CN115160204A - 一种成纤维细胞活化蛋白抑制剂及其制备方法和应用 - Google Patents
一种成纤维细胞活化蛋白抑制剂及其制备方法和应用 Download PDFInfo
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- CN115160204A CN115160204A CN202210519756.1A CN202210519756A CN115160204A CN 115160204 A CN115160204 A CN 115160204A CN 202210519756 A CN202210519756 A CN 202210519756A CN 115160204 A CN115160204 A CN 115160204A
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- difluoropyrrolidin
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Abstract
本发明提供了一类成纤维细胞活化蛋白(FAP)抑制剂，属于药物化学技术领域。其结构通式如式I所示，
所述FAP抑制剂对FAP具有良好的抑制活性和选择性。本发明提供的FAP抑制剂合成方法主要通过亲核取代、酯水解、酰胺缩合，条件温和，步骤简单，纯化便捷。特别地，抑制剂(9h)具有较低的细胞毒性，较好的药理活性以及对FAP较强的选择性，将9h应用于博来霉素诱导的肺纤维化小鼠，体现出一定程度的肺纤维化抑制作用，可为肺纤维化治疗提供更多制药选择。
Description
技术领域
本发明属于药物化学技术领域，具体涉及一种成纤维细胞活化蛋白抑制剂及其制备方法和应用。
技术背景
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
成纤维细胞活化蛋白(Fibroblast activation protein,FAP,seprase)是一种II型跨膜丝氨酸蛋白酶，常见的组成形式为FAPα单体形式(分子量为95kDa)和由FAPα与FAPβ两个亚单位构成的同型二聚体(分子量为170kDa)。FAP是S9b脯氨酸寡肽酶亚家族成员之一，该家族的其他成员还包括二肽基肽酶(DPPs:DPP4,DPP8,DPP9)和脯氨酸寡肽酶(PREP，POP)。将DPP4和FAP进行序列比对发现，两者有48％的同源性。与其他DPP酶一样，FAP也具有二肽酶活性，而FAP的特异性内肽酶活性是独有的，因此可作为区分FAP和DPP4的一个重要依据。FAP很少存在于正常组织、非恶性肿瘤或癌前病变的上皮损伤中，仅在子宫内膜细胞中表达。而在与活化间质和炎症相关的疾病中，如伤口愈合、类风湿性关节炎、骨关节炎、肝硬化和肺纤维化等，FAP通常表达量较高。此外，FAP在约90％上皮性恶性肿瘤中，包括结直肠、卵巢、乳腺、膀胱和肺中也有表达。FAP高表达会促进肿瘤侵袭、生长，因此FAP也是一个重要的肿瘤治疗靶点。
目前缺乏良好选择性且抑制活性较好的FAP抑制剂且迄今为止的研究中，FAP抑制剂的种类较少，也没有较好的抑制剂获批上市。
发明内容
根据现有技术的不足，本发明的目的是提供一种成纤维细胞活化蛋白抑制剂及其制备方法和应用，所述成纤维细胞活化蛋白抑制剂具有较强的FAP抑制活性和选择性，以及较低的细胞毒性，适用于开发肺纤维化抑制药物。
具体地，本发明的技术方案如下所述：
本发明的第一方面，提供一种FAP抑制剂，其具有式I所示结构：
其中，R1选自单取代或多取代卤素，R2选自氢、卤素、甲基、乙基、萘基。
进一步地，R1选自单取代或多取代氟、氯、溴，优选为氟；或R2选自氢、卤素、甲基、萘基，所述卤素为氟、氯、溴。
进一步地，在本发明的实施方式中，R1为(4S)-F时，R2为H,4-氟；
进一步地，在本发明的实施方式中，R1为4,4-diF时，R2为H,3-氯，4-氟，4-甲基，4-氯；
进一步地，在本发明的实施方式中，R1为4,4-diF时，R2为苯环连接苯形成1-萘基和2-萘基。
在本发明的实施方式中，所述成纤维细胞活化蛋白抑制剂具有以下结构：
3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺；
3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-N-(4-氟苯基)-3-氧代丙酰胺；
(S)-N-(3-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-(对甲苯基)丙酰胺；
(S)-N-(4-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺；
(S)-N-(4-氟苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-1-基)-3-氧代丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-2-基)-3-氧代丙酰胺。
在本发明的第二个方面，提供一种上述第一方面所述的FAP抑制剂的制备方法，其包括：
(1)原料1与氨水反应，得到中间体2；
(2)中间体2与三氟乙酸酐反应得到中间体3；
(3)中间体3脱去保护基裸露出氨基得到中间体4；
(4)原料5和原料6反应得到中间体7；
(5)中间体4与具有通式结构的中间体7反应得到具有I通式结构的成纤维细胞活化蛋白(FAP)抑制剂；
或中间体7由如下方式获得：
(6)原料8和原料9反应得到中间体10；
(7)中间体10水解得到中间体7；
所述合成路线如下所示：
其中R1、R2如上述第一方面所定义。
进一步地，在本发明的实施方式中，步骤(1)中，原料1与氨水在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1-羟基苯并三唑存在下反应，反应完成后经分离纯化后得到中间体2；优选地，反应溶剂为乙腈；
或，步骤(2)中，反应溶剂为四氢呋喃，中间体2和三氟乙酸酐反应，在冰浴中进行，反应完成后经分离纯化后得到中间体3；
或，步骤(3)中，向中间体3中加入提前制备好的盐酸二氧六环溶液，室温下搅拌，反应完成后，旋干溶剂，以适量石油醚和二氯甲烷洗涤残渣，得到中间体4；
或，步骤(4)中，将原料5丙二酸环异丙酯溶于乙腈中，加入原料6，加热回流待丙二酸环异丙酯反应完全，经分离纯化后得到中间体7；优选地，所述分离纯化步骤为将反应后的反应物旋蒸除去溶剂，将残留物溶于适量饱和碳酸氢钠溶液，用乙酸乙酯洗涤有机相杂质，分出水相并以盐酸溶液调节水相pH，再用乙酸乙酯萃取水相中的产物，合并有机相，加入无水硫酸镁干燥，过滤去无水硫酸镁，蒸除溶剂，继续向残渣中加入少量乙酸乙酯加热至溶解，再加入少量石油醚至浑浊，静置析晶，得到中间体7；优选地，盐酸溶液浓度为1M，调节水相pH至2；
