CN115197215A - 取代三环类prmt5抑制剂的晶型 - Google Patents
取代三环类prmt5抑制剂的晶型 Download PDFInfo
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- CN115197215A CN115197215A CN202210381825.7A CN202210381825A CN115197215A CN 115197215 A CN115197215 A CN 115197215A CN 202210381825 A CN202210381825 A CN 202210381825A CN 115197215 A CN115197215 A CN 115197215A
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- cyclopropane
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- dihydro
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Abstract
本发明属于医药化学领域,涉及取代三环类PRMT5抑制剂的晶型及其制备方法与用途,具体涉及式(I)的(R)‑7’‑((2‑乙酰基‑2‑氮杂螺[3.3]庚‑6‑基)氨基)‑2’‑(3‑(3,4‑二氢异喹啉‑2(1H)‑基)‑2‑羟丙基)‑2’,3’‑二氢‑1’H‑螺[环丙烷‑1,4’‑[2,6]萘啶]‑1’‑酮的晶型及其制备方法,所述的晶型可用于制备治疗PRMT5介导的疾病的药物,
Description
技术领域
本发明属于医药化学领域,具体涉及(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的晶型及其制备方法与用途。
背景技术
DNA的修饰在触发细胞生长和发育的不同阶段的基因表达程序中起着核心作用,其中精氨酸甲基化在细胞进程中担任重要角色,包括信号传导,转录,RNA加工,DNA重组和修复。蛋白精氨酸甲基转移酶(PRMTs)通过将甲基从S-腺苷甲硫氨酸(SAM)转移到精氨酸的胍氮来催化特定精氨酸残基的甲基化,根据催化精氨酸甲基化方式的不同可将PRMTs分为三类:I型(PRMT1,2,3,4,6和8)催化单甲基化和不对称二甲基化,II型(PRMT5和PRMT9)催化单甲基化和对称二甲基化,而III型(PRMT7)仅进行单甲基化。
其中,PRMT5与甲基转移酶复合体蛋白50(MEP50)特异性结合,可以对称甲基化组蛋白H3和H4,并调节特定靶基因组的转录。PRMT5催化的组蛋白H3精氨酸8(R8)和H4R3对称二甲基化已显示抑制几种肿瘤抑制基因的表达,例如抑癌基因7(ST7),视网膜母细胞瘤(RB)肿瘤抑制基因家族和受体O型蛋白质酪氨酸磷酸酶(PTPROt)。
除了甲基化组蛋白的能力外,PRMT5还能够甲基化几种重要的转录因子,使其在细胞调节的过程发挥重要作用。PRMT5可以甲基化p53并改变其DNA结合活性,从而引发p53控制的基因表达程序的变化。PRMT5还显示甲基化N-MYC并改变其蛋白质稳定性以及增强其在神经母细胞瘤中的致癌活性。PRMT5还可直接甲基化转录因子,包括E2F-1和NF-κB/p65,诱导其靶基因表达。PRMT5不仅可修饰核转录因子,还可甲基化细胞质蛋白如golgin,核糖体蛋白S10(RPS10)。因此,除了其直接调节其自身靶基因的能力之外,PRMT5还能够通过关键转录因子的对称甲基化间接影响全局基因表达,从而影响细胞生长,增殖和分化。
大量研究已经证实PRMT5在不同类型和侵袭性的癌症中过度表达,包括B细胞和T细胞淋巴瘤,转移性黑素瘤,神经母细胞瘤和成胶质细胞瘤,生殖细胞肿瘤,卵巢癌,鼻咽癌,乳腺癌,结肠直肠癌和胃癌。目前研究表明PRMT5在控制细胞生长和增殖中起重要作用,并且其过表达促进细胞转化。
癌细胞中增强的PRMT5表达与其靶肿瘤抑制基因的转录沉默相关。PRMT5能够通过启动子组蛋白H3R8和H4R3的甲基化以及通过修饰包括E2F1和NF-kB/p65的关键转录因子的特定精氨酸残基引起全局染色质变化来促进癌细胞生长。PRMT5还会与程序性细胞死亡4(PDCD4)相互作用,使其在R110处变为甲基化并且在MCF-7细胞中丧失其肿瘤抑制活性。总的来说,PRMT5过表达可能使其与生长促进蛋白和肿瘤抑制蛋白的相互作用,从而以有利于癌细胞生长,存活与转移。
综上所述,PRMT5抑制剂在治疗肿瘤等相关疾病方面有着明确的机制,有很大潜力可以成为肿瘤治疗领域新的治疗手段,因此,需要开发更安全、更有效的PRMT5抑制剂以满足临床需求。
发明内容
本发明的发明人发现了一种取代三环类PRMT5抑制剂,该抑制剂的化合物结构如下式(I)所示,化学名称为(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮(以下简称“式(I)化合物”):
本发明的发明人研究发现,式(I)化合物或其水合物、溶剂合物或结晶对PRMT5表现出了显著的抑制活性,非常有希望成为PRMT5相关疾病的治疗剂。本领域技术人员知道,药用活性化合物的晶型结构往往影响其化学稳定性、溶解度等性质,因此需要深入研究寻找适合药用的晶型。本发明的目的是提供一种水溶性好、生物利用度高、稳定性高的取代三环类PRMT5抑制剂的晶型。具体地说,本发明提供一种如式(I)所示的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型,
本发明的发明人对式(I)化合物晶型进行了X-射线粉末衍射、差示扫描热分析(DSC)、热重分析(TGA)等检测。
在一些实施方案中,本发明式(I)化合物晶型A的X-射线粉末衍射图谱,参见图1,使用Cu-Ka辐射,以2θ角度表示X-射线粉末衍射图,其在约7.3±0.2、7.9±0.2、9.5±0.2、13.5±0.2、18.8±0.2处有特征峰。
进一步地,本发明式(I)化合物晶型A的X-射线粉末衍射图谱在约7.3±0.2、7.9±0.2、9.5±0.2、13.5±0.2、18.8±0.27.3±0.2、7.9±0.2、9.5±0.2、13.5±0.2、18.8±0.27.3±0.2、7.9±0.2、9.5±0.2、13.5±0.2、18.8±0.2处有特征峰。
