CN114763347A - 一类用于诱导间充质干细胞向软骨分化的化合物及其应用 - Google Patents
一类用于诱导间充质干细胞向软骨分化的化合物及其应用 Download PDFInfo
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- CN114763347A CN114763347A CN202110056472.9A CN202110056472A CN114763347A CN 114763347 A CN114763347 A CN 114763347A CN 202110056472 A CN202110056472 A CN 202110056472A CN 114763347 A CN114763347 A CN 114763347A
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Abstract
本发明为一类用于诱导间充质干细胞向软骨分化的化合物及其应用,公开了一类由结构式(I)‑(VI)表示的小分子化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N‑氧化物、立体异构体或异构体,以及含有此类化合物的药物组合物及其制备方法。该类小分子化合物其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N‑氧化物、立体异构体或异构体,或药物组合物,可通过诱导内源性间充质干细胞成软骨分化而改善骨关节炎或关节损伤相关疾病,可用于体外诱导间充质干细胞成软骨分化,增强干细胞体内软骨修复效果,从而治疗、预防或改善骨关节炎或关节损伤和/或与关节炎或关节损伤相关症状。
Description
技术领域
本发明属于药物化学领域,涉及一类用于诱导间充质干细胞向软骨分化的化合物及其应用。
背景技术
骨关节炎(Osteoarthrosis,OA)是一种严重影响患者生活质量的关节退行性疾病,主要病理特点包括关节软骨变性破坏、软骨下骨硬化或囊性变、关节边缘骨质增生、滑膜病变等,从而导致活动程度受限,在中老年人和运动员中多发。据世界卫生组织(WHO)统计,55岁以上人群中OA的患病率约为80%,60岁以上的老年人几乎都患有不同程度的骨关节疾病(Bijlsma JW.et al.doi:10.1016/S0140-6736(11)60243-2.(2011).)。此外,OA给人们带来沉重的社会负担,如在高收入国家OA治疗的医疗费用已经达到了国民生产总值的1.0~2.5%。研究表明,关节软骨血液供应不足和较低的增殖代谢能力导致其自我修复能力很差。因此,治疗退行性关节疾病以及关节软骨的再生修复是目前临床治疗所面临的重大挑战。
目前临床上OA的治疗目的是缓解疼痛,延缓疾病进展,矫正畸形,从而改善或恢复关节功能,提高患者生活质量。除了基础治疗(运动治疗、物理治疗等),临床上的药物主要是以非甾体类抗炎药物(Nonsteroidal antiinflammatory drugs,NSAIDs),或者是镇痛或缓解疼痛类的药物为主;较为严重的OA患者将会采取外科手术治疗,比如微骨折、关节置换等。但这些治疗方法都存在不同程度的不足,临床结果大多不理想,不能从根本上做到关节软骨的再生修复。因此,亟需研发安全有效的治疗手段来治疗OA。
研究表明,组织再生修复具有使受损关节恢复原有的结构和机械性能的潜力。而干细胞疗法更是再生医学中有效的治疗手段。干细胞是具有自我复制和多向分化潜能的原始细胞,它们是人体中状态较为原始的细胞,被医学界成为“万能细胞”。其中间充质干细胞(Mesenchymal Stem Cells,MSCs)因其具有自我更新能力和多向分化潜能被认为是一种潜在的细胞来源,MSCs相对较容易从多种组织中收集,如骨髓、脂肪、脐带或滑膜等,它们具有较高的增殖率,软骨分化能力和免疫抑制活性(Kabat M.et al.doi:10.1002/sctm.19-0202.(2020).)。同时,MSCs相对于胚胎干细胞、诱导多能性干细胞等,具有致瘤性低、免疫原性低等优点。因此,MSCs逐渐成为再生医学中的优质种子细胞。
MSCs是多能干细胞,可以在体外进行分离和增殖,其可以分化成几种不同类型的细胞,也具有三系分化能力,通过分化过程,专门的细胞类型由专门程度较低的细胞类型形成,例如来自MSCs的软骨细胞。MSCs可分化成包括但不限于成骨细胞、软骨细胞和脂肪细胞;也可通过诱导在体外分化成软骨细胞。说明MSCs在关节软骨损伤再生修复方面具有极大的应用潜能。目前,已经有多项研究报道,无论是自体原位的MSCs(Murphy MP.etal.doi:10.1038/s41591-020-1013-2.(2020).),还是移植外源的MSCs(Johnson K.etal.doi:10.1126/science.1215157.(2012).),均表明MSCs在关节软骨损伤修复中起到重要作用。2012年获得韩国FDA批准的脐带血间充质干细胞产品Cartistem是目前世界上唯一通过国家级批准的干细胞治疗OA的药物,上市以来,Cartistem销售额呈指数级增长,更说明了OA干细胞治疗市场前景广阔。但是,MSCs在组织再生修复中依然存在较多问题,在关节软骨再生修复中面临的主要问题是:移植到体内的MSCs真正分化为软骨细胞的效率非常低。在临床细胞疗法中,可以在未损伤部位获取病人自体的软骨细胞,进行体外培养,然后移植回病人关节腔,同样可以有效修复损伤软骨。但是这种方法会对病人本身造成二次伤害,且细胞数量有限。
胶原蛋白是表皮的主要结构成分,胶原蛋白对于皮肤健康非常重要,且广泛地应用于皱纹和皮肤衰老的表皮治疗并且可以作为烧伤患者的愈合助剂。胶原蛋白一般在成纤维细胞中表达产生,表皮治疗或者愈合助剂所施用的一般为人和牛的胶原蛋白。
基于以上临床问题,希望通过在体外诱导MSCs向软骨分化为软骨前体细胞,然后进行移植,从而提高MSCs在关节软骨损伤中的治疗效果。因此,开发一类药物,通过在体外将干细胞诱导分化为软骨前体细胞再进行移植,或者通过关节腔注射诱导关节部位原位的MSCs分化成软骨细胞,从而规避干细胞药物存在的问题。小分子药物相对于生物制剂,具有规避免疫排斥、稳定、低成本、且跨物种风险低等优势。但目前针对软骨修复的药物研发仍然稀少,对化合物结构多样性的探究与生物学机制的摸索并不充分。因此,设计合成新型高效的诱导MSCs成软骨分化促软骨修复的小分子诱导剂对于OA治疗的基础研究与临床应用具有重大的研发空间和研究意义。
Johnson等在2012年发现一种小分子化合物Kartogenin(KGN)能够促进骨髓间充质干细胞(BMSCs)分化为软骨细胞,且关节腔注射KGN可以修复小鼠骨关节软骨损伤(Johnson K.et al.doi:10.1126/science.1215157.(2012).)。但是后续研究发现,单独使用KGN不能在体外诱导3D培养的干细胞软骨形成光滑、紧密的球形细胞结构。只有在TGFβ3诱导分化1周后,再用KGN诱导2周,才能增强干细胞成球能力且呈现软骨表型(Shi D.etal.doi:10.1021/acsnano.5b06663.(2016).)。因此,KGN诱导MSCs软骨分化效率和效果还有很大提升空间,研发能够高效诱导间充质干细胞向软骨分化的小分子药物,鉴定诱导干细胞向软骨分化的新靶标,增强MSCs治疗OA的效果具有巨大的发展前景。且基于目前国内外OA及软骨损伤发病率的持续上升,发病年龄趋于低龄化,以及医药产业在治疗OA及软骨损伤方面的匮乏,亟需开发安全高效的药物来满足社会的需求。
发明内容
为了解决现有技术存在的不足,本发明的目的是提供了一类用于治疗和/或改善和/或预防骨关节炎或关节损伤,或用于诱导间充质干细胞分化成软骨细胞的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物,可通过在基质和生物相容性骨架中施用于所需的对象;可用于抑制关节软骨损伤,改善骨关节稳态;和/或在体外诱导间充质干细胞向软骨细胞分化;和/或在体外诱导间充质干细胞分化成软骨前体细胞用于关节软骨的再生修复。
本发明提供了一类如下式(I)所示的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,所述化合物的结构如下所示:
其中:
k为0、1、2;优选地,为1、2;
m为0、1、2、3或4;优选地,为0、1;
n为0、1、2、3或4;优选地,为0、1、2;
R1独立地为H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基、C1-6烷基羟基、O-C1-6烷基-O-C1-6烷基等;优选地,为卤素、O-C1-6烷基-O-C1-6烷基。
A环选自C3-7环烷基,C7-C10桥环,含一个或多个O、N、S原子的杂环烷基,芳基,含一个或多个O、N、S原子的五元或六元杂环芳基,含一个或多个O、N、S原子的苯并杂环等。所述芳基选自苯基,蒽基,萘基,菲基等;优选地,为苯环;所述杂环芳基选自吡啶基,呋喃基,吡唑基,吡喃基,哒嗪基,嘧啶基,咪唑基,噻唑基,异噻唑基,哌嗪基,喹啉基,吡嗪基,异喹啉基,噻吩基,噁唑基,异噁唑基,吲哚基,苯并呋喃基,苯并噻吩基,嘌呤基,咔唑基,吖啶基,吩基嗪,吩噻嗪基,喋啶基等;优选地,为噻吩;其中,所述环烷基,杂环烷基,芳基,杂环芳基的环上的基团被0-2个R2基团取代;
所述R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基等;优选地,为卤素。
上述式(I)中,当m=0时,所述化合物可由下述结构(II)表示:
