CN108969770B - 二肽修饰的1-甲基-3-羟甲基-四氢-β-咔啉，其合成和应用 - Google Patents
二肽修饰的1-甲基-3-羟甲基-四氢-β-咔啉，其合成和应用 Download PDFInfo
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Abstract
二肽修饰的1‑甲基‑3‑羟甲基‑四氢‑β‑咔啉，其合成和应用。本发明公开了下式的(1S,3S)‑1‑甲基‑3‑(AA‑Val‑甲氧基)‑2,3,4,9‑四氢‑β‑咔啉(式中AA为Gly残基,L‑Ala残基,L‑Leu残基,L‑Ile残基,L‑Pro残基,L‑Phe残基,L‑Val残基和L‑Lys残基),公开了它们的制备方法,公开了它们的抗肿瘤活性,进一步公开了它们抑制P‑选择素表达的活性。因而本发明公开了它们在制备抗肿瘤药物和抑制P‑选择素抑制剂中的应用。
Description
技术领域
本发明涉及下式的(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉,涉及它们的制备方法,涉及它们的抗肿瘤活性,进一步涉及它们抑制P-选择素表达的活性。因而本发明涉及它们在制备抗肿瘤药物和P-选择素抑制剂中的应用。本发明属于生物医药领域。
背景技术
P-选择素在多种恶性肿瘤中都高表达，并参与多种恶性肿瘤的发生和发展过程。以P- 选择素为靶的抗肿瘤药物正受到越来越多的关注。发明人曾经公开五环化合物(下式左面的结构)在0.1μmol/kg/天剂量下有抗肿瘤活性。不过，它们对P-选择素没有抑制作用。分析五环化合物的结构特征，使发明人提取到(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉这个关键核。于是，发明往(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉的羟甲基上引入氨基酸和二肽。最终发现，往(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉的羟甲基上引入AA-Val 二肽(下式右面的结构)可得到一类新的以P-选择素为靶的抗肿瘤剂。根据这个发现，发明人提出了本发明。
发明内容
本发明的第一个内容是提供下式的(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉，式中AA为Gly残基,L-Ala残基,L-Leu残基,L-Ile残基,L-Pro残基,L-Phe残基,L-Val 残基和L-Lys残基。
本发明的第二个内容是提供制备(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉的方法，该方法包括：
(1)在H2SO4水溶液(1M)中L-Trp和乙醛发生Pictet-Spengler缩合制备(1S,3S)-1-甲基-1,2,3,4- 四氢-β-咔啉-3-羧酸；
(2)(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸先在SOCl2中酰氯化,后与甲醇反应制备 (1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧甲酯；
(3)用氢化铝锂还原(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧甲酯制备(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉；
(4)用(Boc)2O与(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉制备N-Boc-[(1S,3S)-1-甲基-3- 羟甲基-1,2,3,4-四氢-β-咔啉]；
(5)制备Boc-AA-Val-OBzl(AA为Gly残基,L-Ala残基,L-Leu残基,L-Ile残基,L-Pro残基,L-Phe 残基,L-Lys残基和L-Val残基)；
(6)在钯碳的催化下将Boc-AA-Val-OBzl氢解制备Boc-AA-Val；
(7)N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]与Boc-AA-Val反应制备 N-Boc-[(1S,3S)-1-甲基-3-(Boc-AA-Val-氧甲基)-1,2,3,4-四氢-β-咔啉]；
(8)N-Boc-[(1S,3S)-1-甲基-3-(Boc-AA-Val-氧甲基)-1,2,3,4-四氢-β-咔啉]在氯化氢的乙酸乙酯溶液(4M)中脱Boc制备(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉。
本发明的第三个内容是测定(1S,3S)-1-甲基-3-(甲氧AA-Val)-2,3,4,9-四氢-β-咔啉的抗肿瘤活性和P-选择素抑制活性。
附图说明