再一步地,本发明式(I)化合物晶型A的X-射线粉末衍射图谱在约7.3±0.2、7.9±0.2、9.5±0.2、12.4±0.2、13.5±0.2、17.0±0.2、17.6±0.2、18.2±0.2、18.8±0.2、19.8±0.2处有特征峰。
更进一步地,本发明式(I)化合物晶型A的X-射线粉末衍射图谱在约7.3±0.2、7.9±0.2、9.5±0.2、12.4±0.2、13.5±0.2、17.0±0.2、17.6±0.2、18.2±0.2、18.8±0.2、19.8±0.2、21.4±0.2、24.8±0.2、26.6±0.2处有特征峰。
更进一步地,本发明式(I)化合物晶型A的X-射线粉末衍射图谱在约7.3±0.2、7.9±0.2、9.5±0.2、11.2±0.2、12.4±0.2、13.5±0.2、14.5±0.2、17.0±0.2、17.6±0.2、18.2±0.2、18.8±0.2、19.8±0.2、21.4±0.2、22.6±0.2、23.21±0.2、23.9±0.2、24.8±0.2、26.6±0.2、27.1±0.2、28.3±0.2、29.8±0.2、30.1±0.2、31.0±0.2、34.2±0.2处有特征峰。
非限制性地,在一个具体的实施方案中,本发明式(I)化合物晶型A具有如图1所示的X-射线粉末衍射图谱。
非限制性地,在一个具体的实施方案中,本发明式(I)化合物晶型A的DSC图谱(参见图2)显示样品在147.7℃有尖锐的吸热峰。
非限制性地,在一个具体的实施方案中,本发明式(I)化合物晶型A具有如图3所示的热重分析(TGA)图谱,结果显示,其在100℃前未见明显失重,在150℃附近存在0.428%失重。
本发明提供一种所述的式(I)所示的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A的制备方法,包括将(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮置于有机溶剂中,然后过滤的步骤。在一个具体的实施方案中,根据本发明的式(I)所示的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A的制备方法,包括将(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮置于有机溶剂中,升温回流溶解,随后降温析晶,过滤的步骤,其中所述的原料(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的存在形态没有特别限定,可以使用任意晶体或无定型物,其中所述的有机溶剂选自乙酸乙酯、丙酮、四氢呋喃和乙腈。
按照本发明的方法制备得到的晶型A不含有或者含有较低含量的残留溶剂,符合国家药典规定的有关医药产品残留溶剂的限量要求,可以较好的作为医药活性成分使用。
本发明另一方面提供式(I)化合物无定型物。
非限制性地,本发明式(I)化合物无定型物的一个典型实例具有如图4所示的X-射线粉末衍射图谱。
本发明提供本发明的式(I)所示的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮无定型物的制备方法,包括将(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮溶于有机溶剂中,然后减压浓缩至干。其中所述的原料(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的存在形态没有特别限定,可以使用任意晶体或无定型物,其中所述的有机溶剂选自二氯甲烷、甲醇、乙醇、乙酸乙酯、丙酮、四氢呋喃和乙腈。
本发明的另一个方面提供一种晶体组合物,其中(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A占晶体组合物重量50%以上,优选80%以上,更优选90%以上,最优选95%以上。
本发明的另一个方面提供一种药物组合物,其含有(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型及药学上可接受的载体,优选为含有(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A及药学上可接受的载体。
本发明的另一个方面提供(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型或包含上述化合物晶型的药物组合物,尤其是(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A或包含上述化合物晶型A的药物组合物用于治疗PRMT5介导的疾病的方法以及在制备治疗PRMT5介导的疾病的药物中的用途,其中所述的PRMT5介导的疾病包括但不限于:增殖性疾病、代谢疾病或血液疾病。在一些实施方案中,本发明所述的PRMT5介导的疾病为癌症。
在一些实施方案中,本发明所述的PRMT5介导的疾病包括但不限于:听神经瘤、腺癌、肾上腺癌、肛门癌、血管肉瘤(例如,淋巴管肉瘤、淋巴管内皮肉瘤、血管肉瘤)、附件癌、良性单克隆性丙种球蛋白病、胆癌(例如,胆管癌)、膀胱癌、乳癌(例如,乳房腺癌、乳房乳头状癌、乳腺癌、乳房髓样癌、三阴性乳腺癌)、脑癌(例如,脑膜瘤;神经胶质瘤,例如星形细胞瘤、少突神经胶质瘤;成神经管细胞瘤)、支气管癌、类癌瘤、宫颈癌(例如宫颈腺癌)、绒毛膜癌、脊索瘤、颅咽管瘤、结肠直肠癌(例如,结肠癌、直肠癌、结肠直肠腺癌)、上皮癌、室管膜瘤、内皮肉瘤(例如,卡波西氏肉瘤(Kaposi's sarcoma)、多发性特发性出血性肉瘤)、子宫内膜癌(例如,子宫癌、子宫肉瘤)、食道癌(例如,食道腺癌、巴瑞特氏腺癌(Barrett’sadenocarinoma))、尤因肉瘤(Ewing