CN105949180B - 治疗中枢神经系统退行性疾病的化合物及其应用 - Google Patents
治疗中枢神经系统退行性疾病的化合物及其应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
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Abstract
本发明涉及治疗中枢神经系统退行性疾病的化合物、药物组合物及其应用，该化合物具有式(Ⅰ)的结构，这些化合物可作为ROCK抑制剂，而被制成适当的药物剂型用于治疗神经退行性疾病。
Description
技术领域
本发明属于医药领域，涉及治疗中枢神经系统退行性疾病的化合物、药物组合物及其应用。本发明还涉及一系列具有多功能的ROCK抑制剂的制备方法及其应用，它们可被制成适当的药物剂型用于治疗神经退行性疾病。
背景技术
中枢神经系统退行性疾病（包括阿尔茨海默病、帕金森氏症、亨廷顿舞蹈病等）的发病机理尚未完全阐明，但是一般认为这些疾病是由多因素促成，主要包括各种信号转导通路的紊乱、氧化应激和自由基的形成、神经兴奋性毒性和炎症反应、蛋白的错误折叠、多巴胺能神经和胆碱神经递质失衡等。这种多因素促成性，使得中枢神经系统退行性疾病的发病机理研究及药物研发困难重重。
随着系统生物学的不断发展，人们发现复杂疾病(如中枢退行性疾病、肿瘤等)并不是由单一信号通路所介导，而是由整个疾病网络所调控。基于疾病的网络药理学(Network pharmacology)理论，调控疾病网络某一节点的单靶点药物不能满足治疗复杂疾病的需求。“一病一靶点、一药一靶点”的传统治疗模式，不是治疗复杂疾病的有效策略。针对诱发疾病的多个靶点、多个节点蛋白同时干预，从而调控整个疾病机制网络的多靶点药物(MTDs)有望成为治疗中枢神经系统退行性疾病更优的策略。
近年来多项研究结果显示，Rho/ROCK信号通路在中枢神经系统退行性疾病的发病过程中起到了非常重要的作用，ROCK抑制剂有望成为治疗中枢神经退行性疾病的新药物。研究发现，ROCK抑制剂发挥作用可能主要通过以下途径：减少Aβ 淀粉样蛋白的产生；促进神经元再生和存活；扩张脑血管，改善大脑血流分布；调节神经元突触的可塑性；促进轴突的生长；促进神经干细胞向神经元的分化；另外ROCK抑制剂还能调节中枢神经系统氧化应激和炎症反应。虽然ROCK抑制剂在治疗中枢神经系统退行性疾病中表现出了良好的前景，但是现有的ROCK抑制剂治疗靶点单一，难以透过血脑屏障，易出现低血压和消化道出血等副作用，限制了它在这方面的应用。
硫辛酸（lipoic acid，LA）被誉为“万能抗氧剂”，是已知天然抗氧剂中效果最强的一种。它是丙酮酸脱氢酶的辅助因子，也是代谢性抗氧剂，在生物体内可以转化为还原型的二氢硫辛酸（dihydrolipoic acid，DHLA），它的抗氧化能力包括：清除自由基和活性氧；抑制脂质过氧化；螯合金属离子；与其他体内的抗氧剂作用。硫辛酸具有分子量低和两亲性的特点，容易透过血脑屏障，可以在中枢神经系统发挥抗氧化作用，因此被认为是治疗神经退行性疾病的有用小分子化合物。但是硫辛酸在体内不稳定，作用靶点较为单一，仅具有抗氧化作用，近年来多将其与其他药物联用或将其作为设计多靶点药物的药效团。
发明内容
基于以上研究和多靶点药物研发理论，我们将具有ROCK激酶抑制作用的药效团和硫辛酸偶联，合成了一系列多功能ROCK抑制剂。
本发明的一系列化合物的特点在于存在两个主要的单元：抗氧化部分和ROCK激酶抑制部分，通过偶联，将这两部分杂合在一个分子中，以达到多靶点，同时改善化合物血脑屏障透过性的目的，从而有效治疗中枢神经系统退行性疾病。
本发明涉及式(Ⅰ)的化合物或其互变异构体、药用盐、前药或溶剂化物：
式（I）
其中，
R1选自-H、-F、-Cl、-Br、-I、-OCH3、-OCH2CH3、-OCH2CH2CH3、-OCH2CH2CH2CH3、、、、、、、和；
R2选自和。
本发明的另一方面提供一种药物组合物，其包含本发明所述的任一化合物或其药学上可接受的盐，以及药学上可接受的载体。
本发明的另一方面提供本发明的任一化合物或其药学上可接受的盐在制备治疗中枢神经系统退行性疾病的药物中的应用。优选地，所述中枢神经系统退行性疾病为阿尔茨海默病、帕金森氏症、肌萎缩侧索硬化症或亨廷顿舞蹈病。
本发明的另一方面提供一种多功能ROCK抑制剂，其包含本发明所述的任一化合物或其药学上可接受的盐。
除非另外指明，本发明的化合物还意欲包括区别仅在于存在一个或多个同位素富集的原子的化合物。例如，用氘或氚替换氢，或者用13C或14C-富集的碳原子替换碳原子，或15N-富集的氮原子替换氮原子的化合物属于本发明的范围内。