或，步骤(5)中，将中间体7溶于有机溶剂中，依次加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、N,N-二异丙基乙胺，在室温下搅拌活化后，加入中间体4，搅拌反应，经纯化得式I化合物；优选地，所述有机溶剂为二氯甲烷和N，N-二甲基甲酰胺的混合溶剂，更优选地，所述二氯甲烷和N，N-二甲基甲酰胺的体积比为2:1；
或，步骤(6)中，将原料9溶于二氯甲烷，在冰浴下逐滴加入用二氯甲烷稀释的原料8氯甲酰乙酸乙酯，后反应，撤去冰浴后继续室温反应，反应完成后经提纯得到中间体10；优选地，冰浴反应时间为15min，或室温反应时间为3h；
或，步骤(7)中，将中间体10溶于甲醇中，向反应液中加入氢氧化钠水溶液，室温搅拌反应过夜，反应完成后经提纯得到中间体7。
本发明的第三方面，提供一种药物组合物，其包含上述第一方面中所述的FAP抑制剂。
本发明所述“组合物”指包括治疗有效量的规定成分的药物产品，以及直接或间接地由规定量的规定成分的组合产生的任何产品。
在本发明的第四方面，提供了一种药物制剂，其包含上述第一方面中所述的FAP抑制剂和至少一种药学上可接受的辅料或载体。
本发明的FAP抑制剂或含有它的药物组合物或药物制剂可以单位剂量形式给药。给药剂型可以是液体剂型、固体剂型。液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混旋剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂、包合物、填埋剂、贴剂、擦剂等。
本发明的药物组合或药物制剂中还可以含有常用的载体，这些载体包括但不局限于：离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白如人血清蛋白、缓冲物质(如磷酸盐、甘油、山梨酯、山梨酸钾、饱和植物脂肪酸的部分甘油酯混合物、水、盐)或电解质、硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、胶态氧化硅、三硅酸镁、聚乙烯吡咯烷酮、纤维素物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜂蜡、羊毛酯等。载体在药物组合物或药物制剂中的含量可以是1wt％-98wt％，通常大约占到80wt％。为方便起见，局部麻醉剂、防腐剂、缓冲剂等可直接溶于载体中。
所述药学上可接受的辅料，包括但不限于赋形剂，所述赋形剂可以为粘合剂、填充剂、润滑剂、崩解剂、缓冲剂、稳定剂、防腐剂等等。所述辅料指药物组合物或药物制剂中除有效成分之外的成分，其对受试者无毒，且与药物活性成分可稳定共存或采用适当手段后稳定共存。
口服片剂和胶囊可以含有粘合剂，比如糖浆、阿拉伯胶、山梨醇、黄芪胶或聚乙烯吡咯烷酮；可以含有填充剂，比如乳糖、蔗糖、玉米淀粉、磷酸钙、山梨醇、氨基乙酸；可以含有润滑剂，如硬脂酸镁，滑石，聚乙二醇，硅土；可以含有崩解剂，如马铃薯淀粉，或可接受的增润剂，如月桂醇钠硫酸盐。片剂可以用制药学上公知的方法包衣。
口服液可以制成水和油的悬浮液，溶液，乳浊液，糖浆，也可以制成干品，用前补充水或其它合适的媒质。这种液体制剂可以包含常规的添加剂，如悬浮剂、山梨醇、纤维素甲醚、葡萄糖糖浆、凝胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶、氢化的食用油脂、乳化剂、如卵磷脂、山梨聚糖单油酸盐、阿拉伯树胶；或非水载体(可能包含可食用油)，如杏仁油、油脂如甘油、乙二醇、或乙醇；防腐剂，如对羟基苯甲酸甲酯或丙酯，山梨酸。如需要可添加调味剂或着色剂。
栓剂可包含常规的栓剂基质，如可可黄油或其它甘油酯。
对于胃肠外投药，液态剂型通常由化合物和一种消毒的载体制成。载体首选水。依照所选载体和药物浓度的不同，化合物既可溶于载体中也可制成悬浮溶液，在制成注射用溶液时先将化合物溶于水中，过滤消毒后装入封口瓶或安瓿中。
在本发明的第五方面，提供了上述第一方面中所述的FAP抑制剂和/或第三方面中所述的药物组合物在制备FAP抑制剂药物中的应用。
在本发明的第六方面，提供了上述第一方面中所述的FAP抑制剂和/或第三方面中所述的药物组合物和/或第四方面制剂在制备治疗作用于FAP介导的疾病药物中的应用，所述疾病包括类风湿性关节炎、骨关节炎、肝硬化和肺纤维化、肝病、动脉粥样硬化、癌症等。
技术方案的有益效果：
(1)本发明所述的成纤维细胞活化蛋白(FAP)抑制剂对FAP有较好的抑制活性以及较强的选择性，且细胞毒性低，特别是化合物9h相较于其他化合物对FAP有更好的抑制活性，将9h应用于博来霉素诱导的肺纤维化小鼠，体现出一定程度的肺纤维化抑制作用，可将其用于治疗肺纤维化药物的制备。
(2)本发明所述合成方法合成步骤简单，原料便宜，条件温和。
附图说明
图1为FAP抑制剂的细胞毒性实验结果；
图2为实施例8中给药期间小鼠体重变化；
图3为实施例8中小鼠肺组织Masson染色结果；为小鼠肺组织Masson染色结果：(A)阴性对照组；(B)肺纤维化模型组；(C)纤维化干预低浓度组；(D)纤维化干预高浓度组；(E)阳性对照药组。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。本发明所使用的试剂或原料均可通过常规途径购买获得，如无特殊说明，本发明所使用的试剂或原料均按照本领域常规方式使用或者按照产品说明书使用。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1：制备通式I化合物(8a-8b)
制备3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺(8a)的合成方法：3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺(中间体2)的合成：
将起始原料(2S,4S)-4-氟-1-叔丁氧羰基吡咯烷-2-甲酸(500mg，2.14mmol)溶于10mL乙腈中，然后冰浴下依次加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI，900mg，2.61mmol)和1-羟基苯并三唑(HOBT，400mg，2.61mmol)后，升至室温搅拌1h，冰浴下逐渐加入市售氨水2mL，产生大量白色浑浊，撤去冰浴，室温下继续搅拌至反应完全。向反应液中加入25mL乙腈后产生大量不溶物，过滤保留滤液，旋蒸除去溶剂，粗产物以硅胶柱层析分离纯化(DCM:MeOH＝80:1)，得微黄色固体400mg，产率80.3％。1H NMR(400MHz,CDCl3)δ6.63(br s,0.5H,rotomer),6.16(br s,0.5H,rotomer),5.43(br s,1H),5.23(dd,J＝52.4,3.6Hz,1H),4.38(br s,1H),4.12–3.33(m,2H),3.03–2.09(m,2H),1.48(s,9H).ESI-MS:m/z[M+H]+calcd for C10H18FN2O3 +233.1,found 232.8.
