CN111909240B - 一种pd-1/pd-l1多肽类抑制剂及其医药用途 - Google Patents
一种pd-1/pd-l1多肽类抑制剂及其医药用途 Download PDFInfo
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Abstract
本发明公开了一种环肽类化合物或其药学上可接受的盐、酯、溶剂化物、前药、外消旋体或异构体，以及含有上述化合物作为有效成分的药物组合物，并通过试验验证了它们在抑制PD‑1/PD‑L1通路中的活性，具有制备为预防和治疗以PD‑1/PD‑L1通路为靶点的肿瘤药物的应用；所述肿瘤包括血液癌症、神经系统癌症、胃肠癌、食道癌、泌尿系统癌症、肺癌、肝癌和皮肤癌等。
Description
技术领域
本发明属于药物化学领域，特别是涉及一类PD-1/PD-L1的多肽类化合物或其药学上可接受的盐、酯或溶剂化合物，以及其在制备PD-1/PD-L1抑制剂药物中的用途。
背景技术
PD-1又称CD279，是一种相对分子量为55000～60000的Ⅰ型跨膜蛋白，属免疫球蛋白超家族成员，其结构主要包括胞外免疫球蛋白可变区(IgV)样结构域、疏水的跨膜区以及胞内区。胞内区包括C端和N端氨基酸残基，含有2个独立的磷酸化作用位点，分别为免疫受体酪氨酸抑制基序(immunoreceptor tyrosine based inhibitory motif,ITIM)和免疫受体酪氨酸转换基序(immunoreceptor tyrosine based switch motif,ITSM)。PD-1主要表达于活化的CD4+T细胞、CD8+T细胞、B细胞、NK细胞、单核细胞和树突状细胞等免疫细胞上，促进T细胞的成熟。PD-1的配体属B7家族成员，包括PD-L1(又名B7-H1，CD274)和PD-L2(又名B7-DC)，两者均高表达于胎盘组织；低表达于脾脏、淋巴结、胸腺；脑组织中无表达。其中，PD-L1同是Ⅰ型跨膜蛋白，主要表达于抗原提呈细胞、B细胞、T细胞、上皮细胞、肌细胞、内皮细胞等。PD-1及PD-L1共同组成PD-1/PD-L1信号通路，抑制生长因子的生成和细胞增殖，并对T细胞的活化及调控免疫应答起到重要作用。PD-1/PD-L1通路激活后，在癌症、妊娠、组织移植以及自身免疫病中抑制免疫系统。目前PD-1/PD-L1抑制剂的开发主要集中在单克隆抗体领域，已有Nivolumab,Lambrolizumab,Atezolizumab,Durvalumab,Avelumab等单抗在国内外上市，可用于治疗非小细胞肺癌，黑色素瘤等常规治疗方法效果不佳的疾病具有明显的治疗效果。与单抗研究开发相比，该领域的肽类抑制剂却进展缓慢。所以研究开发抑制PD-1/PD-L1相互作用的肽类抑制剂具有重要的临床意义。
发明内容
发明目的：本发明提供了一类环肽类化合物或其药学上可以接收的盐、酯或溶剂化合物，还提供了含有有效量的上述环肽类化合物或其药学上可以接收的盐、酯或溶剂化合物的药物组合物，本发明还提供了它们的制药应用。
技术方案：本发明所述的环肽类化合物或其药学上可接受的盐、酯、溶剂化物、前药、外消旋体或异构体，如通式I所示：
其中，A选自：
中的一种。
本发明所述的一种药物组合物，含有环肽类化合物或其药学上可接受的盐、酯或溶剂化物，以及药学上可接受的载体。所述药物组合物可以为片剂、胶囊、颗粒剂、散剂、糖浆剂、口服液或注射剂。
