CN117567552A - IL-2激动剂的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物、及制备和应用 - Google Patents

IL-2激动剂的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物、及制备和应用 Download PDF

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CN117567552A
CN117567552A CN202311541264.3A CN202311541264A CN117567552A CN 117567552 A CN117567552 A CN 117567552A CN 202311541264 A CN202311541264 A CN 202311541264A CN 117567552 A CN117567552 A CN 117567552A
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张筱宜
杨一帆
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Abstract

本发明公开了以下结构的3S‑2‑Thr‑四氢异喹啉‑3‑甲酰‑AA四种化合物(式中AA为L‑Gln残基,L‑Asn残基,L‑Leu残基及L‑Ala残基),公开了它们的制备方法以及在治疗炎症中的应用，并进一步公开了所述3S‑2‑Thr‑四氢异喹啉‑3‑甲酰‑AA在制备IL‑2激动剂中的应用。实验证明,本发明所述化合物制备的IL‑2激动剂有良好的抗炎作用。因而提出本发明为抗炎提供了有效的技术手段。

Description

IL-2激动剂的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物、及 制备和应用
技术领域
本发明涉及四种3S-2-Thr-四氢异喹啉-3-甲酰-AA化合物，涉及它们的制备方法，涉及它们在作为IL-2激动剂中的应用，以及其在制备抗炎症药物中的应用。本发明属于生物医药领域。
背景技术
炎症可触发器官纤维化。中枢神经系统的炎症可触发脑纤维化。癌转移是炎症触发的另一种致命性疾病。冠状动脉炎症或血管炎症与动脉粥样硬化性心血管疾病相关。炎症的另一个致命问题是与多种肿瘤发病相联系。开发无毒副作用的抗炎剂越来越受到重视。
与炎症发病相关的炎症因子有IL-2,IL-8,IL-10以及TNF-ɑ。为了寻找IL-10激动剂,发明人将以下结构的2-Gly-四氢异喹啉-3S-甲酰-AA(AA为L-Gln残基,L-Asn残基,L-Leu残基及L-Ala残基)与IL-2,IL-8,IL-10以及TNF-ɑ的活性部位对接。分子对接表明,2-Thr-四氢异喹啉-3S-甲酰-AA(AA为L-Gln残基,L-Asn残基,L-Leu残基及L-Ala残基)与IL-2的对接自由能最低。活性评价表明,2-Thr-四氢异喹啉-3S-甲酰-AA(AA为L-Gln残基,L-Asn残基,L-Leu残基及L-Ala残基)能有效上调血液IL-2浓度。活性评价还表明,2-Thr-四氢异喹啉-3S-甲酰-AA(AA为L-Gln残基,L-Asn残基,L-Leu残基及L-Ala残基)有良好的抗炎作用。根据这些发现,申请人提出了本发明。
发明内容
本发明要解决的技术问题是提供四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物,并确认了这四种化合物在制备IL-2激动剂中的应用，本发明所述化合物制备的IL-2激动剂具有优秀的抗炎作用。
为了达到所述目的,本发明采用了以下技术手段：
第一个技术手段是提供了具有下式结构的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物：
式中AA分别选自：L-Gln残基,L-Asn残基,L-Leu残基或L-Ala残基。
第二个技术手段是将所述的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物分别与IL-2,IL-8,IL-10以及TNF-ɑ的活性部位对接,筛选IL-2的激动剂。
第三个技术手段是提出上述四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物的制备方法,该制备方法包括以下步骤:
1)制备3S-四氢异喹啉-3-羧酸；
2)制备3S-四氢异喹啉-3-羧酸甲酯；
3)制备3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸甲酯；
4)制备3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸；
5)制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-AA-OBzl；
6)制备3S-2-(Boc-Thr)-四氢异喹啉-3S-甲酰-AA；
7)制备3S-2-Thr-四氢异喹啉-3S-甲酰-AA。
第四个技术手段是确认上式结构的四种2-Thr-四氢异喹啉-3S-甲酰-AA上调血液IL-2浓度。
第五个技术手段是确认上式结构的四种2-Thr-四氢异喹啉-3S-甲酰-AA在制备抗炎剂中的应用。
相较于现有技术，本发明的有益效果是：
本发明制备得到了具有上式结构的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物，通过实验发现，该类化合物与IL-2的对接自由能最低，具有有效上调血液IL-2浓度的作用。因此，本发明的提出对抗炎的治疗提供了新的治疗药物，丰富了人们对于治疗炎症的药物选择。
附图说明
