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下载到 MS-PowerPointCite This: 引用此文：J. Med. Chem. J.Med.Chem. 2023, 66, 13, 8725-8744
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Discovery of 1H-Imidazo[4,5-b]pyridine Derivatives as Potent and Selective BET Inhibitors for the Management of Neuropathic Pain
发现 1H-咪唑并[4,5-b]吡啶衍生物作为治疗神经性疼痛的强效选择性 BET 抑制剂
- Xuetao Chen 陈雪涛Xuetao Chen 陈雪涛Jiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
中国药科大学药物设计与优化重点实验室、天然药物国家重点实验室，江苏南京 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 Xuetao Chen 陈雪涛的更多作品 - ,
- Danyan Cao 曹丹燕Danyan CaoDepartment of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, ChinaMore by Danyan Cao
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- Chihong Liu 刘志宏Chihong LiuJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Chihong Liu
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- Fanying Meng 孟凡英Fanying MengJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Fanying Meng
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- Zijian Zhang 张子健Zijian ZhangJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Zijian Zhang
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- Rujun XuRujun XuJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Rujun Xu
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- Yuanyuan Tong 汤媛媛Yuanyuan TongJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Yuanyuan Tong
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- Yabing Xin 辛亚兵Yabing XinJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Yabing Xin
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- Weikun Zhang 张伟坤Weikun ZhangJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Weikun Zhang
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- Wenjing Kang 康文静Wenjing KangJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Wenjing Kang
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- Qichao Bao 鲍启超Qichao BaoJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaMore by Qichao Bao
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- Jingkang Shen 沈靖康Jingkang Shen 沈靖康Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
中国科学院上海药物研究所药物化学部，上海市祖冲之路 555 号，邮编：201203More by Jingkang Shen 沈靖康的更多作品 - ,
- Bing Xiong* 熊兵*Bing Xiong*Email: bxiong@simm.ac.cnDepartment of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, ChinaUniversity of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, ChinaMore by Bing Xiong
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- Qidong You* 尤启东*Qidong You*Email: youqd@163.comJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Qidong You
- , and 和
- Zhengyu Jiang* 蒋征宇*Zhengyu Jiang*Email: jiangzhengyucpu@163.comJiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaMore by Zhengyu Jiang
Copyright © 2023 American Chemical Society
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版权所有 © 2023 美国化学学会
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Abstract 摘要
Neuropathic pain (NP) is an intolerable pain syndrome that arises from continuous inflammation and excitability after nerve injury. Only a few NP therapeutics are currently available, and all of them do not provide adequate pain relief. Herein, we report the discovery of a selective and potent inhibitor of the bromodomain and extra-terminal (BET) proteins for reducing neuroinflammation and excitability to treat NP. Starting with the screening hit 1 from an in-house compound library, iterative optimization resulted in the potent BET inhibitor DDO-8926 with a unique binding mode and a novel chemical structure. DDO-8926 exhibits excellent BET selectivity and favorable drug-like properties. In mice with spared nerve injury, DDO-8926 significantly alleviated mechanical hypersensitivity by inhibiting pro-inflammatory cytokine expression and reducing excitability. Collectively, these results implicate that DDO-8926 is a promising agent for the treatment of NP.
神经病理性疼痛（NP）是一种难以忍受的疼痛综合征，由神经损伤后的持续炎症和兴奋引起。目前只有少数几种 NP 治疗药物，而且所有这些药物都不能充分缓解疼痛。在此，我们报告发现了一种选择性强效溴化多域和末端外（BET）蛋白抑制剂，可用于降低神经炎症和兴奋性以治疗 NP。从内部化合物库中的筛选结果 1 开始，经过迭代优化，最终产生了具有独特结合模式和新颖化学结构的强效 BET 抑制剂 DDO-8926。DDO-8926 具有出色的 BET 选择性和良好的类药物特性。在神经损伤小鼠中，DDO-8926 通过抑制促炎细胞因子的表达和降低兴奋性，显著减轻了机械过敏性。总之，这些结果表明 DDO-8926 是一种治疗 NP 的有前途的药物。
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Neuropathic pain (NP) is a rather stubborn pain syndrome arising from nerve injury. (1,2) NP affects 7–10% of the general population and severely debilitates patients’ life quality. (3,4) Pharmacological treatments for NP recommended by the International Association for the Study of Pain (IASP) are divided into first-line, second-line, and third-line interventions. (5) First-line treatments mainly contain tricyclic antidepressants, gabapentin, and pregabalin. (6−9) Lidocaine patches, tramadol, and strong opioids are recommended as second- or third-line interventions because of their low tolerability or safety profile, and their clinical use is limited. (10−12) Over the past century, only a few therapies for NP relief have been introduced to the market, and these have not resulted in adequate pain relief for patients. Currently, there remains an unmet need for effective and safe management of this disabling disease.
神经病理性疼痛（NP）是由神经损伤引起的一种相当顽固的疼痛综合征。(1,2）7%-10%的普通人群患有神经病理性疼痛，严重影响患者的生活质量。(3,4）国际疼痛研究协会（IASP）推荐的 NP 药物治疗分为一线、二线和三线干预。(5）一线治疗主要包括三环类抗抑郁药、加巴喷丁和普瑞巴林。(6-9）利多卡因贴片、曲马多和强阿片类药物因其耐受性或安全性较低，被推荐作为二线或三线干预措施，其临床应用有限。(10-12）在过去的一个世纪中，仅有少数几种用于缓解 NP 的疗法推向市场，但这些疗法并未为患者带来充分的疼痛缓解。目前，有效、安全地治疗这种致残性疾病的需求仍未得到满足。
神经病理性疼痛（NP）是由神经损伤引起的一种相当顽固的疼痛综合征。(1,2）7%-10%的普通人群患有神经病理性疼痛，严重影响患者的生活质量。(3,4）国际疼痛研究协会（IASP）推荐的 NP 药物治疗分为一线、二线和三线干预。(5）一线治疗主要包括三环类抗抑郁药、加巴喷丁和普瑞巴林。(6-9）利多卡因贴片、曲马多和强阿片类药物因其耐受性或安全性较低，被推荐作为二线或三线干预措施，其临床应用有限。(10-12）在过去的一个世纪中，仅有少数几种用于缓解 NP 的疗法推向市场，但这些疗法并未为患者带来充分的疼痛缓解。目前，有效、安全地治疗这种致残性疾病的需求仍未得到满足。
Epigenetic modifications are able to modulate gene expression changes, including in pain circuits. (13−17) There is growing evidence that histone acetylation plays a key role in the development and maintenance of chronic pain. (18−21) Serving as epigenetic “readers” of acetylated lysines on histone tails, the bromodomain and extra-terminal (BET) domain family proteins could regulate the expression of genes involved in chronic pain by enriching the corresponding transcription factors at the promoter regions of these genes. (22−25) Recently, BET bromodomain (BET BD) inhibition has also been revealed to alleviate nerve injury-induced NP by ameliorating nerve inflammation and modulating the expression of critical ion channels to reduce neuronal excitability. (26−30) The BET protein family is composed of bromodomain-containing protein 4 (BRD4), BRD3, BRD2, and testes-specific BRDT, each of which contains two tandem BDs, BD1 and BD2, and an extra-terminal domain. (31−34) BD1 and BD2 are essential domains for the BET proteins to perform their functions. (35−38) Each BD consists of approximately 110 amino acids and forms four reverse parallel α-helices (αZ, αA, αB, and αC) and two hydrophobic loops (ZA loop and BC loop). (39−42) The end of the α-helices and the surfaces of the hydrophobic loops together form the acetylated lysine binding pocket, containing a conserved asparagine that forms a hydrogen bond interaction with the acetylated lysine, a hydrophobic residue called the gatekeeper that controls the entry of the acetylated lysine, a hydrophobic WPF shelf adjacent to the ZA loop, and a ZA channel. (43−47)
表观遗传修饰能够调节基因表达变化，包括疼痛回路中的基因表达变化。(13-17）越来越多的证据表明，组蛋白乙酰化在慢性疼痛的发展和维持中起着关键作用。(18-21）作为组蛋白尾部乙酰化赖氨酸的表观遗传 "阅读器"，溴域和外端（BET）域家族蛋白可以通过富集这些基因启动子区域的相应转录因子来调控慢性疼痛相关基因的表达。(22-25）最近，研究还发现，BET溴端域（BET BD）抑制可通过改善神经炎症和调节关键离子通道的表达来降低神经元的兴奋性，从而减轻神经损伤引起的 NP。(26-30）BET 蛋白家族由含溴结构域蛋白 4（BRD4）、BRD3、BRD2 和睾丸特异性 BRDT 组成，每个 BET 蛋白家族都包含两个串联 BD，即 BD1 和 BD2，以及一个末端外结构域。(31-34）BD1 和 BD2 是 BET 蛋白发挥其功能的重要结构域。(35-38）每个 BD 由大约 110 个氨基酸组成，形成四个反向平行的 α 螺旋（αZ、αA、αB 和 αC）和两个疏水环（ZA 环和 BC 环）。(39-42）α-螺旋的末端和疏水环的表面共同形成了乙酰化赖氨酸结合袋，其中包含一个与乙酰化赖氨酸形成氢键相互作用的保守天冬酰胺、一个控制乙酰化赖氨酸进入的称为守门员的疏水残基、一个邻近ZA环的疏水WPF架和一个ZA通道。(43-47)
表观遗传修饰能够调节基因表达变化，包括疼痛回路中的基因表达变化。(13-17）越来越多的证据表明，组蛋白乙酰化在慢性疼痛的发展和维持中起着关键作用。(18-21）作为组蛋白尾部乙酰化赖氨酸的表观遗传 "阅读器"，溴域和外端（BET）域家族蛋白可以通过富集这些基因启动子区域的相应转录因子来调控慢性疼痛相关基因的表达。(22-25）最近，研究还发现，BET溴端域（BET BD）抑制可通过改善神经炎症和调节关键离子通道的表达来降低神经元的兴奋性，从而减轻神经损伤引起的 NP。(26-30）BET 蛋白家族由含溴结构域蛋白 4（BRD4）、BRD3、BRD2 和睾丸特异性 BRDT 组成，每个 BET 蛋白家族都包含两个串联 BD，即 BD1 和 BD2，以及一个末端外结构域。(31-34）BD1 和 BD2 是 BET 蛋白发挥其功能的重要结构域。(35-38）每个 BD 由大约 110 个氨基酸组成，形成四个反向平行的 α 螺旋（αZ、αA、αB 和 αC）和两个疏水环（ZA 环和 BC 环）。(39-42）α-螺旋的末端和疏水环的表面共同形成了乙酰化赖氨酸结合袋，其中包含一个与乙酰化赖氨酸形成氢键相互作用的保守天冬酰胺、一个控制乙酰化赖氨酸进入的称为守门员的疏水残基、一个邻近ZA环的疏水WPF架和一个ZA通道。(43-47)
BET inhibitors have promising therapeutic potential for a variety of diseases by blocking the BDs that recognize and bind the acetylated lysines at histone tails, thereby inhibiting the expression of pathogenic factors. (48) Since the first BET inhibitor, MS417, (49) was discovered, various BET inhibitors have emerged, some of them being in clinical trials, (50) including MK-8628, (51,52) CPI-0610, (53,54) BMS-986158, (55) NHWD-870, (56) ABBV-075, (57) ABBV-744, (58,59) RVX-208, (60) and so forth. Here, we identified a potent and selective BET inhibitor that significantly alleviate nerve injury-induced NP and could be developed as a promising therapeutic option for NP.
BET 抑制剂通过阻断识别并结合组蛋白尾部乙酰化赖氨酸的 BDs，从而抑制致病因子的表达，对多种疾病具有良好的治疗潜力。(48）自第一个 BET 抑制剂 MS417（49）被发现以来，各种 BET 抑制剂不断涌现，其中一些已进入临床试验阶段，（50）包括 MK-8628、（51,52）CPI-0610、（53,54）BMS-986158、（55）NHWD-870、（56）ABBV-075、（57）ABBV-744、（58,59）RVX-208（60）等。在此，我们发现了一种强效且具有选择性的 BET 抑制剂，它能显著缓解神经损伤引起的 NP，可作为治疗 NP 的一种有前途的选择。
BET 抑制剂通过阻断识别并结合组蛋白尾部乙酰化赖氨酸的 BDs，从而抑制致病因子的表达，对多种疾病具有良好的治疗潜力。(48）自第一个 BET 抑制剂 MS417（49）被发现以来，各种 BET 抑制剂不断涌现，其中一些已进入临床试验阶段，（50）包括 MK-8628、（51,52）CPI-0610、（53,54）BMS-986158、（55）NHWD-870、（56）ABBV-075、（57）ABBV-744、（58,59）RVX-208（60）等。在此，我们发现了一种强效且具有选择性的 BET 抑制剂，它能显著缓解神经损伤引起的 NP，可作为治疗 NP 的一种有前途的选择。
Results and Discussion 结果与讨论
Hit Identification and Optimization
命中识别和优化
An in-house compound library of about 600 small molecules originating from commercially purchased or homemade in the laboratory was screened for new hits. The inhibitory activity of these compounds was then evaluated by using competitive fluorescence-polarization (FP) assay to obtain compound 1, which was found to inhibit recombinant human BRD4 BD1 and BD2 with Ki values of 4.3 and 8.1 μM, respectively (Figure 1A). The one-dimensional Carr–Purcell–Meiboom–Gill (CPMG) experiment, which exploits the differences in NMR relaxation properties between ligands and macromolecular complexes, has long been considered a powerful tool for monitoring the binding of small ligands to large proteins. (61,62) The CPMG experiment was then carried out between BRD4 BD1 and 1, and the result showed that BRD4 BD1 significantly reduced the 1H resonances of 1 signal intensity (Figure 1B), suggesting that compound 1 does bind to BRD4 BD1.
我们在内部化合物库中筛选了约 600 种小分子化合物，这些化合物来自市场购买或实验室自制。然后通过竞争性荧光极化（FP）测定法评估了这些化合物的抑制活性，得到了化合物1，发现它能抑制重组人BRD4 BD1和BD2，K值分别为4.3和8.1 μM（图1A）。一维 Carr-Purcell-Meiboom-Gill （CPMG）实验利用配体和大分子复合物之间核磁共振弛豫特性的差异，一直被认为是监测小配体与大蛋白质结合的有力工具。(61,62）随后在 BRD4 BD1 和 1 之间进行了 CPMG 实验，结果表明 BRD4 BD1 显著降低了 1 的 1 H 共振信号强度。H 共振信号强度（图 1B），表明化合物 1 确实与 BRD4 BD1 结合。
我们在内部化合物库中筛选了约 600 种小分子化合物，这些化合物来自市场购买或实验室自制。然后通过竞争性荧光极化（FP）测定法评估了这些化合物的抑制活性，得到了化合物1，发现它能抑制重组人BRD4 BD1和BD2，K值分别为4.3和8.1 μM（图1A）。一维 Carr-Purcell-Meiboom-Gill （CPMG）实验利用配体和大分子复合物之间核磁共振弛豫特性的差异，一直被认为是监测小配体与大蛋白质结合的有力工具。(61,62）随后在 BRD4 BD1 和 1 之间进行了 CPMG 实验，结果表明 BRD4 BD1 显著降低了 1 的 1 H 共振信号强度。H 共振信号强度（图 1B），表明化合物 1 确实与 BRD4 BD1 结合。
Figure 1 图 1
Figure 1. Hit identification and hit-to-lead optimization. (A) Screening of the in-house compound library to find new hits. (B) The 1H CPMG spectra of hit compound 1 and BRD4 BD1 were superimposed on the 1H NMR spectrum of 1 (up) and the 1H NMR spectrum of 1 (down). (C) The SAR of the pyrrolopyridine core modifications resulted in the lead compound 9. See Table S1 for SD values.
图 1.新药鉴定和新药对先导的优化。(A) 筛选内部化合物库以寻找新的命中点。(B) 将命中化合物 1 和 BRD4 BD1 的 {{0}和 BRD4 BD1 的{{1}H NMR 光谱叠加。｝1 的 1 H NMR 光谱（向上）和 1 H NMR 光谱（向下）叠加。1（下）的 1 H NMR 光谱叠加。(C) 吡咯并吡啶核心修饰的 SAR 结果为先导化合物 9。SD 值见表 S1。
Then, we optimized the scaffold of the hit 1 by positioning analogue scanning, which is considered to be an effective strategy for early hit-to-lead optimization. (63) Compounds 2–4 were obtained by exploring the relative positions between the phenyl and pyrroloxypyrazole scaffold, and the results showed that the phenyl at the 4-position (3) was the most potent and the least active at the 6-position (2). Based on compound 3, compounds 5–7 were prepared by systematically exchanging the N atom positions, all with reduced activity compared to compound 3. Subsequently, an additional N atom was introduced into the pyrrolopyridine motif of compound 3, and compounds 8–10 were obtained by scanning the N atom positions. The results showed that compound 9 showed a 2-fold increase in potency against BRD4 BD1 compared to compound 3. The triazole analogues 11–12 were obtained by arranging the N atom position in the imidazolopyridine motif of compound 9. Compound 11 was almost inactive, and 12 also showed markedly reduced activity. Finally, other heterocyclic substitutions were also explored, and the activity of the isoxazole analogue 13 and the dihydroimidazolone analogue 14 was significantly reduced compared to compound 9 (Figure 1C).
然后，我们通过定位模拟扫描优化了命中 1 的支架，这被认为是早期命中到先导优化的有效策略。(63）通过探索苯基和吡咯氧吡唑支架之间的相对位置，我们得到了化合物 2-4，结果表明 4 位的苯基（3）活性最强，而 6 位的苯基（2）活性最低。在化合物 3 的基础上，通过系统地交换 N 原子位置制备了化合物 5-7，与化合物 3 相比，所有化合物的活性都有所降低。随后，在化合物 3 的吡咯并吡啶基团中引入了一个额外的 N 原子，通过扫描 N 原子位置得到了化合物 8-10。结果表明，与化合物 3 相比，化合物 9 对 BRD4 BD1 的药效提高了 2 倍。通过排列化合物 9 中咪唑吡啶基团的 N 原子位置，得到了三唑类似物 11-12。化合物 11 几乎没有活性，化合物 12 的活性也明显降低。最后，还研究了其他杂环取代物，与化合物 9 相比，异噁唑类似物 13 和二氢咪唑啉酮类似物 14 的活性明显降低（图 1C）。
然后，我们通过定位模拟扫描优化了命中 1 的支架，这被认为是早期命中到先导优化的有效策略。(63）通过探索苯基和吡咯氧吡唑支架之间的相对位置，我们得到了化合物 2-4，结果表明 4 位的苯基（3）活性最强，而 6 位的苯基（2）活性最低。在化合物 3 的基础上，通过系统地交换 N 原子位置制备了化合物 5-7，与化合物 3 相比，所有化合物的活性都有所降低。随后，在化合物 3 的吡咯并吡啶基团中引入了一个额外的 N 原子，通过扫描 N 原子位置得到了化合物 8-10。结果表明，与化合物 3 相比，化合物 9 对 BRD4 BD1 的药效提高了 2 倍。通过排列化合物 9 中咪唑吡啶基团的 N 原子位置，得到了三唑类似物 11-12。化合物 11 几乎没有活性，化合物 12 的活性也明显降低。最后，还研究了其他杂环取代物，与化合物 9 相比，异噁唑类似物 13 和二氢咪唑啉酮类似物 14 的活性明显降低（图 1C）。
Optimization of Compound 9
化合物 9 的优化
We investigated the binding mode of 9 to BRD4 BD1 by molecular docking. As expected, the imidazopyridine core of lead compound 9 could act as an acetylated lysine mimic forming hydrogen bond interactions with Asn140 and Tyr97 mediated by a water molecule (Figure 2). In addition, the three methoxy groups on the phenyl ring point to the ZA channel (5′ position), the outer solvent region of the pocket (4′ position), and the WPF shelf (3′ position), respectively, as the trimethoxyphenyl substituent of 9 may engage the WPF shelf, which suggests that the introduction of an aryl substituent can enhance binding. Thus, compounds 15–20 containing different aryl substituents were designed and synthesized. The results showed that the activities of 15–20 were significantly increased compared with those of 9, and among them, compound 16 with the 1,3,5-trimethylpyrazole substituent (Ki = 0.14 μM) was even more effective by about 10-fold (Figure 2).
我们通过分子对接研究了 9 与 BRD4 BD1 的结合模式。不出所料，先导化合物 9 的咪唑吡啶核心可以作为乙酰化赖氨酸的模拟物，在水分子的介导下与 Asn140 和 Tyr97 形成氢键相互作用（图 2）。此外，苯基环上的三个甲氧基分别指向ZA通道（5′位）、口袋的外溶剂区（4′位）和WPF架（3′位），因为9的三甲氧基苯基取代基可能与WPF架接触，这表明引入芳基取代基可以增强结合力。因此，我们设计并合成了含有不同芳基取代基的化合物 15-20。结果表明，与 9 相比，15-20 的活性明显提高，其中含有 1,3,5-三甲基吡唑取代基（K = 0.14 μM）的化合物 16 的活性更是提高了约 10 倍（图 2）。
我们通过分子对接研究了 9 与 BRD4 BD1 的结合模式。不出所料，先导化合物 9 的咪唑吡啶核心可以作为乙酰化赖氨酸的模拟物，在水分子的介导下与 Asn140 和 Tyr97 形成氢键相互作用（图 2）。此外，苯基环上的三个甲氧基分别指向ZA通道（5′位）、口袋的外溶剂区（4′位）和WPF架（3′位），因为9的三甲氧基苯基取代基可能与WPF架接触，这表明引入芳基取代基可以增强结合力。因此，我们设计并合成了含有不同芳基取代基的化合物 15-20。结果表明，与 9 相比，15-20 的活性明显提高，其中含有 1,3,5-三甲基吡唑取代基（K = 0.14 μM）的化合物 16 的活性更是提高了约 10 倍（图 2）。
Figure 2 图 2
Figure 2. Docking of 9 suggested that the trimethoxyphenyl substituent may engage the WPF shelf, which led to the design of several aryl derivatives. Docking analysis of compound 9 (yellow) with BRD4 BD1 (left, PDB ID: 3MXF). Docking analysis of compound 15 (yellow) with BRD4 BD1 (right, PDB ID: 3MXF). Salmon indicates the WPF shelf, red indicates Asn140, orange indicates the BC loop, and green indicates the ZA channel. See Table S1 for SD values.
图 2.对 9 号化合物的 Docking 结果表明，三甲氧基苯基取代基可能会与 WPF 架接合，因此设计出了几种芳基衍生物。化合物 9（黄色）与 BRD4 BD1（左图，PDB ID：3MXF）的对接分析。化合物 15（黄色）与 BRD4 BD1（右，PDB ID：3MXF）的对接分析。鲑鱼色表示 WPF 架，红色表示 Asn140，橙色表示 BC 环，绿色表示 ZA 通道。SD 值见表 S1。
Subsequently, the SAR of the phenyl ring substituents was evaluated, resulting in compounds 21–24 (Figure 3A). The results showed a significant increase in the potency of 21 (BRD4 (1) Ki = 0.031 μM) compared to 16, while the activity of 22 was distinctly decreased (BRD4 (1) Ki = 0.63 μM). This suggests that the effect of fluorine substitution on the activity is not due to electronic factors but by changing the conformation of the compound to be more suitable for BRD4. Next, we replaced the fluorine atom of compound 21 with a larger volume of chlorine atom, leading to compound 23 with a decreased activity. Finally, the introduction of a difluoro substituent at the 4- and 6-positions of the phenyl ring yielded compound 24, whose activity was also reduced compared to compound 21.
随后，对苯环取代基的 SAR 进行了评估，得出了化合物 21-24（图 3A）。结果表明，与 16 号化合物相比，21 号化合物的效力明显提高（BRD4 (1) K = 0.031 μM），而 22 号化合物的活性则明显降低（BRD4 (1) K = 0.63 μM）。这表明氟取代对活性的影响不是由于电子因素，而是通过改变化合物的构象使其更适合 BRD4。接着，我们用更大体积的氯原子取代了化合物 21 中的氟原子，从而得到了活性降低的化合物 23。最后，我们在苯环的 4 位和 6 位引入了二氟取代基，得到了化合物 24，与化合物 21 相比，其活性也有所降低。
随后，对苯环取代基的 SAR 进行了评估，得出了化合物 21-24（图 3A）。结果表明，与 16 号化合物相比，21 号化合物的效力明显提高（BRD4 (1) K = 0.031 μM），而 22 号化合物的活性则明显降低（BRD4 (1) K = 0.63 μM）。这表明氟取代对活性的影响不是由于电子因素，而是通过改变化合物的构象使其更适合 BRD4。接着，我们用更大体积的氯原子取代了化合物 21 中的氟原子，从而得到了活性降低的化合物 23。最后，我们在苯环的 4 位和 6 位引入了二氟取代基，得到了化合物 24，与化合物 21 相比，其活性也有所降低。
Figure 3 图 3
Figure 3. SAR of substitutions to the phenyl ring and the co-crystal structure of compound 21 complexed with BRD4 BD1 and BD2 proteins. (A) SAR of substitutions to the phenyl ring. See Table S1 for SD values. (B) Crystal structure of BRD4 BD1 (light pink) bound to 21 (yellow) (PDB ID: 8IBQ). (C) Crystal structure of BRD4 BD2 (lime green) bound to 21 (yellow) (PDB ID: 8IDH). The ligands and side chains of important residues are shown in a stick model; the hydrogen bond (green) and π–π conjugation (pink) are indicated by the dashed line. (D) Crystal structure of 21 bound to BRD4 BD1 (surface representation: salmon indicates the WPF shelf, cyan indicates Asn140, orange indicates the BC loop, and green indicates the ZA channel).
图 3.取代苯环的 SAR 以及化合物 21 与 BRD4 BD1 和 BD2 蛋白复合物的共晶体结构。(A) 苯环取代的 SAR。SD 值见表 S1。(B）BRD4 BD1（浅粉色）与 21（黄色）结合的晶体结构（PDB ID：8IBQ）。(C) 与 21（黄色）结合的 BRD4 BD2（青绿色）的晶体结构（PDB ID：8IDH）。配体和重要残基的侧链以棒状模型显示；氢键（绿色）和 π-π 共轭（粉红色）用虚线表示。(D) 21 与 BRD4 BD1 结合的晶体结构（表面表示：鲑鱼色表示 WPF 架，青色表示 Asn140，橙色表示 BC 环，绿色表示 ZA 通道）。
To understand the structural basis of this new chemotype for BRD4, we solved the co-crystal structure of 21 bound to BRD4 BD1 and BRD4 BD2. As shown in Figure 3B,C, the imidazolopyridine core of compound 21 acts as a mimic of the acetylated lysine analogue, forming hydrogen bonds with Asn140 (BD1)/Asn433 (BD2) and Tyr97 (BD1)/Tyr390 (BD2). The phenyl ring of 21 forms a π–π stacking interaction with Pro82/Pro375. In addition, the 1,3,5-trimethylpyrazole motif extends into the hydrophobic pocket between the WPF shelf and the BC loop.
为了了解 BRD4 这种新化学型的结构基础，我们解析了 21 与 BRD4 BD1 和 BRD4 BD2 结合的共晶体结构。如图 3B 和 C 所示，化合物 21 的咪唑吡啶核心是乙酰化赖氨酸类似物的模拟物，与 Asn140（BD1）/Asn433（BD2）和 Tyr97（BD1）/Tyr390（BD2）形成氢键。21 的苯基环与 Pro82/Pro375 形成 π-π 堆叠作用。此外，1,3,5-三甲基吡唑基团延伸到 WPF 架和 BC 环之间的疏水袋中。
为了了解 BRD4 这种新化学型的结构基础，我们解析了 21 与 BRD4 BD1 和 BRD4 BD2 结合的共晶体结构。如图 3B 和 C 所示，化合物 21 的咪唑吡啶核心是乙酰化赖氨酸类似物的模拟物，与 Asn140（BD1）/Asn433（BD2）和 Tyr97（BD1）/Tyr390（BD2）形成氢键。21 的苯基环与 Pro82/Pro375 形成 π-π 堆叠作用。此外，1,3,5-三甲基吡唑基团延伸到 WPF 架和 BC 环之间的疏水袋中。
Having validated the structural basis of how these compounds bind BDs, we then extended the structure into the ZA channel to further enhance the binding. Multiple BET inhibitors effectively enhance the activity by occupying the ZA channel with their sulfonamide substituents. (57,64,65) A series of sulfonamide-substituted derivatives are summarized in Table 1. The alkyl sulfonamide-substituted compounds (25–28) brought more than a 2-fold improvement in potency compared to compound 21. However, compounds 29 and 30 substituted with aryl sulfonamides only showed comparable activity to 21. The binding affinity of 25–30 to BRD4 BD1 was tested by microscale thermophoresis (MST) analysis. The results revealed that 25–30 were significantly bound to BRD4 BD1, among which 26 (DDO-8926) was the optimal compound (Kd = 0.024 μM). The bromodomain protein selectivity of DDO-8926 was evaluated by the BROMOscan (Eurofins DiscoverX) at 1 μM. Pleasingly, DDO-8926 showed excellent selectivity over the non-BET bromodomains (Figure 4 and Table S2).
在验证了这些化合物如何与 BD 结合的结构基础后，我们将该结构扩展到了ZA 通道，以进一步增强其结合力。多种 BET 抑制剂通过磺酰胺取代基占据ZA 通道，从而有效提高了活性。(表 1 总结了一系列磺酰胺取代衍生物。与化合物 21 相比，烷基磺酰胺取代的化合物（25-28）的效力提高了 2 倍多。然而，由芳基磺酰胺取代的化合物 29 和 30 只显示出与化合物 21 相当的活性。通过微尺度热泳（MST）分析检测了 25-30 与 BRD4 BD1 的结合亲和力。结果表明，25-30 与 BRD4 BD1 有明显的结合，其中 26（DDO-8926）是最佳化合物（K d = 0.024 μM）。DDO-8926 的溴域蛋白选择性由 BROMOscan（Eurofins DiscoverX）在 1 μM 时进行评估。令人欣喜的是，DDO-8926 对非 BET 溴域表现出了极佳的选择性（图 4 和表 S2）。
在验证了这些化合物如何与 BD 结合的结构基础后，我们将该结构扩展到了ZA 通道，以进一步增强其结合力。多种 BET 抑制剂通过磺酰胺取代基占据ZA 通道，从而有效提高了活性。(表 1 总结了一系列磺酰胺取代衍生物。与化合物 21 相比，烷基磺酰胺取代的化合物（25-28）的效力提高了 2 倍多。然而，由芳基磺酰胺取代的化合物 29 和 30 只显示出与化合物 21 相当的活性。通过微尺度热泳（MST）分析检测了 25-30 与 BRD4 BD1 的结合亲和力。结果表明，25-30 与 BRD4 BD1 有明显的结合，其中 26（DDO-8926）是最佳化合物（K d = 0.024 μM）。DDO-8926 的溴域蛋白选择性由 BROMOscan（Eurofins DiscoverX）在 1 μM 时进行评估。令人欣喜的是，DDO-8926 对非 BET 溴域表现出了极佳的选择性（图 4 和表 S2）。
Figure 4 图 4
Figure 4. Selectivity assessment of DDO-8926 against a panel of bromodomains using a DiscoverX BROMOscan platform at 1 μM. Percent control = [(test compound signal – positive control signal)/(negative control signal – positive control signal)] × 100.
图 4.使用 DiscoverX BROMOscan 平台评估 DDO-8926 在 1 μM 时对一组溴结构域的选择性。控制百分比 = [（测试化合物信号 - 阳性对照信号）/（阴性对照信号 - 阳性对照信号）] × 100。× 100.
Table 1. Introduction and Optimization of Substituents for Targeting the ZA Channel
表 1.针对ZA 通道的取代基介绍和优化
表 1.针对ZA 通道的取代基介绍和优化
a
a 通过 MST 测试了所选化合物与 BRD4 BD1 的结合亲和力。
The binding affinity of the selected compounds to BRD4 BD1 was tested by MST.
a 通过 MST 测试了所选化合物与 BRD4 BD1 的结合亲和力。
Drug-like Property Evaluation of DDO-8926
DDO-8926 的类药物特性评估
After confirming the outstanding selectivity of DDO-8926 within the BET family proteins, drug-like property evaluations were carried out. As shown in Table 2, DDO-8926 exhibited reasonable water solubility and favorable stability in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and rat liver microsomes (RLM). In addition, DDO-8926 showed almost no inhibitory effect on hERG ion channels (Figure S1, IC50 > 30 μM) and the five major CYP450 enzymes (Table S4, IC50 > 10 μM). This suggested a low risk of potential cardiotoxicity and drug–drug interactions for DDO-8926. The mouse plasma protein binding (PPB) rate of DDO-8926 was 99.0% (fraction unbound factor = 0.01) (Table S5). The in vivo pharmacokinetic (PK) profiles of DDO-8926 were tested in mice. DDO-8926 showed a high drug exposure by intravenous (iv) or intraperitoneal (ip) administration, and a half-life of 1.78 h by iv administration (Figure 5). In addition, the PK of DDO-8926 was also tested in rats. DDO-8926 showed a half-life of 2.36 h by iv administration and an acceptable oral bioavailability in rat (F = 21.43%) at a dose of 25 mg/kg (Figure S2). All the above results together indicated that DDO-8926 exhibited favorable drug-like properties.
在确认 DDO-8926 在 BET 家族蛋白中具有出色的选择性之后，我们对其进行了类药物特性评估。如表 2 所示，DDO-8926 在模拟胃液（SGF）、模拟肠液（SIF）和大鼠肝微粒体（RLM）中表现出合理的水溶性和良好的稳定性。此外，DDO-8926 对 hERG 离子通道（图 S1，IC 50 > 30 μM）和五种主要的 CYP450 酶（表 S4，IC 50 > 10 μM）几乎没有抑制作用。这表明 DDO-8926 的潜在心脏毒性和药物间相互作用风险较低。DDO-8926的小鼠血浆蛋白结合率（PPB）为99.0%（未结合因子=0.01）（表S5）。在小鼠体内测试了 DDO-8926 的体内药代动力学（PK）特征。DDO-8926在静脉注射（iv）或腹膜内注射（ip）时显示出较高的药物暴露量，静脉注射的半衰期为1.78小时（图5）。此外，还在大鼠体内测试了 DDO-8926 的 PK。DDO-8926 的静脉注射半衰期为 2.36 小时，大鼠口服生物利用度（F = 21.43%）为 25 毫克/千克（图 S2）。所有上述结果都表明，DDO-8926 具有良好的类药物特性。
在确认 DDO-8926 在 BET 家族蛋白中具有出色的选择性之后，我们对其进行了类药物特性评估。如表 2 所示，DDO-8926 在模拟胃液（SGF）、模拟肠液（SIF）和大鼠肝微粒体（RLM）中表现出合理的水溶性和良好的稳定性。此外，DDO-8926 对 hERG 离子通道（图 S1，IC 50 > 30 μM）和五种主要的 CYP450 酶（表 S4，IC 50 > 10 μM）几乎没有抑制作用。这表明 DDO-8926 的潜在心脏毒性和药物间相互作用风险较低。DDO-8926的小鼠血浆蛋白结合率（PPB）为99.0%（未结合因子=0.01）（表S5）。在小鼠体内测试了 DDO-8926 的体内药代动力学（PK）特征。DDO-8926在静脉注射（iv）或腹膜内注射（ip）时显示出较高的药物暴露量，静脉注射的半衰期为1.78小时（图5）。此外，还在大鼠体内测试了 DDO-8926 的 PK。DDO-8926 的静脉注射半衰期为 2.36 小时，大鼠口服生物利用度（F = 21.43%）为 25 毫克/千克（图 S2）。所有上述结果都表明，DDO-8926 具有良好的类药物特性。
Figure 5 图 5
Figure 5. In vivo PK parameters of DDO-8926 in male mice. The values shown are the means ± SEM (n = 4).
图 5.雄性小鼠体内 DDO-8926 的 PK 参数。所示数值为平均值 ± SEM（n = 4）。
Table 2. Solubility, Stability in SIF, SGF and RLM, and hERG Channel Inhibitory Activity of DDO-8926
表 2.DDO-8926 的溶解度、在 SIF、SGF 和 RLM 中的稳定性以及 hERG 通道抑制活性
表 2.DDO-8926 的溶解度、在 SIF、SGF 和 RLM 中的稳定性以及 hERG 通道抑制活性
| parameters 参数 | DDO-8926 |
|---|---|
| solubility (μg/mL)a 溶解度（微克/毫升） a | 129.8 |
| SIFb | 85.4 |
| SGFb | 86.5 |
| RLM T1/2 (h) RLM T 1/2 (h) | 1.81 |
| RLM Clint (μL/min/mg) RLM Cl int (微升/分钟/毫克） | 6.38 |
| CYP450 (1A2, 2C9, 2C19, 2D6, 3A4, (IC50, μM)) CYP450 (1A2, 2C9, 2C19, 2D6, 3A4, (IC 50 , μM)) | >10 |
| hERG (IC50, μM) hERG（IC 50 ，μM） | >30 |
| PPB (mouse, %)c PPB （小鼠，%） c | 99.0 |
a
a 采用 HPLC 方法进行溶解度测试。
Solubility testing by the HPLC method.
a 采用 HPLC 方法进行溶解度测试。
b
b 培养 24 小时后的剩余量（%）。
Remaining (%) after 24 h incubation.
b 培养 24 小时后的剩余量（%）。
c
c 小鼠血浆蛋白结合
Plasma protein binding in mice.
c 小鼠血浆蛋白结合
Toxicity Evaluation of DDO-8926 in Mice
DDO-8926 对小鼠的毒性评估
A subacute toxicity assay was performed to investigate the safety of DDO-8926. An oral PK experiment of DDO-8926 (500 mg/kg) was first performed on mice to confirm adequate exposure and effective target engagement of DDO-8926. The results showed that the maximum plasma drug level of DDO-8926 could reach 23,812 ng/mL (unbound plasma drug level: 238 ng/mL (557 nM)), and the plasma drug level could be maintained at more than 4800 ng/mL (unbound plasma drug level: 112 nM) for 4 h (Figure S3A). This suggests that oral administration of DDO-8926 (500 mg/kg) had adequate unbound plasma drug level and effective target engagement. Next, healthy C57BL/6 mice were treated with DDO-8926 at a dose of 500 mg/kg for 2 weeks (po, q.d.). During the dosing period, smooth hair, no significant behavioral abnormalities, and weight loss were observed in the normal and administered groups of mice (Figure S3B). As with normal mice, hematoxylin–eosin (H&E) staining of organs from DDO-8926-treated mice for 14 days showed no significant tissue damage (Figure S3C). On day 14, the mice’s blood was collected and subjected to whole blood cell tests. Compared to the control group, the platelets in the drug-treated mice decreased by 39%, which was below the lowest of the normal range, but the platelets could return to normal levels naturally after 10 days of withdrawal from the drug (Figure S3D). These results indicate that DDO-8926 has a minor impact on normal organs and body weight in mice and that longer continuous administration leads to a decrease in platelets.
为了研究 DDO-8926 的安全性，我们进行了亚急性毒性试验。首先对小鼠进行了 DDO-8926（500 毫克/千克）的口服 PK 实验，以确认 DDO-8926 的充分暴露和有效靶向参与。结果表明，DDO-8926的最大血浆药物浓度可达23812纳克/毫升（非结合血浆药物浓度：238纳克/毫升（557毫微克）），且血浆药物浓度可在4800纳克/毫升（非结合血浆药物浓度：112毫微克）以上维持4小时（图S3A）。这表明口服DDO-8926（500 mg/kg）具有足够的非结合血浆药物水平和有效的靶向参与。接下来，健康的 C57BL/6 小鼠以 500 毫克/千克的剂量连续 2 周服用 DDO-8926（po，q.d.）。在给药期间，正常组和给药组小鼠的毛发光滑，没有明显的行为异常和体重减轻（图 S3B）。与正常小鼠一样，DDO-8926 治疗小鼠 14 天的器官苏木精-伊红（H&E）染色显示没有明显的组织损伤（图 S3C）。第 14 天，采集小鼠血液并进行全血细胞检测。与对照组相比，药物治疗组小鼠的血小板减少了 39%，低于正常范围的最低值，但停药 10 天后血小板可自然恢复到正常水平（图 S3D）。这些结果表明，DDO-8926 对小鼠的正常器官和体重影响较小，而长时间连续给药会导致血小板减少。
为了研究 DDO-8926 的安全性，我们进行了亚急性毒性试验。首先对小鼠进行了 DDO-8926（500 毫克/千克）的口服 PK 实验，以确认 DDO-8926 的充分暴露和有效靶向参与。结果表明，DDO-8926的最大血浆药物浓度可达23812纳克/毫升（非结合血浆药物浓度：238纳克/毫升（557毫微克）），且血浆药物浓度可在4800纳克/毫升（非结合血浆药物浓度：112毫微克）以上维持4小时（图S3A）。这表明口服DDO-8926（500 mg/kg）具有足够的非结合血浆药物水平和有效的靶向参与。接下来，健康的 C57BL/6 小鼠以 500 毫克/千克的剂量连续 2 周服用 DDO-8926（po，q.d.）。在给药期间，正常组和给药组小鼠的毛发光滑，没有明显的行为异常和体重减轻（图 S3B）。与正常小鼠一样，DDO-8926 治疗小鼠 14 天的器官苏木精-伊红（H&E）染色显示没有明显的组织损伤（图 S3C）。第 14 天，采集小鼠血液并进行全血细胞检测。与对照组相比，药物治疗组小鼠的血小板减少了 39%，低于正常范围的最低值，但停药 10 天后血小板可自然恢复到正常水平（图 S3D）。这些结果表明，DDO-8926 对小鼠的正常器官和体重影响较小，而长时间连续给药会导致血小板减少。
Antinociceptive Efficacy of DDO-8926 on the Spared Nerve Injury Model of NP in Mice
DDO-8926 对小鼠无神经损伤模型的抗痛觉作用
First, we evaluated the drug distribution of DDO-8926 (30 mg/kg, ip) into the plasma and the intracerebral space in C57BL/6 mice. The results showed that total plasma drug concentration peaked at 0.5 h (Cmax = 161,57 ng/mL) after a single intraperitoneal injection of DDO-8926 (30 mg/kg), while the total brain homogenate drug concentration peaked at 1 h (Cmax = 2392 ng/mL). Based on Poulin’s method, (66) the fraction unbound factor (fub) in the brain was calculated to be 0.0198 in combination with the plasma protein unbound factor (fup = 0.01), and the unbound drug concentration–time curve in the brain was fitted. The results showed that the unbound-brain drug concentration of the peak was 47 ng/mL (110 nM) (Figure 6A). This suggests that DDO-8926 has sufficient unbound drug exposure in the brain at 30 mg/kg. Next, the in vivo efficacy of DDO-8926 was evaluated in a spared nerve injury (SNI) model, as the study of this animal model is a valuable source of information in the field of NP. Female C57BL/6 mice (7–8 weeks old) were randomly divided into four groups, sham mice and SNI mice were operated according to the method reported by Decosterd and Woolf. (67) Drug treatment (DDO-8926, ip or gabapentin, ip) or SNI (vehicle) started at 1 day post-operation (dpo); then, the drug was administered once daily (Figure 6B). In a pilot study, we found that the peak analgesic effect was reached at 1 h after DDO-8926 treatment, so we chose to perform the mechanical pain response experiment at 1 h after drug treatment. Administration of DDO-8926 at 30 mg/kg started to relieve mechanical hypersensitivity on 5 dpo and reached a therapeutic effect comparable to that of gabapentin at 100 mg/kg on dpo 13–15 (Figure 6C). We tested mechanical hypersensitivity within 4 h after administration on day 15. The results showed that the time to peak and the maximal effect of 30 mg/kg DDO-8926 versus 100 mg/kg gabapentin were comparable (Figure 6D). These results suggest that the compound DDO-8926 effectively alleviates mechanical hypersensitivity after SNI.
首先，我们评估了DDO-8926（30 mg/kg，ip）在C57BL/6小鼠血浆和脑内的药物分布情况。结果显示，单次腹腔注射DDO-8926（30 mg/kg）后，血浆药物总浓度在0.5 h达到峰值（C max = 161,57 ng/mL），而脑匀浆药物总浓度在1 h达到峰值（C max = 2392 ng/mL）。根据 Poulin 方法 (66) 计算出脑中的非结合因子（f ub ）与血浆蛋白非结合因子（f up = 0.01）之和为 0.0198，并拟合出脑中非结合药物浓度-时间曲线。结果显示，峰值的脑内未结合药物浓度为 47 ng/mL（110 nM）（图 6A）。这表明 DDO-8926 在 30 毫克/千克的剂量下在脑内有足够的非结合药物暴露。接下来，在幸免神经损伤（SNI）模型中评估了 DDO-8926 的体内疗效，因为这种动物模型的研究是 NP 领域的宝贵信息来源。雌性 C57BL/6 小鼠（7-8 周大）被随机分为四组，假小鼠和 SNI 小鼠按照 Decosterd 和 Woolf 报道的方法进行操作。(67）药物治疗（DDO-8926，ip 或加巴喷丁，ip）或 SNI（载体）从手术后 1 天（dpo）开始；然后，每天给药一次（图 6B）。在一项试验研究中，我们发现 DDO-8926 治疗后 1 小时达到镇痛效果峰值，因此我们选择在药物治疗后 1 小时进行机械痛反应实验。30 mg/kg剂量的DDO-8926从5 dpo开始缓解机械过敏，并在dpo 13-15达到与100 mg/kg剂量的加巴喷丁相当的治疗效果（图6C）。我们在第 15 天测试了用药后 4 小时内的机械过敏性。结果显示，30 毫克/千克的 DDO-8926 与 100 毫克/千克的加巴喷丁达到峰值的时间和最大疗效相当（图 6D）。这些结果表明，化合物 DDO-8926 能有效缓解 SNI 后的机械超敏反应。
首先，我们评估了DDO-8926（30 mg/kg，ip）在C57BL/6小鼠血浆和脑内的药物分布情况。结果显示，单次腹腔注射DDO-8926（30 mg/kg）后，血浆药物总浓度在0.5 h达到峰值（C max = 161,57 ng/mL），而脑匀浆药物总浓度在1 h达到峰值（C max = 2392 ng/mL）。根据 Poulin 方法 (66) 计算出脑中的非结合因子（f ub ）与血浆蛋白非结合因子（f up = 0.01）之和为 0.0198，并拟合出脑中非结合药物浓度-时间曲线。结果显示，峰值的脑内未结合药物浓度为 47 ng/mL（110 nM）（图 6A）。这表明 DDO-8926 在 30 毫克/千克的剂量下在脑内有足够的非结合药物暴露。接下来，在幸免神经损伤（SNI）模型中评估了 DDO-8926 的体内疗效，因为这种动物模型的研究是 NP 领域的宝贵信息来源。雌性 C57BL/6 小鼠（7-8 周大）被随机分为四组，假小鼠和 SNI 小鼠按照 Decosterd 和 Woolf 报道的方法进行操作。(67）药物治疗（DDO-8926，ip 或加巴喷丁，ip）或 SNI（载体）从手术后 1 天（dpo）开始；然后，每天给药一次（图 6B）。在一项试验研究中，我们发现 DDO-8926 治疗后 1 小时达到镇痛效果峰值，因此我们选择在药物治疗后 1 小时进行机械痛反应实验。30 mg/kg剂量的DDO-8926从5 dpo开始缓解机械过敏，并在dpo 13-15达到与100 mg/kg剂量的加巴喷丁相当的治疗效果（图6C）。我们在第 15 天测试了用药后 4 小时内的机械过敏性。结果显示，30 毫克/千克的 DDO-8926 与 100 毫克/千克的加巴喷丁达到峰值的时间和最大疗效相当（图 6D）。这些结果表明，化合物 DDO-8926 能有效缓解 SNI 后的机械超敏反应。
Figure 6 图 6
Figure 6. DDO-8926 attenuates NP hypersensitivity. (A) Distribution of DDO-8926 in the plasma and brain of mice. (B) Experimental scheme depicting the in vivo analgesic evaluation of DDO-8926. (C) Analgesic effect of gabapentin (100 mg/kg, ip) and DDO-8926 (30 mg/kg, ip) on SNI-induced NP response tested by ipsilateral stimulation. (D) Analgesic effects of gabapentin (100 mg/kg, ip) and DDO-8926 (30 mg/kg, ip) at 15 days post-operation (dpo). Results were expressed as mean ± SEM (n = 6), (ns) < 0.1, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001 compared with the SNI (vehicle) group, one-way ANOVA with the Tukey–Kramer post-test.
图 6.DDO-8926 可减轻 NP 超敏反应。(A) DDO-8926 在小鼠血浆和大脑中的分布。(B) 描述 DDO-8926 体内镇痛评估的实验方案。(C）加巴喷丁（100 毫克/千克，ip）和 DDO-8926（30 毫克/千克，ip）对同侧刺激测试 SNI 诱导的 NP 反应的镇痛效果。(D) 加巴喷丁（100 毫克/千克，ip）和 DDO-8926（30 毫克/千克，ip）在手术后 15 天（dpo）的镇痛效果。结果以平均值 ± SEM 表示（n = 6），(ns) < 0.1，(*) P < 0.05，(**) P < 0.01，(***) P < 0.001，(****) 与 SNI（车辆）组相比，P < 0.0001，单因素方差分析，Tukey-Kramer 后检验。
DDO-8926 Regulates Genes Involved in the Generation and Transfer of Action Potentials and Inflammatory Response
DDO-8926 可调控参与动作电位的产生和传递以及炎症反应的基因
Following these encouraging results, to clarify the analgesic mechanism of DDO-8926 after SNI, we used RNA sequencing to analyze the effects of DDO-8926 on the expression of genes in the L4–L6 segment of the spinal cord on 15 dpo. Volcano plots were used to show differentially expressed genes (DEGs). 147 genes downregulated and 39 genes upregulated in the SNI group compared to the sham group. 233 genes downregulated and 111 genes upregulated were detected in the DDO-8926-treated group compared to the SNI group. 27 genes downregulated and 187 genes upregulated in the DDO-8926-treated group compared to the sham mice (Figure 7A). For a clearer understanding of DEGs, gene ontology (GO) functional enrichment was performed. The results showed that the DEGs induced by DDO-8926 were mainly related to ion transport, regulation of action potential, glial cell migration and apoptotic processes, and inflammatory response (Figure 7B). To focus on key differential gene sets, gene sets for ion transport, glial cell activation and migration, action potential regulation, and inflammatory response were evaluated by gene set enrichment analysis (GSEA). The structure showed that treatment with DDO-8926 produced statistically significant changes in these gene sets (Figure 7C–F). Overall, DDO-8926 can affect action potential generation and transfer, glial cell activation and migration, and inflammatory response after SNI.