图1(1S,3S)-1-甲基-3-(甲氧AA-Val)-2,3,4,9-四氢-β-咔啉；i)乙醛,浓硫酸；ii)甲醇,SOCl2； iii)LiAlH4；iv)(Boc)2O,三乙胺；v)二环己基碳二亚胺(DCC)；vi)氯化氢的乙酸乙酯溶液 (4M)。4a和5a中AA为Gly残基,4b和5b中AA为L-Ala残基,4c和5c中AA为L-Leu 残基,4d和5d中AA为L-Ile残基,4e和5e中AA为L-Pro残基,4f和5f中AA为L-Phe 残基,4g和5g中AA为L-Val残基,4h中AA为L-Lys(Boc)残基,5h中AA为L-Lys残基。
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(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸
往5.0g(24.5mmol)L-色氨酸和400mL蒸馏水的悬浮液中缓慢滴入0.2mL浓硫酸(98％)。超声使全部溶解之后，再滴加4.5mL(49.0mmol)乙醛(40％)。反应混合物室温搅拌12h，用浓氨水调pH至7，静置1h。反应混合物过滤，滤饼用蒸馏水充分洗，晾干，得到4.51g(80％) 标题化合物,为无色粉末。ESI-MS(m/z):231[M+H]+；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝10.99(s,1H),9.18(s,1H),7.44(d,J＝7.5Hz,1H),7.33(t,J＝8.0Hz,1H),7.08(t,J＝8.0Hz, 1H),6.99(t,J＝7.5Hz,1H),4.22(q,J＝4.8Hz,1H),3.69(dd,J＝10.5Hz,J＝5.0Hz,1H), 3.14(dd,J＝10.5Hz,J＝2.4Hz,1H),2.83(ddd,J＝10.5Hz,J＝5.0Hz,J＝2.4Hz,1H),1.38 (d,J＝5.0Hz,3H)。
实施例2制备(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯
冰水浴下向100mL甲醇中缓慢滴入5mL氯化亚砜并搅拌30min。向得到的溶液中加入2.0 g(8.7mmol)(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸，并室温搅拌14h。减压除氯化氢气，反应混合物用饱和NaHCO3水溶液调pH至7。充分静置，滤出的沉淀用100mL乙酸乙酯溶解。得到的溶液依次用饱和NaHCO3水溶液洗(30mL×3)及饱和NaCl水溶液洗(30mL×3)。乙酸乙酯相用Na2SO4干燥8h，过滤，滤液减压浓缩，得到1.35g(65％)标题化合物。ESI-MS(m/z): 245[M+H]+；IR(cm-1):3202,2970,2937,2452,1750,1682,1559,1500,1457,1320,1237, 1149,1015；1H NMR(300MHz,DMSO-d6):d＝10.04(s,1H),7.42(t,J＝6.4Hz,1H),7.20(t, J＝8.6Hz,1H),7.11(d,J＝7.5Hz,1H),7.04(d,J＝6.6Hz,1H),4.28(q,J＝6.6Hz,1H),3.78 (s,3H),3.77(m,J＝6.9Hz,1H),3.08(d,J＝11.2Hz,1H),2.81(t,J＝11.2Hz,1H),2.25(s,1H), 1.42(d,J＝6.0Hz,3H)。
实施例3制备(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉
将1.0g(4.10mmol)(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯溶于30mL无水四氢呋喃中。冰水浴下于50min内向该溶液中加入0.15g(12.30mmol)氢化铝锂。反应混合物室温搅拌4h，加0.5mL氢氧化钠水溶液(10％)并室温搅拌1h淬灭反应。过滤，滤饼用无水四氢呋喃反复洗。收集的滤液减压浓缩，得到0.50g(48％)标题化合物。ESI-MS(m/z):217[M+H]+；IR(cm-1):3202,2970,2937,2452,1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.72(s,1H),7.34(d,J＝7.8Hz,1H),7.28(d,J＝7.8Hz,1H),7.01(t,J＝13.8Hz,1H),6.95(t,J＝15.9Hz,1H),4.74(t,J＝6.6Hz,1H), 4.07(m,1H),3.54(dd,J1＝22.8Hz,J2＝5.1Hz,1H),3.47(dd,J1＝22.8Hz,J2＝5.1Hz,1H), 3.02(m,1H),2.30(dd,J1＝3.6Hz,J2＝2.4Hz,1H),2.24(dd,J1＝3.6Hz,J2＝2.4Hz,1H), 1.39(d,J＝6.6Hz,3H)。
实施例4制备N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]
