CN118619923A - 一类四氢异喹啉类pd-l1抑制剂的制备方法与应用 - Google Patents
一类四氢异喹啉类pd-l1抑制剂的制备方法与应用 Download PDFInfo
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- CN118619923A CN118619923A CN202410652871.5A CN202410652871A CN118619923A CN 118619923 A CN118619923 A CN 118619923A CN 202410652871 A CN202410652871 A CN 202410652871A CN 118619923 A CN118619923 A CN 118619923A
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- tetrahydroisoquinoline
- ester
- methyl
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- 239000012271 PD-L1 inhibitor Substances 0.000 title claims abstract description 14
- 229940121656 pd-l1 inhibitor Drugs 0.000 title claims abstract description 14
- UWYZHKAOTLEWKK-UHFFFAOYSA-N 1,2,3,4-tetrahydroisoquinoline Chemical compound C1=CC=C2CNCCC2=C1 UWYZHKAOTLEWKK-UHFFFAOYSA-N 0.000 title claims description 24
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 108010074708 B7-H1 Antigen Proteins 0.000 claims abstract description 25
- 102000008096 B7-H1 Antigen Human genes 0.000 claims abstract description 25
- 239000003814 drug Substances 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 17
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- DRSHXJFUUPIBHX-UHFFFAOYSA-N COc1ccc(cc1)N1N=CC2C=NC(Nc3cc(OC)c(OC)c(OCCCN4CCN(C)CC4)c3)=NC12 Chemical compound COc1ccc(cc1)N1N=CC2C=NC(Nc3cc(OC)c(OC)c(OCCCN4CCN(C)CC4)c3)=NC12 DRSHXJFUUPIBHX-UHFFFAOYSA-N 0.000 claims description 12
- 230000000903 blocking effect Effects 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 230000003993 interaction Effects 0.000 claims description 9
- 239000003513 alkali Substances 0.000 claims description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 7
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 6
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 claims description 6
- -1 amine compound Chemical class 0.000 claims description 6
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- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 4