本申请公开了所述环肽类化合物或其药学上可接受的盐、酯、溶剂化物、前药、外消旋体或异构体在制备预防和治疗肿瘤药物中的应用。进一步的，所述药物为以PD-1/PD-L1通路为靶点的肽类抑制剂。
所述肿瘤包括血液癌症、神经系统癌症、胃肠癌、食道癌、泌尿系统癌症、肺癌、肝癌和皮肤癌。更具体的，所述肿瘤包括淋巴瘤、非小细胞肺癌、小细胞肺癌、头颈部细胞癌、神经胶质瘤、成神经细胞瘤、肺鳞癌、肺腺癌、膀胱癌、胃癌、结肠癌、大肠癌、肾癌、胆管癌、胃癌、食管鳞癌、卵巢癌、胰腺癌、乳腺癌、前列腺癌、肝癌、脑癌、黑色素瘤、多发性骨髓瘤、皮肤癌、上皮细胞癌、白血病和宫颈癌等癌症，包括在其它远离肿瘤原发部位的组织或器官的转移病变。
所述药物可以作为癌症免疫治疗药物、癌症化疗药物或癌症靶向治疗药物。
本申请公开了所述环肽类化合物或其药学上可接受的盐、酯、溶剂化物、前药、外消旋体或异构体在制备PD-1/PD-L1通路抑制剂中的应用。
有益效果：本发明公开的环肽类化合物或其药学上可接受的盐、酯、溶剂化物、前药、外消旋体或异构体，以及含有上述化合物作为有效成分的药物组合物，并通过试验验证了它们在抑制PD-1/PD-L1通路中的活性，具有制备为预防和治疗以PD-1/PD-L1通路为靶点的肿瘤药物的应用。
具体实施方式
下面结合部分化合物具体实例对本发明作进一步阐述，但本发明不局限于这些实施例。
本申请所述环肽类化合物的合成可参照以下方法：
1.固相合成一般方法
固相氨基酸的合成均在固相反应合成管中进行，使用rink amide-AM树脂(Merrifield聚合物负载(2,4-二甲氧基苯基)(4-烷氧基苯基)甲烷，其中4-烷氧基为与树脂连接的位置和化学键类型，负载量0.7mmol/g)。使用DMF和DCM溶解反应所用试剂后，沿管壁加入到反应管中，通入氮气后振荡所需时间。之后，反应液从反应管下侧通过真空泵抽去。反应中所用的溶剂和试剂为：DMF＝N,N-二甲基甲酰胺；DCM＝二氯甲烷；HATU＝1-[二(二甲胺基)亚甲基]-3-氧代-1H-1,2,3-三氮唑并[4,5-b]吡啶六氟磷酸盐；DEPBT＝3-(二乙氧基磷酰氧基)-1,2,3-苯并三嗪-4-酮；TBTU＝O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼；DIPEA＝二异丙基乙胺。树脂的溶胀过程如下文“树脂溶胀步骤”所述。缩合步骤如下文“缩合步骤”所述。使用的氨基酸衍生物和末端羧酸如下所示(侧链保护基置于括号中)：Fmoc-L-Gly-OH，Fmoc-L-Cys(Trt)-OH，Fmoc-L-Leu-OH，Fmoc-L-[N-Me]Nle-OH，Fmoc-L-Trp(CH2COOtBu)-OH，Fmoc-L-Trp(Boc)-OH，Fmoc-L-Dab(Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH，ClCH2COOH。
在一些实施例中，以下氨基酸和末端羧酸也会使用：Fmoc-L-Trp(Cbz)-OH，Fmoc-L-Dab(Cbz)-OH，Fmoc-L-[O-Bn]Hyp-OH，Fmoc-L-Dap(Cbz)-OH，Fmoc-L-[O-Bn]Tyr-OH，丙烯酸。
在一些实施例中，如下氨基酸衍生物也会被使用：2-芴甲氧羰基氨基-4-丁烯酸
2.树脂溶胀步骤
将Rink Amide-AM树脂(286mg，0.2mmol)加入到10mL的固相合成反应管中，加入8mL的DCM，静置半小时。之后将DCM经真空泵抽去即可完成树脂的溶胀。
3.缩合步骤