(2S，4S)-2-氰基-4-氟吡咯烷-1-羧酸叔丁酯(中间体3)的合成中间体3的合成以中间体2(1.8g，7.19mmol)为原料,用5mL干燥的四氢呋喃溶解2，在冰浴下逐渐加入三氟乙酸酐(1.4g，14.39mmol)后，升至室温搅拌3h，旋干四氢呋喃，粗产物以硅胶柱层析分离纯化(纯DCM)，得米黄色油状物1.3g，产率77.8％。1H NMR(400MHz,CDCl3)δ5.31(d,J＝51.5Hz,1H),4.70(dd,J＝43.9,9.0Hz,1H),4.09–3.66(m,1H),3.58(ddd,J＝37.4,13.1,3.2Hz,1H),2.65(dd,J＝19.7,10.5Hz,1H),2.51–2.17(m,1H),1.53(s,9H).
(2S，4S)-4-氟吡咯烷-2-腈盐酸盐(中间体4)的合成
目标化合物4的合成以中间体3(1.2g，5.60mmol)为原料,向3中加入提前制备好的盐酸二氧六环溶液4mL，室温下搅拌3h，旋干溶剂，以石油醚和二氯甲烷洗涤残渣，得0.5g白色固体，产率60％。1H NMR(400MHz,MeOD)δ5.57(dd,J＝29.2,25.7Hz,1H),4.99–4.91(m,1H),3.85–3.71(m 1H),3.55(ddd,J＝36.1,13.6,3.3Hz,1H),2.88–2.60(m,2H).ESI-MS:m/z[M+H]+calcd for C5H8FN2 +115.06,found 115.0.
3-氧代-3-(苯氨基)丙酸(7a)的合成
将起始原料丙二酸环异丙酯(332mg，2.33mmol)先溶于2mL乙腈中，然后加入苯胺(650mg，6.91mmol)，80℃加热回流5h待丙二酸环异丙酯反应完全。旋蒸除去溶剂，将残留物溶于适量饱和碳酸氢钠溶液，用乙酸乙酯洗涤有机相杂质(3*25mL)，分出水相并以1M盐酸溶液调节水相pH至2。再用乙酸乙酯(3*30mL)萃取水相中的产物，合并有机相，加入无水硫酸镁干燥，过滤去无水硫酸镁，蒸除溶剂。继续向残渣中加入少量乙酸乙酯加热至溶解，再加入少量石油醚至浑浊，静置析晶。得白色固体300mg，产率71.9％。1H NMR(400MHz,DMSO-d6)δ10.12(s,1H),7.58(d,J＝7.8Hz,2H),7.31(t,J＝7.9Hz,2H),7.06(t,J＝7.4Hz,1H),3.35(s,2H).ESI-MS:m/z[M-H]-calcd for C9H8O3 -178.1,found 178.1.
3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺(8a)的合成目标化合物8a的合成以中间体7a(138.1mg，771.07μmol)为原料，将7a溶解于2mL二氯甲烷和1mLN，N-二甲基甲酰胺中，然后依次加入HATU(319mg，838.97μmol)、DIPEA(271.4mg，2.10mmol)，室温下搅拌0.5h后加入4(100mg，666.49μmol)至固体全部溶解，继续搅拌10h。40℃旋蒸除去溶剂，反应液中加入乙酸乙酯(3*25mL)和1M盐酸溶液萃取，合并有机相，然后用饱和碳酸氢钠水溶液洗涤，再分别用水和饱和氯化钠水溶液洗涤三次，分出有机相，以无水硫酸镁干燥，过滤无水硫酸镁，旋蒸除去溶剂。粗产物经硅胶制备板(DCM:MeOH＝30:1)纯化得白色固体40mg，产率20.7％。1H NMR(400MHz,CDCl3)δ9.61(s,0.8H),9.33(s,0.2H),7.54(d,J＝7.7Hz,2H),7.31(t,J＝7.9Hz,2H),7.11(t,J＝7.4Hz,1H),5.45–5.27(m,1H),5.21(d,J＝9.0Hz,0.2H),4.92(d,J＝9.3Hz,0.8H),4.07–3.92(m,1H),3.90–3.73(m,1H),3.69–3.41(m,2H),2.67(t,J＝15.4Hz,1H),2.33(dddd,J＝40.5,14.7,9.4,3.4Hz,1H).13CNMR(101MHz,CDCl3)δ167.66,163.14,137.48,129.05,124.70,120.20,117.01,92.62,90.75,53.76,53.52,44.99,41.97,36.24,36.02.ESI-MS:m/z[M-H]-calcd forC14H13FN3O2 -274.1,found 274.2.
制备3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-N-(4-氟苯基)-3-氧代丙酰胺(8b)的合成方法
3-((4-氟苯基)氨基)-3-氧丙酸(7b)的合成
将起始原料丙二酸环异丙酯(259.4mg，1.79mmol)溶于2mL乙腈中，然后加入4-氟苯胺(500mg，4.49mmol)，合成步骤按照7a的合成方法进行，得白色固体250mg，产率50.7％。1HNMR(400MHz,DMSO-d6)δ12.67(s,1H),10.18(s,1H),7.60(dd,J＝9.1,5.0Hz,2H),7.15(t,J＝8.9Hz,2H),3.34(s,2H).ESI-MS:m/z[M-H]-calcd for C9H7FNO3 -196.1,found 196.1.