属于“药用盐、衍生物、溶剂化物、前药”是指任何药用盐，酯，溶剂化物，或经施用于接受者后能够提供（直接或间接）本文所述化合物的其他化合物。然而，应当理解非药用盐也属于本发明的范围内，因为那些可能用于植被药用盐，盐，前药和衍生物的植被可以通过本领域已知的方法进行。例如，本发明提供的化合物的药用盐可以通过常规方法由母体化合物合成，该母体化合物含有碱或酸部分。通常，该盐例如通过将游离酸或碱形式的这些化合物与化学计算量的适当碱或酸在水中或在有机溶剂中或在两者的混合物中制备。通常，非水性介质如乙醚，乙酸乙酯，乙醇，异丙醇或乙腈是优选的。酸加成盐的实例包括无机酸加成盐例如，盐酸盐，氢溴酸盐，氢碘酸盐，硫酸盐，硝酸盐，和有机酸加成盐，如例如乙酸盐，马来酸盐，富马酸盐，柠檬酸盐，草酸盐，琥珀酸盐，酒石酸盐，苹果酸盐，扁桃酸盐和对甲苯磺酸盐。碱加成盐的实例包括无机盐如钠、钾、钙、铵、镁、铝和锂盐；以及有机碱如乙二胺、乙醇胺、N,N-二烷基乙醇胺、三乙醇胺、葡糖胺和碱性氨基酸盐。
优选的衍生物或前药是相对于母体物质，当将这些化合物使用于患者时提高本发明化合物的生物利用度（例如通过使得口服给药的化合物更容易被吸收到血液中）或增强母体化合物向生物区室（例如脑或淋巴系统）的传递的那些。
式（Ⅰ）化合物前药的任何化合物属于本发明的范围内，术语“前药”以其最广泛的意义使用并且包括在体内转化为本发明化合物的那些衍生物。这些衍生物对于本领域技术人员是显而易见的，并且根据分子中存在的官能团，包括不限于本发明化合物的下列衍生物：酯；氨基酸酯；磷酸；金属盐硫酸；氨基甲酸酯和酰胺。
本发明的化合物可以是作为有利化合物或作为溶剂化物的晶体形式，意欲将两种形式都包括在本发明的范围内。溶剂化的方法是本领域公知的。适当的溶剂化物是药用溶剂化物。在一个具体实施方案中，溶剂化物是水合物。
反应路线列举了制备本发明的化合物的方法。
反应路线1
在0℃条件下将钠氢(60%)加入包含取代基的醇中，反应15分钟后加入化合物a，将反应温度升至室温，搅拌过夜，反应完毕，加水和有机溶剂（乙酸乙酯或者二氯甲烷）萃取得到粗产物b；
将化合物2、4-吡啶硼酸嚬呐醇酯、K2CO3、Pd(pph3)4、混合溶剂（甲苯：水：乙醇=3：2：1）加入到15ml封管中，抽真空，使混合物在真空、100℃条件反应6-10小时，反应完毕，除去溶剂后，通过快速色谱法纯化粗产物得化合物c；
化合物c在甲醇溶液中通过雷尼镍、水合肼还原，反应完毕过滤得到化合物d；
化合物d和硫辛酸在EDCI、DMAP、Et3N（或HATU、DIEA）存在的条件下在二氯甲烷（或DMF）溶液中偶联反应6-12小时，反应完毕通过萃取、快速色谱法纯化粗产物得目标化合物1-8。
反应路线2
将化合物b、N-BOC-吡唑硼酸频哪醇酯、Cs2CO3、PdCl(dppf)、混合溶剂（1,4-二氧六环：水=10：1）加入到15ml封管中，抽真空，使混合物在真空、70℃条件反应6-8小时，反应完毕，除去溶剂后，通过快速色谱法纯化粗产物得化合物e；
化合物e在甲醇溶液中通过钯碳/氢气还原，反应完毕过滤得到化合物f；
化合物f和硫辛酸在HATU、DIEA存在的条件下在DMF溶液中偶联反应4-12小时，反应完毕通过萃取、快速色谱法纯化粗产物得化合物g；
化合物g在二氯甲烷溶液中，三氟乙酸存在的条件下脱掉BOC保护基，饱和碳酸氢钠萃取得到目标化合物9-16。
如果需要，可以通过常规方法如结晶法或色谱法纯化反应产物。当用于制备本发明化合物的上述方法产生立体异构体的混合物时，这些异构体可以通过常规技术如制备色谱法分离。如果存在手性中心，化合物可能以外消旋形式制备，或者可以通过对映特异性合成或通过拆分来制备单个的对映异构体。
一种优选的药用形式是结晶形式，包括药物组合物中的这种形式。如果是盐和溶剂化物，另外的离子或溶剂部分也应当是非毒性。本发明的化合物可以存在不同的多晶型物，意欲本发明包括所有这些形式。
由上述发明式(Ⅰ)表示的典型化合物，其盐，它们的溶剂化物或前药显示较强的血脑屏障透过率，相当的ROCK抑制作用、提高内源性抗氧化蛋白谷胱甘肽的水平、神经保护作用以及温和的血管扩张作用。因此，本发明另一方面涉及治疗、改善或预防神经退行性疾病的方法，该方法包含向需要这种治疗的对象施用治疗有效量的式(Ⅰ)的化合物或其药物组合物。在优选的方法中，所述对象为哺乳动物，例如人。
本发明的化合物和组合物可以与其它药物一起使用以提供联合治疗。其它药物可以形成相同组合物的一部分，或者可以作为同时或不同时给药的分开的组合物提供。
附图说明
图1：化合物对L-glutamate诱导HT22细胞死亡的保护作用。###P<0.001，与正常组比较； * P<0.05，** P<0.01，*** P<0.001与谷氨酸组比较。
图2：化合物15对细胞内ROS（A,B）和GSH（C）水平的影响。A (a) CT; (b) Glu; (c)1µM 15 + Glu; (d) 3µM 15 + Glu; (e) 10µM 15 + Glu; (f) 10µM LA + Glu; 标尺:100 μm ###P<0.001，与正常组比较； *** P<0.001， ** P<0.01与谷氨酸组比较。
