CN111484543B - 一种脂肽类tlr2激动剂及其药用组合物和应用 - Google Patents
一种脂肽类tlr2激动剂及其药用组合物和应用 Download PDFInfo
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Abstract
本发明属于药物化学领域,其涉及TLR2激动剂,具体涉及一种脂肽类化合物及其药用组合物作为TLR2激动剂,用于开发疫苗佐剂或抗肿瘤药物。本发明公开了具有式(I)结构的脂肽类化合物或者其药学上可接受的盐或非对映异构体或其前药分子及其制备方法,所述脂肽类化合物或者其药学上可接受的盐或非对映异构体或其前药分子可进一步制备新型高效TLR2激动剂,以及用于制备免疫佐剂或抗肿瘤药物。
Description
技术领域
本发明属于药物化学领域,尤其涉及TLR2激动剂,具体涉及一种脂肽类化合物及其药用组合物作为TLR2激动剂,用于开发疫苗佐剂或抗肿瘤药物。
背景技术
现有技术公开了恶性肿瘤是人类面临的一种重大疾病,极大的危害了人类的健康。在中国,无论是农村还是城市,肿瘤都是中国居民的主要死亡原因。据统计,中国癌症发病率位居全球发病率的74位,发病人数全球第一,癌症死亡率位居全球29 位;随着癌症发病率与死亡率的逐年增加,使人民的生命与财产遭受了巨大损失。目前,针对恶性肿瘤的传统治疗手段是外科手术结合放化疗,实践显示,若干的早期癌症通过手术可以达到根治的目的,但是仍存在癌症晚期患者根治性手术便无法进行;此外,现有的化疗药物在杀伤癌细胞的同时对人体正常细胞也有损害,进行化疗时往往出现不同程度的副作用,如恶心、呕吐、脱发等副反应。近年来,肿瘤的分子靶向治疗飞速发展,多种小分子激酶抑制剂进入临床研究阶段,但是部分患者在用药后较短时间内发生耐药,因此,耐药问题成为该类抑制剂在临床应用上最严重的威胁。
随着科技的进步,肿瘤的细胞免疫治疗取得了显著成就;有研究显示,Toll样受体(Toll like receptors,TLRs)作为一类经典的模式识别受体,通过识别病原体的相关分子模式启动信号的转导。有研究在人和小鼠体内分别发现了10和12种TLRs,依据其细胞的定位,主要分为两类:一类位于细胞表面,包括TLR1、TLR2、TLR4等,配体为微生物膜结构中的脂类、脂蛋白等,也可以被小分子化合物或抗体激活;另一类为胞内囊泡结构,包括TLR3,TLR9等,配体为微生物的核酸结构;其中TLR2广泛高表达于树突状细胞(DC)和T细胞表面,以异源二聚体TLR2/1和TLR2/6形式发挥作用。研究表明,在小鼠非小细胞肺癌模型中,TLR2/1激动剂Pam3CSK4(该化合物由美国Invivogen公司研发,目前处于临床前研究阶段)可明显上调杀伤性T细胞的杀伤能力和特异性增殖能力,显著降低调节性T细胞的增殖抑制功能;体内注射 Pam3CSK4后完全抑制荷瘤小鼠的肿瘤生长,促进肿瘤消退,并诱导小鼠体内长期的抗肿瘤免疫记忆(Zhang Y;Luo F;Cai Y;et al.TLR2/1 agonist induces tumorregression by reciprocalmodulation of effector and regulatory Tcells.Immunol,2011,186,4:1963– 1969)。还有实验结果表明,具有选择性的高效TLR2激动剂对人类战胜恶性肿瘤提供了有效的武器。遗憾的是目前尚没有TLR2激动剂获得美国FDA批准进入市场。回顾靶向TLR2的激动剂研究历程,研发进展缓慢的原因主要有2个方面:一方面,靶向 TLR2的分子多为微生物的脂类或脂蛋白结构,该类分子结构复杂,分子量大(如 Pam3CSK4,该结构包含三条棕榈酰脂肪链,分子量接近1400,手性碳多达7个);另一方面,TLR2的天然配体毒性较大(A.Lahiri.;P.Das.;D.Chakravortty,;Engagement of TLRsignaling as adjuvant:Towards smarter vaccine and beyond.Vaccine,2008,26,6777–6783.),以上因素限制了TLR2激动剂的发展与应用。因此,寻找结构简单、激动活性高、选择性好、毒性低的新型TLR2激动剂依然是该领域的重要课题。
基于现有技术的现状,本申请的发明人拟提供新的TLR2激动剂,具体涉及一种脂肽类化合物及其药用组合物作为TLR2激动剂,用于开发疫苗佐剂或抗肿瘤药物。
发明内容
本发明的目的是针对现有技术存在的不足,提供一种结构简单且高效的新型脂肽类TLR2激动剂,尤其涉及一种脂肽类化合物及其药用组合物作为TLR2激动剂用于发疫苗佐剂或抗肿瘤药物。
本发明的目的通过以下技术方案得以实现:
本发明提供了一类具有激活TLR2信号通路活性的化合物及其药学上可接受的盐或非对映异构体或其前药分子,其具有式(Ⅰ)结构式:
其中,
R1选自C1-C20直链或者支链烷基、含氧烷烃、含氮烷烃、含硫烷烃或H;
R2选自C1-C20直链或者支链烷基、含氧烷烃、含氮烷烃、含硫烷烃或H;
R3选自C1-C20直链或者支链烷基、含氧烷烃、含氮烷烃、含硫烷烃或H;
R4选自羟基,或赖氨酸、天冬氨酸、丙氨酸、天冬酰胺、甘氨酸中的一种或多种;
X、Y、Z选自C,NH,O。
优选地,所述的一类具有激活TLR2信号通路活性的化合物及其药学上可接受的盐或非对映异构体或其前药分子,其具有式(Ⅱ)结构:
R1、R2、R3如上所述;
X、Y、Z如上所述。
优选地,所述的一类具有激活TLR2信号通路活性的化合物及其药学上可接受的盐或非对映异构体或其前药分子,其具有式(Ⅲ)结构:
R1、R3如上所述;
X、Y、Z如上所述。
优选地,所述的一类具有激活TLR2信号通路活性的化合物及其药学上可接受的盐或非对映异构体或其前药分子,其具有式(IV)、式(V)或式(VI)所示的结构:
R3如上所述;
X、Y如上所述。
优选地,所述的一类具有激活TLR2信号通路活性的化合物及其药学上可接受的盐或非对映异构体或其前药分子,其具有式(VII)、式(VIII)、式(IX)或式(X) 所示的结构:
R3如上所述。
优选地,所述的一类具有激活TLR2信号通路活性的化合物及其药学上可接受的盐或非对映异构体或其前药分子,其包括下列化合物:
本发明的另一目的是提供一种疫苗佐剂或抗肿瘤药物的药用组合物。
具体的技术方案如下:
一种疫苗佐剂或抗肿瘤药物的药用组合物,其由上述脂肽类化合物或者其药学上可接受的盐或立体异构体或其前药分子与药学上可接受的载体组成。
本发明的另一目的是提供上述脂肽类化合物的应用。
具体的技术方案如下:
上述脂肽类化合物及其药学上可接受的盐或者立体异构体或其前药分子在制备激活TLR2及TLR2介导的信号通路下游核转录因子NFκB等表达的免疫佐剂或抗肿瘤药物中的应用。
本发明的突出效果为:
制备的脂肽类化合物能够选择性的靶向TLR2,有效激活核转录因子NFκB,对其他TLRs无作用。该类化合物具有很好的抗原提呈细胞活化效果和理化性质,尤其能够活化TLR2高表达的THP-1细胞和BV2细胞,与先导物Pam3CSK4活性相近,且化合物结构较Pam3CSK4更简单,分子量更小。
本发明为TLR2激动剂的研发提供了新的思路,具有发展成为临床使用的疫苗佐剂或抗肿瘤药物的潜力。
具体实施方式
下面结合具体实施例是对本发明做进一步说明。但该实施例并非用于限制本发明的保护范围。下述实施例中所述实验方法,如无特殊说明,均为常规试剂;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1
制备S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-棕榈酰基-L-半胱氨酰-D-丝氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-6-69) S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N-palmitoyl-L-cysteinyl-D-seryl-L-lysyl-L- lysyl-L-lysyl-L-lysine
步骤1:(2R,2'R)-3,3'-二硫烷二基二(2-((叔-丁氧基羰基)氨基)丙酸)(DXM-1-5) (2R,2'R)-3,3'-disulfanediylbis(2-((tert-butoxycarbonyl)amino)propanoicacid)
将L-胱氨酸(1.00g,4.16mmol),三乙胺(1.73mL,12.49mmol)依次加入水(15mL)中,冰浴冷却至0℃,缓慢加入二碳酸二叔丁酯(2.73g,12.49mmol),室温反应12h。 TLC监测反应结束,向反应液中加入乙酸乙酯(150mL),依次用1MHCl(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,得到白色固体(1.71g,收率93.2%);
1H NMR(400MHz,DMSO-d6)δ7.17–7.05(m,2H),4.19–4.13(m,2H),3.18–3.14(m,2H),2.87–2.82(m,2H),1.37(s,18H).ESI-MS:[M-H]-m/z 439.0.