- LXQMHOKEXZETKB-UHFFFAOYSA-N 1-amino-2-methylpropan-2-ol Chemical compound CC(C)(O)CN LXQMHOKEXZETKB-UHFFFAOYSA-N 0.000 claims description 4
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- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 claims description 2
- KJJPLEZQSCZCKE-UHFFFAOYSA-N 2-aminopropane-1,3-diol Chemical compound OCC(N)CO KJJPLEZQSCZCKE-UHFFFAOYSA-N 0.000 claims description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
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- ILBDVRCFTYQLOE-UHFFFAOYSA-N 3-methylpyrrolidin-3-ol Chemical compound CC1(O)CCNC1 ILBDVRCFTYQLOE-UHFFFAOYSA-N 0.000 claims description 2
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- QOSSAOTZNIDXMA-UHFFFAOYSA-N N,N′-Dicyclohexylcarbodiimide Substances C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 2
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- SRJOCJYGOFTFLH-UHFFFAOYSA-N isonipecotic acid Chemical compound OC(=O)C1CCNCC1 SRJOCJYGOFTFLH-UHFFFAOYSA-N 0.000 claims description 2
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Abstract
本发明属于化学医药领域，公开了一类具有四氢异喹啉骨架的PD‑L1抑制剂、制备方法与应用，为PD‑L1抑制剂研究提供了一种全新的、有前景的骨架。本发明基于均相时间分辨荧光实验、表面等离子共振实验及细胞共孵育实验验证了设计合成的四氢异喹啉类化合物能有效阻断PD‑1/PD‑L1作用，在肿瘤免疫治疗上具有应用前景。
Description
技术领域
本发明属于化学药物领域，涉及一系列含四氢异喹啉结构的PD-L1抑制剂的制备方法、结构特征及其医药用途，特别是通过阻断PD-1/PD-L1相互作用来预防和/或治疗肿瘤疾病方面的应用。
背景技术
癌症，又称恶性肿瘤，一般是由正常细胞突变为肿瘤细胞再不断增殖形成肿瘤肿织，损害机体机能，从而影响人体健康。
PD-1(又称CD279)是CD28超家族成员之一，属于免疫球蛋白超家族I型跨膜糖蛋白，相对分子质量为55000，具有单一的胞外免疫球蛋白可变区、跨膜区和胞内区，分别含有免疫受体酪氨酸抑制基序(ITIM)和免疫受体酪氨酸开关基序(ITSM)。PD-1是一种共抑制受体，主要表达于活化的T细胞，也表达于B细胞、自然杀伤(NK)细胞、髓系细胞、单核细胞、中性粒细胞和树突状细胞(DC)，在干扰素及其他炎症因子刺激应答的肿瘤组织和其他组织中可能会迅速上调。
PD-L1(也称为CD274或B7-H1)是PD-1的主要配体，属于B7家族，不仅可由T细胞、B细胞、巨噬细胞、DC和肥大细胞等免疫细胞表达，也可由正常组织细胞和异常肿瘤细胞表达。PD-L2也是能与PD-1结合的天然配体，但其表达范围较窄，主要在活化的巨噬细胞、树突细胞、肥大细胞中表达上调。
在正常情况下，PD-1和PD-L1的结合可以预防自身免疫性疾病的发生，但是在肿瘤微环境中，肿瘤细胞过表达PD-L1与PD-1结合时，阻断T细胞受体和下游信号通路，从而抑制T细胞的活化和增殖，进而扰乱效应T细胞的功能，使肿瘤细胞发生免疫逃逸，若阻断PD-1/PD-L1结合，可使T细胞重新恢复活力，杀死肿瘤细胞。
1,2,3,4-四氢喹啉(THQ)和1,2,3,4-四氢异喹啉(THIQ)是化学式为C9H10N的杂环化合物。杂环化合物，特别是具有氮杂原子的杂环化合物是生物学上的一类活性骨架，约占杂环类药物的60％。