sarcoma)、眼癌(例如,眼内黑素瘤、成视网膜细胞瘤)、家族性嗜酸性粒细胞增多症、胆囊癌、胃癌(例如,胃腺癌)、胃肠道间质瘤(GIST)、头颈部癌(例如,头颈部鳞状细胞癌、口腔癌(例如,口腔鳞状细胞癌(OSCC)、咽喉癌(例如,喉癌、咽癌、鼻咽癌、口咽癌))、造血系统癌(例如,白血病如急性淋巴细胞性白血病(ALL)(例如,B-细胞ALL、T-细胞ALL)、急性髓细胞性白血病(AML)(例如,B-细胞AML、T-细胞AML)、慢性粒细胞性白血病(CML)(例如,B-细胞CML、T-细胞CML)以及慢性淋巴细胞性白血病(CLL)(例如,B-细胞CLL、T-细胞CLL);淋巴瘤如霍奇金淋巴瘤(HL)(例如,B-细胞HL、T-细胞HL)以及非霍奇金淋巴瘤(NHL)(例如,B-细胞NHL如弥漫性大细胞淋巴瘤(DLCL)(例如,弥漫性大B-细胞淋巴瘤(DLBCL))、滤泡性淋巴瘤、慢性淋巴细胞性白血病/小淋巴细胞性淋巴瘤(CLL/SLL)、套细胞淋巴瘤(MCL)、边缘带B-细胞淋巴瘤(例如,粘膜相关淋巴样组织(MALT)淋巴瘤、结节边缘带B-细胞淋巴瘤、脾边缘带B-细胞淋巴瘤)、原发性纵隔B-细胞淋巴瘤、伯基特淋巴瘤(Burkittlymphoma)、淋巴浆细胞淋巴瘤(即,“沃尔丹斯特伦巨球蛋白血症(macroglobulinemia)”)、毛细胞白血病(HCL)、免疫母细胞性大细胞淋巴瘤、前体B-成淋巴细胞性淋巴瘤以及原发性中枢神经系统(CNS)淋巴瘤;以及T-细胞NHL如前体T-成淋巴细胞性淋巴瘤/白血病、外周T-细胞淋巴瘤(PTCL)(例如,皮肤T-细胞淋巴瘤(CTCL)(例如,蕈样真菌病(mycosis fungiodes)、西泽里综合征(Sezary syndrome))、血管免疫母细胞性T-细胞淋巴瘤、结节外天然杀伤T-细胞淋巴瘤、肠病类型T-细胞淋巴瘤、皮下脂膜炎样T-细胞淋巴瘤、间变性大细胞淋巴瘤);如上所描述的一种或多种白血病/淋巴瘤的混合物;以及多发性骨髓瘤(MM))、重链病(例如,α链病、γ链病、μ链病)、成血管细胞瘤、炎性肌纤维母细胞瘤、免疫细胞淀粉样变性、肾癌(例如,肾母细胞瘤又称韦尔姆斯氏瘤(Wilms’tumor)、肾细胞癌)、肝癌(例如,肝细胞癌(HCC)、恶性肝细胞瘤)、肺癌(例如,支气管癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、肺腺癌)、平滑肌肉瘤(LMS)、肥大细胞增多症(例如,全身性肥大细胞增多症)、骨髓发育不良综合征(MDS)、间皮瘤、骨髓增殖性疾病(MPD)(例如,真性红细胞增多症(PV)、特发性血小板增多症(ET)、特发性骨髓外化生(AMM)又称为骨髓纤维变性(MF)、慢性特发性骨髓纤维变性、慢性骨髓性白血病(CML)、慢性嗜中性白血病(CNL)、嗜酸性白细胞增多综合征(HES))、成神经细胞瘤、神经纤维瘤(例如,1型或2型多发性神经纤维瘤(NF)、施旺细胞瘤病(schwannomatosis))、神经内分泌癌(例如,胃肠胰腺神经内分泌肿瘤(GEP-NET)、类癌瘤)、骨肉瘤、卵巢癌(例如,囊腺癌、卵巢胚胎性癌、卵巢腺癌、卵巢透明细胞癌、卵巢浆液性囊腺癌)、乳头状腺癌、胰腺癌(例如,胰腺腺癌、管内乳头状粘液瘤(IPMN)、胰岛细胞肿瘤)、阴茎癌(例如,阴茎和阴囊佩吉特氏病(Paget’s disease))、松果体瘤、原发性神经外胚层瘤(PNT)、前列腺癌(例如,前列腺腺癌)、直肠癌、横纹肌肉瘤、唾液管癌、皮肤癌(例如,鳞状细胞癌(SCC)、角化棘皮瘤(KA)、黑素瘤、基底细胞癌(BCC))、小肠癌(例如,附件癌)、软组织肉瘤(例如,恶性纤维组织细胞瘤(MFH)、脂肪肉瘤、恶性外周神经鞘瘤(MPNST)、软骨肉瘤、纤维肉瘤、粘液肉瘤)、皮脂腺癌、汗腺癌、滑膜瘤、睾丸癌(例如,精原细胞瘤、睾丸胚胎性癌)、甲状腺癌(例如,甲状腺乳头状癌、乳头状甲状腺癌(PTC)、髓样甲状腺癌)、尿道癌、阴道癌以及外阴癌(例如,外阴佩吉特氏病)、髓母细胞瘤、腺样囊性癌、黑色素瘤、胶质母细胞癌。
在一些实施方案中,本发明所述的PRMT5介导的疾病包括代谢性病症如糖尿病或肥胖症。
在一些实施方案中,本发明所述的PRMT5介导的疾病包括血红蛋白病如镰状细胞病或β-地中海贫血。
在一些实施方案中,本发明所述的PRMT5介导的疾病包括炎性和自身免疫性疾病。
在一些优选的实施方案中,本发明提供本发明通式I所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药,或包含其的药物组合物用于治疗PRMT5介导的疾病的方法以及在制备治疗PRMT5介导的疾病的药物中的用途,其中所述的PRMT5介导的疾病包括但不限于:乳腺癌、食道癌、膀胱癌、肺癌、造血系统癌、淋巴瘤、髓母细胞瘤、成神经管细胞瘤、直肠腺癌、结肠癌、胃癌、胰腺癌、肝癌、腺样囊性癌、前列腺癌、肺癌、头颈部鳞状细胞癌、脑癌、肝细胞癌、黑色素瘤、少突神经胶质瘤、胶质母细胞癌、睾丸癌、卵巢透明细胞癌、卵巢浆液性囊腺癌、甲状腺癌、多发性骨髓瘤(AML)、肾细胞癌、套细胞淋巴瘤、三阴性乳腺癌、非小细胞肺癌、血红蛋白病、糖尿病和肥胖症。
在这里需要特别说明的是,X-射线粉末衍射图谱对于特定的晶型具有特征性。判断是否与已知晶型相同时,应该注意的是峰的相对位置(即2θ)而不是它们的相对强度。这是由于谱图(尤其在低角度)的相对强度会因为晶体条件、粒径或其它测定条件的差异产生的优势取向效果而变化,衍射峰的相对强度对于晶型的确定并非是特征性的。另外,同一个晶型的2θ值可能存在轻微误差,约为±0.2°。因此,在确定每种结晶结构时,应该将此误差考虑在内。在XRPD图谱中通常用2θ角或晶面距d值表示峰位置,两者之间具有简单的换算关系:d=λ/2sinθ,其中d值代表晶面间距,λ代表X射线的波长,θ为衍射角。还应特别指出的是,在混合物的鉴定中,由于含量下降等因素会造成部分衍射线缺失,此时,无需依赖高纯试样中观察到的全部谱带,一条谱带也可能对给定的晶体是特征性的。
DSC测定当晶体由于其晶体结构发生变化或晶体熔融而吸收或释放热时的转变温度。对于同种化合物的同种晶型,在连续的分析中,热转变温度和熔点误差典型的在约5℃之内。当我们说一个化合物具有一给定的DSC峰或熔点时,这是指该DSC峰或熔点±5℃。需要指出的是对于混合物而言,其DSC峰或熔点可能会在更大的范围内变动。此外,由于在物质熔化的过程中伴有分解,因此熔化温度与升温速率相关。
除非另外定义,本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。
本发明化合物中的“氢”、“碳”、“氧”包括其所有同位素。同位素应理解为包括具有相同原子数但具有不同质量数的那些原子。举例来说,氢的同位素包括氕、氚和氘,碳的同位素包括13C和14C,氧的同位素包括16O和18O等。
附图说明
图1为式(I)化合物晶型A的X-射线衍射谱图;
图2为式(I)化合物晶型A的DSC图谱;
图3为式(I)化合物晶型A的热重分析(TGA)图谱;
图4为式(I)化合物无定型物的X-射线衍射谱图。
具体实施方式