3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-N-(4-氟苯基)-3-氧代丙酰胺(8b)的合成
目标化合物8b的合成以中间体7b(114mg，578.18μmol)为原料，HATU(219.6mg，577.55μmol)、DIPEA(193.8mg，1.49mmol)和4(80mg，531.28μmol)，合成步骤按照8a的合成方法进行，得白色固体48mg，产率31.1％。1H NMR(400MHz,CDCl3)δ9.67(s,0.8H),9.20(s,0.2H),7.52(dd,J＝8.5,4.9Hz,2H),7.02(t,J＝8.6Hz,2H),5.45(d,J＝51.0Hz,1H),5.10(d,J＝8.9Hz,0.2H),4.96(d,J＝9.3Hz,0.8H),4.03(dd,J＝23.0,12.1Hz,1H),3.84(ddd,J＝35.6,12.2,3.6Hz,1H),3.53(dd,J＝47.5,30.2Hz,2H),2.74(t,J＝15.5Hz,1H),2.46–2.30(m,1H).13C NMR(101MHz,CDCl3)δ167.81,162.52,122.02,121.94,115.77,115.54,92.39,90.59,53.75,53.50,44.99,41.25,36.29,36.08.ESI-MS:m/z[M-H]-calcd forC14H12F2N3O2 -292.1,found 292.4.
实施例2：制备通式I结构化合物(8c-8g)
制备(S)-N-(3-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺(8c)的合成方法
(S)-2-氨基甲酰基-4,4-二氟吡咯烷-1-羧酸叔丁酯(中间体2b)的合成
中间体2b的合成以(S)-1-(叔丁氧基羰基)-4,4-二氟-2-羧酸吡咯烷(50mg，198.96μmol)为原料，按照实施例1的中间体2的合成方法进行合成，得米黄色固体47mg，产率94％。1H NMR(400MHz,CDCl3)δ6.78(s,1H),5.72(s,2H),4.52(s,1H),3.88(dd,J＝23.1,11.3Hz,1H),3.70(d,J＝6.8Hz,1H),2.92(d,J＝14.5Hz,1H),2.57(dd,J＝33.8,27.8Hz,2H),1.48(s,12H).ESI-MS:m/z[M+Na]+calcd for C10H16F2N2NaO3 +273.1,found 272.9.
(S)-2-氰基-4,4-二氟吡咯烷-1-羧酸叔丁酯(3b)的合成
目标化合物3b的合成以中间体2b(2g，8.61mmol)为原料，三氟乙酸酐(1.67g，17.22mmol)，合成步骤按照实施例1的中间体3的合成方法进行，得米黄色油状物1.53g，产率83.1％。1H NMR(400MHz,CDCl3)δ4.73(d,J＝34.4Hz,1H),3.79(d,2H),2.97–2.45(m,2H),1.52(s,9H).
(S)-4,4-二氟吡咯烷-2-腈盐酸盐(4b)的合成
目标化合物4b的合成以中间体3b(500mg，2.15mmol)为原料，合成步骤按照4的合成方法进行，得米黄色油状物250mg，产率68.8％。1H NMR(400MHz,MeOD)δ5.19–5.03(m,1H),4.02–3.75(m,2H),2.99(ddd,J＝18.6,12.6,7.9Hz,2H).ESI-MS:m/z[M+H]+calcd forC5H8FN2 +133.1,found 133.0.
3-((3-氯苯基)氨基)-3-氧代丙酸(7c)的合成
将起始原料丙二酸环异丙酯(244.8mg，1.70mmol)溶于2mL乙腈中，然后加入3-氯苯胺(650mg，5.10mmol)，合成步骤按照7a的合成方法进行，得薄片状棕黄色结晶50mg，产率13.7％。1H NMR(400MHz,DMSO-d6)δ12.69(s,1H),10.33(s,1H),7.81(s,1H),7.42(d,J＝8.3Hz,1H),7.35(t,J＝8.0Hz,1H),7.13(d,J＝7.9Hz,1H),3.36(s,2H).ESI-MS:m/z[M-H]-calcd for C9H7ClNO3 -212.0,found 212.0.
(S)-N-(3-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺(8c)的合成
目标化合物8c的合成以中间体7c(48.5mg，227.04μmol)为原料，HATU(86mg，226.18μmol)、DIPEA(88.01mg，680.98μmol)和4b(38mg，226.15μmol)，合成步骤按照8a的合成方法进行，得白色固体40mg，产率53.7％。1H NMR(400MHz,CDCl3)δ9.54(s,0.8H),9.18(s,0.2H),7.69(s,1H),7.35(d,J＝8.1Hz,1H),7.23(d,J＝8.0Hz,1H),7.11(t,J＝8.1Hz,1H),5.29(dd,J＝8.0,2.8Hz,0.2H),4.97(t,J＝6.5Hz,0.8H),4.12–3.95(m,2H),3.67–3.43(m,2H),2.80(ddd,J＝15.0,8.9,6.2Hz,2H).13C NMR(101MHz,CDCl3)δ167.72,162.44,138.46,134.59,129.99,124.74,120.17,118.03,115.98,53.50,53.17,52.85,44.28,41.80,37.75,37.49,37.24.ESI-MS:m/z[M-H]-calcd for C14H11ClF2N3O2 -326.1,found 326.4.
制备(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺(8d)的合成方法
3-((4-氯苯基)氨基)-3-氧代丙酸(7d)的合成
将起始原料丙二酸环异丙酯(188.3mg，1.31mmol)溶于2mL乙腈中，然后加入4-氯苯胺(500mg，3.92mmol)，合成步骤按照7a的合成方法进行，得白色固体110mg，产率39.4％。1HNMR(400MHz,MeOD)δ7.57(d,J＝8.9Hz,2H),7.30(d,J＝8.9Hz,2H),3.31(s,2H).ESI-MS:m/z[M-H]-calcd for C9H7ClNO3 -212.0,found 212.1.