其中:
k为0、1、2;优选地,为1、2;
n为0、1、2、3或4;优选地,为0、1、2;
R1独立地为H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基、C1-6烷基羟基、O-C1-6烷基-O-C1-6烷基等;优选地,为卤素、O-C1-6烷基-O-C1-6烷基。
A环选自C3-7环烷基,C7-C10桥环,含一个或多个O、N、S原子的杂环烷基,芳基,含一个或多个O、N、S原子的五元或六元杂环芳基,含一个或多个O、N、S原子的苯并杂环等。
所述芳基选自苯基,蒽基,萘基,菲基等;优选地,为苯环;所述杂环芳基选自吡啶基,呋喃基,吡唑基,吡喃基,哒嗪基,嘧啶基,咪唑基,噻唑基,异噻唑基,哌嗪基,喹啉基,吡嗪基,异喹啉基,噻吩基,噁唑基,异噁唑基,吲哚基,苯并呋喃基,苯并噻吩基,嘌呤基,咔唑基,吖啶基,吩基嗪,吩噻嗪基,喋啶基等;优选地,为噻吩;其中,所述环烷基,杂环烷基,芳基,杂环芳基的环上的基团被0-2个R2基团取代;
所述R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基等;优选地,为卤素。
其中:
n为0、1、2、3或4;优选地,为0、1、2;
A环选自C3-7环烷基,C7-C10桥环,含一个或多个O、N、S原子的杂环烷基,芳基,含一个或多个O、N、S原子的五元或六元杂环芳基,含一个或多个O、N、S原子的苯并杂环等。
所述芳基选自苯基,蒽基,萘基,菲基等;优选地,为苯环;所述杂环芳基选自吡啶基,呋喃基,吡唑基,吡喃基,哒嗪基,嘧啶基,咪唑基,噻唑基,异噻唑基,哌嗪基,喹啉基,吡嗪基,异喹啉基,噻吩基,噁唑基,异噁唑基,吲哚基,苯并呋喃基,苯并噻吩基,嘌呤基,咔唑基,吖啶基,吩基嗪,吩噻嗪基,喋啶基等;优选地,为噻吩;其中,所述环烷基,杂环烷基,芳基,杂环芳基的环上的基团被0-2个R2基团取代;
所述R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基等中的一种或几种;优选地,为卤素。
上述式(III)中,当A环为苯环时,所述化合物可由下述结构(IV)表示:
其中:
n为0、1、2、3或4;优选地,为0、1、2;
j为0、1、2;
R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基等。
前述式(III)中,当A环为噻吩环时,所述化合物可由下述结构(V)表示:
其中:
n为0、1、2、3或4;优选地,为0、1、2;
j为0、1、2;
R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基等。
上述式(V)中,当n=0时,所述化合物可由下述结构(VI)表示:
其中:
j为0、1、2;
R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基等。
如式(I)—(VI)所述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,所述化合物具体可以选自:
5-溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺;
3-氯-N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(噻吩-2-基)乙酰胺;
5-溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)呋喃-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)苯甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-苯基乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)环丙烷甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(2-氟苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)环戊烷甲酰胺;
4,5-二溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)呋喃-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-氯苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)吡啶酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)异喹啉-1-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(2-氯苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(对甲苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-氟苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-3-苯基丙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-4-苯基丁酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(3-氯苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-溴苯基)乙酰胺;
N-(3-溴苯基)噻吩-2-甲酰胺;
N-(3-(三氟甲基)苯基)噻吩-2-甲酰胺;
N-(3-氰基苯基)噻吩-2-甲酰胺;
N-(3-甲氧基苯基)噻吩-2-甲酰胺;
N-(4-氰基苯基)噻吩-2-甲酰胺;
N-(2-氰基苯基)噻吩-2-甲酰胺;
N-(3-溴苄基)噻吩-2-甲酰胺;
N-(4-氰基苄基)噻吩-2-甲酰胺;
N-(3-氰基苄基)噻吩-2-甲酰胺。
本发明还提供了一种药物组合物,所述药物组合物包含了本发明前述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体以及药学上可接受的赋形剂。
在某些实施方案中,该药物组合物进一步包含了能有效预防、治疗、改善哺乳动物的关节炎或关节损伤和/或与关节炎或关节损伤相关症状的额外化合物。
在某些实施方案中,该额外的化合物选自NSAIDS、镇痛药、血管生成素样3蛋白(ANGPTL3)或其软骨形成变体、口服鲑鱼降钙素、SD-6010(iNOS抑制剂)、维生素D3(胆骨化醇)、凋亡/胱天蛋白酶抑制剂(恩利卡生)、胶原水解物、FGF18、BMP7、鳄梨大豆不皂化物(avocado soy unsaponifiables)(ASU)、透明质酸等中的一种或几种;优选地,为FGF18、BMP7与透明质酸。
本发明还提供了前述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物在制备预防和/或治疗和/或改善哺乳动物的关节炎或关节损伤和/或与关节炎或关节损伤相关症状的药物中的用途;
所述关节炎是骨关节炎、创伤性关节炎、自身免疫性关节炎等中的一种或几种。
所述化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体、药物组合物可通过在基质和生物相容性骨架中施用于所需的对象;所施用的组合物中还包含透明质酸。
本发明还提供了预防、治疗、或改善哺乳动物的关节炎或关节损伤的方法,该方法包括对所述哺乳动物施用治疗有效量的本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物,其中,所述哺乳动物为具有关节损伤或关节炎或处于其风险中。
本发明还提供了预防、治疗或改善人类患者的关节炎或关节损伤的方法,该方法包含:对所述患者的关节施用包含有效量的本发明化合物或组合物,由此预防、治疗或改善患者的关节炎或关节损伤。
所述患者具有关节炎或关节损伤;或不具有关节炎或关节损伤,但处于其风险中;
其中,所述关节炎是骨关节炎、创伤性关节炎或自身免疫性关节炎等;所施用的组合物中还包含透明质酸。
本发明还提供了一种前述化合物用于诱导间充质干细胞(MSCs)分化成软骨细胞的方法。所述方法包括使间充质干细胞接触足量的本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物,由此诱导干细胞分化成软骨细胞。
所述间充质干细胞包括骨髓间充质干细胞,脐带间充质干细胞,脂肪间充质干细胞,滑膜间充质干细胞等中的一种或几种;优选地,为脐带间充质干细胞。
所述诱导间充质干细胞(MSCs)分化成软骨细胞的方法可以在体外进行,也可以在哺乳动物体内进行,且干细胞存在于哺乳动物中。
在一些实施方案中,该哺乳动物是驯养动物或家畜。在某些实施方案中,该哺乳动物是人、鼠、兔、狗、猫或马。
上述方法中所述接触在基质或生物相容性骨架中进行;所述接触也可以通过所述化合物结合一种或多种另外的软骨形成因子进行;所述软骨形成因子选自血管生成素样3蛋白(ANGPTL3)、口服鲑鱼降钙素、SD-6010(iNOS抑制剂)、维生素D3(胆骨化醇)、胶原蛋白水解物、FGF18、BMP7、醋酸芦沙拉肽、鳄梨大豆不皂化物(ASU)、类固醇和类固醇抗炎药(NSAID)、透明质酸等中的一种或几种。