下面代表性的实施例是为了更好地说明本发明,而非用于限制本发明的保护范围。以下实施例中使用的材料如无特殊说明均为商购获得。
一、实验所用的测试仪器
1.X-射线粉末衍射谱(XRPD)
仪器型号:Bruker D8 Advance X-射线衍射仪;
测试条件:光管电压:40kV;光管电流:40mA;
狭缝:1.0/1.0/Ni/0.2;步长:0.02°;
靶型:Cu靶;角度范围:3.00-40.00°;扫描时间:0.3秒。
2.热重分析(TGA)
仪器型号:NETZSCH TG 209型热重分析仪
温度范围:30–400℃
升温速率:10℃/min
3.差示扫描量热(DSC)
仪器型号:NETZSCH DSC 204型差热分析仪
温度范围:25–300℃
升温速率:10℃/min
二、式(I)化合物的制备
实施例1:(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的制备
步骤1、(R)-2-(环氧乙烷-2-基甲基)-1,2,3,4-四氢异喹啉顺丁烯二酸盐的制备
于50L玻璃反应釜中加入无水乙醇(4.26kg)和R-(-)-环氧氯丙烷(0.75kg,8.11mol)。随后滴加1,2,3,4-四氢异喹啉(0.90kg,6.77mol),控制反应体系内温不高于30℃。滴毕,维持25℃±5℃反应6h,加入碳酸钾(1.87kg,13.53mol),维持25℃±5℃反应2h,反应完全后,过滤,以无水乙醇(2.13kg)淋洗反应釜,淋洗液用于漂洗滤饼。随后将滤液转移至50L玻璃反应釜中,缓慢加入顺丁烯二酸(0.86kg,7.41mol),室温搅拌0.5h后冷却至5℃±5℃,析晶8h以上,过滤,以无水乙醇(0.71kg)淋洗反应釜,淋洗液用于漂洗滤饼,滤饼于40℃±5℃真空干燥12小时以上,得固体1.59kg,收率77.1%。
步骤2、(R)-7'-氯-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-1'-酮的制备
于50L玻璃反应釜中加入乙酸乙酯(6.44kg)和(R)-2-(环氧乙烷-2-基甲基)-1,2,3,4-四氢异喹啉顺丁烯二酸盐(1.43kg,4.68mol),室温下搅拌均匀,随后加入三乙胺(1.18kg,11.70mol),室温搅拌0.5h后加入纯化水(7.14kg),搅拌1小时后分层,有机层用纯化水(7.14kg)洗2次,无水硫酸钠干燥,过滤,滤液浓缩至干得红色液体。
于10L玻璃反应釜中加入N,N-二甲基甲酰胺(4.91kg)、上述红色液体、7'-氯-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-1'-酮(0.65kg,3.12mol)和碳酸铯(2.03kg,6.24mol),随后加热将反应体系升温至95℃±5℃,反应1h,反应完全后,将反应体系冷却至25℃±5℃,过滤,滤饼用乙酸乙酯(4.69kg)漂洗。将滤液转移至50L玻璃反应釜中,加入水(10.40kg)洗涤,静置分层,水层用乙酸乙酯(4.69kg)萃取,静置分层,合并有机层,加入水(10.40kg)洗涤,静置分层,有机层加入无水硫酸钠干燥脱水,过滤,浓缩至干,加入乙酸乙酯(1.17kg)转移至10L玻璃反应釜中,加入正庚烷(1.33kg),冷却至10℃±5℃打浆3小时以上,过滤,用正庚烷(0.44kg)淋洗反应釜,淋洗液用于漂洗滤饼,滤饼于40℃±5℃真空干燥12小时以上,得固体0.95kg,收率76.6%。
步骤3、(R)-6-((2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-1'-氧代-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-7'-基)氨基)-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯的制备
在10L玻璃反应釜中加入四氢呋喃(8.16kg)、(R)-7'-氯-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-1'-酮(0.92kg,2.31mol)、tert-butyl 6-氨基-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯(0.59kg,2.78mol)、三(二亚苄基丙酮)二钯(0.04kg,0.046mol),2-二-叔丁膦基-2',4',6'-三异丙基联苯(0.08kg,0.184mol)和叔丁醇钠(0.44kg,4.62mol),随后用氮气置换空气,接着将反应液升温至65℃±5℃反应1h,反应结束后,将反应液冷却至25℃±5℃,过滤,滤饼用二氯甲烷(12.19kg)漂洗。将滤液转移至50L玻璃反应釜中,以纯化水(9.20kg)洗涤有机层3次,随后于有机层加入无水硫酸钠(1.84kg)干燥脱水,过滤,浓缩至干,加入乙酸乙酯(5.81kg)转移至50L玻璃反应釜中,加入正庚烷(8.81kg),室温打浆8小时以上,过滤,滤饼于35℃±5℃真空干燥12小时以上,得固体1.17kg,收率88.3%。
步骤4、(R)-7'-((2-氮杂螺[3.3]庚-6-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-1'-酮的制备
在10L玻璃反应釜中加入二氯甲烷(6.04kg)和(R)-6-((2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-1'-氧代-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-7'-基)氨基)-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯(1.14kg,2.00mol),随后将反应体系降温至5℃±5℃,滴加三氟乙酸(3.50kg),并控制反应体系内温不高于30℃。加毕,维持25℃±5℃反应1h,反应完全后,浓缩反应液,加入纯化水(5.70kg)溶解后转移至50L玻璃反应釜,加入甲基叔丁基醚(4.22kg),搅拌分层,有机层用纯化水(5.70kg)洗2次,合并水层转移至50L玻璃反应釜,加入乙腈(4.48kg),在25℃±5℃下用2M氢氧化钠水溶液调节pH值至8~9。随后加入二氯甲烷萃取(30.21kg),有机层加入无水硫酸钠,过滤,将滤液转移至50L玻璃反应釜,加入巯基硅胶(0.06kg,),在25℃±5℃下搅拌0.5h,过滤,浓缩至干,固体于35℃±5℃真空干燥12小时以上,得固体0.898kg,收率95.4%。