由于四氢喹啉骨架在药物化学和药物发现中的重要性，在过去的几十年中，具有喹啉结构的各种化合物，如生物碱、治疗药物和合成类似物显示了多种生物活性，包括抗炎、抗疟疾、抗细菌、抗哮喘、抗高血压、抗肿瘤，以及血小板衍生生长因子受体(PDGFR)酪氨酸激酶抑制。除此之外，大量的喹啉类化合物被报道用于不同的生物学过程，如拓扑异构酶抑制、酪氨酸激酶抑制、抑制微管蛋白聚合，以及细胞周期停滞在G2期等。由于四氢异喹啉类化合物具有广泛生物活性，1,2,3,4四氢喹啉/异喹啉骨架一直是研发新生物活性分子的一类重要的母核。
发明内容
本发明的目的在于提供一类四氢异喹啉类PD-L1抑制剂及其制备方法。
本发明的另一目的在于提供上述四氢异喹啉类PD－L1抑制剂在制备抗肿瘤药物中的应用。
本发明的技术方案：
一类四氢异喹啉类PD-L1抑制剂，具有通式Ⅰ所示的结构：
其中：R选自乙醇胺、L-丝氨醇、2-氨基-2-甲基-1,3-丙二醇、三羟甲基氨基甲烷、1-氨基-2-甲基-2-丙醇、3-甲胺基-1,2-丙二醇、N,N-二甲基乙二胺、N-乙酰基乙二胺、4-羟基哌啶、哌啶-4-甲酰胺、哌嗪、1-乙酰哌嗪、吗啉、DL-3-吡咯烷醇、3-羟基-3-甲基-吡咯烷、N-甲基哌嗪、四氢吡咯、顺式-4-羟基-D-脯氨酸、L-脯氨酸、4-哌啶甲酸、2-哌啶甲酸、4-哌嗪甲酸、D-丙氨酸、L-苏氨酸、L-丝氨酸、L-甲硫氨酸、牛磺酸、(2R,4R)-4-羟基吡咯烷-2-羧酸甲酯、L-苏氨酸甲酯、L-丝氨酸甲酯、L-脯氨酸甲酯、D-丙氨酸甲酯中的一种。
一类四氢异喹啉类PD-L1抑制剂的制备方法，制备方法如下：
(1)在20～25℃温度条件下，以甲醇为溶剂，将化合物Ⅲ在碱性条件下反应2-4小时得到化合物Ⅱ；碱包括碳酸钾、碳酸氢钾、氢氧化钠，化合物Ⅲ与碱的摩尔比为1：2～1：5；
(2)在0～90℃温度条件下，化合物Ⅱ通过缩合反应与胺类化合物在碱性条件下，进行反应12～24小时得到目标化合物；若R基团中存在酯基时，在20～25℃温度条件下，以甲醇为溶剂，将酯在碱性条件下水解2-4小时得到相对应的酸；
其中：反应体系所用溶剂为二氯甲烷、四氢呋喃、乙腈、N，N-二甲基甲酰胺中的一种或两种以上组合；
反应体系所用碱包括碳酸钾、碳酸氢钠、三乙胺、N,N-二异丙基乙胺及胺类反应物，化合物Ⅱ与碱的摩尔比为1：2～1：5；
反应体系所用缩合剂包括EDCI、DCC、HOBt、HATU、HBTU中的一种或两种以上组合，化合物Ⅱ与缩合剂的摩尔比为1：1～1：5；
化合物Ⅱ与胺类化合物的摩尔比为1：1.1～1：4，酯与碱的摩尔比为1：2～1：5。
一种药物的组合物，其中包含：通式Ⅰ所示化合物或其衍生物；至少一种药学上可接受的盐；可药用载体。
一种四氢异喹啉类PD-L1抑制剂在制备阻断PD-1/PD-L1相互作用来预防和/或治疗肿瘤疾病的药物中的用途。
本发明的一类新型的四氢异喹啉类PD-L1抑制剂，其可通过恢复T细胞的免疫功能从而杀伤肿瘤细胞，具有治疗肿瘤疾病的用途。
附图说明
图1药物/HepG2/Jurkat细胞共培养48小时后HepG2的存活率。
具体实施方式
以下结合附图，通过实施例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。本发明所制备的化合物，其结构经核磁及质谱等分析手段确证。
实施例1
化合物Ⅰf的制备
第一步，将60mg(0.14mmol)化合物Ⅲ加入到25mL单口烧瓶中，5mL甲醇溶解，加入1M NaOH溶液将pH调至12，磁力搅拌，3小时后TLC检测。向反应液中加入1M HCl溶液pH调至2～3，产生白色固体，过滤得30mg白色固体即为产物，产率55％。
1H NMR(600MHz,DMSO-d6)δ7.66(d,J＝7.7Hz,2H),7.61(d,J＝7.8Hz,2H),7.49–
7.40(m,4H),7.35(t,J＝7.4Hz,1H),6.87(s,1H),6.78(s,1H),4.14(dd,J＝9.5,4.4Hz,1H),3.68(s,3H),3.43(s,2H),3.22(dd,J＝13.6,4.4Hz,1H),3.12(dt,J＝12.1,5.9Hz,1H),2.90(dd,J＝13.6,9.4Hz,1H),2.82(dt,J＝11.8,5.7Hz,1H),2.62(ddt,J＝19.4,13.1,5.8Hz,2H).
13C NMR(101MHz,DMSO)δ172.27,155.36,139.88,138.75,136.19,132.60,130.46,128.96,126.77,126.53,123.79,123.17,108.85,55.41,55.25,40.15,38.78,35.08,24.55.