任意适量的本发明化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可以诱导MSCs分化成软骨细胞,根据活性成分的特定应用和效能,本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物的使用量范围为0.1mg至10000mg,例如1.0mg至1000mg、例如10mg至5000mg的量存在等。在对膝盖的关节内注射中,本发明的化合物可以以0.1μM至100μM的浓度存在。
本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可以用于预防、治疗或改善任意类型的关节软骨损伤,也包括来源于创伤事件或肌腱或韧带撕裂的损伤。施用本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可预防或改善关节炎或关节损伤,例如,其中在关节手术之前或过程中存在关节炎或关节损伤或关节损伤的遗传或家族史;也可用于治疗关节损伤;所述关节损伤可以是创伤性关节损伤;也可以是因年龄或不活动导致的损伤;还可以是因自身免疫性疾病导致的损害。在本发明的一些实施方案中,本发明的化合物、组合物和方法可以用于治疗、改善或预防骨关节炎;在一些实施方案中,所述化合物、组合物和方法用于改善或预防处于具有或获得性关节炎风险中的受试者中的关节炎;在一些实施方案中,所述化合物、组合物和方法用于改善或预防处于具有或获得性关节损伤的风险中的受试者的关节损伤,如实施例6、实施例7和实施例8。
本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体、药物组合物可用于刺激例如因跌打损伤或软骨病导致的受损的软骨组织中软骨细胞增殖和软骨产生的方法。具体地,本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可用于治疗关节中的软骨损伤,例如关节连接面上的软骨损伤,例如脊骨、肩嘈中腕、指、髋、膝、踝关节和足关节;可以得益于治疗的疾病或障碍的实例包括骨关节炎、类风湿性关节炎、其它的自身免疫病或骨软骨炎病。此外,软骨损伤或破坏还会作为一些遗传性或代谢性障碍的结果发生,软骨畸形通常在人的侏儒症中观察到,和/或治疗软骨损伤或破坏通常是重建外科手术的结果;因此,本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物无论是单独使用或与其它方法结合使用,都是对患者有用的疗法。
本发明可通过直接注入关节腔、全身施用(口服或静脉内)或直接注入软骨缺陷部位来施用本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物,其可以单独施用或与延长释放的适合的载体复合施用;也可以将化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物在生物相容性材质或骨架中施用。本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物还可以与手术操作结合在受损关节处使用。本发明的实施,可以在手术操作之前、过程中或与之结合、和/或手术操作之后进行。例如,本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可以用于使培养物中的软骨细胞群扩充,以便自体或异体软骨细胞植入(ACI);可以在施用本发明化合物和组合物的同时治疗植入软骨细胞。在这些方法中,例如,可以任选地在本发明化合物和组合物和/或另外的生长因子的存在下通过关节内镜从受损关节的带有少量未受损负荷的区域中收集软骨细胞,且在体外孵育以便增加细胞数量,然后移植。然后任选地将扩充的培养物与本发明的化合物和组合物混合,并且放入关节间隙或直接放入缺陷。具体地,可以将扩充的培养物与本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物一起悬浮于基质或膜中并放入关节间隙。本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物还可以与一种或多种包含软骨形成细胞的骨膜或软骨膜移植物结合使用,有助于将植入的软骨细胞或软骨细胞前体细胞保持就位。此外,使用本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物结合其它方法也可以修复软骨损伤,所述其它方法包括但不限于灌洗关节、刺激骨髓、磨削性关节成形术、软骨下钻孔术或近端软骨下骨微小骨折等;在施用本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物,并且软骨生长后,再进行手术治疗可以有益于适当地使新形成的软骨表面形成轮廓。
本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可以促进人表皮成纤维细胞中的胶原蛋白表达;本发明由此提供增加成纤维细胞中胶原蛋白产生的方法,通过使成纤维细胞接触本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物来进行,由此增加成纤维细胞中胶原蛋白的产生。所述接触可以通过将所述化合物直接注入待治疗的区域在体内进行,也可以在体外进入成纤维细胞群。
附图说明
图1所示为GCP003可以浓度梯度及时间梯度促进HUCMSC向软骨细胞分化。其中,图1A显示阿尔新蓝染色效果,于24孔板中悬滴培养5天,颜色越深表示分化效果越好。图1B和1C分别显示,不同浓度的GCP003诱导hUCMSCs向软骨细胞分化的marker基因的表达和蛋白表达。图1D显示,阿尔新蓝染色效果颜色越深表示分化效果越好。图1E显示,不同分化时间点GCP003诱导hUCMSCs向软骨细胞分化的marker基因的表达。*P<0.05,**P<0.01,***P<0.001。
图2所示为GCP003能促进HUCMSC 3D成球软骨分化。其中,图2A.将hUCMSCs体外离心成球,用分化培养基诱导分化21天,发现GCP003诱导hUCMSCs成球大小和成球重量都优于KGN。图2B.检测小球培养期间,培养基上清中GAG的含量,发现GCP003在诱导分化期间可以产生更多的GAG。图2C.对细胞小球切片后进行免疫荧光染色,发现GCP003诱导的小球可以产生跟多的Collagen II信号,这也显示GCP003具有更好的分化效果。图2D.对细胞小球切片后,阿尔新蓝染色。结果显示,GCP003和KGN软骨基质含量相对于Control组更高,仔细观察切片发现GCP003和KGN分化小球的细胞密度较Control更均匀稠密,且分泌的软骨细胞外基质更多,尤其是GCP003中的分布更加均匀有序。Scale bars:100X 200μm,200X 100μm。
图3所示为GCP003可以促进hBMSCs成软骨分化。其中,图3A.2.5万hBMSCs悬滴培养于24孔板,之后加入GCP003和KGN诱导分化7天,阿尔新蓝染色检测分化效果。图3B.收集培养7天的培养基上清检测GAG含量。图3C.GCP003和KGN诱导hBMSCs分化成球培养。图3D.GCP003和KGN诱导hBMSCs分化基因表达。图3E.GCP003和KGN可诱导临床来源的hBMSCs成软骨基因表达。*P<0.05,**P<0.01,***P<0.001。
图4所示为经GCP003诱导7天后的hUCMSCs,可以促进大鼠关节DMM术后修复。经GCP003诱导7天后hUCMSCs,对DMM模型SD大鼠进行关节腔注射,然后收取关节样本,进行切片番红固绿染色。可以观察关节软骨、软骨下骨和骨组织的结构,从而显示关节部位软骨损伤情况。Scale bars:20X 1000μm n=6。
图5所示为经GCP003诱导7天后hUCMSCs,用水凝胶复合之后可以促进大鼠关节软骨缺损修复。图中显示,SD大鼠关节软骨缺损膜型,各组大鼠关节在治疗8周后修复图像,进行番红固绿染色以及损伤修复的评分。
图6所示为GCP003可促进C57小鼠关节DMM术后修复以及关节软骨缺损后修复。其中,图6A.小鼠膝关节番红固绿染色。图6B.软骨损伤评分统计结果。图6C.行为学测痛。将手术后8周的小鼠膝关节进行石蜡切片以及番红固绿染色。图6D.C57BL/6小鼠关节软骨缺损膜型,各组小鼠关节在关节腔注射4周后修复效果。用番红固绿染色。n=6,Sacle bars:10X200μm。*P<0.05,**P<0.01,***P<0.001。
具体实施方式
结合以下具体实施例和附图,对发明作进一步的详细说明。实施本发明的过程、条件、实验方法等,除以下专门提及的内容之外,均为本领域的普遍知识和公知常识,本发明没有特别限制内容。
本发明提供了用于诱导间充质干细胞分化为软骨细胞以及用于预防、治疗、改善哺乳动物关节炎或关节损伤和/或与关节炎或关节损伤相关症状的化合物以及这类化合物的制备方法。本发明还提供了这类化合物药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体以及包含至少一种这样的化合物或该化合物药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体的药物组合物。
本发明提供的化合物可使用本领域技术人员已知的标准合成反应或使用本领域已知的方法来合成。可以以线性顺序采用反应来提供所述化合物,或者可使用这些反应来合成片段,随后通过本领域已知的方法进行连接。