将0.50g(2.31mmol)(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉溶于30mL无水四氢呋喃中。冰浴下往该溶液中加0.61g(2.77mmol)(Boc)2O，之后滴三乙胺调pH至10。室温搅拌8h。期间每隔1h减压抽气。反应混合物减压浓缩，残留物用50mL乙酸乙酯溶解。得到的溶液依次用1N的HCl水溶液洗(30mL×3)及饱和NaCl水溶液洗(30mL×3)。乙酸乙酯相用无水Na2SO4干燥8h，过滤，滤液减压浓缩，残留物用硅胶柱层析纯化(二氯甲烷 /丙酮＝150/1)，得到0.51g(70％)标题化合物。ESI-MS(m/z):317[M+H]+；IR(cm-1):3202, 2970,2937,2452,1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz, DMSO-d6):δ(ppm)＝10.88(s,1H),7.40(d,J＝7.5Hz,1H),7.29(d,J＝8.1Hz,1H),7.05(t, J＝6.9Hz,1H),6.93(t,J＝7.2Hz,1H),5.02(d,J＝4.8Hz,1H),4.89(m,1H),4.63(s,1H),2.98(d, J＝15.6Hz,1H),2.65(dd,J1＝15.6Hz,J2＝9.6Hz,1H),1.45(m,12H)。
实施例5制备Boc-AA-Val
用二环己基碳二亚胺(DCC)法由Boc-AA(AA为Gly残基,L-Ala残基,L-Leu残基,L-Ile 残基,L-Pro残基,L-Phe残基,L-Lys残基和L-Val残基)和Val-OBzl偶合制备 Boc-AA-Val-OBzl。在钯碳的催化下将Boc-AA-Val-OBzl氢解制备Boc-AA-Val(AA为Gly 残基,L-Ala残基,L-Leu残基,L-Ile残基,L-Pro残基,L-Phe残基,L-Lys残基和L-Val残基)。
实施例6制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Gly-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4a)
将2.6g(9.5mmol)Boc-Gly-Val溶于50mL无水二氯甲烷中。冰浴下依次向该溶液中加 4-二甲氨基吡啶0.3g(2.5mmol)4-二甲氨基吡啶和0.8g(3.8mmol)二环己基碳二亚胺，搅拌30min。向反应混合物中加1.0g(3.2mmol)N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]，室温搅拌24h。过滤，滤液减压浓缩，残留物用100mL乙酸乙酯溶解。得到的溶液依次用1N的HCl水溶液洗(30mL×3)及饱和NaCl水溶液洗(30mL×3)。乙酸乙酯相用无水Na2SO4干燥8h，过滤，滤液减压浓缩，残留物用薄层板纯化，得到0.5g(25％) 标题化合物。ESI-MS(m/z):595[M+Na]+；δ(ppm)＝11.29(s,1H),7.45(d,J＝8Hz,1H),7.38 (d,J＝8Hz,1H),7.13(t,J＝7.5Hz,1H),7.00(t,J＝7.5Hz,1H),4.84(s,1H),4.58(m,1 H),4.50(m,2H),3.72(m,1H),3.59(s,2H)2.99(m,2H),2.22(m,1H),1.78(d,J＝7Hz, 3H),1.38(m,24H)。
实施例7制备(1S,3S)-1-甲基-3-(Gly-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5a)
冰浴下将0.5g(0.87mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-Gly-Val-氧甲基)-1,2,3,4-四氢 -β-咔啉]用5mL氯化氢乙酸乙酯溶液(4M)溶解，搅拌30min。室温搅拌4h。减压除氯化氢气，加入乙醚反复磨洗，得到0.3g(85％)标题化合物，为无色粉末。ESI-MS(m/z):373 [M+H]+；Mp 216-217℃；
IR(cm-1):3202,2970,2937,2452,1750, 1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.29(s,1H),8.13(s,3H),7.45(d,J＝8Hz,1H),7.38(d,J＝8Hz,1H),7.13(t,J＝7.5Hz,1H), 7.00(t,J＝7.5Hz,1H),4.84(s,1H),4.58(m,1H),4.50(m,2H),3.72(m,1H),3.59(s,2H) 2.99(m,2H),2.22(m,1H),1.78(d,J＝7Hz,3H),0.93(d,J＝7Hz,3H),0.89(d,J＝7Hz,3H)；13C NMR(75MHz,DMSO-d6):δ(ppm)＝171.61,136.24,127.41,122.22,119.30,111.12,108.35, 68.08,55.66,49.82,43.72,30.54,23.16,17.53。
实施例8制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Ala-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4b)