图3：化合物15对预收缩血管环的舒张作用。** P<0.01，*** P<0.001与DMSO组比较。
具体实施方式
提供下列实施例进一步举例说明本发明，它们不应当认为是对本发明范围的限定。
实施例1 - 5-(1,2-二硫环戊烷-3-基)-N-(4-(吡啶-4-基)苯基)戊酰胺(1)
按照上述合成路线1合成化合物1，得到淡黄色固体，三步总产率为52%. 1H NMR(400 MHz, CDCl3) δ 8.62 (d, J = 4.7 Hz, 2H), 7.96 (s, 1H), 7.68 (d, J = 8.1Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 5.2 Hz, 2H), 3.64 – 3.49 (m,1H), 3.22 – 3.04 (m, 2H), 2.53 – 2.33 (m, 3H), 1.90 (dt, J = 19.7, 6.8 Hz,1H), 1.84 – 1.62 (m, 4H), 1.61 – 1.43 (m, 2H). 13C NMR (100 MHz, CDCl3) δ171.4, 150.1, 147.7, 139.2, 133.4, 127.6, 121.3, 120.2, 56.4, 40.3, 38.5,37.4, 34.7, 28.9, 25.2. ESI-HRMS for [C19H23N2OS2]+,calcd: 359.1246; found:359.1232. 纯度: 100% （HPLC）。.
实施例2 - [ 5-(1,2-二硫环戊烷-3-基)-N-(2-氟-4-(吡啶-4-基)苯基)戊酰胺(2)
按照上述合成路线1合成化合物2，得到淡黄色固体，3步总产率为32.8%. 1H NMR(400 MHz, CDCl3) δ 8.65 (d, J = 6.0 Hz, 2H), 8.48 (t, J = 8.2 Hz, 1H), 7.51 –7.35 (m, 5H), 3.66 – 3.53 (m, 1H), 3.25 – 3.06 (m, 2H), 2.57 – 2.37 (m, 3H),1.93 (td, J = 13.6, 6.9 Hz, 1H), 1.87 – 1.72 (m, 4H), 1.62 – 1.47 (m, 2H).13CNMR (100 MHz, CDCl3) δ 170.16 (s), 151.50 (d, J = 243.8 Hz), 149.33 (s),145.51 (d, J = 1.8 Hz), 133.04 (d, J = 7.5 Hz), 126.22 (d, J = 10.5 Hz),122.13 (d, J = 3.0 Hz), 121.10 (s), 120.13 (s), 112.21 (d, J = 20.7 Hz),55.32 (s), 39.24 (s), 37.49 (s), 36.43 (s), 33.64 (s), 27.80 (s), 24.07 (s).ESI-HRMS for [C19H22FN2OS2]+,calcd: 377.1158; found: 377.1151. 纯度: 99%（HPLC）.
实施例3 - 5-(1,2-二硫环戊烷-3-基)-N-(2-甲氧基-4-(吡啶-4-基)苯基)戊酰胺(3)
按照上述合成路线1合成化合物3，得到淡黄色固体，三步总产率为22%. 1H NMR(400 MHz, CDCl3) δ 8.62 (dd, J = 4.5, 1.6 Hz, 2H), 8.50 (d, J = 8.4 Hz, 1H),7.83 (s, 1H), 7.47 (dd, J = 4.5, 1.6 Hz, 2H), 7.29 – 7.23 (m, 1H), 7.11 (d, J= 1.8 Hz, 1H), 3.97 (s, 3H), 3.64 – 3.52 (m, 1H), 3.24 – 3.06 (m, 2H), 2.54 –2.39 (m, 3H), 1.97 – 1.85 (m, 2H), 1.83 – 1.69 (m, 4H), 1.60 – 1.47 (m, 2H).13C NMR (100MHz, CDCl3) δ 170.0, 149.2, 147.1, 147.0, 132.3, 127.7, 120.3,119.0, 107.3, 75.7, 55.4, 54.9, 39.3, 37.5, 36.7, 33.7, 27.8, 24.2. ESI-HRMSfor [C20H25N2O2S2]+,calcd: 389.1352; found: 389.1350. 纯度: 99%.