如果需要,可使用包括但不限于过滤、蒸馏、结晶及色谱法等常规技术来分离和纯化反应产物。这类材料可使用包括物理常数和波谱数据在内的常规手段来表征。
本发明所使用的低温反应装置为EYELA(PSL-1810)磁力搅拌低温恒温水槽;EYELA(N-1100)旋转蒸发仪;实验所得化合物的纯度结果均来自于Agilent 1200series LCsystem高效液相色谱分析仪(色谱条件:Zorbax XDB-C18(4.6×150nm,5μm),柱温40℃,流动相为MeOH/H2O,运行流速为1.5mL/min,紫外检测波长为254nm,进样量为10μL);核磁共振波谱仪为Bruker 300型或500型(内标:TMS,使用溶剂为CDCl3或DMSO-d6);反应中间体以及终产物的纯化均使用层析色谱柱(所用硅胶200-300目),所使用的硅胶购买于青岛海洋化工厂。所有溶剂在使用前均经过重新蒸馏,所使用的无水溶剂均是按标准方法干燥处理获得;除特别说明外,所有反应均是在氮气保护下进行并用TLC跟踪反应进度,后处理均经过饱和食盐水洗和无水硫酸钠干燥。
所述哺乳动物部分没有患有关节炎或关节损伤,但其患关节炎或关节损伤的风险增加。
本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可以用于预防、治疗、改善任何类型的关节炎或关节损伤或改善各种软骨病症;施用本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物还可以预防关节炎或关节损伤,例如在存在关节炎或关节损伤的遗传或家族史或先前进行过关节手术的情况下,或在关节炎或关节损伤风险增加的其他情况下。本发明提供的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物治疗或预防的示例性的病况或病症包括但不限于系统性类风湿性关节炎、幼年型慢性关节炎、骨关节炎、椎间盘退变性疾病、脊椎关节病和系统性硬化症(硬皮病)。所述关节炎可以是骨关节炎、创伤性关节炎、椎间盘退变性疾病、迪皮特朗病或腱疾病。
本发明还提供了一种用于刺激已经由于创伤性损伤或软骨病而损伤的软骨组织中的软骨细胞增殖和软骨产生的方法。创伤性损伤可以包括但不限于关节的钝伤或韧带的损伤,如前交叉韧带、内侧副韧带撕裂或半月板撕裂;展现出关节联接表面,因此对治疗特别敏感的组织包括但不限于脊柱、肩、肘、腕、手指关节、髋、膝、踝、足关节等。可以受益于治疗的疾病包括骨关节炎、类风湿性关节炎、其他自身免疫性疾病、分离性骨软骨炎等。另外,软骨畸形在人类侏儒形式中常见,因此本发明能够用于帮助治疗软骨畸形的患者。
本发明所述的“哺乳动物”是指被分类为哺乳动物的任何哺乳动物,包括人、驯养动物和农畜、宠物动物或家畜以及动物园饲养、竞技活动所用的动物;所述“宠物动物”是指狗、猫、啮齿动物和兔。具体地,可以是人、牛、马、狗、绵羊、猪、兔、山羊、猫等。动物的选择根据实际情况确定。
本发明还提供了一种诱导间充质干细胞(MSC)分化为软骨细胞的方法。该方法包括使间充质干细胞接触足量的本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物,由此诱导干细胞分化为软骨细胞。
MSCs是多能干细胞,其能够分化成数种不同类型的细胞,包括但不限于成骨细胞、软骨细胞和脂肪细胞。分化过程是由特化程度较低的细胞类型形成特化的细胞类型,例如由MSCs形成软骨细胞的过程。所述诱导间充质干细胞(MSC)分化为软骨细胞的方法可以在体外进行,也可以在哺乳动物的体内进行,并且所述干细胞存在于该哺乳动物中。在具体的实施例中,所述哺乳动物是人、狗、猫或马中的一种或多种。
诱导MSCs分化为软骨细胞可以使用任何适量的本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物来完成,根据活性组分的特定应用和效力,本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物的使用量范围为0.1mg至10000mg,例如1.0mg至1000mg或例如10mg至500mg等。此外,本发明的化合物也可以以0.1μM–100μM的浓度存在于针对膝盖的关节内注射液中。
实施例1:化合物的制备
实施例1-1、5-溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺(GCP001)的制备
将4-氯-2-硝基苯酚(1.74g,10mmol),2-溴乙基甲基醚(4.17g,30mmol),碳酸钾(4.14g,30mmol)溶于N,N-二甲基甲酰胺(30mL)中,80℃反应2小时。TLC检测反应完全后,用乙酸乙酯萃取反应液,有机相用水洗涤两次,饱和食盐水洗一次,无水硫酸钠干燥。蒸干有机相后,通过柱层析分离纯化得到化合物4-氯-1-(2-甲氧基乙氧基)-2-硝基苯(2.2g,产率95%)。
将4-氯-1-(2-甲氧基乙氧基)-2-硝基苯(2.2g,9.5mmol)溶于甲醇(30mL)中,加入钯碳,通入氢气,室温反应2小时。TLC检测反应完全后,用硅藻土抽滤,滤液旋干得到化合物5-氯-2-(2-甲氧基乙氧基)苯胺(1.8g,产率94%)。
将5-氯-2-(2-甲氧基乙氧基)苯胺(202mg,1mmol),5-溴-2-羧基噻吩(228mg,1.1mmol),N,N-二异丙基乙胺(388mg,3mmol)和HATU(418mg,1.1mmol)溶于3mL的N,N-二甲基甲酰胺中,室温搅拌反应6小时。TLC检测反应完全后,用乙酸乙酯萃取反应液,有机相用水洗涤两次,饱和食盐水洗一次,无水硫酸钠干燥。蒸干有机相后,通过柱层析分离纯化得到产物GCP001(320mg,产率82%)。
1H NMR(CDCl3,300MHz):8.52(s,1H),8.31(s,1H),7.22(d,J=8.4Hz,1H),6.91(d,J=3.9Hz,1H),6.86-6.83(m,1H),6.72(d,J=8.7Hz,1H),4.03(t,J=4.2Hz,2H),3.55(t,J=4.2Hz,2H),3.23(s,3H)。
实施例1-2、3-氯-N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺(GCP002)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为3-氯噻吩-2-羧酸,最终得到化合物GCP002,产率80%。
1H NMR(CDCl3,300MHz):9.62(s,1H),8.58(s,1H),7.51(d,J=5.1Hz,1H),7.06-6.95(m,2H),6.85(d,J=5.1Hz,1H),4.18(t,J=4.2Hz,2H),3.76(t,J=4.2Hz,2H),3.41(s,3H)。
实施例1-3、N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺(GCP003)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为2-噻吩甲酸,最终得到化合物GCP003,产率79%。
1H NMR(CDCl3,300MHz):8.73(s,1H),8.55(s,1H),7.64(d,J=3.9Hz,1H),7.55(d,J=3.9Hz,1H),7.13(t,J=3.9Hz,1H),7.02-6.99(m,1H),6.88(d,J=8.4Hz,1H),4.20(t,J=3.6Hz,2H),3.72(t,J=3.6Hz,2H),3.41(s,3H)。
实施例1-4、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(噻吩-2-基)乙酰胺(GCP004)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为2-噻吩乙酸,最终得到化合物GCP004,产率81%。
1H NMR(CDCl3,300MHz):8.45(s,1H),8.22(s,1H),7.31-7.30(m,1H),7.06-7.05(m,2H),6.98-6.95(m,1H),6.76(d,J=8.7Hz,1H),4.03(t,J=4.5Hz,2H),3.96(s,2H),3.60(t,J=4.2Hz,2H),3.41(s,3H)。
实施例1-5、5-溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)呋喃-2-甲酰胺(GCP005)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为5-溴-2-糠酸,最终得到化合物GCP005,产率81%。
1H NMR(CDCl3,300MHz):8.85(s,1H),8.51(s,1H),7.05-7.04(m,2H),7.03-6.98(m,1H),6.86(d,J=8.1Hz,1H),6.51(d,J=1.8Hz,1H),4.21(t,J=3.0Hz,2H),3.80(t,J=3.0Hz,2H),3.52(s,3H)。
实施例1-6、N-(5-氯-2-(2-甲氧基乙氧基)苯基)苯甲酰胺(GCP006)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为苯甲酸,最终得到化合物GCP006,产率72%。
1H NMR(CDCl3,300MHz):8.90(s,1H),8.64-8.63(m,1H),7.93-7.91(m,2H),7.53-7.49(m,3H),7.06-6.98(m,1H),6.91(d,J=8.4Hz,1H),3.73(t,J=4.2Hz,2H),3.50(t,J=4.2Hz,2H),3.37(s,3H)。