根据这些令人鼓舞的结果，为了明确DDO-8926在SNI后的镇痛机制，我们使用RNA测序分析了DDO-8926对15 dpo时脊髓L4-L6段基因表达的影响。我们使用火山图来显示差异表达基因（DEGs）。与假体组相比，SNI 组有 147 个基因下调，39 个基因上调。与SNI组相比，DDO-8926治疗组检测到233个基因下调，111个基因上调。与假小鼠相比，DDO-8926 处理组有 27 个基因下调，187 个基因上调（图 7A）。为了更清楚地了解 DEGs，进行了基因本体（GO）功能富集。结果显示，DDO-8926 诱导的 DEGs 主要与离子转运、动作电位调节、胶质细胞迁移和凋亡过程以及炎症反应有关（图 7B）。为了聚焦关键的差异基因组，通过基因组富集分析（GSEA）评估了离子转运、胶质细胞活化和迁移、动作电位调控和炎症反应的基因组。该结构显示，用 DDO-8926 处理会使这些基因组发生统计学意义上的显著变化（图 7C-F）。总体而言，DDO-8926 可影响 SNI 后的动作电位产生和传递、神经胶质细胞活化和迁移以及炎症反应。
根据这些令人鼓舞的结果，为了明确DDO-8926在SNI后的镇痛机制，我们使用RNA测序分析了DDO-8926对15 dpo时脊髓L4-L6段基因表达的影响。我们使用火山图来显示差异表达基因（DEGs）。与假体组相比，SNI 组有 147 个基因下调，39 个基因上调。与SNI组相比，DDO-8926治疗组检测到233个基因下调，111个基因上调。与假小鼠相比，DDO-8926 处理组有 27 个基因下调，187 个基因上调（图 7A）。为了更清楚地了解 DEGs，进行了基因本体（GO）功能富集。结果显示，DDO-8926 诱导的 DEGs 主要与离子转运、动作电位调节、胶质细胞迁移和凋亡过程以及炎症反应有关（图 7B）。为了聚焦关键的差异基因组，通过基因组富集分析（GSEA）评估了离子转运、胶质细胞活化和迁移、动作电位调控和炎症反应的基因组。该结构显示，用 DDO-8926 处理会使这些基因组发生统计学意义上的显著变化（图 7C-F）。总体而言，DDO-8926 可影响 SNI 后的动作电位产生和传递、神经胶质细胞活化和迁移以及炎症反应。
Figure 7 图 7
Figure 7. DDO-8926 regulates genes involved in the generation and transfer of action potentials and inflammatory response. (A) These volcano maps show DEGs after SNI surgery and DDO-8926 treatment. (B) GO enrichment analysis of DEGs between vehicle and DDO-8926 treatment. GSEA analysis of (C) ion transfer, (D) glial cell migration, (E) action potential, and (F) inflammatory response between vehicle and DDO-8926 treatment.
图 7.DDO-8926 可调控参与动作电位的产生和传递以及炎症反应的基因。(A）这些火山图显示了 SNI 手术和 DDO-8926 治疗后的 DEGs。(B) 车辆和 DDO-8926 治疗后 DEGs 的 GO 富集分析。车辆和 DDO-8926 治疗之间 (C) 离子转移、(D) 神经胶质细胞迁移、(E) 动作电位和 (F) 炎症反应的 GSEA 分析。
DDO-8926 Reduces the Activation of Microglia and Neurons after SNI in the Dorsal Horn Spinal Cord
DDO-8926 可降低背角脊髓 SNI 后小胶质细胞和神经元的活化程度
Considering the tips of RNA sequencing, we subsequently evaluated the effect of DDO-8926 treatment on glial cells and neurons in the dorsal horn, where nociceptive afferents project. At 15 dpo, immunofluorescence staining for microglia and neurons revealed an increase of IBA1-positive cells and NeuN-positive cells after SNI in the dorsal horn, while these positive cells were significantly reduced after DDO-8926 treatment (Figure 8A,B). Notably, this phenomenon was not observed in GFAP-traced astrocytes (Figure 8C). These results suggested that DDO-8926 treatment may inhibit microglial and neuronal activation after SNI.
考虑到 RNA 测序的提示，我们随后评估了 DDO-8926 处理对背角神经胶质细胞和神经元的影响，背角神经胶质细胞和神经元是痛觉传入的投射点。在 15 dpo 时，小胶质细胞和神经元的免疫荧光染色显示，背角的 IBA1 阳性细胞和 NeuN 阳性细胞在 SNI 处理后有所增加，而这些阳性细胞在 DDO-8926 处理后显著减少（图 8A,B）。值得注意的是，在 GFAP 追踪的星形胶质细胞中没有观察到这种现象（图 8C）。这些结果表明，DDO-8926 治疗可抑制 SNI 后的小胶质细胞和神经元活化。
考虑到 RNA 测序的提示，我们随后评估了 DDO-8926 处理对背角神经胶质细胞和神经元的影响，背角神经胶质细胞和神经元是痛觉传入的投射点。在 15 dpo 时，小胶质细胞和神经元的免疫荧光染色显示，背角的 IBA1 阳性细胞和 NeuN 阳性细胞在 SNI 处理后有所增加，而这些阳性细胞在 DDO-8926 处理后显著减少（图 8A,B）。值得注意的是，在 GFAP 追踪的星形胶质细胞中没有观察到这种现象（图 8C）。这些结果表明，DDO-8926 治疗可抑制 SNI 后的小胶质细胞和神经元活化。
Figure 8 图 8
Figure 8. DDO-8926 reduces microglial and neuronal activation in the spinal cord after SNI and has no effect on astrocytes. Representative microphotographs from the L5 spinal dorsal horn of the ipsilateral hemisections labeled with (A) NeuN, (B) IBA1, and (C) GFAP of Sham, SNI (vehicle), and SNI (DDO-8926 30 mg/kg) groups at 15 dpo. Representative microphotographs (left) and positive cells (right). Results were expressed as mean ± SEM (n = 3), (ns) < 0.1, (***) P < 0.001, and (****) P < 0.0001 compared with the SNI (vehicle) group, one-way ANOVA with the Tukey–Kramer post-test.
图 8.DDO-8926 可减少 SNI 后脊髓中的小胶质细胞和神经元活化，而对星形胶质细胞没有影响。SHAM 组、SNI 组（药物）和 SNI 组（DDO-8926 30 mg/kg）在 15 dpo 时同侧半裂 L5 脊髓背角的代表性显微照片，分别标记有 (A)NeuN、(B) IBA1 和 (C) GFAP。具有代表性的显微照片（左）和阳性细胞（右）。结果以均数 ± SEM 表示（n = 3），与 SNI（载体）组相比，(ns) < 0.1，(***) P < 0.001，(****) P < 0.0001，单因素方差分析，Tukey-Kramer 后检验。
DDO-8926 Regulates the mRNA Level of Proinflammatory Cytokines and Ion Channels and Na+/K+ ATPase Pump after SNI
DDO-8926 可调节促炎细胞因子、离子通道和 Na + 的 mRNA 水平/K + ATPase Pump after SNI
Neuroinflammation is closely associated with active excitability and persistent NP. Therefore, we measured the mRNA expression of pro-inflammatory cytokines at the enlargement of the spinal cord (L5–L6 segment) at dpo 15. The results showed that the vehicle-treated SNI mice increased the expression of IL-1β and TNF-α, which was prevented by DDO-8926 or gabapentin treatment, and the former showed a stronger anti-inflammatory effect (Figure 9A). SNI-induced pain hypersensitivity is manifested by a rise in excitability, which is regulated by the generation and transfer of action potentials. This is consistent with the results of transcriptomics showing an effect on excitability and action potential after SNI. SCNA3A, a subunit belonging to the Nav channel, is associated with the generation of action potentials. At 15 dpo, DDO-8926-treated mice exhibited a significant downregulation of SCNA3A, compared to the vehicle group (Figure 9B). In addition, DDO-8926 treatment significantly inhibited the SNI-induced overexpression of KCNQ2 and KCNQ3, which are subunits of the slow Kv channels that are essential for the maintenance of the membrane potential (Figure 9C). Ion transporters are also an important means of ion transmembrane transport, such as the Na+/K+ ATPase pump. Thus, the mRNA levels of αNKA, a subunit of the Na+/K+ ATPase pump, were detected in the spinal cord. Overexpression of αNKA was observed in vehicle-treated mice, and it was significantly suppressed by DDO-8926 treatment (Figure 9D). The αNKA subunit negatively regulates the Na+/K+ ATPase pump activity, which is antagonized by DDO-8926, leading to hyperpolarization of neurons. The above experimental results showed that DDO-8926 reduced SNI-induced excitability by affecting ion transmembrane transport and Na+/K+ ATPase pump activity. In addition, DDO-8926 also reduced the expression of pro-inflammatory cytokines with protective effects on neuroinflammation after SNI.
神经炎症与活跃的兴奋性和持续的NP密切相关。因此，我们在第15天测量了脊髓扩大处（L5-L6节段）促炎细胞因子的mRNA表达。结果表明，用药物治疗的SNI小鼠IL-1β和TNF-α的表达增加，而DDO-8926或加巴喷丁治疗可阻止IL-1β和TNF-α的表达，前者的抗炎作用更强（图9A）。SNI诱导的痛觉过敏表现为兴奋性升高，而兴奋性是由动作电位的产生和传递调节的。这与转录组学结果显示 SNI 会影响兴奋性和动作电位是一致的。SCNA3A 是 Nav 通道的一个亚基，与动作电位的产生有关。与车辆组相比，DDO-8926 处理的小鼠在 15 dpo 时表现出 SCNA3A 的显著下调（图 9B）。此外，DDO-8926 还能显著抑制 SNI 诱导的 KCNQ2 和 KCNQ3 的过表达，KCNQ2 和 KCNQ3 是慢 Kv 通道的亚基，对维持膜电位至关重要（图 9C）。离子转运体也是离子跨膜转运的重要途径，如 Na + /K + 通道。/K +ATPase 泵。因此，Na + /K + ATPase 泵的一个亚基 αNKA 的 mRNA 水平也会受到影响。/K +ATPase 泵的 mRNA 水平。在用药物治疗的小鼠中观察到αNKA的过表达，DDO-8926治疗显著抑制了αNKA的过表达（图9D）。αNKA亚基对Na + /K + 负调控。/K +ATPase 泵的活性，DDO-8926 可拮抗这种活性，从而导致神经元超极化。上述实验结果表明，DDO-8926 通过影响离子跨膜转运和 Na + /K + ATPase 泵活性，降低了 SNI 诱导的兴奋性。/K +ATPase 泵的活性，从而降低 SNI 诱导的兴奋性。此外，DDO-8926 还能减少促炎细胞因子的表达，对 SNI 后的神经炎症具有保护作用。
神经炎症与活跃的兴奋性和持续的NP密切相关。因此，我们在第15天测量了脊髓扩大处（L5-L6节段）促炎细胞因子的mRNA表达。结果表明，用药物治疗的SNI小鼠IL-1β和TNF-α的表达增加，而DDO-8926或加巴喷丁治疗可阻止IL-1β和TNF-α的表达，前者的抗炎作用更强（图9A）。SNI诱导的痛觉过敏表现为兴奋性升高，而兴奋性是由动作电位的产生和传递调节的。这与转录组学结果显示 SNI 会影响兴奋性和动作电位是一致的。SCNA3A 是 Nav 通道的一个亚基，与动作电位的产生有关。与车辆组相比，DDO-8926 处理的小鼠在 15 dpo 时表现出 SCNA3A 的显著下调（图 9B）。此外，DDO-8926 还能显著抑制 SNI 诱导的 KCNQ2 和 KCNQ3 的过表达，KCNQ2 和 KCNQ3 是慢 Kv 通道的亚基，对维持膜电位至关重要（图 9C）。离子转运体也是离子跨膜转运的重要途径，如 Na + /K + 通道。/K +ATPase 泵。因此，Na + /K + ATPase 泵的一个亚基 αNKA 的 mRNA 水平也会受到影响。/K +ATPase 泵的 mRNA 水平。在用药物治疗的小鼠中观察到αNKA的过表达，DDO-8926治疗显著抑制了αNKA的过表达（图9D）。αNKA亚基对Na + /K + 负调控。/K +ATPase 泵的活性，DDO-8926 可拮抗这种活性，从而导致神经元超极化。上述实验结果表明，DDO-8926 通过影响离子跨膜转运和 Na + /K + ATPase 泵活性，降低了 SNI 诱导的兴奋性。/K +ATPase 泵的活性，从而降低 SNI 诱导的兴奋性。此外，DDO-8926 还能减少促炎细胞因子的表达，对 SNI 后的神经炎症具有保护作用。
Figure 9 图 9
Figure 9. DDO-8926 alleviates inflammation and affects the expression of ion channels and the Na+/K+ ATPase pump in the spinal cord at 15 dpo after SNI. (A) DDO-8926 downregulates the expression of pro-inflammatory factor mRNA in the spinal cord after SNI. DDO-8926 inhibits SNI-induced mRNA expression of (B) voltage-gated sodium channels and (C) voltage-gated potassium channels. (D) DDO-8926 reduces the SNI-induced mRNA expression of the αNKA subunit of the Na+/K+ ATPase pump. Results were expressed as mean ± SEM (n = 6), (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001 compared with the SNI (vehicle) group, one-way ANOVA with the Tukey–Kramer post-test.
图 9.DDO-8926 可减轻炎症并影响离子通道和 Na + /K + 的表达。/K +ATPase 泵的表达。(A) DDO-8926 下调了 SNI 后脊髓中促炎因子 mRNA 的表达。DDO-8926 可抑制 SNI 诱导的 (B) 电压门控钠通道和 (C) 电压门控钾通道的 mRNA 表达。(D) DDO-8926 可减少 SNI 诱导的 Na + /K + αNKA 亚基的 mRNA 表达。/K +ATPase 泵的 mRNA 表达。结果以平均值 ± SEM 表示（n = 6），与 SNI（载体）组相比，(**) P < 0.01，(***) P < 0.001，(****) P < 0.0001，单因素方差分析，Tukey-Kramer 后检验。
Compounds 1–14 were synthesized according to Scheme 1. Intermediate 44 was prepared by condensation reaction of 46 and 47. Target compounds 1–14 were synthesized by Suzuki coupling of 31–44 and 45 in the presence of Pd(dppf)Cl2·CH2Cl2 and cesium carbonate.
化合物 1-14 是按照方案 1 合成的。中间体 44 是通过 46 和 47 的缩合反应制备的。在 Pd(dppf)Cl {{0} 存在下，通过 31-44 和 45 的铃木偶联合成了目标化合物 1-14。｝-CH 2 Cl 2 和碳酸铯。
化合物 1-14 是按照方案 1 合成的。中间体 44 是通过 46 和 47 的缩合反应制备的。在 Pd(dppf)Cl {{0} 存在下，通过 31-44 和 45 的铃木偶联合成了目标化合物 1-14。｝-CH 2 Cl 2 和碳酸铯。
Scheme 1 方案 1
Scheme 1. Reagents and Conditions: (a) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h and (b) THF, 80 °C, 4 h
方案 1.试剂和条件： (a) Cs 2 CO 3 Pd(dppf)Cl 2 -CH 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h.O, 100 °C, 3 h 和 (b) THF, 80 °C, 4 h
方案 1.试剂和条件： (a) Cs 2 CO 3 Pd(dppf)Cl 2 -CH 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h.O, 100 °C, 3 h 和 (b) THF, 80 °C, 4 h
Target compounds 15–20 were synthesized based on Scheme 2. Intermediates 50a–50f were prepared by Suzuki coupling of 48 and 49a–49f in the presence of Pd(dppf)Cl2·CH2Cl2 and cesium carbonate. 50g was obtained by the substitution reaction of 50f and 53. Then, intermediates 50a–50e and 50g were subsequently subjected to reaction with 51 to give boronate intermediates 52a–52e and 52g, respectively. Target compounds 15–20 were synthesized by Suzuki coupling of 52a–52e, 52g, and 42.
根据方案 2 合成了目标化合物 15-20。在 Pd(dppf)Cl {{0} 的存在下，通过 48 和 49a-49f 的铃木偶联制备了中间体 50a-50f。｝-CH 2 Cl 2 和碳酸铯。通过 50f 和 53 的取代反应得到 50g。随后，中间体 50a-50e 和 50g 与 51 反应，分别得到硼酸中间体 52a-52e 和 52g。目标化合物 15-20 是通过 52a-52e、52g 和 42 的铃木偶联合成的。
根据方案 2 合成了目标化合物 15-20。在 Pd(dppf)Cl {{0} 的存在下，通过 48 和 49a-49f 的铃木偶联制备了中间体 50a-50f。｝-CH 2 Cl 2 和碳酸铯。通过 50f 和 53 的取代反应得到 50g。随后，中间体 50a-50e 和 50g 与 51 反应，分别得到硼酸中间体 52a-52e 和 52g。目标化合物 15-20 是通过 52a-52e、52g 和 42 的铃木偶联合成的。
Scheme 2 方案 2
Scheme 2. Reagents and Conditions: (a) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl2·CH2Cl2, Dry 1,4-Dioxane, 100 °C, 3 h; (c) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 7 h; and (d) Cs2CO3, DMF, r.t., 5 h
方案 2.试剂和条件： (a) Cs 2 CO 3 Pd(dppf)Cl 2 -CH 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h; Pd(dppf)Cl 2 -CH 2 Cl 2 O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl { 2 -CH 2 Cl 2 干 1,4-二恶烷，100 °C，3 小时； (c) Cs 2 CO 3 (c) Cs 2 CO 3 , Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-二恶烷，H 2 OO，100 °C，7 小时；以及 (d) Cs 2 CO 3 ，DMF，r.t. ，5 小时
方案 2.试剂和条件： (a) Cs 2 CO 3 Pd(dppf)Cl 2 -CH 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h; Pd(dppf)Cl 2 -CH 2 Cl 2 O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl { 2 -CH 2 Cl 2 干 1,4-二恶烷，100 °C，3 小时； (c) Cs 2 CO 3 (c) Cs 2 CO 3 , Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-二恶烷，H 2 OO，100 °C，7 小时；以及 (d) Cs 2 CO 3 ，DMF，r.t. ，5 小时
Target compounds 21–24 were prepared according to Scheme 3. Commercially available 54a–54d were subjected to Suzuki coupling with 49b to give intermediates 55a–55d, followed by reaction with 51 to give boronate intermediates 56a–56d. 56a–56d were further reacted with 42 to give compounds 21–24.
目标化合物 21-24 是根据方案 3 制备的。市售的 54a-54d 与 49b 进行铃木偶联，得到中间体 55a-55d，然后与 51 反应，得到硼酸盐中间体 56a-56d。56a-56d 与 42 进一步反应，得到化合物 21-24。
目标化合物 21-24 是根据方案 3 制备的。市售的 54a-54d 与 49b 进行铃木偶联，得到中间体 55a-55d，然后与 51 反应，得到硼酸盐中间体 56a-56d。56a-56d 与 42 进一步反应，得到化合物 21-24。
Scheme 3 方案 3
Scheme 3. Reagents and Conditions: (a) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl2·CH2Cl2, Dry 1,4-Dioxane, 100 °C, 3 h; and (c) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 7 h
方案 3.试剂和条件： (a) Cs 2 CO 3 Pd(dppf)Cl 2 -CH 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h; Pd(dppf)Cl 2 -CH 2 Cl 2 O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl { 2 -CH 2 Cl 2 干 1,4-二恶烷，100 °C，3 小时；以及 (c) Cs 2 CO 3 (c) Cs 2 CO 3 , Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 7 h.O, 100 °C, 7 h
方案 3.试剂和条件： (a) Cs 2 CO 3 Pd(dppf)Cl 2 -CH 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h; Pd(dppf)Cl 2 -CH 2 Cl 2 O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl { 2 -CH 2 Cl 2 干 1,4-二恶烷，100 °C，3 小时；以及 (c) Cs 2 CO 3 (c) Cs 2 CO 3 , Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 7 h.O, 100 °C, 7 h
Target compounds 25–30 were synthesized according to Scheme 4. Commercial 58a–58f were condensed with 57 to give intermediates 59a–59f and then reacted with 49b by Suzuki coupling to give intermediates 60a–60f, and 60a–60f were boronated in the presence of 51 to give boronate intermediates 61a–61f and then further reacted with 42 to give compounds 25–30.
目标化合物 25-30 是根据方案 4 合成的。商用 58a-58f 与 57 缩合得到中间体 59a-59f，然后与 49b 通过铃木偶联反应得到中间体 60a-60f，60a-60f 在 51 的存在下被硼化得到硼酸盐中间体 61a-61f，然后进一步与 42 反应得到化合物 25-30。
目标化合物 25-30 是根据方案 4 合成的。商用 58a-58f 与 57 缩合得到中间体 59a-59f，然后与 49b 通过铃木偶联反应得到中间体 60a-60f，60a-60f 在 51 的存在下被硼化得到硼酸盐中间体 61a-61f，然后进一步与 42 反应得到化合物 25-30。
Scheme 4 方案 4
Scheme 4. Reagents and Conditions: (a) Pyridine, CH2Cl2, r.t., 4 h; (b) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h; (c) AcOK, Pd(dppf)Cl2·CH2Cl2, Dry 1,4-Dioxane, 100 °C, 3 h; and (d) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 7 h
方案 4.试剂和条件： (a) 吡啶、CH 2 Cl 2 4 小时； (b) Cs 2 CO 3 (b) Cs 2 CO 3 , Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h; Pd(dppf)Cl 2 -CH 2 Cl 2 O, 100 °C, 3 h; (c) AcOK, Pd(dppf)Cl { 2 -CH 2 Cl 2 干 1,4-二恶烷，100 °C，3 小时；以及 (d) Cs 2 CO 3 (d) Cs 2 CO 3 ， Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 7 h.O, 100 °C, 7 h
方案 4.试剂和条件： (a) 吡啶、CH 2 Cl 2 4 小时； (b) Cs 2 CO 3 (b) Cs 2 CO 3 , Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 3 h; Pd(dppf)Cl 2 -CH 2 Cl 2 O, 100 °C, 3 h; (c) AcOK, Pd(dppf)Cl { 2 -CH 2 Cl 2 干 1,4-二恶烷，100 °C，3 小时；以及 (d) Cs 2 CO 3 (d) Cs 2 CO 3 ， Pd(dppf)Cl 2 -CH 2 Cl 2 1,4-Dioxane, H 2 O, 100 °C, 7 h.O, 100 °C, 7 h
Herein, a novel class of BET selective inhibitors were developed by combining iterative structure–relationship studies and crystal structure-guided drug design. Starting with the discovery of the hit 1 (BRD4 (1) Ki = 4.3 μM), derived from an in-house chemical library, a lead compound 9 (BRD4 (1) Ki = 1.1 μM) with an imidazole-pyridine core was obtained by the positioning analogue scanning method. Subsequently, the structure was expanded to the WPF shelf, leading to the identification of compound 16 (BRD4 (1) Ki = 0.14 μM) with significantly improved potency for BRD4 BDs. The SAR of the phenyl ring substituents was subsequently investigated, resulting in compounds 21 (BRD4 (1) Ki = 0.031 μM) with further enhanced activity. The co-crystal structure of 21-BRD4 shows that the imidazole-pyridine core is anchored at the Kac binding site and forms hydrogen bonding interactions with Asn and Tyr, and the trimethylpyrazole group is adjacent to the WPF shelf and forms a hydrophobic interaction. However, the structure still lacks interaction with the ZA channel. Then, the structure was extended into the ZA channel, and the activity was further enhanced, resulting in the identification of DDO-8926 (BRD4 (1) Ki = 0.015 μM). DDO-8926 exhibited excellent selectivity for BET proteins. DDO-8926 had almost no inhibitory effect on hERG ion channels (IC50 > 30 μM) and CYP450 enzymes (IC50 > 10 μM). In vivo, DDO-8926 at 30 mg/kg relieved nociceptive hypersensitivity after SNI to an extent comparable to gabapentin at 100 mg/kg. Further studies revealed that DDO-8926 inhibited the SNI-induced microglial and neuronal activation and decreased the expression of pro-inflammatory cytokine in the dorsal horn of the spinal cord. Also, it reduced neuronal excitability by inhibiting the generation and transfer of membrane potential. Overall, we have identified a potent and selective BET inhibitor DDO-8926, which has the potential to be used as a NP therapeutic agent for further research.
在此，我们结合迭代结构关系研究和晶体结构指导药物设计，开发了一类新型 BET 选择性抑制剂。从发现来自内部化学文库的热门化合物 1（BRD4 (1) K = 4.3 μM）开始，通过定位模拟扫描法获得了以咪唑吡啶为核心的先导化合物 9（BRD4 (1) K = 1.1 μM）。随后，将该结构扩展到 WPF 架，从而鉴定出化合物 16（BRD4 (1) K = 0.14 μM），其对 BRD4 BD 的效力显著提高。随后研究了苯环取代基的 SAR，结果发现化合物 21（BRD4 (1) K = 0.031 μM）的活性进一步增强。21-BRD4 的共晶体结构显示，咪唑吡啶核心锚定在 Kac 结合位点，与 Asn 和 Tyr 形成氢键相互作用，三甲基吡唑基团与 WPF 架相邻，形成疏水相互作用。然而，该结构仍然缺乏与ZA通道的相互作用。随后，将该结构扩展到ZA通道，活性进一步增强，最终确定了 DDO-8926（BRD4 (1) K = 0.015 μM）。DDO-8926 对 BET 蛋白具有极佳的选择性。DDO-8926 对 hERG 离子通道（IC 50 > 30 μM）和 CYP450 酶（IC 50 > 10 μM）几乎没有抑制作用。在体内，30 毫克/千克的 DDO-8926 可缓解 SNI 后的痛觉过敏反应，缓解程度与 100 毫克/千克的加巴喷丁相当。进一步研究发现，DDO-8926 可抑制 SNI 诱导的小胶质细胞和神经元活化，减少脊髓背角促炎细胞因子的表达。此外，它还通过抑制膜电位的产生和传递来降低神经元的兴奋性。总之，我们发现了一种强效且具有选择性的 BET 抑制剂 DDO-8926，它有望作为一种 NP 治疗剂用于进一步研究。
在此，我们结合迭代结构关系研究和晶体结构指导药物设计，开发了一类新型 BET 选择性抑制剂。从发现来自内部化学文库的热门化合物 1（BRD4 (1) K = 4.3 μM）开始，通过定位模拟扫描法获得了以咪唑吡啶为核心的先导化合物 9（BRD4 (1) K = 1.1 μM）。随后，将该结构扩展到 WPF 架，从而鉴定出化合物 16（BRD4 (1) K = 0.14 μM），其对 BRD4 BD 的效力显著提高。随后研究了苯环取代基的 SAR，结果发现化合物 21（BRD4 (1) K = 0.031 μM）的活性进一步增强。21-BRD4 的共晶体结构显示，咪唑吡啶核心锚定在 Kac 结合位点，与 Asn 和 Tyr 形成氢键相互作用，三甲基吡唑基团与 WPF 架相邻，形成疏水相互作用。然而，该结构仍然缺乏与ZA通道的相互作用。随后，将该结构扩展到ZA通道，活性进一步增强，最终确定了 DDO-8926（BRD4 (1) K = 0.015 μM）。DDO-8926 对 BET 蛋白具有极佳的选择性。DDO-8926 对 hERG 离子通道（IC 50 > 30 μM）和 CYP450 酶（IC 50 > 10 μM）几乎没有抑制作用。在体内，30 毫克/千克的 DDO-8926 可缓解 SNI 后的痛觉过敏反应，缓解程度与 100 毫克/千克的加巴喷丁相当。进一步研究发现，DDO-8926 可抑制 SNI 诱导的小胶质细胞和神经元活化，减少脊髓背角促炎细胞因子的表达。此外，它还通过抑制膜电位的产生和传递来降低神经元的兴奋性。总之，我们发现了一种强效且具有选择性的 BET 抑制剂 DDO-8926，它有望作为一种 NP 治疗剂用于进一步研究。
Experimental Section 实验部分
General Procedures 一般程序
The synthesis of target compounds is shown in Schemes 1–4. Reactions were monitored by thin-layer chromatography on 0.25 mm silica gel plates (GF254) and visualized under UV light. The 1H NMR and 13C NMR spectra were recorded on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane as the internal standard. High-resolution mass spectra (HRMS) were recorded on an Aglient 6545 Q-TOF. The purity (≥95%) of the compounds was verified by HPLC (Shimadzu LC-20AT) performed on a Shimadzu C18 (4.6 mm × 150 mm, 3.5 μm) column using a mixture of solvent methanol/water 65:35 or 80:20 at a flow rate of 1 mL/min and peak detection at 254 nm.
目标化合物的合成过程如图 1-4 所示。反应在 0.25 毫米硅胶板（GF254）上进行薄层色谱监测，并在紫外光下观察。 1 H核磁共振和{{1H NMR 和 13 C NMR 光谱。C NMR 光谱由布鲁克 AV-300 仪器记录，使用氚代溶剂，以四甲基硅烷为内标。高分辨质谱（HRMS）由 Aglient 6545 Q-TOF 仪器记录。化合物的纯度（≥95%）由 HPLC（Shimadzu LC-20AT）验证，HPLC 采用 Shimadzu C18（4.6 mm × 150 mm，3.5 μm）色谱柱，使用甲醇/水 65:35 或 80:20 混合溶剂，流速为 1 mL/min，色谱峰检测波长为 254 nm。
目标化合物的合成过程如图 1-4 所示。反应在 0.25 毫米硅胶板（GF254）上进行薄层色谱监测，并在紫外光下观察。 1 H核磁共振和{{1H NMR 和 13 C NMR 光谱。C NMR 光谱由布鲁克 AV-300 仪器记录，使用氚代溶剂，以四甲基硅烷为内标。高分辨质谱（HRMS）由 Aglient 6545 Q-TOF 仪器记录。化合物的纯度（≥95%）由 HPLC（Shimadzu LC-20AT）验证，HPLC 采用 Shimadzu C18（4.6 mm × 150 mm，3.5 μm）色谱柱，使用甲醇/水 65:35 或 80:20 混合溶剂，流速为 1 mL/min，色谱峰检测波长为 254 nm。
General Procedure A: Suzuki Coupling Reactions for the Synthesis of 1–30, 50a–50f, 55a–55d, and 60a–60f
一般程序 A：合成 1-30、50a-50f、55a-55d 和 60a-60f 的铃木偶联反应
Aryl halides (1.0 equiv) and arylboronic acid or arylboronic acid esters (1.3 equiv) were dissolved in dioxane and water (3:1 v/v), and then cesium carbonate (3.0 equiv) and [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride dichloromethane complex (0.15 equiv) were added. After reaction at 100 °C for 3 h under an argon atmosphere, the reaction was cooled to room temperature, equal volumes of ethyl acetate and water were added and extracted three times, and the organic layer was combined and washed with brine. The organic layer was dried over anhydrous Na2SO4 and purified by column chromatography to obtain 1–30, 50a–50f, 55a–55d, and 60a–60f.
将芳基卤化物（1.0 等量）和芳基硼酸或芳基硼酸酯（1.3 等量）溶于二氧六环和水（3:1 v/v）中，然后加入碳酸铯（3.0 等量）和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物（0.15 等量）。在 100 °C 的氩气环境下反应 3 小时后，将反应物冷却至室温，加入等体积的乙酸乙酯和水并萃取三次，合并有机层并用盐水洗涤。有机层在无水 Na 2 SO {{1} 上干燥。SO 4 干燥，用柱层析法纯化，得到 1-30、50a-50f、55a-55d 和 60a-60f。
将芳基卤化物（1.0 等量）和芳基硼酸或芳基硼酸酯（1.3 等量）溶于二氧六环和水（3:1 v/v）中，然后加入碳酸铯（3.0 等量）和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物（0.15 等量）。在 100 °C 的氩气环境下反应 3 小时后，将反应物冷却至室温，加入等体积的乙酸乙酯和水并萃取三次，合并有机层并用盐水洗涤。有机层在无水 Na 2 SO {{1} 上干燥。SO 4 干燥，用柱层析法纯化，得到 1-30、50a-50f、55a-55d 和 60a-60f。
5-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (1)
5-(3,4,5-三甲氧基苯基)-1H-吡咯并[2,3-b]吡啶 (1)
1 was prepared from 5-bromo-1H-pyrrolo[2,3-b]pyridine 31 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. Yellow solid (317 mg, yield: 73.2%). 1H NMR (300 MHz, DMSO-d6): δ 11.74 (s, 1H), 8.58 (d, J = 2.2 Hz, 1H), 8.26 (dd, J = 2.2, 0.7 Hz, 1H), 7.54 (dd, J = 3.5, 2.5 Hz, 1H), 6.99 (s, 2H), 6.53 (dd, J = 3.4, 1.9 Hz, 1H), 3.91 (s, 6H), 3.73 (s, 3H). HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1238. Purity: 99.36% by HPLC (MeOH/H2O = 65:35, tR = 9.588 min).
根据一般程序 A，由 5-溴-1H-吡咯并[2,3-b]吡啶 31（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 1。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.74（s，1H），8.58（d，J = 2.2 Hz，1H），8.26（dd，J = 2.2，0.7 Hz，1H），7.54（dd，J = 3.5，2.5 Hz，1H），6.99（s，2H），6.53（dd，J = 3.4，1.9 Hz，1H），3.91（s，6H），3.73（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1238。HPLC 测定纯度：99.36%（MeOH/H 2 O = 65:35，t R = 9.588 分钟）。
根据一般程序 A，由 5-溴-1H-吡咯并[2,3-b]吡啶 31（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 1。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.74（s，1H），8.58（d，J = 2.2 Hz，1H），8.26（dd，J = 2.2，0.7 Hz，1H），7.54（dd，J = 3.5，2.5 Hz，1H），6.99（s，2H），6.53（dd，J = 3.4，1.9 Hz，1H），3.91（s，6H），3.73（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1238。HPLC 测定纯度：99.36%（MeOH/H 2 O = 65:35，t R = 9.588 分钟）。
6-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (2)
6-(3,4,5-三甲氧基苯基)-1H-吡咯并[2,3-b]吡啶 (2)
2 was prepared from 6-bromo-1H-pyrrolo[2,3-b]pyridine 32 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. Yellow solid (297 mg, yield: 68.8%). 1H NMR (300 MHz, DMSO-d6): δ 11.78 (s, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.50 (dd, J = 3.5, 2.5 Hz, 1H), 7.42 (s, 2H), 6.49 (dd, J = 3.4, 1.8 Hz, 1H), 3.92 (s, 6H), 3.75 (s, 3H). HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1234. Purity: 99.79% by HPLC (MeOH/H2O = 65:35, tR = 6.204 min).
根据一般程序 A，由 6-溴-1H-吡咯并[2,3-b]吡啶 32（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 2。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.78（s，1H），8.05（d，J = 8.3 Hz，1H），7.73（d，J = 8.3 Hz，1H），7.50（dd，J = 3.5，2.5 Hz，1H），7.42（s，2H），6.49（dd，J = 3.4，1.8 Hz，1H），3.92（s，6H），3.75（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1234。HPLC 测定纯度：99.79%（MeOH/H 2 O = 65:35，t R = 6.204 分钟）。
根据一般程序 A，由 6-溴-1H-吡咯并[2,3-b]吡啶 32（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 2。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.78（s，1H），8.05（d，J = 8.3 Hz，1H），7.73（d，J = 8.3 Hz，1H），7.50（dd，J = 3.5，2.5 Hz，1H），7.42（s，2H），6.49（dd，J = 3.4，1.8 Hz，1H），3.92（s，6H），3.75（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1234。HPLC 测定纯度：99.79%（MeOH/H 2 O = 65:35，t R = 6.204 分钟）。
5-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (3)
5-(3,4,5-三甲氧基苯基)-1H-吡咯并[2,3-b]吡啶 (3)
3 was prepared from 4-bromo-1H-pyrrolo[2,3-b]pyridine 33 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. Yellow solid (327 mg, yield: 75.7%). 1H NMR (300 MHz, DMSO-d6): δ 11.82 (s, 1H), 8.30 (d, J = 5.0 Hz, 1H), 7.57 (t, J = 3.0 Hz, 1H), 7.26 (d, J = 5.0 Hz, 1H), 7.05 (s, 2H), 6.72 (dd, J = 3.5, 1.8 Hz, 1H), 3.91 (s, 6H), 3.77 (s, 3H). HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1232. Purity: 93.09% by HPLC (MeOH/H2O = 65:35, tR = 5.951 min).
根据一般程序 A，由 4-溴-1H-吡咯并[2,3-b]吡啶 33（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 3。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.82（s，1H），8.30（d，J = 5.0 Hz，1H），7.57（t，J = 3.0 Hz，1H），7.26（d，J = 5.0 Hz，1H），7.05（s，2H），6.72（dd，J = 3.5，1.8 Hz，1H），3.91（s，6H），3.77（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1232。通过 HPLC（MeOH/H 2 O = 65:35，t R = 5.951 分钟）检测，纯度为 93.09%。
根据一般程序 A，由 4-溴-1H-吡咯并[2,3-b]吡啶 33（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 3。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.82（s，1H），8.30（d，J = 5.0 Hz，1H），7.57（t，J = 3.0 Hz，1H），7.26（d，J = 5.0 Hz，1H），7.05（s，2H），6.72（dd，J = 3.5，1.8 Hz，1H），3.91（s，6H），3.77（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1232。通过 HPLC（MeOH/H 2 O = 65:35，t R = 5.951 分钟）检测，纯度为 93.09%。
3-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (4)
3-(3,4,5-三甲氧基苯基)-1H-吡咯并[2,3-b]吡啶 (4)
4 was prepared from 4-bromo-1H-pyrrolo[2,3-b]pyridine 34 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. White solid (288 mg, yield: 66.7%). 1H NMR (300 MHz, DMSO-d6): δ 11.92 (s, 1H), 8.42–8.24 (m, 2H), 7.90 (d, J = 2.7 Hz, 1H), 7.19 (dd, J = 8.0, 4.7 Hz, 1H), 6.97 (s, 2H), 3.91 (s, 6H), 3.72 (s, 3H). HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1238. Purity: 95.91% by HPLC (MeOH/H2O = 80:20, tR = 4.630 min).
根据一般程序 A，由 4-溴-1H-吡咯并[2,3-b]吡啶 34（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 4。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.92（s，1H），8.42-8.24（m，2H），7.90（d，J = 2.7 Hz，1H），7.19（dd，J = 8.0，4.7 Hz，1H），6.97（s，2H），3.91（s，6H），3.72（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1238。纯度：95.91%（HPLC，MeOH/H 2 O = 80:20，t R = 4.630 分钟）。
根据一般程序 A，由 4-溴-1H-吡咯并[2,3-b]吡啶 34（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 4。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.92（s，1H），8.42-8.24（m，2H），7.90（d，J = 2.7 Hz，1H），7.19（dd，J = 8.0，4.7 Hz，1H），6.97（s，2H），3.91（s，6H），3.72（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1238。纯度：95.91%（HPLC，MeOH/H 2 O = 80:20，t R = 4.630 分钟）。
4-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-c]pyridine (5)
4-(3,4,5-三甲氧基苯基)-1H-吡咯并[2,3-c]吡啶 (5)
5 was prepared from 4-bromo-1H-pyrrolo[2,3-c]pyridine 35 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. Yellow solid (293 mg, yield: 67.8%). 1H NMR (300 MHz, DMSO-d6): δ 11.80 (s, 1H), 8.76 (s, 1H), 8.28 (s, 1H), 7.70 (t, J = 2.8 Hz, 1H), 6.99 (s, 2H), 6.83–6.65 (m, 1H), 3.89 (s, 6H), 3.75 (s, 3H). HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1233. Purity: 92.46% by HPLC (MeOH/H2O = 80:20, tR = 4.274 min).
根据一般程序 A，由 4-溴-1H-吡咯并[2,3-c]吡啶 35（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 5。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.80（s，1H），8.76（s，1H），8.28（s，1H），7.70（t，J = 2.8 Hz，1H），6.99（s，2H），6.83-6.65（m，1H），3.89（s，6H），3.75（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 sH 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1233。通过 HPLC（MeOH/H2O = 80:20，t R = 4.274 分钟）测定，纯度为 92.46%。
根据一般程序 A，由 4-溴-1H-吡咯并[2,3-c]吡啶 35（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 5。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.80（s，1H），8.76（s，1H），8.28（s，1H），7.70（t，J = 2.8 Hz，1H），6.99（s，2H），6.83-6.65（m，1H），3.89（s，6H），3.75（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 sH 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1233。通过 HPLC（MeOH/H2O = 80:20，t R = 4.274 分钟）测定，纯度为 92.46%。
4-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazole (6)
4-(3,4,5-三甲氧基苯基)-1H-苯并[d]咪唑 (6)
6 was prepared from 4-bromo-1H-benzo[d]imidazole 36 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. Yellow solid (339 mg, yield: 78.4%). 1H NMR (300 MHz, DMSO-d6): δ 12.63 (s, 1H), 8.29 (d, J = 23.4 Hz, 1H), 7.72–7.26 (m, 5H), 6.92 (s, 1H), 3.76 (s, 3H). HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1238. Purity: 98.66% by HPLC (MeOH/H2O = 65:35, tR = 4.214 min).
根据一般程序 A，由 4-溴-1H-苯并[d]咪唑 36（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 6。 1 H NMR（300 MHz，DMSO-d 6 ）：δ12.63（s，1H），8.29（d，J = 23.4 Hz，1H），7.72-7.26（m，5H），6.92（s，1H），3.76（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1238。通过 HPLC（MeOH/H 2 O = 65:35，t R = 4.214 分钟）检测，纯度为 98.66%。
根据一般程序 A，由 4-溴-1H-苯并[d]咪唑 36（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 6。 1 H NMR（300 MHz，DMSO-d 6 ）：δ12.63（s，1H），8.29（d，J = 23.4 Hz，1H），7.72-7.26（m，5H），6.92（s，1H），3.76（s，3H）。HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1238。通过 HPLC（MeOH/H 2 O = 65:35，t R = 4.214 分钟）检测，纯度为 98.66%。
4-(3,4,5-Trimethoxyphenyl)-1H-indazole (7)
4-(3,4,5-三甲氧基苯基)-1H-吲唑 (7)
7 was prepared from 4-bromo-1H-indazole 37 (300 mg, 1.52 mmol) and 45 (419.64 mg, 1.98 mmol) according to general procedure A. Yellow solid (323 mg, yield: 74.7%). 1H NMR (300 MHz, DMSO-d6): δ 12.63 (s, 1H), 8.29 (d, J = 23.4 Hz, 1H), 7.72–7.26 (m, 5H), 6.92 (s, 1H), 3.76 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 153.70, 141.04, 137.65, 135.60, 134.61, 133.35, 126.71, 121.49, 119.86, 109.73, 105.86, 60.57, 56.38. HRMS (ESI): calcd for C16H16N2O3 [M + H]+, 285.1161; found, 285.1237. Purity: 99.90% by HPLC (MeOH/H2O = 65:35, tR = 4.249 min).
根据一般程序 A，由 4-溴-1H-吲唑 37（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 7。 1 H NMR（300 MHz，DMSO-d 6 ）：δ12.63（s，1H），8.29（d，J = 23.4 Hz，1H），7.72-7.26（m，5H），6.92（s，1H），3.76（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.70, 141.04, 137.65, 135.60, 134.61, 133.35, 126.71, 121.49, 119.86, 109.73, 105.86, 60.57, 56.38.HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1237。纯度：99.90%（HPLC，MeOH/H 2 O = 65:35，t R = 4.249 分钟）。
根据一般程序 A，由 4-溴-1H-吲唑 37（300 毫克，1.52 毫摩尔）和 45（419.64 毫克，1.98 毫摩尔）制备 7。 1 H NMR（300 MHz，DMSO-d 6 ）：δ12.63（s，1H），8.29（d，J = 23.4 Hz，1H），7.72-7.26（m，5H），6.92（s，1H），3.76（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.70, 141.04, 137.65, 135.60, 134.61, 133.35, 126.71, 121.49, 119.86, 109.73, 105.86, 60.57, 56.38.HRMS（ESI）：煅烧为 C 16 H { 16 H 16 N 2 O 3 [M + H] + ，285.1161；发现值，285.1237。纯度：99.90%（HPLC，MeOH/H 2 O = 65:35，t R = 4.249 分钟）。
4-(3,4,5-Trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (8)
4-(3,4,5-三甲氧基苯基)-1H-吡唑并[3,4-b]吡啶 (8)
8 was prepared from 4-bromo-1H-pyrazolo[3,4-b]pyridine 38 (200 mg, 1.01 mmol) and 45 (278.37 mg, 1.31 mmol) according to general procedure A. Yellow solid (152 mg, yield: 53.1%). 1H NMR (300 MHz, DMSO-d6): δ 13.81 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 8.45 (s, 1H), 7.44 (d, J = 4.8 Hz, 1H), 7.14 (s, 2H), 3.79 (s, 3H). HRMS (ESI): calcd for C15H15N3O3 [M + H]+, 286.1147; found, 286.1184. Purity: 99.77% by HPLC (MeOH/H2O = 65:35, tR = 9.147 min).
根据一般程序 A，由 4-溴-1H-吡唑并[3,4-b]吡啶 38（200 毫克，1.01 毫摩尔）和 45（278.37 毫克，1.31 毫摩尔）制备 8。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.81（s，1H），8.60（d，J = 4.8 Hz，1H），8.45（s，1H），7.44（d，J = 4.8 Hz，1H），7.14（s，2H），3.79（s，3H）。HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值为 286.1184。HPLC 测定纯度：99.77%（MeOH/H 2 O = 65:35，t R = 9.147 分钟）。
根据一般程序 A，由 4-溴-1H-吡唑并[3,4-b]吡啶 38（200 毫克，1.01 毫摩尔）和 45（278.37 毫克，1.31 毫摩尔）制备 8。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.81（s，1H），8.60（d，J = 4.8 Hz，1H），8.45（s，1H），7.44（d，J = 4.8 Hz，1H），7.14（s，2H），3.79（s，3H）。HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值为 286.1184。HPLC 测定纯度：99.77%（MeOH/H 2 O = 65:35，t R = 9.147 分钟）。
7-(3,4,5-Trimethoxyphenyl)-3H-imidazo[4,5-b]pyridine (9)
7-(3,4,5-三甲氧基苯基)-3H-咪唑并[4,5-b]吡啶 (9)
9 was prepared from 7-bromo-3H-imidazo[4,5-b]pyridine 39 (200 mg, 1.01 mmol) and 45 (278.37 mg, 1.31 mmol) according to general procedure A. Brown solid (121 mg, yield: 42.4%). 1H NMR (300 MHz, DMSO-d6): δ 13.25 (s, 1H), 8.53 (s, 1H), 8.40 (d, J = 5.1 Hz, 1H), 7.75 (s, 2H), 7.65 (d, J = 5.3 Hz, 1H), 3.92 (d, J = 3.3 Hz, 6H), 3.77 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 153.93, 153.02, 149.46, 143.15, 138.91, 133.23, 132.91, 115.35, 112.84, 106.06, 60.60, 56.49. HRMS (ESI): calcd for C15H15N3O3 [M + H]+, 286.1147; found, 286.1187. Purity: 99.09% by HPLC (MeOH/H2O = 65:35, tR = 3.982 min).
根据一般程序 A，由 7-溴-3H-咪唑并[4,5-b]吡啶 39（200 毫克，1.01 毫摩尔）和 45（278.37 毫克，1.31 毫摩尔）制备得到 9。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.25（s，1H），8.53（s，1H），8.40（d，J = 5.1 Hz，1H），7.75（s，2H），7.65（d，J = 5.3 Hz，1H），3.92（d，J = 3.3 Hz，6H），3.77（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.93, 153.02, 149.46, 143.15, 138.91, 133.23, 132.91, 115.35, 112.84, 106.06, 60.60, 56.49.HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值，286.1187。通过 HPLC 测定，纯度为 99.09%（MeOH/H 2 O = 65:35，t R = 3.982 分钟）。
根据一般程序 A，由 7-溴-3H-咪唑并[4,5-b]吡啶 39（200 毫克，1.01 毫摩尔）和 45（278.37 毫克，1.31 毫摩尔）制备得到 9。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.25（s，1H），8.53（s，1H），8.40（d，J = 5.1 Hz，1H），7.75（s，2H），7.65（d，J = 5.3 Hz，1H），3.92（d，J = 3.3 Hz，6H），3.77（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.93, 153.02, 149.46, 143.15, 138.91, 133.23, 132.91, 115.35, 112.84, 106.06, 60.60, 56.49.HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值，286.1187。通过 HPLC 测定，纯度为 99.09%（MeOH/H 2 O = 65:35，t R = 3.982 分钟）。
4-(3,4,5-Trimethoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (10)
4-(3,4,5-三甲氧基苯基)-7H-吡咯并[2,3-d]嘧啶 (10)
10 was prepared from 4-bromo-7H-pyrrolo[2,3-d]pyrimidine 40 (200 mg, 1.52 mmol) and 45 (278.37 mg, 1.31 mmol) according to general procedure A. Yellow solid (169 mg, yield: 59.3%). 1H NMR (300 MHz, DMSO-d6): δ 12.28 (s, 1H), 8.85 (s, 1H), 7.68 (dd, J = 3.6, 2.3 Hz, 1H), 7.46 (s, 2H), 6.96 (dd, J = 3.7, 1.7 Hz, 1H), 3.94 (s, 6H), 3.79 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 155.80, 153.58, 153.12, 151.29, 139.59, 133.91, 128.12, 114.76, 106.22, 100.63, 60.63, 56.41. HRMS (ESI): calcd for C15H15N3O3 [M + H]+, 286.1147; found, 286.1146. Purity: 99.36% by HPLC (MeOH/H2O = 65:35, tR = 9.588 min).
根据一般程序 A，由 4-溴-7H-吡咯并[2,3-d]嘧啶 40（200 毫克，1.52 毫摩尔）和 45（278.37 毫克，1.31 毫摩尔）制备 10。 1 H NMR（300 MHz，DMSO-d 6 ）：δ12.28（s，1H），8.85（s，1H），7.68（dd，J = 3.6，2.3 Hz，1H），7.46（s，2H），6.96（dd，J = 3.7，1.7 Hz，1H），3.94（s，6H），3.79（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 155.80, 153.58, 153.12, 151.29, 139.59, 133.91, 128.12, 114.76, 106.22, 100.63, 60.63, 56.41.HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值，286.1146。纯度：99.36%（HPLC，MeOH/H 2 O = 65:35，t R = 9.588 分钟）。
根据一般程序 A，由 4-溴-7H-吡咯并[2,3-d]嘧啶 40（200 毫克，1.52 毫摩尔）和 45（278.37 毫克，1.31 毫摩尔）制备 10。 1 H NMR（300 MHz，DMSO-d 6 ）：δ12.28（s，1H），8.85（s，1H），7.68（dd，J = 3.6，2.3 Hz，1H），7.46（s，2H），6.96（dd，J = 3.7，1.7 Hz，1H），3.94（s，6H），3.79（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 155.80, 153.58, 153.12, 151.29, 139.59, 133.91, 128.12, 114.76, 106.22, 100.63, 60.63, 56.41.HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值，286.1146。纯度：99.36%（HPLC，MeOH/H 2 O = 65:35，t R = 9.588 分钟）。
8-(3,4,5-Trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (11)
11 was prepared from 8-bromo-[1,2,4]triazolo[4,3-a]pyridine 41 (250 mg, 1.26 mmol) and 45 (347.58 mg, 1.64 mmol) according to general procedure A. White solid (178 mg, yield: 62.4%). 1H NMR (300 MHz, DMSO-d6): δ 9.40 (d, J = 3.5 Hz, 1H), 8.70–8.52 (m, 1H), 7.77 (dd, J = 7.2, 3.6 Hz, 1H), 7.63 (d, J = 3.7 Hz, 2H), 7.19–7.03 (m, 1H), 3.91 (d, J = 3.5 Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 153.26, 138.61, 137.62, 130.60, 127.24, 125.60, 124.47, 114.40, 106.58, 60.62, 56.53. HRMS (ESI): calcd for C15H15N3O3 [M + H]+, 286.1147; found, 286.1186. Purity: 98.10% by HPLC (MeOH/H2O = 80:20, tR = 3.880 min).