图1为3S-2-Thr-四氢异喹啉-3S-甲酰-AA的合成路线图:i)CH2O,H2SO4；ii)SOCl2,甲醇；iii)DCC,HOBt,N-甲基吗啉,Boc-Ala；iv)0℃,NaOH水溶液(2N)；v)钯碳,持续通入氢气；vi)氯化氢的乙酸乙酯溶液(4N)。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1制备3S-四氢异喹啉-3-羧酸(1)
将L-Phe(4.0g,24.2mmol)依次与21.6mL甲醛及36mL浓盐酸(35％)充分搅拌,使均匀混合。将得到的混悬液80℃搅拌,2小时后L-Phe完全溶解,2.5小时后开始有无色沉淀,7小时后薄层层析(CHCl3/CH3OH,5/1)显示L-Phe消失。滤得4.2g(98％)标题化合物,为无色固体,直接用于下步反应。ESI-MS(m/z)176[M-H]-；1HNMR(300MHZ,DMSO-d6):δ/ppm＝12.89(s,1H),7.41(d,J＝7.5Hz,1H),7.31(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),4.36(m,J＝4.5Hz,2H),3.88(t,J＝5.6Hz,1H),3.02(dd,J1＝8.2Hz,J2＝7.2Hz,2H),1.92(s,1H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝173.2,135.9,130.6,127.5,126.9,126.3,56.6,46.4,35.5,30.2。
实施例2制备3S-四氢异喹啉-3-羧酸甲酯(2)
将50mL甲醇冷却至0℃,往冷却的甲醇中缓缓滴加10mLSOCl2,搅拌30分钟,得到冷却的甲醇-SOCl2溶液体系。往冷却的甲醇-SOCl2溶液体系中加3S-四氢异喹啉-3-羧酸(4.2g,23.7mmol),反应混合物于0℃搅拌24小时,薄层层析(CHCl3/CH3OH,5/1)显示3S-四氢异喹啉-3-羧酸消失,反应混合物减压浓缩至干。得到的残留物加30mL甲醇溶解,再减压浓缩至干。该操作重复3次,除净剩余的SOCl2及氯化氢,得到4.3g(95％)标题化合物,为无色固体。ESI-MS(m/e):192[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝7.41(d,J＝7.5Hz,1H),7.31(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),4.36(m,J＝4.5Hz,2H),3.88(t,J＝5.6Hz,1H),3.66(s,3H),3.02(dd,J1＝8.2Hz,J2＝7.2Hz,2H),1.92(s,1H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝173.8,135.9,130.6,127.5,126.9,126.3,56.6,51.9,46.4,35.5,30.2。
实施例3制备3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸甲酯(3)
0℃下往3S-四氢异喹啉-3-羧酸甲酯(4.30g,22.5mmol)与Boc-Thr(4.31g,22.5mmol)和150mL无水四氢呋喃的溶液中加N-羟基苯并三唑(HOBt,3.04g,22.5mmol)及二环己基羰二亚胺(DCC,4.71g,22.5mmol)。反应混合物0℃搅拌30分钟。然后用N-甲基吗啉(NMM)调pH8。反应混合物0℃搅拌6小时,薄层层析(乙酸乙酯/甲醇,20/1)显示3S-四氢-β-咔啉-3-羧酸消失,终止反应。反应混合物过滤,滤液减压浓缩至干,残留物用200mL乙酸乙酯溶解。得到的溶液依次用5％碳酸氢钠水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),5％盐酸水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3)。分离乙酸乙酯层,用无水硫酸钠干燥12小时,过滤,滤液减压浓缩至干,得到8.21g(93％)标题化合,为无色粉末。ESI-MS(m/e):393[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝8.73(s,1H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),5.37(s,1H),5.03(t,J＝6.1Hz,1H),5.00(m,J＝4.5Hz,1H),4.36(d,J＝4.5Hz,1H),4.29(s,2H),3.66(s,3H),3.08(d,J＝6.1Hz,2H),1.53(s,3H),1.52(s,3H),1.51(s,3H),1.49(s,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.85,171.63,167.88,137.09,129.63,129.13,128.45,127.85,127.21,80.02,68.22,67.88,58.14,53.66,46.05,28.57,28.56,28.55,27.49,18.56。
实施例4制备3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸(4)