将6mL 20％的哌啶/DMF溶液加入到反应管中，振荡30分钟。反应液抽干，树脂用无水DMF(10mL)，无水甲醇(10mL)和无水DCM(10mL)分别洗涤3次后，取样经四氯苯醌显色，树脂呈蓝色可指示脱除保护基结束。
脱除保护基结束后，将所需氨基酸(0.6mmol)，HATU(228mg，0.6mmol)，DMF(6mL)和DIPEA(210μL，1.2mmol)依次加入到干燥的圆底烧瓶中，超声助溶使其澄清。将混合液加入到固相合成反应管中，25℃反应3小时，取样经四氯苯醌显色，树脂呈无色透明可指示缩合反应结束。树脂用无水DMF(10mL)，无水甲醇(10mL)和无水DCM(10mL)分别洗涤3次后即可进行下一次缩合。
4.肽游离步骤
完成所需直链肽合成后，将树脂再用无水DMF洗涤1次，后抽至干。向干燥的树脂加入切割液(三氟乙酸：硫代苯甲醚：1,2-乙二硫醇：苯甲醚＝90:5:2.5:2.5，体积比)。混合物在10℃下振摇3小时。反应结束后，将切割液抽滤出来，浓缩至原体积1/2，浓缩液逐滴加入到10倍体积的-20℃无水乙醚中。抽滤形成的沉淀即得粗肽，可不经处理直接进行下一步反应。
5.RP-HPLC分离步骤
将所得的粗品肽溶于一定量的纯净水中，使用三乙胺或2M HCl调节PH至7，加入乙腈至澄清，冻干得粗品固体。加入一定量的乙腈使其完全溶解，经0.33μM滤器过滤。使用Aglient Eclipase XDB-C18柱进行分离，流动相A：0.1％TFA/H2O；流动相B：0.1％TFA/MeCN。色谱条件为10％B-100％B，60min。
实施例1
根据前述“树脂溶胀步骤”，使树脂溶胀后，根据“缩合步骤”，选用氨基酸分别为Fmoc-L-Gly-OH，Fmoc-L-Cys(Trt)-OH，Fmoc-L-Leu-OH，Fmoc-L-[N-Me]Nle-OH，Boc-L-Trp-OH，Fmoc-L-Trp(Boc)-OH，Fmoc-L-Dab(Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH。缩合完成后，根据前述“肽游离步骤”，选择对应的氨基酸可得直链肽前体粗品。取60mg该粗品溶于乙腈：0.1M碳酸铵＝1：1(体积比)总共600mL溶液中，该反应在室温搅拌5小时。将反应液旋干，经冷冻干燥后得到粉末状混合物。将该混合物溶于乙腈：水＝1:1(体积比)中，根据前述“RP-HPLC分离步骤”可得环肽前体纯品。取该前体纯品5mg，溶于乙腈：水＝1：1(体积比)总共0.6mL中，向该溶液中加入0.07mL 30％H2O2，在室温下搅拌3小时。反应结束后，过量的双氧水用抗坏血酸破坏，溶液旋干，冷冻干燥可得粗品。根据前述“RP-HPLC分离步骤”可得目标产物。tR＝18.760min。HRESIMS:[M+H]+＝1916.9560。
实施例2
根据前述“实施例1”中的方法可得，tR＝27.251min。HRESIMS:[M+H]+＝1916.9601。
实施例3
根据前述实施例1的合成，将氧化步骤更换为：取该前体纯品5mg，溶于甲醇：水＝1：1(体积比)共500μL中。该混合液中加入过氧单磺酸钾0.5mg，室温搅拌。反应经RP-HPLC可判断反应结束。向反应液中加入抗坏血酸以破坏过氧化物，反应液冻干后根据前述“RP-HPLC分离步骤”可得产物。tR＝20.230min。HRESIMS:[M+Na]+＝1896.9402。
实施例4