根据一般程序 A，由 8-溴-[1,2,4]三唑并[4,3-a]吡啶 41（250 毫克，1.26 毫摩尔）和 45（347.58 毫克，1.64 毫摩尔）制备 11。 1 H NMR（300 MHz，DMSO-d 6 ）：δ9.40（d，J = 3.5 Hz，1H），8.70-8.52（m，1H），7.77（dd，J = 7.2，3.6 Hz，1H），7.63（d，J = 3.7 Hz，2H），7.19-7.03（m，1H），3.91（d，J = 3.5 Hz，6H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.26, 138.61, 137.62, 130.60, 127.24, 125.60, 124.47, 114.40, 106.58, 60.62, 56.53.HRMS（ESI）：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值为 286.1186。纯度：98.10%（HPLC，MeOH/H 2 O = 80:20，t R = 3.880 分钟）。
根据一般程序 A，由 8-溴-[1,2,4]三唑并[4,3-a]吡啶 41（250 毫克，1.26 毫摩尔）和 45（347.58 毫克，1.64 毫摩尔）制备 11。 1 H NMR（300 MHz，DMSO-d 6 ）：δ9.40（d，J = 3.5 Hz，1H），8.70-8.52（m，1H），7.77（dd，J = 7.2，3.6 Hz，1H），7.63（d，J = 3.7 Hz，2H），7.19-7.03（m，1H），3.91（d，J = 3.5 Hz，6H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.26, 138.61, 137.62, 130.60, 127.24, 125.60, 124.47, 114.40, 106.58, 60.62, 56.53.HRMS（ESI）：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值为 286.1186。纯度：98.10%（HPLC，MeOH/H 2 O = 80:20，t R = 3.880 分钟）。
5-(3,4,5-Trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (12)
12 was prepared from 5-bromo-[1,2,4]triazolo[4,3-a]pyridine 42 (250 mg, 1.26 mmol) and 45 (347.58 mg, 1.64 mmol) according to general procedure A. Brown solid (165 mg, yield: 57.8%). 1H NMR (300 MHz, DMSO-d6): δ 9.39 (s, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.49 (dd, J = 9.2, 6.8 Hz, 1H), 7.11 (s, 2H), 7.09–7.05 (m, 1H), 3.90 (s, 6H), 3.79 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 153.85, 149.79, 139.22, 137.20, 135.61, 128.68, 128.44, 114.43, 114.06, 106.26, 60.59, 56.59. HRMS (ESI): calcd for C15H15N3O3 [M + H]+, 286.1147; found, 286.1188. Purity: 98.94% by HPLC (MeOH/H2O = 65:35, tR = 9.958 min).
12 由 5-溴-[1,2,4]三唑并[4,3-a]吡啶 42（250 毫克，1.26 毫摩尔）和 45（347.58 毫克，1.64 毫摩尔）按一般程序 A 制备而成。 1 H NMR（300 MHz，DMSO-d 6 ）：δ9.39（s，1H），7.82（d，J = 9.2 Hz，1H），7.49（dd，J = 9.2，6.8 Hz，1H），7.11（s，2H），7.09-7.05（m，1H），3.90（s，6H），3.79（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.85, 149.79, 139.22, 137.20, 135.61, 128.68, 128.44, 114.43, 114.06, 106.26, 60.59, 56.59.HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值，286.1188。HPLC 测定纯度：98.94%（MeOH/H 2 O = 65:35，t R = 9.958 分钟）。
12 由 5-溴-[1,2,4]三唑并[4,3-a]吡啶 42（250 毫克，1.26 毫摩尔）和 45（347.58 毫克，1.64 毫摩尔）按一般程序 A 制备而成。 1 H NMR（300 MHz，DMSO-d 6 ）：δ9.39（s，1H），7.82（d，J = 9.2 Hz，1H），7.49（dd，J = 9.2，6.8 Hz，1H），7.11（s，2H），7.09-7.05（m，1H），3.90（s，6H），3.79（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 153.85, 149.79, 139.22, 137.20, 135.61, 128.68, 128.44, 114.43, 114.06, 106.26, 60.59, 56.59.HRMS (ESI)：煅烧为 C 15 H { 15 H 15 N 3 O 3 [M + H] + ，286.1147；发现值，286.1188。HPLC 测定纯度：98.94%（MeOH/H 2 O = 65:35，t R = 9.958 分钟）。
4-(3,4,5-Trimethoxyphenyl)benzo[d]isoxazole (13)
4-(3,4,5-三甲氧基苯基)苯并[d]异恶唑 (13)
13 was prepared from 4-bromobenzo[d]isoxazole 43 (250 mg, 1.26 mmol) and 45 (347.58 mg, 1.64 mmol) according to general procedure A. Brown solid (216 mg, yield: 76.1%). 1H NMR (300 MHz, DMSO-d6): δ 11.21 (d, J = 4.5 Hz, 1H), 7.57 (td, J = 7.9, 2.7 Hz, 1H), 7.14–6.82 (m, 4H), 3.88 (s, 6H), 3.81–3.74 (m, 3H). 13C NMR (75 MHz, DMSO-d6): δ 161.70, 153.21, 146.31, 138.17, 134.51, 134.03, 120.71, 115.10, 106.68, 98.65, 60.57, 56.46. HRMS (ESI): calcd for C16H15NO4 [M + Na]+, 309.0901; found, 309.0929. Purity: 97.92% by HPLC (MeOH/H2O = 65:35, tR = 5.070 min).
13 由 4-溴苯并[d]异恶唑 43（250 毫克，1.26 毫摩尔）和 45（347.58 毫克，1.64 毫摩尔）按一般程序 A 制备而成。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.21（d，J = 4.5 Hz，1H），7.57（td，J = 7.9，2.7 Hz，1H），7.14-6.82（m，4H），3.88（s，6H），3.81-3.74（m，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 161.70, 153.21, 146.31, 138.17, 134.51, 134.03, 120.71, 115.10, 106.68, 98.65, 60.57, 56.46.HRMS（ESI）：煅烧为 C 16 H { 15 H 15 NO 4 [M + Na] + ，309.0901；发现值为 309.0929。HPLC 测定纯度：97.92%（MeOH/H 2 O = 65:35，t R = 5.070 分钟）。
13 由 4-溴苯并[d]异恶唑 43（250 毫克，1.26 毫摩尔）和 45（347.58 毫克，1.64 毫摩尔）按一般程序 A 制备而成。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.21（d，J = 4.5 Hz，1H），7.57（td，J = 7.9，2.7 Hz，1H），7.14-6.82（m，4H），3.88（s，6H），3.81-3.74（m，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 161.70, 153.21, 146.31, 138.17, 134.51, 134.03, 120.71, 115.10, 106.68, 98.65, 60.57, 56.46.HRMS（ESI）：煅烧为 C 16 H { 15 H 15 NO 4 [M + Na] + ，309.0901；发现值为 309.0929。HPLC 测定纯度：97.92%（MeOH/H 2 O = 65:35，t R = 5.070 分钟）。
7-Bromo-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (44)
4-Bromopyridine-2,3-diamine 46 (300 mg, 1.6 mmol) and di(1H-imidazole-1-yl)methanone 47 (517.4 mg, 3.19 mmol) were dissolved by tetrahydrofuran (10 mL) and heated at 80 °C for 4 h. Tetrahydrofuran was removed by concentration under reduced pressure, and 10 mL of dichloromethane was added to dissolve the residue, a large amount of red powder was precipitated, and 44 (283 mg, yield: 82.9%) was obtained after filtration. Without further purification, it was directly fed to the next step.
将 4-溴吡啶-2,3-二胺 46（300 毫克，1.6 毫摩尔）和二(1H-咪唑-1-基)甲酮 47（517.4 毫克，3.19 毫摩尔）溶于四氢呋喃（10 毫升），在 80 °C 下加热 4 小时。减压浓缩除去四氢呋喃，加入 10 mL 二氯甲烷溶解残留物，析出大量红色粉末，过滤后得到 44（283 mg，产率：82.9%）。无需进一步纯化，直接进入下一步。
将 4-溴吡啶-2,3-二胺 46（300 毫克，1.6 毫摩尔）和二(1H-咪唑-1-基)甲酮 47（517.4 毫克，3.19 毫摩尔）溶于四氢呋喃（10 毫升），在 80 °C 下加热 4 小时。减压浓缩除去四氢呋喃，加入 10 mL 二氯甲烷溶解残留物，析出大量红色粉末，过滤后得到 44（283 mg，产率：82.9%）。无需进一步纯化，直接进入下一步。
4-(3,4,5-Trimethoxyphenyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (14)
14 was prepared from 7-bromo-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one 44 (283 mg, 1.33 mmol) and 45 (366.1 mg, 1.73 mmol) according to general procedure A. Pale yellow solid (293 mg, yield: 73.4%). 1H NMR (300 MHz, DMSO-d6): δ 11.47 (s, 1H), 11.08 (s, 1H), 7.95 (d, J = 5.5 Hz, 1H), 7.11 (d, J = 5.5 Hz, 1H), 6.87 (s, 2H), 3.91 (s, 6H), 3.74 (s, 3H). Purity: 97.69% by HPLC (MeOH/H2O = 65:35, tR = 3.891 min).
根据一般程序 A，由 7-溴-1,3-二氢-2H-咪唑并[4,5-b]吡啶-2-酮 44（283 毫克，1.33 毫摩尔）和 45（366.1 毫克，1.73 毫摩尔）制备 14。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.47（s，1H），11.08（s，1H），7.95（d，J = 5.5 Hz，1H），7.11（d，J = 5.5 Hz，1H），6.87（s，2H），3.91（s，6H），3.74（s，3H）。纯度：97.69%（HPLC，MeOH/H 2 O = 65:35，t R = 3.891 分钟）。
根据一般程序 A，由 7-溴-1,3-二氢-2H-咪唑并[4,5-b]吡啶-2-酮 44（283 毫克，1.33 毫摩尔）和 45（366.1 毫克，1.73 毫摩尔）制备 14。 1 H NMR（300 MHz，DMSO-d 6 ）：δ11.47（s，1H），11.08（s，1H），7.95（d，J = 5.5 Hz，1H），7.11（d，J = 5.5 Hz，1H），6.87（s，2H），3.91（s，6H），3.74（s，3H）。纯度：97.69%（HPLC，MeOH/H 2 O = 65:35，t R = 3.891 分钟）。
4-(3-Bromophenyl)-1-(cyclopropylmethyl)-3,5-dimethyl-1H-pyrazole (50g)
4-(3-溴苯基)-1-(环丙基甲基)-3,5-二甲基-1H-吡唑（50 克）
50f was prepared from 48 (500 mg, 1.77 mmol) and 49f (510.3 mg, 2.30 mmol) according to general procedure A. Brown solid (356 mg, yield: 80.2%). 50f (500 mg, 1.99 mmol) and (bromomethyl)cyclopropane 53 (403.19 mg, 2.99 mmol) were dissolved in DMF (15 mL), followed by addition of cesium carbonate (1.30 g, 3.98 mmol). The mixture was stirred at room temperature overnight. Equal volumes of ethyl acetate and water were added and extracted three times, and the organic layer was combined and washed with brine. The organic layer was dried over anhydrous Na2SO4 and purified by column chromatography (PE/EA = 1:1) to obtain colorless oil 50g (467 mg, yield: 76.9%).
根据一般步骤 A，由 48（500 毫克，1.77 毫摩尔）和 49f（510.3 毫克，2.30 毫摩尔）制备 50f，得到棕色固体（356 毫克，产率：80.2%）。将 50f（500 毫克，1.99 毫摩尔）和（溴甲基）环丙烷 53（403.19 毫克，2.99 毫摩尔）溶于 DMF（15 毫升），然后加入碳酸铯（1.30 克，3.98 毫摩尔）。混合物在室温下搅拌过夜。加入等体积的乙酸乙酯和水并萃取三次，合并有机层并用盐水洗涤。有机层用无水 Na 2 干燥。SO 4 ，并通过柱层析（PE/EA = 1:1）纯化，得到无色油 50 克（467 毫克，产率：76.9%）。
根据一般步骤 A，由 48（500 毫克，1.77 毫摩尔）和 49f（510.3 毫克，2.30 毫摩尔）制备 50f，得到棕色固体（356 毫克，产率：80.2%）。将 50f（500 毫克，1.99 毫摩尔）和（溴甲基）环丙烷 53（403.19 毫克，2.99 毫摩尔）溶于 DMF（15 毫升），然后加入碳酸铯（1.30 克，3.98 毫摩尔）。混合物在室温下搅拌过夜。加入等体积的乙酸乙酯和水并萃取三次，合并有机层并用盐水洗涤。有机层用无水 Na 2 干燥。SO 4 ，并通过柱层析（PE/EA = 1:1）纯化，得到无色油 50 克（467 毫克，产率：76.9%）。
General Procedure B: Preparation of Boronic Esters for the Synthesis of 52a–52g, 56a–56d, and 61a–61f
一般程序 B：制备用于合成 52a-52g、56a-56d 和 61a-61f 的硼酸酯
Aryl halides (1.0 equiv) and 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.3 equiv) were dissolved in anhydrous dioxaborolane, followed by the addition of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (0.1 equiv) and potassium acetate (2 equiv). After reaction at 100 °C for 3 h under an argon atmosphere, the reaction was cooled to room temperature, equal volumes of ethyl acetate and water were added and extracted three times, and the organic layer was combined and washed with brine. The organic layer was concentrated under reduced pressure to obtain arylboronic acid esters, which were directly proceeded to the next step without further purification.
将芳基卤化物（1.0 等量）和 4,4,4′,4′,5,5,5′,5′-八甲基-2,2′-双(1,3,2-二氧杂硼烷)（1.3当量）溶于无水二氧硼戊环，然后加入[1,1-双（二苯基膦）二茂铁]二氯化钯二氯甲烷络合物（0.1当量）和醋酸钾（2当量）。在 100 °C 的氩气环境下反应 3 小时后，将反应物冷却至室温，加入等体积的乙酸乙酯和水并萃取三次，合并有机层并用盐水洗涤。有机层在减压下浓缩，得到芳基硼酸酯，直接进入下一步，无需进一步纯化。
将芳基卤化物（1.0 等量）和 4,4,4′,4′,5,5,5′,5′-八甲基-2,2′-双(1,3,2-二氧杂硼烷)（1.3当量）溶于无水二氧硼戊环，然后加入[1,1-双（二苯基膦）二茂铁]二氯化钯二氯甲烷络合物（0.1当量）和醋酸钾（2当量）。在 100 °C 的氩气环境下反应 3 小时后，将反应物冷却至室温，加入等体积的乙酸乙酯和水并萃取三次，合并有机层并用盐水洗涤。有机层在减压下浓缩，得到芳基硼酸酯，直接进入下一步，无需进一步纯化。
4-(3-(1H-Imidazo[4,5-b]pyridin-7-yl)phenyl)-3,5-dimethylisoxazole (15)
50a was prepared from 48 (500 mg, 1.77 mmol) and 49a (512.54 mg, 2.30 mmol) according to general procedure A. Brown solid (400 mg, yield: 89.8%). 52a was prepared from 51 (438 mg, 1.73 mmol) and 50a (334 mg, 1.33 mmol) according to general procedure B. Brown oil (320 mg, yield: 77.3%). 15 was prepared from 42 (258 mg, 1.30 mmol) and 52a (506.7 mg, 1.69 mmol) according to general procedure A. Pale yellow solid (197 mg, yield: 60.6%). 1H NMR (300 MHz, DMSO-d6): δ 13.29 (s, 1H), 8.53 (s, 1H), 8.48–8.37 (m, 2H), 8.29 (d, J = 7.9 Hz, 1H), 7.72–7.61 (m, 2H), 7.53 (d, J = 7.7 Hz, 1H), 3.41 (s, 3H), 2.35 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 165.88, 158.72, 149.29, 144.73, 144.19, 137.37, 136.70, 132.37, 130.68, 130.09, 129.73, 128.34, 116.32, 115.74, 11.94, 11.10. HRMS (ESI): calcd for C17H14N4O [M + H]+, 291.1201; found, 291.1239. Purity: 97.01% by HPLC (MeOH/H2O = 65:35, tR = 4.241 min).
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49a（512.54 毫克，2.30 毫摩尔）制备出 50a。根据一般程序 B，由 51（438 毫克，1.73 毫摩尔）和 50a（334 毫克，1.33 毫摩尔）制备 52a。根据一般程序 A，由 42（258 毫克，1.30 毫摩尔）和 52a（506.7 毫克，1.69 毫摩尔）制备 15。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.29（s，1H），8.53（s，1H），8.48-8.37（m，2H），8.29（d，J = 7.9 Hz，1H），7.72-7.61（m，2H），7.53（d，J = 7.7 Hz，1H），3.41（s，3H），2.35（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 165.88, 158.72, 149.29, 144.73, 144.19, 137.37, 136.70, 132.37, 130.68, 130.09, 129.73, 128.34, 116.32, 115.74, 11.94, 11.10.HRMS（ESI）：煅烧为 C 17 H { 14 H 14 N 4 O [M + H] + ，291.1201；发现值，291.1239。纯度：97.01%（HPLC，MeOH/H 2 O = 65:35，t R = 4.241 分钟）。
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49a（512.54 毫克，2.30 毫摩尔）制备出 50a。根据一般程序 B，由 51（438 毫克，1.73 毫摩尔）和 50a（334 毫克，1.33 毫摩尔）制备 52a。根据一般程序 A，由 42（258 毫克，1.30 毫摩尔）和 52a（506.7 毫克，1.69 毫摩尔）制备 15。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.29（s，1H），8.53（s，1H），8.48-8.37（m，2H），8.29（d，J = 7.9 Hz，1H），7.72-7.61（m，2H），7.53（d，J = 7.7 Hz，1H），3.41（s，3H），2.35（s，3H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 165.88, 158.72, 149.29, 144.73, 144.19, 137.37, 136.70, 132.37, 130.68, 130.09, 129.73, 128.34, 116.32, 115.74, 11.94, 11.10.HRMS（ESI）：煅烧为 C 17 H { 14 H 14 N 4 O [M + H] + ，291.1201；发现值，291.1239。纯度：97.01%（HPLC，MeOH/H 2 O = 65:35，t R = 4.241 分钟）。
7-(3-(1,3,5-Trimethyl-1H-pyrazol-4-yl)phenyl)-1H-imidazo[4,5-b]pyridine (16)
50b was prepared from 48 (500 mg, 1.77 mmol) and 49b (533 mg, 2.30 mmol) according to general procedure A. Yellow solid (413 mg, yield: 88.1%). 52b was prepared from 51 (498 mg, 1.96 mmol) and 50b (400 mg, 1.51 mmol) according to general procedure B. Red oil (390 mg, yield: 82.8%). 16 was prepared from 42 (192 mg, 0.96 mmol) and 52b (390 mg, 1.25 mmol) according to general procedure A. Pale yellow solid (232 mg, yield: 79.7%). 1H NMR (300 MHz, chloroform-d): δ 8.58 (d, J = 5.4 Hz, 1H), 8.50 (s, 1H), 8.10 (d, J = 7.9 Hz, 1H), 8.02 (s, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 5.1 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 3.86 (s, 3H), 2.36 (d, J = 2.5 Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 149.31, 144.71, 144.19, 143.87, 137.91, 136.51, 136.34, 134.84, 130.24, 129.87, 129.34, 126.75, 118.33, 115.78, 36.33, 13.02, 10.51. HRMS (ESI): calcd for C18H17N5 [M + H]+, 304.1518; found, 304.1502. Purity: 99.88% by HPLC (MeOH/H2O = 65:35, tR = 6.782 min).
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49b（533 毫克，2.30 毫摩尔）制备 50b。根据一般程序 B，由 51（498 毫克，1.96 毫摩尔）和 50b（400 毫克，1.51 毫摩尔）制备 52b。根据一般步骤 A，由 42（192 毫克，0.96 毫摩尔）和 52b（390 毫克，1.25 毫摩尔）制备出 16。 1 H NMR（300 MHz，氯仿-d）：δ8.58（d，J = 5.4 Hz，1H），8.50（s，1H），8.10（d，J = 7.9 Hz，1H），8.02（s，1H），7.65（t，J = 7.7 Hz，1H），7.56（d，J = 5.1 Hz，1H），7.42（d，J = 7.7 Hz，1H），3.86（s，3H），2.36（d，J = 2.5 Hz，6H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 149.31, 144.71, 144.19, 143.87, 137.91, 136.51, 136.34, 134.84, 130.24, 129.87, 129.34, 126.75, 118.33, 115.78, 36.33, 13.02, 10.51.HRMS (ESI)：煅烧为 C 18 H { 17 H 17 N 5 [M + H] + ，304.1518；发现值为 304.1502。HPLC 测定纯度：99.88%（MeOH/H 2 O = 65:35，t R = 6.782 分钟）。
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49b（533 毫克，2.30 毫摩尔）制备 50b。根据一般程序 B，由 51（498 毫克，1.96 毫摩尔）和 50b（400 毫克，1.51 毫摩尔）制备 52b。根据一般步骤 A，由 42（192 毫克，0.96 毫摩尔）和 52b（390 毫克，1.25 毫摩尔）制备出 16。 1 H NMR（300 MHz，氯仿-d）：δ8.58（d，J = 5.4 Hz，1H），8.50（s，1H），8.10（d，J = 7.9 Hz，1H），8.02（s，1H），7.65（t，J = 7.7 Hz，1H），7.56（d，J = 5.1 Hz，1H），7.42（d，J = 7.7 Hz，1H），3.86（s，3H），2.36（d，J = 2.5 Hz，6H）。 13 C NMR（75 MHz，DMSO-d 6 ）：δ 149.31, 144.71, 144.19, 143.87, 137.91, 136.51, 136.34, 134.84, 130.24, 129.87, 129.34, 126.75, 118.33, 115.78, 36.33, 13.02, 10.51.HRMS (ESI)：煅烧为 C 18 H { 17 H 17 N 5 [M + H] + ，304.1518；发现值为 304.1502。HPLC 测定纯度：99.88%（MeOH/H 2 O = 65:35，t R = 6.782 分钟）。
7-(3-(1,4-Dimethyl-1H-pyrazol-5-yl)phenyl)-1H-imidazo[4,5-b]pyridine (17)
50c was prepared from 48 (500 mg, 1.77 mmol) and 49c (510 mg, 2.30 mmol) according to general procedure A. Yellow solid (398 mg, yield: 89.7%). 52c was prepared from 51 (522 mg, 2.05 mmol) and 50c (400 mg, 1.59 mmol) according to general procedure B. Red oil (412 mg, yield: 86.7%). 17 was prepared from 42 (203.9 mg, 1.03 mmol) and 52c (399.3 mg, 1.34 mmol) according to general procedure A. Pale yellow solid (192 mg, yield: 62.7%). 1H NMR (300 MHz, chloroform-d): δ 8.59 (d, J = 5.2 Hz, 1H), 8.43 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.17 (s, 1H), 7.73 (t, J = 7.7 Hz, 1H), 7.57 (d, J = 5.1 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.47 (s, 1H), 3.92 (s, 3H), 2.14 (s, 3H). HRMS (ESI): calcd for C17H15N5 [M + H]+, 290.1361; found, 290.1367. Purity: 97.66% by HPLC (MeOH/H2O = 65:35, tR = 4.975 min).
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49c（510 毫克，2.30 毫摩尔）制备 50c。根据一般程序 B，由 51（522 毫克，2.05 毫摩尔）和 50c（400 毫克，1.59 毫摩尔）制备 52c。根据一般程序 A，由 42（203.9 毫克，1.03 毫摩尔）和 52c（399.3 毫克，1.34 毫摩尔）制备出 17。 1 H NMR（300 MHz，氯仿-d）：δ8.59（d，J = 5.2 Hz，1H），8.43（s，1H），8.28（d，J = 7.9 Hz，1H），8.17（s，1H），7.73（t，J = 7.7Hz，1H）、7.57（d，J = 5.1Hz，1H）、7.49（d，J = 7.6Hz，1H）、7.47（s，1H）、3.92（s，3H）、2.14（s，3H）。HRMS (ESI)：煅烧为 C 17 H { 15 H 15 N 5 [M + H] + ，290.1361；发现值，290.1367。纯度：97.66%（HPLC，MeOH/H 2 O = 65:35，t R = 4.975 分钟）。
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49c（510 毫克，2.30 毫摩尔）制备 50c。根据一般程序 B，由 51（522 毫克，2.05 毫摩尔）和 50c（400 毫克，1.59 毫摩尔）制备 52c。根据一般程序 A，由 42（203.9 毫克，1.03 毫摩尔）和 52c（399.3 毫克，1.34 毫摩尔）制备出 17。 1 H NMR（300 MHz，氯仿-d）：δ8.59（d，J = 5.2 Hz，1H），8.43（s，1H），8.28（d，J = 7.9 Hz，1H），8.17（s，1H），7.73（t，J = 7.7Hz，1H）、7.57（d，J = 5.1Hz，1H）、7.49（d，J = 7.6Hz，1H）、7.47（s，1H）、3.92（s，3H）、2.14（s，3H）。HRMS (ESI)：煅烧为 C 17 H { 15 H 15 N 5 [M + H] + ，290.1361；发现值，290.1367。纯度：97.66%（HPLC，MeOH/H 2 O = 65:35，t R = 4.975 分钟）。
7-(2′,6′-Dimethyl-[1,1′-biphenyl]-3-yl)-1H-imidazo[4,5-b]pyridine (18)
50d was prepared from 48 (500 mg, 1.77 mmol) and 49d (533 mg, 2.30 mmol) according to general procedure A. Yellow solid (430 mg, yield: 93.2%). 52d was prepared from 51 (505.6 mg, 1.99 mmol) and 50d (400 mg, 1.53 mmol) according to general procedure B. Brown oil (417 mg, yield: 88.3%). 18 was prepared from 42 (203.9 mg, 1.03 mmol) and 52d (417 mg, 1.35 mmol) according to general procedure A. Pale yellow solid (229.1 mg, yield: 69.2%). 1H NMR (300 MHz, chloroform-d): δ 8.56 (d, J = 5.1 Hz, 1H), 8.45 (s, 1H), 8.38–8.31 (m, 1H), 7.91 (s, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 5.1 Hz, 1H), 7.36–7.31 (m, 1H), 7.21 (q, J = 5.8 Hz, 3H), 2.17 (s, 6H). HRMS (ESI): calcd for C20H17N3 [M + H]+, 300.1456; found, 300.1497. Purity: 99.64% by HPLC (MeOH/H2O = 65:35, tR = 13.178 min).
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49d（533 毫克，2.30 毫摩尔）制备 50d。根据一般程序 B，由 51（505.6 毫克，1.99 毫摩尔）和 50d（400 毫克，1.53 毫摩尔）制备 52d。根据一般程序 A，由 42（203.9 毫克，1.03 毫摩尔）和 52d（417 毫克，1.35 毫摩尔）制备出 18。 1 H NMR（300 MHz，氯仿-d）：δ8.56（d，J = 5.1 Hz，1H），8.45（s，1H），8.38-8.31（m，1H），7.91（s，1H），7.68（t，J = 7.6 Hz，1H），7.55（d，J = 5.1 Hz，1H），7.36-7.31（m，1H），7.21（q，J = 5.8 Hz，3H），2.17（s，6H）。HRMS (ESI)：煅烧为 C 20 H { 17 H 17 N 3 [M + H] + ，300.1456；发现值，300.1497。HPLC 测定纯度：99.64%（MeOH/H 2 O = 65:35，t R = 13.178 分钟）。
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49d（533 毫克，2.30 毫摩尔）制备 50d。根据一般程序 B，由 51（505.6 毫克，1.99 毫摩尔）和 50d（400 毫克，1.53 毫摩尔）制备 52d。根据一般程序 A，由 42（203.9 毫克，1.03 毫摩尔）和 52d（417 毫克，1.35 毫摩尔）制备出 18。 1 H NMR（300 MHz，氯仿-d）：δ8.56（d，J = 5.1 Hz，1H），8.45（s，1H），8.38-8.31（m，1H），7.91（s，1H），7.68（t，J = 7.6 Hz，1H），7.55（d，J = 5.1 Hz，1H），7.36-7.31（m，1H），7.21（q，J = 5.8 Hz，3H），2.17（s，6H）。HRMS (ESI)：煅烧为 C 20 H { 17 H 17 N 3 [M + H] + ，300.1456；发现值，300.1497。HPLC 测定纯度：99.64%（MeOH/H 2 O = 65:35，t R = 13.178 分钟）。
8-(3-(1H-Imidazo[4,5-b]pyridin-7-yl)phenyl)quinoline (19)
8-(3-(1H-咪唑并[4,5-b]吡啶-7-基)苯基)喹啉 (19)
50e was prepared from 48 (500 mg, 1.77 mmol) and 49e (586 mg, 2.30 mmol) according to general procedure A. Brown solid (410 mg, yield: 81.6%). 52e was prepared from 51 (464.7 mg, 1.83 mmol) and 50e (400 mg, 1.41 mmol) according to general procedure B. Red oil (372 mg, yield: 83.3%). 19 was prepared from 42 (178.2 mg, 0.91 mmol) and 52e (372 mg, 1.17 mmol) according to general procedure A. Pale yellow solid (201.7 mg, yield: 69.6%). 1H NMR (300 MHz, chloroform-d): δ 9.03 (dd, J = 4.2, 1.8 Hz, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.37 (t, J = 1.7 Hz, 1H), 8.31–8.23 (m, 2H), 8.10 (d, J = 7.7 Hz, 1H), 7.87 (ddd, J = 14.3, 7.6, 1.5 Hz, 2H), 7.79 (dt, J = 7.7, 1.4 Hz, 1H), 7.66 (t, J = 7.7 Hz, 2H), 7.52–7.44 (m, 2H). HRMS (ESI): calcd for C21H14N4 [M + H]+, 323.1252; found, 323.1290. Purity: 94.81% by HPLC (MeOH/H2O = 65:35, tR = 13.287 min).
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49e（586 毫克，2.30 毫摩尔）制备 50e。根据一般程序 B，由 51（464.7 毫克，1.83 毫摩尔）和 50e（400 毫克，1.41 毫摩尔）制备 52e。根据一般程序 A，由 42（178.2 毫克，0.91 毫摩尔）和 52e（372 毫克，1.17 毫摩尔）制备 19。 1 H NMR（300 MHz，氯仿-d）：δ9.03（dd，J = 4.2，1.8 Hz，1H），8.49（d，J = 5.2 Hz，1H），8.37（t，J = 1.7 Hz，1H），8.31-8.23（m，2H），8.10（d，J = 7.7Hz，1H），7.87（ddd，J = 14.3，7.6，1.5 Hz，2H），7.79（dt，J = 7.7，1.4 Hz，1H），7.66（t，J = 7.7 Hz，2H），7.52-7.44（m，2H）。HRMS (ESI)：煅烧为 C 21 H 14 N 4 [M + H] + ，323.1252；发现值，323.1290。通过 HPLC（MeOH/H 2 O = 65:35，t R = 13.287 分钟）测定，纯度为 94.81%。
根据一般程序 A，由 48（500 毫克，1.77 毫摩尔）和 49e（586 毫克，2.30 毫摩尔）制备 50e。根据一般程序 B，由 51（464.7 毫克，1.83 毫摩尔）和 50e（400 毫克，1.41 毫摩尔）制备 52e。根据一般程序 A，由 42（178.2 毫克，0.91 毫摩尔）和 52e（372 毫克，1.17 毫摩尔）制备 19。 1 H NMR（300 MHz，氯仿-d）：δ9.03（dd，J = 4.2，1.8 Hz，1H），8.49（d，J = 5.2 Hz，1H），8.37（t，J = 1.7 Hz，1H），8.31-8.23（m，2H），8.10（d，J = 7.7Hz，1H），7.87（ddd，J = 14.3，7.6，1.5 Hz，2H），7.79（dt，J = 7.7，1.4 Hz，1H），7.66（t，J = 7.7 Hz，2H），7.52-7.44（m，2H）。HRMS (ESI)：煅烧为 C 21 H 14 N 4 [M + H] + ，323.1252；发现值，323.1290。通过 HPLC（MeOH/H 2 O = 65:35，t R = 13.287 分钟）测定，纯度为 94.81%。
7-(3-(1-(Cyclopropylmethyl)-3,5-dimethyl-1H-pyrazol-4-yl)phenyl)-1H-imidazo[4,5-b]pyridine (20)
7-(3-(1-(环丙基甲基)-3,5-二甲基-1H-吡唑-4-基)苯基)-1H-咪唑并[4,5-b]吡啶 (20)
52g was prepared from 51 (432.6 mg, 1.70 mmol) and 50g (400 mg, 1.31 mmol) according to general procedure B. Red oil (383 mg, yield: 83.0%). 20 was prepared from 42 (168.2 mg, 0.85 mmol) and 52g (387.7 mg, 1.10 mmol) according to general procedure A. Pale yellow solid (149 mg, yield: 51.3%). 1H NMR (300 MHz, DMSO-d6): δ 13.24 (s, 1H), 8.50 (s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.30 (s, 1H), 8.17 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 6.1 Hz, 2H), 7.41 (d, J = 7.8 Hz, 1H), 3.95 (d, J = 6.9 Hz, 2H), 2.35 (s, 3H), 2.25 (s, 3H), 1.27 (s, 1H), 0.55 (d, J = 7.6 Hz, 2H), 0.41 (d, J = 4.9 Hz, 2H). HRMS (ESI): calcd for C21H21N5 [M + H]+, 344.1831; found, 344.1871. Purity: 97.29% by HPLC (MeOH/H2O = 80:20, tR = 3.165 min).
根据一般步骤 B，由 51（432.6 毫克，1.70 毫摩尔）和 50 克（400 毫克，1.31 毫摩尔）制备 52 克。根据一般程序 A，由 42（168.2 毫克，0.85 毫摩尔）和 52g（387.7 毫克，1.10 毫摩尔）制备 20。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.24（s，1H），8.50（s，1H），8.44（d，J = 5.1 Hz，1H），8.30（s，1H），8.17（d，J = 7.8 Hz，1H），7.61（d，J = 6.1 Hz，2H），7.41（d，J = 7.8Hz，1H）、3.95（d，J = 6.9Hz，2H）、2.35（s，3H）、2.25（s，3H）、1.27（s，1H）、0.55（d，J = 7.6Hz，2H）、0.41（d，J = 4.9Hz，2H）。HRMS (ESI)：煅烧为 C 21 H { 21 H 21 N 5 [M + H] + ，344.1831；发现值，344.1871。纯度：97.29%（HPLC，MeOH/H 2 O = 80:20，t R = 3.165 分钟）。
根据一般步骤 B，由 51（432.6 毫克，1.70 毫摩尔）和 50 克（400 毫克，1.31 毫摩尔）制备 52 克。根据一般程序 A，由 42（168.2 毫克，0.85 毫摩尔）和 52g（387.7 毫克，1.10 毫摩尔）制备 20。 1 H NMR（300 MHz，DMSO-d 6 ）：δ13.24（s，1H），8.50（s，1H），8.44（d，J = 5.1 Hz，1H），8.30（s，1H），8.17（d，J = 7.8 Hz，1H），7.61（d，J = 6.1 Hz，2H），7.41（d，J = 7.8Hz，1H）、3.95（d，J = 6.9Hz，2H）、2.35（s，3H）、2.25（s，3H）、1.27（s，1H）、0.55（d，J = 7.6Hz，2H）、0.41（d，J = 4.9Hz，2H）。HRMS (ESI)：煅烧为 C 21 H { 21 H 21 N 5 [M + H] + ，344.1831；发现值，344.1871。纯度：97.29%（HPLC，MeOH/H 2 O = 80:20，t R = 3.165 分钟）。
7-(2-Fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)-1H-imidazo[4,5-b]pyridine (21)
55a was prepared from 54a (530 mg, 1.76 mmol) and 49b (540 mg, 2.29 mmol) according to general procedure A. White solid (400 mg, yield: 80.2%). 56a was prepared from 55a (400 mg, 1.41 mmol) and 51 (466 mg, 1.84 mmol) according to general procedure B. Reddish brown oil (366 mg, yield: 78.5%). 21 was prepared from 42 (128 mg, 0.65 mmol) and 56a (279.5 mg, 0.85 mmol) according to general procedure A. White solid (138 mg, yield: 66.0%). 1H NMR (300 MHz, chloroform-d): δ 8.59 (s, 1H), 8.40 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.55 (s, 1H), 7.38 (p, J = 7.5 Hz, 2H), 3.84 (s, 3H), 2.26 (s, 3H), 2.23 (s, 3H). HRMS (ESI): calcd for C18H16FN5 [M + H]+, 322.1423; found, 322.1475. Purity: 98.83% by HPLC (MeOH/H2O = 80:20, tR = 5.051 min).
根据一般程序 A，由 54a（530 毫克，1.76 毫摩尔）和 49b（540 毫克，2.29 毫摩尔）制备 55a。根据一般程序 B，由 55a（400 毫克，1.41 毫摩尔）和 51（466 毫克，1.84 毫摩尔）制备 56a。根据一般程序 A，由 42（128 毫克，0.65 毫摩尔）和 56a（279.5 毫克，0.85 毫摩尔）制备出 21。 1 H NMR（300 MHz，氯仿-d）：δ8.59（s，1H），8.40（s，1H），7.89（d，J = 7.6 Hz，1H），7.55（s，1H），7.38（p，J = 7.5 Hz，2H），3.84（s，3H），2.26（s，3H），2.23（s，3H）。HRMS（ESI）：煅烧为 C 18 H { 16 H 16 FN 5 [M + H] + ，322.1423；发现值，322.1475。纯度：98.83%（HPLC，MeOH/H 2 O = 80:20，t R = 5.051 分钟）。
根据一般程序 A，由 54a（530 毫克，1.76 毫摩尔）和 49b（540 毫克，2.29 毫摩尔）制备 55a。根据一般程序 B，由 55a（400 毫克，1.41 毫摩尔）和 51（466 毫克，1.84 毫摩尔）制备 56a。根据一般程序 A，由 42（128 毫克，0.65 毫摩尔）和 56a（279.5 毫克，0.85 毫摩尔）制备出 21。 1 H NMR（300 MHz，氯仿-d）：δ8.59（s，1H），8.40（s，1H），7.89（d，J = 7.6 Hz，1H），7.55（s，1H），7.38（p，J = 7.5 Hz，2H），3.84（s，3H），2.26（s，3H），2.23（s，3H）。HRMS（ESI）：煅烧为 C 18 H { 16 H 16 FN 5 [M + H] + ，322.1423；发现值，322.1475。纯度：98.83%（HPLC，MeOH/H 2 O = 80:20，t R = 5.051 分钟）。
7-(4-Fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)-1H-imidazo[4,5-b]pyridine (22)
55b was prepared from 54b (530 mg, 1.76 mmol) and 49b (540 mg, 2.29 mmol) according to general procedure A. White solid (372 mg, yield: 74.6%). 56b was prepared from 55b (400 mg, 1.41 mmol) and 51 (466 mg, 1.84 mmol) according to general procedure B. Reddish brown oil (401 mg, yield: 85.9%). 22 was prepared from 42 (128 mg, 0.65 mmol) and 56b (279.5 mg, 0.85 mmol) according to general procedure A. Pale yellow solid (113 mg, yield: 54.1%). 1H NMR (300 MHz, chloroform-d): δ 8.62 (s, 1H), 8.40 (s, 1H), 8.21 (s, 1H), 8.06 (d, J = 6.9 Hz, 1H), 7.52 (s, 1H), 7.38 (d, J = 9.0 Hz, 1H), 3.85 (s, 3H), 2.29 (d, J = 5.9 Hz, 6H). HRMS (ESI): calcd for C18H16FN5 [M + H]+, 322.1423; found, 322.1323. Purity: 97.70% by HPLC (MeOH/H2O = 80:20, tR = 3.788 min).
根据一般程序 A，由 54b（530 毫克，1.76 毫摩尔）和 49b（540 毫克，2.29 毫摩尔）制备 55b。根据一般程序 B，由 55b（400 毫克，1.41 毫摩尔）和 51（466 毫克，1.84 毫摩尔）制备 56b。根据一般程序 A，由 42（128 毫克，0.65 毫摩尔）和 56b（279.5 毫克，0.85 毫摩尔）制备 22。 1 H NMR（300 MHz，氯仿-d）：δ8.62（s，1H），8.40（s，1H），8.21（s，1H），8.06（d，J = 6.9 Hz，1H），7.52（s，1H），7.38（d，J = 9.0 Hz，1H），3.85（s，3H），2.29（d，J = 5.9 Hz，6H）。HRMS (ESI)：煅烧为 C 18 H { 16 H 16 FN 5 [M + H] + ，322.1423；发现值，322.1323。纯度：97.70%（HPLC，MeOH/H 2 O = 80:20，t R = 3.788 分钟）。
根据一般程序 A，由 54b（530 毫克，1.76 毫摩尔）和 49b（540 毫克，2.29 毫摩尔）制备 55b。根据一般程序 B，由 55b（400 毫克，1.41 毫摩尔）和 51（466 毫克，1.84 毫摩尔）制备 56b。根据一般程序 A，由 42（128 毫克，0.65 毫摩尔）和 56b（279.5 毫克，0.85 毫摩尔）制备 22。 1 H NMR（300 MHz，氯仿-d）：δ8.62（s，1H），8.40（s，1H），8.21（s，1H），8.06（d，J = 6.9 Hz，1H），7.52（s，1H），7.38（d，J = 9.0 Hz，1H），3.85（s，3H），2.29（d，J = 5.9 Hz，6H）。HRMS (ESI)：煅烧为 C 18 H { 16 H 16 FN 5 [M + H] + ，322.1423；发现值，322.1323。纯度：97.70%（HPLC，MeOH/H 2 O = 80:20，t R = 3.788 分钟）。
7-(2-Chloro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)-1H-imidazo[4,5-b]pyridine (23)
55c was prepared from 54c (530 mg, 1.67 mmol) and 49b (512 mg, 2.17 mmol) according to general procedure A. White solid (381 mg, yield: 76.2%). 56c was prepared from 55c (400 mg, 1.34 mmol) and 51 (441 mg, 1.74 mmol) according to general procedure B. Reddish brown oil (351 mg, yield: 75.8%). 23 was prepared from 42 (128 mg, 0.65 mmol) and 56c (292.9 mg, 0.85 mmol) according to general procedure A. Brown solid (96 mg, yield: 43.7%). 1H NMR (300 MHz, chloroform-d): δ 8.59 (s, 1H), 8.38 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.48 (s, 2H), 7.35 (d, J = 7.3 Hz, 1H), 3.84 (s, 3H), 2.21 (s, 3H), 2.19 (s, 3H). HRMS (ESI): calcd for C18H16ClN5 [M + H]+, 338.11; found, 338.1129 Purity: 98.18% by HPLC (MeOH/H2O = 65:35, tR = 3.454 min).
根据一般程序 A，由 54c（530 毫克，1.67 毫摩尔）和 49b（512 毫克，2.17 毫摩尔）制备 55c。根据一般程序 B，由 55c（400 毫克，1.34 毫摩尔）和 51（441 毫克，1.74 毫摩尔）制备 56c。根据一般程序 A，由 42（128 毫克，0.65 毫摩尔）和 56c（292.9 毫克，0.85 毫摩尔）制备出 23。 1 H NMR（300 MHz，氯仿-d）：δ8.59（s，1H），8.38（s，1H），7.61（d，J = 7.5 Hz，1H），7.48（s，2H），7.35（d，J = 7.3 Hz，1H），3.84（s，3H），2.21（s，3H），2.19（s，3H）。HRMS (ESI)：煅烧为 C 18 H { 16 H 16 ClN 5 [M + H] + 纯度：98.18%，HPLC（MeOH/H 2 O = 65:35，t R = 3.454 分钟）。
根据一般程序 A，由 54c（530 毫克，1.67 毫摩尔）和 49b（512 毫克，2.17 毫摩尔）制备 55c。根据一般程序 B，由 55c（400 毫克，1.34 毫摩尔）和 51（441 毫克，1.74 毫摩尔）制备 56c。根据一般程序 A，由 42（128 毫克，0.65 毫摩尔）和 56c（292.9 毫克，0.85 毫摩尔）制备出 23。 1 H NMR（300 MHz，氯仿-d）：δ8.59（s，1H），8.38（s，1H），7.61（d，J = 7.5 Hz，1H），7.48（s，2H），7.35（d，J = 7.3 Hz，1H），3.84（s，3H），2.21（s，3H），2.19（s，3H）。HRMS (ESI)：煅烧为 C 18 H { 16 H 16 ClN 5 [M + H] + 纯度：98.18%，HPLC（MeOH/H 2 O = 65:35，t R = 3.454 分钟）。
7-(2,4-Difluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)-1H-imidazo[4,5-b]pyridine (24)
55d was prepared from 54d (530 mg, 1.66 mmol) and 49b (510 mg, 2.16 mmol) according to general procedure A. White solid (391 mg, yield: 78.1%). 56d was prepared from 55d (400 mg, 1.36 mmol) and 51 (438 mg, 1.73 mmol) according to general procedure B. Reddish brown oil (376 mg, yield: 79.2%). 24 was prepared from 42 (128 mg, 0.65 mmol) and 56d (294.2 mg, 0.85 mmol) according to general procedure A. Pale yellow solid (105 mg, yield: 47.6%).1H NMR (300 MHz, chloroform-d): δ 8.59 (s, 1H), 8.38 (s, 1H), 7.89 (t, J = 8.3 Hz, 1H), 7.51 (s, 1H), 7.12 (t, J = 9.9 Hz, 1H), 3.83 (s, 3H), 2.26 (s, 3H), 2.25 (s, 3H). HRMS (ESI): calcd for C18H15F2N5 [M + H]+, 340.1329; found, 340.1375. Purity: 95.81% by HPLC (MeOH/H2O = 80:20, tR = 6.516 min).
General Procedure C: The Synthesis of Intermediates 59a–59f
3,5-Dibromo-4-fluoroaniline 57 (1.0 equiv) and pyridine (2.5 equiv) were dissolved in dichloromethane, followed by slow dropwise addition of different sulfonyl chlorides 58a–58f (1.1 equiv) at room temperature. The reaction mixture was stirred at room temperature for 4 h, and the initial raw materials disappeared. The reaction mixture was neutralized with 1 M hydrochloric acid to pH 7 and then extracted three times with ethyl acetate. The organic layer was washed with brine and dried with Na2SO4. The solvent was removed under reduced pressure, and column chromatography was performed to obtain intermediates 59a–59f.
N-(4-Fluoro-3-(1H-imidazo[4,5-b]pyridin-7-yl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)methanesulfonamide (25)
59a was prepared from 57 (2.0 g, 7.44 mmol) and 58a (937 mg, 8.18 mmol) according to general procedure C. Yellow solid (2.47 g, yield: 95.7%). 60a was prepared from 49b (531 mg, 2.25 mmol) and 59a (600 mg, 1.73 mmol) according to general procedure A. Light yellow solid (532 mg, yield: 81.8%). 61a was prepared from 51 (351 mg, 1.38 mmol) and 60a (400 mg, 1.06 mmol) according to general procedure B. Light yellow oil (351 mg, yield: 78.0%). 25 was prepared from 42 (108 mg, 0.51 mmol) and 61a (300 mg, 0.71 mmol) according to general procedure A. Brown solid (193 mg, yield: 65.7%). 1H NMR (300 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.96 (d, J = 9.5 Hz, 1H), 8.52 (s, 1H), 8.45 (d, J = 5.0 Hz, 1H), 7.67 (dd, J = 5.9, 2.8 Hz, 1H), 7.42 (dd, J = 5.0, 1.8 Hz, 1H), 7.22 (dd, J = 6.1, 2.8 Hz, 1H), 3.75 (s, 3H), 3.12 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H). HRMS (ESI): calcd for C19H19FN6O2S [M + H]+, 415.1308; found, 415.1351. Purity: 98.61% by HPLC (MeOH/H2O = 80:20, tR = 4.298 min).
N-(4-Fluoro-3-(1H-imidazo[4,5-b]pyridin-7-yl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)ethanesulfonamide (26)
59b was prepared from 57 (2.0 g, 7.44 mmol) and 58b (1.04 g, 8.18 mmol) according to general procedure C. Yellow solid (2.56 g, yield: 95.3%). 60b was prepared from 49b (510 mg, 2.16 mmol) and 59b (600 mg, 1.66 mmol) according to general procedure A. Yellow solid (541 mg, yield: 83.4%). 61b was prepared from 51 (338 mg, 1.33 mmol) and 60b (400 mg, 1.02 mmol) according to general procedure B. Light yellow oil (347 mg, yield: 77.4%). 26 was prepared from 42 (104 mg, 0.53 mmol) and 61b (300 mg, 0.68 mmol) according to general procedure A. Light yellow solid (203 mg, yield: 69.1%). 1H NMR (300 MHz, DMSO-d6): δ 13.32 (s, 1H), 9.99 (s, 1H), 8.57–8.41 (m, 2H), 7.68 (dd, J = 5.9, 2.8 Hz, 1H), 7.43–7.32 (m, 1H), 7.22 (dd, J = 6.0, 2.8 Hz, 1H), 3.75 (s, 3H), 3.21 (dd, J = 8.7, 5.8 Hz, 2H), 2.23 (s, 3H), 2.13 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): δ 151.86, 148.90, 144.62, 144.46, 144.26, 137.79, 134.62, 133.85, 132.92, 125.41, 125.18, 123.97, 122.34, 118.34, 112.06, 45.68, 36.40, 12.75, 10.51, 8.56. HRMS (ESI): calcd for C20H21FN6O2S [M + H]+, 429.1464; found, 429.1521. Purity: 99.09% by HPLC (MeOH/H2O = 80:20, tR = 5.010 min).
N-(4-Fluoro-3-(1H-imidazo[4,5-b]pyridin-7-yl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)propane-1-sulfonamide (27)
59c was prepared from 57 (2.0 g, 7.44 mmol) and 58c (1.17 g, 8.18 mmol) according to general procedure C. Yellow solid (2.61 g, yield: 93.6%). 60c was prepared from 49b (491 mg, 2.08 mmol) and 59c (600 mg, 1.60 mmol) according to general procedure A. Light yellow solid (554 mg, yield: 85.7%). 61c was prepared from 51 (327 mg, 1.29 mmol) and 60c (400 mg, 0.98 mmol) according to general procedure B. Light yellow oil (331 mg, yield: 74.1%). 27 was prepared from 42 (101 mg, 0.51 mmol) and 61c (300 mg, 0.66 mmol) according to general procedure A. Light yellow solid (164 mg, yield: 55.8%). 1H NMR (300 MHz, chloroform-d): δ 8.97 (s, 1H), 8.54 (d, J = 5.1 Hz, 1H), 8.38 (s, 1H), 7.77 (s, 1H), 7.48 (d, J = 4.5 Hz, 1H), 3.78 (s, 3H), 3.15 (dd, J = 9.4, 6.3 Hz, 2H), 2.16 (s, 3H), 2.14 (s, 3H), 1.90 (h, J = 7.4 Hz, 2H), 1.03 (d, J = 7.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): δ 155.01, 151.75, 144.78, 144.55, 144.29, 137.77, 134.70, 125.17, 124.95, 124.01, 123.28, 123.04, 122.93, 118.45, 111.98, 52.89, 36.40, 17.36, 13.06, 12.72, 10.49. HRMS (ESI): calcd for C21H23FN6O2S [M + Na]+, 465.1621; found, 465.1484. Purity: 98.84% by HPLC (MeOH/H2O = 80:20, tR = 3.631 min).