步骤2:二苯甲基3,3'-二硫烷二基(2R,2'R)-二(2-((叔-丁氧基羰基)氨基)丙酸酯) (DXM-1-10)
dibenzyl 3,3'-disulfanediyl(2R,2'R)-bis(2-((tert-butoxycarbonyl)amino)propanoate)
将化合物DXM-1-5(1.82g,4.14mmol),无水碳酸钾(1.43g,10.4mmol),溴化苄(1.10 mL,9.15mmol)依次加入N,N-二甲基甲酰胺(20mL)中,室温反应10h。TLC监测反应结束,向反应液中加入乙酸乙酯(150mL),依次用水(20mL×2)、饱和氯化钠溶液 (20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=1:10)得到白色固体(2.14g,收率83.2%);
1H NMR(400MHz,CDCl3)δ7.35(s,10H),5.41–5.39(m,2H),5.17(s,4H),4.62–4.60(m,2H),3.16–3.09(m,4H),1.43(s,18H).ESI-MS:[M+Na]+m/z 643.2.
步骤3:苯甲基(tert-butoxycarbonyl)-L-半胱氨酸酸酯(DXM-1-15)
benzyl(tert-butoxycarbonyl)-L-cysteinate
N2保护下,将化合物DXM-1-10(0.05g,0.08mmol)溶解于四氢呋喃(1mL)中,缓慢加入三丁基膦(0.02mL,0.09mmol),室温反应0.5h,加入水(0.01mL,0.40mmol),室温反应1h。TLC监测反应结束,向反应液中加入乙酸乙酯(15mL),依次用水(2mL ×2)、饱和氯化钠溶液(2mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=1:10)得到白色固体(0.05g,收率95.1%);
1H NMR(400MHz,CDCl3)δ7.37(s,5H),5.42(s,1H),5.21(dd,J=32.4,12.2Hz,2H),4.64(d,J=6.2Hz,1H),3.06–2.88(m,2H),1.45(s,9H),1.29(t,J=8.7Hz,1H). ESI-MS:[M+Na]+m/z 333.8.
步骤4:苯甲基N-(叔-丁氧基羰基)-S-(2-羟基乙基)-L-半胱氨酸酸酯(DXM-5-6)
benzyl N-(tert-butoxycarbonyl)-S-(2-hydroxyethyl)-L-cysteinate
将化合物DXM-1-15(2.00g,6.43mmol),碳酸钾(1.33g,9.64mmol)依次加入无水丙酮(20mL)中,缓慢加入2-碘乙醇(0.60mL,7.72mmol),室温反应14h。TLC监测反应结束,向反应液中加入乙酸乙酯(150mL),依次用水(20mL×2)、饱和氯化钠溶液 (20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=6:10)得到黄色液体(2.10g,收率92.0%);
1H NMR(400MHz,CDCl3)δ7.35(s,5H),5.49(d,J=7.5Hz,1H),5.28(s,1H),5.18(q,J=12.3Hz,2H),4.60–4.54(m,1H),3.70–3.60(m,2H),3.00–2.91(m,2H),2.68– 2.62(m,2H),1.43(s,9H).ESI-MS:[M+Na]+m/z 378.2.
步骤5:苯甲基N-(叔-丁氧基羰基)-S-(2-((甲磺酰)氧代)乙基)-L-半胱氨酸酸酯(DXM-5-15)
benzyl N-(tert-butoxycarbonyl)-S-(2-((methylsulfonyl)oxy)ethyl)-L-cysteinate
将化合物DXM-5-6(2.00g,5.63mmol),DIPEA(4.85mL,28.17mmol)依次加入无水二氯甲烷(100mL)中,冰浴冷却至0℃,缓慢滴加甲基磺酰氯(2.18mL,28.17mmol),室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(150mL),依次用水(20 mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,得到黄色液体,粗品未经纯化直接进行下一步;
ESI-MS:[M+H]+m/z 434.1.
步骤6:苯甲基S-(2-叠氮乙基)-N-(叔-丁氧基羰基)-L-半胱氨酸酸酯(DXM-5-16)
benzyl S-(2-azidoethyl)-N-(tert-butoxycarbonyl)-L-cysteinate
将化合物DXM-5-15(0.50g,1.15mmol),叠氮化钠(0.38g,5.77mmol)依次加入N,N-二甲基甲酰胺(80mL)中,70℃反应8h。TLC监测反应结束,向反应液中加入乙酸乙酯(100mL),依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=1:10)得到黄色液体(0.35g,收率79.0%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),5.41(d,J=33.9Hz,1H),5.19(q,J=12.2Hz,2H),4.62–4.54(m,1H),3.42–3.32(m,2H),3.10–2.95(m,2H),2.68–2.59(m,2H), 1.44(s,9H).ESI-MS:[M+Na]+m/z 403.2.
步骤7:苯甲基S-(2-氨基乙基)-N-(叔-丁氧基羰基)-L-半胱氨酸酸酯(DXM-7-5)
benzyl S-(2-aminoethyl)-N-(tert-butoxycarbonyl)-L-cysteinate
N2保护下,将化合物DXM-5-16(0.70g,1.84mmol),三苯基膦(0.72g,2.75mmol) 依次加入四氢呋喃(20mL)中,室温反应2.5h,加入水(5mL),室温反应2.5h。TLC 监测反应结束,向反应液中加入乙酸乙酯(100mL),依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=50:1)得到白色固体(0.49g,收率75.1%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),5.63(d,J=33.1Hz,1H),5.18(q,J=12.2Hz,2H),4.63–4.50(m,1H),3.03–2.90(m,2H),2.83–2.75(m,2H),2.61–2.48(m,2H), 1.61–1.51(m,2H),1.43(s,9H).ESI-MS:[M+H]+m/z 355.2.
步骤8:苯甲基N-(叔-丁氧基羰基)-S-(2-(((十六烷氧基)羰基)氨基)乙基)-L-半胱氨酸酸酯(DXM-5-25)
benzyl N-(tert-butoxycarbonyl)-S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-L-cysteinate
将化合物DXM-7-5(1.00g,2.82mmol),DIPEA(1.15mL,7.05mmol)依次加入无水二氯甲烷(100mL)中,冰浴冷却至0℃,缓慢滴加氯甲酸十六烷基酯(1.85mL,5.64 mmol),室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(150mL),依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=1:10)得到白色固体(1.46g,收率83.2%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),5.38(d,J=7.8Hz,2H),5.19(q,J=12.1Hz,2H),5.11–5.03(m,1H),4.61–4.52(m,1H),4.03(t,J=6.1Hz,2H),3.34–3.22(m,2H),3.06–2.88(m,2H),2.65–2.54(m,2H),1.62(t,J=11.1Hz,4H),1.44(s,10H),1.25(s,26H),0.87(t,J=5.9Hz,3H).ESI-MS:[M+H]+m/z 623.4.
步骤9:苯甲基S-(2-(((十六烷氧基)羰基)氨基)乙基)-L-半胱氨酸酸酯(DXM-5-27)
benzyl S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-L-cysteinate
将化合物DXM-5-25(1.00g,1.61mmol),三氟乙酸(2mL)依次加入无水二氯甲烷(8mL) 中,室温反应2h。TLC监测反应结束,向反应液中加入二氯甲烷(150mL),依次用饱和碳酸氢钠溶液(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=4:10)得到黄色固体 (0.76g,收率90.2%);
1H NMR(400MHz,CDCl3)δ7.37(s,5H),5.17(s,2H),4.03(t,J=5.6Hz,2H),3.75–3.66(m,1H),3.38–3.24(m,2H),2.98–2.88(m,1H),2.87–2.77(m,1H),2.68–2.59 (m,2H),1.89–1.79(m,2H),1.64–1.53(m,2H),1.25(s,26H),0.87(t,J=5.7Hz,3H). ESI-MS:[M+H]+m/z 523.4.