根据前述“树脂溶胀步骤”，“缩合步骤”和“肽游离步骤”，将实施例1中的氨基酸衍生物Fmoc-L-Cys(Trt)-OH更换为2-芴甲氧羰基氨基-4-丁烯酸，末端羧酸更换为丙烯酸可得直链肽前体粗品。取该粗品10mg，溶于二氯甲烷中，加入Grubb’s 2nd催化剂(0.2equiv)，回流12小时。将粗品溶于DMSO中，过滤以除去催化剂。溶液冻干，“RP-HPLC分离步骤”可得Z/E混合物。tR＝15.245min。HRESIMS:[M+Na]+＝1896.9801。
实施例5
将实施例4中所得化合物取10mg，溶于0.5mL甲醇中，加入1mg Pd/C，反应在氢气氛下，45℃反应8小时。反应液加入二氯甲烷稀释，旋干后根据“RP-HPLC分离步骤”可得纯品。tR＝15.258min。HRESIMS:[M+2Na]+/2＝1869.0002。
实施例6
具体合成方法为：
将预先活化的4A分子筛5g，LiOH(384mg，16mmol)加入到无水DMF(50mL)中，室温搅拌20分钟。然后将6-1(2.01g,8mmol)加入到混合液中，室温下搅拌40分钟。再将烯丙基溴(830μL,9.6mmol)加入到反应液中，该反应在室温下搅拌12小时。反应结束后，反应液用乙酸乙酯稀释，抽滤，浓缩至干可得粗品。经硅胶柱层析分离(正己烷:乙酸乙酯＝20:1)可得目标化合物6-2(1.82g)，产率78％。1H-NMR：δ(DMSO-d6,ppm)7.13(d,J＝7.3Hz,2H),6.86(d,J＝7.3Hz,2H),5.87–5.84(m,1H),5.20-5.17(m,2H),3.86–3.82(m,1H),3.66,(s,3H),3,39-3.10(m,3H),1.42(s,9H).HRESIMS:[M+H]+＝292.1830
取化合物6-2(1.37g，4.7mmol)溶于30mL甲醇中，反应液移至0℃下，加入氢氧化锂(124mg，5.17mmol)该反应在0℃下反应1小时。反应结束后，反应液加入乙酸乙酯稀释，加入2M盐酸溶液调节pH至3，两相分离，有机层氯化钠饱和溶液洗1次。浓缩至干可得化合物6-2，可不经处理直接进行下一步反应。HRESIMS:[M+H]+＝277.3644
取化合物6-3粗品1.4g，溶于25mL的10％碳酸钠溶液25mL和丙酮50mL中，反应液移至冰浴下，加入Fmoc-OSu(2.05g，6.06mmol)，反应在室温下搅拌18小时。反应结束后，反应液用2M盐酸溶液调节pH至3，加入乙酸乙酯稀释，两相分离，有机相水洗3次，氯化钠饱和溶液洗1次，浓缩至干，经硅胶柱层析分离(DCM:MeOH＝300:1)可得化合物6-4(1.22g)。两步产率61％。.1H-NMR：δ(DMSO-d6,ppm)7.90(d,J＝7.1Hz,2H),7.55(d.J＝7.0Hz,2H),7.41-7.29(m,4H),7.11(d,J＝7.3Hz,2H),6.87(d,J＝7.3Hz,2H),5.87–5.84(m,1H),5.20-5.17(m,2H),4.71-4.59(m,3H),3.86–3.82(m,1H),3,39-3.10(m,3H),1.42(s,9H)。
实施例7
根据前述“树脂溶胀步骤”，“缩合步骤”和“肽游离步骤”，将实施例1中的Fmoc-L-Cys(STrt)-OH更换为2-芴甲氧羰基氨基-4-丁烯酸，Fmoc-L-Tyr(OtBu)-OH更换为实施例6，可得直链肽前体粗品。取该粗品10mg，溶于二氯甲烷中，加入Grubb’s 2nd催化剂(0.2equiv)，回流12小时。将粗品溶于DMSO中，过滤以除去催化剂。溶液冻干，根据“RP-HPLC分离步骤”可得目标产物tR＝22.254min。HRESIMS:[M+Na]+＝1874.9908。