实施例 4 - 5-(1,2-二硫环戊烷-3-基)-N-(3-氟-4-(吡啶-4-基)苯基)戊酰胺(4)
按照上述合成路线1合成化合物4，淡黄色固体，3步总产率为45.2%. 1H NMR (400MHz, CDCl3) δ 8.66 (s, 1H), 8.63 (d, J = 5.8 Hz, 2H), 7.68 (d, J = 12.9 Hz,1H), 7.47 (d, J = 4.7 Hz, 2H), 7.40 (t, J = 8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz,1H), 3.59 – 3.48 (m, 1H), 3.21 – 3.02 (m, 2H), 2.46 – 2.36 (m, 3H), 1.87 (td,J = 13.6, 6.9 Hz, 1H), 1.81 – 1.61 (m, 4H), 1.56 – 1.43 (m, 2H). 13C NMR (100MHz, CDCl3) δ 171.8, 161.3, 158.8, 149.7, 143.5, 140.8, 140.6, 130.3, 130.2,123.5, 123.4, 121.1, 121.0, 115.6, 108.1, 107.8, 56.4, 40.3, 38.5, 37.3,34.6, 28.9, 25.2. ESI-HRMS for [C19H22FN2OS2]+,calcd: 377.1152; found:377.1136. 纯度: 98%.
实施例5 - 5-(1,2-二硫环戊烷-3-基)-N-(3-甲氧基-4-(吡啶-4-基)苯基)戊酰胺(5)
按照上述合成路线1合成化合物5，得到黄色固体，3步总产率为78.8%. 1H NMR(400 MHz, CDCl3) δ 8.59 (dd, J = 4.6, 1.6 Hz, 2H), 7.78 (s, 1H), 7.65 (s,1H), 7.47 (dd, J = 4.5, 1.6 Hz, 2H), 7.28 (d, J = 8.3 Hz, 1H), 6.96 (dd, J =8.3, 2.0 Hz, 1H), 3.84 (s, 3H), 3.58 (td, J = 12.7, 6.4 Hz, 1H), 3.26 – 3.06(m, 2H), 2.51 – 2.39 (m, 3H), 1.91 (dt, J = 19.7, 6.9 Hz, 1H), 1.84 – 1.69(m, 4H), 1.58 – 1.48 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 171.5, 157.2, 149.2,146.2, 140.3, 130.5, 124.2, 122.9, 111.7, 103.4, 56.4, 55.6, 40.3, 38.5,37.4, 34.7, 28.9, 25.2. ESI-HRMS for [C20H25N2O2S2]+,calcd: 389.1352; found:389.1342. 纯度: 99%.
实施例6 - N-(2-(2-(二甲基氨基)乙氧基)-4-(吡啶-4-基)苯基)-5-(1,2-二硫环戊烷基-3-)戊酰胺(6)
按照上述合成路线1合成化合物6，得到淡黄色固体，4步总产率为67.8%. 1H NMR(400 MHz, CDCl3) δ 9.34 (s, 1H), 8.54 (dd, J = 4.5, 1.6 Hz, 2H), 8.40 (d, J =8.5 Hz, 1H), 7.39 (dd, J = 4.5, 1.6 Hz, 2H), 7.24 (dd, J = 8.5, 2.0 Hz, 1H),7.14 (d, J = 2.0 Hz, 1H), 4.12 (t, J = 5.2 , 2H), 3.57 – 3.48 (m, 1H), 3.17 –2.99 (m, 2H), 2.63 (t , J = 5.2 , 2H), 2.41 – 2.34 (m, 3H), 2.29 (s, 6H),1.85 (td, J = 13.8, 7.0 Hz, 1H), 1.76 – 1.62 (m, 4H), 1.53 – 1.40 (m, 2H). 13CNMR (100 MHz, CDCl3) δ 170.7, 149.1, 147.0, 146.8, 132.2, 130.0, 120.2,112.1, 67.0, 57.0, 55.4, 44.2, 39.2, 37.5, 36.2, 33.7, 28.0, 24.3. ESI-HRMSfor [C23H32N3O2S2]+,calcd: 446.1930; found: 446.1929. Purity: 99%.
实施例7- 5-(1,2-二硫环戊烷-3-基)-N-(2-((1-甲基哌啶-4-基)甲氧基)-4-(吡啶-4-基)苯基)戊酰胺(7)
按照上述合成路线1合成化合物7，棕黄色固体，4步总产率为64.8%. 1H NMR (400MHz, CDCl3) δ 8.62 (d, J = 4.6 Hz, 2H), 8.48 (d, J = 8.0 Hz, 1H), 7.81 (s,1H), 7.47 (d, J = 4.7 Hz, 2H), 7.10 (s, 1H), 3.99 (d, J = 5.6 Hz, 2H), 3.64 –3.53 (m, 1H), 3.21– 3.08 (m, 2H), 3.06 – 2.91 (m, 3H), 2.51– 2.43 (m, 3H),2.33 (s, 3H), 2.08 – 1.72 (m, 17H). 13C NMR (100 MHz, CDCl3) δ 171.0, 150.2,148.2, 147.5, 133.4, 128.7, 121.4, 120.2, 120.0, 109.4, 73.2, 56.4, 55.2,46.2, 40.3, 38.5, 37.8, 35.2, 34.7, 29.0, 28.9, 25.3. ESI-HRMS for[C26H36N3O2S2]+,calcd: 486.2243; found: 486.2234. 纯度: 98%.