步骤5、(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的制备
于50L玻璃反应釜中加入二氯甲烷(11.79kg)、(R)-7'-((2-氮杂螺[3.3]庚-6-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-[2,6]萘啶]-1'-酮(0.89kg,1.88mol)和N,N-二异丙基乙胺(0.49kg,3.76mol),随后将反应体系冷却至0℃±5℃,滴加乙酸酐(0.16kg,1.69mol)的二氯甲烷(1.18kg)溶液,并控制反应体系内温不高于5℃,加毕维持0℃±5℃反应0.5h,反应完全后加入氢氧化钠(0.014kg)水(8.90kg)溶液,搅拌静置分层,有机层用纯化水(8.90kg)洗涤2次,无水硫酸钠干燥脱水,过滤,浓缩至干,加二氯甲烷(1.18kg)溶解后通过柱层析(洗脱剂:甲醇/二氯甲烷=1/20)分离纯化,柱液收集浓缩至干,固体于35℃±5℃真空干燥12小时以上,得固体0.655kg,收率67.6%。
步骤6、(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的精制
在20L玻璃反应釜中加入乙酸乙酯(2.62kg),加入以上步骤中制得的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮(0.58kg),氮气氛围下升温至75℃±5℃搅拌3小时。随后将反应体系降温至25℃±5℃,搅拌析晶3小时,过滤,用乙酸乙酯(0.26kg)淋洗反应釜,淋洗液用于漂洗滤饼,滤饼于35℃±5℃真空干燥12小时以上,得固体0.49kg,收率84.5%。1H NMR(400MHz,DMSO-d6)δ7.71(s,1H),7.00-7.15(m,4H),6.87(m,1H),4.78(s,1H),4.16(s,1H),4.04-4.10(m,3H),3.88(m,1H),3.70-3.80(m,3H),3.61-3.63(m,2H),2.80-2.83(m,2H),2.70-2.72(m,2H),2.18-2.20(m,1H),1.98-2.02(m,4H),1.72(s,3H),1.24-1.30(m,4H),0.94-1.0(m,4H).LC-MS m/z:[M+H]+=516.3.
实施例2:(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A的制备
称取100mg式(I)化合物,加入0.5mL乙酸乙酯,回流3小时,随后降温至25℃±5℃,搅拌析晶3小时,过滤,取固体进行XRPD表征,表征结果显示本次实验晶型为晶型A。
式(I)化合物晶型A的X-射线衍射谱图(参见图1),使用Cu-Ka辐射,以2θ角度表示X-射线粉末衍射图,在约7.3、7.9、9.5、11.2、12.4、13.5、14.5、17.0、17.6、18.2、18.8、19.8、21.4、22.6、23.21、23.9、24.8、26.6、27.1、28.3、29.8、30.1、31.0、34.2处有特征峰。将图1中的2θ以及峰的相对强度列于表1。
式(I)化合物晶型A的DSC表征结果示于图2中,测试结果显示,样品在147.7℃有尖锐的吸热峰。
式(I)化合物晶型A的TGA表征结果示于图3中,结果显示,其在100℃前未见明显失重,在150℃附近存在0.428%失重。
表1:式(I)化合物晶型A的XRPD图谱详情
实施例3:(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮无定型物的制备
分别称取适量式(I)化合物,溶于5倍体积的二氯甲烷中,加热溶清后,减压浓缩至干,取固体进行XRPD表征,表征结果显示本次实验晶型为无定型物,其X-射线衍射谱图如图4所示。
实验例1:化合物体外激酶活性评价
1.实验材料
化合物:将(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮用DMSO配制成10mM母液,最终稀释为10个浓度进行检测,终浓度为10000.00nM、3333.33nM、1111.11nM、370.37nM、123.46nM、41.15nM、13.72nM、4.57nM、1.52nM、0.51nM。
试剂与耗材:PRMT5,购自于Active Motif公司,货号为31921;多肽底物H4(1-21)S1ac,购自于吉尔生化(上海)有限公司,货号为342095;[3H]-SAM,购自于PerkinElmer公司,货号为NET155V001MC;SAM,购自于Sigma,货号为A7007-100MG;SAH,购自于Sigma公司,货号为A9384-25MG;DTT,购自于生工生物工程(上海)股份有限公司,货号为A620058-0005。Corning-3657,购自于Corning公司,货号为3657;Echo Qualified 384-Well,购自于Labcyte公司,货号为P-05525;FlashPlate,购自于Perkin Elmer,货号为SMP410J001PK。
仪器:闪烁计数仪,购自于PerkinElmer公司,型号为MicroBeta2;超声波纳升液体处理系统,购自于Labcyte公司,型号为Echo 550。
2.实验方法
2.1.反应缓冲液和反应终止液配制:1倍反应缓冲液体成分为10mM Tris-HCl,pH8.0;0.01%Tween-20;1mM DTT。反应终止液成分为125μM的3H-SAM溶液。
2.2化合物配制
2.2.1化合物稀释
化合物用100%DMSO溶解成10mM母液,再将化合物在Echo384孔板上稀释到所需要的浓度。
2.2.2转移化合物到384反应板
用Echo550仪器从上述稀释好Echo384孔板中转移250nL化合物到384孔反应板中。
2.3酶学反应
2.3.1配制1.67倍酶溶液
将PRMT5加入1倍反应缓冲液,形成1.67倍酶溶液。
2.3.2配制2.5倍的底物溶液
将多肽底物和[3H]-SAM加入1倍反应缓冲液,形成2.5倍底物溶液(终浓度分别为100nM和250nM)。
2.3.3向384孔板中加入酶溶液