实施例8
将实施例7中所得化合物取10mg，溶于0.5mL甲醇中，加入1mg Pd/C，反应在氢气氛下，45℃反应8小时。反应液加入二氯甲烷稀释，旋干后根据前述“RP-HPLC分离步骤”可得纯品。tR＝22.243min。HRESIMS:[M+Na]+＝1877.0246。
实施例9
根据前述“树脂溶胀步骤”，“缩合步骤”和“肽游离步骤”，将实施例1中使用的氨基酸衍生物Fmoc-L-Trp(Boc)-OH，Fmoc-L-Dab(Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-[O-tBu]Tyr-OH分别替换为Fmoc-L-Trp(Cbz)-OH，Fmoc-L-Dab(Cbz)-OH，Fmoc-L-[O-Bn]Hyp-OH，Fmoc-L-Dap(Cbz)-OH，Fmoc-L-[O-Bn]Tyr-OH可得直连肽前体。取该粗品10mg，溶于无水DMF 150mL中，反应液移至-20℃。预先混合HATU(41mg,109μmol),DEPBT(33mg,109μmol),TBTU(35mg,109μmol)，加入到反应液中。反应液在-20℃搅拌1小时后，在0℃下搅拌1小时，然后室温下搅拌3天。经HPLC鉴定反应结束后，反应液加入乙酸乙酯稀释，水洗1次。有机层浓缩至干，根据前述“RP-HPLC分离步骤”可得目标产物。tR＝24.236min。HRESIMS:[M+Na]+＝1919.0022
实施例10
体外抑制活性的测定
1、均相时间荧光法测定实施例抑制PD-1/PD-L1相互作用的评估：
PD-1和PD-L1的相互作用可使用两种蛋白的胞外域部分的重组蛋白测定。PD-1和PD-L1蛋白质细胞外结构域表达为具有检测标签的融合蛋白，对于PD-1，标签是免疫球蛋白(PD-1-Ig)的Fc部分，对于PD-L1，它是6组氨酸基序(PD-L1-His)。人PD-1(25-167)，具有免疫球蛋白G(Ig)表位标签[hPD-1(25-167)-3S-IG]的C末端人Fc结构域和人PD-L1(18-239)具有C末端His表位标签[hPD-L1(18-239)-TVMV-His1]在HEK293T细胞中表达，并通过蛋白A亲和层析和尺寸排阻层析依次纯化。
相互作用研究均在由额外添加0.1％(含)牛血清白蛋白和0.05％(v/v)Tween-20的dPBS组成的HTRF测定缓冲液中进行。对于hPD-L1-His结合测定，将抑制剂与PD-L1-His(终浓度10nM)在4ul测定缓冲液中预孵育15分钟，然后加入PD-1-Ig(终浓度20nM)。在1uL测定缓冲液中并进一步培养15分钟。使用铕钙磷酸盐标记的抗Ig(终浓度1nM)和异烟酞菁(APC)标记的抗His(最终20nM)实现HTRF检测。将抗体在HTRF检测缓冲液中稀释并取5ul。使反应混合物平衡30分钟，并使用EnVision荧光计获得所得信号(665nm/620nm)。
各个环肽类化合物抑制PD-1/PD-L1相互作用测定结果如表1所示。
表1
A：IC50<100nM；B：100nM<IC50<10μM；C：10μM<IC50<100μM
| 实施例 | IC50 |
| 1 | 0.33μM |
| 2 | 0.13μM |
| 3 | A |
| 4 | A |
| 5 | A |
| 7(Z/E) | A |
| 8 | A |
| 9 | A |
如上表所示，实施例中的环肽类化合物均有一定的抑制PD-1/PD-L1相互作用的能力。