按实施例6的方法从2.7g(9.5mmol)Boc-Ala-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1- 甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.5g(28％)标题化合物。ESI-MS(m/z):609 [M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.28(s,1H),7.44(d,J＝7.5Hz,1H),7.39 (d,J＝7.5Hz,1H),7.13(t,J＝7.2Hz,1H),7.01(t,J＝7.2Hz,1H),4.84(s,1H),4.57(m,1H), 4.50(m,2H),4.20(m,1H),4.01(m,2H),2.99(m,2H),2.28(m,1H),1.79(d,J＝6.6Hz,3H), 1.38(m,27H)。
实施例9制备(1S,3S)-1-甲基-3-(Ala-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5b)
按实施例7的方法从0.5g(0.85mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-Ala-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4b)得到0.28g(85％)标题化合物，为无色粉末。ESI-MS(m/z):387 [M+H]+；Mp:162-163℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.28(s,1H),8.22(s,3H),7.44(d,J＝7.5Hz,1H),7.39(d,J＝7.5Hz,1H),7.13(t,J＝7.2Hz,1H), 7.01(t,J＝7.2Hz,1H),4.84(s,1H),4.57(m,1H),4.50(m,2H),4.20(m,1H),4.01(m,2H), 2.99(m,2H),2.28(m,1H),1.79(d,J＝6.6Hz,3H),1.38(d,J＝6.9Hz,3H),0.94(d,J＝7.2Hz,3H), 0.88(d,J＝7.2Hz,3H)；13C NMR(75MHz,DMSO-d6):δ(ppm)＝171.81,136.25,127.42,122.21, 119.80,111.15,108.32,68.05,55.92,50.09,48.84,30.52,23.21,20.54,17.53。
实施例10制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Leu-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4c)
按实施例6的方法从3.1g(9.5mmol)Boc-Leu-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1- 甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.6g(30％)标题化合物。ESI-MS(m/z):651 [M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.27(s,1H),7.42(d,J＝7.2Hz,1H),7.38(d, J＝7.2Hz,1H),7.13(t,J＝7Hz,1H),7.02(t,J＝7Hz,1H),4.86(m,1H),4.60(m,1H),4.40(m, 2H),4.14(m,1H),3.91(d,J＝5.4Hz,1H),3.00(m,2H),2.24(m,1H),1.80(d,J＝6.3Hz,3H), 1.48(m,33H)。
实施例11制备(1S,3S)-1-甲基-3-(Leu-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5c)
按实施例7的方法从0.6g(0.96mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-Leu-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4c)得到0.4g(90％)标题化合物，为无色粉末。ESI-MS(m/z):443 [M+H]+；Mp 203-204℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.27(s,1H),8.27(s,3H),7.42(d,J＝7.2Hz,1H),7.38(d,J＝7.2Hz,1H),7.13(t,J＝7Hz,1H), 7.02(t,J＝7Hz,1H),4.86(m,1H),4.60(m,1H),4.40(m,2H),4.14(m,1H),3.91(d,J＝5.4Hz, 1H),3.00(m,2H),2.24(m,1H),1.80(d,J＝6.3Hz,3H),1.65(m,3H),0.94(m,12H)；13C NMR (75MHz,DMSO-d6):δ(ppm)＝171.62,134.12,127.45,122.21,119.08,111.13,108.34,68.07, 55.97,51.65,48.89,30.56,22.23,17.53。