0℃下将3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸甲酯(7.45g,19mmol)和50mL甲醇的溶液与5mLNaOH水溶液(3N)混合。反应混合物0℃搅拌6小时,薄层层析(乙酸乙酯/甲醇,20/1)监测到3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸甲酯消失,终止反应。反应混合物用稀盐酸调pH至2,滤液减压浓缩至干,残留物用200mL乙酸乙酯溶解。得到的溶液依次用5％碳酸氢钠水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),5％盐酸水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3)。分离乙酸乙酯层,用无水硫酸钠干燥12小时,过滤,滤液减压浓缩至干,得到6.68g(93％)标题化合,为无色粉末。ESI-MS(m/e):377[M-H]-；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.91(s,1H),8.73(s,1H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),5.37(s,1H),5.03(t,J＝6.1Hz,1H),5.00(m,J＝4.5Hz,1H),4.36(d,J＝4.5Hz,1H),4.29(s,2H),3.08(d,J＝6.1Hz,2H),1.53(s,3H),1.52(s,3H),1.51(s,3H),1.49(s,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.85,171.63,167.88,137.09,129.63,129.13,128.45,127.85,127.21,80.02,68.22,67.88,58.14,46.05,28.57,28.56,28.55,27.49,18.56。
实施例5制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Gln-OBzl(5a)
采用实施例3的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸(3.78g,10mmol)和Gln-OBzl(2.36g,10mmol)得到4.95g(83％)标题化合,为无色粉末。ESI-MS(m/z):597[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),7.17(d,J＝8.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),6.96(t,J＝7.5Hz,1H),6.18(s,2H),5.37(s,1H),5.09(t,J＝6.3Hz,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),3.16(d,J＝6.3Hz,2H),2.18(m,J＝4.9Hz,2H),2.01(t,J＝4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,159.34,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,27.49,18.56。
实施例6制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Gln(6a)
将3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Gln-OBzl(4.77g,8mmol)溶于50mL甲醇,加入240mgPd/C,通入氢气,氢解48小时。滤除Pd/C,减压浓缩除去甲醇,残留物用石油醚磨洗(30mL×3)得到3.85g(95％)标题化合,为无色粉末。ESI-MS(m/z):507[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.92(s,1H),8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),5.37(s,1H),5.09(t,J＝6.3Hz,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),3.16(d,J＝6.3Hz,2H),2.18(m,J＝4.9Hz,2H),2.01(t,J＝4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,159.34,134.09,127.59,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,28.57,28.56,28.55,28.36,27.49,18.56。
实施例7制备3S-2-Thr-四氢异喹啉-3-甲酰-Gln(7a)
将3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Gln(3.54g,7mmol)溶于100mL浓度为4N的氯化氢的无水乙酸乙酯溶液,0℃搅拌50分钟。向反应混合物加入无水乙醚,减压浓缩,直至氯化氢气完全抽净。得2.71g(95％)标题化合,为无色粉末。FT-ICR-MS(m/e):407.1931[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.92(s,1H),8.23(s,1H),8.22(s,2H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),5.37(s,1H),5.09(t,J＝6.3Hz,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),3.16(d,J＝6.3Hz,2H),2.18(m,J＝4.9Hz,2H),2.01(t,J＝4.9Hz,2H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,134.09,127.59,127.45,127.33,127.32,126.85,67.56,65.40,55.58,46.34,46.14,31.46,28.36,27.49,18.56。