ESI-MS calculated for C25H25NO3:387.1834；found:388.1907[M+H]+
第二步，称取0.1149g(0.30mmol)化合物Ⅱ、72μL(0.89mmol)DL-3-吡咯烷醇、0.1596g(0.41mmol)HBTU于25mL单口烧瓶中，加入10mL DCM，室温下磁力搅拌12小时，TLC监测反应进度，反应完全。反应液依次用去离子水、饱和NaHCO3、饱和NaCl溶液洗涤，有机相用无水Na2SO4干燥，过滤，减压旋蒸得无色油状物0.1250g。硅胶柱层析分离(DCM:MeOH＝11:1)得纯品白色固体110mg，产率83％。
1H NMR(400MHz,DMSO-d6)δ7.66(d,J＝7.4Hz,2H),7.62(d,J＝8.2Hz,2H),7.47(d,J＝7.5Hz,2H),7.44–7.39(m,2H),7.35(t,J＝7.3Hz,1H),6.83(s,1H),6.76(s,1H),4.96(d,J＝39.5Hz,1H),4.28(d,J＝28.6Hz,1H),4.17(dd,J＝9.4,4.4Hz,1H),3.67(s,3H),3.55(pd,J＝5.3,4.9,1.7Hz,2H),3.47(s,1H),3.43(s,1H),3.40(q,J＝4.8,3.9Hz,1H),3.30–3.27(m,1H),3.23(dd,J＝13.6,4.4Hz,1H),3.14(ddd,J＝12.0,6.8,5.1Hz,1H),2.91(dd,J＝13.6,9.4Hz,1H),2.87–2.79(m,1H),2.63(dq,J＝13.3,6.8,6.0Hz,2H),2.00–1.68(m,2H).
13C NMR(101MHz,DMSO)δ168.76,154.98,140.13,138.89,137.95,137.63,131.00,130.15,128.92,127.19,126.50,126.19,108.69,69.60,56.62,55.37,54.54,53.84,43.52,41.41,34.03,32.48,28.08.
ESI-MS calculated for C25H25NO3:456.2413,；found:457.2487[M+H]+
实施例2
化合物Ⅰt的制备
第一步，同实施例1。
第二步，称取0.1083g(0.28mmol)化合物Ⅱ、0.1534g(1.10mmol)D-丙氨酸甲酯盐酸盐、0.1402g(0.37mmol)HBTU和0.14mL(1.04mmol)三乙胺于25mL单口烧瓶中，加入10mLDCM，室温下磁力搅拌12小时反应完全。反应液依次用去离子水、饱和NaHCO3溶液、饱和NaCl溶液洗涤，有机相用无水Na2SO4干燥，过滤，减压旋蒸得无色油状物0.1343g。硅胶柱层析分离(DCM:MeOH＝20:1)得纯品白色固体78mg，产率59％。
1H NMR(400MHz,DMSO-d6)δ8.30(d,J＝7.0Hz,1H),7.66(d,J＝7.2Hz,2H),7.61(d,J＝7.9Hz,2H),7.43(dd,J＝13.3,7.8Hz,4H),7.35(t,J＝7.4Hz,1H),6.88(s,1H),6.75(s,1H),4.27(p,J＝7.2Hz,1H),4.16(dd,J＝9.3,4.4Hz,1H),3.67(s,3H),3.62(s,3H),3.36(s,2H),3.22(dd,J＝13.6,4.4Hz,1H),3.13(dt,J＝12.1,5.9Hz,1H),2.90(dd,J＝13.5,9.4Hz,1H),2.86–
2.80(m,1H),2.61(q,J＝6.7,6.2Hz,2H),1.28(d,J＝7.3Hz,3H).
13C NMR(101MHz,DMSO)δ173.18,170.02,155.07,140.03,138.21,137.98,136.03,130.83,130.21,128.91,127.24,126.55,126.49,125.37,122.84,108.76,56.26,55.32,51.78,47.61,40.77,39.40,35.78,27.09,17.04.
ESI-MS calculated for C29H32N2O4:472.2362；found:473.2441[M+H]+
第三步，将酯在碱性条件下进行水解，室温下磁力搅拌2小时后，TLC检测反应完全，减压旋去有机溶剂，再加入1M稀盐酸调节pH至弱酸性，析出白色固体即为水解产物Ⅰt，产率96％。
1H NMR(400MHz,DMSO-d6)δ7.67(d,J＝7.7Hz,4H),7.55–7.43(m,4H),7.37(d,J＝7.4Hz,1H),7.04(d,J＝4.3Hz,1H),6.50(s,1H),4.70(s,1H),4.33–4.13(m,2H),3.76(dd,J＝10.5,5.7Hz,1H),3.64–3.54(m,1H),3.52(s,3H),3.39(q,J＝7.2Hz,3H),3.26(d,J＝7.0Hz,2H),3.22–3.13(m,2H),3.07–2.96(m,1H),2.92–2.81(m,1H),2.29(ddt,J＝13.8,10.0,5.0Hz,1H),1.84–1.73(m,1H),1.22(s,1H).