N-(4-Fluoro-3-(1H-imidazo[4,5-b]pyridin-7-yl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl) Cyclopropanesulfonamide (28)
59d was prepared from 57 (2.0 g, 7.44 mmol) and 58d (1.15 g, 8.18 mmol) according to general procedure C. Yellow solid (2.58 g, yield: 93.0%). 60d was prepared from 49b (493 mg, 2.09 mmol) and 59d (600 mg, 1.61 mmol) according to general procedure A. Yellow solid (547 mg, yield: 84.5%). 61d was prepared from 51 (328 mg, 1.29 mmol) and 60d (400 mg, 0.99 mmol) according to general procedure B. Light yellow oil (324 mg, yield: 72.5%). 28 was prepared from 42 (102 mg, 0.52 mmol) and 61d (300 mg, 0.67 mmol) according to general procedure A. Yellow solid (176 mg, yield: 59.8%). 1H NMR (300 MHz, DMSO-d6): δ 9.92 (s, 1H), 8.55 (s, 1H), 8.47 (d, J = 5.0 Hz, 1H), 7.63 (s, 1H), 7.39 (dd, J = 5.0, 1.4 Hz, 1H), 7.25 (dd, J = 6.2, 2.8 Hz, 1H), 3.75 (s, 3H), 2.75 (tt, J = 7.2, 5.2 Hz, 1H), 2.23 (s, 3H), 2.14 (s, 3H), 1.00 (dq, J = 4.4, 2.5 Hz, 4H). HRMS (ESI): calcd for C21H21FN6O2S [M + H]+, 441.1464; found, 441.1495. Purity: 98.54% by HPLC (MeOH/H2O = 80:20, tR = 4.971 min).
N-(4-Fluoro-3-(1H-imidazo[4,5-b]pyridin-7-yl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)-1-methyl-1H-pyrazole-4-sulfonamide (29)
59e was prepared from 57 (2.0 g, 7.44 mmol) and 58e (1.48 g, 8.18 mmol) according to general procedure C. Yellow solid (2.79 g, yield: 90.8%). 60e was prepared from 49b (446 mg, 1.89 mmol) and 59e (600 mg, 1.45 mmol) according to general procedure A. Brown solid (542 mg, yield: 84.4%). 61e was prepared from 51 (298.5 mg, 1.18 mmol) and 60e (400 mg, 0.90 mmol) according to general procedure B. Brown oil (324 mg, yield: 73.2%). 29 was prepared from 42 (93 mg, 0.47 mmol) and 61e (300 mg, 0.61 mmol) according to general procedure A. Dark brown solid (182 mg, yield: 61.8%). 1H NMR (300 MHz, DMSO-d6): δ 13.31 (s, 1H), 10.59 (s, 1H), 8.56–8.41 (m, 2H), 7.91 (dd, J = 4.7, 2.3 Hz, 1H), 7.58 (dd, J = 5.9, 2.8 Hz, 1H), 7.33 (dd, J = 5.0, 1.7 Hz, 1H), 7.14 (ddd, J = 15.1, 6.1, 2.8 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 3.93 (s, 3H), 3.73 (s, 3H), 2.15 (s, 3H), 2.05 (d, J = 2.9 Hz, 3H). HRMS (ESI): calcd for C22H21FN8O2S [M + H]+, 481.1526; found, 481.1567. Purity: 97.26% by HPLC (MeOH/H2O = 80:20, tR = 2.999 min).
N-(4-Fluoro-3-(1H-imidazo[4,5-b]pyridin-7-yl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl)pyridine-2-sulfonamide (30)
59f was prepared from 57 (2.0 g, 7.44 mmol) and 58f (1.45 g, 8.18 mmol) according to general procedure C. Yellow solid (2.63 g, yield: 86.3%). 60f was prepared from 49b (449 mg, 1.90 mmol) and 59f (600 mg, 1.46 mmol) according to general procedure A. Brown solid (517 mg, yield: 80.4%). 61f was prepared from 51 (301 mg, 1.20 mmol) and 60f (400 mg, 0.91 mmol) according to general procedure B. Brown oil (342 mg, yield: 77.2%). 30 was prepared from 42 (94 mg, 0.47 mmol) and 61f (300 mg, 0.62 mmol) according to general procedure A. Dark brown solid (158 mg, yield: 53.6%). 1H NMR (300 MHz, DMSO-d6): δ 13.35 (s, 1H), 10.73 (s, 1H), 9.09–8.78 (m, 2H), 8.49 (d, J = 25.0 Hz, 2H), 8.24 (d, J = 7.9 Hz, 1H), 7.81–7.51 (m, 2H), 7.33 (s, 1H), 7.02 (s, 1H), 3.72 (s, 3H), 2.09 (s, 3H), 1.99 (s, 3H). HRMS (ESI): calcd for C23H20FN7O2S [M + H]+, 478.1417; found, 478.1446. Purity: 96.35% by HPLC (MeOH/H2O = 80:20, tR = 5.427 min).
Protein Expression and Purification
The expression and purification of the BRD4 BD1 and BD2 proteins were performed according to our prior report. (68) BRD4 BD1 (K57–E168) and BRD4 BD2 (E352–M457) were expressed from the pET-28b (+) and pET-22b (+) vectors, respectively, in Escherichia coli BL21 (DE3) cells. Protein expression was induced by 1 mM IPTG at 37 °C for 6 h. Cells were collected and then lysed by an ultrasonic cracker in 25 mM Tris buffer containing 300 mM NaCl (pH = 7.4), 0.1% Tween 20, and 0.1% PMSF. The proteins were purified in gradient concentration imidazole buffer using an ÄKTA TM pure 25 (GE Healthcare, Life Sciences) automated purification system. Then, the proteins were further purified on a Superdex 75 column equilibrated with 10 mM HEPES (pH 7.4), 100 mM NaCl, and 5 mM DTT. Last, 15% SDS-PAGE was used for confirming the molecular weight of the purified proteins.
FP-Based Competitive Assay
An FP competition assay was performed to evaluate the inhibition activities of the compounds against BRD4 BD1 and BRD4 BD2. Procedures were performed according to our prior report. (68) Briefly, 5-FAM-labeled (+)-JQ1 was selected as a fluorescent probe. BRD4 BD1 (16 nM), BRD4 BD2 (12 nM), and FAM-(+)-JQ1 (3.7 nM) were diluted to the needed concentrations in the assay buffer. The experiment was implemented in 384-well black flat-bottomed polystyrene plates (Corning #3575). The FP values were detected using a Spectra Max multimode microplate reader (Molecular Devices) with excitation and emission wavelengths at 485 and 535 nm, respectively. For each assay, the polarization value of the blank wells (probe only) was recorded as Pmin; the polarization value of the negative wells (probe and protein) was recorded as Pmax, and the polarization value of the test wells (compound, probe, and protein) was 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 8.0 software. The binding affinity of FAM-(+)-JQ1 to BRD4 BD1 and BRD4 BD2 by BLI is 21.0 and 13.8 nM, respectively. Ki was calculated as previously reported. (69,70)
MST Assay
The binding affinity of compounds for BRD4 BD1 and BRD4 BD2 was tested by MST. BRD4 BD1 and BRD4 BD2 proteins were labeled using the Monolith TM RED-NHS second-generation protein labeling kit (NanoTemper). The labeled proteins were diluted into MST buffer and mixed with different concentrations of compounds at a ratio of 1:1, and the temperature gradient field was set by an infrared laser on the Monolith N.115 (NanoTemper Technologies) analyzer. The movement of molecules in the temperature gradient field was tracked by fluorescent dye labeling, tryptophan autofluorescence, and fluorescent fusion protein signals. The affinity of small molecules to BRD4 BD1 or BRD4 BD2 was analyzed by PR Therm Control software.
NMR-Based CPMG Assay
The NMR-based CPMG assay was applied to confirm BRD4 BD1 and the test compound interactions based on the protocols previously described. (71) The assay was conducted on a Bruker AVANCE III-600 MHz (proton frequency) spectrometer equipped with a cryogenically cooled probe (Bruker BioSpin, Germany) at 25 °C. Samples composed of 200 μM test compound and 10 μM BRD4 BD1 in the assay buffer (2% DMSO in D2O PBS) were used for NMR date collection.
Crystallization and Data Collection
Aliquots of the purified BRD4 BD1 and BRD4 BD1 proteins were set up for crystallization using the vapor diffusion method. The complex structure of BRD4 BD1 or BRD4 BD2 and its ligand was grown at 18 °C in 1 μL of the protein with an equal volume of reservoir solution containing 1.8–3.8 M sodium formate and 10% glycerol. Crystals grew to diffracting quality within 1–3 weeks. Data on cocrystalline structures were collected at 100 K on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF) (Shanghai, China).
Aqueous Solubility Study
The solubility was measured using an HPLC assay. The DDO-8926 stock solution was first prepared and then diluted to 6.86–5000 μM. Standard curves were established by the corresponding absorption peak areas of different concentrations. DDO-8926 was added into 1 mL of buffer solution to prepare a saturated solution at 37 °C. The saturated solution was centrifuged, and the supernatant was taken for HPLC (Shimadzu LC-20AT). The saturated solubility was calculated using the standard curve equation. The absorption peak area was determined using a Shimadzu LC-20AT system.
Simulated Gastric and Intestinal Fluid Stability
The SGF and SIF stabilities were measured according to the previous method. (72) A solution of DDO-8926 (10 μL, 10 mM in DMSO) was added to 990 μL of SGF or SIF. This mixture was incubated at 37 °C. Samples were taken (90 μL) after 0.25, 0.5, 1, 2, 4, 8, and 24 h and analyzed by HPLC (Shimadzu LC-20AT). This assay was carried out in triplicates.
RLM Stability
Procedures were performed according to the previous method. (72) DDO-8926 was preincubated with RLMs (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.
CYP450 Enzyme Inhibition Assay
The assay was conducted in phosphate buffer (100 mM, pH 7.4). For each assay, equal volumes (25 μL) of diluted microsomes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) (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/M (SHIMADZU LCMS-8050) to determine the metabolites. The inhibition rate was calculated using the following equation: inhibition rate (%) = [1 – (formation of the metabolite in the presence of test compound)/(formation of the metabolite of negative control)] × 100%.
Mouse Plasma Protein Binding Rate
The prepared membranes were loaded into the dialysis device, and the device was installed again following manufacturer’s guidelines. The final concentration for the test compound was 2 or 2.5 μM. 50 μL of the spiked plasma solution suspension was immediately transferred to a 96-well plate to act as T = 0 control sample. The samples are treated in the same way as the samples after incubation. At the same time, the remaining spiked plasma solution sample in the plastic plate or a separate plastic tube was incubated for 5 h at 37 °C in a constant temperature shaking box. At T = 5 h, 50 μL of the original spiked plasma solution suspension was transferred to the 96-well plate for analysis. Cells with were loaded with 150 μL of plasma sample and dialyzed against equal volume of PBS. The unit was covered with a gas-permeable lid and incubated for 5 h at 37 °C at 100 rpm in a constant temperature shaking box. At the end of incubation, the lid was removed, and 50 μL of post-dialysis samples was pipetted from both buffer and plasma solution chambers into a separated 96-well plate for analysis. 50 μL of the plasma solution was added to the buffer samples, and an equal volume of PBS was added to the collected plasma solution samples. The plate was shaken at 1000 rpm for 2 min, and 600 μL of 50% ACN/MeOH containing an appropriate internal standard was added to the precipitate protein to release the compound. Then, 100 μL of the supernatant was transferred to new 96-well plates for analysis. 100 μL of distilled water was added to each sample and mixed for analysis by LC–MS/MS.
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. Male SD rats (200–250g) or C57BL/6 mice (18–22 g) (GemPharmatech, Jiangsu, China) were randomly grouped (n = 4 for each group) and administered DDO-8926 intravenously, intraperitoneally, and orally. Blood samples 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. Brain tissue sample was taken at 0.083, 0.25, 0.5, 1.0, 2.0, and 4 h, and a 1 g/mL methanol solution of brain homogenate was prepared to measure the total brain drug levels. The samples were analyzed by LC–MS/MS (SHIMADZU LCMS-8050). The unbound brain drug levels were calculated using the plasma protein unbound factor (fup = 0.01) based on Pollin’s method. (66) The equation for the calculation of fub is as follows: fub = 1/(1 + (((1 – fup)/fub)RAt)), where fub and fup = the fraction unbound in the brain and plasma, respectively; RAt = the ratio of albumin concentration found in the tissue over the plasma and brain, and RAt is 0.50 in Poulin’s method.
Toxicity Evaluation in Mice
All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. Male C57BL/6 mice (18–22 g) were randomly divided into two groups (n = 12). Mice in the administration group were treated with DDO-8926 (500 mg/kg, po, q.d.) for 2 weeks. Mice in the control group were treated (po, q.d.) with a vehicle. Body weight was monitored every other day. At day 14, blood samples from half of the mice were taken into EDTA-K2 anticoagulation tubes (250 μL); these mice were then sacrificed, and the heart, liver, spleen, lung, and kidney organs were collected. After 10 days of withdrawal, blood samples from the other half of the mice were also taken into EDTA-K2 anticoagulation tubes (250 μL). Blood samples in EDTA K2 tubes were subjected to whole blood cell tests by means of an automated blood counter system (ADVIA 2120). The organs were fixed in 4% formalin and embedded in paraffin for sectioning, and the sections were analyzed by H&E staining.
In Vivo Efficacy Evaluation in SNI Mice
All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. Male C57BL/6 mice aged 7–8 weeks and weighed 18–22 g were housed at 25 °C with free access to food and water under 12 h light/12 h dark cycle. Animals were acclimated to experimental environments and experimenters and randomly divided into four groups. The animal were anesthetized with an intraperitoneal injection of 4 mL/kg of a mixture of ketamine (17 mg/mL) and xylazine (2.5 mg/mL) in saline. The right leg from the knee to the hip was shaved. After skin incision, the sciatic nerve was exposed at the mid-thigh level and freed of adhering tissue. Then, three terminal branches were gently separated proximal to the poplitea fossa; at this level, the tibial and common peroneal nerves were tightly ligated with 5–0 silk and sectioned distal to the ligation. The sham procedure consisted of the same surgery without ligation and transection of the nerves. Care was taken to avoid damage of the sural nerve. The shaved skin layer was sutured and disinfected, and the animals were kept in a warm environment until recovery from anesthesia. Mice received DDO-8926 (30 mg/kg, ip) 2 h post-operation and daily onward until specified. The vehicle-treated animals received only the vehicle solution. The positive control group received gabapentin (100 mg/kg, ip) as mentioned above. Mice that received different treatments were mixed and housed together in animal cages. Animal welfare was followed after injury by using protocols held in the animal facility, including disinfection of surgical wounds, weight monitoring, fur appearance.
Behavioral Tests
The experimenter placed each mouse in a transparent plexiglas box (10 × 10 × 13 cm). Mice in sham, SNI, and drug treatment groups were familiarized with the application of Von-Frey Hairs (RWD Life Science Co.) under the paw. Recordings of pain sensitivity were then performed before and after surgery. The plantar side of the paw ipsilateral or contralateral to the surgery was stimulated with calibrated Von Frey monofilaments. Monofilaments were perpendicularly applied to the glabrous skin with sufficient force to cause filament bending. Ten stimuli were made with each of a series of Von Frey Hairs comprising the first 11 monofilaments supplied by the manufacturer (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, 1.4, 2.0, and 4.0 g). The withdrawal threshold was calculated as the average of three trials with time interval in between.
RNA Extraction and qRT-PCR
Animals used for the functional assessment were sacrificed at 15 dpo (n = 6 per group). Then, the spinal cord L5-L6 segments were removed and snap-frozen. Tissue was homogenized with the QIAzol lysis reagent (QIAGEN), and RNA was extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s guidelines. RNA was quantified with a spectrophotometer (Nano-Drop Technologies) and reverse-transcribed using an Applied Biosystems kit (Thermo Fisher Scientific). Then, the expression of target sequences was quantified by qRT-PCR using the SYBR Green QPCR Master Mix (Agilent Technologies) and the corresponding primers.
Immunofluorescence
A 5 mm length of the lumbar spinal cord, containing the L5 segment, was perfused with 4% paraformaldehyde. The tissue was dehydrated, embedded, and sectioned in a cryostat (Leica CM1950, Leica Microsystems). For immunohistochemical staining, the sections were rehydrated in PBS and blocked with 5% normal donkey serum in PBS 0.3% Triton-X100 for 1 h at room temperature. The sections were then incubated overnight at 4 °C with primary antibodies against a neuronal-specific nuclear protein (NeuN) (1:300, Abcam), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, ab178846-Abcam), and glial fibrillary acidic protein (GFAP) (1:500, E4L7M, XP Rabbit mAb, CST). After rinsing in PBS containing 0.01% Triton-X-100, the sections were incubated in secondary antibodies labeled with Alexa Fluor 488 (1:400; Invitrogen) at room temperature for 2 h. Nuclei were labeled with DAPI staining (1:1000; D9564-10MG-Sigma). A LSM 800 laser scanning confocal microscope (Carl Zeiss, Inc Germany) with peak excitation wavelengths of 488 nm and 340 nm was used to acquire representative images with 20× objectives. ZEN software (Carl Zeiss, Inc Germany) was used to analyze the images. ImageJ software was used to determine the number of positive cells after defining a threshold for background correction.
Molecular Docking
Compounds were imported to Schrödinger 2019. a. The crystal structure of BRD4 BD1 was downloaded from the Protein Data Bank (PDB code 3MXF). The protein was prepared by the Protein Preparation Wizard in the Schrödinger module according to the default values. b. The grid file for BRD4 BD1 was generated using the Receptor Grid Generation in the Schrödinger module with default parameters. c. Compounds were prepared using the LigPrep suite with a standard setting. d. The prepared ligands are flexibly docked using the Ligand Docking (Glide) suite with default settings in the standard precision mode. Pymol was applied to display the interaction between the compound and BRD4 BD1.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00372.
Activity results of compounds, bromodomain selectivity of the compound, crystallography information for the complex structures, CYP450 isozyme and hERG ion channel inhibition assay, toxicity evaluation of DDO-8926 in mice, in vivo PK parameters of DDO-8926 in rats, mouse plasma binding rate, and compound structure characterization (PDF)
Molecular formula strings (CSV)
Predicated binding modes of 9 and 15 (ZIP)
Atomic coordinates have been deposited in the Protein Data Bank (PDB code: 8IBQ and 8IDH). Authors will release the atomic coordinates upon article publication.
Terms & Conditions
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- Bing Xiong - Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China; University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China;
https://orcid.org/0000-0001-9776-8136;
- Qidong You - Jiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, 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;
- Zhengyu Jiang - Jiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, 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;
- Xuetao Chen - Jiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China;https://orcid.org/0000-0003-4137-3949
X.C., D.C., and C.L. contributed equally. 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 the National Natural Science Foundation of China (81930100 and 82173680), the Jiangsu Province Funds for Distinguished Young Scientists (grant BK20220087), the Young Elite Scientists Sponsorship Program by CAST (no. YESS20180146, China), the Jiangsu Funding Program for Excellent Postdoctoral Talent (2023ZB486), and supported by the Fundamental Research Funds for the Central Universities (2632023GR14).
| BDs | bromodomain |
| BET | bromodomain and extra-terminal domain |
| CPMG | Carr–Purcell–Meiboom–Gill |
| DEGs | differentially expressed genes |
| FP | fluorescence polarization |
| GO | gene ontology |
| GSEA | gene set enrichment analysis |
| IASP | International Association for the Study of Pain |
| MST | microscale thermophoresis |
| NP | neuropathic pain |
| PK | pharmacokinetic |
| qRT-PCR | quantitative real-time polymerase chain reaction |
| RML | rat liver microsomes |
| SGF | simulated gastric fluid |
| SIF | simulated intestinal fluid |
| SNI | spared nerve injury |
This article references 72 other publications.
- 1Richner, M.; Pallesen, L. T.; Ulrichsen, M.; Poulsen, E. T.; Holm, T. H.; Login, H.; Castonguay, A.; Lorenzo, L. E.; Gonçalves, N. P.; Andersen, O. M.; Lykke-Hartmann, K.; Enghild, J. J.; Rønn, L. C. B.; Malik, I. J.; De Koninck, Y.; Bjerrum, O. J.; Vægter, C. B.; Nykjær, A. Sortilin gates neurotensin and BDNF signaling to control peripheral neuropathic pain. Sci. Adv. 2019, 5, 9946, DOI: 10.1126/sciadv.aav9946Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlaktbrF&md5=160fb0a2d84197e1b356d67450e5f1f5Sortilin gates neurotensin and BDNF signaling to control peripheral neuropathic painRichner, Mette; Pallesen, Lone T.; Ulrichsen, Maj; Poulsen, Ebbe T.; Holm, Thomas H.; Login, Hande; Castonguay, Annie; Lorenzo, Louis-Etienne; Goncalves, Nadia P.; Andersen, Olav M.; Lykke-Hartmann, Karin; Enghild, Jan J.; Roenn, Lars C. B.; Malik, Ibrahim J.; De Koninck, Yves; Bjerrum, Ole J.; Vaegter, Christian B.; Nykjaer, AndersScience Advances (2019), 5 (6), eaav9946CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)Neuropathic pain is a major incurable clin. problem resulting from peripheral nerve trauma or disease. A central mechanism is the reduced expression of the potassium chloride cotransporter 2 (KCC2) in dorsal horn neurons induced by brain-derived neurotrophic factor (BDNF), causing neuronal disinhibition within spinal nociceptive pathways. Here, we demonstrate how neurotensin receptor 2 (NTSR2) signaling impairs BDNF-induced spinal KCC2 down-regulation, showing how these two pathways converge to control the abnormal sensory response following peripheral nerve injury. We establish how sortilin regulates this convergence by scavenging neurotensin from binding to NTSR2, thus modulating its inhibitory effect on BDNF-mediated mech. allodynia. Using sortilindeficient mice or receptor inhibition by antibodies or a small-mol. antagonist, we lastly demonstrate that we are able to fully block BDNF-induced pain and alleviate injury-induced neuropathic pain, validating sortilin as a clin. relevant target.
- 2Wang, Z.; Liu, F.; Wei, M.; Qiu, Y.; Ma, C.; Shen, L.; Huang, Y. Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathway. J. Neuroinflammation 2018, 15, 179, DOI: 10.1186/s12974-018-1215-4Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFWnsrrF&md5=902791b068f987ed7308b57aded1f583Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathwayWang, Zhiyao; Liu, Fan; Wei, Min; Qiu, Yue; Ma, Chao; Shen, Le; Huang, YuguangJournal of Neuroinflammation (2018), 15 (), 179/1-179/12CODEN: JNOEB3; ISSN:1742-2094. (BioMed Central Ltd.)Background: microRNA-146a-5p (miRNA-146a-5p) is a key mol. in the neg. regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain. Methods: The sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quant. real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mech. stimuli and radiant heat were used to evaluate mech. allodynia and thermal hyperalgesia. Results: We found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mech. and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mech. and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further. Conclusions: miRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.
- 3Wang, K.; Wang, S.; Chen, Y.; Wu, D.; Hu, X.; Lu, Y.; Wang, L.; Bao, L.; Li, C.; Zhang, X. Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain. Cell Res. 2021, 31, 904– 918, DOI: 10.1038/s41422-021-00479-9Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXms1GktbY%253D&md5=2cb67ef42ce5b7cbd4faa8381250f0eeSingle-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic painWang, Kaikai; Wang, Sashuang; Chen, Yan; Wu, Dan; Hu, Xinyu; Lu, Yingjin; Wang, Liping; Bao, Lan; Li, Changlin; Zhang, XuCell Research (2021), 31 (8), 904-918CODEN: CREEB6; ISSN:1001-0602. (Nature Portfolio)Peripheral nerve injury could lead to chronic neuropathic pain. Understanding transcriptional changes induced by nerve injury could provide fundamental insights into the complex pathogenesis of neuropathic pain. Gene expression profiles of dorsal root ganglia (DRG) in neuropathic pain condition have been studied. However, little is known about transcriptomic changes in individual DRG neurons after peripheral nerve injury. Here we performed single-cell RNA sequencing on dissocd. mouse DRG cells after spared nerve injury (SNI). In addn. to DRG neuron types that are found under physiol. conditions, we identified three SNI-induced neuronal clusters (SNIICs) characterized by the expression of Atf3/Gfra3/Gal (SNIIC1), Atf3/Mrgprd (SNIIC2) and Atf3/S100b/Gal (SNIIC3). These SNIICs originated from Cldn9+/Gal+, Mrgprd+ and Trappc3l+ DRG neurons, resp. Interestingly, SNIIC2 switched to SNIIC1 by increasing Gal and reducing Mrgprd expression 2 days after nerve injury. Inferring the gene regulatory networks after nerve injury, we revealed that activated transcription factors Atf3 and Egr1 in SNIICs could enhance Gal expression while activated Cpeb1 in SNIIC2 might suppress Mrgprd expression within 2 days after SNI. Furthermore, we mined the transcriptomic changes in the development of neuropathic pain to identify potential analgesic targets. We revealed that cardiotrophin-like cytokine factor 1, which activates astrocytes in the dorsal horn of spinal cord, was upregulated in SNIIC1 neurons and contributed to SNI-induced mech. allodynia. Therefore, our results provide a new landscape to understand the dynamic course of neuron type changes and their underlying mol. mechanisms during the development of neuropathic pain.
- 4Colloca, L.; Ludman, T.; Bouhassira, D.; Baron, R.; Dickenson, A. H.; Yarnitsky, D.; Freeman, R.; Truini, A.; Attal, N.; Finnerup, N. B.; Eccleston, C.; Kalso, E.; Bennett, D. L.; Dworkin, R. H.; Raja, S. N. Neuropathic pain. Nat. Rev. Dis. Prim. 2017, 3, 17002, DOI: 10.1038/nrdp.2017.2Google ScholarThere is no corresponding record for this reference.
- 5Finnerup, N. B.; Attal, N.; Haroutounian, S.; McNicol, E.; Baron, R.; Dworkin, R. H.; Gilron, I.; Haanpaa, M.; Hansson, P.; Jensen, T. S.; Kamerman, P. R.; Lund, K.; Moore, A.; Raja, S. N.; Rice, A. S.; Rowbotham, M.; Sena, E.; Siddall, P.; Smith, B. H.; Wallace, M. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. J. Vasc. Surg. 2015, 62, 1091– 1173, DOI: 10.1016/j.jvs.2015.08.010Google ScholarThere is no corresponding record for this reference.
- 6Mathieson, S.; Lin, C.-W. C.; Underwood, M.; Eldabe, S. Pregabalin and gabapentin for pain. BMJ 2020, 369, m1315, DOI: 10.1136/bmj.m1315Google ScholarThere is no corresponding record for this reference.
- 7Tesfaye, S.; Sloan, G.; Petrie, J.; White, D.; Bradburn, M.; Julious, S.; Rajbhandari, S.; Sharma, S.; Rayman, G.; Gouni, R.; Alam, U.; Cooper, C.; Loban, A.; Sutherland, K.; Glover, R.; Waterhouse, S.; Turton, E.; Horspool, M.; Gandhi, R.; Maguire, D.; Jude, E. B.; Ahmed, S. H.; Vas, P.; Hariman, C.; McDougall, C.; Devers, M.; Tsatlidis, V.; Johnson, M.; Rice, A. S. C.; Bouhassira, D.; Bennett, D. L.; Selvarajah, D.; group, O.-D. t. Comparison of amitriptyline supplemented with pregabalin, pregabalin supplemented with amitriptyline, and duloxetine supplemented with pregabalin for the treatment of diabetic peripheral neuropathic pain (OPTION-DM): a multicentre, double-blind, randomised crossover trial. Lancet 2022, 400, 680– 690, DOI: 10.1016/s0140-6736(22)01472-6Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlCit7nO&md5=0b135694aa3408206fd069978b111399Comparison of amitriptyline supplemented with pregabalin, pregabalin supplemented with amitriptyline, and duloxetine supplemented with pregabalin for the treatment of diabetic peripheral neuropathic pain (OPTION-DM): a multicentre, double-blind, randomised crossover trialTesfaye, Solomon; Sloan, Gordon; Petrie, Jennifer; White, David; Bradburn, Mike; Julious, Stephen; Rajbhandari, Satyan; Sharma, Sanjeev; Rayman, Gerry; Gouni, Ravikanth; Alam, Uazman; Cooper, Cindy; Loban, Amanda; Sutherland, Katie; Glover, Rachel; Waterhouse, Simon; Turton, Emily; Horspool, Michelle; Gandhi, Rajiv; Maguire, Deirdre; Jude, Edward B.; Ahmed, Syed H.; Vas, Prashanth; Hariman, Christian; McDougall, Claire; Devers, Marion; Tsatlidis, Vasileios; Johnson, Martin; Rice, Andrew S. C.; Bouhassira, Didier; Bennett, David L.; Selvarajah, DineshLancet (2022), 400 (10353), 680-690CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)Diabetic peripheral neuropathic pain (DPNP) is common and often distressing. Most guidelines recommend amitriptyline, duloxetine, pregabalin, or gabapentin as initial analgesic treatment for DPNP, but there is little comparative evidence on which one is best or whether they should be combined. We aimed to assess the efficacy and tolerability of different combinations of first-line drugs for treatment of DPNP. OPTION-DM was a multicentre, randomised, double-blind, crossover trial in patients with DPNP with mean daily pain numerical rating scale (NRS) of 4 or higher (scale is 0-10) from 13 UK centers. Participants were randomly assigned (1:1:1:1:1:1), with a predetd. randomisation schedule stratified by site using permuted blocks of size six or 12, to receive one of six ordered sequences of the three treatment pathways: amitriptyline supplemented with pregabalin (A-P), pregabalin supplemented with amitriptyline (P-A), and duloxetine supplemented with pregabalin (D-P), each pathway lasting 16 wk. Monotherapy was given for 6 wk and was supplemented with the combination medication if there was suboptimal pain relief (NRS >3), reflecting current clin. practice. Both treatments were titrated towards max. tolerated dose (75 mg per day for amitriptyline, 120 mg per day for duloxetine, and 600 mg per day for pregabalin). The primary outcome was the difference in 7-day av. daily pain during the final week of each pathway. This trial is registered with ISRCTN, ISRCTN17545443. Between Nov 14, 2017, and July 29, 2019, 252 patients were screened, 140 patients were randomly assigned, and 130 started a treatment pathway (with 84 completing at least two pathways) and were analyzed for the primary outcome. The 7-day av. NRS scores at week 16 decreased from a mean 6·6 (SD 1·5) at baseline to 3·3 (1·8) at week 16 in all three pathways. The mean difference was -0·1 (98·3% CI -0·5 to 0·3) for D-P vs. A-P, -0·1 (-0·5 to 0·3) for P-A vs. A-P, and 0·0 (-0·4 to 0·4) for P-A vs. D-P, and thus not significant. Mean NRS redn. in patients on combination therapy was greater than in those who remained on monotherapy (1·0 [SD 1·3] vs 0·2 [1·5]). Adverse events were predictable for the monotherapies: we obsd. a significant increase in dizziness in the P-A pathway, nausea in the D-P pathway, and dry mouth in the A-P pathway. To our knowledge, this was the largest and longest ever, head-to-head, crossover neuropathic pain trial. We showed that all three treatment pathways and monotherapies had similar analgesic efficacy. Combination treatment was well tolerated and led to improved pain relief in patients with suboptimal pain control with a monotherapy.
- 8Tanabe, M.; Ono, K.; Honda, M.; Ono, H. Gabapentin and pregabalin ameliorate mechanical hypersensitivity after spinal cord injury in mice. Eur. J. Pharmacol. 2009, 609, 65– 68, DOI: 10.1016/j.ejphar.2009.03.020Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkslyht74%253D&md5=690dd4d5fe2caee8086d08ce70c87be5Gabapentin and pregabalin ameliorate mechanical hypersensitivity after spinal cord injury in miceTanabe, Mitsuo; Ono, Koto; Honda, Motoko; Ono, HidekiEuropean Journal of Pharmacology (2009), 609 (1-3), 65-68CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)The antiepileptic drugs gabapentin and pregabalin exhibit well-established analgesic effects in patients with several neuropathic conditions. In the present study, we examd. their effects on mech. hypersensitivity in mice subjected to wt.-drop spinal cord injury. Hindlimb motor function and mech. hypersensitivity were evaluated using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale and the von Frey test, resp., for 4 wk after spinal cord injury. Despite gradual recovery of hindlimb motor function after spinal cord injury, mice exhibited continuous development of mech. hypersensitivity. Gabapentin (30 and 100 mg/kg) and pregabalin (10 and 30 mg/kg), administered i.p. on the 28th day after spinal cord injury, reduced mech. hypersensitivity in a dose-dependent manner. These results suggest that gabapentin and pregabalin could be useful therapeutic tools for patients with neuropathic pain after spinal cord injury.
- 9Kremer, M.; Salvat, E.; Muller, A.; Yalcin, I.; Barrot, M. Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insights. Neuroscience 2016, 338, 183– 206, DOI: 10.1016/j.neuroscience.2016.06.057Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFKktLrK&md5=e654dec57f8820a5a51f15894da8c938Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insightsKremer, Melanie; Salvat, Eric; Muller, Andre; Yalcin, Ipek; Barrot, MichelNeuroscience (Amsterdam, Netherlands) (2016), 338 (), 183-206CODEN: NRSCDN; ISSN:0306-4522. (Elsevier B.V.)Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory system. It is generally chronic and challenging to treat. The recommended pharmacotherapy for neuropathic pain includes the use of some antidepressants, such as tricyclic antidepressants (TCAs) (amitriptyline...) or serotonin and noradrenaline re-uptake inhibitors (duloxetine...), and/or anticonvulsants such as the gabapentinoids gabapentin or pregabalin. Antidepressant drugs are not acute analgesics but require a chronic treatment to relieve neuropathic pain, which suggests the recruitment of secondary downstream mechanisms as well as long-term mol. and neuronal plasticity. Noradrenaline is a major actor for the action of antidepressant drugs in a neuropathic pain context. Mechanistic hypotheses have implied the recruitment of noradrenergic descending pathways as well as the peripheral recruitment of noradrenaline from sympathetic fibers sprouting into dorsal root ganglia; and importance of both α2 and β2 adrenoceptors have been reported. These monoamine re-uptake inhibitors may also indirectly act as anti-proinflammatory cytokine drugs; and their therapeutic action requires the opioid system, particularly the mu (MOP) and/or delta (DOP) opioid receptors. Gabapentinoids, which target the voltage-dependent calcium channels α2δ-1 subunit, inhibit calcium currents, thus decreasing the excitatory transmitter release and spinal sensitization. Gabapentinoids also activate the descending noradrenergic pain inhibitory system coupled to spinal α2 adrenoceptors. Gabapentinoid treatment may also indirectly impact on neuroimmune actors, like proinflammatory cytokines. These drugs are effective against neuropathic pain both with acute administration at high dose and with repeated administration. This review focuses on mechanistic knowledge concerning chronic antidepressant treatment and gabapentinoid treatment in a neuropathic pain context.
- 10Kuehn, B. M. Gabapentin Increasingly Implicated in Overdose Deaths. JAMA 2022, 327, 2387, DOI: 10.1001/jama.2022.10100Google ScholarThere is no corresponding record for this reference.
- 11Anand, P.; Bley, K. Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch. Br. J. Anaesth. 2011, 107, 490– 502, DOI: 10.1093/bja/aer260Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFKiu77M&md5=a0ca0882470e2df01319b1ecb54a72caTopical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patchAnand, P.; Bley, K.British Journal of Anaesthesia (2011), 107 (4), 490-502CODEN: BJANAD; ISSN:0007-0912. (Oxford University Press)A review. Topical capsaicin formulations are used for pain management. Safety and modest efficacy of low-concn. capsaicin formulations, which require repeated daily self-administration, are supported by meta-analyses of numerous studies. A high-concn. capsaicin 8% patch (Qutenza) was recently approved in the EU and USA. A single 60-min application in patients with neuropathic pain produced effective pain relief for up to 12 wk. Advantages of the high-concn. capsaicin patch include longer duration of effect, patient compliance, and low risk for systemic effects or drug-drug interactions. The mechanism of action of topical capsaicin has been ascribed to depletion of substance P. However, exptl. and clin. studies show that depletion of substance P from nociceptors is only a correlate of capsaicin treatment and has little, if any, causative role in pain relief. Rather, topical capsaicin acts in the skin to attenuate cutaneous hypersensitivity and reduce pain by a process best described as defunctionalization' of nociceptor fibers. Defunctionalization is due to a no. of effects that include temporary loss of membrane potential, inability to transport neurotrophic factors leading to altered phenotype, and reversible retraction of epidermal and dermal nerve fiber terminals. Peripheral neuropathic hypersensitivity is mediated by diverse mechanisms, including altered expression of the capsaicin receptor TRPV1 or other key ion channels in affected or intact adjacent peripheral nociceptive nerve fibers, aberrant re-innervation, and collateral sprouting, all of which are defunctionalized by topical capsaicin. Evidence suggests that the utility of topical capsaicin may extend beyond painful peripheral neuropathies.
- 12Romanelli, M. N.; Borgonetti, V.; Galeotti, N. Dual BET/HDAC inhibition to relieve neuropathic pain: Recent advances, perspectives, and future opportunities. Pharmacol. Res. 2021, 173, 105901, DOI: 10.1016/j.phrs.2021.105901Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisF2gsL3N&md5=bfb84167b792d2d6c17deebf97f2b879Dual BET/HDAC inhibition to relieve neuropathic pain: Recent advances, perspectives, and future opportunitiesRomanelli, Maria Novella; Borgonetti, Vittoria; Galeotti, NicolettaPharmacological Research (2021), 173 (), 105901CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)A review. Despite the intense research on developing new therapies for neuropathic pain states, available treatments have limited efficacy and unfavorable safety profiles. Epigenetic alterations have a great influence on the development of cancer and neurol. diseases, as well as neuropathic pain. Histone acetylation has prevailed as one of the well investigated epigenetic modifications in these diseases. Altered spinal activity of histone deacetylase (HDAC) and Bromo and Extra terminal domain (BET) have been described in neuropathic pain models and restoration of these aberrant epigenetic modifications showed pain-relieving activity. Over the last decades HDACs and BETs have been the focus of drug discovery studies, leading to the development of numerous small-mol. inhibitors. Clin. trials to evaluate their anticancer activity showed good efficacy but raised toxicity concerns that limited translation to the clinic. To maximize activity and minimize toxicity, these compds. can be applied in combination of sub-maximal doses to produce additive or synergistic interactions (combination therapy). Recently, of particular interest, dual BET/HDAC inhibitors (multi-target drugs) have been developed to assure simultaneous modulation of BET and HDAC activity by a single mol. This review will summarize the most recent advances with these strategies, describing advantages and limitations of single drug treatment vs combination regimens. This review will also provide a focus on dual BET/HDAC drug discovery investigations as future therapeutic opportunity for human therapy of neuropathic pain.
- 13Mauceri, D. Role of Epigenetic Mechanisms in Chronic Pain. Cells 2022, 11, 2613, DOI: 10.3390/cells11162613Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlSlt7rO&md5=c11bade9fb3025beabae68ee572f64edRole of Epigenetic Mechanisms in Chronic PainMauceri, DanielaCells (2022), 11 (16), 2613CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)Pain is an unpleasant but essential-to-life sensation, usually resulting from tissue damage. When pain persists long after the injury has resolved, it becomes pathol. The precise mol. and cellular mechanisms causing the transition from acute to chronic pain are not fully understood. A key aspect of pain chronicity is that several plasticity events happen along the neural pathways involved in pain. These long-lasting adaptive changes are enabled by alteration in the expression of relevant genes. Among the different modulators of gene transcription in adaptive processes in the nervous system, epigenetic mechanisms play a pivotal role. In this review, I will first outline the main classes of epigenetic mediators and then discuss their implications in chronic pain.
- 14Ghosh, K.; Pan, H. L. Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic Pain. ACS Chem. Neurosci. 2022, 13, 432– 441, DOI: 10.1021/acschemneuro.1c00841Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFyqtbw%253D&md5=67079ccaf63f5fbeaa7b0903ae417539Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic PainGhosh, Krishna; Pan, Hui-LinACS Chemical Neuroscience (2022), 13 (4), 432-441CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)A review. Neuropathic pain is a challenging clin. problem and remains difficult to treat. Altered gene expression in peripheral sensory nerves and neurons by nerve injury is well documented and contributes critically to the synaptic plasticity in the spinal cord and the initiation and maintenance of chronic pain. However, our understanding of the epigenetic mechanisms regulating the transcription of pro-nociceptive (e.g., NMDA receptors and α2δ-1) and antinociceptive (e.g., potassium channels and opioid and cannabinoid receptors) genes are still limited. In this review, we summarize recent studies detg. the roles of histone modifications (including methylation, acetylation, and ubiquitination), DNA methylation, and noncoding RNAs in neuropathic pain development. We review the epigenetic writer, reader, and eraser proteins that participate in the transcriptional control of the expression of key ion channels and neurotransmitter receptors in the dorsal root ganglion after traumatic nerve injury, which is commonly used as a preclin. model of neuropathic pain. A better understanding of epigenetic reprogramming involved in the transition from acute to chronic pain could lead to the development of new treatments for neuropathic pain.
- 15Descalzi, G.; Ikegami, D.; Ushijima, T.; Nestler, E. J.; Zachariou, V.; Narita, M. Epigenetic mechanisms of chronic pain. Trends Neurosci. 2015, 38, 237– 246, DOI: 10.1016/j.tins.2015.02.001Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFGnurY%253D&md5=7002edc8fdae56befdc74cfb986459a9Epigenetic mechanisms of chronic painDescalzi, Giannina; Ikegami, Daigo; Ushijima, Toshikazu; Nestler, Eric J.; Zachariou, Venetia; Narita, MinoruTrends in Neurosciences (2015), 38 (4), 237-246CODEN: TNSCDR; ISSN:0166-2236. (Elsevier Ltd.)Neuropathic and inflammatory pain promote a large no. of persisting adaptations at the cellular and mol. level, allowing even transient tissue or nerve damage to elicit changes in cells that contribute to the development of chronic pain and assocd. symptoms. There is evidence that injury-induced changes in chromatin structure drive stable changes in gene expression and neural function, which may cause several symptoms, including allodynia, hyperalgesia, anxiety, and depression. Recent findings on epigenetic changes in the spinal cord and brain during chronic pain may guide fundamental advances in new treatments. Here, we provide a brief overview of epigenetic regulation in the nervous system and then discuss the still-limited literature that directly implicates epigenetic modifications in chronic pain syndromes.
- 16Ghosh, K.; Pan, H.-L. Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic Pain. ACS Chem. Neurosci. 2022, 13, 432– 441, DOI: 10.1021/acschemneuro.1c00841Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFyqtbw%253D&md5=67079ccaf63f5fbeaa7b0903ae417539Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic PainGhosh, Krishna; Pan, Hui-LinACS Chemical Neuroscience (2022), 13 (4), 432-441CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)A review. Neuropathic pain is a challenging clin. problem and remains difficult to treat. Altered gene expression in peripheral sensory nerves and neurons by nerve injury is well documented and contributes critically to the synaptic plasticity in the spinal cord and the initiation and maintenance of chronic pain. However, our understanding of the epigenetic mechanisms regulating the transcription of pro-nociceptive (e.g., NMDA receptors and α2δ-1) and antinociceptive (e.g., potassium channels and opioid and cannabinoid receptors) genes are still limited. In this review, we summarize recent studies detg. the roles of histone modifications (including methylation, acetylation, and ubiquitination), DNA methylation, and noncoding RNAs in neuropathic pain development. We review the epigenetic writer, reader, and eraser proteins that participate in the transcriptional control of the expression of key ion channels and neurotransmitter receptors in the dorsal root ganglion after traumatic nerve injury, which is commonly used as a preclin. model of neuropathic pain. A better understanding of epigenetic reprogramming involved in the transition from acute to chronic pain could lead to the development of new treatments for neuropathic pain.
- 17Spering, M.; Carrasco, M. Acting without seeing: eye movements reveal visual processing without awareness. Trends Neurosci. 2015, 38, 247– 258, DOI: 10.1016/j.tins.2015.02.002Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjt1aqsrs%253D&md5=bda910509011a55b3a1a3c3d9d10f51aActing without seeing: eye movements reveal visual processing without awarenessSpering, Miriam; Carrasco, MarisaTrends in Neurosciences (2015), 38 (4), 247-258CODEN: TNSCDR; ISSN:0166-2236. (Elsevier Ltd.)Visual perception and eye movements are considered to be tightly linked. Diverse fields, ranging from developmental psychol. to computer science, utilize eye tracking to measure visual perception. However, this prevailing view has been challenged by recent behavioral studies. Here, we review converging evidence revealing dissocns. between the contents of perceptual awareness and different types of eye movement. Such dissocns. reveal situations in which eye movements are sensitive to particular visual features that fail to modulate perceptual reports. We also discuss neurophysiol., neuroimaging, and clin. studies supporting the role of subcortical pathways for visual processing without awareness. Our review links awareness to perceptual-eye movement dissocns. and furthers our understanding of the brain pathways underlying vision and movement with and without awareness.
- 18Ligon, C. O.; Moloney, R. D.; Greenwood-Van Meerveld, B. Targeting Epigenetic Mechanisms for Chronic Pain: A Valid Approach for the Development of Novel Therapeutics. J. Pharmacol. Exp. Ther. 2016, 357, 84– 93, DOI: 10.1124/jpet.115.231670Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XpvVeksLs%253D&md5=a962565b89196b152e4fcd57ed7b784eTargeting epigenetic mechanisms for chronic pain: a valid approach for the development of novel therapeuticsLigon, Casey O.; Moloney, Rachel D.; Greenwood-Van Meerveld, BeverleyJournal of Pharmacology and Experimental Therapeutics (2016), 357 (1), 84-93CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)Chronic pain is a multifaceted and complex condition. Broadly classified into somatic, visceral, or neuropathic pain, it is poorly managed despite its prevalence. Current drugs used for the treatment of chronic pain are limited by tolerance with long-term use, abuse potential, and multiple adverse side effects. The persistent nature of pain suggests that epigenetic machinery may be a crit. factor driving chronic pain. In this review, we discuss the latest insights into epigenetic processes, including DNA methylation, histone modifications, and microRNAs, and we describe their involvement in the pathophysiol. of chronic pain and whether epigenetic modifications could be applied as future therapeutic targets for chronic pain. We provide evidence from exptl. models and translational research in human tissue that have enhanced our understanding of epigenetic processes mediating nociception, and we then speculate on the potential future use of more specific and selective agents that target epigenetic mechanisms to attenuate pain.
- 19Odell, D. W. Epigenetics of pain mediators. Curr. Opin. Anaesthesiol. 2018, 31, 402– 406, DOI: 10.1097/aco.0000000000000613Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MbhvValug%253D%253D&md5=9b241240be57e986799a3aa35fe63770Epigenetics of pain mediatorsOdell Daniel WCurrent opinion in anaesthesiology (2018), 31 (4), 402-406 ISSN:.PURPOSE OF REVIEW: The field of epigenetics continues its influential rise as a means to better understand an organism's unique developmental identity over a lifespan. Whereas a genome is constant and unchanging, an epigenome is dynamic and alterable. Epigenetic changes are in response to innumerable internal and external influences including environmental changes such as diet, exercise, disease, toxins, and stress. Epigenetics is of particular interest in the medical research community both for the potential to cause disease and as a target for therapeutic interventions. This article provides a succinct explanation of the potential for epigenetics to influence the understanding of pain as well as a review of relevant research on the topic. RECENT FINDINGS: Studies on epigenetics and pain remain largely preclinical and investigate the theoretical ability of epigenetics to alter the nociceptive pathways both in the periphery and centrally. Significant evidence now exists for the ability of epigenetics to modify broadly categorized pain types, including inflammatory, neuropathic, visceral, and cancer related. SUMMARY: Both patients and providers recognize that novel medications for the treatment of both acute and chronic pain conditions are sorely needed. The understanding of epigenetics and its influence on nociception remains in relative infancy but early evidence is strong for potential therapeutic benefits to treat these conditions.
- 20Denk, F.; McMahon, S. B. Chronic pain: emerging evidence for the involvement of epigenetics. Neuron 2012, 73, 435– 444, DOI: 10.1016/j.neuron.2012.01.012Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitFOjtLw%253D&md5=3570ae634b3d1ddced5616599f57aaa8Chronic Pain: Emerging Evidence for the Involvement of EpigeneticsDenk, Franziska; McMahon, Stephen B.Neuron (2012), 73 (3), 435-444CODEN: NERNET; ISSN:0896-6273. (Cell Press)A review. Epigenetic processes, such as histone modifications and DNA methylation, have been assocd. with many neural functions including synaptic plasticity, learning, and memory. Here, we critically examine emerging evidence linking epigenetic mechanisms to the development or maintenance of chronic pain states. Although in its infancy, research in this area potentially unifies several pathophysiol. processes underpinning abnormal pain processing and opens up a different avenue for the development of novel analgesics.
- 21Sun, C.; An, Q.; Li, R.; Chen, S.; Gu, X.; An, S.; Wang, Z. Calcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic pain. CNS Neurosci. Ther. 2021, 27, 1409– 1424, DOI: 10.1111/cns.13720Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XpvVSnsQ%253D%253D&md5=3c179a50b0cded1236c3b67ebae5548cCalcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic painSun, Chenyan; An, Qi; Li, Ruidi; Chen, Shuhui; Gu, Xinpei; An, Shuhong; Wang, ZhaojinCNS Neuroscience & Therapeutics (2021), 27 (11), 1409-1424CODEN: CNTNAB; ISSN:1755-5930. (Wiley-Blackwell)Calcitonin gene-related peptide (CGRP) as a regulator of astrocyte activation may facilitate spinal nociceptive processing. Histone H3 lysine 9 acetylation (H3K9ac) is considered an important regulator of cytokine and chemokine gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K9ac in the activation of astrocytes, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. Astroglial cells (C6) were treated with CGRP and differentially enrichments of H3K9ac on gene promoters were examd. using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on astrocyte activation and H3K9ac signaling in CCI-induced neuropathic pain. Specific inhibitors were employed to delineate the involved signaling. Intrathecal injection of CGRP and CCI increased the no. of astrocytes displaying H3K9ac in the spinal dorsal horn of rats. Treatment of CGRP was able to up-regulate H3K9ac and glial fibrillary acidic protein (GFAP) expression in astroglial cells. ChIP-seq data indicated that CGRP significantly altered H3K9ac enrichments on gene promoters in astroglial cells following CGRP treatment, including 151 gaining H3K9ac and 111 losing this mark, which mostly enriched in proliferation, autophagy, and macrophage chemotaxis processes. qRT-PCR verified expressions of representative candidate genes (ATG12, ATG4C, CX3CR1, MMP28, MTMR14, HMOX1, RET) and RTCA verified astrocyte proliferation. Addnl., CGRP treatment increased the expression of H3K9ac, CX3CR1, and IL-1β in the spinal dorsal horn. CGRP antagonist and HAT inhibitor attenuated mech. and thermal hyperalgesia in CCI rats. Such analgesic effects were concurrently assocd. with the reduced levels of H3K9ac, CX3CR1, and IL-1β in the spinal dorsal horn of CCI rats. Our findings highly indicate that CGRP is assocd. with the development of neuropathic pain through astrocytes-mediated neuroinflammatory responses via H3K9ac in spinal dorsa horn following nerve injury. This study found that CGRP act on their astrocytic receptors and lead to H3K9 acetylation (H3K9ac), which are mainly assocd. with proliferation-, autophagy-, and inflammation-related gene expression. The no. of astrocytes with H3K9ac expression is increased after nerve injury. Inhibition of CGRP attenuates the development of neuropathic pain, which was accompanied by the suppression of H3K9ac, CX3CR1, and IL-1β expression in CCI rats.