实施例8 - 5-(1,2-二硫环戊烷-3-基)-N-(4-吡啶-4-基)2-(2,2,2,-三氟乙氧基)苯基)戊酰胺(8)
按照上述合成路线1合成化合物8，得到淡黄色固体，4步总产率为40.4%. 1H NMR(400 MHz, CDCl3) δ 8.64 (d, J = 4.9 Hz, 2H), 8.52 (d, J = 8.3 Hz, 1H), 7.73(s, 1H), 7.45 (d, J = 5.2 Hz, 2H), 7.36 (d, J = 8.1 Hz, 1H), 7.13 (s, 1H),4.52 (dd, J = 15.7, 7.8 Hz, 2H), 3.69 – 3.52 (m, 1H), 3.29 – 3.04 (m, 2H),2.59 – 2.37 (m, 3H), 1.92 (dt, J = 19.7, 6.8 Hz, 1H), 1.88 – 1.67 (m, 4H),1.64 – 1.45 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 170.0, 149.3, 146.2, 145.2,132.7, 128.2, 123.5, 121.2, 120.7, 120.2, 120.1, 109.7, 66.5, 66.1, 65.8,65.4, 55.3, 39.2, 37.5, 36.7, 33.7, 27.8, 24.1. ESI-HRMS for [C21H24F3N2O2S2]+,calcd: 457.1226; found: 457.1225. 纯度: 99%.
实施例9 - N-(4-(1H-吡唑-4-基)苯基)-5-(1,2-二硫环戊烷-3-基)戊酰胺(9)
按照上述合成路线2合成化合物9，得到白色固体，4步总产率为5.6%. 1H NMR(400 MHz, DMSO) δ 12.81 (s, 1H), 9.86 (s, 1H), 7.97 (s, 2H), 7.57 (d, J = 8.6Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 3.74 – 3.56 (m, 1H), 3.26 – 3.05 (m, 2H),2.41 (td, J = 12.5, 6.3 Hz, 1H), 2.30 (t, J = 7.3 Hz, 2H), 1.87 (dt, J =19.7, 6.8 Hz, 1H), 1.77 – 1.55 (m, 4H), 1.46 – 1.37 (m, 2H). 13C NMR (100 MHz,DMSO) δ 170.9, 137.2, 127.7, 125.3, 120.9, 119.4, 56.1, 38.1, 36.2, 34.1,28.3, 24.9. ESI-HRMS for [C17H22N3OS2]+,calcd: 348.1199; found: 348.1195. 纯度: 97%.
实施例10 - 5-(1,2-二硫环戊烷-3-基)-N-(2-氟-4-(1H-吡唑-4-基)苯基)戊酰胺(10)
按照上述合成路线2合成化合物10，得到淡黄色固体，4步总产率为7%. 1H NMR(400 MHz, DMSO) δ 12.94 (s, 1H), 9.60 (s, 1H), 8.06 (s, 2H), 7.77 (t, J = 8.4Hz, 1H), 7.49 (dd, J = 12.3, 1.8 Hz, 1H), 7.38 (dd, J = 8.3, 1.5 Hz, 1H),3.62 (td, J = 12.3, 6.2 Hz, 1H), 3.22 – 3.05 (m, 2H), 2.45 - 2.31 (m, 3H),1.87 (dq, J = 13.5, 6.8 Hz, 1H), 1.75 – 1.52 (m, 4H), 1.48 – 1.33 (m, 5H). 13CNMR (100 MHz, DMSO) δ 171.41 (s), 154.14 (d, J = 243.8 Hz), 130.44 (d, J =7.6 Hz), 124.73 (s), 123.62 (d, J = 12.2 Hz), 120.62 (d, J = 2.8 Hz), 119.98(d, J = 1.9 Hz), 111.82 (d, J = 20.8 Hz), 56.10 (s), 38.07 (s), 35.52 (s),34.08 (s), 28.23 (s), 24.87 (s). ESI-HRMS for [C17H21FN3OS2]+,calcd:366.1105; found: 366.1098. 纯度: 97%.
实施例11 - 5-(1,2-二硫环戊烷-3-基)-N-(2-甲氧基-4-(1H-吡唑-4-基)苯基)戊酰胺(11)
按照上述合成路线2合成化合物11，得到白色固体，4步总产率为14.3%. 1H NMR(400 MHz, DMSO) δ 12.90 (s, 1H), 9.02 (s, 1H), 8.34 – 7.76 (m, 3H), 7.23 (s,1H), 7.13 (d, J = 8.2 Hz, 1H), 3.88 (s, 3H), 3.62 (td, J = 12.5, 6.2 Hz, 1H),3.26 – 3.06 (m, 2H), 2.47 – 2.32 (m, 3H), 1.87 (dq, J = 13.5, 6.8 Hz, 1H),1.77 – 1.50 (m, 4H), 1.48 – 1.33 (m, 2H).13C NMR (100 MHz, DMSO) δ 171.6,150.5, 129.7, 125.8, 122.9, 121.7, 117.3, 108.6, 56.7, 56.2, 38.6, 36.3,34.6, 28.8, 25.5. ESI-HRMS for [C18H24N3O2S2]+,calcd: 378.1304; found:378.1306. Purity: 99%.