3C NMR(101MHz,DMSO)δ173.32,168.66,155.13,139.88,138.80,136.05,130.54,128.99,127.41,126.81,126.56,123.48,108.79,68.85,57.42,55.26,54.99,54.60,38.89,38.69,37.16,34.65,24.30.
ESI-MS calculated for C28H30N2O4:458.2206；found:459.2279[M+H]+
实施例3
均相时间分辨荧光试验(HTRF)检测化合物阻断PD-1/PD-L1蛋白结合的能力。
测试原理：采用铕标记的Anti-Tag1(HTRF供体)和XL665标记的Anti-Tag2(HTRF受体)检测PD-L1与PD-1的相互作用。当PD-L1和PD-1结合使供体抗体和受体抗体接近时，激发供体抗体引发荧光共振能量转移(FRET)向受体抗体，受体抗体在665nm处特异性发光。这个特定的信号与PD-1/PD-L1相互作用的程度成正比。因此，阻断PD-1/PD-L1相互作用的化合物或抗体将导致HTRF信号的降低。
仪器与耗材：PD-1/PD-L1 binding assay kit(PerkinElmer,cat.no.64PD1PEH)购自上海优宁维生物科技股份有限公司，DMSO购自Gentihold公司。
实验步骤：用DMSO溶解合成小分子或BMS-1配制10mM母液，放置于-20℃冻存待用。将Anti-Tag2-XL665与Anti-Tag1-Eu3+按照1:1配置成混合液，微孔白板中每孔中加入2μL待测物、4μL PD-1蛋白、4μL PD-L1蛋白及10μL抗体混合液，常温下离心混匀后孵育1小时，酶标仪检测665nm和620nm处的荧光信号。根据以下公式，计算发射信号比率和抑制率，计算平均值，结果如表1，化合物结构如下所示：
Emission Ratio(ER)＝665nm Emission signal/615nm Emission signal*106
抑制率＝(ERpositive-ERsample)/(ERpositive-ERnegative)*100％
表1化合物在50μM浓度下阻断PD-1/PD-L1相互作用的HTRF结果
实验结果：大部分目标化合物在分子水平上可显著阻断PD-1/PD-L1相互作用，值得注意的是，结构改造引入甲基后，化合物的阻断活性普遍增加。例如，化合物Ⅰf引入甲基后变为化合物Ⅰg，其阻断活性增强；类似地，化合物Ⅰj和Ⅰi引入甲基后也表现出更高的活性。酯的阻断活性优于相应的酸，这提示酯基可能在与靶点的结合中起到关键作用。选择阻断活性前八的化合物(Ⅰb、Ⅰd、Ⅰe、Ⅰf、Ⅰg、Ⅰm、Ⅰq、Ⅰr)进一步分析，探究其阻断机制。
实施例4
表面等离子共振(SPR)检测小分子与PD-L1蛋白的结合
测试原理：表面等离子共振(SPR)是一种光学现象，可被用来实时跟踪在天然状态下生物分子间的相互作用。这种方法对生物分子无任何损伤，且不需任何标记物。引起表面等离子共振的入射角称为SPR角。先将一种生物分子(靶分子)键合在生物传感器表面，再将含有另一种能与靶分子产生相互作用的生物分子(分析物)的溶液注入并流经生物传感器表面。当有分析物结合到芯片表面后，导致芯片表面折射率的改变，从而引起SPR角度的变化，而这种变化被实时记录在传感图上。