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺(8d)的合成
目标化合物8d的合成以中间体7a(65mg，362.76μmol)为原料，HATU(126.5mg，332.69μmol)、DIPEA(117.1mg，906.07μmol)和4b(50.7mg，301.73μmol)，合成步骤按照8a的合成方法进行，得白色固体25mg，产率28.3％。1H NMR(400MHz,CDCl3)δ9.33(s,0.8H),8.89(s,0.2H),7.54(d,J＝7.9Hz,2H),7.33(t,J＝7.9Hz,2H),7.14(t,J＝7.3Hz,1H),5.37(d,J＝8.5Hz,0.2H),4.97(t,J＝6.5Hz,0.8H),4.02(ddd,J＝37.1,15.8,9.0Hz,2H),3.69–3.41(m,2H),2.80(tt,J＝17.1,4.1Hz,2H).13C NMR(101MHz,CDCl3)δ167.66,162.50,137.25,129.00,124.86,120.22,115.97,53.54,53.22,52.90,44.22,42.08,37.77,37.52,37.27.ESI-MS:m/z[M+H]+calcd for C14H14F2N3O2 +294.1,found294.1.
制备(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-(对甲苯基)丙酰胺(8e)的合成方法
3-氧代-3-(对甲苯胺基)丙酸(7e)的合成
将起始原料丙二酸环异丙酯(430.4mg，2.98mmol)溶于2mL乙腈中，然后加入对甲基苯胺(800mg，3.92mmol)，合成步骤按照7a的合成方法进行，得白色固体150mg，产率26.1％。1H NMR(400MHz,DMSO-d6)δ12.59(s,1H),10.02(s,1H),7.45(d,J＝14.3Hz,2H),7.11(d,J＝8.3Hz,2H),3.32(s,2H),2.25(s,3H).ESI-MS:m/z[M-H]-calcd for C10H10NO3 -192.1,found192.0.
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-(对甲苯基)丙酰胺(8e)的合成
目标化合物8e的合成以中间体7e(73mg，377.84μmol)为原料，HATU(143mg，377.84μmol)、DIPEA(145.7mg，1.13mmol)和4b(63.8mg，377.84μmol)，合成步骤按照8a的合成方法进行，得白色固体50mg，产率43.1％。1H NMR(400MHz,DMSO-d6)δ10.11(s,1H),7.46(d,J＝8.3Hz,2H),7.12(d,J＝8.2Hz,2H),5.10(dd,J＝9.2,2.7Hz,1H),4.26–4.07(m,2H),3.58–3.47(m,2H),2.97–2.79(m,2H),2.26(s,3H).13C NMR(101MHz,DMSO-d6)δ162.93,157.58,129.96,129.81,124.72,115.28,111.25,48.79,48.47,48.15,39.41,37.29,33.03,32.78,32.52,16.13.ESI-MS:m/z[M-H]-calcd for C14H11ClF2N3O2 -306.1,found 306.9.
制备(S)-N-(4-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺(8f)的合成方法
目标化合物8f的合成以中间体7d(124.5mg，582.83μmol)为原料，HATU(221.2mg，582.83μmol)、DIPEA(205mg，1.58mmol)和4b(89.3mg，529.85μmol)，合成步骤按照8a的合成方法进行，得白色固体51mg，产率29.4％。1H NMR(400MHz,CDCl3)δ9.47(s,0.8H),9.10(s,0.2H),7.49(d,J＝8.8Hz,2H),7.28(d,J＝8.8Hz,2H),5.28(d,J＝2.9Hz,0.2H),4.96(t,J＝6.6Hz,0.8H),4.10–3.95(m,2H),3.50(dd,J＝38.9,21.8Hz,2H),2.80(ddd,J＝14.9,9.1,6.2Hz,2H).13C NMR(101MHz,CDCl3)δ167.48,162.10,135.83,129.84,129.06,121.36,115.72,53.55,53.23,52.91,44.24,41.53,37.90,37.58,37.26.ESI-MS:m/z[M-H]-calcdfor C14H11ClF2N3O2 -326.1,found 326.1.
制备(S)-N-(4-氟苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺(8g)的合成方法
目标化合物8g的合成以中间体7b(124.5mg，582.83μmol)为原料，HATU(221.2mg，582.83μmol)、DIPEA(205mg，1.58mmol)和4b(89.3mg，529.85μmol)，合成步骤按照8a的合成方法进行，得白色固体51mg，产率30.9％。1H NMR(400MHz,DMSO-d6)δ10.24(s,1H),7.59(dd,J＝9.1,5.0Hz,2H),7.16(t,J＝8.9Hz,2H),5.10(dd,J＝9.2,2.9Hz,1H),4.20(dd,J＝27.5,25.1Hz,2H),3.53(q,J＝15.4Hz,2H),2.89(ddd,J＝25.9,15.6,6.4Hz,2H).13C NMR(101MHz,CDCl3)δ167.71,162.19,122.03,121.96,115.82,115.59,53.56,53.24,52.92,44.24,41.61,37.81,37.56,37.31,29.70.
实施例3：制备通式I结构化合物(9h-i)
制备(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-1-基)-3-氧代丙酰胺(9h)的合成方法
3-(萘-1-基氨基)-3-氧代丙酸乙酯(7h)的合成
将起始原料1-萘胺(300mg，2.08mmol)先溶于10mL二氯甲烷中，然后将氯甲酰乙酸乙酯(626mg，4.16mmol)以4mL二氯甲烷稀释后在冰浴下逐渐滴加入反应液搅拌15min后撤去冰浴，室温下继续搅拌3h。旋蒸除去溶剂。向残留物中加入40mL乙酸乙酯，用饱和碳酸钠溶液洗涤至水相无色，再以饱和氯化钠溶液洗涤有机相三次，无水硫酸镁干燥1h，过滤除去无水硫酸镁，蒸干溶剂，粗产物以硅胶柱层析分离纯化(DCM:MeOH＝80:1)，得白色固体400mg，产率74.4％。1H NMR(400MHz,CDCl3)δ9.94(s,1H),8.13(d,J＝7.5Hz,1H),8.01(d,J＝8.4Hz,1H),7.87(d,J＝8.0Hz,1H),7.68(d,J＝8.2Hz,1H),7.56(t,J＝7.5Hz,1H),7.49(dt,J＝13.3,7.7Hz,2H),4.33(q,J＝7.1Hz,2H),3.61(s,2H),1.37(t,J＝7.1Hz,3H).ESI-MS:m/z[M-H]-calcd for C15H14NO3 -256.1,found 256.3.