- 22Wang, J.; Chen, J.; Jin, H.; Lin, D.; Chen, Y.; Chen, X.; Wang, B.; Hu, S.; Wu, Y.; Wu, Y.; Zhou, Y.; Tian, N.; Gao, W.; Wang, X.; Zhang, X. BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in rats. J. Cell Mol. Med. 2019, 23, 3214– 3223, DOI: 10.1111/jcmm.14196Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXosF2mtrk%253D&md5=7d45d05ede6e1d90e2150df5cd796228BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in ratsWang, Jianle; Chen, Jiaoxiang; Jin, Haiming; Lin, Dongdong; Chen, Yu; Chen, Ximiao; Wang, Ben; Hu, Sunli; Wu, Yan; Wu, Yaosen; Zhou, Yifei; Tian, Naifeng; Gao, Weiyang; Wang, Xiangyang; Zhang, XiaoleiJournal of Cellular and Molecular Medicine (2019), 23 (5), 3214-3223CODEN: JCMMC9; ISSN:1582-4934. (Wiley-Blackwell)The pathophysiol. of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro-inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF-κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro-inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro-inflammatory cytokine prodn. in microglia which ultimately promotes functional recovery after SCI.
- 23Li, Y.; Xiang, J.; Zhang, J.; Lin, J.; Wu, Y.; Wang, X. Inhibition of Brd4 by JQ1 Promotes Functional Recovery From Spinal Cord Injury by Activating Autophagy. Front. Cell. Neurosci. 2020, 14, 555591, DOI: 10.3389/fncel.2020.555591Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisF2gu7s%253D&md5=b1ba672645e6013ed780830b7b319711Inhibition of Brd4 by JQ1 promotes functional recovery from spinal cord injury by activating autophagyLi, Yao; Xiang, Jie; Zhang, Jing; Lin, Jiahao; Wu, Yaosen; Wang, XiangyangFrontiers in Cellular Neuroscience (2020), 14 (), 555591CODEN: FCNRAH; ISSN:1662-5102. (Frontiers Media S.A.)Spinal cord injury (SCI) is a destructive neurol. disorder that is characterized by impaired sensory and motor function. Inhibition of bromodomain protein 4 (Brd4) has been shown to promote the maintenance of cell homeostasis by activating autophagy. However, the role of Brd4 inhibition in SCI and the underlying mechanisms are poorly understood. Thus, the goal of the present study was to evaluate the effects of sustained Brd4 inhibition using the bromodomain and extraterminal domain (BET) inhibitor JQ1 on the regulation of apoptosis, oxidative stress and autophagy in a mouse model of SCI. First, we obsd. that Brd4 expression at the lesion sites of mouse spinal cords increased after SCI. Treatment with JQ1 significantly decreased the expression of Brd4 and improved functional recovery for up to 28 day after SCI. In addn., JQ1-mediated inhibition of Brd4 reduced oxidative stress and inhibited the expression of apoptotic proteins to promote neural survival. Our results also revealed that JQ1 treatment activated autophagy and restored autophagic flux, while the pos. effects of JQ1 were abrogated by autophagy inhibitor 3-MA intervention, indicating that autophagy plays a crucial role in therapeutic effects Brd4 induced by inhibition of the functional recovery SCI. In the mechanistic anal., we obsd. that modulation of the AMPK-mTORULK1 pathway is involved in the activation of autophagy mediated by Brd4 inhibition. Taken together, the results of our investigation provides compelling evidence that Brd4 inhibition by JQ1 promotes functional recovery after SCI and that Brd4 may serve as a potential target for SCI treatment.
- 24Rudman, M. D.; Choi, J. S.; Lee, H. E.; Tan, S. K.; Ayad, N. G.; Lee, J. K. Bromodomain and extraterminal domain-containing protein inhibition attenuates acute inflammation after spinal cord injury. Exp. Neurol. 2018, 309, 181– 192, DOI: 10.1016/j.expneurol.2018.08.005Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsF2gur3P&md5=72e3d3bfcad4101012dc35edc101342cBromodomain and extraterminal domain-containing protein inhibition attenuates acute inflammation after spinal cord injuryRudman, Michelle D.; Choi, James S.; Lee, Ha Eun; Tan, Sze Kiat; Ayad, Nagi G.; Lee, Jae K.Experimental Neurology (2018), 309 (), 181-192CODEN: EXNEAC; ISSN:0014-4886. (Elsevier Inc.)Inflammation is a major contributor to the secondary damage that occurs after spinal cord injury (SCI). The inflammatory response is coordinated by many different signaling modalities including the epigenetic modification of promoters and enhancers. Bromodomain and extraterminal domain-contg. proteins (BETs; Brd2, Brd3, Brd4, BrdT) are epigenetic readers that bind acetylated histones to promote transcription of pro-inflammatory genes. BET inhibition is anti-inflammatory in animal models of cancer, rheumatoid arthritis, and coronary artery disease. However, the role of BETs in neuroinflammation remains largely unexplored. In this study, we investigated the role of BETs in promoting inflammation in neural cells and the ability of the BET inhibitor JQ1 to decrease inflammation acutely after SCI. Expression of BET mRNA was assessed via qPCR in purified primary mouse macrophages, astrocytes, neurons, oligodendrocytes, and microglia, as well as in naive, sham-injured, and contusion-injured mouse spinal cord. Brd2, Brd3, and Brd4 mRNA were expressed in all purified primary neural cells and in the uninjured and injured mouse spinal cord. BET inhibition significantly attenuated proinflammatory signaling in all activated cell populations in vitro. To investigate the effects of BET modulation after SCI, the BET inhibitor JQ1 was injected i.p. (30 mg/kg, bidaily) 3 h after spinal cord contusion in adult female C57BL/6 mice. By 3 days post-injury, BET inhibition significantly decreased pro-inflammatory cytokine expression and leukocyte recruitment to the injury site. However, this decrease did not lead to locomotor improvements or smaller lesion size. Taken together, our data implicate BETs as regulators of multiple key pro-inflammatory cytokines, and suggest that BETs can be pharmacol. inhibited to reduce inflammation acutely after SCI.
- 25Ferri, E.; Petosa, C.; McKenna, C. E. Bromodomains: Structure, function and pharmacology of inhibition. Biochem. Pharmacol. 2016, 106, 1– 18, DOI: 10.1016/j.bcp.2015.12.005Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGrt7%252FM&md5=a3f075bad4b77cc890fcb708490f4c9dBromodomains: Structure, function and pharmacology of inhibitionFerri, Elena; Petosa, Carlo; McKenna, Charles E.Biochemical Pharmacology (Amsterdam, Netherlands) (2016), 106 (), 1-18CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)Bromodomains are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation. The human proteome comprises 46 bromodomain-contg. proteins with a total of 61 bromodomains, which, despite highly conserved structural features, recognize a wide array of natural peptide ligands. Over the past five years, bromodomains have attracted great interest as promising new epigenetic targets for diverse human diseases, including inflammation, cancer, and cardiovascular disease. The demonstration in 2010 that two small mol. compds., JQ1 and I-BET762, potently inhibit proteins of the bromodomain and extra-terminal (BET) family with translational potential for cancer and inflammatory disease sparked intense efforts in academia and pharmaceutical industry to develop novel bromodomain antagonists for therapeutic applications. Several BET inhibitors are already in clin. trials for hematol. malignancies, solid tumors and cardiovascular disease. Currently, the field faces the challenge of single-target selectivity, esp. within the BET family, and of overcoming problems related to the development of drug resistance. At the same time, new trends in bromodomain inhibitor research are emerging, including an increased interest in non-BET bromodomains and a focus on drug synergy with established antitumor agents to improve chemotherapeutic efficacy. This review presents an updated view of the structure and function of bromodomains, traces the development of bromodomain inhibitors and their potential therapeutic applications, and surveys the current challenges and future directions of this vibrant new field in drug discovery.
- 26Takahashi, K.; Yi, H.; Liu, C.-H.; Liu, S.; Kashiwagi, Y.; Patin, D. J.; Hao, S. Spinal bromodomain-containing protein 4 contributes to neuropathic pain induced by HIV glycoprotein 120 with morphine in rats. Neuroreport 2018, 29, 441– 446, DOI: 10.1097/wnr.0000000000000992Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlvVKnt7w%253D&md5=52aaa5455a044daa0ce449e82b1986b4Spinal bromodomain-containing protein 4 contributes to neuropathic pain induced by HIV glycoprotein 120 with morphine in ratsTakahashi, Keiya; Yi, Hyun; Liu, Ching-Hang; Liu, Shue; Kashiwagi, Yuta; Patin, Dennis J.; Hao, ShuanglinNeuroReport (2018), 29 (6), 441-446CODEN: NERPEZ; ISSN:0959-4965. (Lippincott Williams & Wilkins)The symptoms of HIV-sensory neuropathy are dominated by neuropathic pain. Recent data show that repeated use of opiates enhances the chronic pain states in HIV patients. Limited attention has so far been devoted to exploring the exact pathogenesis of HIV painful disorder and opiate abuse in vivo, for which there is no effective treatment. Bromodomain-contg. protein 4 (Brd4) is a member of the bromodomain and extraterminal domain protein (BET) family and functions as a chromatin 'reader' that binds acetylated lysines in histones in brain neurons to mediate the transcriptional regulation underlying learning and memory. Here, we established a neuropathic pain model of interaction of intrathecal HIV envelope glycoprotein 120 (gp120) and chronic morphine in rats. The combination of gp120 and morphine (gp120/M, for 5 days) induced persistent mech. allodynia compared with either gp120 or morphine alone. Mech. allodynia reached the lowest values at day 10 from gp120/M application, beginning to recover from day 21. In the model, gp120/M induced overexpression of Brd4 mRNA and protein at day 10 using RT-qPCR and western blots, resp. Immunohistochem. studies showed that Brd4 at day 10 was expressed in the neurons of spinal cord dorsal horn. BET inhibitor I-BET762 dose-dependently increased the mech. threshold in the gp120/M pain state. The present study provides preclin. evidence for treating HIV neuropathic pain with opioids using the BET inhibitor.
- 27Palomes-Borrajo, G.; Badia, J.; Navarro, X.; Penas, C. Nerve Excitability and Neuropathic Pain is Reduced by BET Protein Inhibition After Spared Nerve Injury. J. Pain 2021, 22, 1617– 1630, DOI: 10.1016/j.jpain.2021.05.005Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XmslGgtg%253D%253D&md5=8ad7d5c77a6c757a79e51e6e6e3f8d4bNerve Excitability and Neuropathic Pain is Reduced by BET Protein Inhibition After Spared Nerve InjuryPalomes-Borrajo, Georgina; Badia, Jordi; Navarro, Xavier; Penas, ClaraJournal of Pain (2021), 22 (12), 1617-1630CODEN: JPOAB5; ISSN:1526-5900. (Elsevier Inc.)Neuropathic pain is a common disability produced by enhanced neuronal excitability after nervous system injury. The pathophysiol. changes that underlie the generation and maintenance of neuropathic pain require modifications of transcriptional programs. In particular, there is an induction of pro-inflammatory neuromodulators levels, and changes in the expression of ion channels and other factors intervening in the detn. of the membrane potential in neuronal cells. We have previously found that inhibition of the BET proteins epigenetic readers reduced neuroinflammation after spinal cord injury. Within the present study we aimed to det. if BET protein inhibition may also affect neuroinflammation after a peripheral nerve injury, and if this would beneficially alter neuronal excitability and neuropathic pain. For this purpose, C57BL/6 female mice underwent spared nerve injury (SNI), and were treated with the BET inhibitor JQ1, or vehicle. Electrophysiol. and algesimetry tests were performed on these mice. We also detd. the effects of JQ1 treatment after injury on neuroinflammation, and the expression of neuronal components important for the maintenance of axon membrane potential. We found that treatment with JQ1 affected neuronal excitability and mech. hyperalgesia after SNI in mice. BET protein inhibition regulated cytokine expression and reduced microglial reactivity after injury. In addn., JQ1 treatment altered the expression of SCN3A, SCN9A, KCNA1, KCNQ2, KCNQ3, HCN1 and HCN2 ion channels, as well as the expression of the Na+/K+ ATPase pump subunits. In conclusion, both, alteration of inflammation, and neuronal transcription, could be the responsible epigenetic mechanisms for the redn. of excitability and hyperalgesia obsd. after BET inhibition. Inhibition of BET proteins is a promising therapy for reducing neuropathic pain after neural injury.
- 28Vasavda, C.; Xu, R.; Liew, J.; Kothari, R.; Dhindsa, R. S.; Semenza, E. R.; Paul, B. D.; Green, D. P.; Sabbagh, M. F.; Shin, J. Y.; Yang, W.; Snowman, A. M.; Albacarys, L. K.; Moghekar, A.; Pardo-Villamizar, C. A.; Luciano, M.; Huang, J.; Bettegowda, C.; Kwatra, S. G.; Dong, X.; Lim, M.; Snyder, S. H. Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain. Sci. Adv. 2022, 8, eabo5633 DOI: 10.1126/sciadv.abo5633Google ScholarThere is no corresponding record for this reference.
- 29Borgonetti, V.; Galeotti, N. Combined inhibition of histone deacetylases and BET family proteins as epigenetic therapy for nerve injury-induced neuropathic pain. Pharmacol. Res. 2021, 165, 105431, DOI: 10.1016/j.phrs.2021.105431Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmvVKksbc%253D&md5=7a8db7ff3af849ab329f0da8b25f5990Combined inhibition of histone deacetylases and BET family proteins as epigenetic therapy for nerve injury-induced neuropathic painBorgonetti, Vittoria; Galeotti, NicolettaPharmacological Research (2021), 165 (), 105431CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)Current treatments for neuropathic pain have often moderate efficacy and present unwanted effects showing the need to develop effective therapies. Accumulating evidence suggests that histone acetylation plays essential roles in chronic pain and the analgesic activity of histone deacetylases (HDACs) inhibitors is documented. Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that interact with acetylated lysine residues on histones, but little is known about their implication in neuropathic pain. Thus, the current study was aimed to investigate the effect of the combination of HDAC and BET inhibitors in the spared nerve injury (SNI) model in mice. Intranasal administration of i-BET762 (BET inhibitor) or SAHA (HDAC inhibitor) attenuated thermal and mech. hypersensitivity and this antiallodynic activity was improved by co-administration of both drugs. Spinal cord sections of SNI mice showed an increased expression of HDAC1 and Brd4 proteins and combination produced a stronger redection compared to each epigenetic agent alone. SAHA and i-BET762, administered alone or in combination, counteracted the SNI-induced microglia activation by inhibiting the expression of IBA1, CD11b, inducible nitric oxide synthase (iNOS), the activation of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription-1 (STAT1) with comparable efficacy. Conversely, the epigenetic inhibitors showed a modest effect on spinal proinflammatory cytokines content that was significantly potentiated by their combination. Present results indicate a key role of acetylated histones and their recruitment by BET proteins on microglia-mediated spinal neuroinflammation. Targeting neuropathic pain with the combination of HDAC and BET inhibitors may represent a promising new therapeutic option.
- 30Borgonetti, V.; Meacci, E.; Pierucci, F.; Romanelli, M. N.; Galeotti, N. Dual HDAC/BRD4 Inhibitors Relieves Neuropathic Pain by Attenuating Inflammatory Response in Microglia After Spared Nerve Injury. Neurotherapeutics 2022, 19, 1634– 1648, DOI: 10.1007/s13311-022-01243-6Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvVSmsbnN&md5=71d3a419e62de54d4ff5fc4a39a5aa7bDual HDAC/BRD4 Inhibitors Relieves Neuropathic Pain by Attenuating Inflammatory Response in Microglia After Spared Nerve InjuryBorgonetti, Vittoria; Meacci, Elisabetta; Pierucci, Federica; Romanelli, Maria Novella; Galeotti, NicolettaNeurotherapeutics (2022), 19 (5), 1634-1648CODEN: NEURNV; ISSN:1878-7479. (Springer)Despite the effort on developing new treatments, therapy for neuropathic pain is still a clin. challenge and combination therapy regimes of two or more drugs are often needed to improve efficacy. Accumulating evidence shows an altered expression and activity of histone acetylation enzymes in chronic pain conditions and restoration of these aberrant epigenetic modifications promotes pain-relieving activity. Recent studies showed a synergistic activity in neuropathic pain models by combination of histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) inhibitors. On these premises, the present study investigated the pharmacol. profile of new dual HDAC/BRD4 inhibitors, named SUM52 and SUM35, in the spared nerve injury (SNI) model in mice as innovative strategy to simultaneously inhibit HDACs and BETs. Intranasal administration of SUM52 and SUM35 attenuated thermal and mech. hypersensitivity in the absence of locomotor side effects. Both dual inhibitors showed a preferential interaction with BRD4-BD2 domain, and SUM52 resulted the most active compd. SUM52 reduced microglia-mediated spinal neuroinflammation in spinal cord sections of SNI mice as showed by redn. of IBA1 immunostaining, inducible nitric oxide synthase (iNOS) expression, p65 nuclear factor-κB (NF-κB) and p38 MAPK over-phosphorylation. A robust decrease of the spinal proinflammatory cytokines content (IL-6, IL-1ss) was also obsd. after SUM52 treatment. Present results, showing the pain-relieving activity of HDAC/BRD4 dual inhibitors, indicate that the simultaneous modulation of BET and HDAC activity by a single mol. acting as multi-target agent might represent a promise for neuropathic pain relief.
- 31Liu, Z.; Chen, H.; Wang, P.; Li, Y.; Wold, E. A.; Leonard, P. G.; Joseph, S.; Brasier, A. R.; Tian, B.; Zhou, J. Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation. J. Med. Chem. 2020, 63, 5242– 5256, DOI: 10.1021/acs.jmedchem.0c00035Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVOhsbs%253D&md5=95cb6c3f150a5ee9e65a76fd36a559fdDiscovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological EvaluationLiu, Zhiqing; Chen, Haiying; Wang, Pingyuan; Li, Yi; Wold, Eric A.; Leonard, Paul G.; Joseph, Sarah; Brasier, Allan R.; Tian, Bing; Zhou, JiaJournal of Medicinal Chemistry (2020), 63 (10), 5242-5256CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Arylquinazolinones and arylchromenones such as I (X = CH2, MeN) were prepd. as selective inhibitors of bromodomain-contg. protein 4 (BRD4) for potential use as orally bioavailable antiinflammatory agents. I inhibited BRD4 with IC50 values of 67-84 nM and were selective for BRD1 over binding domains of BRD2, BRD3, and BRDT and over CBP; I inhibited the expression of Toll-like receptor (TLR3)-induced inflammatory genes in vitro and inhibited airway inflammation in mice. The pharmacokinetics (t1/2, AUC, Cmax, and clearance), metabolic stability of I (X = NMe) in murine and human cells, and inhibition of cytochrome P450 enzymes and hERG by I (X = MeN) were detd. The structure of I (X = NMe) bound to human BRD4 binding domain 1 was detd. by X-ray crystallog.
- 32Hu, J.; Tian, C. Q.; Damaneh, M. S.; Li, Y.; Cao, D.; Lv, K.; Yu, T.; Meng, T.; Chen, D.; Wang, X.; Chen, L.; Li, J.; Song, S. S.; Huan, X. J.; Qin, L.; Shen, J.; Wang, Y. Q.; Miao, Z. H.; Xiong, B. Structure-Based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein Inhibitors. J. Med. Chem. 2019, 62, 8642– 8663, DOI: 10.1021/acs.jmedchem.9b01094Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslegsbvK&md5=af9fa23078f8280cc36d3c097d6fbf34Structure-Based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein InhibitorsHu, Jianping; Tian, Chang-Qing; Damaneh, Mohammadali Soleimani; Li, Yanlian; Cao, Danyan; Lv, Kaikai; Yu, Ting; Meng, Tao; Chen, Danqi; Wang, Xin; Chen, Lin; Li, Jian; Song, Shan-Shan; Huan, Xia-Juan; Qin, Lihuai; Shen, Jingkang; Wang, Ying-Qing; Miao, Ze-Hong; Xiong, BingJournal of Medicinal Chemistry (2019), 62 (18), 8642-8663CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)BRD4 has recently emerged as a promising drug target. Therefore, identifying novel inhibitors with distinct properties could enrich their use in anticancer treatment. Guided by the cocrystal structure of hit compd. 4 harboring a five-membered-ring linker motif, we quickly identified lead compd. 7, which exhibited good antitumor effects in an MM.1S xenograft model by oral administration. Encouraged by its high potency and interesting scaffold, we performed further lead optimization to generate a novel potent series of bromodomain and extra-terminal (BET) inhibitors with a (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one structure. Among them, compd. 19 was found to have the best balance of activity, stability, and antitumor efficacy. After confirming its low brain penetration, we conducted comprehensive preclin. studies, including a multiple-species pharmacokinetics profile, extensive cellular mechanism studies, hERG assay, and in vivo antitumor growth effect testing, and we found that compd. 19 is a potential BET protein drug candidate for the treatment of cancer.
- 33Zhang, M.; Zhang, Y.; Song, M.; Xue, X.; Wang, J.; Wang, C.; Zhang, C.; Li, C.; Xiang, Q.; Zou, L.; Wu, X.; Wu, C.; Dong, B.; Xue, W.; Zhou, Y.; Chen, H.; Wu, D.; Ding, K.; Xu, Y. Structure-Based Discovery and Optimization of Benzo[ d]isoxazole Derivatives as Potent and Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC). J. Med. Chem. 2018, 61, 3037– 3058, DOI: 10.1021/acs.jmedchem.8b00103Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXls12rs78%253D&md5=2dbbfc3d1916482c817ad13cd3ef9edaStructure-Based Discovery and Optimization of Benzo[d]isoxazole Derivatives as Potent and Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC)Zhang, Maofeng; Zhang, Yan; Song, Ming; Xue, Xiaoqian; Wang, Junjian; Wang, Chao; Zhang, Cheng; Li, Chenchang; Xiang, Qiuping; Zou, Lingjiao; Wu, Xishan; Wu, Chun; Dong, Baijun; Xue, Wei; Zhou, Yulai; Chen, Hongwu; Wu, Donghai; Ding, Ke; Xu, YongJournal of Medicinal Chemistry (2018), 61 (7), 3037-3058CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The bromodomain and extra-terminal (BET) family proteins have gained increasing interest as drug targets for treatment of castration-resistant prostate cancer (CRPC). Here, the authors describe the design, optimization, and evaluation of benzo[d]isoxazole-contg. compds. as potent BET bromodomain inhibitors. Cocrystal structures of the representative inhibitors in complex with BRD4(1) provided solid structural basis for compd. optimization. The two most potent compds., 6i (Y06036) and 7m (Y06137), bound to the BRD4(1) bromodomain with Kd values of 82 and 81 nM, resp. They also exhibited high selectivity over other non-BET subfamily members. The compds. potently inhibited cell growth, colony formation, and the expression of AR, AR regulated genes, and MYC in prostate cancer cell lines. Compds. 6i and 7m also demonstrated therapeutic effects in a C4-2B CRPC xenograft tumor model in mice. These potent and selective BET inhibitors represent a new class of compds. for the development of potential therapeutics against CRPC.
- 34Huang, Y.; Liu, N.; Pan, Z.; Li, Z.; Sheng, C. BET-HDAC Dual Inhibitors for Combinational Treatment of Breast Cancer and Concurrent Candidiasis. J. Med. Chem. 2023, 66, 1239– 1253, DOI: 10.1021/acs.jmedchem.2c01191Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXlsValsg%253D%253D&md5=e4118948754bbbc36d4981d1d521b3d8BET-HDAC Dual Inhibitors for Combinational Treatment of Breast Cancer and Concurrent CandidiasisHuang, Yahui; Liu, Na; Pan, Zhizhi; Li, Zhuang; Sheng, ChunquanJournal of Medicinal Chemistry (2023), 66 (2), 1239-1253CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Breast cancer is susceptible to Candida infections, and candidiasis has an enhancing effect on the progression and metastasis of tumor. Breast cancer and concurrent candidiasis represent a significant challenge in clin. therapy. Herein, a series of novel small mol. inhibitors simultaneously targeting bromodomain and extra-terminal (BET) and histone deacetylase (HDAC) were designed for combinational treatment of breast cancer and resistant Candida albicans infections. Among them, compds. 13c I and 17b II exhibited excellent and balanced inhibitory activity against both BET family proteins BRD4 and HDAC1. As compared with BRD4 or HDAC1 inhibitors, dual inhibitors 13c I and 17b II displayed improved in vivo antitumor efficacy in MDA-MB-231 breast cancer xenograft models. Notably, they synergized with fluconazole (FLC) to effectively reduce the kidney fungal burden in a murine model of disseminated candidiasis. Thus, the BET-HDAC dual inhibitors represented a novel therapeutic strategy for combinational treatment of breast cancer and concurrent candidiasis.
- 35Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J. P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Muller, S.; Pawson, T.; Gingras, A. C.; Arrowsmith, C. H.; Knapp, S. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 2012, 149, 214– 231, DOI: 10.1016/j.cell.2012.02.013Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltVamurs%253D&md5=05583a01a1cd4c3b59fc59c00e4c4777Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain FamilyFilippakopoulos, Panagis; Picaud, Sarah; Mangos, Maria; Keates, Tracy; Lambert, Jean-Philippe; Barsyte-Lovejoy, Dalia; Felletar, Ildiko; Volkmer, Rudolf; Muller, Susanne; Pawson, Tony; Gingras, Anne-Claude; Arrowsmith, Cheryl H.; Knapp, StefanCell (Cambridge, MA, United States) (2012), 149 (1), 214-231CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resoln. crystal structures, covering all BRD families. These proteins are: ASH1L, ATAD2, BAZ2B, BPTF, BRD1, BRD3(1), BRD3(2), BRD4(1), BRD4(2), BRD9, BRDT(1), CECR2, EP300, CREBBP, GCN5L2, KIAA1240, PB1(1), PB1(2), PB1(3), PB1(4), PB1(5), PB1(6), PCAF, PHIP(2), TAF1(2), WDR9(2), BRD4(1). Comprehensive crossfamily structural anal. identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-contg. peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.
- 36Zhao, Y.; Bai, L.; Liu, L.; McEachern, D.; Stuckey, J. A.; Meagher, J. L.; Yang, C. Y.; Ran, X.; Zhou, B.; Hu, Y.; Li, X.; Wen, B.; Zhao, T.; Li, S.; Sun, D.; Wang, S. Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethy lisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain Inhibitor. J. Med. Chem. 2017, 60, 3887– 3901, DOI: 10.1021/acs.jmedchem.7b00193Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVKru7s%253D&md5=915019e36ab5f5b83b69b1bbc291abb0Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain InhibitorZhao, Yujun; Bai, Longchuan; Liu, Liu; McEachern, Donna; Stuckey, Jeanne A.; Meagher, Jennifer L.; Yang, Chao-Yie; Ran, Xu; Zhou, Bing; Hu, Yang; Li, Xiaoqin; Wen, Bo; Zhao, Ting; Li, Siwei; Sun, Duxin; Wang, ShaomengJournal of Medicinal Chemistry (2017), 60 (9), 3887-3901CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 9H-pyrimido[4,5-b]indole-contg. compds. was designed and synthesized to obtain potent and orally bioavailable BET inhibitors. By incorporation of an indole or a quinoline moiety to the 9H-pyrimido[4,5-b]indole core, we identified a series of small mols. showing high binding affinities to BET proteins and low nanomolar potencies in inhibition of cell growth in acute leukemia cell lines. One such compd., 4-(6-methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (I) has excellent microsomal stability and good oral pharmacokinetics in rats and mice. Orally administered, I achieves significant antitumor activity in the MV4;11 leukemia and MDA-MB-231 triple-neg. breast cancer xenograft models in mice. Detn. of the cocrystal structure of I with BRD4 BD2 provides a structural basis for its high binding affinity to BET proteins. Testing its binding affinities against other bromodomain-contg. proteins shows that I is a highly selective inhibitor of BET proteins. These data show that I is a potent, selective, and orally active BET inhibitor.
- 37He, S.; Dong, G.; Li, Y.; Wu, S.; Wang, W.; Sheng, C. Potent Dual BET/HDAC Inhibitors for Efficient Treatment of Pancreatic Cancer. Angew. Chem., Int. Ed. 2020, 59, 3028, DOI: 10.1002/anie.201915896Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFegtLw%253D&md5=78db62cb533611939d9abd0e654ff5f3Potent Dual BET/HDAC Inhibitors for Efficient Treatment of Pancreatic CancerHe, Shipeng; Dong, Guoqiang; Li, Yu; Wu, Shanchao; Wang, Wei; Sheng, ChunquanAngewandte Chemie, International Edition (2020), 59 (8), 3028-3032CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)As one of the most aggressive and lethal human malignancies with extremely poor prognosis, there is an urgent demand of more effective therapy for the treatment of pancreatic cancer. Reported here is a new, effective therapeutic strategy and the design of small-mol. inhibitors that simultaneously target bromodomain and extra-terminal (BET) and histone deacetylase (HDAC), potentially serving as promising therapeutic agents for pancreatic cancer. A highly potent dual inhibitor (13 a) is identified to possess excellent and balanced activities against BRD4 BD1 (IC50=11 nM) and HDAC1 (IC50=21 nM). Notably, this compd. shows higher in vitro and in vivo antitumor potency than the BET inhibitor (+)-JQ1 and the HDAC inhibitor vorinostat, either alone or and in combination, highlighting the advantages of BET/HDAC dual inhibitors for more effective treatment of pancreatic cancer.
- 38Liu, Z.; Li, Y.; Chen, H.; Lai, H.-T.; Wang, P.; Wu, S.-Y.; Wold, E. A.; Leonard, P. G.; Joseph, S.; Hu, H.; Chiang, C.-M.; Brasier, A. R.; Tian, B.; Zhou, J. Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site. J. Med. Chem. 2022, 65, 2388– 2408, DOI: 10.1021/acs.jmedchem.1c01851Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFOjtw%253D%253D&md5=b21df874c6323304ccceea1b265711e5Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding SiteLiu, Zhiqing; Li, Yi; Chen, Haiying; Lai, Hsien-Tsung; Wang, Pingyuan; Wu, Shwu-Yuan; Wold, Eric A.; Leonard, Paul G.; Joseph, Sarah; Hu, Haitao; Chiang, Cheng-Ming; Brasier, Allan R.; Tian, Bing; Zhou, JiaJournal of Medicinal Chemistry (2022), 65 (3), 2388-2408CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Bromodomain-contg. protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this crit. system and the progress toward clin. development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural anal. of ZL0590 in complex with human BRD4 BD1 and the assocd. mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biol. underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.
- 39Zeng, L.; Zhou, M.-M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 2002, 513, 124– 128, DOI: 10.1016/s0014-5793(01)03309-9Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitFejtbo%253D&md5=79070a63cd171f846ab577aa9f7092bbBromodomain: an acetyl-lysine binding domainZeng, Lei; Zhou, Ming-MingFEBS Letters (2002), 513 (1), 124-128CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)A review with 43 refs. Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins assocd. with chromatin and in nearly all nuclear histone acetyltransferases, have been recently discovered to function as acetyllysine-binding domains. More recent structural studies of bromodomain/peptide ligand complexes have enriched the understanding of differences in ligand selectivity of bromodomains. These new findings demonstrate that bromodomain/acetyllysine recognition can serve as a pivotal mechanism for regulating protein-protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation, and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site.
- 40Liu, Z.; Wang, P.; Chen, H.; Wold, E. A.; Tian, B.; Brasier, A. R.; Zhou, J. Drug Discovery Targeting Bromodomain-Containing Protein 4. J. Med. Chem. 2017, 60, 4533– 4558, DOI: 10.1021/acs.jmedchem.6b01761Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXis1yiurg%253D&md5=6d7ba5844073fac8c916b1249af402e5Drug Discovery Targeting Bromodomain-Containing Protein 4Liu, Zhiqing; Wang, Pingyuan; Chen, Haiying; Wold, Eric A.; Tian, Bing; Brasier, Allan R.; Zhou, JiaJournal of Medicinal Chemistry (2017), 60 (11), 4533-4558CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. BRD4, the most extensively studied member of BET family, is an epigenetic regulator that localizes to DNA via binding acetylated histones and controls the expression of therapeutically important gene regulatory networks through recruiting transcription factors to form mediator complexes, phosphorylating RNA polymerase II and by its intrinsic histone acetyltransferase activity. Disrupting the protein-protein interactions between BRD4 and acetyl-lysine has been shown to effectively block cell proliferation in cancer, cytokine prodn. in acute inflammation, etc. To date, significant efforts have been devoted to the development of BRD4 inhibitors, and consequently, a dozen have progressed into human clin. trials. Herein, the authors summarize the advances in drug discovery and development of BRD4 inhibitors by focusing on their chemotypes, in vitro and in vivo activity, selectivity, relevant mechanisms of action and therapeutic potential. Opportunities and challenges to achieve selective and efficacious BRD4 inhibitors as a viable therapeutic strategy for human diseases are also highlighted.
- 41Tang, P.; Zhang, J.; Liu, J.; Chiang, C. M.; Ouyang, L. Targeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological Development. J. Med. Chem. 2021, 64, 2419– 2435, DOI: 10.1021/acs.jmedchem.0c01487Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXksFGmsr8%253D&md5=f63a3dad4fb1fab28fe245da72c2fa1cTargeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological DevelopmentTang, Pan; Zhang, Jifa; Liu, Jie; Chiang, Cheng-Ming; Ouyang, LiangJournal of Medicinal Chemistry (2021), 64 (5), 2419-2435CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Bromodomain and extraterminal (BET) proteins bind acetylated lysine residues in histones and nonhistone proteins via tandem bromodomains and regulate chromatin dynamics, cellular processes, and disease procession. Thus targeting BET proteins is a promising strategy for treating various diseases, esp. malignant tumors and chronic inflammation. Many pan-BET small-mol. inhibitors have been described, and some of them are in clin. evaluation. Nevertheless, the limited clin. efficacy of the current BET inhibitors is also evident and has inspired the development of new technologies to improve their clin. outcomes and minimize unwanted side effects. In this Review, we summarize the latest protein characteristics and biol. functions of BRD4 as an example of BET proteins, analyze the clin. development status and preclin. resistance mechanisms, and discuss recent advances in BRD4-selective inhibitors, dual-target BET inhibitors, proteolysis targeting chimera degraders, and protein-protein interaction inhibitors.
- 42Cui, H.; Carlson, A. S.; Schleiff, M. A.; Divakaran, A.; Johnson, J. A.; Buchholz, C. R.; Zahid, H.; Vail, N. R.; Shi, K.; Aihara, H.; Harki, D. A.; Miller, G. P.; Topczewski, J. J.; Pomerantz, W. C. K. 4-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved Selectivity. J. Med. Chem. 2021, 64, 10497– 10511, DOI: 10.1021/acs.jmedchem.1c00933Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1WmtbnK&md5=6dfafd833706b5a418e5998f73c2eb944-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved SelectivityCui, Huarui; Carlson, Angela S.; Schleiff, Mary A.; Divakaran, Anand; Johnson, Jorden A.; Buchholz, Caroline R.; Zahid, Huda; Vail, Nora R.; Shi, Ke; Aihara, Hideki; Harki, Daniel A.; Miller, Grover P.; Topczewski, Joseph J.; Pomerantz, William C. K.Journal of Medicinal Chemistry (2021), 64 (14), 10497-10511CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein-protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3-triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure-activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, resp. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 (I) and 26 (II) are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.
- 43Chen, J.; Tang, P.; Wang, Y.; Wang, J.; Yang, C.; Li, Y.; Yang, G.; Wu, F.; Zhang, J.; Ouyang, L. Targeting Bromodomain-Selective Inhibitors of BET Proteins in Drug Discovery and Development. J. Med. Chem. 2022, 65, 5184– 5211, DOI: 10.1021/acs.jmedchem.1c01835Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XnvVKhsr0%253D&md5=bd2bb412aad23fa184778d41fc957a21Targeting Bromodomain-Selective Inhibitors of BET Proteins in Drug Discovery and DevelopmentChen, Juncheng; Tang, Pan; Wang, Yuxi; Wang, Jiaxing; Yang, Chengcan; Li, Yang; Yang, Gaoxia; Wu, Fengbo; Zhang, Jifa; Ouyang, LiangJournal of Medicinal Chemistry (2022), 65 (7), 5184-5211CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Blocking the interactions between bromodomain and extraterminal (BET) proteins and acetylated lysines of histones by small mols. has important implications for the treatment of cancers and other diseases. Many pan-BET inhibitors have shown satisfactory results in clin. trials, but their potential for poor tolerability and toxicity persist. However, recently reported studies illustrate that some BET bromodomain (BET-BD1 or BET-BD2)-selective inhibitors have advantage over pan-inhibitors, including reduced toxicity concerns. Furthermore, some selective BET inhibitors have similar or even better therapeutic efficacy in inflammatory diseases or cancers. Therefore, the development of selective BET inhibitors has become a hot spot for medicinal chemists. Here, we summarize the known selective BET-BD1 and BET-BD2 inhibitors and review the methods for enhancing the selectivity and potency of these inhibitors based on their different modes of interactions with BET-BD1 or BET-BD2. Finally, we discuss prospective strategies that selectively target the bromodomains of BET proteins.
- 44Liang, D.; Yu, Y.; Ma, Z. Novel strategies targeting bromodomain-containing protein 4 (BRD4) for cancer drug discovery. Eur. J. Med. Chem. 2020, 200, 112426, DOI: 10.1016/j.ejmech.2020.112426Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtV2ktLvL&md5=dd01654a37d6e9045f92caf772a08df3Novel strategies targeting bromodomain-containing protein 4 (BRD4) for cancer drug discoveryLiang, Dailin; Yu, Yifan; Ma, ZonghuiEuropean Journal of Medicinal Chemistry (2020), 200 (), 112426CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)A review. As epigenetic readers of the histone code, BRD4 is the most extensively and thoroughly studied member of BET family, which plays a crit. role in many human diseases including cancer, inflammation, HIV infections, CNS disorders, and cardiovascular diseases and has been proved to be a promising therapeutic target for these diseases. To date, many small-mol. BRD4 inhibitors have been discovered, and some of them are in clin. trials for the treatment of different diseases. Due to the lack of selectivity of these small mols. for BRD4 BD1, BRD4 BD2 and/or other BET proteins, they exert some toxic side effects, including dizziness, nausea, and vomit. Now, novel strategies are urgent needed to improve the selectivity and reduce the side effects of current BRD4 inhibitors. Herein, in this article, we made a summary of the recent development of novel strategies targeting BRD4. Opportunities for these strategies to achieve selective and efficacious BRD4 inhibitors for treating human diseases are also highlighted.
- 45Hajmirza, A.; Emadali, A.; Gauthier, A.; Casasnovas, O.; Gressin, R.; Callanan, M. B. BET Family Protein BRD4: An Emerging Actor in NFκB Signaling in Inflammation and Cancer. Biomedicines 2018, 6, 16, DOI: 10.3390/biomedicines6010016Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjslGqsLo%253D&md5=5ee66c89aa4f18d643cd13a43e1ad7e3BET family protein BRD4: an emerging actor in NFκB signaling in inflammation and cancerHajmirza, Azadeh; Emadali, Anouk; Gauthier, Arnaud; Casasnovas, Olivier; Gressin, Remy; Callanan, Mary B.Biomedicines (2018), 6 (1), 16/1-16/9CODEN: BIOMID; ISSN:2227-9059. (MDPI AG)NFκB (Nuclear Factor-κ-light-chain-enhancer of activated B cells) signaling elicits global transcriptional changes by activating cognate promoters and through genome-wide remodeling of cognate regulatory elements called "super enhancers". BET (Bromodomain and Extra-Terminal domain) protein family inhibitor studies have implicated BET protein member BRD4 and possibly other BET proteins in NFκB-dependent promoter and super-enhancer modulation. Members of the BET protein family are known to bind acetylated chromatin to facilitate access by transcriptional regulators to chromatin, as well as to assist the activity of transcription elongation complexes via CDK9/pTEFb. BET family member BRD4 has been shown to bind non-histone proteins and modulate their activity. One such protein is RELA, the NFκB co-activator. Specifically, BRD4 binds acetylated RELA, which increases its transcriptional transactivation activity and stability in the nucleus. In aggregate, this establishes an intimate link between NFκB and BET signaling, at least via BRD4. The present review provides a brief overview of the structure and function of BET family proteins and then examines the connections between NFκB and BRD4 signaling, using the inflammatory response and cancer cell signaling as study models. We also discuss the potential of BET inhibitors for relief of aberrant NFκB signaling in cancer, focusing on non-histone, acetyl-lysine binding functions.
- 46Chiang, C. M. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4. F1000 Biol. Rep. 2009, 1, 98, DOI: 10.3410/b1-98Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3MrlvVygtQ%253D%253D&md5=d575a30cf8b3d918367d6580c2c0d64aBrd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4Chiang Cheng-MingF1000 biology reports (2009), 1 (), 98 ISSN:.Bromodomain-containing protein 4 (Brd4) contains two tandem bromodomains (BD1 and BD2) that bind preferentially to acetylated lysine residues found in histones and nonhistone proteins. This molecular recognition allows Brd4 to associate with acetylated chromatin throughout the cell cycle and regulates transcription at targeted loci. Recruitment of positive transcription elongation factor b, and possibly the general initiation cofactor Mediator as well, plays an important role in Brd4-regulated transcription. Selective contacts with acetyl-lysines in nucleosomal histones and chromatin-binding factors likely provide a molecular switch modulating the steps from chromatin targeting to transcriptional regulation, thus further expanding the 'acetylation code' for combinatorial regulation in eukaryotes.
- 47Duan, Y.; Guan, Y.; Qin, W.; Zhai, X.; Yu, B.; Liu, H. Targeting Brd4 for cancer therapy: inhibitors and degraders. Medchemcomm 2018, 9, 1779– 1802, DOI: 10.1039/c8md00198gGoogle Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVGisbrP&md5=b244437266ec6a0d3398bf63897beca4Targeting Brd4 for cancer therapy: inhibitors and degradersDuan, Yingchao; Guan, Yuanyuan; Qin, Wenping; Zhai, Xiaoyu; Yu, Bin; Liu, HongminMedChemComm (2018), 9 (11), 1779-1802CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Bromodomain-contg. protein 4 (Brd4) plays an important role in mediating the expression of genes involved in cancers and non-cancer diseases such as inflammatory diseases and acute heart failure. Inactivating Brd4 or downregulating its expression inhibits cancer development, leading to the current interest in Brd4 as a promising anticancer drug target. Numerous Brd4 inhibitors have been studied in recent years and some of them are currently in various phases of clin. trials. Recently, selective degrdn. of target proteins by small bifunctional mols. (PROTACs) has emerged as an attractive drug discovery approach owing to the advantages it could offer over traditional small-mol. inhibitors. A no. of Brd4 degraders have been reported and showed more efficient anticancer activities than just protein inhibition. In this review, we will discuss recent findings in the discovery and development of small-mol. inhibitors and degraders that target Brd4 as a potential anticancer agent.
- 48Xue, X.; Zhang, Y.; Wang, C.; Zhang, M.; Xiang, Q.; Wang, J.; Wang, A.; Li, C.; Zhang, C.; Zou, L.; Wang, R.; Wu, S.; Lu, Y.; Chen, H.; Ding, K.; Li, G.; Xu, Y. Benzoxazinone-containing 3,5-dimethylisoxazole derivatives as BET bromodomain inhibitors for treatment of castration-resistant prostate cancer. Eur. J. Med. Chem. 2018, 152, 542– 559, DOI: 10.1016/j.ejmech.2018.04.034Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVShurjM&md5=dc7275ea2931fa922f265aa03a6d8449Benzoxazinone-containing 3,5-dimethylisoxazole derivatives as BET bromodomain inhibitors for treatment of castration-resistant prostate cancerXue, Xiaoqian; Zhang, Yan; Wang, Chao; Zhang, Maofeng; Xiang, Qiuping; Wang, Junjian; Wang, Anhui; Li, Chenchang; Zhang, Cheng; Zou, Lingjiao; Wang, Rui; Wu, Shuang; Lu, Yongzhi; Chen, Hongwu; Ding, Ke; Li, Guohui; Xu, YongEuropean Journal of Medicinal Chemistry (2018), 152 (), 542-559CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Design, synthesis and evaluation of a new series of benzoxazinone-contg. 3,5-dimethylisoxazole derivs. I (R1 = n-Bu, CH2-cyclopropyl, Bn, etc.) as selective BET inhibitors was reported. One of the new compds., (R)-I (R1 = cyclopropylmethyl), binds to BRD4(1) with a Kd value of 110 nM and blocks bromodomain and acetyl lysine interactions with an IC50 value of 100 nM. It also exhibits selectivity for BET over non-BET bromodomain proteins and demonstrates reasonable anti-proliferation and colony formation inhibition effect in prostate cancer cell lines such as 22Rv1 and C4-2B. The BRD4 inhibitor (R)-I (R1 = cyclopropylmethyl) also significantly suppresses the expression of ERG, Myc and AR target gene PSA at the mRNA level in prostate cancer cells. Treatment with (R)-I (R1 = cyclopropylmethyl) significantly suppresses the tumor growth of prostate cancer (TGI=70%) in a 22Rv1-derived xenograft model. These data suggest that (R)-I (R1 = cyclopropylmethyl) is a promising lead compd. for the development of a new class of therapeutics for the treatment of CRPC.
- 49Miyoshi, S.; Ooike, S.; Iwata, K.; Hikawa, H.; Sugahara, K. Antitumor agent. WO 2009084693 A1, 2009.Google ScholarThere is no corresponding record for this reference.
- 50Cochran, A. G.; Conery, A. R.; Sims, R. J. Bromodomains: a new target class for drug development. Nat. Rev. Drug Discovery 2019, 18, 609– 628, DOI: 10.1038/s41573-019-0030-7Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlWitbnL&md5=88e56eec8edeb7c325d28156ec131ed8Bromodomains: a new target class for drug developmentCochran, Andrea G.; Conery, Andrew R.; Sims, III, Robert J.Nature Reviews Drug Discovery (2019), 18 (8), 609-628CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-mol. targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chem. probe discovery to understand the biol. potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compds. have since advanced to human clin. trials. Here, we review the current state of BET inhibitor biol. in relation to clin. development, and we discuss the next wave of bromodomain inhibitors with clin. potential in oncol. and non-oncol. indications. The lessons learned from BET inhibitor programs should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.
- 51Coudé, M.-M.; Braun, T.; Berrou, J.; Dupont, M.; Bertrand, S.; Masse, A.; Raffoux, E.; Itzykson, R.; Delord, M.; Riveiro, M. E.; Herait, P.; Baruchel, A.; Dombret, H.; Gardin, C. BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells. Oncotarget 2015, 6, 17698– 17712, DOI: 10.18632/oncotarget.4131Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MflsVChsA%253D%253D&md5=0db953a0a24b12ec2918a02d1a2df00bBET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cellsCoude Marie-Magdelaine; Braun Thorsten; Berrou Jeannig; Dupont Melanie; Bertrand Sibyl; Masse Aline; Raffoux Emmanuel; Itzykson Raphael; Baruchel Andre; Dombret Herve; Gardin Claude; Coude Marie-Magdelaine; Braun Thorsten; Gardin Claude; Raffoux Emmanuel; Itzykson Raphael; Dombret Herve; Delord Marc; Riveiro Maria E; Herait Patrice; Baruchel AndreOncotarget (2015), 6 (19), 17698-712 ISSN:.The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.
- 52Vázquez, R.; Riveiro, M. E.; Astorgues-Xerri, L.; Odore, E.; Rezai, K.; Erba, E.; Panini, N.; Rinaldi, A.; Kwee, I.; Beltrame, L.; Bekradda, M.; Cvitkovic, E.; Bertoni, F.; Frapolli, R.; D’Incalci, M. The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus. Oncotarget 2016, 8, 7598– 7613, DOI: 10.18632/oncotarget.13814Google ScholarThere is no corresponding record for this reference.
- 53Albrecht, B. K.; Gehling, V. S.; Hewitt, M. C.; Vaswani, R. G.; Cote, A.; Leblanc, Y.; Nasveschuk, C. G.; Bellon, S.; Bergeron, L.; Campbell, R.; Cantone, N.; Cooper, M. R.; Cummings, R. T.; Jayaram, H.; Joshi, S.; Mertz, J. A.; Neiss, A.; Normant, E.; O’Meara, M.; Pardo, E.; Poy, F.; Sandy, P.; Supko, J.; Sims, R. J., 3rd; Harmange, J. C.; Taylor, A. M.; Audia, J. E. Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials. J. Med. Chem. 2016, 59, 1330– 1339, DOI: 10.1021/acs.jmedchem.5b01882Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFKjs7g%253D&md5=5e1c5aab26aa58844a9f6670a7bbd78bIdentification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical TrialsAlbrecht, Brian K.; Gehling, Victor S.; Hewitt, Michael C.; Vaswani, Rishi G.; Cote, Alexandre; Leblanc, Yves; Nasveschuk, Christopher G.; Bellon, Steve; Bergeron, Louise; Campbell, Robert; Cantone, Nico; Cooper, Michael R.; Cummings, Richard T.; Jayaram, Hariharan; Joshi, Shivangi; Mertz, Jennifer A.; Neiss, Adrianne; Normant, Emmanuel; O'Meara, Michael; Pardo, Eneida; Poy, Florence; Sandy, Peter; Supko, Jeffrey; Sims, Robert J.; Harmange, Jean-Christophe; Taylor, Alexander M.; Audia, James E.Journal of Medicinal Chemistry (2016), 59 (4), 1330-1339CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clin. trials for hematol. malignancies (CPI-0610).
- 54Siu, K. T.; Eda, H.; Santo, L.; Ramachandran, J.; Koulnis, M.; Mertz, J.; Sims, R. J.; Cooper, M.; Raje, N. S. Effect of the BET Inhibitor, Cpi-0610, Alone and in Combination with Lenalidomide in Multiple Myeloma. Blood 2015, 126, 4255, DOI: 10.1182/blood.v126.23.4255.4255Google ScholarThere is no corresponding record for this reference.