2、实施例细胞层面抑制PD-1/PD-L1相互作用评估：
该实验中使用重组鼠源PD-L1作为细胞实验中PD-L1的来源。小鼠脾细胞是将小鼠脾脏在40um细胞滤网中捣碎后，在室温下用1mL ACK裂解缓冲液进一步处理5分钟得到。用9mL RPMI完全培养基洗涤后，将细胞重悬于15mL管中的3mL 1xPBS中。小心地将3mLHistopaque加入管底部，不干扰覆盖的脾细胞悬浮液。在室温下以800×g离心20分钟后，收集不透明的脾细胞层。得到的脾细胞再用冷PBS溶液冲洗两次，使用台盼蓝染色计数总细胞数用于之后的细胞层面测试。脾细胞在RPMI完全培养基(RPMI+10％胎牛血清+1mM丙酮酸钠+10,000u/mL青霉素和10,000ug/mL链霉素)中培养，并保持在37℃下含5％CO2的CO2培养箱中。
CFSE是一种被动扩散到细胞内并与细胞内蛋白结合的染料。1×106细胞/mL的脾细胞用5uM CFSE在预热的1x PBS/0.1％BSA溶液中于37℃处理10分钟。过量CFSE将5倍体积的0℃培养基淬灭至细胞中并在冰上孵育5分钟。用0℃完全RPMI培养基进一步洗涤CFSE标记的脾细胞三次。将CFSE标记的1×105脾细胞加入到含有MDA-MB231细胞(在高葡萄糖DMEM培养基中培养1×10个细胞)或重组人PD-L1(100ng/mL)和测试化合物的孔中。用抗小鼠CD3和抗小鼠CD28抗体(各1ug/mL)刺激脾细胞，并将培养物在37℃，5％CO2下进一步培养72小时。收获细胞并用冰冷的FACS缓冲液洗涤三次，并通过流式细胞术用488nm激发和521nm发射滤光片分析百分比增殖。
使用FACS程序分析脾细胞增殖百分比，并且在扣除背景增殖值(％)并将刺激的脾细胞增殖(％，阳性对照)标准化为100％后以计算化合物回复脾细胞增殖百分比。
刺激的脾细胞：脾细胞+anti-CD3/CD28
刺激背景增殖：脾细胞+anti-CD3/CD28+PD-L1
化合物增殖：脾细胞+anti-CD3/CD28+PD-L1+化合物
通过添加所需浓度来检测化合物效果。在配体(PD-L1)存在下化合物对anti-CD3/CD28刺激的脾细胞的表达。
各个环肽类化合物在5nM浓度下抑制PD-1/PD-L1相互作用测定结果如表2所示。
表2
A：>90％；B：70％<IC50<90％；C：50％<IC50<70％；D：<50％
| 实施例 | 抑制率 |
| 1 | 92％ |
| 2 | 96％ |
| 3 | A |
| 4 | A |
| 5 | A |
| 7(Z/E) | A |
| 8 | A |
| 9 | A |
如上表所示，实施例中的环肽类化合物均有一定的抑制PD-1/PD-L1相互作用的能力。
Claims (6)
1.通式I所示的环肽类化合物或其药学上可接受的盐：
其中，A选自：
2.一种药物组合物，其特征在于，含有权利要求1所述的环肽类化合物或其药学上可接受的盐，以及药学上可接受的载体。
3.权利要求1所述环肽类化合物或其药学上可接受的盐在制备预防和治疗肿瘤药物中的应用；所述肿瘤为实体瘤。
4.根据权利要求3所述应用，其特征在于，所述肿瘤包括神经系统癌症、胃肠癌、食道癌、泌尿系统癌症、肺癌、肝癌和皮肤癌。
5.根据权利要求3所述应用，其特征在于，所述药物为以PD-1/PD-L1通路为靶点的肽类抑制剂。
6.权利要求1所述环肽类化合物或其药学上可接受的盐在制备PD-1/PD-L1通路抑制剂中的应用。
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