步骤10:苯甲基S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酸酸酯(DXM-5-28)
benzyl S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N-palmitoyl-L-cysteinate
将化合物DXM-5-27(1.00g,1.91mmol),DIPEA(0.67mL,3.83mmol)依次加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,缓慢滴加棕榈酰氯(1.16mL,3.83mmol),室温反应5h。TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL ×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=200:1)得到白色固体(1.05g,收率71.9%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),6.40(d,J=7.0Hz,1H),5.19(q,J=12.1Hz,2H),5.01(t,J=5.8Hz,1H),4.91–4.83(m,1H),4.03(t,J=6.7Hz,2H),3.31–3.22(m, 2H),3.07–2.99(m,1H),2.99–2.91(m,1H),2.57(t,J=6.2Hz,2H),2.24(t,J=7.6Hz, 2H),1.67–1.55(m,4H),1.25(s,50H),0.87(t,J=6.7Hz,6H).ESI-MS:[M+H]+m/z 761.6.
步骤11:S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酸 (DXM-5-29)
S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N-palmitoyl-L-cysteine
将化合物DXM-5-28(1.00g,1.32mmol),氢氧化钯(0.37g,2.64mmol)依次加入甲醇(30mL)中,氢气置换内部气体,50℃反应1h。TLC监测反应结束,过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=20:1)得到白色固体(0.68g,收率 77.2%);
1H NMR(400MHz,CDCl3)δ6.99–6.79(m,1H),5.44–5.19(m,1H),4.80–4.61(m, 1H),4.17–3.90(m,2H),3.46–3.24(m,2H),3.18–2.95(m,2H),2.77–2.51(m,2H), 2.31–2.13(m,2H),1.61(s,4H),1.25(s,50H),0.96–0.76(m,6H).ESI-MS:[M-H]-m/z 669.5.
步骤12:苯甲基N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(叔-丁氧基羰基)-L-赖氨酸酸酯 (DXM-5-58)
benzyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysinate
将N-alpha-芴甲氧羰基-N-epsilon-叔丁氧羰基-L-赖氨酸(2.50g,5.34mmol),无水碳酸钾(0.90g,6.40mmol),溴化苄(0.76mL,6.40mmol)依次加入N,N-二甲基甲酰胺(100mL)中,室温反应12h。TLC监测反应结束,依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=60:1)得到白色固体(2.87g,收率96.2%);
1H NMR(400MHz,CDCl3)δ7.77(d,J=7.2Hz,2H),7.60(d,J=7.1Hz,2H),7.45–7.28(m,9H),5.44(d,J=8.1Hz,1H),5.18(q,J=12.0Hz,2H),4.56–4.49(m,1H),4.45 –4.34(m,2H),4.26–4.17(m,1H),3.11–2.98(m,2H),1.92–1.80(m,1H),1.77–1.62 (m,3H),1.43(s,9H),1.38–1.23(m,2H).ESI-MS:[M+H]+m/z 559.3.
步骤13:苯甲基N6-(叔-丁氧基羰基)-L-赖氨酸酸酯(DXM-5-58-2)
benzyl N6-(tert-butoxycarbonyl)-L-lysinate
将化合物DXM-5-58(1.00g,1.79mmol),二乙胺(0.93mL,8.96mmol)依次加入无水乙腈(50mL)中,室温反应2h。TLC监测反应结束,反应液减压浓缩,得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+Na]+m/z 359.2.
步骤14:苯甲基N2-(N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酸酸酯(DXM-5-60)
benzyl N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl)- N6-(tert-butoxycarbonyl)-L-lysinate
将N-alpha-芴甲氧羰基-N-epsilon-叔丁氧羰基-L-赖氨酸(1.00g,2.13mmol)加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,依次加入HATU(0.97g,2.56mmol)、DIPEA(0.45mL,2.56mmol),0℃反应0.5h,加入化合物DXM-5-58-2(0.86g,2.56mmol),室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL ×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=80:1)得到白色固体(1.40g,收率83.3%);
1H NMR(400MHz,CDCl3)δ7.62(d,J=7.2Hz,2H),7.48–7.42(m,2H),7.30–7.12 (m,10H),6.63–6.46(m,1H),5.54–5.38(m,1H),5.02(q,J=10.9Hz,2H),4.65–4.39 (m,3H),4.29–4.16(m,2H),4.12–4.00(m,2H),3.03–2.80(m,4H),1.78–1.66(m, 2H),1.60–1.46(m,2H),1.33–1.23(m,24H),1.19–1.05(m,2H).ESI-MS:[M+Na]+ m/z 809.4.
步骤15:苯甲基N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-L-赖氨酰)-L-赖氨酸酸酯(DXM-5-60-2)
benzyl N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-L-lysyl)-L-lysinate
将化合物DXM-5-60(1.00g,1.27mmol),二乙胺(0.66mL,6.36mmol)依次加入无水乙腈(50mL)中,室温反应2h。TLC监测反应结束,反应液减压浓缩,得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+Na]+m/z 587.4.
步骤16:苯甲基N2-(N2-(N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酸酸酯(DXM-5-61)
benzyl N2-(N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L- lysyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(tert-butoxycarbonyl)-L-lysinate
将N-alpha-芴甲氧羰基-N-epsilon-叔丁氧羰基-L-赖氨酸(1.00g,2.13mmol)加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,依次加入HATU(0.97g,2.56mmol)、DIPEA(0.45mL,2.56mmol),0℃反应0.5h,加入化合物DXM-5-60-2(1.44g,2.56mmol),室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL ×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=60:1)得到白色固体(1.75g,收率80.6%);
1H NMR(400MHz,CDCl3)δ7.85(d,J=7.2Hz,2H),7.74–7.64(m,2H),7.54–7.37 (m,4H),5.24(q,J=11.9Hz,2H),5.06–4.82(m,2H),4.71–4.60(m,1H),4.54–4.40 (m,2H),4.35–4.22(m,1H),3.30–2.98(m,6H),2.00–1.86(m,3H),1.84–1.71(m, 3H),1.62–1.54(m,6H),1.51(s,27H),1.47–1.37(m,6H).ESI-MS:[M+Na]+m/z 1037.6.
步骤17:苯甲基N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-L-赖氨酰)-L-赖氨酰)-L-赖氨酸酸酯(DXM-5-61-2)
benzyl N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert- butoxycarbonyl)-L-lysyl)-L-lysyl)-L-lysinate
将化合物DXM-5-61(1.00g,0.99mmol),二乙胺(0.51mL,4.93mmol)依次加入无水乙腈(50mL)中,室温反应2h。TLC监测反应结束,反应液减压浓缩,得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+Na]+m/z 815.5.
步骤18:苯甲基N2-(N2-(N2-(N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(叔-丁氧基羰基)-L- 赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酸酸酯(DXM-5-62)
benzyl N2-(N2-(N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl) -L-lysyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(tert-bu toxycarbonyl)-L-lysinate
将N-alpha-芴甲氧羰基-N-epsilon-叔丁氧羰基-L-赖氨酸(1.00g,2.13mmol)加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,依次加入HATU(0.97g,2.56mmol)、DIPEA(0.45mL,2.56mmol),0℃反应0.5h,加入化合物DXM-5-61-2(2.03g,2.56mmol),室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL ×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=50:1)得到白色固体(2.18g,收率82.1%);
1H NMR(400MHz,CDCl3)δ9.21–8.98(m,1H),8.87–8.70(m,1H),7.75(d,J=7.6 Hz,2H),7.60(d,J=8.7Hz,2H),7.44–7.22(m,10H),6.23–5.79(m,2H),5.25–5.04 (m,2H),4.86–4.59(m,2H),4.51–4.39(m,1H),4.39–4.29(m,3H),4.25–4.16(m, 1H),3.30–2.83(m,8H),2.07–1.65(m,8H),1.64–1.51(m,8H),1.46–1.39(m,36H), 1.30–1.20(m,8H).ESI-MS:[M+Na]+m/z 1265.7.
步骤19:苯甲基N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-L-赖氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酸酸酯(DXM-5-62-2)
benzyl N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert- butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-L-lysyl)-L-lysyl)-L-lysyl)-L-lysinate
将化合物DXM-5-62(1.00g,0.81mmol),二乙胺(0.42mL,4.02mmol)依次加入无水乙腈(50mL)中,室温反应2h。TLC监测反应结束,反应液减压浓缩,得白色固体,粗品未经纯化直接进行下一步、
ESI-MS:[M+Na]+m/z 1043.6.