- 55Gavai, A. V.; Norris, D.; Delucca, G.; Tortolani, D.; Tokarski, J. S.; Dodd, D.; O’Malley, D.; Zhao, Y.; Quesnelle, C.; Gill, P.; Vaccaro, W.; Huynh, T.; Ahuja, V.; Han, W.-C.; Mussari, C.; Harikrishnan, L.; Kamau, M.; Poss, M.; Sheriff, S.; Yan, C.; Marsilio, F.; Menard, K.; Wen, M.-L.; Rampulla, R.; Wu, D.-R.; Li, J.; Zhang, H.; Li, P.; Sun, D.; Yip, H.; Traeger, S. C.; Zhang, Y.; Mathur, A.; Zhang, H.; Huang, C.; Yang, Z.; Ranasinghe, A.; Everlof, G.; Raghavan, N.; Tye, C. K.; Wee, S.; Hunt, J. T.; Vite, G.; Westhouse, R.; Lee, F. Y. Discovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug Design. J. Med. Chem. 2021, 64, 14247– 14265, DOI: 10.1021/acs.jmedchem.1c00625Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVCrtr3E&md5=bb9d5466a765ae78c4ca72b329a0495aDiscovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug DesignGavai, Ashvinikumar V.; Norris, Derek; Delucca, George; Tortolani, David; Tokarski, John S.; Dodd, Dharmpal; O'Malley, Daniel; Zhao, Yufen; Quesnelle, Claude; Gill, Patrice; Vaccaro, Wayne; Huynh, Tram; Ahuja, Vijay; Han, Wen-Ching; Mussari, Christopher; Harikrishnan, Lalgudi; Kamau, Muthoni; Poss, Michael; Sheriff, Steven; Yan, Chunhong; Marsilio, Frank; Menard, Krista; Wen, Mei-Li; Rampulla, Richard; Wu, Dauh-Rurng; Li, Jianqing; Zhang, Huiping; Li, Peng; Sun, Dawn; Yip, Henry; Traeger, Sarah C.; Zhang, Yingru; Mathur, Arvind; Zhang, Haiying; Huang, Christine; Yang, Zheng; Ranasinghe, Asoka; Everlof, Gerry; Raghavan, Nirmala; Tye, Ching Kim; Wee, Susan; Hunt, John T.; Vite, Gregory; Westhouse, Richard; Lee, Francis Y.Journal of Medicinal Chemistry (2021), 64 (19), 14247-14265CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an addnl. lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) (I) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematol. and solid tumor models, BMS-986158 was selected as a candidate for clin. evaluation.
- 56Yin, M.; Guo, Y.; Hu, R.; Cai, W. L.; Li, Y.; Pei, S.; Sun, H.; Peng, C.; Li, J.; Ye, R.; Yang, Q.; Wang, N.; Tao, Y.; Chen, X.; Yan, Q. Potent BRD4 inhibitor suppresses cancer cell-macrophage interaction. Nat. Commun. 2020, 11, 1833, DOI: 10.1038/s41467-020-15290-0Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntFOiu70%253D&md5=20b5ac123381a3ca75164a5e53a64c18Potent BRD4 inhibitor suppresses cancer cell-macrophage interactionYin, Mingzhu; Guo, Ying; Hu, Rui; Cai, Wesley L.; Li, Yao; Pei, Shiyao; Sun, Hongyin; Peng, Cong; Li, Jiali; Ye, Rui; Yang, Qiaohong; Wang, Nenghui; Tao, Yongguang; Chen, Xiang; Yan, QinNature Communications (2020), 11 (1), 1833CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Small mol. inhibitor of the bromodomain and extraterminal domain (BET) family proteins is a promising option for cancer treatment. However, current BET inhibitors are limited by their potency or oral bioavailability. Here we report the discovery and characterization of NHWD-870, a BET inhibitor that is more potent than three major clin. stage BET inhibitors BMS-986158, OTX-015, and GSK-525762. NHWD-870 causes tumor shrinkage or significantly suppresses tumor growth in nine xenograft or syngeneic models. In addn. to its ability to downregulate c-MYC and directly inhibit tumor cell proliferation, NHWD-870 blocks the proliferation of tumor assocd. macrophages (TAMs) through multiple mechanisms, partly by reducing the expression and secretion of macrophage colony-stimulating factor CSF1 by tumor cells. NHWD-870 inhibits CSF1 expression through suppressing BRD4 and its target HIF1α. Taken together, these results reveal a mechanism by which BRD4 inhibition suppresses tumor growth, and support further development of NHWD-870 to treat solid tumors.
- 57McDaniel, K. F.; Wang, L.; Soltwedel, T.; Fidanze, S. D.; Hasvold, L. A.; Liu, D.; Mantei, R. A.; Pratt, J. K.; Sheppard, G. S.; Bui, M. H.; Faivre, E. J.; Huang, X.; Li, L.; Lin, X.; Wang, R.; Warder, S. E.; Wilcox, D.; Albert, D. H.; Magoc, T. J.; Rajaraman, G.; Park, C. H.; Hutchins, C. W.; Shen, J. J.; Edalji, R. P.; Sun, C. C.; Martin, R.; Gao, W.; Wong, S.; Fang, G.; Elmore, S. W.; Shen, Y.; Kati, W. M. Discovery of N-(4-(2,4-Difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/Mivebresib), a Potent and Orally Available Bromodomain and Extraterminal Domain (BET) Family Bromodomain Inhibitor. J. Med. Chem. 2017, 60, 8369– 8384, DOI: 10.1021/acs.jmedchem.7b00746Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFOjtrbF&md5=16ea9e9ade8a22a6e99aeae16fd1bf9cDiscovery of N-(4-(2,4-Difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/Mivebresib), a Potent and Orally Available Bromodomain and Extraterminal Domain (BET) Family Bromodomain InhibitorMcDaniel, Keith F.; Wang, Le; Soltwedel, Todd; Fidanze, Steven D.; Hasvold, Lisa A.; Liu, Dachun; Mantei, Robert A.; Pratt, John K.; Sheppard, George S.; Bui, Mai H.; Faivre, Emily J.; Huang, Xiaoli; Li, Leiming; Lin, Xiaoyu; Wang, Rongqi; Warder, Scott E.; Wilcox, Denise; Albert, Daniel H.; Magoc, Terrance J.; Rajaraman, Ganesh; Park, Chang H.; Hutchins, Charles W.; Shen, Jianwei J.; Edalji, Rohinton P.; Sun, Chaohong C.; Martin, Ruth; Gao, Wenqing; Wong, Shekman; Fang, Guowei; Elmore, Steven W.; Shen, Yu; Kati, Warren M.Journal of Medicinal Chemistry (2017), 60 (20), 8369-8384CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examn. in clin. studies, particularly in oncol. settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metab. and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a crit. asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Addnl. structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clin. candidate I (ABBV-075/mivebresib), which demonstrates excellent potency in biochem. and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.
- 58Sheppard, G. S.; Wang, L.; Fidanze, S. D.; Hasvold, L. A.; Liu, D.; Pratt, J. K.; Park, C. H.; Longenecker, K.; Qiu, W.; Torrent, M.; Kovar, P. J.; Bui, M.; Faivre, E.; Huang, X.; Lin, X.; Wilcox, D.; Zhang, L.; Shen, Y.; Albert, D. H.; Magoc, T. J.; Rajaraman, G.; Kati, W. M.; McDaniel, K. F. Discovery of N-Ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]- 6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET Bromodomain Inhibitor with Selectivity for the Second Bromodomain. J. Med. Chem. 2020, 63, 5585– 5623, DOI: 10.1021/acs.jmedchem.0c00628Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnvVeisrk%253D&md5=675827659c3c7b6099ccd667f4c60f95Discovery of N-Ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]-6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET Bromodomain Inhibitor with Selectivity for the Second BromodomainSheppard, George S.; Wang, Le; Fidanze, Steven D.; Hasvold, Lisa A.; Liu, Dachun; Pratt, John K.; Park, Chang H.; Longenecker, Kenton; Qiu, Wei; Torrent, Maricel; Kovar, Peter J.; Bui, Mai; Faivre, Emily; Huang, Xiaoli; Lin, Xiaoyu; Wilcox, Denise; Zhang, Lu; Shen, Yu; Albert, Daniel H.; Magoc, Terrance J.; Rajaraman, Ganesh; Kati, Warren M.; McDaniel, Keith F.Journal of Medicinal Chemistry (2020), 63 (10), 5585-5623CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clin. development for oncol. bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (~ 40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compd. 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clin. development compd. 46 (ABBV-744).
- 59Faivre, E. J.; McDaniel, K. F.; Albert, D. H.; Mantena, S. R.; Plotnik, J. P.; Wilcox, D.; Zhang, L.; Bui, M. H.; Sheppard, G. S.; Wang, L.; Sehgal, V.; Lin, X.; Huang, X.; Lu, X.; Uziel, T.; Hessler, P.; Lam, L. T.; Bellin, R. J.; Mehta, G.; Fidanze, S.; Pratt, J. K.; Liu, D.; Hasvold, L. A.; Sun, C.; Panchal, S. C.; Nicolette, J. J.; Fossey, S. L.; Park, C. H.; Longenecker, K.; Bigelow, L.; Torrent, M.; Rosenberg, S. H.; Kati, W. M.; Shen, Y. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer. Nature 2020, 578, 306– 310, DOI: 10.1038/s41586-020-1930-8Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1Smsr0%253D&md5=800d9106c33084390fd8cbf1ac7f1766Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancerFaivre, Emily J.; McDaniel, Keith F.; Albert, Daniel H.; Mantena, Srinivasa R.; Plotnik, Joshua P.; Wilcox, Denise; Zhang, Lu; Bui, Mai H.; Sheppard, George S.; Wang, Le; Sehgal, Vasudha; Lin, Xiaoyu; Huang, Xiaoli; Lu, Xin; Uziel, Tamar; Hessler, Paul; Lam, Lloyd T.; Bellin, Richard J.; Mehta, Gaurav; Fidanze, Steve; Pratt, John K.; Liu, Dachun; Hasvold, Lisa A.; Sun, Chaohong; Panchal, Sanjay C.; Nicolette, John J.; Fossey, Stacey L.; Park, Chang H.; Longenecker, Kenton; Bigelow, Lance; Torrent, Maricel; Rosenberg, Saul H.; Kati, Warren M.; Shen, YuNature (London, United Kingdom) (2020), 578 (7794), 306-310CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clin. activity in monotherapy cancer trials. A reduced no. of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematol. and gastrointestinal defects were obsd. after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities assocd. with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacol. inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compds. that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chem. campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunopptn. followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-contg. super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.
- 60Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Martin, S.; Savitsky, P.; Diez-Dacal, B.; Philpott, M.; Bountra, C.; Lingard, H.; Fedorov, O.; Muller, S.; Brennan, P. E.; Knapp, S.; Filippakopoulos, P. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 19754– 19759, DOI: 10.1073/pnas.1310658110Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFKms7zN&md5=c918702c9fdb91241bc837732730f7f6RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomainPicaud, Sarah; Wells, Christopher; Felletar, Ildiko; Brotherton, Deborah; Martin, Sarah; Savitsky, Pavel; Diez-Dacal, Beatriz; Philpott, Martin; Bountra, Chas; Lingard, Hannah; Fedorov, Oleg; Muller, Susanne; Brennan, Paul E.; Knapp, Stefan; Filippakopoulos, PanagisProceedings of the National Academy of Sciences of the United States of America (2013), 110 (49), 19754-19759,S19754/1-S19754/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compd. currently in phase II clin. trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.
- 61Ayotte, Y.; Marando, V. M.; Vaillancourt, L.; Bouchard, P.; Heffron, G.; Coote, P. W.; Larda, S. T.; LaPlante, S. R. Exposing Small-Molecule Nanoentities by a Nuclear Magnetic Resonance Relaxation Assay. J. Med. Chem. 2019, 62, 7885– 7896, DOI: 10.1021/acs.jmedchem.9b00653Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFyru7%252FJ&md5=32a5015f45e3f64352be7d3f423e6d2bExposing Small-Molecule Nanoentities by a Nuclear Magnetic Resonance Relaxation AssayAyotte, Yann; Marando, Victoria M.; Vaillancourt, Louis; Bouchard, Patricia; Heffron, Gregory; Coote, Paul W.; Larda, Sacha T.; La Plante, Steven R.Journal of Medicinal Chemistry (2019), 62 (17), 7885-7896CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Small mols. can self-assemble in aq. soln. into a wide range of nanoentity types and sizes (dimers, n-mers, micelles, colloids, etc.), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to nonspecific binding, off-target effects, and false positives and negatives. Here, we demonstrate the use of the spin-spin relaxation Carr-Purcell-Meiboom-Gill NMR expt., which is sensitive to mol. tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone-tumbling mols. vs. nanoentities of various sizes. The technique is highly sensitive to chem. exchange between single-mol. and aggregate states and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in soln. behavior for compds. within structurally related series, demonstrating structure-nanoentity relationships. This practical expt. is a valuable tool to support drug discovery efforts.
- 62Urick, A. K.; Calle, L. P.; Espinosa, J. F.; Hu, H.; Pomerantz, W. C. Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to (1)H CPMG Ligand-Observed NMR. ACS Chem. Biol. 2016, 11, 3154– 3164, DOI: 10.1021/acschembio.6b00730Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFSlsbzJ&md5=44a2c33abe8f630fd7326cc08d7d685eProtein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to 1H CPMG Ligand-Observed NMRUrick, Andrew K.; Calle, Luis Pablo; Espinosa, Juan F.; Hu, Haitao; Pomerantz, William C. K.ACS Chemical Biology (2016), 11 (11), 3154-3164CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)To evaluate its potential as a ligand discovery tool, the authors compare a newly developed 1D-protein-obsd. fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-obsd. 1H CPMG NMR screen. The authors selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small mol. inhibitors of related epigenetic regulatory proteins. The authors compare the two methods' hit sensitivity, triaging ability, expt. speed, material consumption, and the potential for false positives and negatives. To this end, the authors screened 930 fragment mols. against Brd4 in mixts. of five and followed up these studies with mixt. deconvolution and affinity characterization of the top hits. In selected examples, the authors also compare the environmental responsiveness of the 19F chem. shift to 1H in 1D-protein obsd. 1H NMR expts. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titrn. calorimetry. The authors conclude that PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used 1D protein-obsd. NMR may also be effective; however the 19F chem. shift remains significantly more responsive.
- 63Pennington, L. D.; Aquila, B. M.; Choi, Y.; Valiulin, R. A.; Muegge, I. Positional Analogue Scanning: An Effective Strategy for Multiparameter Optimization in Drug Design. J. Med. Chem. 2020, 63, 8956– 8976, DOI: 10.1021/acs.jmedchem.9b02092Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslKgt70%253D&md5=75a473c6d4d0dd0f46de618ad8253c80Positional Analogue Scanning: An Effective Strategy for Multiparameter Optimization in Drug DesignPennington, Lewis D.; Aquila, Brian M.; Choi, Younggi; Valiulin, Roman A.; Muegge, IngoJournal of Medicinal Chemistry (2020), 63 (17), 8956-8976CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Minimizing the no. and duration of design cycles needed to optimize hit or lead compds. into high-quality chem. probes or drug candidates is an ongoing challenge in biomedical research. Small structure modifications to hit or lead compds. can have meaningful impacts on pharmacol. profiles due to significant effects on mol. and physicochem. properties and intra- and intermol. interactions. Rapid pharmacol. profiling of an efficiently prepd. series of positional analogs stemming from the systematic exchange of methine groups with heteroatoms or other substituents in arom. or heteroarom. ring-contg. hit or lead compds. is one approach toward minimizing design cycles (e.g., exchange of arom. or heteroarom. CH groups with N atoms or CF, CMe, or COH groups). In this Perspective, positional analog scanning is shown to be an effective strategy for multiparameter optimization in drug design, whereby substantial improvements in a variety of pharmacol. parameters can be achieved.
- 64Li, Y.; Zhao, J.; Gutgesell, L. M.; Shen, Z.; Ratia, K.; Dye, K.; Dubrovskyi, O.; Zhao, H.; Huang, F.; Tonetti, D. A.; Thatcher, G. R. J.; Xiong, R. Novel Pyrrolopyridone Bromodomain and Extra-Terminal Motif (BET) Inhibitors Effective in Endocrine-Resistant ER+ Breast Cancer with Acquired Resistance to Fulvestrant and Palbociclib. J. Med. Chem. 2020, 63, 7186– 7210, DOI: 10.1021/acs.jmedchem.0c00456Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVSht7jM&md5=b44638b7c6db7998abd72b50f09382caNovel Pyrrolopyridone Bromodomain and Extra-Terminal Motif (BET) Inhibitors Effective in Endocrine-Resistant ER+ Breast Cancer with Acquired Resistance to Fulvestrant and PalbociclibLi, Yangfeng; Zhao, Jiong; Gutgesell, Lauren M.; Shen, Zhengnan; Ratia, Kiira; Dye, Katherine; Dubrovskyi, Oleksii; Zhao, Huiping; Huang, Fei; Tonetti, Debra A.; Thatcher, Gregory R. J.; Xiong, RuiJournal of Medicinal Chemistry (2020), 63 (13), 7186-7210CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Acquired resistance to fulvestrant and palbociclib is a new challenge to treatment of estrogen receptor pos. (ER+) breast cancer. ER is expressed in most resistance settings; thus, bromodomain and extra-terminal protein inhibitors (BETi) that target BET-amplified ER-mediated transcription have therapeutic potential. Novel pyrrolopyridone BETi leveraged novel interactions with L92/L94 confirmed by a cocrystal structure of 27 with BRD4. Optimization of BETi using growth inhibition in fulvestrant-resistant (MCF-7:CFR) cells was confirmed in endocrine-resistant, palbociclib-resistant, and ESR1 mutant cell lines. 27 was more potent in MCF-7:CFR cells than six BET inhibitors in clin. trials. Transcriptomic anal. differentiated 27 from the benchmark BETi, JQ-1, showing downregulation of oncogenes and upregulation of tumor suppressors and apoptosis. The therapeutic approach was validated by oral administration of 27 in orthotopic xenografts of endocrine-resistant breast cancer in monotherapy and in combination with fulvestrant. Importantly, at an equiv. dose in rats, thrombocytopenia was mitigated.
- 65Wang, L.; Pratt, J. K.; Soltwedel, T.; Sheppard, G. S.; Fidanze, S. D.; Liu, D.; Hasvold, L. A.; Mantei, R. A.; Holms, J. H.; McClellan, W. J.; Wendt, M. D.; Wada, C.; Frey, R.; Hansen, T. M.; Hubbard, R.; Park, C. H.; Li, L.; Magoc, T. J.; Albert, D. H.; Lin, X.; Warder, S. E.; Kovar, P.; Huang, X.; Wilcox, D.; Wang, R.; Rajaraman, G.; Petros, A. M.; Hutchins, C. W.; Panchal, S. C.; Sun, C.; Elmore, S. W.; Shen, Y.; Kati, W. M.; McDaniel, K. F. Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors. J. Med. Chem. 2017, 60, 3828– 3850, DOI: 10.1021/acs.jmedchem.7b00017Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsVygu7k%253D&md5=91750e660bafec08b8bcbe6bcd5e818fFragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain InhibitorsWang, Le; Pratt, John K.; Soltwedel, Todd; Sheppard, George S.; Fidanze, Steven D.; Liu, Dachun; Hasvold, Lisa A.; Mantei, Robert A.; Holms, James H.; McClellan, William J.; Wendt, Michael D.; Wada, Carol; Frey, Robin; Hansen, T. Matthew; Hubbard, Robert; Park, Chang H.; Li, Leiming; Magoc, Terrance J.; Albert, Daniel H.; Lin, Xiaoyu; Warder, Scott E.; Kovar, Peter; Huang, Xiaoli; Wilcox, Denise; Wang, Rongqi; Rajaraman, Ganesh; Petros, Andrew M.; Hutchins, Charles W.; Panchal, Sanjay C.; Sun, Chaohong; Elmore, Steven W.; Shen, Yu; Kati, Warren M.; McDaniel, Keith F.Journal of Medicinal Chemistry (2017), 60 (9), 3828-3850CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Members of the BET family of bromodomain contg. proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A 2-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a Ph pyridazinone fragment with a weak binding affinity (I, Ki = 160 μM). SAR investigation of fragment I, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochem. and cell based assays, e.g. II. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.
- 66Poulin, P.; Theil, F. P. Prediction of Pharmacokinetics Prior to In Vivo Studies. 1. Mechanism-Based Prediction of Volume of Distribution. J. Pharm. Sci. 2002, 91, 129– 156, DOI: 10.1002/jps.10005Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xmslyhtg%253D%253D&md5=17006eeba58f27fefc9ff13e50ee4010Prediction of pharmacokinetics prior to in vivo studies. 1. Mechanism-based prediction of volume of distributionPoulin, Patrick; Theil, Frank-PeterJournal of Pharmaceutical Sciences (2002), 91 (1), 129-156CODEN: JPMSAE; ISSN:0022-3549. (Wiley-Liss, Inc.)In drug discovery and nonclin. development the vol. of distribution at steady state (Vss) of each novel drug candidate is commonly detd. under in vivo conditions. Therefore, it is of interest to predict Vss without conducting in vivo studies. The traditional description of Vss corresponds to the sum of the products of each tissue:plasma partition coeff. (Pt:p) and the resp. tissue vol. in addn. to the plasma vol. Because data on vols. of tissues and plasma are available in the literature for mammals, the other input parameters needed to est. Vss are the Pt:p's, which can potentially be predicted with established tissue compn.-based equations. In vitro data on drug lipophilicity and plasma protein binding are the input parameters used in these equations. Such a mechanism-based approach would be particularly useful to provide first-cut ests. of Vss prior to any in vivo studies and to explore potential unexpected deviations between sets of predicted and in vivo Vss data, when the in vivo data become available during the drug development process. The objective of the present study was to use tissue compn.-based equations to predict rat and human Vss prior to in vivo studies for 123 structurally unrelated compds. (acids, bases, and neutrals). The predicted data were compared with in vivo data obtained from the literature or at Roche. Overall, the av. ratio of predicted-to-exptl. rat and human Vss values was 1.06 (SD=0.817, r=0.78, n=147). In fact, 80% of all predicted values were within a factor of two of the corresponding exptl. values. The drugs can therefore be sepd. into two groups. The first group contains 98 drugs for which the predicted Vss were within a factor of two of those exptl. detd. (av. ratio of 1.01, SD=0.39, r=0.93, n=118), and the second group includes 25 other drugs for which the predicted and exptl. Vss differ by a factor larger than two (av. ratio of 1.32, SD=1.74, r=0.42, n=29). Thus, addnl. relevant distribution processes were neglected in predicting Vss of drugs of the second group. This was true esp. in the case of some cationic-amphiphilic bases. The present study is the first attempt to develop and validate a mechanistic distribution model for predicting rat and human Vss of drugs prior to in vivo studies.
- 67Decosterd, I.; Woolf, C. J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000, 87, 149– 158, DOI: 10.1016/s0304-3959(00)00276-1Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3cvosVSntw%253D%253D&md5=c7a759c4b4bde22be4e7f76df85a334bSpared nerve injury: an animal model of persistent peripheral neuropathic painDecosterd Isabelle; Woolf Clifford JPain (2000), 87 (2), 149-158 ISSN:0304-3959.Peripheral neuropathic pain is produced by multiple etiological factors that initiate a number of diverse mechanisms operating at different sites and at different times and expressed both within, and across different disease states. Unraveling the mechanisms involved requires laboratory animal models that replicate as far as possible, the different pathophysiological changes present in patients. It is unlikely that a single animal model will include the full range of neuropathic pain mechanisms. A feature of several animal models of peripheral neuropathic pain is partial denervation. In the most frequently used models a mixture of intact and injured fibers is created by loose ligation of either the whole (Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87-107) or a tight ligation of a part (Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990;43:205-218) of a large peripheral nerve, or a tight ligation of an entire spinal segmental nerve (Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355-363). We have developed a variant of partial denervation, the spared nerve injury model. This involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact. The spared nerve injury model differs from the Chung spinal segmental nerve, the Bennett chronic constriction injury and the Seltzer partial sciatic nerve injury models in that the co-mingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the non-injured skin territories adjacent to the denervated areas. The spared nerve injury model results in early (<24 h), prolonged (>6 months), robust (all animals are responders) behavioral modifications. The mechanical (von Frey and pinprick) sensitivity and thermal (hot and cold) responsiveness is increased in the ipsilateral sural and to a lesser extent saphenous territories, without any change in heat thermal thresholds. Crush injury of the tibial and common peroneal nerves produce similar early changes, which return, however to baseline at 7-9 weeks. The spared nerve injury model may provide, therefore, an additional resource for unraveling the mechanisms responsible for the production of neuropathic pain.
- 68Chen, X.; Meng, F.; Zhang, J.; Zhang, Z.; Ye, X.; Zhang, W.; Tong, Y.; Ji, X.; Xu, R.; Xu, X. L.; You, Q. D.; Jiang, Z. Y. Discovery of 2-((2-methylbenzyl)thio)-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5 -carbonitrile as a novel and effective bromodomain and extra-terminal (BET) inhibitor for the treatment of sepsis. Eur. J. Med. Chem. 2022, 238, 114423, DOI: 10.1016/j.ejmech.2022.114423Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Gqur7F&md5=d9b2210f9d07e9042ff549dcb3e6864eDiscovery of 2-((2-methylbenzyl)thio)-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile as a novel and effective bromodomain and extra-terminal (BET) inhibitor for the treatment of sepsisChen, Xuetao; Meng, Fanying; Zhang, Jingtian; Zhang, Zijian; Ye, Xuan; Zhang, Weikun; Tong, Yuanyuan; Ji, Xinrui; Xu, Rujun; Xu, Xiao-Li; You, Qi-Dong; Jiang, Zheng-YuEuropean Journal of Medicinal Chemistry (2022), 238 (), 114423CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Sepsis has long been a major health problem worldwide. It threatens the lives of hospitalized patients and has been one of the leading causes of death in hospitalized patients over the past decades. BRD4 has been regarded as a potential target for sepsis therapy, for its crit. role in the transcriptional expression of NF-κB pathway-dependent inflammatory factors. In this study, compd. 1 was obtained through virtual screening, and candidate compd. 27 was obtained through several rounds of iterative SAR anal. 27 decreased LPS-induced NO prodn. and expression of the pro-inflammatory factors IL-6, IL-1β and TNF-α. In vivo, 27 effectively protected mice from LPS-induced sepsis, increased survival rate and decreased the level of pro-inflammatory factors in serum. Collectively, we reported here 27, a BRD4 inhibitor with a new scaffold, as a potential candidate for the treatment of sepsis.
- 69Zhang, R.; Mayhood, T.; Lipari, P.; Wang, Y.; Durkin, J.; Syto, R.; Gesell, J.; McNemar, C.; Windsor, W. Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction. Anal. Biochem. 2004, 331, 138– 146, DOI: 10.1016/s0003-2697(04)00223-4Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXls1Wlu70%253D&md5=d965d43de2f290c1454ad82bb8781d83Fluorescence polarization assay and inhibitor design for MDM2/p53 interactionZhang, Rumin; Mayhood, Todd; Lipari, Philip; Wang, Yaolin; Durkin, James; Syto, Rosalinda; Gesell, Jennifer; McNemar, Charles; Windsor, WilliamAnalytical Biochemistry (2004), 331 (1), 138-146CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)MDM2 is an important neg. regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-mol. inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.
- 70Ran, X.; Zhao, Y.; Liu, L.; Bai, L.; Yang, C. Y.; Zhou, B.; Meagher, J. L.; Chinnaswamy, K.; Stuckey, J. A.; Wang, S. Structure-Based Design of gamma-Carboline Analogues as Potent and Specific BET Bromodomain Inhibitors. J. Med. Chem. 2015, 58, 4927– 4939, DOI: 10.1021/acs.jmedchem.5b00613Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVagu7rM&md5=e6e150169812e439c1d7d91416eb3ee9Structure-Based Design of γ-Carboline Analogues as Potent and Specific BET Bromodomain InhibitorsRan, Xu; Zhao, Yujun; Liu, Liu; Bai, Longchuan; Yang, Chao-Yie; Zhou, Bing; Meagher, Jennifer L.; Chinnaswamy, Krishnapriya; Stuckey, Jeanne A.; Wang, ShaomengJournal of Medicinal Chemistry (2015), 58 (12), 4927-4939CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Small-mol. inhibitors of bromodomain and extra terminal proteins (BET), including BRD2, BRD3, and BRD4 proteins have therapeutic potential for the treatment of human cancers and other diseases and conditions. In this paper, the authors report the design, synthesis, and evaluation of γ-carboline-contg. compds. as a new class of small-mol. BET inhibitors. The most potent inhibitor I (RX-37) obtained from this study binds to BET bromodomain proteins (BRD2, BRD3, and BRD4) with Ki values of 3.2-24.7 nM and demonstrates high selectivity over other non-BET bromodomain-contg. proteins. Compd. I potently and selectively inhibits cell growth in human acute leukemia cell lines harboring the rearranged mixed lineage leukemia 1 gene. The authors have detd. a cocrystal structure of I in complex with BRD4 BD2 at 1.4 Å resoln., which provides a solid structural basis for the compd.'s high binding affinity and for its further structure-based optimization. Compd. I represents a promising lead compd. for the development of a new class of therapeutics for the treatment of human cancer and other conditions.
- 71Jiang, F.; Guo, A. P.; Xu, J. C.; You, Q. D.; Xu, X. L. Discovery of a Potent Grp94 Selective Inhibitor with Anti-Inflammatory Efficacy in a Mouse Model of Ulcerative Colitis. J. Med. Chem. 2018, 61, 9513– 9533, DOI: 10.1021/acs.jmedchem.8b00800Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFSrtr7F&md5=65fa429395e1d57fc8bd6e5c51f52ccbDiscovery of a Potent Grp94 Selective Inhibitor with Anti-Inflammatory Efficacy in a Mouse Model of Ulcerative ColitisJiang, Fen; Guo, An-ping; Xu, Jia-chen; You, Qi-Dong; Xu, Xiao-LiJournal of Medicinal Chemistry (2018), 61 (21), 9513-9533CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)As the endoplasmic reticulum paralog of Hsp90, Grp94 chaperones a small set of client proteins assocd. with some diseases, including cancer, primary open-angle glaucoma, and inflammatory disorders. Grp94-selective inhibition has been a potential therapeutic strategy for these diseases. In this study, inspired by the conclusion that ligand-induced "Phe199 shift" effect is the structural basis of Grp94-selective inhibition, a series of novel Grp94 selective inhibitors incorporating "benzamide" moiety were developed, among which compd. I manifested the most potent Grp94 inhibitory activity with an IC50 value of 2 nM and over 1000-fold selectivity to Grp94 against Hsp90α. In a DSS-induced mouse model of ulcerative colitis (UC), compd. I exhibited significant anti-inflammatory efficacy. This work provides a potent Grp94 selective inhibitor as probe compd. for the biol. study of Grp94 and represents the first study that confirms the potential therapeutic efficacy of Grp94-selective inhibitors against UC.
- 72Zhu, P. J.; Yu, Z. Z.; Lv, Y. F.; Zhao, J. L.; Tong, Y. Y.; You, Q. D.; Jiang, Z. Y. Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability. J. Med. Chem. 2021, 64, 11330– 11353, DOI: 10.1021/acs.jmedchem.1c00682Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1ylt7vJ&md5=2fce800fbc1f6de0d676a5054787584dDiscovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral BioavailabilityZhu, Peng-Ju; Yu, Ze-Zhou; Lv, Yi-Fei; Zhao, Jing-Long; Tong, Yuan-Yuan; You, Qi-Dong; Jiang, Zheng-YuJournal of Medicinal Chemistry (2021), 64 (15), 11330-11353CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1) protein is a key neg. 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 compds. as Mcl-1 inhibitors. Stepwise optimizations of hit compd. 11 with primary Mcl-1 inhibition (52%@30μM) led to the discovery of the most potent compd. 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 physicochem. 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 compd. 40 is a promising antitumor agent that deserves further preclin. evaluations.
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- Yangfeng Li, Zhengnan Shen, Kiira Ratia, Jiong Zhao, Fei Huang, Oleksii Dubrovyskyii, Divakar Indukuri, Jiqiang Fu, Omar Lozano Ramos, Gregory R. J. Thatcher, Rui Xiong. Structure-Guided Design and Synthesis of Pyridinone-Based Selective Bromodomain and Extra-Terminal Domain (BET)-First Bromodomain (BD1) Inhibitors. Journal of Medicinal Chemistry 2024, 67
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, 2712-2731. https://doi.org/10.1021/acs.jmedchem.3c01837
Abstract 摘要
Figure 1 图 1
Figure 1. Hit identification and hit-to-lead optimization. (A) Screening of the in-house compound library to find new hits. (B) The 1H CPMG spectra of hit compound 1 and BRD4 BD1 were superimposed on the 1H NMR spectrum of 1 (up) and the 1H NMR spectrum of 1 (down). (C) The SAR of the pyrrolopyridine core modifications resulted in the lead compound 9. See Table S1 for SD values.
图 1.新药鉴定和新药对先导的优化。(A) 筛选内部化合物库以寻找新的命中点。(B) 将命中化合物 1 和 BRD4 BD1 的 {{0}和 BRD4 BD1 的{{1}H NMR 光谱叠加。｝1 的 1 H NMR 光谱（向上）和 1 H NMR 光谱（向下）叠加。1（下）的 1 H NMR 光谱叠加。(C) 吡咯并吡啶核心修饰的 SAR 结果为先导化合物 9。SD 值见表 S1。Figure 2
Figure 2. Docking of 9 suggested that the trimethoxyphenyl substituent may engage the WPF shelf, which led to the design of several aryl derivatives. Docking analysis of compound 9 (yellow) with BRD4 BD1 (left, PDB ID: 3MXF). Docking analysis of compound 15 (yellow) with BRD4 BD1 (right, PDB ID: 3MXF). Salmon indicates the WPF shelf, red indicates Asn140, orange indicates the BC loop, and green indicates the ZA channel. See Table S1 for SD values.
Figure 3
Figure 3. SAR of substitutions to the phenyl ring and the co-crystal structure of compound 21 complexed with BRD4 BD1 and BD2 proteins. (A) SAR of substitutions to the phenyl ring. See Table S1 for SD values. (B) Crystal structure of BRD4 BD1 (light pink) bound to 21 (yellow) (PDB ID: 8IBQ). (C) Crystal structure of BRD4 BD2 (lime green) bound to 21 (yellow) (PDB ID: 8IDH). The ligands and side chains of important residues are shown in a stick model; the hydrogen bond (green) and π–π conjugation (pink) are indicated by the dashed line. (D) Crystal structure of 21 bound to BRD4 BD1 (surface representation: salmon indicates the WPF shelf, cyan indicates Asn140, orange indicates the BC loop, and green indicates the ZA channel).
Figure 4
Figure 4. Selectivity assessment of DDO-8926 against a panel of bromodomains using a DiscoverX BROMOscan platform at 1 μM. Percent control = [(test compound signal – positive control signal)/(negative control signal – positive control signal)] × 100.
Figure 5
Figure 5. In vivo PK parameters of DDO-8926 in male mice. The values shown are the means ± SEM (n = 4).
Figure 6
Figure 6. DDO-8926 attenuates NP hypersensitivity. (A) Distribution of DDO-8926 in the plasma and brain of mice. (B) Experimental scheme depicting the in vivo analgesic evaluation of DDO-8926. (C) Analgesic effect of gabapentin (100 mg/kg, ip) and DDO-8926 (30 mg/kg, ip) on SNI-induced NP response tested by ipsilateral stimulation. (D) Analgesic effects of gabapentin (100 mg/kg, ip) and DDO-8926 (30 mg/kg, ip) at 15 days post-operation (dpo). Results were expressed as mean ± SEM (n = 6), (ns) < 0.1, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001 compared with the SNI (vehicle) group, one-way ANOVA with the Tukey–Kramer post-test.
Figure 7
Figure 7. DDO-8926 regulates genes involved in the generation and transfer of action potentials and inflammatory response. (A) These volcano maps show DEGs after SNI surgery and DDO-8926 treatment. (B) GO enrichment analysis of DEGs between vehicle and DDO-8926 treatment. GSEA analysis of (C) ion transfer, (D) glial cell migration, (E) action potential, and (F) inflammatory response between vehicle and DDO-8926 treatment.
Figure 8
Figure 8. DDO-8926 reduces microglial and neuronal activation in the spinal cord after SNI and has no effect on astrocytes. Representative microphotographs from the L5 spinal dorsal horn of the ipsilateral hemisections labeled with (A) NeuN, (B) IBA1, and (C) GFAP of Sham, SNI (vehicle), and SNI (DDO-8926 30 mg/kg) groups at 15 dpo. Representative microphotographs (left) and positive cells (right). Results were expressed as mean ± SEM (n = 3), (ns) < 0.1, (***) P < 0.001, and (****) P < 0.0001 compared with the SNI (vehicle) group, one-way ANOVA with the Tukey–Kramer post-test.
Figure 9
Figure 9. DDO-8926 alleviates inflammation and affects the expression of ion channels and the Na+/K+ ATPase pump in the spinal cord at 15 dpo after SNI. (A) DDO-8926 downregulates the expression of pro-inflammatory factor mRNA in the spinal cord after SNI. DDO-8926 inhibits SNI-induced mRNA expression of (B) voltage-gated sodium channels and (C) voltage-gated potassium channels. (D) DDO-8926 reduces the SNI-induced mRNA expression of the αNKA subunit of the Na+/K+ ATPase pump. Results were expressed as mean ± SEM (n = 6), (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001 compared with the SNI (vehicle) group, one-way ANOVA with the Tukey–Kramer post-test.
Scheme 1
Scheme 1. Reagents and Conditions: (a) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h and (b) THF, 80 °C, 4 hScheme 2
Scheme 2. Reagents and Conditions: (a) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl2·CH2Cl2, Dry 1,4-Dioxane, 100 °C, 3 h; (c) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 7 h; and (d) Cs2CO3, DMF, r.t., 5 hScheme 3
Scheme 3. Reagents and Conditions: (a) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h; (b) AcOK, Pd(dppf)Cl2·CH2Cl2, Dry 1,4-Dioxane, 100 °C, 3 h; and (c) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 7 hScheme 4
Scheme 4. Reagents and Conditions: (a) Pyridine, CH2Cl2, r.t., 4 h; (b) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 3 h; (c) AcOK, Pd(dppf)Cl2·CH2Cl2, Dry 1,4-Dioxane, 100 °C, 3 h; and (d) Cs2CO3, Pd(dppf)Cl2·CH2Cl2, 1,4-Dioxane, H2O, 100 °C, 7 hReferences
ARTICLE SECTIONSThis article references 72 other publications.
- 1Richner, M.; Pallesen, L. T.; Ulrichsen, M.; Poulsen, E. T.; Holm, T. H.; Login, H.; Castonguay, A.; Lorenzo, L. E.; Gonçalves, N. P.; Andersen, O. M.; Lykke-Hartmann, K.; Enghild, J. J.; Rønn, L. C. B.; Malik, I. J.; De Koninck, Y.; Bjerrum, O. J.; Vægter, C. B.; Nykjær, A. Sortilin gates neurotensin and BDNF signaling to control peripheral neuropathic pain. Sci. Adv. 2019, 5, 9946, DOI: 10.1126/sciadv.aav99461https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlaktbrF&md5=160fb0a2d84197e1b356d67450e5f1f5Sortilin gates neurotensin and BDNF signaling to control peripheral neuropathic painRichner, Mette; Pallesen, Lone T.; Ulrichsen, Maj; Poulsen, Ebbe T.; Holm, Thomas H.; Login, Hande; Castonguay, Annie; Lorenzo, Louis-Etienne; Goncalves, Nadia P.; Andersen, Olav M.; Lykke-Hartmann, Karin; Enghild, Jan J.; Roenn, Lars C. B.; Malik, Ibrahim J.; De Koninck, Yves; Bjerrum, Ole J.; Vaegter, Christian B.; Nykjaer, AndersScience Advances (2019), 5 (6), eaav9946CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)Neuropathic pain is a major incurable clin. problem resulting from peripheral nerve trauma or disease. A central mechanism is the reduced expression of the potassium chloride cotransporter 2 (KCC2) in dorsal horn neurons induced by brain-derived neurotrophic factor (BDNF), causing neuronal disinhibition within spinal nociceptive pathways. Here, we demonstrate how neurotensin receptor 2 (NTSR2) signaling impairs BDNF-induced spinal KCC2 down-regulation, showing how these two pathways converge to control the abnormal sensory response following peripheral nerve injury. We establish how sortilin regulates this convergence by scavenging neurotensin from binding to NTSR2, thus modulating its inhibitory effect on BDNF-mediated mech. allodynia. Using sortilindeficient mice or receptor inhibition by antibodies or a small-mol. antagonist, we lastly demonstrate that we are able to fully block BDNF-induced pain and alleviate injury-induced neuropathic pain, validating sortilin as a clin. relevant target.
- 2Wang, Z.; Liu, F.; Wei, M.; Qiu, Y.; Ma, C.; Shen, L.; Huang, Y. Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathway. J. Neuroinflammation 2018, 15, 179, DOI: 10.1186/s12974-018-1215-42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFWnsrrF&md5=902791b068f987ed7308b57aded1f583Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathwayWang, Zhiyao; Liu, Fan; Wei, Min; Qiu, Yue; Ma, Chao; Shen, Le; Huang, YuguangJournal of Neuroinflammation (2018), 15 (), 179/1-179/12CODEN: JNOEB3; ISSN:1742-2094. (BioMed Central Ltd.)Background: microRNA-146a-5p (miRNA-146a-5p) is a key mol. in the neg. regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain. Methods: The sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quant. real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mech. stimuli and radiant heat were used to evaluate mech. allodynia and thermal hyperalgesia. Results: We found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mech. and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mech. and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further. Conclusions: miRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.
- 3Wang, K.; Wang, S.; Chen, Y.; Wu, D.; Hu, X.; Lu, Y.; Wang, L.; Bao, L.; Li, C.; Zhang, X. Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain. Cell Res. 2021, 31, 904– 918, DOI: 10.1038/s41422-021-00479-93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXms1GktbY%253D&md5=2cb67ef42ce5b7cbd4faa8381250f0eeSingle-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic painWang, Kaikai; Wang, Sashuang; Chen, Yan; Wu, Dan; Hu, Xinyu; Lu, Yingjin; Wang, Liping; Bao, Lan; Li, Changlin; Zhang, XuCell Research (2021), 31 (8), 904-918CODEN: CREEB6; ISSN:1001-0602. (Nature Portfolio)Peripheral nerve injury could lead to chronic neuropathic pain. Understanding transcriptional changes induced by nerve injury could provide fundamental insights into the complex pathogenesis of neuropathic pain. Gene expression profiles of dorsal root ganglia (DRG) in neuropathic pain condition have been studied. However, little is known about transcriptomic changes in individual DRG neurons after peripheral nerve injury. Here we performed single-cell RNA sequencing on dissocd. mouse DRG cells after spared nerve injury (SNI). In addn. to DRG neuron types that are found under physiol. conditions, we identified three SNI-induced neuronal clusters (SNIICs) characterized by the expression of Atf3/Gfra3/Gal (SNIIC1), Atf3/Mrgprd (SNIIC2) and Atf3/S100b/Gal (SNIIC3). These SNIICs originated from Cldn9+/Gal+, Mrgprd+ and Trappc3l+ DRG neurons, resp. Interestingly, SNIIC2 switched to SNIIC1 by increasing Gal and reducing Mrgprd expression 2 days after nerve injury. Inferring the gene regulatory networks after nerve injury, we revealed that activated transcription factors Atf3 and Egr1 in SNIICs could enhance Gal expression while activated Cpeb1 in SNIIC2 might suppress Mrgprd expression within 2 days after SNI. Furthermore, we mined the transcriptomic changes in the development of neuropathic pain to identify potential analgesic targets. We revealed that cardiotrophin-like cytokine factor 1, which activates astrocytes in the dorsal horn of spinal cord, was upregulated in SNIIC1 neurons and contributed to SNI-induced mech. allodynia. Therefore, our results provide a new landscape to understand the dynamic course of neuron type changes and their underlying mol. mechanisms during the development of neuropathic pain.
- 4Colloca, L.; Ludman, T.; Bouhassira, D.; Baron, R.; Dickenson, A. H.; Yarnitsky, D.; Freeman, R.; Truini, A.; Attal, N.; Finnerup, N. B.; Eccleston, C.; Kalso, E.; Bennett, D. L.; Dworkin, R. H.; Raja, S. N. Neuropathic pain. Nat. Rev. Dis. Prim. 2017, 3, 17002, DOI: 10.1038/nrdp.2017.2There is no corresponding record for this reference.
- 5Finnerup, N. B.; Attal, N.; Haroutounian, S.; McNicol, E.; Baron, R.; Dworkin, R. H.; Gilron, I.; Haanpaa, M.; Hansson, P.; Jensen, T. S.; Kamerman, P. R.; Lund, K.; Moore, A.; Raja, S. N.; Rice, A. S.; Rowbotham, M.; Sena, E.; Siddall, P.; Smith, B. H.; Wallace, M. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. J. Vasc. Surg. 2015, 62, 1091– 1173, DOI: 10.1016/j.jvs.2015.08.010There is no corresponding record for this reference.
- 6Mathieson, S.; Lin, C.-W. C.; Underwood, M.; Eldabe, S. Pregabalin and gabapentin for pain. BMJ 2020, 369, m1315, DOI: 10.1136/bmj.m1315There is no corresponding record for this reference.
- 7Tesfaye, S.; Sloan, G.; Petrie, J.; White, D.; Bradburn, M.; Julious, S.; Rajbhandari, S.; Sharma, S.; Rayman, G.; Gouni, R.; Alam, U.; Cooper, C.; Loban, A.; Sutherland, K.; Glover, R.; Waterhouse, S.; Turton, E.; Horspool, M.; Gandhi, R.; Maguire, D.; Jude, E. B.; Ahmed, S. H.; Vas, P.; Hariman, C.; McDougall, C.; Devers, M.; Tsatlidis, V.; Johnson, M.; Rice, A. S. C.; Bouhassira, D.; Bennett, D. L.; Selvarajah, D.; group, O.-D. t. Comparison of amitriptyline supplemented with pregabalin, pregabalin supplemented with amitriptyline, and duloxetine supplemented with pregabalin for the treatment of diabetic peripheral neuropathic pain (OPTION-DM): a multicentre, double-blind, randomised crossover trial. Lancet 2022, 400, 680– 690, DOI: 10.1016/s0140-6736(22)01472-67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlCit7nO&md5=0b135694aa3408206fd069978b111399Comparison of amitriptyline supplemented with pregabalin, pregabalin supplemented with amitriptyline, and duloxetine supplemented with pregabalin for the treatment of diabetic peripheral neuropathic pain (OPTION-DM): a multicentre, double-blind, randomised crossover trialTesfaye, Solomon; Sloan, Gordon; Petrie, Jennifer; White, David; Bradburn, Mike; Julious, Stephen; Rajbhandari, Satyan; Sharma, Sanjeev; Rayman, Gerry; Gouni, Ravikanth; Alam, Uazman; Cooper, Cindy; Loban, Amanda; Sutherland, Katie; Glover, Rachel; Waterhouse, Simon; Turton, Emily; Horspool, Michelle; Gandhi, Rajiv; Maguire, Deirdre; Jude, Edward B.; Ahmed, Syed H.; Vas, Prashanth; Hariman, Christian; McDougall, Claire; Devers, Marion; Tsatlidis, Vasileios; Johnson, Martin; Rice, Andrew S. C.; Bouhassira, Didier; Bennett, David L.; Selvarajah, DineshLancet (2022), 400 (10353), 680-690CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)Diabetic peripheral neuropathic pain (DPNP) is common and often distressing. Most guidelines recommend amitriptyline, duloxetine, pregabalin, or gabapentin as initial analgesic treatment for DPNP, but there is little comparative evidence on which one is best or whether they should be combined. We aimed to assess the efficacy and tolerability of different combinations of first-line drugs for treatment of DPNP. OPTION-DM was a multicentre, randomised, double-blind, crossover trial in patients with DPNP with mean daily pain numerical rating scale (NRS) of 4 or higher (scale is 0-10) from 13 UK centers. Participants were randomly assigned (1:1:1:1:1:1), with a predetd. randomisation schedule stratified by site using permuted blocks of size six or 12, to receive one of six ordered sequences of the three treatment pathways: amitriptyline supplemented with pregabalin (A-P), pregabalin supplemented with amitriptyline (P-A), and duloxetine supplemented with pregabalin (D-P), each pathway lasting 16 wk. Monotherapy was given for 6 wk and was supplemented with the combination medication if there was suboptimal pain relief (NRS >3), reflecting current clin. practice. Both treatments were titrated towards max. tolerated dose (75 mg per day for amitriptyline, 120 mg per day for duloxetine, and 600 mg per day for pregabalin). The primary outcome was the difference in 7-day av. daily pain during the final week of each pathway. This trial is registered with ISRCTN, ISRCTN17545443. Between Nov 14, 2017, and July 29, 2019, 252 patients were screened, 140 patients were randomly assigned, and 130 started a treatment pathway (with 84 completing at least two pathways) and were analyzed for the primary outcome. The 7-day av. NRS scores at week 16 decreased from a mean 6·6 (SD 1·5) at baseline to 3·3 (1·8) at week 16 in all three pathways. The mean difference was -0·1 (98·3% CI -0·5 to 0·3) for D-P vs. A-P, -0·1 (-0·5 to 0·3) for P-A vs. A-P, and 0·0 (-0·4 to 0·4) for P-A vs. D-P, and thus not significant. Mean NRS redn. in patients on combination therapy was greater than in those who remained on monotherapy (1·0 [SD 1·3] vs 0·2 [1·5]). Adverse events were predictable for the monotherapies: we obsd. a significant increase in dizziness in the P-A pathway, nausea in the D-P pathway, and dry mouth in the A-P pathway. To our knowledge, this was the largest and longest ever, head-to-head, crossover neuropathic pain trial. We showed that all three treatment pathways and monotherapies had similar analgesic efficacy. Combination treatment was well tolerated and led to improved pain relief in patients with suboptimal pain control with a monotherapy.
- 8Tanabe, M.; Ono, K.; Honda, M.; Ono, H. Gabapentin and pregabalin ameliorate mechanical hypersensitivity after spinal cord injury in mice. Eur. J. Pharmacol. 2009, 609, 65– 68, DOI: 10.1016/j.ejphar.2009.03.0208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkslyht74%253D&md5=690dd4d5fe2caee8086d08ce70c87be5Gabapentin and pregabalin ameliorate mechanical hypersensitivity after spinal cord injury in miceTanabe, Mitsuo; Ono, Koto; Honda, Motoko; Ono, HidekiEuropean Journal of Pharmacology (2009), 609 (1-3), 65-68CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)The antiepileptic drugs gabapentin and pregabalin exhibit well-established analgesic effects in patients with several neuropathic conditions. In the present study, we examd. their effects on mech. hypersensitivity in mice subjected to wt.-drop spinal cord injury. Hindlimb motor function and mech. hypersensitivity were evaluated using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale and the von Frey test, resp., for 4 wk after spinal cord injury. Despite gradual recovery of hindlimb motor function after spinal cord injury, mice exhibited continuous development of mech. hypersensitivity. Gabapentin (30 and 100 mg/kg) and pregabalin (10 and 30 mg/kg), administered i.p. on the 28th day after spinal cord injury, reduced mech. hypersensitivity in a dose-dependent manner. These results suggest that gabapentin and pregabalin could be useful therapeutic tools for patients with neuropathic pain after spinal cord injury.