3-(萘-1-基氨基)-3-氧代丙酸(8h)的合成
目标化合物8h的合成以中间体7h(400mg，1.55mmol)为原料，将7h溶解于5mL甲醇中，向反应液中倾入提前配置好的氢氧化钠溶液(124mg，3.10mmol),室温下搅拌过夜。旋蒸除去有机溶剂，剩余水溶液中加入乙酸乙酯(3*25mL)洗涤有机相杂质，分出水相，并加入适量1M盐酸溶液至pH为3，出现大量固体，直接过滤保留滤饼，烘干得300mg，产率84％。1H NMR(400MHz,MeOD)δ8.10(d,J＝7.5Hz,1H),7.95–7.87(m,1H),7.78(d,J＝8.3Hz,1H),7.67(d,J＝7.3Hz,1H),7.60–7.45(m,3H),3.61(s,2H).ESI-MS:m/z[M-H]-calcd for C13H10NO3 -228.1,found 228.1.
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-1-基)-3-氧代丙酰胺(9h)的合成
目标化合物9h的合成以中间体8h(133.6mg，582.83μmol)为原料，将8h溶解于10mL二氯甲烷中，然后依次加入HATU(221.6mg，582.83μmol)、DIPEA(205.4mg，1.59mmol),室温下搅拌0.5h后加入4a(89mg，529.85μmol)至固体全部溶解，继续搅拌3h。40℃旋蒸除去溶剂，残留物中加入乙酸乙酯(3*25mL)和1M盐酸溶液萃取，合并有机相，然后用饱和碳酸氢钠水溶液洗涤，再分别用水和饱和氯化钠水溶液洗涤三次，分出有机相，以无水硫酸镁干燥，过滤无水硫酸镁，旋蒸除去溶剂。粗产物经硅胶制备板(DCM:MeOH＝35:1)纯化得白色固体35mg，产率19.2％。1H NMR(400MHz,CDCl3)δ10.02(s,0.8H),9.70(s,0.2H),7.96(dd,J＝11.2,8.0Hz,2H),7.80(d,J＝8.0Hz,1H),7.63(d,J＝8.1Hz,1H),7.53–7.37(m,3H),5.17–5.11(m,0.2H),4.94(dd,J＝8.1,4.8Hz,0.8H),4.03–3.87(m,2H),3.54(dd,J＝46.6,29.3Hz,2H),2.71(dt,J＝8.8,6.0Hz,2H).13C NMR(101MHz,CDCl3)δ168.04,162.83,134.07,132.04,128.68,126.72,126.18,125.85,125.66,120.86,119.97,115.90,53.49,53.17,52.85,44.25,41.28,37.78,37.53,37.28,31.75.ESI-MS:m/z[M-H]-calcd forC18H14F2N3O2 -342.1,found 342.5.制备(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-2-基)-3-氧代丙酰胺(9i)的合成方法
3-(萘-2-基氨基)-3-氧代丙酸乙酯(7i)的合成
将起始原料2-萘胺(300mg，2.08mmol)合成步骤按照7h的合成方法进行得白色固体250mg，产率46.4％。1H NMR(400MHz,CDCl3)δ9.44(s,1H),8.24(s,1H),7.79(t,J＝8.1Hz,3H),7.56–7.37(m,3H),4.29(q,J＝7.1Hz,2H),3.53(s,2H),1.35(t,J＝7.1Hz,3H).ESI-MS:m/z[M-H]-calcd for C15H14NO3 -256.1,found 256.2.
3-(萘-2-基氨基)-3-氧代丙酸3-(naphthalen-2-ylamino)-3-oxopropanoicacid(8i)的合成
目标化合物8i的合成以中间体7i(400mg，1.55mmol)为原料,合成步骤按照8h的合成方法进行，得白色固体205mg，产率57.5％。1H NMR(400MHz,MeOD)δ8.25(s,1H),7.82(dd,J＝15.2,8.3Hz,3H),7.58(dd,J＝8.8,2.1Hz,1H),7.53–7.39(m,2H),3.52(s,2H).ESI-MS:m/z[M-H]-calcd for C13H10NO3 -228.1,found 228.1.
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-2-基)-3-氧代丙酰胺(9i)的合成
目标化合物9i的合成以中间体8i(133.6mg，582.83μmol)为原料,合成步骤按照9h的合成方法进行，得白色固体15mg，产率13.7％。1H NMR(400MHz,CDCl3)δ9.54(s,1H),8.10(s,1H),7.67(t,J＝8.3Hz,3H),7.51–7.27(m,3H),5.31–5.22(m,0.2H),4.84(dd,J＝7.9,4.9Hz,0.8H),3.92(ddd,J＝23.3,14.8,10.1Hz,2H),3.70–3.35(m,2H),2.66(ddd,J＝19.1,18.3,12.4Hz,2H).13C NMR(101MHz,CDCl3)δ167.78,162.77,134.80,133.69,130.83,128.83,127.70,127.57,126.59,125.26,119.97,117.06,116.08,53.48,53.16,52.84,44.21,42.07,37.71,37.45,37.20.ESI-MS:m/z[M-H]-calcd for C18H14F2N3O2 -342.1,found342.1.