实施例8制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Asn-OBzl(5b)
采用实施例3的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸(3.78g,10mmol)和Asn-OBzl(2.22g,10mmol)得到4.84g(83％)标题化合,为无色粉末。ESI-MS(m/z):583[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),7.17(d,J＝8.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),6.96(t,J＝7.5Hz,1H),6.18(s,2H),5.37(s,1H),5.09(t,J＝6.3Hz,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),3.16(d,J＝6.3Hz,2H),2.18(d,J＝4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,159.34,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,18.56。
实施例9制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Asn(6b)
采用实施例6的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Asn-OBzl(4.31g,8mmol)得到4.42g(95％)标题化合,为无色粉末。ESI-MS(m/z):493[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.28(s,1H),8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),5.37(s,1H),5.09(t,J＝6.3Hz,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),3.16(d,J＝6.3Hz,2H),2.18(d,J＝4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,159.34,134.97,128.68,127.81,127.59,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,18.56。
实施例10制备3S-2-Thr-四氢异喹啉-3-甲酰-Asn(7b)
采用实施例7的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Asn(3.13g,7mmol)得到2.31g(95％)标题化合,为无色粉末。FT-ICR-MS(m/e):393.1774[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.28(s,1H),8.23(s,1H),8.22(s,2H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),5.37(s,1H),5.09(t,J＝6.3Hz,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),3.16(d,J＝6.3Hz,2H),2.18(d,J＝4.9Hz,2H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,159.34,134.97,128.68,127.81,127.59,126.85,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.36,18.56。
实施例11制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Leu-OBzl(5c)
采用实施例3的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸(3.78g,10mmol)和Leu-OBzl(2.22g,10mmol)得到4.99g(86％)标题化合,为无色粉末。ESI-MS(m/z):582[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝8.23(s,1H),8.22(s,1H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),7.17(d,J＝8.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),6.96(t,J＝7.5Hz,1H),6.18(s,2H),5.37(s,1H),5.00(t,J＝4.9Hz,1H),4.89(t,J＝4.8Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),2.18(d,J＝4.9Hz,2H),1.99(m,J＝5.6Hz,2H),1.98(m,J＝5.6Hz,1H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H),1.04(d,J＝4.7Hz,3H),1.02(d,J＝4.7Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,44.46,31.46,30.05,28.57,28.56,28.55,28.36,27.36,22.63,22.42,18.56。