步骤20:苯甲基N2-(N2-(N2-(N2-(N-(((9H-芴-9-基)甲氧基)羰基)-O-(叔-丁基)-D-丝氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酸酸酯(DXM-5-64)
Benzyl N2-(N2-(N2-(N2-(N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(tert-butyl)-D- seryl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(tert- butoxycarbonyl)-L-lysyl)-N6-(tert-butoxycarbonyl)-L-lysinate
将FMOC-O-叔丁基-L-丝氨酸(1.00g,2.61mmol)加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,依次加入HATU(1.19g,3.13mmol)、DIPEA(0.55mL,3.13mmol),0 ℃反应0.5h,加入化合物DXM-5-62-2(3.20g,3.13mmol),室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=60:1)得到白色固体(2.61g,收率72.1%)、
1H NMR(400MHz,CDCl3)δ7.70–7.62(m,2H),7.62–7.46(m,2H),7.33–7.17(m, 9H),6.01–5.95(m,1H),5.14–4.94(m,2H),4.86–4.43(m,3H),4.41–3.88(m,5H), 3.72–3.54(m,1H),3.51–3.28(m,1H),3.17–2.67(m,8H),1.73–1.49(m,8H),1.46– 1.27(m,44H),1.27–1.13(m,8H),1.08(s,9H).ESI-MS:[M+Na]+m/z 1409.6,.
步骤21:苯甲基N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(O-(叔-丁基)-D-丝氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酸酸酯(DXM-5-64-2)
benzyl N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert- butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(O-(tert-butyl)-D-seryl)-L-lysyl)-L- lysyl)-L-lysyl)-L-lysinate
将化合物DXM-5-64(1.00g,0.72mmol),二乙胺(0.38mL,3.61mmol)依次加入无水乙腈(50mL)中,室温反应2h。TLC监测反应结束,反应液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=10:1)得到黄色固体(0.57g,收率68.1%);
1H NMR(400MHz,CDCl3)δ7.40–7.29(m,2H),5.20–5.04(m,1H),5.01–4.26(m, 4H),3.63–3.39(m,4H),3.27–2.79(m,4H),2.04–1.88(m,2H),1.88–1.75(m,2H), 1.71–1.57(m,4H),1.40(s,52H),1.15(s,9H).
ESI-MS:[M+Na]+m/z 1186.7.
步骤22:苯甲基N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-O-(叔-丁基)-N-(S-(2-(((十六烷氧基)羰基)氨基)乙基)-N- 棕榈酰<十六烷酰>-L-半胱氨酰)-D-丝氨酰-L-赖氨酰-L-赖氨酰)-L-赖氨酰)-L-赖氨酸酸酯(DXM-6-65)
benzyl N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2 -(N6-(tert-butoxycarbonyl)-O-(tert-butyl)-N-(S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N- palmitoyl-L-cysteinyl)-D-seryl-L-lysyl-L-lysyl)-L-lysyl)-L-lysinate
将化合物DXM-5-29(0.02g,0.03mmol)加入无水二氯甲烷(10mL)中,冰浴冷却至0℃,依次加入HOBT(0.005g,0.04mmol)、EDCI(0.007g,0.04mmol)、DIPEA(0.005 mL,0.04mmol),0℃反应1h。加入化合物5-64-2(0.03g,0.03mmol),室温反应12 h。TLC监测反应结束,向反应液中加入二氯甲烷(50mL),依次用水(10mL×2)、饱和氯化钠溶液(10mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=60:1)得到白色固体(0.02g,收率52.1%);
1H NMR(400MHz,CDCl3)δ7.34(d,J=3.9Hz,5H),5.21–5.08(m,2H),4.97–4.74 (m,1H),4.57–4.21(m,3H),4.18–3.92(m,3H),3.88–3.57(m,1H),3.50–3.25(m, 2H),3.22–2.86(m,8H),2.81–2.61(m,2H),2.36–2.15(m,2H),2.09–1.94(m,4H), 1.86–1.72(m,4H),1.64–1.55(m,4H),1.54–1.31(m,52H),1.24(s,50H),1.16(s,9H). ESI-MS:[M+2Na]2+m/z916.8.
步骤23:N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)- N2-(N6-(叔-丁氧基羰基)-O-(叔-丁基)-N-(S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰)-D-丝氨酰-L-赖氨酰-L-赖氨酰)-L-赖氨酰)-L-赖氨酸 (DXM-6-67)
N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)- N2-(N6-(tert-butoxycarbonyl)-O-(tert-butyl)-N-(S-(2-(((hexadecyloxy)carbonyl)amino)eth yl)-N-palmitoyl-L-cysteinyl)-D-seryl-L-lysyl-L-lysyl)-L-lysyl)-L-lysine
将化合物DXM-6-65(0.02g,0.01mmol),氢氧化钯(0.002g,0.02mmol)依次加入甲醇(5mL)中,氢气置换内部气体,50℃反应1h。TLC监测反应结束,过滤,滤液减压浓缩,得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+2H]2+m/z 814.4.
步骤24:S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰-D- 丝氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-6-69)
S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N-palmitoyl-L-cysteinyl-D-seryl-L-lysyl-L-l ysyl-L-lysyl-L-lysine
将化合物DXM-6-67(0.02g,0.02mmol)加入无水二氯甲烷(10mL)中,冰浴冷却至0℃,缓慢滴加1M氯化氢-二氧六环(1mL),0℃反应2h。TLC监测反应结束,反应液减压浓缩,HPLC纯化(流动相A:甲醇,流动相B:水+0.1%甲酸),得白色固体(0.004 g,收率27.2%)。1HNMR(600MHz,CD3OD)δ5.34(t,J=4.7Hz,1H),4.55–4.46(m, 1H),4.43–4.29(m,5H),4.02(t,J=6.3Hz,2H),4.00–3.93(m,1H),3.86–3.78(m, 1H),3.62–3.60(m,2H),3.07–3.01(m,1H),2.99–2.95(m,8H),2.90–2.83(m,1H), 2.72–2.64(m,2H),1.96–1.93(m,2H),1.85–1.75(m,4H),1.74–1.69(m,8H),1.65– 1.59(m,4H),1.55–1.49(m,6H),1.41–1.35(m,8H),1.31–1.27(m,48H),0.90(t,J= 7.0Hz,6H).13C NMR(150MHz,CD3OD)δ174.94,174.43,174.30,174.09,174.00, 159.35,66.09,64.21,62.69,55.86,54.99,54.77,54.54,53.18,40.62,40.57,36.88,33.05, 32.16,31.76,30.79,30.76,30.70,30.44,28.08,27.93,27.89,27.79,27.00,26.90,23.71, 14.43.ESI-MS:[M+2H]2+m/z 636.0.HRMS(ESI):m/zCalcd for C65H127N11O11S [M+H]+1270.9510,found 1270.9511.
实施例2
制备N-乙酰基-S-(2-(((十六烷氧基)羰基)氨基)乙基)-L-半胱氨酰-D-丝氨酰-L-赖氨酰 -L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-7-40)
N-acetyl-S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-L-cysteinyl-D-seryl-L-lysyl-L-lysyl -L-lysyl-L-lysine
合成方法如实施例1。
ESI-MS:[M+2H]2+m/z 538.2.
实施例3
N-棕榈酰<十六烷酰>-S-(2-(((十四烷氧基)羰基)氨基)乙基)-L-半胱氨酰-D-丝氨酰-L- 赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-7-49)
N-palmitoyl-S-(2-(((tetradecyloxy)carbonyl)amino)ethyl)-L-cysteinyl-D-seryl-L-lysyl-L-ly syl-L-lysyl-L-lysine
合成方法如实施例1。
白色固体,收率51.0%。1H NMR(600MHz,DMSO-d6)δ12.74–12.28(m,1H),8.15–8.08(m,14H),7.17–7.07(m,1H),5.21–5.11(m,1H),4.50–4.42(m,1H),4.31–4.19 (m,4H),4.17–4.14(m,1H),3.89(t,J=6.1Hz,2H),3.69–3.62(m,1H),3.59–3.53(m, 1H),3.15–3.08(m,2H),2.90–2.82(m,1H),2.79–2.69(m,10H),2.57(t,J=6.7Hz, 2H),2.18–2.07(m,2H),1.72–1.65(m,4H),1.62–1.52(m,14H),1.51–1.45(m,4H), 1.39–1.32(m,8H),1.23–1.21(m,44H),0.84(t,J=6.8Hz,6H).13C NMR(150MHz, DMSO-d6)δ173.25,172.75,172.55,171.49,171.33,170.15,169.92,156.26,63.69,62.78, 61.59,55.20,52.52,52.17,51.66,38.41,35.23,35.09,31.26,31.11,30.86,30.14,29.03, 28.99,28.84,28.68,28.54,27.16,26.52,26.37,25.34,25.08,22.06,13.90.ESI-MS: [M+2H]2+m/z 622.0.HRMS(ESI):m/z Calcd for C63H123N11O11S[M+H]+1242.9197, found 1242.9206.
