, 61, 5332– 5349, DOI: 10.1021/acs.jmedchem.8b0054979https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVKktbzL&md5=11a4b00f8d9a883a387acb553510ba2aClick Chemistry-Based Discovery of [3-Hydroxy-5-(1H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia-Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of AnemiaWu, Yue; Jiang, Zhensheng; Li, Zhihong; Gu, Jing; You, Qidong; Zhang, XiaojinJournal of Medicinal Chemistry (2018), 61 (12), 5332-5349CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)As a gene assocd. with anemia, the erythropoiesis gene is physiol. expressed under hypoxia regulated by †hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study, we applied click chem. to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time, and a series of triazole compds. showed preferable inhibitory activity in fluorescence polarization assays. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the Hb of cisplatin-induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity obsd. in vivo. These results confirm that triazole compd. 15i is a promising candidate for the treatment of renal anemia.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00682.
Molecular formula strings and SAR data (CSV)
Predicated binding modes of 13, 16, 40, and 43 (ZIP)
Predicated drug-like properties, hERG channel inhibition assay, hematoxylin and eosin (H&E) staining, TUNEL analysis, statistical analysis and 1H NMR, 13C NMR, HRMS and HPLC spectra for representative target compounds (PDF)
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