实施例12制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Leu(6c)
采用实施例6的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Leu-OBzl(4.65g,8mmol)得到3.73g(95％)标题化合,为无色粉末。ESI-MS(m/z):492[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.13(s,1H),8.23(s,1H),8.22(s,1H),7.56(d,J＝7.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),5.37(s,1H),5.00(t,J＝4.9Hz,1H),4.89(t,J＝4.8Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),2.18(d,J＝4.9Hz,2H),1.99(m,J＝5.6Hz,2H),1.98(m,J＝5.6Hz,1H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H),1.04(d,J＝4.7Hz,3H),1.02(d,J＝4.7Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,134.97,129.63,127.45,127.33,127.32,126.85,80.02,67.56,55.58,46.34,44.46,31.46,30.05,28.57,28.56,28.55,28.36,27.36,22.63,22.42,18.56。
实施例13制备3S-2-Thr-四氢异喹啉-3-甲酰-Leu(7c)
采用实施例7的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Leu(3.44g,7mmol)得到2.61g(95％)标题化合,为无色粉末。FT-ICR-MS(m/e):392.2185[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝11.13(s,1H),8.23(s,1H),8.12(s,2H),7.56(d,J＝7.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),5.37(s,1H),5.00(t,J＝4.9Hz,1H),4.89(t,J＝4.8Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.45(s,2H),2.18(d,J＝4.9Hz,2H),1.99(m,J＝5.6Hz,2H),1.98(m,J＝5.6Hz,1H),1.21(d,J＝4.5Hz,3H),1.04(d,J＝4.7Hz,3H),1.02(d,J＝4.7Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,134.97,129.63,127.45,127.33,127.32,126.85,67.56,55.58,46.34,44.46,31.46,30.05,28.36,27.36,22.63,22.42,18.56。
实施例14制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Ala-OBzl(5d)
采用实施例3的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸(3.78g,10mmol)和Ala-OBzl(1.79g,10mmol)得到4.64g(86％)标题化合,为无色粉末。ESI-MS(m/z):540[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝8.23(s,1H),8.22(s,1H),7.56(d,J＝7.5Hz,1H),7.33(t,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.21(t,J＝7.5Hz,1H),7.17(d,J＝8.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),7.02(t,J＝7.4Hz,1H),6.96(t,J＝7.5Hz,1H),6.18(s,2H),5.37(s,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.16(m,J＝5.6Hz,1H),4.45(s,2H),2.18(d,J＝4.9Hz,2H),1.44(d,J＝5.6Hz,3H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,44.46,28.57,28.56,28.55,28.36,22.63,22.42,18.56。
实施例15制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Ala(6d)