实施例1-7、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-苯基乙酰胺(GCP007)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为苯甲酸,最终得到化合物GCP007,产率77%。
1H NMR(CDCl3,300MHz):8.44(s,1H),8.00(s,1H),7.40-7.25(m,5H),6.95-6.91(m,1H),6.73(d,J=8.4Hz,1H),6.82(d,J=8.4Hz,1H),3.83(t,J=4.2Hz,2H),3.61(t,J=4.2Hz,2H),3.41(s,3H)。
实施例1-8、N-(5-氯-2-(2-甲氧基乙氧基)苯基)环丙烷甲酰胺(GCP008)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为环丙甲酸,最终得到化合物GCP008,产率73%。
1H NMR(CDCl3,300MHz):9.36(s,1H),8.07(d,J=1.8Hz,1H),7.12-7.08(m,2H),4.18(s,J=4.5Hz,2H),3.72(t,J=4.5Hz,2H),3.33(s,3H),1.02-1.00(m,1H),0.83-0.79(m,4H)。
实施例1-9、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(2-氟苯基)乙酰胺(GCP009)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为2-氟苯乙酸,最终得到化合物GCP009,产率73%。
1H NMR(CDCl3,300MHz):9.17(s,1H),8.07(s,1H),7.41-7.33(m,2H),7.23-7.16(m,2H),7.10(d,J=0.9Hz,2H),4.16(t,J=4.5Hz,2H),3.84(s,2H),3.67(t,J=4.5Hz,2H),3.32(s,3H)。
实施例1-10、N-(5-氯-2-(2-甲氧基乙氧基)苯基)环戊烷甲酰胺(GCP010)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为环戊酸,最终得到化合物GCP010,产率81%。
1H NMR(CDCl3,300MHz):8.92(s,1H),8.07(s,1H),7.11-7.07(m,2H),4.15(t,J=4.5Hz,2H),3.69(t,J=4.5Hz,2H),3.32(s,3H),2.50-2.47(m,1H),1.85-1.56(m,8H)。
实施例1-11、4,5-二溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)呋喃-2-甲酰胺(GCP011)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为4,5-二溴-2-糠酸,最终得到化合物GCP011,产率84%。
1H NMR(CDCl3,300MHz):9.30(s,1H),7.99(s,1H),7.60(s,1H),7.22-7.18(m,2H),4.20(t,J=4.2Hz,2H),3.67(t,J=4.2Hz,2H),3.34(s,3H)。
实施例1-12、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-氯苯基)乙酰胺(GCP012)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为对氯苯乙酸,最终得到化合物GCP012,产率84%。
1H NMR(CDCl3,300MHz):9.14(s,1H),8.02(s,1H),7.41-7.35(m,4H),7.08-7.04(m,2H),4.13(t,J=4.5Hz,2H),3.77(s,2H),3.64(t,J=4.5Hz,2H),3.30(s,3H)。
实施例1-13、N-(5-氯-2-(2-甲氧基乙氧基)苯基)吡啶酰胺(GCP013)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为2-吡啶甲酸,最终得到化合物GCP013,产率84%。
1H NMR(CDCl3,300MHz):10.65(s,1H),8.72-8.71(m,1H),8.51(d,J=2.1Hz,1H),8.20-8.07(m,2H),7.73-7.68(m,1H),7.21-7.16(m,1H),4.27(t,J=4.2Hz,2H),3.54(t,J=4.2Hz,2H),3.39(s,3H)。
实施例1-14、N-(5-氯-2-(2-甲氧基乙氧基)苯基)异喹啉-1-甲酰胺(GCP014)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为异喹啉-3-甲酸,最终得到化合物GCP014,产率79%。
1H NMR(CDCl3,300MHz):10.93(s,1H),9.47(d,J=8.1Hz,1H),8.62(d,J=8.1Hz,1H),8.54(d,J=1.6Hz,1H),8.18-8.11(m,2H),7.89-7.82(m,2H),7.18(s,2H),4.27(t,J=4.2Hz,2H),3.60(t,J=4.2Hz,2H),3.39(s,3H)。
实施例1-15、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(2-氯苯基)乙酰胺(GCP015)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为邻氯苯乙酸,最终得到化合物GCP015,产率71%。
1H NMR(CDCl3,300MHz):8.47(s,1H),8.13(s,1H),7.47-7.42(m,2H),7.32-7.25(m,2H),6.98-6.95(m,1H),6.78(t,J=8.7Hz,2H),4.06(t,J=4.5Hz,2H),3.90(s,2H),3.62(t,J=4.5Hz,2H),3.42(s,3H)。
实施例1-16、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(对甲苯基)乙酰胺(GCP016)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为对甲基苯乙酸,最终得到化合物GCP016,产率71%。
1H NMR(CDCl3,300MHz):8.46(s,1H),8.00(s,1H),7.27-7.16(m,4H),6.97-6.93(m,1H),6.73(d,J=8.7Hz,1H),3.99(t,J=4.5Hz,2H),3.72(s,2H),3.53(t,J=8.1Hz,2H),3.40(s,3H),2.37(s,3H)。
实施例1-17、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-氟苯基)乙酰胺(GCP017)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为对氟苯乙酸,最终得到化合物GCP017,产率71%。
1H NMR(CDCl3,300MHz):8.46(s,1H),8.01(s,1H),7.35-7.31(m,2H),7.13-7.10(m,2H),7.09-6.96(m,2H),7.13(d,J=8.7Hz,1H),4.03(t,J=4.5Hz,2H),3.75(s,2H),3.56(t,J=4.5Hz,2H),3.41(s,3H)。
实施例1-18、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-3-苯基丙酰胺(GCP018)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为苯丙酸,最终得到化合物GCP018,产率71%。
1H NMR(CDCl3,300MHz):8.49(s,1H),8.14(s,1H),7.34-7.21(m,5H),7.00-6.99(m,1H),6.83(d,J=8.7Hz,1H),4.11(t,J=4.5Hz,2H),3.69(s,3H),3.42(s,2H),3.07(t,J=8.1Hz,2H),2.94(t,J=8.1Hz,2H)。
实施例1-19、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-4-苯基丁酰胺(GCP019)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为苯丁酸,最终得到化合物GCP019,产率71%。
1H NMR(CDCl3,300MHz):8.51(s,1H),8.17(s,1H),7.33-7.30(m,3H),7.26-6.23(m,2H),7.02-6.98(m,1H),6.90(d,J=8.7Hz,1H),4.16(t,J=4.5Hz,2H),3.72(t,J=4.5Hz,2H),3.40(s,3H),2.76(t,J=7.5Hz,2H),2.42(t,J=7.5Hz,2H),2.10(t,J=7.5Hz,2H)。