实施例12制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Ile-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4d)
按实施例6的方法从3.1g(9.5mmol)Boc-Ile-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.6g(30％)标题化合物。ESI-MS(m/z):651[M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.28(s,1H),7.44(d,J＝8Hz,1H),7.38(d,J＝8Hz, 1H),7.13(t,J＝7.5Hz,1H),7.01(t,J＝7.5Hz,1H),4.83(m,1H),4.61(m,1H),4.58(m,2H),4.08 (m,1H),3.91(m,1H),2.98(m,2H),2.25(m,1H),2.06(m,1H),1.78(d,J＝8.1Hz,3H),1.49(m, 32H)。
实施例13制备(1S,3S)-1-甲基-3-(Ile-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5d)
按实施例7的方法从0.6g(0.96mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-Ile-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4d)得到0.4g(90％)标题化合物，为无色粉末。ESI-MS(m/z):443 [M+H]+；Mp 203-204℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.28(s,1H),8.22(s,3H),7.44(d,J＝8Hz,1H),7.38(d,J＝8Hz,1H),7.13(t,J＝7.5Hz,1H), 7.01(t,J＝7.5Hz,1H),4.83(m,1H),4.61(m,1H),4.58(m,2H),4.08(m,1H),3.91(m,1H), 2.98(m,2H),2.25(m,1H),2.06(m,1H),1.78(d,J＝8.1Hz,3H),0.94(m,14H)；13C NMR (75MHz,DMSO-d6):δ(ppm)＝171.62,134.12,127.45,122.21,119.08,111.13,108.34,68.07, 55.97,51.65,48.89,30.56,22.23,17.53。
实施例14制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Pro-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4e)
按实施例6的方法从3.0g(9.5mmol)Boc-Pro-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1- 甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.4g(22％)标题化合物。ESI-MS(m/z):635 [M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.29(s,1H),7.45(d,J＝6Hz,1H),7.40(d, J＝6Hz,1H),7.14(t,J＝7.2Hz,1H),7.00(t,J＝7.2Hz,1H),4.85(m,1H),4.58(m,1H),4.50(m, 2H),4.00(m,1H),3.30(m,2H),2.98(d,J＝6.6Hz,2H),2.35(m,1H),1.86(d,J＝6Hz,3H), 1.80(m,4H),1.37(d,24H)。
实施例15制备(1S,3S)-1-甲基-3-(Pro-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5e)