实施例12 - 5-(1,2-二硫环戊烷-3-基)-N-(3-氟-4-(1H-吡唑-4-基)苯基)戊酰胺(12)
按照上述合成路线2合成化合物12，得到淡黄色固体，4步总产率为15.3%. 1H NMR(400 MHz, DMSO) δ 13.00 (s, 1H), 10.08 (s, 1H), 7.98 (s, 2H), 7.73 – 7.57 (m,2H), 7.28 (dd, J = 8.5, 1.8 Hz, 1H), 3.67 – 3.56 (m, 1H), 3.24 – 3.06 (m,2H), 2.47 – 2.37 (m, 1H), 2.33 (dd, J = 18.4, 11.0 Hz, 2H), 1.87 (td, J =13.4, 6.8 Hz, 1H), 1.74 – 1.52 (m, 4H), 1.46 – 1.35 (m, 2H).13C NMR (100 MHz,DMSO) δ 171.8, 159.9, 157.4, 139.0, 138.9, 128.3, 128.2, 115.5, 115.5, 115.4,115.3, 115.1, 115.0, 107.0, 106.7, 56.6, 38.6, 36.7, 34.6, 28.8, 25.2. ESI-HRMS for [C17H21FN3OS2]+,calcd: 366.1105; found: 366.1090. Purity: 98%.
实施例13 - 5-(1,2-二硫环戊烷-3-基)-N-(3-甲氧基-4-(1H-吡唑-4-基)苯基)戊酰胺(13)
按照上述合成路线2合成化合物13，得到橘黄色固体，4步总产率为4.3%. 1H NMR(400 MHz, DMSO) δ 12.78 (s, 1H), 9.89 (s, 1H), 7.96 (s, 2H), 7.50 (d, J = 8.3Hz, 1H), 7.44 (d, J = 1.6 Hz, 1H), 7.15 (dd, J = 8.4, 1.8 Hz, 1H), 3.82 (s,3H), 3.67 – 3.57 (m, 1H), 3.23 – 3.07 (m, 2H), 2.41 (dt, J = 18.7, 6.3 Hz,1H), 2.30 (t, J = 7.3 Hz, 2H), 1.87 (td, J = 13.5, 6.8 Hz, 1H), 1.75 – 1.54(m, 4H), 1.46 – 1.36 (m, 2H). 13C NMR (100 MHz, DMSO) δ 171.0, 155.4, 138.4,127.0, 116.8, 116.2, 111.2, 102.7, 56.1, 55.2, 38.1, 36.3, 34.1, 28.3, 24.8.ESI-HRMS for [C18H24N3O2S2]+,calcd: 378.1304; found: 378.1301. Purity: 98%.
实施例14N-(2-(2-(二甲基氨基)乙氧基)-4-(1H-吡唑-4-基)苯基)-5-(1,2-二硫环戊烷-3-基)戊酰胺(14)
按照上述合成路线2合成化合物14，得到淡黄色固体，4步总产率为16.1%. 1H NMR(400 MHz, DMSO) δ 12.89 (s, 1H), 9.34 (s, 1H), 8.14 (s, 2H), 7.92 (d, J = 8.0Hz, 1H), 7.31 (s, 1H), 7.18 (d, J = 7.9 Hz, 1H), 4.16 (t, J = 4.8 Hz, 2H),3.71 – 3.53 (m, 1H), 3.22 – 3.11 (m, 2H), 2.63 (t, J = 4.8 Hz, 2H), 2.46 –2.38 (m, 1H), 2.38 – 2.30 (m, 2H), 2.27 (s, 6H), 1.87 (dt, J = 19.2, 6.5 Hz,1H), 1.80 – 1.50 (m, 4H), 1.50 – 1.30 (m, 2H).13C NMR (100 MHz, DMSO) δ 171.3,149.4, 136.9, 129.6, 127.5, 125.9, 122.5, 121.5, 118.5, 112.3, 68.3, 58.0,56.6, 55.4, 45.7, 38.6, 36.6, 34.7, 28.8, 25.5. ESI-HRMS for [C21H31N4O2S2]+,calcd: 435.1883; found: 435.1883. 纯度: 99%.