实施例4：酶水平中抑制剂的抑制活性
首先对所合成的FAP抑制剂的IC50值进行测定：先用不同浓度的抑制剂与FAP进行孵育，随后加入荧光底物Z-Gly-Pro-AMC，FAP能在特定条件下识别该底物并释放出荧光团7-氨基-4-甲基香豆素(AMC)。利用这一特性，将相应的荧光强度的数据转化为对应化合物的IC50值。
实验步骤如下：
1.用分析纯DMSO将FAP抑制剂配制成浓度为10mM的浓储待用。
2.用50mM Tris,140mM NaCl,pH为7.5的缓冲液将待测化合物浓储稀释至以下9个浓度梯度：160μM、80μM、10μM、2μM、400nM、100nM、25nM、2.50nM、0.25nM。
3.取一管提前分装好的冻存于-78℃冰箱中的FAP，用上述缓冲液配制成浓度为100ng/mL的溶液。于96孔板中，每孔加入50μL不同浓度的待测化合物溶液，同时设对照孔和空白孔。接着用排枪向所有孔中加入50μL的FAP溶液。将96孔板在恒温37℃摇床中孵育30min，振荡使得溶液混合均匀。
4.提前将Z-Gly-Pro-AMC用缓冲液配制成浓度为60μM的溶液，取出96孔板，用排枪快速向每孔中加入100μL探针溶液，继续放回恒温37℃摇床中孵育30min，振荡使得溶液混合均匀。
5.打开POLARstar Omega酶标仪，将96孔板取出放入酶标仪开始测试。记录下最大吸收处的荧光强度数值。以上化合物的酶水平抑制试验均重复三次并计算SD值，数据导入Graphpad Prism作图，得出各化合物的半数抑制浓度IC50值。
由图1可见，通过活性筛选实验，化合物8a-9i均表现出对FAP的抑制活性，其中9h,8e,8f,9i表现出相当或更加优秀的抑制活性。
实施例5：酶水平中抑制剂对DPP4和DPP2的抑制活性
实验步骤同实施例4:将待测化合物浓储用25mM Tris缓冲液稀释至以下8个浓度梯度：500μM、250μM、50μM、25μM、12.50μM、6.25μM、3.13μM、1.56μM。用上述缓冲液将DPP4配制成浓度为100ng/mL的溶液。将H-Gly-Pro-AMC用缓冲液配制成浓度为80μM的溶液。用25mMTris,50mM NaCl,pH为7.4缓冲液将DPP2配制成浓度为400ng/mL，将H-Gly-Pro-AMC用缓冲液配制成浓度为80μM的溶液。
表1酶水平中抑制剂的抑制活性及选择性
由表1可见，所有FAP抑制剂对DPP4均无明显抑制作用，化合物8a-d,9h对DPP2也无明显抑制作用，对FAP表现出较好的选择性，值得进一步研究。
实施例6：化合物细胞毒的测定
取生长状态良好的对数生长期U87MG-Fluc细胞接种在全新的96孔板中(8000个细胞/孔；100μL/孔)，培养12小时后至细胞贴壁。弃去96孔板中培养基，加入用DMEM培养基配制成7个浓度的抑制剂：250μM、125μM、62.50μM、31.25μM、15.63μM、7.81μM、3.91μM，每个浓度设置6个复孔，孵育24h后加入配置好的CCK8工作液(20μL/孔)，在振荡器孵育30min后用酶标仪测定450nm的吸光度，用Graphpad Prism处理数据。
由图1可见，所有化合物在孵育24h后细胞的存活率在125μM下大于50％，说明化合物细胞毒性小。
实施例7：细胞水平中抑制剂对FAP抑制活性的研究
运用本实验室开发的FAP生物发光探针可选择性检测FAP的含量，利用该探针和表达萤火虫萤光素酶的U87MG-Fluc进行体外评估所合成的化合物抑制FAP活性。实验方法如下：取生长状态良好的对数生长期U87MG-Fluc细胞接种在全新的96孔板中(40000个细胞/孔；100μL/孔)，培养12小时后至细胞贴壁。弃去96孔板中培养基，加入用DMEM培养基配制成不同浓度梯度的抑制剂：400μM、200μM、20μM、2μM、400nM、80nM、20nM、8nM、4nM，在摇床中使细胞与抑制剂继续孵育1h。将生物发光探针配制成40μM混合均匀的溶液。取出96孔板，用排枪快速连续地向每孔中加入混合均匀的探针溶液，然后立即放入活体成像仪捕捉生物发光。导出活体成像仪自带软件分析出的各孔生物发光强度值。通过软件Graphpad Prism作图，得出各待测抑制剂的半数抑制浓度IC50值。
表2细胞水平中抑制活性
由表2可知，通过细胞水平筛选实验，化合物8a-9i均表现出对FAP的抑制活性，其中化合物8a,8b,9h体现了相当或更加优秀的抑制活性。故我们选择9h用于小鼠体内研究。
实施例8：FAP抑制剂对肺纤维化模型小鼠作用的研究
适应性饲养7天后，将小鼠分为5组，造模前禁食不进水24小时。造模前将小鼠进行编号分组，称重。分组情况如下：
阴性对照组(气管内注入生理盐水，同时口服灌胃生理盐水14天)；
肺纤维化模型组(气管内注入博莱霉素，同时口服灌胃生理盐水14天)；
阳性对照药组(气管内注入博莱霉素，口服灌胃阳性对照药1mg/kg/天)；
纤维化干预低浓度组(气管内注入博莱霉素，口服灌胃化合物9h溶液5mg/kg/天)；
纤维化干预高浓度组(气管内注入博莱霉素，口服灌胃化合物9h溶液30mg/kg/天)。
肺纤维化模型小鼠的建立：用1％的戊巴比妥钠对小鼠进行麻醉并对颈部皮肤进行消毒，然后沿中线将颈部皮肤剪开，暴露出气管。用微量进样器从口腔由气管内注入30μL博来霉素溶液，随后提起小鼠左右摇晃，使得药物在肺部均匀分布。小鼠呼吸正常后缝合皮肤并创口消毒，放回笼中，加强护理。阴性对照组小鼠气管内注入等量生理盐水。
造模后，阳性对照药组每日口服一次阳性对照化合物溶液(1mg/kg)200μL；纤维化干预低浓度组每日口服一次化合物9h溶液(5mg/kg)200μL，高浓度组每日口服化合物9h溶液(30mg/kg)；阴性对照组和肺纤维化模型组每日口服生理盐水200μL。
第14天处死小鼠，并测量其重量，取材，进行后续病理评价，如图3所示，实验期间记录小鼠造模当天和实验结束时体重变化，结果如图2所示。生理盐水组小鼠在14天后体重维持稳定，基本无变化；肺纤维化模型组在实验期间，体重急剧下降，平均体重削减为初始体重的四分之三。三组药物治疗组与模型组相比，均呈现一定程度的体重回升。从小鼠体重可以说明，9h和SP-13786均能在一定程度上缓解小鼠肺纤维化病变程度。