向384孔反应板孔中加入15μL的1.67倍酶溶液。对于无酶活对照孔,用15μL的1倍反应缓冲液替代酶溶液。1000rpm离心1min,室温下孵育15分钟。
2.3.4向384孔板中加入底物溶液启动酶学反应
向384孔反应板每孔中加入10μL的2.5倍底物溶液。1000rpm离心1min。25℃反应60分钟。
2.3.5酶学反应的终止
向384孔反应板每孔中加入5μL的反应终止液终止反应。从试验板中每孔取25uL转移到Flashplate中,在室温下放置1h。然后用0.1%的Tween-20溶液洗Flashpate板3次。
2.4 MicroBeta 2读取数据
2.4抑制率计算
从Microbeta 2上复制数据。把数据转化成抑制率数据。其中最大值是指DMSO对照的转化率,最小值是指无酶活对照的转化率。抑制率(%)=(最大值-样本值)/(最大值-最小值)×100%。
将数据导入GraphPad,并使用“log(inhibitor)vs.response--Variable slope”进行曲线拟合,得到IC50。(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮对PRMT5酶的IC50值为49.70nM。
实验例2:化合物体外细胞活性评价
1.实验材料
受试化合物:将(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮用DMSO配制成10mM母液,最终稀释为8个浓度进行检测,Z-138细胞实验的化合物终浓度为33333.00nM、6666.60nM、1333.32nM、266.66nM、53.33nM、10.67nM、2.13nM、0.43nM,MDA-MB-468和NCI-H358细胞实验化合物终浓度为50000nM、10000nM、2000nM、400nM、80nM、16nM、3.2nM、0.64nM。
人套细胞淋巴瘤细胞Z-138、三阴性乳腺癌细胞MDA-MB-468、人非小细胞肺癌细胞NCI-H358购于美国典型培养物保藏中心(ATCC)。
试剂:Iscove's Modified Dulbecco's Medium(IMEM培养基),货号为ATCC 30-2005;Leibovitz'sL-15Medium(L-15培养基),货号为Gibco 11415-064;1640培养基,货号为Gibco 22400089;马血清,货号为Gibco 16050122;Fetal Bovine Serum,货号为Gibco10099-141;青链霉素,货号为Gibco 15140-122;丙酮酸钠,货号为Gibco 11360070;CellTiter-Glo Luminescent Cell Viability Assay,货号为Promega G7571。CCK-8增殖抑制检测试剂盒,货号为KeyGEN KGA317。
2.实验方法
2.1细胞复苏:
2.1.1 Z-138细胞复苏:从液氮罐中取出Z-138细胞冻存管置于37℃水浴锅中,轻轻摇动使其尽快解冻。解冻后取出冻存管,用酒精棉球消毒后旋开盖子,吸出细胞液注入离心管,并加入1mL含10%马血清的完全培养基,混匀后置于离心机中,1000rpm,离心5min。之后弃上清液,加入完全培养基反复吹打至细胞完全吹散、重悬。以适宜浓度接种于培养皿中。置37℃,5%CO2、95%潮湿空气的CO2培养箱中培养。
2.1.2 MDA-MB-468细胞复苏:从液氮罐中取出MDA-MB-468细胞冻存管置于37℃水浴锅中,轻轻摇动使其尽快解冻。解冻后取出冻存管,用酒精棉球消毒后旋开盖子,吸出细胞液注入离心管,并加入1mL含10%FBS的L-15培养基,混匀后置于离心机中,1000rpm,离心5min。之后弃上清液,加入完全培养基反复吹打至细胞完全吹散、重悬。以适宜浓度接种于培养皿中。置37℃,95%潮湿空气的无CO2培养箱中培养。
2.1.3NCI-H358细胞复苏:从液氮罐中取出NCI-H358细胞冻存管置于37℃水浴锅中,轻轻摇动使其尽快解冻。解冻后取出冻存管,用酒精棉球消毒后旋开盖子,吸出细胞液注入离心管,并加入1mL含10%FBS的1640培养基,混匀后置于离心机中,1000rpm,离心5min。之后弃上清液,加入完全培养基反复吹打至细胞完全吹散、重悬。以适宜浓度接种于培养皿中。置37℃,5%CO2,95%潮湿空气的CO2培养箱中培养。
2.2细胞培养和传代:
2.2.1 Z-138细胞培养和传代:细胞生长至约80-90%,将培养基(IMDM培养基+10%马血清+1%青链霉素)转移至15mL离心管中,1000rpm,离心5min。去除上清,用完全培养基重悬细胞,按所需密度接种于培养皿中,置于37℃、5%CO2、95%潮湿空气的培养箱中培养,视细胞生长情况每2-3天补一次培养液或进行传代。
2.2.2 MDA-MB-468细胞培养和传代:细胞生长至约80-90%,将培养基(Leibovitz's L-15Medium培养基+10%FBS+1%青链霉素)转移至15mL离心管中,1000rpm,离心5min。去除上清,用完全培养基重悬细胞,按所需密度接种于培养皿中,置于37℃、95%潮湿空气的无CO2培养箱中培养,视细胞生长情况每2-3天换一次培养液或进行传代。
2.2.3NCI-H358细胞培养和传代:细胞生长至约80-90%,将培养基(1640培养基+10%FBS+1%青链霉素+1mM丙酮酸钠)转移至15mL离心管中,1000rpm,离心5min。去除上清,用完全培养基重悬细胞,按所需密度接种于培养皿中,置于37℃、5%CO2、95%潮湿空气的CO2培养箱中培养,视细胞生长情况每2-3天换一次培养液或进行传代。
2.3实验步骤:
实验第一天:
Z-138细胞传代后以1000个/孔的密度重悬于完全培养基中,接种于96孔培养板内:96孔板最外面一圈36个孔以200μL PBS填充,以防边缘培养基蒸发较快导致内部板孔的培养条件差异过大;内部60个孔的最左列为空白孔,不加细胞,以等体积的PBS填充;其余的54个孔以排枪进行细胞铺板,每孔为含对应细胞的100μL培养基,铺板完成后,拍打96孔板使细胞均匀悬浮,放入5%CO2培养箱内37℃培养24h。
MDA-MB-468细胞传代后以对应的密度重悬于完全培养基中,2000个/孔,接种于96孔培养板内:细胞外面一圈以200μL PBS填充,以防边缘培养基蒸发较快导致内部板孔的培养条件差异过大;内部60个孔的最左列为空白孔,不加细胞,以等体积的PBS填充;其余的54个孔以排枪进行细胞铺板,每孔100μL,放入无CO2培养箱内37℃培养24h。