按实施例7的方法从0.4g(0.65mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-Pro-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4e)得到0.2g(80％)标题化合物，为无色粉末。ESI-MS(m/z):413 [M+H]+；Mp 220-221℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.29(s,1H),8.55(s,3H),7.45(d,J＝6Hz,1H),7.40(d,J＝6Hz,1H),7.14(t,J＝7.2Hz,1H), 7.00(t,J＝7.2Hz,1H),4.85(m,1H),4.58(m,1H),4.50(m,2H),4.00(m,1H),3.30(m,2H), 2.98(d,J＝6.6Hz,2H),2.35(m,1H),1.86(d,J＝6Hz,3H),1.80(m,4H),0.94(d,J＝7Hz,3H), 0.94(d,J＝7.2Hz,3H)；13C NMR(75MHz,DMSO-d6):δ(ppm)＝171.62,136.21,127.43,122.21, 119.07,111.12,108.34,68.05,55.92,49.87,45.52,32.25,24.98,17.54。
实施例16制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Phe-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4f)
按实施例6的方法从3.4g(9.5mmol)Boc-Phe-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1- 甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.6g(31％)标题化合物，ESI-MS(m/z):685 [M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.29(s,1H),7.30(m,8H),4.84(s,1H), 4.50(m,3H),4.00(m,1H),4.15(m,1H),3.00(d,J＝6.6Hz,2H),2.25(m,1H),1.90(s,1H), 1.78(d,J＝7.2Hz,3H),1.35(m,25H)。
实施例17制备(1S,3S)-1-甲基-3-(Phe-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5f)
按实施例7的方法从0.6g(0.91mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-PheVal-氧甲基)-1,2,3,4-四氢-β-咔啉](4f)得到0.3g(82％)标题化合物，为无色粉末。ESI-MS(m/z):463 [M+H]+；Mp 226-227℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.29(s,1H),10.45(s,1H),9.85(m,1H),9.50(t,J＝9Hz,1H),8.35(s,3H),7.30(m,8H), 4.84(s,1H),4.50(m,3H),4.00(m,1H),4.15(m,1H),3.00(d,J＝6.6Hz,2H),2.25(m,1H), 1.90(s,1H),1.78(d,J＝7.2Hz,3H),1.35(m,1H),0.97(m,6H)；13C NMR(75MHz,DMSO-d6): δ(ppm)＝171.82,139.52,134.15,127.43,122.21,120.12,119.07,111.12,108.34,68.09,55.92, 49.83,40.35,30.54,23.12,17.53。
实施例18制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-Val-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4g)
按实施例6的方法从3.0g(9.5mmol)Boc-Val-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1- 甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.5g(25％)标题化合物。ESI-MS(m/z):637[M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.28(s,1H),7.44(d,J＝7.8Hz,1H),7.38(d,J＝8.4 Hz,1H),7.13(t,J＝7.2Hz,1H),7.02(t,J＝7.8Hz,1H),4.85(s,1H),4.50(m,3H),4.32(m,1 H),4.00(m,2H),3.30(m,2H),2.20(m,2H),1.90(s,1H),1.78(d,J＝7.2Hz,3H),1.39(m, 32H)。
实施例19制备(1S,3S)-1-甲基-3-(Val-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5g)