实施例4
N-棕榈酰<十六烷酰>-S-(2-(棕榈酰<十六烷酰>氧代)乙基)-L-半胱氨酰-D-丝氨酰-L- 赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-5-70)
N-palmitoyl-S-(2-(palmitoyloxy)ethyl)-L-cysteinyl-D-seryl-L-lysyl-L-lysyl-L-lysyl-L -lysine
合成方法如实施例1。
白色固体,收率52.3%。1H NMR(600MHz,CD3OD)δ5.37–5.31(m,1H),4.45–4.29 (m,5H),4.26–4.23(m,1H),4.01–3.93(m,1H),3.87–3.78(m,1H),3.63–3.59(m, 2H),3.09–3.03(m,1H),3.01–2.93(m,9H),2.89–2.83(m,2H),2.36–2.30(m,2H), 1.98–1.92(m,2H),1.82–1.67(m,12H),1.64–1.58(m,4H),1.55–1.48(m,6H),1.33 –1.26(m,56H),0.92–0.88(m,6H).13C NMR(150MHz,CD3OD)δ175.31,174.94, 174.37,174.08,174.00,173.63,64.40,64.20,62.75,57.34,54.92,54.76,54.54,53.18, 40.62,40.56,36.88,35.04,33.05,31.77,30.77,30.45,30.23,27.90,26.92,26.05,23.71, 14.43.ESI-MS:[M+2H]2+m/z621.6.HRMS(ESI):m/z Calcd for C64H124N10O11S [M+H]+1241.9245,found 1241.9236.
实施例5
S-(2-棕榈酰氨基乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰-D-丝氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-8-9)
S-(2-palmitamidoethyl)-N-palmitoyl-L-cysteinyl-D-seryl-L-lysyl-L-lysyl-L-lysyl-L-lysine
合成方法如实施例1。
白色固体,收率49.9%.1H NMR(600MHz,CD3OD)δ5.34(t,J=4.8Hz,1H),4.55–4.47(m,1H),4.45–4.27(m,5H),3.75–3.72(m,2H),3.62–3.60(m,2H),3.26–3.22 (m,2H),3.07–2.85(m,10H),2.75–2.65(m,2H),2.37–2.25(m,2H),2.21(t,J=6.4 Hz,2H),1.97–1.88(m,4H),1.84–1.76(m,4H),1.73–1.71(m,4H),1.57–1.48(m, 8H),1.40–1.38(m,8H),1.33–1.26(m,48H),0.90(t,J=6.9Hz,6H).13C NMR(150 MHz,CD3OD)δ176.63,176.51,174.91,174.37,174.10,173.98,173.78,64.22,62.72, 57.50,55.89,55.00,54.78,54.54,53.19,43.94,40.63,39.86,37.20,36.87,33.03,32.14, 31.75,30.76,30.43,27.90,27.05,26.91,23.79,23.69,18.84,17.42,14.42.ESI-MS: [M+2H]2+m/z 621.0,[M+H]+m/z 1241.6.HRMS(ESI):m/z Calcd for C64H125N11O10S[M+2H]2+620.9739,found620.9711.
实施例6
S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰-D-丝氨酰-L- 赖氨酰-L-赖氨酰-L-赖氨酸(DXM-8-39)
S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N-palmitoyl-L-cysteinyl-D-seryl-L-lysyl-L- lysyl-L-lysine
合成方法如实施例1。
白色固体,收率42.2%。1H NMR(600MHz,CD3OD)δ5.33(t,J=4.7Hz,1H),4.49–4.44(m,1H),4.43–4.28(m,4H),4.08–3.98(m,2H),3.97–3.90(m,1H),3.85–3.77 (m,1H),3.65–3.62(m,2H),3.02–2.92(m,7H),2.88–2.83(m,1H),2.73–2.63(m, 2H),2.31–2.20(m,2H),1.97–1.87(m,3H),1.75–1.66(m,7H),1.63–1.58(m,4H), 1.54–1.49(m,4H),1.31–1.27(m,54H),0.89(t,J=6.8Hz,6H).13C NMR(150MHz, CD3OD)δ176.72,174.91,174.29,174.22,174.09,173.96,173.63,159.29,66.07,64.12, 62.73,57.44,57.30,55.07,54.92,54.76,54.53,41.31,40.78,40.74,36.87,33.01,32.03, 31.86,31.77,30.76,30.72,30.52,30.40,30.25,30.20,27.97,27.84,26.97,26.89,23.67, 14.40.ESI-MS:[M+2H]2+m/z 572.0.HRMS(ESI):m/z Calcd for C59H115N9O10S [M+2H]2+571.9317,found571.9292.
实施例7
N-棕榈酰<十六烷酰>-S-(2-(((戊氧基)羰基)氨基)乙基)-L-半胱氨酰-D-丝氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-7-65)
N-palmitoyl-S-(2-(((pentyloxy)carbonyl)amino)ethyl)-L-cysteinyl-D-seryl-L-lysyl-L-lysyl- L-lysyl-L-lysine
合成方法如实施例1。
白色固体,收率46.2%。1H NMR(600MHz,CD3OD)δ5.33(t,1H),4.56–4.47(m,1H),4.45–4.35(m,4H),4.33–4.30(m,1H),4.10–3.92(m,3H),3.89–3.79(m,1H),3.67– 3.61(m,2H),3.02–2.94(m,9H),2.89–2.83(m,1H),2.74–2.64(m,2H),2.36–2.21 (m,2H),1.96–1.87(m,4H),1.85–1.77(m,4H),1.76–1.70(m,8H),1.57–1.49(m, 8H),1.33–1.25(m,32H),0.92–0.87(m,6H).13C NMR(150MHz,CD3OD)δ176.84, 176.65,174.88,174.32,174.06,173.97,159.17,66.01,63.99,62.66,57.38,55.33,55.00, 54.79,54.50,53.20,41.28,40.61,40.53,36.80,32.95,32.07,31.64,30.69,30.43,30.34, 29.81,29.10,27.81,26.84,23.72,23.62,23.33,14.39.ESI-MS:[M+2H]2+m/z 559.0. HRMS(ESI):m/z Calcdfor C54H105N11O11S[M+H]+1116.7789,found 1116.7782.
实施例8
S-(2-(3-十六烷基脲基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰-D-丝氨酰-L-赖氨酰 -L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-8-10)
S-(2-(3-hexadecylureido)ethyl)-N-palmitoyl-L-cysteinyl-D-seryl-L-lysyl-L-lysyl-L-lysyl-L -lysine
步骤1:十六烷基氨基甲酰氯(DXM-7-69-1)
hexadecylcarbamic chloride
将固体光气(0.14g,0.47mmol)加入无水甲苯(15mL)中,缓慢滴加十六胺(0.23g,0.95 mmol)的甲苯(15mL)溶液,110℃反应6h。减压蒸除溶剂,得黄色液体,粗品未经纯化,溶于二氯甲烷(15mL)中备用;
步骤2:苯甲基N-(叔-丁氧基羰基)-S-(2-(3-十六烷基脲基)乙基)-L-半胱氨酸酸酯 (DXM-7-69)
benzyl N-(tert-butoxycarbonyl)-S-(2-(3-hexadecylureido)ethyl)-L-cysteinate
将DXM-7-5(0.40g,1.13mmol)、三乙胺(0.66mL,4.75mmol)依次加入无水二氯甲烷(30mL)中,冰浴冷却至0℃,滴加上述备液,室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(50mL),依次用水(10mL×2)、饱和氯化钠溶液(10mL ×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=100:1)得到黄色固体(0.43g,收率72.1%);
1H NMR(400MHz,CDCl 3)δ7.36(s,5H),5.37(d,J=8.3Hz,1H),5.22(s,1H),5.20–5.15(m,2H),4.70–4.63(m,1H),4.57–4.48(m,1H),3.49–3.33(m,1H),3.33–3.20 (m,1H),3.19–3.07(m,2H),3.02–2.89(m,1H),2.89–2.79(m,1H),2.78–2.66(m, 1H),2.65–2.48(m,1H),1.76(s,2H),1.44(s,9H),1.24(s,26H),0.87(t,J=6.6Hz,3H). ESI-MS:[M+H]+m/z622.0.