NCI-H358细胞传代后以对应的密度重悬于完全培养基中,1000个/孔,接种于96孔培养板内:细胞外面一圈以200μL PBS填充,以防边缘培养基蒸发较快导致内部板孔的培养条件差异过大;内部60个孔的最左列为空白孔,不加细胞,以等体积的PBS填充;其余的54个孔以排枪进行细胞铺板,每孔100μL,放入5%CO2培养箱内37℃培养24h。
实验第二天:
Z-138细胞在原培养基(100μL)的基础上,加入50μL的(3×)药物,每个浓度组设置两个复孔,继续放入CO2培养箱培养7天。化合物配制如下:提前称取化合物1-2mg,使用DMSO配置成10mM母液。使用完全培养基稀释药物,药物终浓度以33333.00nM为起始最高浓度,按1:4梯度依次稀释至7个浓度梯度:6666.60nM、1333.32nM、266.66nM、53.33nM、10.67nM、2.13nM、0.43nM。①取10mM的母液1:4稀释成对应的药液,共8个浓度(10μL母液+40μLDMSO);②取5μL①的药物加入的495μL完全培养基,配制成对应的浓度(3×)(稀释100倍)。
MDA-MB-468细胞在原培养基(100μL)的基础上,加入100μL的(2×)药物,每个浓度组设置两个复孔,继续放入无CO2培养箱培养7天。化合物配制如下:提前称取化合物1-2mg,使用DMSO配置成10mM母液。使用完全培养基稀释药物,药物终浓度以50000nM为起始最高浓度,按1:4梯度依次稀释至7个浓度梯度:50000nM、10000nM、2000nM、400nM、80nM、16nM、3.2nM、0.64nM。①取10mM的母液1:4稀释成对应的药液,共8个浓度(10μL药液+40μLDMSO);②取5μL①的药物加入的495μL完全培养基,配制成对应的浓度(2×)(稀释100倍)。
NCI-H358细胞在原培养基(100μL)的基础上,加入100μL的(2×)药物,每个浓度组设置两个复孔,继续放入5%CO2培养箱培养7天。化合物配制如下:提前称取化合物1-2mg,使用DMSO配置成10mM母液。使用完全培养基稀释药物,药物终浓度以50000nM为起始最高浓度,按1:4梯度依次稀释至7个浓度梯度:50000nM、10000nM、2000nM、400nM、80nM、16nM、3.2nM、0.64nM。①取10mM的母液1:4稀释成对应的药液,共8个浓度(10μL药液+40μLDMSO);②取5μL①的药物加入的495μL完全培养基,配制成对应的浓度(2×)(稀释100倍)。
实验第八天:
Z-138细胞在药物处理7天后,提前30min将CellTiter-Glo Luminescent CellViabillity Assay取出,平衡至室温。空白孔将PBS吸弃,加入150μL的完全培养基,然后空白孔、给药孔和DMSO孔都加入75uL的Celltiter-Glo reagent,室温震荡2min。继续室温孵育10min后,每孔各吸取180μL转移至不透明白板,去除气泡后,检测化学发光信号,震荡,Read进样检测条件为500ms。根据酶标仪导出的A.U.值,计算每个孔相对于溶剂对照孔的抑制率:Inhibition(%)=100-(A.U.实验孔–A.U.空白孔)/(A.U.溶剂对照孔-A.U.空白孔)*100。根据不同药物浓度及其所对应的抑制率,使用GraghPad 5.0软件进行IC50曲线绘制,分析数据,得出最终IC50值,实验结果见表2。
MDA-MB-468和NCI-H358细胞药物处理7天后吸弃孔内培养基,加入100μL已加入CCK-8的完全培养基(CCK-8:完全培养基=1:10),第一竖排PBS作为空白对照孔,同步加入100μL的CCK-8,然后放入培养箱培养40min-2h左右,根据CCK-8的显色深浅决定最佳检测时间(DMSO组的OD值在1.0左右最佳)。待CCK-8显色至橙色,并且出现肉眼可分辨的一定梯度,将96孔板从培养箱中取出,置于室温中平衡5-10分钟;打开酶标仪软件,调整好检测参数,检测450nm处的吸光度(OD值);将培养板的盖子取下,直接将培养板水平放置于板槽内,开始读数;读数完成后,保存程序,导出数据,关闭软件和电脑。根据酶标仪导出的OD值,计算每个孔相对于溶剂对照孔的抑制率:Inhibition(%)=100-(OD实验孔–OD空白孔)/(OD溶剂对照孔-OD空白孔)*100。根据不同药物浓度及其所对应的抑制率,使用GraghPad 5.0软件进行IC50曲线绘制,分析数据,得出最终IC50值,实验结果见表2。
表2
从以上实验可以看出,本发明的式(I)的化合物对人套细胞淋巴瘤细胞Z-138、三阴性乳腺癌细胞MDA-MB-468、人非小细胞肺癌细胞NCI-H358均表现出了良好的抑制活性,非常有希望成为淋巴瘤、三阴性乳腺癌、非小细胞肺癌治疗剂。
实验例3:电生理手动膜片钳检测化合物对hERG钾通道的作用
hERG钾离子通道是药物安全筛查的标准。hERG钾离子通道被阻滞会导致心脏中毒和心室复极延长,严重时可能会导致猝死。具有hERG钾通道抑制作用的药物可能是临床用药的潜在祸患。因此,化合物对hERG钾离子通道抑制作用弱则安全性高。药物导致的QT间期延长与致命性室性心律失常和猝死的危险性增加有关。
1实验材料
主要试剂:青霉素-链霉素溶液(100×)、DMEM/F12购自Gibco公司;胎牛血清购自PAA公司;DMSO、EGTA、MgATP购自Sigma公司;KCl、CaCl2·2H2O、MgCl2·6H2O、NaCl购自Sinopharm公司;葡萄糖购自General-reagent公司;HEPES购自Solarbio公司;奎尼丁购自aladdin公司。
仪器:TI-S-FLU显微镜购自Nikon公司;SMZ-140/143显微镜购自Motic公司;EPC-10放大器、Patchmaster V2X60购自HEKA公司;TMC-36防震台购自TMC公司;MP-225、MPC-200操控器、ROE-200微操纵仪、P-97电极拉制仪购自Sutter公司;VC3-8PP灌流给药系统购自ALA公司。
2实验方法
测试溶剂配制:细胞外液配制(mM):137NaCl、4KCl、1.8CaCl2、1MgCl2、10葡萄糖和10HEPES(pH 7.4);细胞内液配制(mM):130KCl、1MgCl2、5EGTA、5MgATP和10HEPES(pH7.2);阴性对照配制:细胞外液+0.3%DMSO;阳性对照:奎尼丁。