图3小鼠Masson染色结果显示，生理盐水组小鼠肺组织无病变，肺结构完整(纤维化程度：0)；模型组小鼠呈现明显的肺组织病变，肺结构损坏，弥漫性肺纤维化形成并伴随大量纤维胶原增生(纤维化程度：3)；经过药物治疗的三组小鼠，肺纤维化程度与模型组相比较轻，其中低浓度干预组小鼠肺纤维化较严重，组织中可见较多胶原纤维增生(纤维化程度：2)；高浓度干预组小鼠和阳性对照药组小鼠的肺纤维化得到缓解，肺组织结构基本完整，肺组织中仅观察到少量胶原纤维增生(纤维化程度：1)。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对发明保护范围的限制，所属领域技术人员应该明白，在本发明的技术方案基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围内。
Claims (10)
1.一种FAP抑制剂，其特征在于，其具有式I所示结构：
其中，R1选自单取代或多取代卤素，R2选自氢、卤素、甲基、乙基、萘基。
2.如权利要求1所述FAP抑制剂，其特征在于，R1选自单取代或多取代氟、氯、溴，优选为氟；
或R2选自氢、卤素、甲基、萘基，所述卤素为氟、氯、溴。
3.如权利要求1所述FAP抑制剂，其特征在于，R1为(4S)-F时，R2为H,4-氟；
或，R1为4,4-diF时，R2为H,3-氯，4-氟，4-甲基，4-氯，萘基。
4.如权利要求1所述FAP抑制剂，其特征在于，所述成纤维细胞活化蛋白抑制剂具有以下结构：
3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺；
3-((2S，4S)-2-氰基-4-氟吡咯烷-1-基)-N-(4-氟苯基)-3-氧代丙酰胺；
(S)-N-(3-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-苯基丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代-N-(对甲苯基)丙酰胺；
(S)-N-(4-氯苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺；
(S)-N-(4-氟苯基)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-3-氧代丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-1-基)-3-氧代丙酰胺；
(S)-3-(2-氰基-4,4-二氟吡咯烷-1-基)-N-(萘-2-基)-3-氧代丙酰胺。
5.如权利要求1-4任一项所述FAP抑制剂的制备方法，其特征在于，其包括：
(1)中间体1与氨水反应，得到中间体2；
(2)中间体2与三氟乙酸酐反应得到中间体3；
(3)中间体3脱去保护基裸露出氨基得到中间体4；
(4)原料5和原料6反应得到中间体7；
(5)中间体4与具有通式结构的中间体7反应得到具有I通式结构的成纤维细胞活化蛋白(FAP)抑制剂；
或中间体7由如下方式获得：
(6)原料8和原料9反应得到中间体10；
(7)中间体10水解得到中间体7；
所述合成路线如下所示：
其中R1、R2如上述权利要求1所定义。
6.如权利要求5所述FAP抑制剂的制备方法，其特征在于，步骤(1)中，原料1与氨水在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1-羟基苯并三唑存在下反应，反应完成后经分离纯化后得到中间体2；优选地，反应溶剂为乙腈；
或，步骤(2)中，反应溶剂为四氢呋喃，反应在冰浴中进行，反应完成后经分离纯化后得到中间体3；
或，步骤(3)中，向中间体3中加入提前制备好的盐酸二氧六环溶液，室温下搅拌，反应完成后，旋干溶剂，以适量石油醚和二氯甲烷洗涤残渣，得到中间体4；
或，步骤(4)中，将原料5丙二酸环异丙酯溶于乙腈中，加入原料6，加热回流待丙二酸环异丙酯反应完全，经分离纯化后得到中间体7；优选地，所述分离纯化步骤为将反应后的反应物旋蒸除去溶剂，将残留物溶于适量饱和碳酸氢钠溶液，用乙酸乙酯洗涤有机相杂质，分出水相并以盐酸溶液调节水相pH，再用乙酸乙酯萃取水相中的产物，合并有机相，加入无水硫酸镁干燥，过滤去无水硫酸镁，蒸除溶剂，继续向残渣中加入少量乙酸乙酯加热至溶解，再加入少量石油醚至浑浊，静置析晶，得到中间体7；优选地，盐酸溶液浓度为1M，调节水相pH至2；
或，步骤(5)中，将中间体7溶于有机溶剂中，依次加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、N,N-二异丙基乙胺，在室温下搅拌活化后，加入中间体4，搅拌反应，经纯化得式I化合物；优选地，所述有机溶剂为二氯甲烷和N，N-二甲基甲酰胺的混合溶剂，更优选地，所述二氯甲烷和N，N-二甲基甲酰胺的体积比为1:1；
或，步骤(6)中，将原料9溶于二氯甲烷，在冰浴下逐滴加入用二氯甲烷稀释的原料8氯甲酰乙酸乙酯，后反应，撤去冰浴后继续室温反应，反应完成后经提纯得到中间体10；优选地，冰浴反应时间为15min，或室温反应时间为3h；
或，步骤(7)中，将中间体10溶于甲醇中，向反应液中加入氢氧化钠，室温搅拌反应过夜，反应完成后经提纯得到中间体7。
7.一种药物组合物，其特征在于，包含权利要求1所述的FAP抑制剂。
8.一种药物制剂，其特征在于，其包含权利要求1所述的FAP抑制剂和至少一种药学上可接受的辅料或载体。
9.如权利要求1所述的FAP抑制剂和/或权利要求7所述的药物组合物和/或权利要求8所述药物制剂在制备治疗作用于FAP介导的疾病药物中的应用。
10.如权利要求9所述应用，其特征在于，所述疾病包括类风湿性关节炎、骨关节炎、肝硬化和肺纤维化、肝病、动脉粥样硬化和癌症。
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