实施例15 - 5-(1,2-二硫环戊烷-3-基)-N-(2-((1-甲基哌啶-4-基)甲氧基-4-(1H-吡唑-4-基)苯基)戊酰胺(15)
按照上述合成路线2合成化合物15，得到白色固体，4步总产率为24.6%. 1H NMR(400 MHz, CDCl3) δ 8.34 (d, J = 8.2 Hz, 1H), 7.80 (s, 2H), 7.73 (s, 1H), 7.09(dd, J = 8.3, 1.7 Hz, 1H), 6.97 (d, J = 1.7 Hz, 1H), 3.96 (d, J = 5.9 Hz,2H), 3.64 – 3.52 (m, 1H), 3.24 – 3.08 (m, 2H), 3.06 – 2.96 (m, 2H), 2.50 –2.41 (m, 4H), 2.36 (s, 3H), 2.15 – 2.05 (m, 3H), 1.96 – 1.82 (m, 5H), 1.82 –1.68 (m, 5H), 1.66 – 1.50 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 170.8, 147.6,131.0, 128.3, 126.3, 122.5, 120.5, 118.5, 108.7, 73.1, 56.4, 55.2, 46.1,40.3, 38.5, 37.7, 35.2, 34.7, 29.7, 28.9, 25.4. ESI-HRMS for [C24H35N4O2S2]+,calcd: 475.2196; found: 475.2179. 纯度: 99%.
实施例16 - N-(4-(1H-吡唑-4-基)苯基) -2-（2,2,2-三氟乙氧基）苯基)-5-(1,2-二硫环戊烷基-3-)戊酰胺(16)
按照上述合成路线2合成化合物16，得到白色固体，4步总产率为8.7%. 1H NMR(400 MHz, DMSO) δ 12.91 (s, 1H), 9.01 (s, 1H), 8.09 (s, 2H), 7.66 (d, J = 7.0Hz, 1H), 7.40 (s, 1H), 7.25 (d, J = 7.8 Hz, 1H), 4.81 (d, J = 8.6 Hz, 2H),3.68 – 3.60 (m, 1H), 3.25 – 2.98 (m, 2H), 2.46 – 2.22 (m, 3H), 1.88 (dd, J =12.0, 6.2 Hz, 1H), 1.80 – 1.52 (m, 4H), 1.49 – 1.36 (m, 2H). 13C NMR (100 MHz,DMSO) δ 171.7, 149.7, 130.7, 125.9, 125.8, 125.2, 123.1, 121.2, 119.2, 111.2,66.6, 66.3, 65.9, 65.6, 56.6, 38.6, 36.3, 34.7, 28.7, 25.5. ESI-HRMS for[C19H23F3N3O2S2]+,calcd: 446.1178; found: 446.1161. 纯度: 98%.
实施例17 - 化合物对多种蛋白激酶活性抑制作用
采用基于荧光共振能量转移的Z’-LYTE激酶法试剂盒 （Invitrogen, Carlsbad,CA）测定化合物对ROCK 2，PKA (protein kinase A, PKA), PKG (protein kinase G,PKG)激酶的IC50值。将待测化合物配置成10 mM， 再逐级3倍往下稀释，一共设置10个浓度。384孔板中每孔加入10 μL反应液(包含2 μM溶解在50mM HEPES, pH 7.5, 0.01% Brij-35,10 mM MgCl2, 1 mM EGTA的短肽底物，和适量的ROCK 1，ROCK 2，PKA, PKG)和一系列3倍稀释的待测化合物。ATP的终浓度为75 μM。1 h的孵育之后，反应终止，根据说明书计算荧光比值。采用Prism 5.0拟合得到量效曲线，并计算IC50值。结果见表1，由实验结果可以看出，化合物7, 14和15表现出一定的ROCK抑制作用，而且与法舒地尔相比，这些化合物对ROCK2的选择性更好。
表1. 待测化合物对不同蛋白激酶的抑制作用
| 化合物 | ROCK2 IC50(μM) | PKA IC50(μM) | PKG IC50(μM) |
| 1 | 156 | ＞5000 | 76 |
| 2 | 70 | ＞5000 | 60 |
| 3 | 92 | ＞5000 | 105 |
| 6 | 49 | ＞5000 | 14 |
| 7 | 5±1 | 252 | 5.9±0.8 |
| 9 | 64 | ＞5000 | 81 |
| 10 | 46 | ＞5000 | 168 |
| 11 | 37 | ＞5000 | 147 |
| 14 | 10±2 | 256±55 | 33±3 |
| 15 | 0.8±0.1 | 363±26 | 24±3 |
| fasudil | 0.46±0.06 | 5.3±1 | 2.5±0.7 |
实施例18 - 化合物抑制L-glutamate诱导细胞毒性作用