- 9Kremer, M.; Salvat, E.; Muller, A.; Yalcin, I.; Barrot, M. Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insights. Neuroscience 2016, 338, 183– 206, DOI: 10.1016/j.neuroscience.2016.06.0579https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFKktLrK&md5=e654dec57f8820a5a51f15894da8c938Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insightsKremer, Melanie; Salvat, Eric; Muller, Andre; Yalcin, Ipek; Barrot, MichelNeuroscience (Amsterdam, Netherlands) (2016), 338 (), 183-206CODEN: NRSCDN; ISSN:0306-4522. (Elsevier B.V.)Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory system. It is generally chronic and challenging to treat. The recommended pharmacotherapy for neuropathic pain includes the use of some antidepressants, such as tricyclic antidepressants (TCAs) (amitriptyline...) or serotonin and noradrenaline re-uptake inhibitors (duloxetine...), and/or anticonvulsants such as the gabapentinoids gabapentin or pregabalin. Antidepressant drugs are not acute analgesics but require a chronic treatment to relieve neuropathic pain, which suggests the recruitment of secondary downstream mechanisms as well as long-term mol. and neuronal plasticity. Noradrenaline is a major actor for the action of antidepressant drugs in a neuropathic pain context. Mechanistic hypotheses have implied the recruitment of noradrenergic descending pathways as well as the peripheral recruitment of noradrenaline from sympathetic fibers sprouting into dorsal root ganglia; and importance of both α2 and β2 adrenoceptors have been reported. These monoamine re-uptake inhibitors may also indirectly act as anti-proinflammatory cytokine drugs; and their therapeutic action requires the opioid system, particularly the mu (MOP) and/or delta (DOP) opioid receptors. Gabapentinoids, which target the voltage-dependent calcium channels α2δ-1 subunit, inhibit calcium currents, thus decreasing the excitatory transmitter release and spinal sensitization. Gabapentinoids also activate the descending noradrenergic pain inhibitory system coupled to spinal α2 adrenoceptors. Gabapentinoid treatment may also indirectly impact on neuroimmune actors, like proinflammatory cytokines. These drugs are effective against neuropathic pain both with acute administration at high dose and with repeated administration. This review focuses on mechanistic knowledge concerning chronic antidepressant treatment and gabapentinoid treatment in a neuropathic pain context.
- 10Kuehn, B. M. Gabapentin Increasingly Implicated in Overdose Deaths. JAMA 2022, 327, 2387, DOI: 10.1001/jama.2022.10100There is no corresponding record for this reference.
- 11Anand, P.; Bley, K. Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch. Br. J. Anaesth. 2011, 107, 490– 502, DOI: 10.1093/bja/aer26011https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFKiu77M&md5=a0ca0882470e2df01319b1ecb54a72caTopical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patchAnand, P.; Bley, K.British Journal of Anaesthesia (2011), 107 (4), 490-502CODEN: BJANAD; ISSN:0007-0912. (Oxford University Press)A review. Topical capsaicin formulations are used for pain management. Safety and modest efficacy of low-concn. capsaicin formulations, which require repeated daily self-administration, are supported by meta-analyses of numerous studies. A high-concn. capsaicin 8% patch (Qutenza) was recently approved in the EU and USA. A single 60-min application in patients with neuropathic pain produced effective pain relief for up to 12 wk. Advantages of the high-concn. capsaicin patch include longer duration of effect, patient compliance, and low risk for systemic effects or drug-drug interactions. The mechanism of action of topical capsaicin has been ascribed to depletion of substance P. However, exptl. and clin. studies show that depletion of substance P from nociceptors is only a correlate of capsaicin treatment and has little, if any, causative role in pain relief. Rather, topical capsaicin acts in the skin to attenuate cutaneous hypersensitivity and reduce pain by a process best described as defunctionalization' of nociceptor fibers. Defunctionalization is due to a no. of effects that include temporary loss of membrane potential, inability to transport neurotrophic factors leading to altered phenotype, and reversible retraction of epidermal and dermal nerve fiber terminals. Peripheral neuropathic hypersensitivity is mediated by diverse mechanisms, including altered expression of the capsaicin receptor TRPV1 or other key ion channels in affected or intact adjacent peripheral nociceptive nerve fibers, aberrant re-innervation, and collateral sprouting, all of which are defunctionalized by topical capsaicin. Evidence suggests that the utility of topical capsaicin may extend beyond painful peripheral neuropathies.
- 12Romanelli, M. N.; Borgonetti, V.; Galeotti, N. Dual BET/HDAC inhibition to relieve neuropathic pain: Recent advances, perspectives, and future opportunities. Pharmacol. Res. 2021, 173, 105901, DOI: 10.1016/j.phrs.2021.10590112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisF2gsL3N&md5=bfb84167b792d2d6c17deebf97f2b879Dual BET/HDAC inhibition to relieve neuropathic pain: Recent advances, perspectives, and future opportunitiesRomanelli, Maria Novella; Borgonetti, Vittoria; Galeotti, NicolettaPharmacological Research (2021), 173 (), 105901CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)A review. Despite the intense research on developing new therapies for neuropathic pain states, available treatments have limited efficacy and unfavorable safety profiles. Epigenetic alterations have a great influence on the development of cancer and neurol. diseases, as well as neuropathic pain. Histone acetylation has prevailed as one of the well investigated epigenetic modifications in these diseases. Altered spinal activity of histone deacetylase (HDAC) and Bromo and Extra terminal domain (BET) have been described in neuropathic pain models and restoration of these aberrant epigenetic modifications showed pain-relieving activity. Over the last decades HDACs and BETs have been the focus of drug discovery studies, leading to the development of numerous small-mol. inhibitors. Clin. trials to evaluate their anticancer activity showed good efficacy but raised toxicity concerns that limited translation to the clinic. To maximize activity and minimize toxicity, these compds. can be applied in combination of sub-maximal doses to produce additive or synergistic interactions (combination therapy). Recently, of particular interest, dual BET/HDAC inhibitors (multi-target drugs) have been developed to assure simultaneous modulation of BET and HDAC activity by a single mol. This review will summarize the most recent advances with these strategies, describing advantages and limitations of single drug treatment vs combination regimens. This review will also provide a focus on dual BET/HDAC drug discovery investigations as future therapeutic opportunity for human therapy of neuropathic pain.
- 13Mauceri, D. Role of Epigenetic Mechanisms in Chronic Pain. Cells 2022, 11, 2613, DOI: 10.3390/cells1116261313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlSlt7rO&md5=c11bade9fb3025beabae68ee572f64edRole of Epigenetic Mechanisms in Chronic PainMauceri, DanielaCells (2022), 11 (16), 2613CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)Pain is an unpleasant but essential-to-life sensation, usually resulting from tissue damage. When pain persists long after the injury has resolved, it becomes pathol. The precise mol. and cellular mechanisms causing the transition from acute to chronic pain are not fully understood. A key aspect of pain chronicity is that several plasticity events happen along the neural pathways involved in pain. These long-lasting adaptive changes are enabled by alteration in the expression of relevant genes. Among the different modulators of gene transcription in adaptive processes in the nervous system, epigenetic mechanisms play a pivotal role. In this review, I will first outline the main classes of epigenetic mediators and then discuss their implications in chronic pain.
- 14Ghosh, K.; Pan, H. L. Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic Pain. ACS Chem. Neurosci. 2022, 13, 432– 441, DOI: 10.1021/acschemneuro.1c0084114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFyqtbw%253D&md5=67079ccaf63f5fbeaa7b0903ae417539Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic PainGhosh, Krishna; Pan, Hui-LinACS Chemical Neuroscience (2022), 13 (4), 432-441CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)A review. Neuropathic pain is a challenging clin. problem and remains difficult to treat. Altered gene expression in peripheral sensory nerves and neurons by nerve injury is well documented and contributes critically to the synaptic plasticity in the spinal cord and the initiation and maintenance of chronic pain. However, our understanding of the epigenetic mechanisms regulating the transcription of pro-nociceptive (e.g., NMDA receptors and α2δ-1) and antinociceptive (e.g., potassium channels and opioid and cannabinoid receptors) genes are still limited. In this review, we summarize recent studies detg. the roles of histone modifications (including methylation, acetylation, and ubiquitination), DNA methylation, and noncoding RNAs in neuropathic pain development. We review the epigenetic writer, reader, and eraser proteins that participate in the transcriptional control of the expression of key ion channels and neurotransmitter receptors in the dorsal root ganglion after traumatic nerve injury, which is commonly used as a preclin. model of neuropathic pain. A better understanding of epigenetic reprogramming involved in the transition from acute to chronic pain could lead to the development of new treatments for neuropathic pain.
- 15Descalzi, G.; Ikegami, D.; Ushijima, T.; Nestler, E. J.; Zachariou, V.; Narita, M. Epigenetic mechanisms of chronic pain. Trends Neurosci. 2015, 38, 237– 246, DOI: 10.1016/j.tins.2015.02.00115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFGnurY%253D&md5=7002edc8fdae56befdc74cfb986459a9Epigenetic mechanisms of chronic painDescalzi, Giannina; Ikegami, Daigo; Ushijima, Toshikazu; Nestler, Eric J.; Zachariou, Venetia; Narita, MinoruTrends in Neurosciences (2015), 38 (4), 237-246CODEN: TNSCDR; ISSN:0166-2236. (Elsevier Ltd.)Neuropathic and inflammatory pain promote a large no. of persisting adaptations at the cellular and mol. level, allowing even transient tissue or nerve damage to elicit changes in cells that contribute to the development of chronic pain and assocd. symptoms. There is evidence that injury-induced changes in chromatin structure drive stable changes in gene expression and neural function, which may cause several symptoms, including allodynia, hyperalgesia, anxiety, and depression. Recent findings on epigenetic changes in the spinal cord and brain during chronic pain may guide fundamental advances in new treatments. Here, we provide a brief overview of epigenetic regulation in the nervous system and then discuss the still-limited literature that directly implicates epigenetic modifications in chronic pain syndromes.
- 16Ghosh, K.; Pan, H.-L. Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic Pain. ACS Chem. Neurosci. 2022, 13, 432– 441, DOI: 10.1021/acschemneuro.1c0084116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFyqtbw%253D&md5=67079ccaf63f5fbeaa7b0903ae417539Epigenetic Mechanisms of Neural Plasticity in Chronic Neuropathic PainGhosh, Krishna; Pan, Hui-LinACS Chemical Neuroscience (2022), 13 (4), 432-441CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)A review. Neuropathic pain is a challenging clin. problem and remains difficult to treat. Altered gene expression in peripheral sensory nerves and neurons by nerve injury is well documented and contributes critically to the synaptic plasticity in the spinal cord and the initiation and maintenance of chronic pain. However, our understanding of the epigenetic mechanisms regulating the transcription of pro-nociceptive (e.g., NMDA receptors and α2δ-1) and antinociceptive (e.g., potassium channels and opioid and cannabinoid receptors) genes are still limited. In this review, we summarize recent studies detg. the roles of histone modifications (including methylation, acetylation, and ubiquitination), DNA methylation, and noncoding RNAs in neuropathic pain development. We review the epigenetic writer, reader, and eraser proteins that participate in the transcriptional control of the expression of key ion channels and neurotransmitter receptors in the dorsal root ganglion after traumatic nerve injury, which is commonly used as a preclin. model of neuropathic pain. A better understanding of epigenetic reprogramming involved in the transition from acute to chronic pain could lead to the development of new treatments for neuropathic pain.
- 17Spering, M.; Carrasco, M. Acting without seeing: eye movements reveal visual processing without awareness. Trends Neurosci. 2015, 38, 247– 258, DOI: 10.1016/j.tins.2015.02.00217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjt1aqsrs%253D&md5=bda910509011a55b3a1a3c3d9d10f51aActing without seeing: eye movements reveal visual processing without awarenessSpering, Miriam; Carrasco, MarisaTrends in Neurosciences (2015), 38 (4), 247-258CODEN: TNSCDR; ISSN:0166-2236. (Elsevier Ltd.)Visual perception and eye movements are considered to be tightly linked. Diverse fields, ranging from developmental psychol. to computer science, utilize eye tracking to measure visual perception. However, this prevailing view has been challenged by recent behavioral studies. Here, we review converging evidence revealing dissocns. between the contents of perceptual awareness and different types of eye movement. Such dissocns. reveal situations in which eye movements are sensitive to particular visual features that fail to modulate perceptual reports. We also discuss neurophysiol., neuroimaging, and clin. studies supporting the role of subcortical pathways for visual processing without awareness. Our review links awareness to perceptual-eye movement dissocns. and furthers our understanding of the brain pathways underlying vision and movement with and without awareness.
- 18Ligon, C. O.; Moloney, R. D.; Greenwood-Van Meerveld, B. Targeting Epigenetic Mechanisms for Chronic Pain: A Valid Approach for the Development of Novel Therapeutics. J. Pharmacol. Exp. Ther. 2016, 357, 84– 93, DOI: 10.1124/jpet.115.23167018https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XpvVeksLs%253D&md5=a962565b89196b152e4fcd57ed7b784eTargeting epigenetic mechanisms for chronic pain: a valid approach for the development of novel therapeuticsLigon, Casey O.; Moloney, Rachel D.; Greenwood-Van Meerveld, BeverleyJournal of Pharmacology and Experimental Therapeutics (2016), 357 (1), 84-93CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)Chronic pain is a multifaceted and complex condition. Broadly classified into somatic, visceral, or neuropathic pain, it is poorly managed despite its prevalence. Current drugs used for the treatment of chronic pain are limited by tolerance with long-term use, abuse potential, and multiple adverse side effects. The persistent nature of pain suggests that epigenetic machinery may be a crit. factor driving chronic pain. In this review, we discuss the latest insights into epigenetic processes, including DNA methylation, histone modifications, and microRNAs, and we describe their involvement in the pathophysiol. of chronic pain and whether epigenetic modifications could be applied as future therapeutic targets for chronic pain. We provide evidence from exptl. models and translational research in human tissue that have enhanced our understanding of epigenetic processes mediating nociception, and we then speculate on the potential future use of more specific and selective agents that target epigenetic mechanisms to attenuate pain.
- 19Odell, D. W. Epigenetics of pain mediators. Curr. Opin. Anaesthesiol. 2018, 31, 402– 406, DOI: 10.1097/aco.000000000000061319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MbhvValug%253D%253D&md5=9b241240be57e986799a3aa35fe63770Epigenetics of pain mediatorsOdell Daniel WCurrent opinion in anaesthesiology (2018), 31 (4), 402-406 ISSN:.PURPOSE OF REVIEW: The field of epigenetics continues its influential rise as a means to better understand an organism's unique developmental identity over a lifespan. Whereas a genome is constant and unchanging, an epigenome is dynamic and alterable. Epigenetic changes are in response to innumerable internal and external influences including environmental changes such as diet, exercise, disease, toxins, and stress. Epigenetics is of particular interest in the medical research community both for the potential to cause disease and as a target for therapeutic interventions. This article provides a succinct explanation of the potential for epigenetics to influence the understanding of pain as well as a review of relevant research on the topic. RECENT FINDINGS: Studies on epigenetics and pain remain largely preclinical and investigate the theoretical ability of epigenetics to alter the nociceptive pathways both in the periphery and centrally. Significant evidence now exists for the ability of epigenetics to modify broadly categorized pain types, including inflammatory, neuropathic, visceral, and cancer related. SUMMARY: Both patients and providers recognize that novel medications for the treatment of both acute and chronic pain conditions are sorely needed. The understanding of epigenetics and its influence on nociception remains in relative infancy but early evidence is strong for potential therapeutic benefits to treat these conditions.
- 20Denk, F.; McMahon, S. B. Chronic pain: emerging evidence for the involvement of epigenetics. Neuron 2012, 73, 435– 444, DOI: 10.1016/j.neuron.2012.01.01220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitFOjtLw%253D&md5=3570ae634b3d1ddced5616599f57aaa8Chronic Pain: Emerging Evidence for the Involvement of EpigeneticsDenk, Franziska; McMahon, Stephen B.Neuron (2012), 73 (3), 435-444CODEN: NERNET; ISSN:0896-6273. (Cell Press)A review. Epigenetic processes, such as histone modifications and DNA methylation, have been assocd. with many neural functions including synaptic plasticity, learning, and memory. Here, we critically examine emerging evidence linking epigenetic mechanisms to the development or maintenance of chronic pain states. Although in its infancy, research in this area potentially unifies several pathophysiol. processes underpinning abnormal pain processing and opens up a different avenue for the development of novel analgesics.
- 21Sun, C.; An, Q.; Li, R.; Chen, S.; Gu, X.; An, S.; Wang, Z. Calcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic pain. CNS Neurosci. Ther. 2021, 27, 1409– 1424, DOI: 10.1111/cns.1372021https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XpvVSnsQ%253D%253D&md5=3c179a50b0cded1236c3b67ebae5548cCalcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic painSun, Chenyan; An, Qi; Li, Ruidi; Chen, Shuhui; Gu, Xinpei; An, Shuhong; Wang, ZhaojinCNS Neuroscience & Therapeutics (2021), 27 (11), 1409-1424CODEN: CNTNAB; ISSN:1755-5930. (Wiley-Blackwell)Calcitonin gene-related peptide (CGRP) as a regulator of astrocyte activation may facilitate spinal nociceptive processing. Histone H3 lysine 9 acetylation (H3K9ac) is considered an important regulator of cytokine and chemokine gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K9ac in the activation of astrocytes, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. Astroglial cells (C6) were treated with CGRP and differentially enrichments of H3K9ac on gene promoters were examd. using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on astrocyte activation and H3K9ac signaling in CCI-induced neuropathic pain. Specific inhibitors were employed to delineate the involved signaling. Intrathecal injection of CGRP and CCI increased the no. of astrocytes displaying H3K9ac in the spinal dorsal horn of rats. Treatment of CGRP was able to up-regulate H3K9ac and glial fibrillary acidic protein (GFAP) expression in astroglial cells. ChIP-seq data indicated that CGRP significantly altered H3K9ac enrichments on gene promoters in astroglial cells following CGRP treatment, including 151 gaining H3K9ac and 111 losing this mark, which mostly enriched in proliferation, autophagy, and macrophage chemotaxis processes. qRT-PCR verified expressions of representative candidate genes (ATG12, ATG4C, CX3CR1, MMP28, MTMR14, HMOX1, RET) and RTCA verified astrocyte proliferation. Addnl., CGRP treatment increased the expression of H3K9ac, CX3CR1, and IL-1β in the spinal dorsal horn. CGRP antagonist and HAT inhibitor attenuated mech. and thermal hyperalgesia in CCI rats. Such analgesic effects were concurrently assocd. with the reduced levels of H3K9ac, CX3CR1, and IL-1β in the spinal dorsal horn of CCI rats. Our findings highly indicate that CGRP is assocd. with the development of neuropathic pain through astrocytes-mediated neuroinflammatory responses via H3K9ac in spinal dorsa horn following nerve injury. This study found that CGRP act on their astrocytic receptors and lead to H3K9 acetylation (H3K9ac), which are mainly assocd. with proliferation-, autophagy-, and inflammation-related gene expression. The no. of astrocytes with H3K9ac expression is increased after nerve injury. Inhibition of CGRP attenuates the development of neuropathic pain, which was accompanied by the suppression of H3K9ac, CX3CR1, and IL-1β expression in CCI rats.
- 22Wang, J.; Chen, J.; Jin, H.; Lin, D.; Chen, Y.; Chen, X.; Wang, B.; Hu, S.; Wu, Y.; Wu, Y.; Zhou, Y.; Tian, N.; Gao, W.; Wang, X.; Zhang, X. BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in rats. J. Cell Mol. Med. 2019, 23, 3214– 3223, DOI: 10.1111/jcmm.1419622https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXosF2mtrk%253D&md5=7d45d05ede6e1d90e2150df5cd796228BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in ratsWang, Jianle; Chen, Jiaoxiang; Jin, Haiming; Lin, Dongdong; Chen, Yu; Chen, Ximiao; Wang, Ben; Hu, Sunli; Wu, Yan; Wu, Yaosen; Zhou, Yifei; Tian, Naifeng; Gao, Weiyang; Wang, Xiangyang; Zhang, XiaoleiJournal of Cellular and Molecular Medicine (2019), 23 (5), 3214-3223CODEN: JCMMC9; ISSN:1582-4934. (Wiley-Blackwell)The pathophysiol. of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro-inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF-κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro-inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro-inflammatory cytokine prodn. in microglia which ultimately promotes functional recovery after SCI.
- 23Li, Y.; Xiang, J.; Zhang, J.; Lin, J.; Wu, Y.; Wang, X. Inhibition of Brd4 by JQ1 Promotes Functional Recovery From Spinal Cord Injury by Activating Autophagy. Front. Cell. Neurosci. 2020, 14, 555591, DOI: 10.3389/fncel.2020.55559123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisF2gu7s%253D&md5=b1ba672645e6013ed780830b7b319711Inhibition of Brd4 by JQ1 promotes functional recovery from spinal cord injury by activating autophagyLi, Yao; Xiang, Jie; Zhang, Jing; Lin, Jiahao; Wu, Yaosen; Wang, XiangyangFrontiers in Cellular Neuroscience (2020), 14 (), 555591CODEN: FCNRAH; ISSN:1662-5102. (Frontiers Media S.A.)Spinal cord injury (SCI) is a destructive neurol. disorder that is characterized by impaired sensory and motor function. Inhibition of bromodomain protein 4 (Brd4) has been shown to promote the maintenance of cell homeostasis by activating autophagy. However, the role of Brd4 inhibition in SCI and the underlying mechanisms are poorly understood. Thus, the goal of the present study was to evaluate the effects of sustained Brd4 inhibition using the bromodomain and extraterminal domain (BET) inhibitor JQ1 on the regulation of apoptosis, oxidative stress and autophagy in a mouse model of SCI. First, we obsd. that Brd4 expression at the lesion sites of mouse spinal cords increased after SCI. Treatment with JQ1 significantly decreased the expression of Brd4 and improved functional recovery for up to 28 day after SCI. In addn., JQ1-mediated inhibition of Brd4 reduced oxidative stress and inhibited the expression of apoptotic proteins to promote neural survival. Our results also revealed that JQ1 treatment activated autophagy and restored autophagic flux, while the pos. effects of JQ1 were abrogated by autophagy inhibitor 3-MA intervention, indicating that autophagy plays a crucial role in therapeutic effects Brd4 induced by inhibition of the functional recovery SCI. In the mechanistic anal., we obsd. that modulation of the AMPK-mTORULK1 pathway is involved in the activation of autophagy mediated by Brd4 inhibition. Taken together, the results of our investigation provides compelling evidence that Brd4 inhibition by JQ1 promotes functional recovery after SCI and that Brd4 may serve as a potential target for SCI treatment.
- 24Rudman, M. D.; Choi, J. S.; Lee, H. E.; Tan, S. K.; Ayad, N. G.; Lee, J. K. Bromodomain and extraterminal domain-containing protein inhibition attenuates acute inflammation after spinal cord injury. Exp. Neurol. 2018, 309, 181– 192, DOI: 10.1016/j.expneurol.2018.08.00524https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsF2gur3P&md5=72e3d3bfcad4101012dc35edc101342cBromodomain and extraterminal domain-containing protein inhibition attenuates acute inflammation after spinal cord injuryRudman, Michelle D.; Choi, James S.; Lee, Ha Eun; Tan, Sze Kiat; Ayad, Nagi G.; Lee, Jae K.Experimental Neurology (2018), 309 (), 181-192CODEN: EXNEAC; ISSN:0014-4886. (Elsevier Inc.)Inflammation is a major contributor to the secondary damage that occurs after spinal cord injury (SCI). The inflammatory response is coordinated by many different signaling modalities including the epigenetic modification of promoters and enhancers. Bromodomain and extraterminal domain-contg. proteins (BETs; Brd2, Brd3, Brd4, BrdT) are epigenetic readers that bind acetylated histones to promote transcription of pro-inflammatory genes. BET inhibition is anti-inflammatory in animal models of cancer, rheumatoid arthritis, and coronary artery disease. However, the role of BETs in neuroinflammation remains largely unexplored. In this study, we investigated the role of BETs in promoting inflammation in neural cells and the ability of the BET inhibitor JQ1 to decrease inflammation acutely after SCI. Expression of BET mRNA was assessed via qPCR in purified primary mouse macrophages, astrocytes, neurons, oligodendrocytes, and microglia, as well as in naive, sham-injured, and contusion-injured mouse spinal cord. Brd2, Brd3, and Brd4 mRNA were expressed in all purified primary neural cells and in the uninjured and injured mouse spinal cord. BET inhibition significantly attenuated proinflammatory signaling in all activated cell populations in vitro. To investigate the effects of BET modulation after SCI, the BET inhibitor JQ1 was injected i.p. (30 mg/kg, bidaily) 3 h after spinal cord contusion in adult female C57BL/6 mice. By 3 days post-injury, BET inhibition significantly decreased pro-inflammatory cytokine expression and leukocyte recruitment to the injury site. However, this decrease did not lead to locomotor improvements or smaller lesion size. Taken together, our data implicate BETs as regulators of multiple key pro-inflammatory cytokines, and suggest that BETs can be pharmacol. inhibited to reduce inflammation acutely after SCI.
- 25Ferri, E.; Petosa, C.; McKenna, C. E. Bromodomains: Structure, function and pharmacology of inhibition. Biochem. Pharmacol. 2016, 106, 1– 18, DOI: 10.1016/j.bcp.2015.12.00525https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGrt7%252FM&md5=a3f075bad4b77cc890fcb708490f4c9dBromodomains: Structure, function and pharmacology of inhibitionFerri, Elena; Petosa, Carlo; McKenna, Charles E.Biochemical Pharmacology (Amsterdam, Netherlands) (2016), 106 (), 1-18CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)Bromodomains are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation. The human proteome comprises 46 bromodomain-contg. proteins with a total of 61 bromodomains, which, despite highly conserved structural features, recognize a wide array of natural peptide ligands. Over the past five years, bromodomains have attracted great interest as promising new epigenetic targets for diverse human diseases, including inflammation, cancer, and cardiovascular disease. The demonstration in 2010 that two small mol. compds., JQ1 and I-BET762, potently inhibit proteins of the bromodomain and extra-terminal (BET) family with translational potential for cancer and inflammatory disease sparked intense efforts in academia and pharmaceutical industry to develop novel bromodomain antagonists for therapeutic applications. Several BET inhibitors are already in clin. trials for hematol. malignancies, solid tumors and cardiovascular disease. Currently, the field faces the challenge of single-target selectivity, esp. within the BET family, and of overcoming problems related to the development of drug resistance. At the same time, new trends in bromodomain inhibitor research are emerging, including an increased interest in non-BET bromodomains and a focus on drug synergy with established antitumor agents to improve chemotherapeutic efficacy. This review presents an updated view of the structure and function of bromodomains, traces the development of bromodomain inhibitors and their potential therapeutic applications, and surveys the current challenges and future directions of this vibrant new field in drug discovery.
- 26Takahashi, K.; Yi, H.; Liu, C.-H.; Liu, S.; Kashiwagi, Y.; Patin, D. J.; Hao, S. Spinal bromodomain-containing protein 4 contributes to neuropathic pain induced by HIV glycoprotein 120 with morphine in rats. Neuroreport 2018, 29, 441– 446, DOI: 10.1097/wnr.000000000000099226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlvVKnt7w%253D&md5=52aaa5455a044daa0ce449e82b1986b4Spinal bromodomain-containing protein 4 contributes to neuropathic pain induced by HIV glycoprotein 120 with morphine in ratsTakahashi, Keiya; Yi, Hyun; Liu, Ching-Hang; Liu, Shue; Kashiwagi, Yuta; Patin, Dennis J.; Hao, ShuanglinNeuroReport (2018), 29 (6), 441-446CODEN: NERPEZ; ISSN:0959-4965. (Lippincott Williams & Wilkins)The symptoms of HIV-sensory neuropathy are dominated by neuropathic pain. Recent data show that repeated use of opiates enhances the chronic pain states in HIV patients. Limited attention has so far been devoted to exploring the exact pathogenesis of HIV painful disorder and opiate abuse in vivo, for which there is no effective treatment. Bromodomain-contg. protein 4 (Brd4) is a member of the bromodomain and extraterminal domain protein (BET) family and functions as a chromatin 'reader' that binds acetylated lysines in histones in brain neurons to mediate the transcriptional regulation underlying learning and memory. Here, we established a neuropathic pain model of interaction of intrathecal HIV envelope glycoprotein 120 (gp120) and chronic morphine in rats. The combination of gp120 and morphine (gp120/M, for 5 days) induced persistent mech. allodynia compared with either gp120 or morphine alone. Mech. allodynia reached the lowest values at day 10 from gp120/M application, beginning to recover from day 21. In the model, gp120/M induced overexpression of Brd4 mRNA and protein at day 10 using RT-qPCR and western blots, resp. Immunohistochem. studies showed that Brd4 at day 10 was expressed in the neurons of spinal cord dorsal horn. BET inhibitor I-BET762 dose-dependently increased the mech. threshold in the gp120/M pain state. The present study provides preclin. evidence for treating HIV neuropathic pain with opioids using the BET inhibitor.
- 27Palomes-Borrajo, G.; Badia, J.; Navarro, X.; Penas, C. Nerve Excitability and Neuropathic Pain is Reduced by BET Protein Inhibition After Spared Nerve Injury. J. Pain 2021, 22, 1617– 1630, DOI: 10.1016/j.jpain.2021.05.00527https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XmslGgtg%253D%253D&md5=8ad7d5c77a6c757a79e51e6e6e3f8d4bNerve Excitability and Neuropathic Pain is Reduced by BET Protein Inhibition After Spared Nerve InjuryPalomes-Borrajo, Georgina; Badia, Jordi; Navarro, Xavier; Penas, ClaraJournal of Pain (2021), 22 (12), 1617-1630CODEN: JPOAB5; ISSN:1526-5900. (Elsevier Inc.)Neuropathic pain is a common disability produced by enhanced neuronal excitability after nervous system injury. The pathophysiol. changes that underlie the generation and maintenance of neuropathic pain require modifications of transcriptional programs. In particular, there is an induction of pro-inflammatory neuromodulators levels, and changes in the expression of ion channels and other factors intervening in the detn. of the membrane potential in neuronal cells. We have previously found that inhibition of the BET proteins epigenetic readers reduced neuroinflammation after spinal cord injury. Within the present study we aimed to det. if BET protein inhibition may also affect neuroinflammation after a peripheral nerve injury, and if this would beneficially alter neuronal excitability and neuropathic pain. For this purpose, C57BL/6 female mice underwent spared nerve injury (SNI), and were treated with the BET inhibitor JQ1, or vehicle. Electrophysiol. and algesimetry tests were performed on these mice. We also detd. the effects of JQ1 treatment after injury on neuroinflammation, and the expression of neuronal components important for the maintenance of axon membrane potential. We found that treatment with JQ1 affected neuronal excitability and mech. hyperalgesia after SNI in mice. BET protein inhibition regulated cytokine expression and reduced microglial reactivity after injury. In addn., JQ1 treatment altered the expression of SCN3A, SCN9A, KCNA1, KCNQ2, KCNQ3, HCN1 and HCN2 ion channels, as well as the expression of the Na+/K+ ATPase pump subunits. In conclusion, both, alteration of inflammation, and neuronal transcription, could be the responsible epigenetic mechanisms for the redn. of excitability and hyperalgesia obsd. after BET inhibition. Inhibition of BET proteins is a promising therapy for reducing neuropathic pain after neural injury.
- 28Vasavda, C.; Xu, R.; Liew, J.; Kothari, R.; Dhindsa, R. S.; Semenza, E. R.; Paul, B. D.; Green, D. P.; Sabbagh, M. F.; Shin, J. Y.; Yang, W.; Snowman, A. M.; Albacarys, L. K.; Moghekar, A.; Pardo-Villamizar, C. A.; Luciano, M.; Huang, J.; Bettegowda, C.; Kwatra, S. G.; Dong, X.; Lim, M.; Snyder, S. H. Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain. Sci. Adv. 2022, 8, eabo5633 DOI: 10.1126/sciadv.abo5633There is no corresponding record for this reference.
- 29Borgonetti, V.; Galeotti, N. Combined inhibition of histone deacetylases and BET family proteins as epigenetic therapy for nerve injury-induced neuropathic pain. Pharmacol. Res. 2021, 165, 105431, DOI: 10.1016/j.phrs.2021.10543129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmvVKksbc%253D&md5=7a8db7ff3af849ab329f0da8b25f5990Combined inhibition of histone deacetylases and BET family proteins as epigenetic therapy for nerve injury-induced neuropathic painBorgonetti, Vittoria; Galeotti, NicolettaPharmacological Research (2021), 165 (), 105431CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)Current treatments for neuropathic pain have often moderate efficacy and present unwanted effects showing the need to develop effective therapies. Accumulating evidence suggests that histone acetylation plays essential roles in chronic pain and the analgesic activity of histone deacetylases (HDACs) inhibitors is documented. Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that interact with acetylated lysine residues on histones, but little is known about their implication in neuropathic pain. Thus, the current study was aimed to investigate the effect of the combination of HDAC and BET inhibitors in the spared nerve injury (SNI) model in mice. Intranasal administration of i-BET762 (BET inhibitor) or SAHA (HDAC inhibitor) attenuated thermal and mech. hypersensitivity and this antiallodynic activity was improved by co-administration of both drugs. Spinal cord sections of SNI mice showed an increased expression of HDAC1 and Brd4 proteins and combination produced a stronger redection compared to each epigenetic agent alone. SAHA and i-BET762, administered alone or in combination, counteracted the SNI-induced microglia activation by inhibiting the expression of IBA1, CD11b, inducible nitric oxide synthase (iNOS), the activation of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription-1 (STAT1) with comparable efficacy. Conversely, the epigenetic inhibitors showed a modest effect on spinal proinflammatory cytokines content that was significantly potentiated by their combination. Present results indicate a key role of acetylated histones and their recruitment by BET proteins on microglia-mediated spinal neuroinflammation. Targeting neuropathic pain with the combination of HDAC and BET inhibitors may represent a promising new therapeutic option.
- 30Borgonetti, V.; Meacci, E.; Pierucci, F.; Romanelli, M. N.; Galeotti, N. Dual HDAC/BRD4 Inhibitors Relieves Neuropathic Pain by Attenuating Inflammatory Response in Microglia After Spared Nerve Injury. Neurotherapeutics 2022, 19, 1634– 1648, DOI: 10.1007/s13311-022-01243-630https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvVSmsbnN&md5=71d3a419e62de54d4ff5fc4a39a5aa7bDual HDAC/BRD4 Inhibitors Relieves Neuropathic Pain by Attenuating Inflammatory Response in Microglia After Spared Nerve InjuryBorgonetti, Vittoria; Meacci, Elisabetta; Pierucci, Federica; Romanelli, Maria Novella; Galeotti, NicolettaNeurotherapeutics (2022), 19 (5), 1634-1648CODEN: NEURNV; ISSN:1878-7479. (Springer)Despite the effort on developing new treatments, therapy for neuropathic pain is still a clin. challenge and combination therapy regimes of two or more drugs are often needed to improve efficacy. Accumulating evidence shows an altered expression and activity of histone acetylation enzymes in chronic pain conditions and restoration of these aberrant epigenetic modifications promotes pain-relieving activity. Recent studies showed a synergistic activity in neuropathic pain models by combination of histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) inhibitors. On these premises, the present study investigated the pharmacol. profile of new dual HDAC/BRD4 inhibitors, named SUM52 and SUM35, in the spared nerve injury (SNI) model in mice as innovative strategy to simultaneously inhibit HDACs and BETs. Intranasal administration of SUM52 and SUM35 attenuated thermal and mech. hypersensitivity in the absence of locomotor side effects. Both dual inhibitors showed a preferential interaction with BRD4-BD2 domain, and SUM52 resulted the most active compd. SUM52 reduced microglia-mediated spinal neuroinflammation in spinal cord sections of SNI mice as showed by redn. of IBA1 immunostaining, inducible nitric oxide synthase (iNOS) expression, p65 nuclear factor-κB (NF-κB) and p38 MAPK over-phosphorylation. A robust decrease of the spinal proinflammatory cytokines content (IL-6, IL-1ss) was also obsd. after SUM52 treatment. Present results, showing the pain-relieving activity of HDAC/BRD4 dual inhibitors, indicate that the simultaneous modulation of BET and HDAC activity by a single mol. acting as multi-target agent might represent a promise for neuropathic pain relief.
- 31Liu, Z.; Chen, H.; Wang, P.; Li, Y.; Wold, E. A.; Leonard, P. G.; Joseph, S.; Brasier, A. R.; Tian, B.; Zhou, J. Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation. J. Med. Chem. 2020, 63, 5242– 5256, DOI: 10.1021/acs.jmedchem.0c0003531https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVOhsbs%253D&md5=95cb6c3f150a5ee9e65a76fd36a559fdDiscovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological EvaluationLiu, Zhiqing; Chen, Haiying; Wang, Pingyuan; Li, Yi; Wold, Eric A.; Leonard, Paul G.; Joseph, Sarah; Brasier, Allan R.; Tian, Bing; Zhou, JiaJournal of Medicinal Chemistry (2020), 63 (10), 5242-5256CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Arylquinazolinones and arylchromenones such as I (X = CH2, MeN) were prepd. as selective inhibitors of bromodomain-contg. protein 4 (BRD4) for potential use as orally bioavailable antiinflammatory agents. I inhibited BRD4 with IC50 values of 67-84 nM and were selective for BRD1 over binding domains of BRD2, BRD3, and BRDT and over CBP; I inhibited the expression of Toll-like receptor (TLR3)-induced inflammatory genes in vitro and inhibited airway inflammation in mice. The pharmacokinetics (t1/2, AUC, Cmax, and clearance), metabolic stability of I (X = NMe) in murine and human cells, and inhibition of cytochrome P450 enzymes and hERG by I (X = MeN) were detd. The structure of I (X = NMe) bound to human BRD4 binding domain 1 was detd. by X-ray crystallog.
- 32Hu, J.; Tian, C. Q.; Damaneh, M. S.; Li, Y.; Cao, D.; Lv, K.; Yu, T.; Meng, T.; Chen, D.; Wang, X.; Chen, L.; Li, J.; Song, S. S.; Huan, X. J.; Qin, L.; Shen, J.; Wang, Y. Q.; Miao, Z. H.; Xiong, B. Structure-Based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein Inhibitors. J. Med. Chem. 2019, 62, 8642– 8663, DOI: 10.1021/acs.jmedchem.9b0109432https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslegsbvK&md5=af9fa23078f8280cc36d3c097d6fbf34Structure-Based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein InhibitorsHu, Jianping; Tian, Chang-Qing; Damaneh, Mohammadali Soleimani; Li, Yanlian; Cao, Danyan; Lv, Kaikai; Yu, Ting; Meng, Tao; Chen, Danqi; Wang, Xin; Chen, Lin; Li, Jian; Song, Shan-Shan; Huan, Xia-Juan; Qin, Lihuai; Shen, Jingkang; Wang, Ying-Qing; Miao, Ze-Hong; Xiong, BingJournal of Medicinal Chemistry (2019), 62 (18), 8642-8663CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)BRD4 has recently emerged as a promising drug target. Therefore, identifying novel inhibitors with distinct properties could enrich their use in anticancer treatment. Guided by the cocrystal structure of hit compd. 4 harboring a five-membered-ring linker motif, we quickly identified lead compd. 7, which exhibited good antitumor effects in an MM.1S xenograft model by oral administration. Encouraged by its high potency and interesting scaffold, we performed further lead optimization to generate a novel potent series of bromodomain and extra-terminal (BET) inhibitors with a (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one structure. Among them, compd. 19 was found to have the best balance of activity, stability, and antitumor efficacy. After confirming its low brain penetration, we conducted comprehensive preclin. studies, including a multiple-species pharmacokinetics profile, extensive cellular mechanism studies, hERG assay, and in vivo antitumor growth effect testing, and we found that compd. 19 is a potential BET protein drug candidate for the treatment of cancer.
- 33Zhang, M.; Zhang, Y.; Song, M.; Xue, X.; Wang, J.; Wang, C.; Zhang, C.; Li, C.; Xiang, Q.; Zou, L.; Wu, X.; Wu, C.; Dong, B.; Xue, W.; Zhou, Y.; Chen, H.; Wu, D.; Ding, K.; Xu, Y. Structure-Based Discovery and Optimization of Benzo[ d]isoxazole Derivatives as Potent and Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC). J. Med. Chem. 2018, 61, 3037– 3058, DOI: 10.1021/acs.jmedchem.8b0010333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXls12rs78%253D&md5=2dbbfc3d1916482c817ad13cd3ef9edaStructure-Based Discovery and Optimization of Benzo[d]isoxazole Derivatives as Potent and Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC)Zhang, Maofeng; Zhang, Yan; Song, Ming; Xue, Xiaoqian; Wang, Junjian; Wang, Chao; Zhang, Cheng; Li, Chenchang; Xiang, Qiuping; Zou, Lingjiao; Wu, Xishan; Wu, Chun; Dong, Baijun; Xue, Wei; Zhou, Yulai; Chen, Hongwu; Wu, Donghai; Ding, Ke; Xu, YongJournal of Medicinal Chemistry (2018), 61 (7), 3037-3058CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The bromodomain and extra-terminal (BET) family proteins have gained increasing interest as drug targets for treatment of castration-resistant prostate cancer (CRPC). Here, the authors describe the design, optimization, and evaluation of benzo[d]isoxazole-contg. compds. as potent BET bromodomain inhibitors. Cocrystal structures of the representative inhibitors in complex with BRD4(1) provided solid structural basis for compd. optimization. The two most potent compds., 6i (Y06036) and 7m (Y06137), bound to the BRD4(1) bromodomain with Kd values of 82 and 81 nM, resp. They also exhibited high selectivity over other non-BET subfamily members. The compds. potently inhibited cell growth, colony formation, and the expression of AR, AR regulated genes, and MYC in prostate cancer cell lines. Compds. 6i and 7m also demonstrated therapeutic effects in a C4-2B CRPC xenograft tumor model in mice. These potent and selective BET inhibitors represent a new class of compds. for the development of potential therapeutics against CRPC.
- 34Huang, Y.; Liu, N.; Pan, Z.; Li, Z.; Sheng, C. BET-HDAC Dual Inhibitors for Combinational Treatment of Breast Cancer and Concurrent Candidiasis. J. Med. Chem. 2023, 66, 1239– 1253, DOI: 10.1021/acs.jmedchem.2c0119134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXlsValsg%253D%253D&md5=e4118948754bbbc36d4981d1d521b3d8BET-HDAC Dual Inhibitors for Combinational Treatment of Breast Cancer and Concurrent CandidiasisHuang, Yahui; Liu, Na; Pan, Zhizhi; Li, Zhuang; Sheng, ChunquanJournal of Medicinal Chemistry (2023), 66 (2), 1239-1253CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Breast cancer is susceptible to Candida infections, and candidiasis has an enhancing effect on the progression and metastasis of tumor. Breast cancer and concurrent candidiasis represent a significant challenge in clin. therapy. Herein, a series of novel small mol. inhibitors simultaneously targeting bromodomain and extra-terminal (BET) and histone deacetylase (HDAC) were designed for combinational treatment of breast cancer and resistant Candida albicans infections. Among them, compds. 13c I and 17b II exhibited excellent and balanced inhibitory activity against both BET family proteins BRD4 and HDAC1. As compared with BRD4 or HDAC1 inhibitors, dual inhibitors 13c I and 17b II displayed improved in vivo antitumor efficacy in MDA-MB-231 breast cancer xenograft models. Notably, they synergized with fluconazole (FLC) to effectively reduce the kidney fungal burden in a murine model of disseminated candidiasis. Thus, the BET-HDAC dual inhibitors represented a novel therapeutic strategy for combinational treatment of breast cancer and concurrent candidiasis.
- 35Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J. P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Muller, S.; Pawson, T.; Gingras, A. C.; Arrowsmith, C. H.; Knapp, S. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 2012, 149, 214– 231, DOI: 10.1016/j.cell.2012.02.01335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltVamurs%253D&md5=05583a01a1cd4c3b59fc59c00e4c4777Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain FamilyFilippakopoulos, Panagis; Picaud, Sarah; Mangos, Maria; Keates, Tracy; Lambert, Jean-Philippe; Barsyte-Lovejoy, Dalia; Felletar, Ildiko; Volkmer, Rudolf; Muller, Susanne; Pawson, Tony; Gingras, Anne-Claude; Arrowsmith, Cheryl H.; Knapp, StefanCell (Cambridge, MA, United States) (2012), 149 (1), 214-231CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resoln. crystal structures, covering all BRD families. These proteins are: ASH1L, ATAD2, BAZ2B, BPTF, BRD1, BRD3(1), BRD3(2), BRD4(1), BRD4(2), BRD9, BRDT(1), CECR2, EP300, CREBBP, GCN5L2, KIAA1240, PB1(1), PB1(2), PB1(3), PB1(4), PB1(5), PB1(6), PCAF, PHIP(2), TAF1(2), WDR9(2), BRD4(1). Comprehensive crossfamily structural anal. identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-contg. peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.
- 36Zhao, Y.; Bai, L.; Liu, L.; McEachern, D.; Stuckey, J. A.; Meagher, J. L.; Yang, C. Y.; Ran, X.; Zhou, B.; Hu, Y.; Li, X.; Wen, B.; Zhao, T.; Li, S.; Sun, D.; Wang, S. Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethy lisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain Inhibitor. J. Med. Chem. 2017, 60, 3887– 3901, DOI: 10.1021/acs.jmedchem.7b0019336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVKru7s%253D&md5=915019e36ab5f5b83b69b1bbc291abb0Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain InhibitorZhao, Yujun; Bai, Longchuan; Liu, Liu; McEachern, Donna; Stuckey, Jeanne A.; Meagher, Jennifer L.; Yang, Chao-Yie; Ran, Xu; Zhou, Bing; Hu, Yang; Li, Xiaoqin; Wen, Bo; Zhao, Ting; Li, Siwei; Sun, Duxin; Wang, ShaomengJournal of Medicinal Chemistry (2017), 60 (9), 3887-3901CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 9H-pyrimido[4,5-b]indole-contg. compds. was designed and synthesized to obtain potent and orally bioavailable BET inhibitors. By incorporation of an indole or a quinoline moiety to the 9H-pyrimido[4,5-b]indole core, we identified a series of small mols. showing high binding affinities to BET proteins and low nanomolar potencies in inhibition of cell growth in acute leukemia cell lines. One such compd., 4-(6-methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (I) has excellent microsomal stability and good oral pharmacokinetics in rats and mice. Orally administered, I achieves significant antitumor activity in the MV4;11 leukemia and MDA-MB-231 triple-neg. breast cancer xenograft models in mice. Detn. of the cocrystal structure of I with BRD4 BD2 provides a structural basis for its high binding affinity to BET proteins. Testing its binding affinities against other bromodomain-contg. proteins shows that I is a highly selective inhibitor of BET proteins. These data show that I is a potent, selective, and orally active BET inhibitor.
- 37He, S.; Dong, G.; Li, Y.; Wu, S.; Wang, W.; Sheng, C. Potent Dual BET/HDAC Inhibitors for Efficient Treatment of Pancreatic Cancer. Angew. Chem., Int. Ed. 2020, 59, 3028, DOI: 10.1002/anie.20191589637https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFegtLw%253D&md5=78db62cb533611939d9abd0e654ff5f3Potent Dual BET/HDAC Inhibitors for Efficient Treatment of Pancreatic CancerHe, Shipeng; Dong, Guoqiang; Li, Yu; Wu, Shanchao; Wang, Wei; Sheng, ChunquanAngewandte Chemie, International Edition (2020), 59 (8), 3028-3032CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)As one of the most aggressive and lethal human malignancies with extremely poor prognosis, there is an urgent demand of more effective therapy for the treatment of pancreatic cancer. Reported here is a new, effective therapeutic strategy and the design of small-mol. inhibitors that simultaneously target bromodomain and extra-terminal (BET) and histone deacetylase (HDAC), potentially serving as promising therapeutic agents for pancreatic cancer. A highly potent dual inhibitor (13 a) is identified to possess excellent and balanced activities against BRD4 BD1 (IC50=11 nM) and HDAC1 (IC50=21 nM). Notably, this compd. shows higher in vitro and in vivo antitumor potency than the BET inhibitor (+)-JQ1 and the HDAC inhibitor vorinostat, either alone or and in combination, highlighting the advantages of BET/HDAC dual inhibitors for more effective treatment of pancreatic cancer.
- 38Liu, Z.; Li, Y.; Chen, H.; Lai, H.-T.; Wang, P.; Wu, S.-Y.; Wold, E. A.; Leonard, P. G.; Joseph, S.; Hu, H.; Chiang, C.-M.; Brasier, A. R.; Tian, B.; Zhou, J. Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site. J. Med. Chem. 2022, 65, 2388– 2408, DOI: 10.1021/acs.jmedchem.1c0185138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFOjtw%253D%253D&md5=b21df874c6323304ccceea1b265711e5Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding SiteLiu, Zhiqing; Li, Yi; Chen, Haiying; Lai, Hsien-Tsung; Wang, Pingyuan; Wu, Shwu-Yuan; Wold, Eric A.; Leonard, Paul G.; Joseph, Sarah; Hu, Haitao; Chiang, Cheng-Ming; Brasier, Allan R.; Tian, Bing; Zhou, JiaJournal of Medicinal Chemistry (2022), 65 (3), 2388-2408CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Bromodomain-contg. protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this crit. system and the progress toward clin. development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural anal. of ZL0590 in complex with human BRD4 BD1 and the assocd. mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biol. underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.
- 39Zeng, L.; Zhou, M.-M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 2002, 513, 124– 128, DOI: 10.1016/s0014-5793(01)03309-939https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitFejtbo%253D&md5=79070a63cd171f846ab577aa9f7092bbBromodomain: an acetyl-lysine binding domainZeng, Lei; Zhou, Ming-MingFEBS Letters (2002), 513 (1), 124-128CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)A review with 43 refs. Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins assocd. with chromatin and in nearly all nuclear histone acetyltransferases, have been recently discovered to function as acetyllysine-binding domains. More recent structural studies of bromodomain/peptide ligand complexes have enriched the understanding of differences in ligand selectivity of bromodomains. These new findings demonstrate that bromodomain/acetyllysine recognition can serve as a pivotal mechanism for regulating protein-protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation, and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site.