按实施例7的方法从0.5g(0.81mmol)N-Boc-[(1S,3S)-1-甲基-3-(Boc-Val-Val-氧甲基)-1,2,3,4-四氢-β-咔啉](4g)得到0.3g(90％)标题化合物，为无色粉末。ESI-MS(m/z):415 [M+H]+；Mp 216-217℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.28(s,1H),8.23(s,3H),7.44(d,J＝7.8Hz,1H),7.38(d,J＝8.4Hz,1H),7.13(t,J＝7.2Hz,1 H),7.02(t,J＝7.8Hz,1H),4.85(s,1H),4.50(m,3H),4.32(m,1H),4.00(m,2H),3.30(m,2H), 2.20(m,2H),1.90(s,1H),1.78(d,J＝7.2Hz,3H),1.41(m,1H),0.94(m,14H)；13C NMR (75MHz,DMSO-d6):δ(ppm)＝171.12,136.24；127.45,120.15,111.15,108.32,68.09,60.00, 55.91,44.82,33.84,30.52,23.12,17.12。
实施例20制备N-Boc-{(1S,3S)-1-甲基-3-[Boc-Lys(Boc)-Val-氧甲基]-1,2,3,4-四氢-β-咔啉}(4h)
按实施例6的方法从4.2g(9.5mmol)Boc-Lys(Boc)-Val和1.0g(3.2mmol)N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]得到0.49g(21％)标题化合物。ESI-MS(m/z): 766[M+Na]+；1H NMR(300MHz,DMSO-d6):δ(ppm)＝11.30(s,1H),7.44(d,J＝7.8Hz,1H), 7.38(d,J＝8.1Hz,1H),7.13(t,J＝7.2Hz,1H),7.02(t,J＝6.6Hz,1H),4.85(s,1H),4.25(m,1 H),3.90(m,1H),3.00(m,2H),2.25(m,2H),1.90(s,1H),1.78(d,J＝6.6Hz,3H),1.41(m, 33H)。
实施例21制备(1S,3S)-1-甲基-3-(Lys-Val-氧甲基)-1,2,3,4-四氢-β-咔啉(5h)
按实施例7的方法从0.49g(0.66mmol)N-Boc-{(1S,3S)-1-甲基-3-[Boc-Lys(Boc)-Val-氧甲基]-1,2,3,4-四氢-β-咔啉}(4h)得到0.25g(87％)标题化合物，为无色粉末。ESI-MS(m/z):443 [M+H]+；Mp 213-214℃；
IR(cm-1):3202,2970,2937,2452, 1750,1682,1559,1500,1457,1320,1237,1149,1015；1H NMR(300MHz,DMSO-d6):δ(ppm) ＝11.30(s,1H),8.35(d,J＝13.8Hz,3H),7.99(m,3H),7.44(d,J＝7.8Hz,1H),7.38(d,J＝8.1Hz, 1H),7.13(t,J＝7.2Hz,1H),7.02(t,J＝6.6Hz,1H),4.85(s,1H),4.25(m,1H),3.90(m,1H), 3.00(m,2H),2.25(m,2H),1.90(s,1H),1.78(d,J＝6.6Hz,3H),1.41(m,9H),0.94(m,6H)；13C NMR(75MHz,DMSO-d6):δ(ppm)＝171.62,134.12；127.42,120.14,111.12,108.32,68.02, 55.91,48.82,42.16,34.32,30.52,23.16,20.76,17.50。
实施例22评价化合物5a-h的抗肿瘤活性
化合物5a-h用生理盐水溶解，阿霉素用生理盐水溶解作阳性对照，生理盐水作阴性对照。小鼠腹腔注射化合物5a-h的剂量为0.1μmol/kg/天，小鼠腹腔注射阿霉素的剂量为2μmol/kg/ 天，连续给药7天，共给药7次。实验小鼠为ICR雄性小鼠，体重为20±2g。瘤源来自小鼠的S180肉瘤,自行传代维持。
无菌条件下抽取生长旺盛的S180腹水瘤瘤液接种,用生理盐水稀释成(1:2)的液体充分混合,将肿瘤细胞悬液用新鲜配制的0.2％台盼蓝染色,混匀后按白细胞计数方法计数, 染蓝色者为死细胞,不染色者为活细胞。按细胞浓度＝4大方格内活细胞数/4×104×稀释倍数＝细胞数/mL计算细胞浓度。按细胞存活率＝活细胞数/(活细胞数+死细胞数)×100％计算细胞存活率。将存活率大于90％的瘤液用匀浆法制备成2.0×107个/mL的细胞悬液,于鼠腋皮下接种,0.2mL/只,制造S180荷瘤小鼠。肿瘤接种3天后,随机分组，每组12只小鼠。小鼠每日腹腔注射化合物5a-h(剂量为0.1μmol/kg/天)，或每日腹腔注射阿霉素(剂量为2 μmol/kg/天)，或每日腹腔注射蒸馏水(剂量为10mL/kg/天)，连续给药7天，共给药7次。第8 天乙醚麻醉,脱颈椎处死小鼠,收集小鼠血用于测定P-选择素浓度。然后用镊子固定小鼠右腋肿瘤生长部位,剪开皮肤,暴露肿瘤,钝性剥离,称重,瘤重以平均值±SD g表示。实验数据采用t检验和方差分析,结果见表1。可以看出，在0.1μmol/kg剂量下化合物5a-h明显抑制肿瘤生长，活性与剂量比它们高20倍的阿霉素无统计学差异。本发明有突出的技术效果。
表1化合物5a-h的抗肿瘤活性
a)与生理盐水比p<0.01,与阿霉素比p>0.05；n＝12.