步骤3:苯甲基S-(2-(3-十六烷基脲基)乙基)-L-半胱氨酸酸酯(DXM-7-72-1)
benzyl S-(2-(3-hexadecylureido)ethyl)-L-cysteinate
将化合物DXM-7-69(1.00g,1.61mmol),三氟乙酸(2mL)依次加入无水二氯甲烷(20mL)中,室温反应2h。TLC监测反应结束,向反应液中加入二氯甲烷(50mL),依次用饱和碳酸氢钠溶液(10mL×2)、饱和氯化钠溶液(10mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品未经纯化直接进行下一步;
ESI-MS:[M+H]+m/z 522.0.
步骤4:苯甲基S-(2-(3-十六烷基脲基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酸酸酯 (DXM-7-72)
benzyl S-(2-(3-hexadecylureido)ethyl)-N-palmitoyl-L-cysteinate
将化合物DXM-7-72-1(1.00g,1.92mmol),DIPEA(0.67mL,3.84mmol)依次加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,缓慢滴加棕榈酰氯(1.16mL,3.84mmol),室温反应4h。TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20 mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=300:1)得到白色固体(1.19g,收率81.6%);1H NMR(400MHz,CDCl3)δ7.36(s,5H),6.52(s,1H),5.37(s,1H),5.25–5.13(m,2H), 5.04–4.89(m,1H),4.88–4.77(m,1H),3.44–3.33(m,1H),3.31–3.21(m,1H),3.18– 3.06(m,2H),3.03–2.92(m,1H),2.90–2.81(m,1H),2.75–2.67(m,1H),2.62–2.52 (m,1H),2.26(t,J=7.6Hz,2H),1.66–1.58(m,2H),1.49–1.42(m,2H),1.24(s,50H), 0.87(t,J=6.3Hz,6H).ESI-MS:[M+H]+m/z 760.6.
步骤5:S-(2-(3-十六烷基脲基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酸(DXM-7-79) S-(2-(3-hexadecylureido)ethyl)-N-palmitoyl-L-cysteine
将化合物DXM-7-72(0.50g,0.66mmol),氢氧化钯(0.19g,1.32mmol)依次加入甲醇(30mL)中,氢气置换内部气体,50℃反应1h。TLC监测反应结束,过滤,滤液减压浓缩得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+H]+m/z 670.4.
步骤6:苯甲基N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(O-(叔-丁基)-N-(S-(2-(3-十六烷基脲基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰)-D-丝氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酸酸酯 (DXM-8-2)
benzyl N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycar -bonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(O-(tert-butyl)-N-(S-(2-(3-hexadecylureido )ethyl)-N-palmitoyl-L-cysteinyl)-D-seryl)-L-lysyl)-L-lysyl)-L-lysyl)-L-lysinate
将化合物DXM-7-79(0.02g,0.03mmol)加入无水二氯甲烷(10mL)中,冰浴冷却至0℃,依次加入HOBT(0.005g,0.04mmol)、EDCI(0.007g,0.04mmol)、DIPEA(0.005 mL,0.04mmol),0℃反应1h。加入化合物5-64-2(0.03g,0.03mmol),室温反应12 h。TLC监测反应结束,向反应液中加入二氯甲烷(50mL),依次用水(10mL×2)、饱和氯化钠溶液(10mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=60:1)得到白色固体(0.03g,收率47.2%);
1H NMR(400MHz,CDCl3)δ7.34(s,5H),5.15(q,J=12.2Hz,2H),5.09–4.29(m,4H),4.27–3.32(m,4H),3.24–2.94(m,8H),2.92–2.05(m,6H),2.05–1.82(m,10H),1.83 –1.55(m,10H),1.46–1.38(m,38H),1.30–1.20(m,50H),1.17(s,9H),0.86(t,J=6.7 Hz,6H).ESI-MS:[M-2Boc+2H]2+m/z 808.8.
步骤7:N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(N6-(叔-丁氧基羰基)-N2-(O-(叔-丁基)-N-(S-(2-(3-十六烷基脲基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰)-D-丝氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酰)-L-赖氨酸(DXM-8-8)
N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2 -(N6-(tert-butoxycarbonyl)-N2-(O-(tert-butyl)-N-(S-(2-(3-hexadecylureido)ethyl)-N-palmi toyl-L-cysteinyl)-D-seryl)-L-lysyl)-L-lysyl)-L-lysyl)-L-lysine
将化合物DXM-8-2(0.02g,0.01mmol),氢氧化钯(0.002g,0.02mmol)依次加入甲醇(5mL)中,氢气置换内部气体,50℃反应1h。TLC监测反应结束,过滤,滤液减压浓缩,得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+2H]2+m/z 863.8.
步骤8:S-(2-(3-十六烷基脲基)乙基)-N-棕榈酰<十六烷酰>-L-半胱氨酰-D-丝氨酰-L- 赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-8-10)
S-(2-(3-hexadecylureido)ethyl)-N-palmitoyl-L-cysteinyl-D-seryl-L-lysyl-L-lysyl-L-lysyl-L -lysine
将化合物DXM-8-8(0.02g,0.01mmol)加入无水二氯甲烷(10mL)中,冰浴冷却至0℃,缓慢滴加1M氯化氢-二氧六环(1mL),0℃反应2h。TLC监测反应结束,反应液减压浓缩,HPLC纯化(流动相A:甲醇,流动相B:水+0.1%甲酸),得白色固体(0.004g,收率32.7%)。1HNMR(600MHz,CD3OD)δ5.33(t,J=4.8Hz,1H),4.51–4.44(m,1H), 4.42–4.35(m,4H),4.34–4.29(m,1H),3.77–3.72(m,2H),3.67–3.61(m,2H),3.25– 3.22(m,2H),3.11(t,J=6.5Hz,2H),3.01–2.96(m,8H),2.79–2.65(m,2H),1.97– 1.85(m,6H),1.76–1.71(m,8H),1.54–1.48(m,8H),1.41–1.38(m,20H),1.32–1.25 (m,38H),0.89(t,J=7.0Hz,6H).13C NMR(150MHz,CD3OD)δ174.88,174.31,174.17, 174.07,173.98,172.90,171.57,160.92,63.96,62.76,62.68,57.13,55.81,55.11,54.79, 54.49,53.23,43.89,41.20,40.66,40.56,40.49,32.93,31.61,31.11,30.65,30.43,30.33, 30.22,30.19,28.00,27.90,27.78,23.72,23.61,23.54,14.40.ESI-MS:[M+2H]2+m/z 635.9,[M+H]+m/z 1269.9.HRMS(ESI):m/z Calcd for C65H128N12O10S[M+2H]2+ 635.4871,found 635.4876.
实施例9
S-(2-(((十六烷氧基)羰基)氨基)乙基)-N-(十四烷基氨基甲酰)-L-半胱氨酰-D-丝氨酰-L- 赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-7-91)
S-(2-(((hexadecyloxy)carbonyl)amino)ethyl)-N-(tetradecylcarbamoyl)-L-cysteinyl-D-seryl -L-lysyl-L-lysyl-L-lysyl-L-lysine
合成方法如实施例8。
白色固体,收率47.3%。1H NMR(600MHz,CD3OD)δ5.36–5.31(m,1H),4.44–4.27 (m,6H),4.05–3.96(m,2H),3.88–3.81(m,1H),3.77–3.71(m,1H),3.64–3.60(m, 4H),3.17–3.05(m,1H),3.01–2.93(m,10H),2.76–2.64(m,1H),1.84–1.77(m,4H), 1.74–1.68(m,8H),1.56–1.47(m,10H),1.38–1.22(m,54H),0.92–0.86(m,6H).13C NMR(150MHz,CD3OD)δ174.94,174.48,174.33,174.17,174.04,159.32,159.20,66.11, 64.12,57.44,54.97,54.55,53.17,52.49,41.18,40.59,33.02,32.04,31.73,31.21,30.74, 30.56,30.41,30.27,30.20,28.06,27.90,27.79,26.98,23.77,23.68,14.41.ESI-MS: [M+2H]2+m/z636.6.HRMS(ESI):m/z Calcd for C64H126N12O11S[M+H]+1271.9463, found 1271.9457.
实施例10
N-乙酰基-S-((R)-2,3-二(棕榈酰<十六烷酰>氧代)丙基)-L-半胱氨酰-D-丝氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-6-68)
N-acetyl-S-((R)-2,3-bis(palmitoyloxy)propyl)-L-cysteinyl-D-seryl-L-lysyl-L-lysyl-L-lysyl- L-lysine
步骤1:(R)-4-(碘甲基)-2,2-二甲基-1,3-二噁戊环(DXM-1-53)
(R)-4-(iodomethyl)-2,2-dimethyl-1,3-dioxolane
将(S)-甘油醇缩丙酮(0.50g,3.78mmol),三苯基膦(1.20g,4.57mmol),碘单质(1.25 g,4.92mmol),咪唑(0.77g,11.30mmol),依次加入甲苯(20ml)中,90℃反应3h。 TLC检测反应结束,减压蒸除溶剂,向反应液中加入二氯甲烷(150mL),依次用饱和硫代硫酸钠溶液(30mL×2)、饱和氯化钠溶液(30mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷)得到黄色固体(0.75g,收率 82.1%);
1H NMR(400MHz,CDCl3)δ4.33–4.22(m,1H),4.18–4.10(m,1H),3.83–3.74(m, 1H),3.29–3.21(m,1H),3.18–3.10(m,1H),1.46(s,3H),1.35(s,3H).ESI-MS:[M+H]+ m/z242.6.