化合物处理:称取化合物溶解于DMSO中,配制10mM母液,用DMSO稀释母液成次级母液,浓度为3.3,1.1,0.37,0.12mM。取母液和次级母液各90μL稀释至30mL细胞外液中,用于电生理检测。化合物的终浓度为30,10,3.3,1.1,0.37μM,DMSO的终浓度为3:1000。
稳转细胞培养:细胞株来源于过表达hERG钾离子通道HEK-293细胞,是在纽约大学医学院Mohamed Boutjdir博士实验室提供技术支持后科瑞斯生物与之合作建立并验证。细胞在37℃、5%CO2培养箱中培养。当细胞密度达培养皿80%时,先用磷酸盐缓冲液(PBS)预清洗,然后用胰蛋白酶/EDTA消化细胞2-3min,加入细胞培养基停止消化,轻轻把细胞吹下来并转移到离心管中,1000rpm*3min,上清液弃置,加入细胞培养基,轻轻吹打将细胞混匀,随后转移到培养皿中进行传代培养,或将细胞滴于圆形玻片之上并置于培养皿中待细胞贴壁用于实验。
细胞培养基组成:DMEM、15%胎牛血清和1%100×青霉素-链霉素。
电生理手动膜片钳系统实验:将稳转的细胞接种于玻片上,细胞密度低于50%,培养过夜。将实验用细胞转移到一个嵌于倒置显微镜平台的约1mL的浴槽中,灌流细胞外液,灌流速度为2.7mL/min。稳定5分钟后即可开始实验。采用HEKA EPC-10膜片钳放大器和PATCHMASTER采集系统记录膜电流。所有实验均在室温(22-24℃)下完成。实验中使用P-97微电极拉制仪拉直电极(BF150-110-10)。电极内径为1-1.5mm,充满内液后的入水电阻为2-4MΩ。hERG钾通道的电生理刺激方案,是首先将膜电压钳制在-80mV,给予细胞持续2s、+20mV电压刺激,激活hERG钾通道,再复极化至-50mV、持续5s,产生外向尾电流,刺激频率每15s一次。电流值为尾电流的峰值。实验中采用全细胞记录模式记录通道电流。首先灌流细胞外液(大约每分钟2mL)并持续记录,并等待电流稳定(5分钟内电流衰减(Run-Down)小于5%),此时尾电流峰值即为对照电流值。接着灌流含待测药物的细胞外液并持续记录直到药物对hERG电流的抑制作用到达稳定状态,此时尾电流峰值即为加药后电流值。稳定状态的标准以最近的连续3个电流记录线是否重合来判断。达到稳定态势以后如果以细胞外液灌流冲洗后hERG电流回复或接近加药物之前的大小,则可以继续灌流测试其它浓度或药物。30μM奎尼丁被用于实验中作为阳性对照以保证所使用的细胞反应正常。
3参数分析和数据分析统计
本研究通过测量对照组与药物处理组的电流最大值,计算处理组最大电流值所占对照组最大电流值的比率,评估待测化合物在测试浓度下对hERG钾离子通道的作用效果(Mean±SE)。
实验数据使用PATCHMASTER V2X60采集,并采用Origin 8.5软件以及MicrosoftExcel进行分析和统计。实验结果见表3。
表3
从以上实验可以看出,本发明式(I)的化合物对hERG钾通道抑制作用轻,对心脏的毒性低,具有非常好的心脏安全性。
尽管以上已经对本发明作了详细描述,但是本领域技术人员理解,在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述,而应归属于权利要求书。
Claims (10)
1.一种(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A,其特征是X-射线粉末衍射光谱用2θ角度表示在7.3±0.2、7.9±0.2、9.5±0.2、13.5±0.2、18.8±0.2处有特征峰。
2.根据权利要求1所述的晶型A,其特征是X-射线粉末衍射光谱用2θ角度表示在7.3±0.2、7.9±0.2、9.5±0.2、13.5±0.2、18.8±0.2处有特征峰。
3.根据权利要求1所述的晶型A,其特征是X-射线粉末衍射光谱用2θ角度表示在7.3±0.2、7.9±0.2、9.5±0.2、12.4±0.2、13.5±0.2、17.0±0.2、17.6±0.2、18.2±0.2、18.8±0.2、19.8±0.2、21.4±0.2、24.8±0.2、26.6±0.2处有特征峰。
4.根据权利要求1所述的晶型A,其特征是X-射线粉末衍射光谱用2θ角度表示在7.3±0.2、7.9±0.2、9.5±0.2、11.2±0.2、12.4±0.2、13.5±0.2、14.5±0.2、17.0±0.2、17.6±0.2、18.2±0.2、18.8±0.2、19.8±0.2、21.4±0.2、22.6±0.2、23.21±0.2、23.9±0.2、24.8±0.2、26.6±0.2、27.1±0.2、28.3±0.2、29.8±0.2、30.1±0.2、31.0±0.2、34.2±0.2处有特征峰。
5.根据权利要求1所述的晶型A,其具有基本上如图1所示的X-射线粉末衍射光谱。
6.一种如权利要求1-5之任一项所述的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A的制备方法,包括将(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮置于有机溶剂中,然后过滤的步骤。
7.一种(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮无定型物,其具有基本上如图4所示的X-射线粉末衍射光谱。
8.一种晶体组合物,其中权利要求1-5之任一项所述的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A占晶体组合物重量50%以上,优选80%以上,更优选90%以上,最优选95%以上。
9.一种药物组合物,其包含权利要求1-5之任一项所述的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A或权利要求7所述的无定型物和可药用载体。
10.如权利要求1-5之任一项所述的(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮晶型A或如权利要求7所述的无定型物或如权利要求8所述的晶体组合物或如权利要求9所述的药物组合物在制备用于治疗PRMT5介导的疾病的药物中的用途。
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