采用实施例6的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Ala-OBzl(4.31g,8mmol)得到3.41g(95％)标题化合,为无色粉末。ESI-MS(m/z):450[M+H]+；1HNMR(300MHZ,DMSO-d6):δ/ppm＝12.89(s,1H),8.23(s,1H),8.22(s,1H),7.56(d,J＝7.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),6.96(t,J＝7.5Hz,1H),5.37(s,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.16(m,J＝5.6Hz,1H),4.45(s,2H),2.18(d,J＝4.9Hz,2H),1.44(d,J＝5.6Hz,3H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,171.68,134.97,129.63,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,28.57,28.56,28.55,28.36,22.63,22.42,18.56。
实施例16制备3S-2-Thr-四氢异喹啉-3-甲酰-Ala(7d)
采用实施例7的方法从3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-Ala(3.14g,7mmol)得到2.32g(95％)标题化合,为无色粉末。FT-ICR-MS(m/e):350.1716[M+H]+；12.89(s,1H),8.23(s,1H),8.22(s,2H),7.56(d,J＝7.5Hz,1H),7.15(d,J＝7.3Hz,1H),7.14(d,J＝7.3Hz,1H),6.96(t,J＝7.5Hz,1H),5.37(s,1H),5.00(t,J＝4.9Hz,1H),4.58(m,J＝4.5Hz,1H),4.48(d,J＝4.5Hz,1H),4.16(m,J＝5.6Hz,1H),4.45(s,2H),2.18(d,J＝4.9Hz,2H),1.44(d,J＝5.6Hz,3H),1.21(d,J＝4.5Hz,3H)；13CNMR(75MHZ,DMSO-d6):δ/ppm＝172.65,172.45,172.13,134.97,129.63,127.45,127.33,127.32,126.85,67.56,65.40,55.58,46.34,28.36,22.63,22.42,18.56。
实施例17 3S-2-Thr-四氢异喹啉-3-甲酰-AA的分子对接
为了预测3S-2-Thr-四氢异喹啉-3-甲酰-Gln(7a),3S-2-Thr-四氢异喹啉-3-甲酰-Asn(7b),3S-2-Thr-四氢异喹啉-3-甲酰-Leu(7c)及3S-2-Thr-四氢异喹啉-3-甲酰-Ala(7d)对炎症小鼠血液炎症因子的影响本申请完成了7a-d和IL-2,IL-8,IL-10及TNF-ɑ的活性部位的分子对接。IL-2,IL-8,IL-10及TNF-ɑ均来自欧洲蛋白质库。对接时采用DiscoveryStudio的LigandFit将7a-d对接到IL-2,IL-8,IL-10及TNF-ɑ的活性部位。对接时经历了四个步骤。第一步,用flood-filling算法选择腔体,以便选择和确定作为对接的区域的IL-2,IL-8,IL-10及TNF-ɑ的活性位点。第二步,为7a-d选择位点时先通过随机抽样选择可变扭转角的柔性值搜索7a-d的构象,再用三维规则网格检测位点的检测并估算对接IL-2,IL-8,IL-10及TNF-ɑ的活性位点所需能量。第三步,比较IL-2,IL-8,IL-10及TNF-ɑ和7a-d的库仑力,范德华力,结合能,原子间距,氢键能,空间相互作用,亲脂相互作用,溶剂化效应和熵效应的分数,以便得到综合评价结果。第四步,计算7a-d的对接自由能。表1的数据表明,向IL-2,IL-8,IL-10以及TNF-ɑ的活性部位对接时7a-d与IL-2的结合自由能显著性低于与IL-8,IL-10及TNF-ɑ的结合自由能。可见,IL-12是7a-d的潜在的分子靶点。
表1 7a-d与IL-2,IL-8,IL-10以及TNF-ɑ的活性部位的结合能
实施例18评价3S-2-Thr-四氢异喹啉-3-甲酰-AA的抗炎活性
二甲苯引起的小鼠耳肿胀被公认为急性炎症模型,本发明在二甲苯引起的小鼠耳肿胀模型上测定3S-2-Thr-四氢异喹啉-3-甲酰-AA的抗炎活性。阿司匹林是治疗急性炎症的阳性药,本发明选择阿司匹林为阳性对照。ICR雄性小鼠(体重35±2g)在温度为25℃的环境静息2天,自由饮水和进食。之后,随机分组。小鼠或口服生理盐水(剂量为10mL/kg,10只小鼠),或口服阿司匹林(剂量为1110μmol/kg,10只小鼠),或口服3S-2-Thr-四氢异喹啉-3-甲酰-Gln(7a,10nmol/kg,10只小鼠),或口服3S-2-Thr-四氢异喹啉-3-甲酰-Asn(7b,10nmol/kg,10只小鼠),或口服3S-2-Thr-四氢异喹啉-3-甲酰-Leu(7c,10nmol/kg,10只小鼠),或口服3S-2-Thr-四氢异喹啉-3-甲酰-Ala(7d,10nmol/kg,10只小鼠)。口服30分钟后,往小鼠的左耳廓均匀涂抹30μL二甲苯,2小时后小鼠乙醚麻醉断颈椎处死,取血,然后剪下左右两耳,用直径7mm的打孔器在两耳的相同位置取圆形耳片,称重,两耳重量差作为肿胀度。即肿胀度＝左耳圆片重量–右耳圆片重量。
表2的数据表明,在10nmol/kg口服剂量下7a-d有效地抑制二甲苯引发的炎症(与生理盐水比p<0.01)。表2的数据还表明,在10nmol/kg口服剂量下7a-d抑制二甲苯引发的炎症的活性显著性强于在1110μmol/kg口服剂量下阿司匹林抑制二甲苯引发的炎症的活性(与阿司匹林比p<0.01)。可见,7a-d有突出的技术效果。
表2 3S-2-Thr-四氢异喹啉-3-甲酰-AA的抗炎活性
a)与生理盐水及阿司匹林比p<0.01；n＝10.