- 40Liu, Z.; Wang, P.; Chen, H.; Wold, E. A.; Tian, B.; Brasier, A. R.; Zhou, J. Drug Discovery Targeting Bromodomain-Containing Protein 4. J. Med. Chem. 2017, 60, 4533– 4558, DOI: 10.1021/acs.jmedchem.6b0176140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXis1yiurg%253D&md5=6d7ba5844073fac8c916b1249af402e5Drug Discovery Targeting Bromodomain-Containing Protein 4Liu, Zhiqing; Wang, Pingyuan; Chen, Haiying; Wold, Eric A.; Tian, Bing; Brasier, Allan R.; Zhou, JiaJournal of Medicinal Chemistry (2017), 60 (11), 4533-4558CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. BRD4, the most extensively studied member of BET family, is an epigenetic regulator that localizes to DNA via binding acetylated histones and controls the expression of therapeutically important gene regulatory networks through recruiting transcription factors to form mediator complexes, phosphorylating RNA polymerase II and by its intrinsic histone acetyltransferase activity. Disrupting the protein-protein interactions between BRD4 and acetyl-lysine has been shown to effectively block cell proliferation in cancer, cytokine prodn. in acute inflammation, etc. To date, significant efforts have been devoted to the development of BRD4 inhibitors, and consequently, a dozen have progressed into human clin. trials. Herein, the authors summarize the advances in drug discovery and development of BRD4 inhibitors by focusing on their chemotypes, in vitro and in vivo activity, selectivity, relevant mechanisms of action and therapeutic potential. Opportunities and challenges to achieve selective and efficacious BRD4 inhibitors as a viable therapeutic strategy for human diseases are also highlighted.
- 41Tang, P.; Zhang, J.; Liu, J.; Chiang, C. M.; Ouyang, L. Targeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological Development. J. Med. Chem. 2021, 64, 2419– 2435, DOI: 10.1021/acs.jmedchem.0c0148741https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXksFGmsr8%253D&md5=f63a3dad4fb1fab28fe245da72c2fa1cTargeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological DevelopmentTang, Pan; Zhang, Jifa; Liu, Jie; Chiang, Cheng-Ming; Ouyang, LiangJournal of Medicinal Chemistry (2021), 64 (5), 2419-2435CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Bromodomain and extraterminal (BET) proteins bind acetylated lysine residues in histones and nonhistone proteins via tandem bromodomains and regulate chromatin dynamics, cellular processes, and disease procession. Thus targeting BET proteins is a promising strategy for treating various diseases, esp. malignant tumors and chronic inflammation. Many pan-BET small-mol. inhibitors have been described, and some of them are in clin. evaluation. Nevertheless, the limited clin. efficacy of the current BET inhibitors is also evident and has inspired the development of new technologies to improve their clin. outcomes and minimize unwanted side effects. In this Review, we summarize the latest protein characteristics and biol. functions of BRD4 as an example of BET proteins, analyze the clin. development status and preclin. resistance mechanisms, and discuss recent advances in BRD4-selective inhibitors, dual-target BET inhibitors, proteolysis targeting chimera degraders, and protein-protein interaction inhibitors.
- 42Cui, H.; Carlson, A. S.; Schleiff, M. A.; Divakaran, A.; Johnson, J. A.; Buchholz, C. R.; Zahid, H.; Vail, N. R.; Shi, K.; Aihara, H.; Harki, D. A.; Miller, G. P.; Topczewski, J. J.; Pomerantz, W. C. K. 4-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved Selectivity. J. Med. Chem. 2021, 64, 10497– 10511, DOI: 10.1021/acs.jmedchem.1c0093342https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1WmtbnK&md5=6dfafd833706b5a418e5998f73c2eb944-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved SelectivityCui, Huarui; Carlson, Angela S.; Schleiff, Mary A.; Divakaran, Anand; Johnson, Jorden A.; Buchholz, Caroline R.; Zahid, Huda; Vail, Nora R.; Shi, Ke; Aihara, Hideki; Harki, Daniel A.; Miller, Grover P.; Topczewski, Joseph J.; Pomerantz, William C. K.Journal of Medicinal Chemistry (2021), 64 (14), 10497-10511CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein-protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3-triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure-activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, resp. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 (I) and 26 (II) are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.
- 43Chen, J.; Tang, P.; Wang, Y.; Wang, J.; Yang, C.; Li, Y.; Yang, G.; Wu, F.; Zhang, J.; Ouyang, L. Targeting Bromodomain-Selective Inhibitors of BET Proteins in Drug Discovery and Development. J. Med. Chem. 2022, 65, 5184– 5211, DOI: 10.1021/acs.jmedchem.1c0183543https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XnvVKhsr0%253D&md5=bd2bb412aad23fa184778d41fc957a21Targeting Bromodomain-Selective Inhibitors of BET Proteins in Drug Discovery and DevelopmentChen, Juncheng; Tang, Pan; Wang, Yuxi; Wang, Jiaxing; Yang, Chengcan; Li, Yang; Yang, Gaoxia; Wu, Fengbo; Zhang, Jifa; Ouyang, LiangJournal of Medicinal Chemistry (2022), 65 (7), 5184-5211CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Blocking the interactions between bromodomain and extraterminal (BET) proteins and acetylated lysines of histones by small mols. has important implications for the treatment of cancers and other diseases. Many pan-BET inhibitors have shown satisfactory results in clin. trials, but their potential for poor tolerability and toxicity persist. However, recently reported studies illustrate that some BET bromodomain (BET-BD1 or BET-BD2)-selective inhibitors have advantage over pan-inhibitors, including reduced toxicity concerns. Furthermore, some selective BET inhibitors have similar or even better therapeutic efficacy in inflammatory diseases or cancers. Therefore, the development of selective BET inhibitors has become a hot spot for medicinal chemists. Here, we summarize the known selective BET-BD1 and BET-BD2 inhibitors and review the methods for enhancing the selectivity and potency of these inhibitors based on their different modes of interactions with BET-BD1 or BET-BD2. Finally, we discuss prospective strategies that selectively target the bromodomains of BET proteins.
- 44Liang, D.; Yu, Y.; Ma, Z. Novel strategies targeting bromodomain-containing protein 4 (BRD4) for cancer drug discovery. Eur. J. Med. Chem. 2020, 200, 112426, DOI: 10.1016/j.ejmech.2020.11242644https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtV2ktLvL&md5=dd01654a37d6e9045f92caf772a08df3Novel strategies targeting bromodomain-containing protein 4 (BRD4) for cancer drug discoveryLiang, Dailin; Yu, Yifan; Ma, ZonghuiEuropean Journal of Medicinal Chemistry (2020), 200 (), 112426CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)A review. As epigenetic readers of the histone code, BRD4 is the most extensively and thoroughly studied member of BET family, which plays a crit. role in many human diseases including cancer, inflammation, HIV infections, CNS disorders, and cardiovascular diseases and has been proved to be a promising therapeutic target for these diseases. To date, many small-mol. BRD4 inhibitors have been discovered, and some of them are in clin. trials for the treatment of different diseases. Due to the lack of selectivity of these small mols. for BRD4 BD1, BRD4 BD2 and/or other BET proteins, they exert some toxic side effects, including dizziness, nausea, and vomit. Now, novel strategies are urgent needed to improve the selectivity and reduce the side effects of current BRD4 inhibitors. Herein, in this article, we made a summary of the recent development of novel strategies targeting BRD4. Opportunities for these strategies to achieve selective and efficacious BRD4 inhibitors for treating human diseases are also highlighted.
- 45Hajmirza, A.; Emadali, A.; Gauthier, A.; Casasnovas, O.; Gressin, R.; Callanan, M. B. BET Family Protein BRD4: An Emerging Actor in NFκB Signaling in Inflammation and Cancer. Biomedicines 2018, 6, 16, DOI: 10.3390/biomedicines601001645https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjslGqsLo%253D&md5=5ee66c89aa4f18d643cd13a43e1ad7e3BET family protein BRD4: an emerging actor in NFκB signaling in inflammation and cancerHajmirza, Azadeh; Emadali, Anouk; Gauthier, Arnaud; Casasnovas, Olivier; Gressin, Remy; Callanan, Mary B.Biomedicines (2018), 6 (1), 16/1-16/9CODEN: BIOMID; ISSN:2227-9059. (MDPI AG)NFκB (Nuclear Factor-κ-light-chain-enhancer of activated B cells) signaling elicits global transcriptional changes by activating cognate promoters and through genome-wide remodeling of cognate regulatory elements called "super enhancers". BET (Bromodomain and Extra-Terminal domain) protein family inhibitor studies have implicated BET protein member BRD4 and possibly other BET proteins in NFκB-dependent promoter and super-enhancer modulation. Members of the BET protein family are known to bind acetylated chromatin to facilitate access by transcriptional regulators to chromatin, as well as to assist the activity of transcription elongation complexes via CDK9/pTEFb. BET family member BRD4 has been shown to bind non-histone proteins and modulate their activity. One such protein is RELA, the NFκB co-activator. Specifically, BRD4 binds acetylated RELA, which increases its transcriptional transactivation activity and stability in the nucleus. In aggregate, this establishes an intimate link between NFκB and BET signaling, at least via BRD4. The present review provides a brief overview of the structure and function of BET family proteins and then examines the connections between NFκB and BRD4 signaling, using the inflammatory response and cancer cell signaling as study models. We also discuss the potential of BET inhibitors for relief of aberrant NFκB signaling in cancer, focusing on non-histone, acetyl-lysine binding functions.
- 46Chiang, C. M. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4. F1000 Biol. Rep. 2009, 1, 98, DOI: 10.3410/b1-9846https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3MrlvVygtQ%253D%253D&md5=d575a30cf8b3d918367d6580c2c0d64aBrd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4Chiang Cheng-MingF1000 biology reports (2009), 1 (), 98 ISSN:.Bromodomain-containing protein 4 (Brd4) contains two tandem bromodomains (BD1 and BD2) that bind preferentially to acetylated lysine residues found in histones and nonhistone proteins. This molecular recognition allows Brd4 to associate with acetylated chromatin throughout the cell cycle and regulates transcription at targeted loci. Recruitment of positive transcription elongation factor b, and possibly the general initiation cofactor Mediator as well, plays an important role in Brd4-regulated transcription. Selective contacts with acetyl-lysines in nucleosomal histones and chromatin-binding factors likely provide a molecular switch modulating the steps from chromatin targeting to transcriptional regulation, thus further expanding the 'acetylation code' for combinatorial regulation in eukaryotes.
- 47Duan, Y.; Guan, Y.; Qin, W.; Zhai, X.; Yu, B.; Liu, H. Targeting Brd4 for cancer therapy: inhibitors and degraders. Medchemcomm 2018, 9, 1779– 1802, DOI: 10.1039/c8md00198g47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVGisbrP&md5=b244437266ec6a0d3398bf63897beca4Targeting Brd4 for cancer therapy: inhibitors and degradersDuan, Yingchao; Guan, Yuanyuan; Qin, Wenping; Zhai, Xiaoyu; Yu, Bin; Liu, HongminMedChemComm (2018), 9 (11), 1779-1802CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Bromodomain-contg. protein 4 (Brd4) plays an important role in mediating the expression of genes involved in cancers and non-cancer diseases such as inflammatory diseases and acute heart failure. Inactivating Brd4 or downregulating its expression inhibits cancer development, leading to the current interest in Brd4 as a promising anticancer drug target. Numerous Brd4 inhibitors have been studied in recent years and some of them are currently in various phases of clin. trials. Recently, selective degrdn. of target proteins by small bifunctional mols. (PROTACs) has emerged as an attractive drug discovery approach owing to the advantages it could offer over traditional small-mol. inhibitors. A no. of Brd4 degraders have been reported and showed more efficient anticancer activities than just protein inhibition. In this review, we will discuss recent findings in the discovery and development of small-mol. inhibitors and degraders that target Brd4 as a potential anticancer agent.
- 48Xue, X.; Zhang, Y.; Wang, C.; Zhang, M.; Xiang, Q.; Wang, J.; Wang, A.; Li, C.; Zhang, C.; Zou, L.; Wang, R.; Wu, S.; Lu, Y.; Chen, H.; Ding, K.; Li, G.; Xu, Y. Benzoxazinone-containing 3,5-dimethylisoxazole derivatives as BET bromodomain inhibitors for treatment of castration-resistant prostate cancer. Eur. J. Med. Chem. 2018, 152, 542– 559, DOI: 10.1016/j.ejmech.2018.04.03448https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVShurjM&md5=dc7275ea2931fa922f265aa03a6d8449Benzoxazinone-containing 3,5-dimethylisoxazole derivatives as BET bromodomain inhibitors for treatment of castration-resistant prostate cancerXue, Xiaoqian; Zhang, Yan; Wang, Chao; Zhang, Maofeng; Xiang, Qiuping; Wang, Junjian; Wang, Anhui; Li, Chenchang; Zhang, Cheng; Zou, Lingjiao; Wang, Rui; Wu, Shuang; Lu, Yongzhi; Chen, Hongwu; Ding, Ke; Li, Guohui; Xu, YongEuropean Journal of Medicinal Chemistry (2018), 152 (), 542-559CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Design, synthesis and evaluation of a new series of benzoxazinone-contg. 3,5-dimethylisoxazole derivs. I (R1 = n-Bu, CH2-cyclopropyl, Bn, etc.) as selective BET inhibitors was reported. One of the new compds., (R)-I (R1 = cyclopropylmethyl), binds to BRD4(1) with a Kd value of 110 nM and blocks bromodomain and acetyl lysine interactions with an IC50 value of 100 nM. It also exhibits selectivity for BET over non-BET bromodomain proteins and demonstrates reasonable anti-proliferation and colony formation inhibition effect in prostate cancer cell lines such as 22Rv1 and C4-2B. The BRD4 inhibitor (R)-I (R1 = cyclopropylmethyl) also significantly suppresses the expression of ERG, Myc and AR target gene PSA at the mRNA level in prostate cancer cells. Treatment with (R)-I (R1 = cyclopropylmethyl) significantly suppresses the tumor growth of prostate cancer (TGI=70%) in a 22Rv1-derived xenograft model. These data suggest that (R)-I (R1 = cyclopropylmethyl) is a promising lead compd. for the development of a new class of therapeutics for the treatment of CRPC.
- 49Miyoshi, S.; Ooike, S.; Iwata, K.; Hikawa, H.; Sugahara, K. Antitumor agent. WO 2009084693 A1, 2009.There is no corresponding record for this reference.
- 50Cochran, A. G.; Conery, A. R.; Sims, R. J. Bromodomains: a new target class for drug development. Nat. Rev. Drug Discovery 2019, 18, 609– 628, DOI: 10.1038/s41573-019-0030-750https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlWitbnL&md5=88e56eec8edeb7c325d28156ec131ed8Bromodomains: a new target class for drug developmentCochran, Andrea G.; Conery, Andrew R.; Sims, III, Robert J.Nature Reviews Drug Discovery (2019), 18 (8), 609-628CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-mol. targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chem. probe discovery to understand the biol. potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compds. have since advanced to human clin. trials. Here, we review the current state of BET inhibitor biol. in relation to clin. development, and we discuss the next wave of bromodomain inhibitors with clin. potential in oncol. and non-oncol. indications. The lessons learned from BET inhibitor programs should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.
- 51Coudé, M.-M.; Braun, T.; Berrou, J.; Dupont, M.; Bertrand, S.; Masse, A.; Raffoux, E.; Itzykson, R.; Delord, M.; Riveiro, M. E.; Herait, P.; Baruchel, A.; Dombret, H.; Gardin, C. BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells. Oncotarget 2015, 6, 17698– 17712, DOI: 10.18632/oncotarget.413151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MflsVChsA%253D%253D&md5=0db953a0a24b12ec2918a02d1a2df00bBET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cellsCoude Marie-Magdelaine; Braun Thorsten; Berrou Jeannig; Dupont Melanie; Bertrand Sibyl; Masse Aline; Raffoux Emmanuel; Itzykson Raphael; Baruchel Andre; Dombret Herve; Gardin Claude; Coude Marie-Magdelaine; Braun Thorsten; Gardin Claude; Raffoux Emmanuel; Itzykson Raphael; Dombret Herve; Delord Marc; Riveiro Maria E; Herait Patrice; Baruchel AndreOncotarget (2015), 6 (19), 17698-712 ISSN:.The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.
- 52Vázquez, R.; Riveiro, M. E.; Astorgues-Xerri, L.; Odore, E.; Rezai, K.; Erba, E.; Panini, N.; Rinaldi, A.; Kwee, I.; Beltrame, L.; Bekradda, M.; Cvitkovic, E.; Bertoni, F.; Frapolli, R.; D’Incalci, M. The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus. Oncotarget 2016, 8, 7598– 7613, DOI: 10.18632/oncotarget.13814There is no corresponding record for this reference.
- 53Albrecht, B. K.; Gehling, V. S.; Hewitt, M. C.; Vaswani, R. G.; Cote, A.; Leblanc, Y.; Nasveschuk, C. G.; Bellon, S.; Bergeron, L.; Campbell, R.; Cantone, N.; Cooper, M. R.; Cummings, R. T.; Jayaram, H.; Joshi, S.; Mertz, J. A.; Neiss, A.; Normant, E.; O’Meara, M.; Pardo, E.; Poy, F.; Sandy, P.; Supko, J.; Sims, R. J., 3rd; Harmange, J. C.; Taylor, A. M.; Audia, J. E. Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials. J. Med. Chem. 2016, 59, 1330– 1339, DOI: 10.1021/acs.jmedchem.5b0188253https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFKjs7g%253D&md5=5e1c5aab26aa58844a9f6670a7bbd78bIdentification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical TrialsAlbrecht, Brian K.; Gehling, Victor S.; Hewitt, Michael C.; Vaswani, Rishi G.; Cote, Alexandre; Leblanc, Yves; Nasveschuk, Christopher G.; Bellon, Steve; Bergeron, Louise; Campbell, Robert; Cantone, Nico; Cooper, Michael R.; Cummings, Richard T.; Jayaram, Hariharan; Joshi, Shivangi; Mertz, Jennifer A.; Neiss, Adrianne; Normant, Emmanuel; O'Meara, Michael; Pardo, Eneida; Poy, Florence; Sandy, Peter; Supko, Jeffrey; Sims, Robert J.; Harmange, Jean-Christophe; Taylor, Alexander M.; Audia, James E.Journal of Medicinal Chemistry (2016), 59 (4), 1330-1339CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clin. trials for hematol. malignancies (CPI-0610).
- 54Siu, K. T.; Eda, H.; Santo, L.; Ramachandran, J.; Koulnis, M.; Mertz, J.; Sims, R. J.; Cooper, M.; Raje, N. S. Effect of the BET Inhibitor, Cpi-0610, Alone and in Combination with Lenalidomide in Multiple Myeloma. Blood 2015, 126, 4255, DOI: 10.1182/blood.v126.23.4255.4255There is no corresponding record for this reference.
- 55Gavai, A. V.; Norris, D.; Delucca, G.; Tortolani, D.; Tokarski, J. S.; Dodd, D.; O’Malley, D.; Zhao, Y.; Quesnelle, C.; Gill, P.; Vaccaro, W.; Huynh, T.; Ahuja, V.; Han, W.-C.; Mussari, C.; Harikrishnan, L.; Kamau, M.; Poss, M.; Sheriff, S.; Yan, C.; Marsilio, F.; Menard, K.; Wen, M.-L.; Rampulla, R.; Wu, D.-R.; Li, J.; Zhang, H.; Li, P.; Sun, D.; Yip, H.; Traeger, S. C.; Zhang, Y.; Mathur, A.; Zhang, H.; Huang, C.; Yang, Z.; Ranasinghe, A.; Everlof, G.; Raghavan, N.; Tye, C. K.; Wee, S.; Hunt, J. T.; Vite, G.; Westhouse, R.; Lee, F. Y. Discovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug Design. J. Med. Chem. 2021, 64, 14247– 14265, DOI: 10.1021/acs.jmedchem.1c0062555https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVCrtr3E&md5=bb9d5466a765ae78c4ca72b329a0495aDiscovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug DesignGavai, Ashvinikumar V.; Norris, Derek; Delucca, George; Tortolani, David; Tokarski, John S.; Dodd, Dharmpal; O'Malley, Daniel; Zhao, Yufen; Quesnelle, Claude; Gill, Patrice; Vaccaro, Wayne; Huynh, Tram; Ahuja, Vijay; Han, Wen-Ching; Mussari, Christopher; Harikrishnan, Lalgudi; Kamau, Muthoni; Poss, Michael; Sheriff, Steven; Yan, Chunhong; Marsilio, Frank; Menard, Krista; Wen, Mei-Li; Rampulla, Richard; Wu, Dauh-Rurng; Li, Jianqing; Zhang, Huiping; Li, Peng; Sun, Dawn; Yip, Henry; Traeger, Sarah C.; Zhang, Yingru; Mathur, Arvind; Zhang, Haiying; Huang, Christine; Yang, Zheng; Ranasinghe, Asoka; Everlof, Gerry; Raghavan, Nirmala; Tye, Ching Kim; Wee, Susan; Hunt, John T.; Vite, Gregory; Westhouse, Richard; Lee, Francis Y.Journal of Medicinal Chemistry (2021), 64 (19), 14247-14265CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an addnl. lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) (I) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematol. and solid tumor models, BMS-986158 was selected as a candidate for clin. evaluation.
- 56Yin, M.; Guo, Y.; Hu, R.; Cai, W. L.; Li, Y.; Pei, S.; Sun, H.; Peng, C.; Li, J.; Ye, R.; Yang, Q.; Wang, N.; Tao, Y.; Chen, X.; Yan, Q. Potent BRD4 inhibitor suppresses cancer cell-macrophage interaction. Nat. Commun. 2020, 11, 1833, DOI: 10.1038/s41467-020-15290-056https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntFOiu70%253D&md5=20b5ac123381a3ca75164a5e53a64c18Potent BRD4 inhibitor suppresses cancer cell-macrophage interactionYin, Mingzhu; Guo, Ying; Hu, Rui; Cai, Wesley L.; Li, Yao; Pei, Shiyao; Sun, Hongyin; Peng, Cong; Li, Jiali; Ye, Rui; Yang, Qiaohong; Wang, Nenghui; Tao, Yongguang; Chen, Xiang; Yan, QinNature Communications (2020), 11 (1), 1833CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Small mol. inhibitor of the bromodomain and extraterminal domain (BET) family proteins is a promising option for cancer treatment. However, current BET inhibitors are limited by their potency or oral bioavailability. Here we report the discovery and characterization of NHWD-870, a BET inhibitor that is more potent than three major clin. stage BET inhibitors BMS-986158, OTX-015, and GSK-525762. NHWD-870 causes tumor shrinkage or significantly suppresses tumor growth in nine xenograft or syngeneic models. In addn. to its ability to downregulate c-MYC and directly inhibit tumor cell proliferation, NHWD-870 blocks the proliferation of tumor assocd. macrophages (TAMs) through multiple mechanisms, partly by reducing the expression and secretion of macrophage colony-stimulating factor CSF1 by tumor cells. NHWD-870 inhibits CSF1 expression through suppressing BRD4 and its target HIF1α. Taken together, these results reveal a mechanism by which BRD4 inhibition suppresses tumor growth, and support further development of NHWD-870 to treat solid tumors.
- 57McDaniel, K. F.; Wang, L.; Soltwedel, T.; Fidanze, S. D.; Hasvold, L. A.; Liu, D.; Mantei, R. A.; Pratt, J. K.; Sheppard, G. S.; Bui, M. H.; Faivre, E. J.; Huang, X.; Li, L.; Lin, X.; Wang, R.; Warder, S. E.; Wilcox, D.; Albert, D. H.; Magoc, T. J.; Rajaraman, G.; Park, C. H.; Hutchins, C. W.; Shen, J. J.; Edalji, R. P.; Sun, C. C.; Martin, R.; Gao, W.; Wong, S.; Fang, G.; Elmore, S. W.; Shen, Y.; Kati, W. M. Discovery of N-(4-(2,4-Difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/Mivebresib), a Potent and Orally Available Bromodomain and Extraterminal Domain (BET) Family Bromodomain Inhibitor. J. Med. Chem. 2017, 60, 8369– 8384, DOI: 10.1021/acs.jmedchem.7b0074657https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFOjtrbF&md5=16ea9e9ade8a22a6e99aeae16fd1bf9cDiscovery of N-(4-(2,4-Difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/Mivebresib), a Potent and Orally Available Bromodomain and Extraterminal Domain (BET) Family Bromodomain InhibitorMcDaniel, Keith F.; Wang, Le; Soltwedel, Todd; Fidanze, Steven D.; Hasvold, Lisa A.; Liu, Dachun; Mantei, Robert A.; Pratt, John K.; Sheppard, George S.; Bui, Mai H.; Faivre, Emily J.; Huang, Xiaoli; Li, Leiming; Lin, Xiaoyu; Wang, Rongqi; Warder, Scott E.; Wilcox, Denise; Albert, Daniel H.; Magoc, Terrance J.; Rajaraman, Ganesh; Park, Chang H.; Hutchins, Charles W.; Shen, Jianwei J.; Edalji, Rohinton P.; Sun, Chaohong C.; Martin, Ruth; Gao, Wenqing; Wong, Shekman; Fang, Guowei; Elmore, Steven W.; Shen, Yu; Kati, Warren M.Journal of Medicinal Chemistry (2017), 60 (20), 8369-8384CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examn. in clin. studies, particularly in oncol. settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metab. and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a crit. asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Addnl. structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clin. candidate I (ABBV-075/mivebresib), which demonstrates excellent potency in biochem. and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.
- 58Sheppard, G. S.; Wang, L.; Fidanze, S. D.; Hasvold, L. A.; Liu, D.; Pratt, J. K.; Park, C. H.; Longenecker, K.; Qiu, W.; Torrent, M.; Kovar, P. J.; Bui, M.; Faivre, E.; Huang, X.; Lin, X.; Wilcox, D.; Zhang, L.; Shen, Y.; Albert, D. H.; Magoc, T. J.; Rajaraman, G.; Kati, W. M.; McDaniel, K. F. Discovery of N-Ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]- 6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET Bromodomain Inhibitor with Selectivity for the Second Bromodomain. J. Med. Chem. 2020, 63, 5585– 5623, DOI: 10.1021/acs.jmedchem.0c0062858https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnvVeisrk%253D&md5=675827659c3c7b6099ccd667f4c60f95Discovery of N-Ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]-6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET Bromodomain Inhibitor with Selectivity for the Second BromodomainSheppard, George S.; Wang, Le; Fidanze, Steven D.; Hasvold, Lisa A.; Liu, Dachun; Pratt, John K.; Park, Chang H.; Longenecker, Kenton; Qiu, Wei; Torrent, Maricel; Kovar, Peter J.; Bui, Mai; Faivre, Emily; Huang, Xiaoli; Lin, Xiaoyu; Wilcox, Denise; Zhang, Lu; Shen, Yu; Albert, Daniel H.; Magoc, Terrance J.; Rajaraman, Ganesh; Kati, Warren M.; McDaniel, Keith F.Journal of Medicinal Chemistry (2020), 63 (10), 5585-5623CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clin. development for oncol. bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (~ 40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compd. 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clin. development compd. 46 (ABBV-744).
- 59Faivre, E. J.; McDaniel, K. F.; Albert, D. H.; Mantena, S. R.; Plotnik, J. P.; Wilcox, D.; Zhang, L.; Bui, M. H.; Sheppard, G. S.; Wang, L.; Sehgal, V.; Lin, X.; Huang, X.; Lu, X.; Uziel, T.; Hessler, P.; Lam, L. T.; Bellin, R. J.; Mehta, G.; Fidanze, S.; Pratt, J. K.; Liu, D.; Hasvold, L. A.; Sun, C.; Panchal, S. C.; Nicolette, J. J.; Fossey, S. L.; Park, C. H.; Longenecker, K.; Bigelow, L.; Torrent, M.; Rosenberg, S. H.; Kati, W. M.; Shen, Y. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer. Nature 2020, 578, 306– 310, DOI: 10.1038/s41586-020-1930-859https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1Smsr0%253D&md5=800d9106c33084390fd8cbf1ac7f1766Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancerFaivre, Emily J.; McDaniel, Keith F.; Albert, Daniel H.; Mantena, Srinivasa R.; Plotnik, Joshua P.; Wilcox, Denise; Zhang, Lu; Bui, Mai H.; Sheppard, George S.; Wang, Le; Sehgal, Vasudha; Lin, Xiaoyu; Huang, Xiaoli; Lu, Xin; Uziel, Tamar; Hessler, Paul; Lam, Lloyd T.; Bellin, Richard J.; Mehta, Gaurav; Fidanze, Steve; Pratt, John K.; Liu, Dachun; Hasvold, Lisa A.; Sun, Chaohong; Panchal, Sanjay C.; Nicolette, John J.; Fossey, Stacey L.; Park, Chang H.; Longenecker, Kenton; Bigelow, Lance; Torrent, Maricel; Rosenberg, Saul H.; Kati, Warren M.; Shen, YuNature (London, United Kingdom) (2020), 578 (7794), 306-310CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clin. activity in monotherapy cancer trials. A reduced no. of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematol. and gastrointestinal defects were obsd. after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities assocd. with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacol. inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compds. that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chem. campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunopptn. followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-contg. super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.
- 60Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Martin, S.; Savitsky, P.; Diez-Dacal, B.; Philpott, M.; Bountra, C.; Lingard, H.; Fedorov, O.; Muller, S.; Brennan, P. E.; Knapp, S.; Filippakopoulos, P. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 19754– 19759, DOI: 10.1073/pnas.131065811060https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFKms7zN&md5=c918702c9fdb91241bc837732730f7f6RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomainPicaud, Sarah; Wells, Christopher; Felletar, Ildiko; Brotherton, Deborah; Martin, Sarah; Savitsky, Pavel; Diez-Dacal, Beatriz; Philpott, Martin; Bountra, Chas; Lingard, Hannah; Fedorov, Oleg; Muller, Susanne; Brennan, Paul E.; Knapp, Stefan; Filippakopoulos, PanagisProceedings of the National Academy of Sciences of the United States of America (2013), 110 (49), 19754-19759,S19754/1-S19754/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compd. currently in phase II clin. trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.
- 61Ayotte, Y.; Marando, V. M.; Vaillancourt, L.; Bouchard, P.; Heffron, G.; Coote, P. W.; Larda, S. T.; LaPlante, S. R. Exposing Small-Molecule Nanoentities by a Nuclear Magnetic Resonance Relaxation Assay. J. Med. Chem. 2019, 62, 7885– 7896, DOI: 10.1021/acs.jmedchem.9b0065361https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFyru7%252FJ&md5=32a5015f45e3f64352be7d3f423e6d2bExposing Small-Molecule Nanoentities by a Nuclear Magnetic Resonance Relaxation AssayAyotte, Yann; Marando, Victoria M.; Vaillancourt, Louis; Bouchard, Patricia; Heffron, Gregory; Coote, Paul W.; Larda, Sacha T.; La Plante, Steven R.Journal of Medicinal Chemistry (2019), 62 (17), 7885-7896CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Small mols. can self-assemble in aq. soln. into a wide range of nanoentity types and sizes (dimers, n-mers, micelles, colloids, etc.), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to nonspecific binding, off-target effects, and false positives and negatives. Here, we demonstrate the use of the spin-spin relaxation Carr-Purcell-Meiboom-Gill NMR expt., which is sensitive to mol. tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone-tumbling mols. vs. nanoentities of various sizes. The technique is highly sensitive to chem. exchange between single-mol. and aggregate states and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in soln. behavior for compds. within structurally related series, demonstrating structure-nanoentity relationships. This practical expt. is a valuable tool to support drug discovery efforts.
- 62Urick, A. K.; Calle, L. P.; Espinosa, J. F.; Hu, H.; Pomerantz, W. C. Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to (1)H CPMG Ligand-Observed NMR. ACS Chem. Biol. 2016, 11, 3154– 3164, DOI: 10.1021/acschembio.6b0073062https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFSlsbzJ&md5=44a2c33abe8f630fd7326cc08d7d685eProtein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to 1H CPMG Ligand-Observed NMRUrick, Andrew K.; Calle, Luis Pablo; Espinosa, Juan F.; Hu, Haitao; Pomerantz, William C. K.ACS Chemical Biology (2016), 11 (11), 3154-3164CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)To evaluate its potential as a ligand discovery tool, the authors compare a newly developed 1D-protein-obsd. fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-obsd. 1H CPMG NMR screen. The authors selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small mol. inhibitors of related epigenetic regulatory proteins. The authors compare the two methods' hit sensitivity, triaging ability, expt. speed, material consumption, and the potential for false positives and negatives. To this end, the authors screened 930 fragment mols. against Brd4 in mixts. of five and followed up these studies with mixt. deconvolution and affinity characterization of the top hits. In selected examples, the authors also compare the environmental responsiveness of the 19F chem. shift to 1H in 1D-protein obsd. 1H NMR expts. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titrn. calorimetry. The authors conclude that PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used 1D protein-obsd. NMR may also be effective; however the 19F chem. shift remains significantly more responsive.
- 63Pennington, L. D.; Aquila, B. M.; Choi, Y.; Valiulin, R. A.; Muegge, I. Positional Analogue Scanning: An Effective Strategy for Multiparameter Optimization in Drug Design. J. Med. Chem. 2020, 63, 8956– 8976, DOI: 10.1021/acs.jmedchem.9b0209263https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslKgt70%253D&md5=75a473c6d4d0dd0f46de618ad8253c80Positional Analogue Scanning: An Effective Strategy for Multiparameter Optimization in Drug DesignPennington, Lewis D.; Aquila, Brian M.; Choi, Younggi; Valiulin, Roman A.; Muegge, IngoJournal of Medicinal Chemistry (2020), 63 (17), 8956-8976CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Minimizing the no. and duration of design cycles needed to optimize hit or lead compds. into high-quality chem. probes or drug candidates is an ongoing challenge in biomedical research. Small structure modifications to hit or lead compds. can have meaningful impacts on pharmacol. profiles due to significant effects on mol. and physicochem. properties and intra- and intermol. interactions. Rapid pharmacol. profiling of an efficiently prepd. series of positional analogs stemming from the systematic exchange of methine groups with heteroatoms or other substituents in arom. or heteroarom. ring-contg. hit or lead compds. is one approach toward minimizing design cycles (e.g., exchange of arom. or heteroarom. CH groups with N atoms or CF, CMe, or COH groups). In this Perspective, positional analog scanning is shown to be an effective strategy for multiparameter optimization in drug design, whereby substantial improvements in a variety of pharmacol. parameters can be achieved.
- 64Li, Y.; Zhao, J.; Gutgesell, L. M.; Shen, Z.; Ratia, K.; Dye, K.; Dubrovskyi, O.; Zhao, H.; Huang, F.; Tonetti, D. A.; Thatcher, G. R. J.; Xiong, R. Novel Pyrrolopyridone Bromodomain and Extra-Terminal Motif (BET) Inhibitors Effective in Endocrine-Resistant ER+ Breast Cancer with Acquired Resistance to Fulvestrant and Palbociclib. J. Med. Chem. 2020, 63, 7186– 7210, DOI: 10.1021/acs.jmedchem.0c0045664https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVSht7jM&md5=b44638b7c6db7998abd72b50f09382caNovel Pyrrolopyridone Bromodomain and Extra-Terminal Motif (BET) Inhibitors Effective in Endocrine-Resistant ER+ Breast Cancer with Acquired Resistance to Fulvestrant and PalbociclibLi, Yangfeng; Zhao, Jiong; Gutgesell, Lauren M.; Shen, Zhengnan; Ratia, Kiira; Dye, Katherine; Dubrovskyi, Oleksii; Zhao, Huiping; Huang, Fei; Tonetti, Debra A.; Thatcher, Gregory R. J.; Xiong, RuiJournal of Medicinal Chemistry (2020), 63 (13), 7186-7210CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Acquired resistance to fulvestrant and palbociclib is a new challenge to treatment of estrogen receptor pos. (ER+) breast cancer. ER is expressed in most resistance settings; thus, bromodomain and extra-terminal protein inhibitors (BETi) that target BET-amplified ER-mediated transcription have therapeutic potential. Novel pyrrolopyridone BETi leveraged novel interactions with L92/L94 confirmed by a cocrystal structure of 27 with BRD4. Optimization of BETi using growth inhibition in fulvestrant-resistant (MCF-7:CFR) cells was confirmed in endocrine-resistant, palbociclib-resistant, and ESR1 mutant cell lines. 27 was more potent in MCF-7:CFR cells than six BET inhibitors in clin. trials. Transcriptomic anal. differentiated 27 from the benchmark BETi, JQ-1, showing downregulation of oncogenes and upregulation of tumor suppressors and apoptosis. The therapeutic approach was validated by oral administration of 27 in orthotopic xenografts of endocrine-resistant breast cancer in monotherapy and in combination with fulvestrant. Importantly, at an equiv. dose in rats, thrombocytopenia was mitigated.
- 65Wang, L.; Pratt, J. K.; Soltwedel, T.; Sheppard, G. S.; Fidanze, S. D.; Liu, D.; Hasvold, L. A.; Mantei, R. A.; Holms, J. H.; McClellan, W. J.; Wendt, M. D.; Wada, C.; Frey, R.; Hansen, T. M.; Hubbard, R.; Park, C. H.; Li, L.; Magoc, T. J.; Albert, D. H.; Lin, X.; Warder, S. E.; Kovar, P.; Huang, X.; Wilcox, D.; Wang, R.; Rajaraman, G.; Petros, A. M.; Hutchins, C. W.; Panchal, S. C.; Sun, C.; Elmore, S. W.; Shen, Y.; Kati, W. M.; McDaniel, K. F. Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors. J. Med. Chem. 2017, 60, 3828– 3850, DOI: 10.1021/acs.jmedchem.7b0001765https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsVygu7k%253D&md5=91750e660bafec08b8bcbe6bcd5e818fFragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain InhibitorsWang, Le; Pratt, John K.; Soltwedel, Todd; Sheppard, George S.; Fidanze, Steven D.; Liu, Dachun; Hasvold, Lisa A.; Mantei, Robert A.; Holms, James H.; McClellan, William J.; Wendt, Michael D.; Wada, Carol; Frey, Robin; Hansen, T. Matthew; Hubbard, Robert; Park, Chang H.; Li, Leiming; Magoc, Terrance J.; Albert, Daniel H.; Lin, Xiaoyu; Warder, Scott E.; Kovar, Peter; Huang, Xiaoli; Wilcox, Denise; Wang, Rongqi; Rajaraman, Ganesh; Petros, Andrew M.; Hutchins, Charles W.; Panchal, Sanjay C.; Sun, Chaohong; Elmore, Steven W.; Shen, Yu; Kati, Warren M.; McDaniel, Keith F.Journal of Medicinal Chemistry (2017), 60 (9), 3828-3850CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Members of the BET family of bromodomain contg. proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A 2-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a Ph pyridazinone fragment with a weak binding affinity (I, Ki = 160 μM). SAR investigation of fragment I, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochem. and cell based assays, e.g. II. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.
- 66Poulin, P.; Theil, F. P. Prediction of Pharmacokinetics Prior to In Vivo Studies. 1. Mechanism-Based Prediction of Volume of Distribution. J. Pharm. Sci. 2002, 91, 129– 156, DOI: 10.1002/jps.1000566https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xmslyhtg%253D%253D&md5=17006eeba58f27fefc9ff13e50ee4010Prediction of pharmacokinetics prior to in vivo studies. 1. Mechanism-based prediction of volume of distributionPoulin, Patrick; Theil, Frank-PeterJournal of Pharmaceutical Sciences (2002), 91 (1), 129-156CODEN: JPMSAE; ISSN:0022-3549. (Wiley-Liss, Inc.)In drug discovery and nonclin. development the vol. of distribution at steady state (Vss) of each novel drug candidate is commonly detd. under in vivo conditions. Therefore, it is of interest to predict Vss without conducting in vivo studies. The traditional description of Vss corresponds to the sum of the products of each tissue:plasma partition coeff. (Pt:p) and the resp. tissue vol. in addn. to the plasma vol. Because data on vols. of tissues and plasma are available in the literature for mammals, the other input parameters needed to est. Vss are the Pt:p's, which can potentially be predicted with established tissue compn.-based equations. In vitro data on drug lipophilicity and plasma protein binding are the input parameters used in these equations. Such a mechanism-based approach would be particularly useful to provide first-cut ests. of Vss prior to any in vivo studies and to explore potential unexpected deviations between sets of predicted and in vivo Vss data, when the in vivo data become available during the drug development process. The objective of the present study was to use tissue compn.-based equations to predict rat and human Vss prior to in vivo studies for 123 structurally unrelated compds. (acids, bases, and neutrals). The predicted data were compared with in vivo data obtained from the literature or at Roche. Overall, the av. ratio of predicted-to-exptl. rat and human Vss values was 1.06 (SD=0.817, r=0.78, n=147). In fact, 80% of all predicted values were within a factor of two of the corresponding exptl. values. The drugs can therefore be sepd. into two groups. The first group contains 98 drugs for which the predicted Vss were within a factor of two of those exptl. detd. (av. ratio of 1.01, SD=0.39, r=0.93, n=118), and the second group includes 25 other drugs for which the predicted and exptl. Vss differ by a factor larger than two (av. ratio of 1.32, SD=1.74, r=0.42, n=29). Thus, addnl. relevant distribution processes were neglected in predicting Vss of drugs of the second group. This was true esp. in the case of some cationic-amphiphilic bases. The present study is the first attempt to develop and validate a mechanistic distribution model for predicting rat and human Vss of drugs prior to in vivo studies.
- 67Decosterd, I.; Woolf, C. J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000, 87, 149– 158, DOI: 10.1016/s0304-3959(00)00276-167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3cvosVSntw%253D%253D&md5=c7a759c4b4bde22be4e7f76df85a334bSpared nerve injury: an animal model of persistent peripheral neuropathic painDecosterd Isabelle; Woolf Clifford JPain (2000), 87 (2), 149-158 ISSN:0304-3959.Peripheral neuropathic pain is produced by multiple etiological factors that initiate a number of diverse mechanisms operating at different sites and at different times and expressed both within, and across different disease states. Unraveling the mechanisms involved requires laboratory animal models that replicate as far as possible, the different pathophysiological changes present in patients. It is unlikely that a single animal model will include the full range of neuropathic pain mechanisms. A feature of several animal models of peripheral neuropathic pain is partial denervation. In the most frequently used models a mixture of intact and injured fibers is created by loose ligation of either the whole (Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87-107) or a tight ligation of a part (Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990;43:205-218) of a large peripheral nerve, or a tight ligation of an entire spinal segmental nerve (Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355-363). We have developed a variant of partial denervation, the spared nerve injury model. This involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact. The spared nerve injury model differs from the Chung spinal segmental nerve, the Bennett chronic constriction injury and the Seltzer partial sciatic nerve injury models in that the co-mingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the non-injured skin territories adjacent to the denervated areas. The spared nerve injury model results in early (<24 h), prolonged (>6 months), robust (all animals are responders) behavioral modifications. The mechanical (von Frey and pinprick) sensitivity and thermal (hot and cold) responsiveness is increased in the ipsilateral sural and to a lesser extent saphenous territories, without any change in heat thermal thresholds. Crush injury of the tibial and common peroneal nerves produce similar early changes, which return, however to baseline at 7-9 weeks. The spared nerve injury model may provide, therefore, an additional resource for unraveling the mechanisms responsible for the production of neuropathic pain.
- 68Chen, X.; Meng, F.; Zhang, J.; Zhang, Z.; Ye, X.; Zhang, W.; Tong, Y.; Ji, X.; Xu, R.; Xu, X. L.; You, Q. D.; Jiang, Z. Y. Discovery of 2-((2-methylbenzyl)thio)-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5 -carbonitrile as a novel and effective bromodomain and extra-terminal (BET) inhibitor for the treatment of sepsis. Eur. J. Med. Chem. 2022, 238, 114423, DOI: 10.1016/j.ejmech.2022.11442368https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Gqur7F&md5=d9b2210f9d07e9042ff549dcb3e6864eDiscovery of 2-((2-methylbenzyl)thio)-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile as a novel and effective bromodomain and extra-terminal (BET) inhibitor for the treatment of sepsisChen, Xuetao; Meng, Fanying; Zhang, Jingtian; Zhang, Zijian; Ye, Xuan; Zhang, Weikun; Tong, Yuanyuan; Ji, Xinrui; Xu, Rujun; Xu, Xiao-Li; You, Qi-Dong; Jiang, Zheng-YuEuropean Journal of Medicinal Chemistry (2022), 238 (), 114423CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Sepsis has long been a major health problem worldwide. It threatens the lives of hospitalized patients and has been one of the leading causes of death in hospitalized patients over the past decades. BRD4 has been regarded as a potential target for sepsis therapy, for its crit. role in the transcriptional expression of NF-κB pathway-dependent inflammatory factors. In this study, compd. 1 was obtained through virtual screening, and candidate compd. 27 was obtained through several rounds of iterative SAR anal. 27 decreased LPS-induced NO prodn. and expression of the pro-inflammatory factors IL-6, IL-1β and TNF-α. In vivo, 27 effectively protected mice from LPS-induced sepsis, increased survival rate and decreased the level of pro-inflammatory factors in serum. Collectively, we reported here 27, a BRD4 inhibitor with a new scaffold, as a potential candidate for the treatment of sepsis.
- 69Zhang, R.; Mayhood, T.; Lipari, P.; Wang, Y.; Durkin, J.; Syto, R.; Gesell, J.; McNemar, C.; Windsor, W. Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction. Anal. Biochem. 2004, 331, 138– 146, DOI: 10.1016/s0003-2697(04)00223-469https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXls1Wlu70%253D&md5=d965d43de2f290c1454ad82bb8781d83Fluorescence polarization assay and inhibitor design for MDM2/p53 interactionZhang, Rumin; Mayhood, Todd; Lipari, Philip; Wang, Yaolin; Durkin, James; Syto, Rosalinda; Gesell, Jennifer; McNemar, Charles; Windsor, WilliamAnalytical Biochemistry (2004), 331 (1), 138-146CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)MDM2 is an important neg. regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-mol. inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.
- 70Ran, X.; Zhao, Y.; Liu, L.; Bai, L.; Yang, C. Y.; Zhou, B.; Meagher, J. L.; Chinnaswamy, K.; Stuckey, J. A.; Wang, S. Structure-Based Design of gamma-Carboline Analogues as Potent and Specific BET Bromodomain Inhibitors. J. Med. Chem. 2015, 58, 4927– 4939, DOI: 10.1021/acs.jmedchem.5b0061370https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVagu7rM&md5=e6e150169812e439c1d7d91416eb3ee9Structure-Based Design of γ-Carboline Analogues as Potent and Specific BET Bromodomain InhibitorsRan, Xu; Zhao, Yujun; Liu, Liu; Bai, Longchuan; Yang, Chao-Yie; Zhou, Bing; Meagher, Jennifer L.; Chinnaswamy, Krishnapriya; Stuckey, Jeanne A.; Wang, ShaomengJournal of Medicinal Chemistry (2015), 58 (12), 4927-4939CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Small-mol. inhibitors of bromodomain and extra terminal proteins (BET), including BRD2, BRD3, and BRD4 proteins have therapeutic potential for the treatment of human cancers and other diseases and conditions. In this paper, the authors report the design, synthesis, and evaluation of γ-carboline-contg. compds. as a new class of small-mol. BET inhibitors. The most potent inhibitor I (RX-37) obtained from this study binds to BET bromodomain proteins (BRD2, BRD3, and BRD4) with Ki values of 3.2-24.7 nM and demonstrates high selectivity over other non-BET bromodomain-contg. proteins. Compd. I potently and selectively inhibits cell growth in human acute leukemia cell lines harboring the rearranged mixed lineage leukemia 1 gene. The authors have detd. a cocrystal structure of I in complex with BRD4 BD2 at 1.4 Å resoln., which provides a solid structural basis for the compd.'s high binding affinity and for its further structure-based optimization. Compd. I represents a promising lead compd. for the development of a new class of therapeutics for the treatment of human cancer and other conditions.
- 71Jiang, F.; Guo, A. P.; Xu, J. C.; You, Q. D.; Xu, X. L. Discovery of a Potent Grp94 Selective Inhibitor with Anti-Inflammatory Efficacy in a Mouse Model of Ulcerative Colitis. J. Med. Chem. 2018, 61, 9513– 9533, DOI: 10.1021/acs.jmedchem.8b0080071https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFSrtr7F&md5=65fa429395e1d57fc8bd6e5c51f52ccbDiscovery of a Potent Grp94 Selective Inhibitor with Anti-Inflammatory Efficacy in a Mouse Model of Ulcerative ColitisJiang, Fen; Guo, An-ping; Xu, Jia-chen; You, Qi-Dong; Xu, Xiao-LiJournal of Medicinal Chemistry (2018), 61 (21), 9513-9533CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)As the endoplasmic reticulum paralog of Hsp90, Grp94 chaperones a small set of client proteins assocd. with some diseases, including cancer, primary open-angle glaucoma, and inflammatory disorders. Grp94-selective inhibition has been a potential therapeutic strategy for these diseases. In this study, inspired by the conclusion that ligand-induced "Phe199 shift" effect is the structural basis of Grp94-selective inhibition, a series of novel Grp94 selective inhibitors incorporating "benzamide" moiety were developed, among which compd. I manifested the most potent Grp94 inhibitory activity with an IC50 value of 2 nM and over 1000-fold selectivity to Grp94 against Hsp90α. In a DSS-induced mouse model of ulcerative colitis (UC), compd. I exhibited significant anti-inflammatory efficacy. This work provides a potent Grp94 selective inhibitor as probe compd. for the biol. study of Grp94 and represents the first study that confirms the potential therapeutic efficacy of Grp94-selective inhibitors against UC.
- 72Zhu, P. J.; Yu, Z. Z.; Lv, Y. F.; Zhao, J. L.; Tong, Y. Y.; You, Q. D.; Jiang, Z. Y. Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability. J. Med. Chem. 2021, 64, 11330– 11353, DOI: 10.1021/acs.jmedchem.1c0068272https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1ylt7vJ&md5=2fce800fbc1f6de0d676a5054787584dDiscovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral BioavailabilityZhu, Peng-Ju; Yu, Ze-Zhou; Lv, Yi-Fei; Zhao, Jing-Long; Tong, Yuan-Yuan; You, Qi-Dong; Jiang, Zheng-YuJournal of Medicinal Chemistry (2021), 64 (15), 11330-11353CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Myeloid cell leukemia 1 (Mcl-1) protein is a key neg. 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 compds. as Mcl-1 inhibitors. Stepwise optimizations of hit compd. 11 with primary Mcl-1 inhibition (52%@30μM) led to the discovery of the most potent compd. 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 physicochem. 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 compd. 40 is a promising antitumor agent that deserves further preclin. evaluations.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00372.
Activity results of compounds, bromodomain selectivity of the compound, crystallography information for the complex structures, CYP450 isozyme and hERG ion channel inhibition assay, toxicity evaluation of DDO-8926 in mice, in vivo PK parameters of DDO-8926 in rats, mouse plasma binding rate, and compound structure characterization (PDF)
Molecular formula strings (CSV)
Predicated binding modes of 9 and 15 (ZIP)
Atomic coordinates have been deposited in the Protein Data Bank (PDB code: 8IBQ and 8IDH). Authors will release the atomic coordinates upon article publication.
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