步骤2:苯甲基N-(叔-丁氧基羰基)-S-(((R)-2,2-二甲基-1,3-二噁戊环-4-基)甲基)-L-半胱氨酸酸酯(DXM-5-2)
benzyl N-(tert-butoxycarbonyl)-S-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl)- L-cysteinate
将化合物DXM-1-18(1.00g,3.21mmol)加入无水N,N-二甲基甲酰胺(20mL)中,冰浴冷却至0℃,缓慢加入NaH(0.15g,3.86mmol),0℃反应0.5h,缓慢加入化合物 DXM-1-53(0.82g,3.38mmol),室温反应1h。TLC检测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=1:10) 得到白色固体(1.00g,收率73.5%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),5.53–5.42(m,1H),5.19(q,J=12.7Hz,2H),4.62–4.52(m,1H),4.25–4.14(m,1H),4.08–3.99(m,1H),3.63(s,1H),3.14–2.94(m, 2H),2.76–2.65(m,1H),2.64–2.55(m,1H),1.44(s,9H),1.40(s,3H),1.34(s,3H).
ESI-MS:[M+Na]+m/z 448.0.
步骤3:苯甲基N-(叔-丁氧基羰基)-S-((R)-2,3-二羟基丙基)-L-半胱氨酸酸酯(DXM-1-58)
benzyl N-(tert-butoxycarbonyl)-S-((R)-2,3-dihydroxypropyl)-L-cysteinate
将化合物DXM-5-2(0.54g,1.27mmol),冰醋酸(7mL)依次加入水(3mL)中,室温反应12h。TLC监测反应结束,向反应液中加入二氯甲烷(150mL),依次用饱和碳酸氢钠溶液(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=15:10)得到无色液体(0.32g,收率65.3%);
1H NMR(400MHz,CDCl3)δ7.35(s,5H),5.61–5.50(m,1H),5.17(q,2H),4.61–4.52(m,1H),3.79–3.70(m,1H),3.70–3.65(m,1H),3.65–3.59(m,1H),3.55–3.45(m, 1H),3.06–2.88(m,3H),2.73–2.62(m,1H),2.62–2.52(m,1H),1.43(s,9H).ESI-MS: [M+H]+m/z386.2.
步骤4:(R)-3-(((R)-3-(苄氧基)-2-((叔-丁氧基羰基)氨基)-3-羰基丙基)硫代)丙烷-1,2-二基二棕榈酸酯(DXM-5-3)
(R)-3-(((R)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)thio)propane-1,2-d iyl dipalmitate
将化合物DXM-1-58(0.03g,0.07mmol)加入无水二氯甲烷(20mL)中,冰浴冷却至0℃,依次加入棕榈酸(0.07g,0.27mmol),EDCI(0.05g,0.27mmol),DMAP(0.03g, 0.27mmol),室温反应12h,TLC检测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL×2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=1:7)得到白色固体(0.04g,收率72.0%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),5.41–5.32(m,1H),5.23–5.14(m,2H),5.14 –5.05(m,1H),4.63–4.52(m,1H),4.34–4.22(m,1H),4.18–4.04(m,1H),3.11–2.93 (m,2H),2.72–2.62(m,2H),2.33–2.25(m,4H),1.64–1.55(m,4H),1.44(s,9H),1.25 (s,48H),0.91–0.83(m,6H).ESI-MS:[M+Na]+m/z 884.6.
步骤5:(R)-3-(((R)-2-氨基-3-(苄氧基)-3-羰基丙基)硫代)丙烷-1,2-二基二棕榈酸酯 (DXM-5-5)
(R)-3-(((R)-2-amino-3-(benzyloxy)-3-oxopropyl)thio)propane-1,2-diyldipalmitate
将化合物DXM-5-3(0.04g,0.05mmol),三氟乙酸(1mL)加入无水二氯甲烷(4mL)中,室温反应2h。TLC监测反应结束,向反应液中加入二氯甲烷(20mL),依次用饱和碳酸氢钠溶液(5mL×2)、饱和氯化钠溶液(5mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=4:10)得到白色固体(0.04g,收率90.0%);
1H NMR(400MHz,CDCl3)δ7.43–7.31(m,1H),5.22–5.15(m,2H),5.14–5.09(m, 1H),4.38–4.27(m,1H),4.15(s,1H),3.74–3.68(m,1H),3.02–2.93(m,1H),2.89– 2.80(m,1H),2.75–2.69(m,2H),2.34–2.25(m,4H),1.64–1.55(m,4H),1.25(s,48H), 0.88(t,J=6.0Hz,6H).ESI-MS:[M+H]+m/z 762.6.
步骤6:(R)-3-(((R)-2-乙酰氨基-3-(苄氧基)-3-羰基丙基)硫代)丙烷-1,2-二基二棕榈酸酯(DXM-5-10)
(R)-3-(((R)-2-acetamido-3-(benzyloxy)-3-oxopropyl)thio)propane-1,2-diyl dipalmitate
将化合物DXM-5-5(1.00g,1.31mmol),DIPEA(0.46mL,2.62mmol)依次加入无水二氯甲烷(50mL)中,冰浴冷却至0℃,缓慢滴加乙酰氯(0.19mL,2.62mmol),室温反应6h,TLC监测反应结束,向反应液中加入二氯甲烷(100mL),依次用水(20mL× 2)、饱和氯化钠溶液(20mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相乙酸乙酯:石油醚=5:10)得到白色固体(0.88g,收率82.9%);
1H NMR(400MHz,CDCl3)δ7.36(s,5H),6.50–6.38(m,1H),5.22–5.16(m,2H),5.13 –5.06(m,1H),4.91–4.83(m,1H),4.34–4.22(m,1H),4.11–4.03(m,1H),3.08–3.02 (m,2H),2.68–2.61(m,2H),2.33–2.25(m,5H),2.05(s,3H),1.64–1.54(m,4H),1.25 (s,48H),0.87(t,J=6.2Hz,6H).ESI-MS:[M+H]+m/z 804.6.
步骤7:N-乙酰基-S-((R)-2,3-二(棕榈酰<十六烷酰>氧代)丙基)-L-半胱氨酸(DXM-5-12)
N-acetyl-S-((R)-2,3-bis(palmitoyloxy)propyl)-L-cysteine
将化合物DXM-5-10(1.00g,1.24mmol),氢氧化钯(0.35g,2.48mmol)依次加入甲醇(30mL)中,氢气置换内部气体,50℃反应1h。TLC监测反应结束,过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=20:1)得到白色固体(0.63g,收率 70.4%);
1H NMR(400MHz,cdcl3)δ5.22–5.10(m,4H),4.73–4.49(m,1H),4.42–4.31(m,1H),4.18–4.06(m,1H),3.15–2.97(m,2H),2.79–2.66(m,2H),2.36–2.25(m,4H),2.06(s, 3H),1.59(s,4H),1.25(s,48H),0.87(t,J=6.4Hz,6H).ESI-MS:[M+H]+m/z 714.5.