实施例1-20、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(3-氯苯基)乙酰胺(GCP020)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为间氯苯乙酸,最终得到化合物GCP020,产率71%。
1H NMR(CDCl3,300MHz):8.45(s,1H),8.07(s,1H),7.37-7.33(m,2H),7.27-7.25(m,2H),6.99-6.95(m,1H),6.80(t,J=8.7Hz,2H),4.06(t,J=4.5Hz,2H),3.72(s,2H),3.61(t,J=4.5Hz,2H),3.42(s,3H)。
实施例1-21、N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-溴苯基)乙酰胺(GCP021)的制备
采用与制备化合物GCP001类似的方法,将5-溴-2-羧基噻吩替换为对溴苯乙酸,最终得到化合物GCP021,产率71%。
1H NMR(CDCl3,300MHz):8.44(s,1H),8.03(s,1H),7.53(s,J=8.4Hz,2H),7.27-7.22(m,2H),6.98-6.95(m,2H),6.77(d,J=8.4Hz,2H),4.04(t,J=4.5Hz,2H),3.70(s,2H),3.58(t,J=4.5Hz,2H),3.43(s,3H)。
实施例1-22、N-(3-溴苯基)噻吩-2-甲酰胺(GCP022)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为3-溴苯胺,最终得到化合物GCP022,产率71%。
1H NMR(500MHz,Chloroform-d):7.89–7.85(m,1H),7.67(s,1H),7.63(dd,J=3.7,1.1Hz,1H),7.57(dd,J=5.0,1.1Hz,1H),7.53(ddd,J=8.0,1.9,1.0Hz,1H),7.31–7.25(m,1H),7.24–7.20(m,1H),7.14(dd,J=5.0,3.8Hz,1H)。
实施例1-23、N-(3-(三氟甲基)苯基)噻吩-2-甲酰胺(GCP023)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为间-三氟甲基苯胺,最终得到化合物GCP023,产率70%。
1H NMR(500MHz,Chloroform-d):7.91(s,1H),7.84(d,J=8.1Hz,1H),7.79(s,1H),7.66(dd,J=3.7,1.1Hz,1H),7.59(dd,J=5.0,1.1Hz,1H),7.52–7.45(m,1H),7.40(d,J=7.8Hz,1H),7.15(dd,J=5.0,3.8Hz,1H)。
实施例1-24、N-(3-氰基苯基)噻吩-2-甲酰胺(GCP024)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为间-氰基苯胺,最终得到化合物GCP024,产率70%。
1H NMR(500MHz,Chloroform-d):8.01(s,1H),7.88–7.83(m,2H),7.69–7.65(m,1H),7.60(dd,J=5.0,0.9Hz,1H),7.52–7.38(m,2H),7.15(dd,J=4.9,3.8Hz,1H)。
实施例1-25、N-(3-甲氧基苯基)噻吩-2-甲酰胺(GCP025)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为间-甲氧基苯胺,最终得到化合物GCP025,产率75%。
1H NMR(500MHz,Chloroform-d):7.67(s,1H),7.62(dd,J=3.7,1.1Hz,1H),7.55(dd,J=5.0,1.1Hz,1H),7.44–7.40(m,1H),7.28–7.22(m,1H),7.13(dd,J=5.0,3.8Hz,1H),7.05(dd,J=8.0,1.3Hz,1H),6.74–6.68(m,1H),3.83(s,2H)。
实施例1-26、N-(4-氰基苯基)噻吩-2-甲酰胺(GCP026)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为4-氰基苯胺,最终得到化合物GCP026,产率71%。
1H NMR(500MHz,Chloroform-d):7.84(s,1H),7.77(d,J=8.7Hz,2H),7.69–7.63(m,3H),7.63–7.59(m,1H),7.16(dd,J=4.8,3.9Hz,1H)。
实施例1-27、N-(2-氰基苯基)噻吩-2-甲酰胺(GCP027)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为邻氰基苯胺,最终得到化合物GCP027,产率70%。
1H NMR(500MHz,Chloroform-d):8.55(d,J=8.5Hz,1H),8.26(s,1H),7.71(dd,J=3.8,0.9Hz,1H),7.67–7.57(m,3H),7.24–7.14(m,2H)。
实施例1-28、N-(3-溴苄基)噻吩-2-甲酰胺(GCP028)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为(3-溴苯基)甲胺,最终得到化合物GCP028,产率79%。
1H NMR(500MHz,Chloroform-d):7.52(dd,J=3.7,0.9Hz,1H),7.52–7.48(m,2H),7.45–7.41(m,2H),7.29(d,J=7.7Hz,1H),7.24–7.20(m,1H),7.09(dd,J=4.9,3.8Hz,1H),4.60(d,J=5.9Hz,2H)。
实施例1-29、N-(4-氰基苄基)噻吩-2-甲酰胺(GCP029)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为4-(氨基甲基)苄腈,最终得到化合物GCP029,产率70%。
1H NMR(500MHz,Chloroform-d):7.63(d,J=8.3Hz,2H),7.53(ddd,J=15.7,4.3,1.0Hz,2H),7.46(d,J=8.2Hz,2H),7.10(dd,J=4.9,3.8Hz,1H),6.41(s,1H),4.68(d,J=6.1Hz,2H)。
实施例1-30、N-(3-氰基苄基)噻吩-2-甲酰胺(GCP030)的制备
采用与制备化合物GCP003类似的方法,将5-氟-2-(2-甲氧基乙氧基)苯胺替换为3-(氨基甲基)苄氰,最终得到化合物GCP030,产率70%。
1H NMR(500MHz,Chloroform-d):7.64(s,1H),7.59(dd,J=15.6,7.8Hz,2H),7.55(dd,J=3.7,0.9Hz,1H),7.52(dd,J=5.0,0.9Hz,1H),7.49–7.44(m,1H),7.10(dd,J=4.9,3.8Hz,1H),6.43(s,1H),4.66(d,J=6.1Hz,2H)。
表1实施例1-30(GCP001-030)所示化合物制备汇总
本发明的化合物可使用多种试验来鉴定;最初的筛选鉴定了刺激人间充质干细胞(hMSCs)以发展成软骨细胞结节的化合物,并进行阿尔新蓝染色、3D成球等实验,并分别从mRNA和蛋白水平来确定本发明的化合物对软骨分化特异性,并通过毒性试验确定本发明的化合物的毒性。
实施例2:利用MSCs进行化合物的筛选
实施例2-1、细胞的培养与分化
选用人骨髓间充质干细胞(hBMSCs),脐带间充质干细胞(hUCMSCs)与脂肪间充质干细胞(hADMSCs),在间充质干细胞生长培养基(MSCGM)中培养,培养获得的状态良好的2-8代间充质干细胞可用于实验。利用流式细胞仪鉴定了多种干细胞表面marker抗体,证明其对CD44,CD73,CD90和CD105的阳性率>98%,对CD45的阳性率<0.1%,以确定分离得到的干细胞纯度。
实施例2-2、利用免疫细胞化学染色与定量对化合物进行筛选
为让MSCs向软骨分化,将5000个细胞/孔在Corning 384孔板中铺板。过夜培养,除去培养基,并且用25μL包含1%FBS的DMEM(包含所示剂量的待测试化合物)替换。在37℃生长7天。
为检测潜在的促进向软骨分化的蛋白,用4%PFA固定细胞15分钟,用包含0.1%的triton X-100、0.25g/mL的胶原酶2的PBS渗透10分钟,在室温用包含5%BSA的PBST封闭1小时,然后再4℃与包含1%BSA的PBS中的II型胶原蛋白抗体一起孵育过夜。去除一抗后用PBS将细胞洗涤3次,在室温与荧光团缀合的二级抗体一起孵育1小时,然后用PBS洗涤3次,再与DAPI一起孵育5分钟,然后用PBS洗涤3次。使用ImageXpress Micro根据高容量推定定量。使用专用多波长细胞评分脚本进行数据分析,计算本发明实施例1制备的化合物刺激II型胶原蛋白表达水平。
在化合物活性评价上,使用阴性对照(DMSO)作为MSCs成软骨分化的基础水平。选择显示出染色强度相比阴性对照增加30%或更多的本发明实施例1制备的化合物作为活性命中物。代表性数据在表2中示出(A:与阴性对照相比染色强度增加>200%;B:与阴性对照相比染色强度增加100-200%;C:与阴性对照相比染色强度增加30-100%;D:与阴性对照相比染色强度增加<30%)。