仪器与耗材：仪器Biacore T200、PD-L1蛋白购自SinoBiological、标准胺偶联试剂盒购自Cytiva、CM5传感器芯片购自Cytiva、醋酸钠购自Cytiva、NaOH购自Cytiva、PBS购自Servicebio、吐温-20购自萨恩化学技术(上海)有限公司
实验步骤：用超纯水溶解PD-L1蛋白配置成浓度为0.25mg/mL母液，用DMSO溶解小分子配置10mM小分子母液。选择最适pH醋酸钠(pH＝5.0)溶液稀释蛋白至10μg/mL，利用氨基偶联法手动将PD-L1蛋白偶联到CM5芯片的Flow Cell 2通道上，1×PBS为缓冲液。首先，将EDC和NHS等体积混合活化芯片表面的羧基，流速为10μL/min，进样时长7min，随后将蛋白溶液注射到芯片表面，流速为10μL/min，进样时长11min，最后芯片表面剩余的羧基用乙醇胺封闭，流速为10μL/min，进样时长7min。结束偶联过程，最终PD-L1蛋白偶联量为10000RU。使用含0.5％和1.8％的DMSO母液配置含1％ DMSO的校正曲线，以消除溶液中DMSO的影响。使用含1％吐温20的1.05×PBS溶液稀释待测物母液到适宜浓度梯度，并保证待测物DMSO含量为1％。运行缓冲液为含1％ DMSO和1％吐温20的1.05×PBS溶液。利用LMW kinetics模块进行多动力循环检测，速度为30μL/min，结合和解离时间各为60秒，运行结束后，使用Biaore T200 Evalution分析软件拟合曲线，求算KD值，结果如表2。
表2化合物与PD-L1蛋白结合亲和力
测定了阳性对照BMS-1与hPD-L1的亲和力，以确保芯片表面蛋白的活性。结果显示，BMS-1与hPD-L1结合的KD值为27.3μM，这一结果验证了实验系统的有效性。测定了化合物Ⅲ以及HTRF阻断活性前八的化合物(Ⅰb、Ⅰd、Ⅰe、Ⅰf、Ⅰg、Ⅰm、Ⅰq、Ⅰr)与hPD-L1蛋白的结合亲和力，结果表明这些化合物与hPD-L1的结合能力均优于化合物Ⅲ(KD值为11.3μM)，且化合物Ⅰf、Ⅰg、Ⅰq和Ⅰr的KD值达到了纳摩尔级别，结构改造后活性的显著提升，可作为具有可检测的亲和力的新型hPD-L1抑制剂。
实施例5
细胞毒性实验检测化合物对HepG2细胞及Jurkat细胞的无毒浓度
实验耗材：1640培养基购自gibco公司、PBS购自Solarbio公司、青链霉素(双抗)液购自武汉赛维尔生物科技有限公司、胎牛血清(FBS)购自Gibico公司；0.25％胰酶(含EDTA)购自新赛美公司；CCK-8购自GlpBio公司
实验步骤：设置空白、对照、实验组，以5000个/孔细胞接种到96孔板中，每孔加入100μL细胞悬液，空白组不加细胞，37℃，5％ CO2条件下培养箱培养过夜；实验组加入100μL不同浓度药物，平行4个复孔，空白、对照组加入培养基，后续在细胞培养箱中培养48小时。每孔加入20μL的CCK8，在培养箱中孵育1.5小时，酶标仪检测450nm下的吸光度，结果如表3所示。
表3细胞毒性测定
实验结论：10个化合物(BMS-1、Ⅰb、Ⅰd、Ⅰe、Ⅰf、Ⅰg、Ⅰm、Ⅰ1、Ⅰr及化合物Ⅲ)的细胞共孵育浓度分别为10μM、0.37μM、1.11μM、0.37μM、1.11μM、0.37μM、1.11μM、0.37μM、1.11μM及0.37μM。
实施例6