实施例19评价3S-2-Thr-四氢异喹啉-3-甲酰-AA对炎症因子的影响
将炎症小鼠的血于4℃加入浓度为3.8％(g/100mL)枸橼酸钠的生理盐水溶液(v/v＝9:1),3000g离心15分钟,然后吸取上清液作为IL-2,IL-8,IL-10以及TNF-ɑ酶联免疫测定样本。
ELISA法定量测定血浆样本中IL-2,IL-8,IL-10以及TNF-ɑ时,用纯化的IL-2,IL-8,IL-10以及TNF-ɑ抗体包被微孔板。按照ELISA试剂盒的方法实施操作,即向包被单抗的微孔中依次加入标准品或生理盐水治疗的炎症小鼠的血浆样本及7a-d治疗的炎症小鼠的血浆样本,加生物素化的IL-2,IL-8,IL-10以及TNF-ɑ抗体,以及辣根过氧化物酶标记的亲和素。然后,彻底洗涤微孔板并加底物TMB显色。显色时,先看到过氧化物酶作用诱发的蓝色,后看到在酸作用下蓝色转为黄色。黄色的深度与样本中IL-2,IL-8,IL-10以及TNF-ɑ的浓度正相关。在450nm的波长下用酶标仪测定标准品的吸光度(OD),绘制标准曲线。在450nm的波长下用酶标仪测定生理盐水治疗的炎症小鼠及7a-d治疗的炎症小鼠的血浆样本的吸光度(OD),通过标准曲线计算生理盐水治疗的炎症小鼠及7a-d治疗的炎症小鼠的血浆样本的IL-2,IL-8,IL-10以及TNF-ɑ的浓度。每个样本重复6次。表3的数据表明,7a-d显著性升高炎症小鼠的血浆IL-2的浓度(与生理盐水比p<0.01)。表3的数据还表明,7a-d对炎症小鼠的血浆IL-8,IL-10以及TNF-ɑ的浓度无显著影响(与生理盐水比p>0.05)。可见,小鼠血浆的IL-2的确是7a-d抑制炎症反应的分子靶点。
表3 7a-d对血浆IL-2,IL-8,IL-10以及TNF-ɑ的影响
a)7a-d与生理盐水比p<0.01；b)7a-d与生理盐水比p>0.05；c)7a-d与生理盐水比p>0.05；d)7a-d与生理盐水比p>0.05；n＝10。

Claims (5)

1.具有下式结构的四种3S-2-Thr-四氢异喹啉-3-甲酰-AA化合物,
式中AA选自L-Gln残基,L-Asn残基,L-Leu残基或L-Ala残基。
2.权利要求1所述的四种3S-2-Thr-四氢异喹啉-3-甲酰-AA化合物的制备方法,其特在于，该方法包括以下步骤:
1)制备3S-四氢异喹啉-3-羧酸；
2)制备3S-四氢异喹啉-3-羧酸甲酯；
3)制备3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸甲酯；
4)制备3S-2-(Boc-Thr)-四氢异喹啉-3-羧酸；
5)制备3S-2-(Boc-Thr)-四氢异喹啉-3-甲酰-AA-OBzl；
6)制备3S-2-(Boc-Thr)-四氢异喹啉-3S-甲酰-AA；
7)制备3S-2-Thr-四氢异喹啉-3S-甲酰-AA。
3.权利要求1所述的四种3S-2-Thr-四氢异喹啉-3S-甲酰-AA合化物，其特征在于，所述化合物在制备IL-2激动剂中的应用。
4.权利要求1所述的四种3S-2-Thr-四氢异喹啉-3S-甲酰-AA化合物,其特征在于，所述化合物在制备抗炎药物中的应用。
5.权利要求3所述的应用，其特征在于，所述的IL-2激动剂在制备抗炎药物中的应用。
CN202311541264.3A 2023-11-17 2023-11-17 IL-2激动剂的四种2-Thr-四氢异喹啉-3S-甲酰-AA化合物、及制备和应用 Pending CN117567552A (zh)

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