结果如表2所示,绝大部分本发明实施例1制备的化合物都能够显著促进间充质干细胞中II型胶原蛋白的表达。这些结果表明本发明实施例1制备的化合物能够诱导间充质干细胞成软骨分化。
表2.本发明化合物在诱导软骨分化中的活性
实施例3:GCP003可以在体外促进hUCMSC向软骨分化
为了验证不同浓度的本发明实施例1制备的GCP003对hUCMSCs向软骨细胞分化的影响,本实施例设计了hUCMSCs悬滴培养分化实验(图1A),hUCMSCs悬滴诱导分化,将hUCMSCs(5万细胞/12.5μL/孔)轻轻滴加在24孔板中央,于培养箱中培养2-3小时后补加含有小分子GCP003和KGN的培养基。诱导分化期间隔3天换一次新鲜培养基。选取分化3、5、7天的细胞进行阿尔新蓝染色观测分化情况。PBS洗三次,4%多聚甲醛固定10min,1%阿尔新蓝(3%冰醋酸配)染色过夜。用PBS洗三次染色后的24孔板,用照相机拍照记录。同时进行了基因水平(图1B)和蛋白水平(图1C)的检测,包括软骨marker Collagen II、Aggrecan和Sox9的表达。不同浓度1、3.3、10μM的GCP003诱导hUCMSCs向软骨细胞分化。为了验证GCP003在不同分化时间对hUCMSCs向软骨细胞分化的作用效果,本实施例同样利用hUCMSCs悬滴培养分化实验,进行阿尔新蓝染色(图1D),以及基因水平(图1E)的检测,包括软骨markerCollagen II、Aggrecan表达。
结果显示,在一定时间内,分化时间越长效果越好。于不同分化时间点(3、5、7天)收样,10μM的GCP003诱导的hUCMSCs向软骨细胞分化。
实施例4:GCP003能促进hUCMSC 3D成球软骨分化
为了进一步确认GCP003促进软骨分化效率,本实施例利用了体外干细胞悬浮3D培养的实验,评价成球效果和成软骨分化的效果,包括小球重量和培养小球期间培养基上清中可聚蛋白多糖的含量,可聚蛋白多糖(Aggrecan)是一种蛋白多糖大分子,它由糖胺多糖(Alycosaminoglycan,GAG)和核心蛋白(Core protein)组成。Aggrecan与透明质酸以非共价键结合,形成蛋白多糖聚合体,是软骨细胞外基质(extracellular matrix,ECM)的主要结构成分之一,蛋白多糖的进行性丧失是骨关节疾病的特点之一。
hUCMSCs 3D成球实验诱导分化,首先消化计数,25万/2mL/管(15mL离心管),400g离心10min,放于培养箱中。第二天可观察到悬浮的小球产生,更换含有GCP003和KGN的培养基,3天换一次新鲜培养液,每天要轻轻摇晃离心管使小球悬浮,连续悬浮培养21天。检测分化培养基上清中GAG的含量。分化结束后,将小球取出后用PBS洗1-2遍,然后用体视显微镜拍照,记录球体大小、透明度和形貌。固定、脱水、透明、浸蜡、包埋切片,并通过阿尔新蓝染色及免疫荧光检测分化效果。
结果如图2所示,相对于已经报道的化合物KGN,GCP003可以更明显的促进hUCMSCs体外3D成球的分化效果(图2A)。检测了培养基上清GAG的含量,研究小球在分化期间可聚蛋白多糖的情况(图2B)。随后,将小球切片,利用免疫荧光染色技术检测了软骨markerCollagen II的表达(图2C),阿尔新蓝染色(图2D)的结果也显示,GCP003可以明显的促进软骨分化。体外3D成球实验评估GCP003诱导hUCMSCs成球的能力。
实施例5:GCP003能促进BMSC成软骨分化
前期实验结果显示,GCP003可以促进hBMSCs的迁移。本实施例研究GCP003是否可以促进hBMSCs的分化。本实施例设计了干细胞悬滴分化实验,GAG测定实验,干细胞3D成球培养实验和基因表达实验等。最终确定了GCP003具有诱导hBMSCs分化的能力。图3A显示,10μM的GCP003以促进hBMSCs的分化,阿尔新蓝染色效果类似于KGN。图3B显示,收集诱导分化7天后的培养基上清测定可聚蛋白多糖GAG的含量,结果显示,经过GCP003和KGN诱导分化的BMSCs可释放出更多的GAG。图3C显示,GCP003和KGN诱导hBMSCs成球培养,二者都展示出比对照更好的诱导效果。随后,基因表达的结果图3D也显示,GCP003和KGN诱导的hBMSCs可以促进软骨细胞marker Collagen II和Aggrecan的基因表达。在此基础上,本实施例中还收集了一例临床关节置换的老年人的骨髓间充质干细胞,分离后进行诱导分化,并进行基因表达检测。实验结果显示,GCP003也可以促进老年人的骨髓间充质干细胞向软骨分化(图3E)。
实施例6:经GCP003诱导7天后hUCMSCs,可以促进SD大鼠DMM修复
前期体外实验结果表明,经GCP003诱导7天后hUCMSCs具有较好的分化效果和较低的免疫原性。所以,本实施例建立了SD大鼠急性损伤OA手术,即内侧半月板不稳定手术模型(DMM),该方法通过对SD大鼠(n=6/组)通过手术切断内侧半月板胫骨韧带(MMTL),内侧副韧带(MCL),造成膝关节的失稳从而损伤关节软骨,导致OA的表型形成。在模型建立7周后进行干细胞注射。根据前期的实验结果,本实施例选取了诱导7天后的hUCMSCs进行关节腔注射(图4)。
结果显示,PBS组和单独hUCMSCs组关节损伤都比较严重,注射经过GCP003诱导7天后hUCMSCs的关节腔,显示出比较好的关节软骨修复效果。SD大鼠关节炎膜型(DMM),在干细胞治疗后的效果。
实施例7:复合GCP003诱导后hUCMSCs的水凝胶可以促进大鼠软骨缺损修复
本发明前期实验观察到直接注射干细胞会导致大鼠软骨缺损修复模型(MF)的关节出现异常组织增生的情况,因此探索借助生物材料携带和固定干细胞到软骨缺损部位。即利用水凝胶(Gel)包裹MSCs。本实施例使用4%水合氯醛(0.5mL/100g)麻醉雄性SD大鼠(8周龄),依次切开皮肤和肌肉,暴露膝关节,用2mm克氏针造成直径为2mm、深度为2mm的软骨缺损。大鼠随机分为Sham组、水凝胶组、Gel+MSCs组、Gel+MSCs+GCP003组,术后8周,CO2窒息器处死大鼠,收集关节标本,每个样品的软骨修复状态通过数码相机获得。
结果如图5所示,水凝胶组仍有部分的软骨缺损的存在。在Gel+MSCs(未经诱导分化)组中,可以观察到,缺损部位已经完全被覆盖上一层乳白色的组织层,但是这层组织与周围正常软骨组织差别比较大,推测可能是硬度较低的组织。而且,可以很明显的观察到软骨缺损部位。在Gel+MSCs+GCP003(GCP003诱导后的干细胞组)组中,可以观察到,缺损部位已经完全被覆盖上一层组织,这层组织颜色和周围正常软骨融合的很好,且新生组织较难观察出来,这表明,GCP003诱导后的干细胞能够更好的促进干细胞向软骨组织分化,进而达到修复软骨缺损的目的。
实施例8:GCP003能促进C57BL/6小鼠手术诱发急性损伤OA模型及关节软骨缺损模型修复
为了检测关节腔中注射GCP003是否可以促进小鼠体内关节腔修复,本实施例构建了小鼠的急性损伤OA手术,即内侧半月板不稳定手术模型(DMM)。为评价代测化合物促软骨修复能力,实验共分为四组,依次为Sham假手术组(仅切开皮肤未伤害关节韧带),阴性对照(关节腔注射PBS),KGN给药组(KGN溶于PBS,配成33μM,进行关节腔注射),代测化合物GCP003给药组(代测化合物溶于PBS,配成33μM,进行关节腔注射)。在术后3天,各组进行关节腔注射给药,每周注射一次。在8周后,处死小鼠,取出关节样本,进行包埋切片,观察组织学改善情况并进行统计分析。
进一步对6周大C57BL/6小鼠进行了关节软骨损伤模型(MF),利用1mL一次性无菌注射器针头(26G)诱发软骨损伤,针头提前打磨使其变钝,将10μL透明枪头固定在上述针头上,使尖端露出1mm。术前使用阿弗丁(300μL/25g)麻醉雄性C57BL/6小鼠,尽量减轻手术过程中小鼠的痛苦。用75%的酒精对后肢及手术桌面进行消毒。用外科手术刀在股骨近端内侧暴露关节腔,把髌骨移到侧面,然后弯曲关节以露出股骨滑车沟。使用上述针头在滑车沟中造成纵向软骨损伤,通过软骨损伤部位的出血来确认穿透到软骨下骨(损伤深度1mm),最后,用小鼠缝合线将关节囊和皮肤缝合。实验共分为四组(n=6),依次为假手术Sham组、PBS组、KGN给药组与GCP003给药组。两种药物均用PBS配成33μM的浓度用于关节腔注射。造模完成3天后,进行关节腔注射给药,每周一次。4周后,采用颈椎脱臼法将小鼠处死,取出其股骨,体式显微镜拍照观察之后,固定组织,然后脱钙切片,番红固绿染色观测软骨损伤修复的过程。
结果显示,关节腔注射GCP003和KGN可以明显逆转这种损伤(图6A),统计结果显示经过治疗的小鼠骨关节炎损伤明显减轻(图6B)。同时,对8周的小鼠,进行了疼痛行为学分析,包括:机械痛和热痛,结果显示,关节腔注射GCP003可以明显缓解小鼠机械痛(图6C)。而在关节软骨缺损模型中,可以发现阳性化合物KGN并没有很好的软骨损伤修复效果,而本发明化合物GCP003在给药33μM剂量下能够显著促进软骨缺损部位的软骨修复,表明化合物GCP003具有很好的应对软骨损伤修复效果(图6D)。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求书为保护范围。
Claims (20)
1.一类如式(I)所示的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,其特征在于,所述化合物的结构如下所示:
其中:
k为0、1、2;
m为0、1、2、3或4;
n为0、1、2、3或4;