小鼠海马神经元细胞株HT22，用含10%胎牛血清的DMEM完全培养基，在 37℃，饱和湿度，含体积分数为 5% CO2、95%空气的二氧化碳培养箱中常规培养。取对数生长期细胞，以0.25%胰酶消化后，完全培养基重悬，显微镜下细胞计数板计数并调整细胞浓度为10×104个/ml，接种96孔细胞培养板，100 μL/孔，培养过夜，使细胞贴壁。将96孔板中培养基吸走，待测化合物用DMSO溶解，用完全培养基稀释，加入到96孔板中，100 μL/孔。预孵育30min后，加入2 μL 100mM L-glutamate。模型组不加待测化合物，直接加入2 μL 100mM L-glutamate。孵育24 h后，每孔加入10 μL 5mg/mL MTT，孵育2h，弃去上清，加 DMSO 100 μL/孔，振荡使生成物formazan充分溶解，在酶标仪上测定各孔吸光度值，测定波长570 nm。采用公式 化合物促进细胞的存活率(%)=100%*(A待测化合物-A模型组) / (A模型组-A空白) 计算细胞存活率。结果见图1，可以看出大部分化合物的神经保护作用都较硫辛酸更好，其中化合物11,14和15在10μM的时候几乎能完全逆转由谷氨酸诱导的HT22死亡。
实施例19 - 体外血脑屏障透过率
1)取4 µL 2%（PBL）溶液加于MAIPn4550的96孔板的疏水膜上，滴加过程中注意移液枪头勿接触膜表面以防破坏膜结构；
2)迅速(10 min内)定量吸取200 µL待测样品液（0.1 mg/ml）加入到96孔板中的膜上方作为给药池，膜另一侧加入200 µL PBS (pH=7.4)为接受池，注意保持接受液与膜的充分接触；
3)室温静止10h后，小心移除给药池，每孔取200ul，用UV光谱仪测试接受池内化合物吸光度值（250-500 nm）；
4)吸取100 µL待测样品液与100 µL PBS充分混匀，作为理论平衡溶液，测试其吸光度值（250-500 nm），需要用acceptor板测试；
5)根据公式计算logPe值：
式中Vd是接受池的体积，Va是接受池的体积，A是膜面积，t是渗透时间，[drug]acceptor是接受池的吸光度，[drug]equilibrium是理论平衡吸光度。
注：P e×10-6 cm s-1值大于5.3，则化合物能够透过血脑屏障，小于2.4被定义为不能透过血脑屏障。结果见表2. 由结果可知，大部分化合物都能透过体外模型的血脑屏障。
表2 体外PAMPA-BBB法检测血脑屏障透过率，结果用均值±标准差表示
实施例20 - 化合物对自由基的清除作用
采用DHE染色检测氧自由基的生成情况。HT-22细胞用DMEM +10%胎牛血清，并置于37℃，含5% CO2孵箱中。将细胞以合适的密度种在多聚赖氨酸包被的24孔板；待细胞贴壁后，分别用培养基配制好的PT243加入对应的孔中，处理30 min后，加入2 mM谷氨酸处理10h。收集细胞，并用DHE染料孵育细胞30 min左右，洗净DHE染料，用高内涵系统检测DHE的荧光强度。实验结果显示化合物15能明显减少由谷氨酸诱导的ROS升高，而硫辛酸在10μM的浓度下对ROS的含量无明显影响。
实施例21 - 化合物对内源性抗氧化蛋白谷胱甘肽（Glutathione, GSH）的影响
采用GSH测定的试剂盒检测化合物15对内源性GSH的影响。HT-22细胞用不同浓度的化合物处理30 min后，加入2 mM谷氨酸处理10 h。收集细胞，方法参照南京建成GSH试剂盒进行测定。结果见图2. 可以看出，化合物15能显著升高由谷氨酸诱导的GSH耗竭。而硫辛酸在10μM的浓度下无明显作用。
实施例22 - 血管环实验检测化合物的舒血管作用
快速去除大鼠胸主动脉，并在冰盒上冰的Krebs液中分离结缔组织。通入混合气体(95%O2, 5%CO2)，调节气体速度，使基线保持平稳，将血管挂于5mL Krebs液的浴槽中，每15min换一次Krebs液。待平衡约1h后，换KCl (60mM) 5ml，使血管达到最大的收缩值，并稳定15min，再换KCl (60mM) 5mL，重复做2次；将血管重新平衡后，用累积法加入药物（3 μM，10 μM，20 μM，30 μM）。记录下预收缩时的最大张力以及每个药物加入后的张力，以便比较。结果见图3，可以看出，与法舒地尔相比，化合物15表现出较为温和的血管扩张作用。
Claims (5)
1.式(Ⅰ)的化合物或其药用盐：
式（I）
其中，
R1选自-H、-F、-Cl、-Br、-I、-OCH3、-OCH2CH3、-OCH2CH2CH3、-OCH2CH2CH2CH3、、、、、、、和；
R2选自和。
2.一种药物组合物，其包含权利要求1所述的任一化合物或其药用盐，以及药学上可接受的载体。
3.权利要求1所述的任一化合物或其药用盐在制备治疗中枢神经系统退行性疾病的药物中的应用。
4.根据权利要求3所述的应用，其中所述中枢神经系统退行性疾病为阿尔茨海默病、帕金森氏症、肌萎缩侧索硬化症或亨廷顿舞蹈病。
5.一种ROCK抑制剂，其包含权利要求1所述的任一化合物或其药用盐。
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| CN1310712A (zh) * | 1998-07-31 | 2001-08-29 | 日本曹达株式会社 | 苯基吡咯化合物和其制备方法及抗高血脂药 |
| CN1349523A (zh) * | 1999-04-02 | 2002-05-15 | 科学研究与运用咨询公司 | 新的硫辛酸衍生物、其制备方法、其作为药物的应用和含有这些衍生物的药物组合物 |
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| WO2000053601A1 (en) * | 1999-03-08 | 2000-09-14 | The University Of Mississippi | 1,2-dithiolane derivatives |
| CN1349523A (zh) * | 1999-04-02 | 2002-05-15 | 科学研究与运用咨询公司 | 新的硫辛酸衍生物、其制备方法、其作为药物的应用和含有这些衍生物的药物组合物 |
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