细胞共孵育实验检测小分子化合物的PD-1/PD-L1阻断活性
实验耗材：细胞培养试剂同实施例2、IFN-γ购自PeproTech公司、PHA-P购自Invivogen公司。
实验步骤：设置空白、对照、实验组，以1000个/孔细胞接种到96孔板中，每孔加入100μL细胞悬液，空白组不加细胞，同时用10ng/mL IFN-γ刺激HepG2细胞24小时后将其液体吸出；再往空白、对照、实验组中加入2μg/mL PHA活化48小时的Jurkat细胞，以10000个/孔细胞接种到96孔板中，每孔加入100μL细胞悬液，实验组加入100μL。孵育完毕后将其液体吸出，每孔加入100μL含10％ CCK8的培养基，在培养箱中孵育2小时，酶标仪检测450nm下的吸光度。
结果讨论：如图1所示，HepG2细胞在共培养体系中存活率显著下降，说明在共培养中加入药物说可以有效阻断PD-1/PD-L1信号通路从而激活Jurkat T细胞杀伤肿瘤细胞，化合物Ⅰf在1.11μM浓度下抑制率达到42％。
综上，本发明设计并合成了一类新型的四氢异喹啉类PD-L1抑制剂，其可通过恢复T细胞的免疫功能从而杀伤肿瘤细胞，具有治疗肿瘤疾病的潜力。
Claims (4)
1.一类四氢异喹啉类PD－L1抑制剂，具有通式Ⅰ所示的结构：
其中：R选自乙醇胺、L-丝氨醇、2-氨基-2-甲基-1,3-丙二醇、三羟甲基氨基甲烷、1-氨基-2-甲基-2-丙醇、3-甲胺基-1,2-丙二醇、N,N-二甲基乙二胺、N-乙酰基乙二胺、4-羟基哌啶、哌啶-4-甲酰胺、哌嗪、1-乙酰哌嗪、吗啉、DL-3-吡咯烷醇、3-羟基-3-甲基-吡咯烷、N-甲基哌嗪、四氢吡咯、顺式-4-羟基-D-脯氨酸、L-脯氨酸、4-哌啶甲酸、2-哌啶甲酸、4-哌嗪甲酸、D-丙氨酸、L-苏氨酸、L-丝氨酸、L-甲硫氨酸、牛磺酸、(2R,4R)-4-羟基吡咯烷-2-羧酸甲酯、L-苏氨酸甲酯、L-丝氨酸甲酯、L-脯氨酸甲酯、D-丙氨酸甲酯中的一种。
2.权利要求1所述一类四氢异喹啉PD－L1抑制剂的制备方法，其特征在于：
(1)在20～25℃温度条件下，以甲醇为溶剂，将化合物Ⅲ在碱性条件下反应2-4h得到化合物Ⅱ；碱包括碳酸钾、碳酸氢钾、氢氧化钠，化合物Ⅲ与碱的摩尔比为1：2～1：5；
(2)在0～90℃温度条件下，化合物Ⅱ通过缩合反应与胺类化合物在碱性条件下，进行反应12～24h得到目标化合物；若R基团中存在酯基时，在20～25℃温度条件下，以甲醇为溶剂，将酯在碱性条件下水解2-4h得到相对应的酸；
其中：反应体系所用溶剂为二氯甲烷、四氢呋喃、乙腈、N，N-二甲基甲酰胺中的一种或两种以上组合；
反应体系所用碱包括碳酸钾、碳酸氢钠、三乙胺、N,N-二异丙基乙胺及胺类反应物，化合物Ⅱ与碱的摩尔比为1：2～1：5；
反应体系所用缩合剂包括EDCI、DCC、HOBt、HATU、HBTU中的一种或两种以上组合，化合物Ⅱ与缩合剂的摩尔比为1：1～1：5；
化合物Ⅱ与胺类化合物的摩尔比为1：1.1～1：4，酯与碱的摩尔比为1：2～1：5。
3.一种药物的组合物，其中包含：权利要求1所述的通式Ⅰ所示化合物或其衍生物；至少一种药学上可接受的盐；可药用载体。
4.权利要求3所述的组合物在制备阻断PD-1/PD-L1相互作用来预防和/或治疗肿瘤疾病的药物中的用途。
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