实施例23评价化合物5a-h抑制P-选择素表达的活性
ELISA试剂盒(Rat P-selectin ELISA Kit；Wuhan Huamei Biotech Co.,Ltd.,武汉)用于测定。实施例22收集的小鼠血在12000rpm离心10min，取5μL血清用45μL ELISA试剂盒的稀释液稀释，取15μL该稀释的血清用285μL ELISA试剂盒的稀释液稀释，混匀，得到测定用的血清样本。
从1000pg/mL到0pg/mL设置8个浓度梯度，用ELISA试剂盒中的P-选择素标准品配制标准品溶液，绘制标准曲线。分别设置标准品孔和血清样本孔。每孔加100μL标准品及血清样本。37℃孵育2h。弃去液体，甩干。每孔加100μL生物素标记抗体工作液，覆上新板贴，37℃孵育1h。弃去孔内液体，甩干，洗板5次，每次用洗液200μL/孔浸泡2min，甩干。每孔加100μL辣根过氧化物酶标记亲和素工作液，覆上新板贴，37℃孵育1h。弃去孔内液体，甩干，洗板5次，每次用洗液200μL/孔浸泡2min，甩干。依序每孔加90μL底物溶液，37℃避光显色15-30min。依序每孔加50μ终止液L，终止反应。反应终止后5min内用酶标仪在 450nm波长依序测量各孔光密度值(OD值)。用标准曲线和血清样本孔的OD值计算血清内 P-选择素的浓度(均值±SD ng/mL)。实验数据采用t检验和方差分析,结果见表2。可以看出，化合物5a-h治疗的S180小鼠血清P-选择素浓度显著低于生理盐水治疗的S180小鼠血清P-选择素浓度。也就是说，化合物5a-h是P-选择素的有效抑制剂。本发明有突出的技术效果。
表2化合物5a-h治疗的S180小鼠血清P-选择素浓度(Mean±SD，ng/mL,n＝3)
a)与生理盐水比p<0.01；n＝12。
Claims (4)
1.下式的(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉，
式中AA为Gly残基,L-Ala残基,L-Leu残基,L-Ile残基,L-Pro残基,L-Phe残基,L-Val残基或L-Lys残基。
2.权利要求1所述的(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉的制备方法，该方法包括：
(1)在1M的H2SO4水溶液中L-Trp和乙醛发生Pictet-Spengler缩合制备(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸；
(2)(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧酸先在SOCl2中酰氯化,后与甲醇反应制备(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧甲酯；
(3)用氢化铝锂还原(1S,3S)-1-甲基-1,2,3,4-四氢-β-咔啉-3-羧甲酯制备(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉；
(4)用(Boc)2O与(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉制备N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]；
(5)制备Boc-AA-Val-OBzl；
(6)在钯碳的催化下将Boc-AA-Val-OBzl氢解制备Boc-AA-Val；
(7)N-Boc-[(1S,3S)-1-甲基-3-羟甲基-1,2,3,4-四氢-β-咔啉]与Boc-AA-Val反应制备N-Boc-[(1S,3S)-1-甲基-3-(Boc-AA-Val-氧甲基)-1,2,3,4-四氢-β-咔啉]；
(8)N-Boc-[(1S,3S)-1-甲基-3-(Boc-AA-Val-氧甲基)-1,2,3,4-四氢-β-咔啉]在4M氯化氢的乙酸乙酯溶液中脱Boc制备(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉。
3.权利要求1所述的(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉在制备抗肿瘤药物中的应用。
4.权利要求1所述的(1S,3S)-1-甲基-3-(AA-Val-甲氧基)-2,3,4,9-四氢-β-咔啉在制备P-选择素抑制剂中的应用。
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