R1独立地为H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基、C1-6烷基羟基、O-C1-6烷基-O-C1-6烷基;
A环选自C3-7环烷基,C7-C10桥环,含一个或多个O、N、S原子的杂环烷基,芳基,含一个或多个O、N、S原子的五元或六元杂环芳基,含一个或多个O、N、S原子的苯并杂环;所述芳基选自苯基,蒽基,萘基,菲基;所述杂环芳基选自吡啶基,呋喃基,吡唑基,吡喃基,哒嗪基,嘧啶基,咪唑基,噻唑基,异噻唑基,哌嗪基,喹啉基,吡嗪基,异喹啉基,噻吩基,噁唑基,异噁唑基,吲哚基,苯并呋喃基,苯并噻吩基,嘌呤基,咔唑基,吖啶基,吩基嗪,吩噻嗪基,喋啶基;其中,所述环烷基,杂环烷基,芳基,杂环芳基的环上的基团被0-2个R2基团取代;
所述R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基。
2.如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,其特征在于,当m=0时,其由下述结构(II)表示:
其中:
k为0、1、2;
n为0、1、2、3或4;
R1独立地为H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基、C1-6烷基羟基、O-C1-6烷基-O-C1-6烷基;
A环选自C3-7环烷基,C7-C10桥环,含一个或多个O、N、S原子的杂环烷基,芳基,含一个或多个O、N、S原子的五元或六元杂环芳基,含一个或多个O、N、S原子的苯并杂环;所述芳基选自苯基,蒽基,萘基,菲基;所述杂环芳基选自吡啶基,呋喃基,吡唑基,吡喃基,哒嗪基,嘧啶基,咪唑基,噻唑基,异噻唑基,哌嗪基,喹啉基,吡嗪基,异喹啉基,噻吩基,噁唑基,异噁唑基,吲哚基,苯并呋喃基,苯并噻吩基,嘌呤基,咔唑基,吖啶基,吩基嗪,吩噻嗪基,喋啶基;其中,所述环烷基,杂环烷基,芳基,杂环芳基的环上的基团被0-2个R2基团取代;
所述R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基。
其中:
n为0、1、2、3或4;
A环选自C3-7环烷基,C7-C10桥环,含一个或多个O、N、S原子的杂环烷基,芳基,含一个或多个O、N、S原子的五元或六元杂环芳基,含一个或多个O、N、S原子的苯并杂环;所述芳基选自苯基,蒽基,萘基,菲基;所述杂环芳基选自吡啶基,呋喃基,吡唑基,吡喃基,哒嗪基,嘧啶基,咪唑基,噻唑基,异噻唑基,哌嗪基,喹啉基,吡嗪基,异喹啉基,噻吩基,噁唑基,异噁唑基,吲哚基,苯并呋喃基,苯并噻吩基,嘌呤基,咔唑基,吖啶基,吩基嗪,吩噻嗪基,喋啶基;其中,所述环烷基,杂环烷基,芳基,杂环芳基的环上的基团被0-2个R2基团取代;
所述R2选自H、卤素、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、卤素、C1-6卤代烷基、C1-6烷基氰基、氰基、硝基、羧基、氨基、叠氮基。
7.如权利要求1-6之任一项所述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,其特征在于,所述化合物具体选自:
5-溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺;
3-氯-N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)噻吩-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(噻吩-2-基)乙酰胺;
5-溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)呋喃-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)苯甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-苯基乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)环丙烷甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(2-氟苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)环戊烷甲酰胺;
4,5-二溴-N-(5-氯-2-(2-甲氧基乙氧基)苯基)呋喃-2-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-氯苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)吡啶酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)异喹啉-1-甲酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(2-氯苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(对甲苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-氟苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-3-苯基丙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-4-苯基丁酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(3-氯苯基)乙酰胺;
N-(5-氯-2-(2-甲氧基乙氧基)苯基)-2-(4-溴苯基)乙酰胺;
N-(3-溴苯基)噻吩-2-甲酰胺;
N-(3-(三氟甲基)苯基)噻吩-2-甲酰胺;
N-(3-氰基苯基)噻吩-2-甲酰胺;
N-(3-甲氧基苯基)噻吩-2-甲酰胺;
N-(4-氰基苯基)噻吩-2-甲酰胺;
N-(2-氰基苯基)噻吩-2-甲酰胺;
N-(3-溴苄基)噻吩-2-甲酰胺;
N-(4-氰基苄基)噻吩-2-甲酰胺;
N-(3-氰基苄基)噻吩-2-甲酰胺。
8.一种药物组合物,其特征在于,所述药物组合物包含如权利要求1-7中任一项所述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体以及药学上可接受的赋形剂。
9.如权利要求8所述的药物组合物,其特征在于,所述的药物组合物还包含能有效治疗、改善、预防哺乳动物的关节炎或关节损伤和/或与关节炎或关节损伤相关症状的额外化合物。
10.如权利要求9所述的药物组合物,其特征在于,所述额外化合物选自NSAIDS、镇痛药、血管生成素样3蛋白(ANGPTL3)或其软骨形成变体、口服鲑鱼降钙素、iNOS抑制剂、维生素D3、凋亡/胱天蛋白酶抑制剂、胶原水解物、FGF18、BMP7、鳄梨大豆不皂化物(ASU)、透明质酸中的一种或几种。
11.如权利要求1-7之任一项所述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体、或如权利要求8-10之任一项所述的药物组合物在制备预防和/或治疗和/或改善哺乳动物的关节炎或关节损伤和/或与关节炎或关节损伤相关症状的药物中的用途。
12.如权利要求11所述的用途,其特征在于,所述关节炎是骨关节炎、创伤性关节炎、自身免疫性关节炎中的一种或几种。
13.如权利要求11所述的用途,其特征在于,所述化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体、药物组合物可直接或在基质和生物相容性骨架中通过关节腔施用于所需的对象。
14.一种体外诱导间充质干细胞分化成软骨细胞的方法,其特征在于,所述方法通过使间充质干细胞接触足量如权利要求1-7之任一项所述的化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或如权利要求8-10之任一项所述的药物组合物诱导干细胞定向分化成软骨细胞。
15.如权利要求14所述的方法,其特征在于,所述间充质干细胞选自骨髓间充质干细胞,脐带间充质干细胞,脂肪间充质干细胞,滑膜间充质干细胞中的一种或几种。
16.如权利要求14所述的方法,其特征在于,所述接触在哺乳动物体外或体内进行,并且当在体内进行时,所述干细胞存在于哺乳动物中。
17.如权利要求16所述的方法,其特征在于,所述哺乳动物是人、陪伴动物或家畜、驯养动物或家畜中的一种或几种。
18.如权利要求14所述的方法,其特征在于,所述化合物其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体,或药物组合物可直接或在基质和生物相容性骨架中进行施用于所需的对象。
19.如权利要求14所述的方法,其特征在于,与所述化合物或其药学上可接受的盐、溶剂化物、多晶型物、前药、酯、代谢物、N-氧化物、立体异构体或异构体或药物组合物的接触与一种或多种另外的软骨形成因子结合进行。
20.如权利要求19所述的方法,其特征在于,所述软骨形成因子选自血管生成素样3蛋白(ANGPTL3)、口服鲑鱼降钙素、SD-6010(iNOS抑制剂)、维生素D3(胆骨化醇)、胶原蛋白水解物、FGF18、BMP7、醋酸芦沙拉肽、鳄梨大豆不皂化物(ASU)、类固醇和类固醇抗炎药(NSAID)、透明质酸中的一种或几种。
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