步骤8:10S,13S,16S,19S,22R,25R,29R)-25-乙酰氨基-10-((苄氧基)羰基)-10,13,16,19- 四(4-((叔-丁氧基羰基)氨基)丁基)-22-(叔-丁氧基甲基)-2,2-二甲基 -4,12,15,18,21,24-六羰基-3-氧杂-27-硫杂-5,11,14,17,20,23-六氮杂三十烷 -29,30-二基二棕榈酸酯(DXM-6-21)
(10S,13S,16S,19S,22R,25R,29R)-25-acetamido-10-((benzyloxy)carbonyl)-10,13,16,19-tetr a(4-((tert-butoxycarbonyl)amino)butyl)-22-(tert-butoxymethyl)-2,2-dimethyl-4,12,15,18,2 1,24-hexaoxo-3-oxa-27-thia-5,11,14,17,20,23-hexaazatriacontane-29,30-diyl dipalmitate
将化合物DXM-5-12(0.02g,0.03mmol)加入无水二氯甲烷(10mL)中,冰浴冷却至0℃,依次加入HOBT(0.005g,0.04mmol)、EDCI(0.007g,0.04mmol)、DIPEA(0.005 mL,0.04mmol),0℃反应1h。加入化合物5-64-2(0.03g,0.03mmol),室温反应12 h。TLC监测反应结束,向反应液中加入二氯甲烷(50mL),依次用水(10mL×2)、饱和氯化钠溶液(10mL×2)洗涤,无水硫酸钠干燥后过滤,滤液减压浓缩,粗品柱层析纯化(流动相二氯甲烷:甲醇=50:1)得到白色固体(0.03g,收率49.7%);
1H NMR(400MHz,CDCl3)δ7.33(s,5H),5.21–5.10(m,2H),5.09–4.95(m,2H),4.92 –4.66(m,1H),4.53–4.29(m,3H),4.28–4.10(m,3H),4.11–3.93(m,1H),3.90–3.72 (m,1H),3.72–3.55(m,1H),3.39–2.96(m,8H),2.94–2.50(m,4H),2.38–2.20(m, 4H),2.00(s,3H),1.94–1.70(m,8H),1.63–1.54(m,4H),1.52–1.29(m,52H),1.24(s, 48H),1.13(s,9H),0.85(t,J=6.7Hz,6H).ESI-MS:[M+2H]2+m/z 930.6.
步骤9:N2-(N2-(N-(N-乙酰基-S-((R)-2,3-二(棕榈酰<十六烷酰>氧代)丙基)-L-半胱氨酰)-O-(叔-丁基)-D-丝氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酰)-N6-(叔-丁氧基羰基)-L-赖氨酸 (DXM-6-29)
N2-(N2-(N-(N-acetyl-S-((R)-2,3-bis(palmitoyloxy)propyl)-L-cysteinyl)-O-(tert-butyl)-D-s eryl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(tert-butoxycarbonyl-L-lysyl)-N6-(tert-butoxy carbonyl)-L-lysyl)-N6-(tert-butoxycarbonyl)-L-lysine
将化合物DXM-6-21(0.02g,0.01mmol),氢氧化钯(0.002g,0.02mmol)依次加入甲醇(5mL)中,氢气置换内部气体,50℃反应1h。TLC监测反应结束,过滤,滤液减压浓缩,得白色固体,粗品未经纯化直接进行下一步;
ESI-MS:[M+2Na]2+m/z 907.8;
步骤10:N-乙酰基-S-((R)-2,3-二(棕榈酰<十六烷酰>氧代)丙基)-L-半胱氨酰-D-丝氨酰
-L-赖氨酰-L-赖氨酰-L-赖氨酰-L-赖氨酸(DXM-6-68)
N-acetyl-S-((R)-2,3-bis(palmitoyloxy)propyl)-L-cysteinyl-D-seryl-L-lysyl-L-lysyl-L-lysyl- L-lysine
将化合物DXM-6-29(0.02g,0.01mmol)加入无水二氯甲烷(10mL)中,冰浴冷却至0℃,缓慢滴加1M氯化氢-二氧六环(1mL),0℃反应2h。TLC监测反应结束,反应液减压浓缩,HPLC纯化(流动相A:甲醇,流动相B:水+0.1%甲酸),得白色固体(0.005 g,收率36.7%),纯度95.52%。1H NMR(600MHz,D2O)δ5.20–5.14(m,1H),4.49– 4.32(m,3H),4.26–4.20(m,3H),4.12–4.00(m,3H),3.82–3.73(m,2H),3.67–3.62 (m,1H),3.61–3.57(m,1H),2.99–2.87(m,10H),2.32–2.17(m,4H),1.97(s,3H),1.66 –1.57(m,12H),1.55–1.42(m,8H),1.26–1.15(m,56H),0.82–0.76(m,6H).ESI-MS: [M+2H]2+m/z 657.6.
实施例11评价化合物对BV2细胞分泌IL-6的影响实验
TLR2主要表达于抗原提呈细胞(树突状细胞等),与相应配体结合后激活抗原提呈细胞,增加抗原提呈效率并且诱导巨噬细胞分泌多种细胞因子(如IL-6,TNF-α等),这些细胞因子用于调节肿瘤杀伤性T细胞、调节性T细胞等,进而提高机体的抗原识别能力与抗原杀伤能力;本发明采用的活性测试方法是将化合物与巨噬细胞 (BV2或THP-1)共孵育,采用酶联免疫吸附测定(enzyme linked immunosorbent assay,即ELISA)法检测细胞分泌的细胞因子IL6,IL6分泌量越多,则化合物的活性越强;所采用的BV2细胞为高表达TLR2的小鼠单核巨噬细胞,培养条件为 DMEM+10%FBS;
体外培养小鼠单核巨噬细胞BV2,细胞生长至对数生长期后,收集细胞,1000rpm离心5分钟,弃上清,适量无FBS培养基悬浮,调整细胞浓度至1×106个/ml。接种到96 孔细胞培养板中,每孔100μL,放置细胞培养箱(37℃,5%CO2)中培养24小时后,每孔加入100μL稀释好的药物,使其终浓度分别为0.1μM、0.5μM、1μM、2μM,每个浓度设5复孔,平行设置两个阳性对照孔,两个阴性对照孔。不同浓度化合物与细胞共孵育24h后,取上清于零下20℃保存,待下一步ELISA检测;
将28μL IL-6capture antibody、700μL稀释后的coating buffer加入6272μL去离子水中,混匀后加入96孔板(corning costar 9018)中,每孔100μL,4℃孵育12h,弃去板中液体,每孔加入洗液(PBS+0.05%吐温20)300μL,洗板3次,加入稀释后的 ELISA/ELISPOTDiluent溶液,每孔100μL,室温孵育1h,弃去板中液体,每孔加入洗液300μL,洗板一次,每孔加入50μL细胞上清和50μL ELISA/ELISPOT Diluent 溶液,设置IL-6标准品组,共5个浓度,每孔100μL,用以设置IL-6标准曲线,室温孵育2h,弃去板中液体,每孔加入洗液300μL,洗板三次,将28μL IL-6detection antibody加入6972μL ELISA/ELISPOT Diluent溶液中,混匀后加入96孔板中,每孔 100μL,室温孵育1h,弃去板中液体,每孔加入洗液300μL,洗板三次,将28μL Avidin-HRP加入6972μL ELISA/ELISPOT Diluent溶液中,混匀后加入96孔板中,每孔100μL,室温孵育0.5h。弃去板中液体,每孔加入洗液300μL,洗板六次,加入TMB溶液,每孔100μL,室温孵育15min,置酶标仪450nm检测OD值,根据 IL-6标准曲线将OD值转化为IL-6浓度(pg/mL)。
本发明中,代表性化合物对BV2细胞分泌IL-6的影响结果如表1所示。
表1
实施例12评价化合物对THP-1细胞分泌IL-6的影响实验
采用酶联免疫吸附测定(enzyme linked immunosorbent assay,即ELISA)法检测所述化合物对THP-1细胞分泌IL-6影响。所采用的THP-1细胞为高表达TLR2的人单核细胞,培养条件为RPMI1640+10%FBS。
体外测试操作同实施例11。
本发明中,代表性化合物对THP-1细胞分泌IL-6影响结果如表2所示。
表2
上述表2中,“-”表示未进行实验。
本发明化合物DXM-6-69和DXM-6-68在2μM时刺激BV2细胞分泌IL6分别为428.87pg/mL和568.68pg/mL,活性与先导物Pam3CSK4(510.24pg/mL)相似。此外,由于 TLR2在鼠和人中的结构差异,本发明测试了化合物对人源细胞(THP-1)的作用,结果表明化合物DXM-6-69对鼠和人均有作用,化合物DXM-7-49只对鼠有作用,化合物 DXM-7-91和DXM-7-40只对人有作用,实现了较好的人鼠选择性,且化合物 DXM-7-91与DXM-7-40在2.5μM时的活性与先导物Pam3CSK4活性接近。
Claims (12)
10.一种药用组合物,其包含治疗有效量的权利要求1-9中任一项所述脂肽类化合物或者其药学上可接受的盐以及药学上可接受的载体、气味剂、香味剂、赋形剂或稀释液。
11.根据权利要求10所述的药物组合物,其特征是,所述的化合物或其药学上可接受的盐占该药物组合物总重量的20%~99%。
12.权利要求1-9任一项所述的脂肽类化合物或其药学上可接受的盐在制备激活TLR2及TLR2介导的信号通路下游核转录因子NFκB表达的免疫佐剂或抗肿瘤药物中的应用。
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