CN113087712B - L-氨基酸-6-胶霉毒素酯三氟乙酸盐及其制备方法 - Google Patents

L-氨基酸-6-胶霉毒素酯三氟乙酸盐及其制备方法 Download PDF

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CN113087712B
CN113087712B CN202110386977.1A CN202110386977A CN113087712B CN 113087712 B CN113087712 B CN 113087712B CN 202110386977 A CN202110386977 A CN 202110386977A CN 113087712 B CN113087712 B CN 113087712B
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gliotoxin
amino acid
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cancer
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CN113087712A (zh
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单丽红
刘宏民
安雪
李召翔
孙莹莹
赵瑞云
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Zhengzhou University
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    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
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Abstract

本发明涉及药物化学及微生物制药领域，公开了一种以烟曲霉菌的次级代谢产物胶霉毒素为母核，设计合成的L‑氨基酸‑6‑胶霉毒素酯三氟乙酸盐类化合物（通式I）及其制备方法。其制备方法：（1）以胶霉毒素为起始原料，在不破坏其活性中心二硫键的前提下，其6位羟基与N‑Boc‑氨基酸发生酯化反应，得到胶霉毒素6位氨基酸酯衍生物，然后在过量的三氟乙酸中脱除Boc保护基，成盐得到L‑氨基酸‑6‑胶霉毒素酯三氟乙酸盐类化合物。该类化合物对组蛋白赖氨酸去甲基化酶（LSD1）的抑制活性明显优于其母体胶霉毒素，可用于制备抗肿瘤药物，应用于临床治疗人食道癌、胃癌、肺癌、结直肠癌以及乳腺癌等疾病。其通式如下：

Description

L-氨基酸-6-胶霉毒素酯三氟乙酸盐及其制备方法
技术领域
本发明涉及药物化学及微生物制药领域，具体涉及表聚多硫代二酮哌嗪类天然产物胶霉毒素的衍生物：L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物及其制备方法和应用。
背景技术
多硫代二酮哌嗪类化合物(Epipolythiodioxopiperazines，ETPs)，到目前为止仅在真菌的代谢产物中发现过，是一大类结构多样的生物活性次级代谢产物。其特征是具有二酮哌嗪骨架，存在一个二硫或多硫键的六元环，二硫或多硫官能团是其生物活性的关键部位。胶霉毒素(Gliotoxin，GT)作为第一个被报道的ETP类化合物，于1932年首次从真菌的代谢产物中分离出来。由于其具有抗病毒、抗菌、免疫抑制、酶抑制、血小板聚集抑制和抗肿瘤等多种生物活性，已成为药理学中研究最多的ETPs之一。文献报道胶霉毒素对肿瘤细胞有很强的抑制作用，被认为是具有抗肿瘤活性的先导化合物。但由于胶霉毒素产量甚微，且具有一定的毒副作用，限制了它的应用开发。
Figure BDA0003015475910000011
发明人在文献调研和总结前人工作的基础上，通过一系列方法从本地土壤中分离出一株烟曲霉菌ZSS02(Aspergillus fumigatus ZSS02)，从其次级代谢产物中提取分离得到胶霉毒素，通过进一步优化其发酵条件，使胶霉毒素收率达到208mg/L，远远高于文献报道的收率。
ETP类化合物具有优越的药理活性，二硫键及多硫键结构作为此类化合物抗肿瘤活性中心已经得到证实，但是其较差的稳定性和较大的毒副作用制约了其在临床上的应用和发展。此外，有关此类化合物的衍生物合成及构效关系研究也相对较为薄弱。因此，在胶霉毒素结构的基础上设计合成系列衍生物并探讨其构效关系，并进一步从中寻找化学性质更稳定，抗肿瘤活性更高的新的化学实体，具有重要的理论意义和实际应用价值。
组蛋白赖氨酸去甲基化酶(lysine specific demethylase 1，LSD1)于2004年首次被发现，是一种依赖黄素的去甲基化酶，也是第一个组蛋白去甲基化酶。现已证实LSD1在多种肿瘤中过表达，并与肿瘤的侵袭性特征和较差的预后相关。在动物模型中，LSD1的药理抑制和基因耗竭已被证明可以抑制癌细胞的增殖、分化、侵袭和转移。因此，LSD1是一个重要的致癌驱动因素，是潜在的预后不良的肿瘤生物标志物和治疗靶点。
发明内容
本发明目的在于提供一系列L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物，一方面通过提高对LSD1的抑制活性以提高其抗肿瘤作用，一方面增加其稳定性并降低毒副作用，为其在临床上的应用提供可能。
本发明的另一目的在于提供其制备方法以及其在制备抗肿瘤药物中的应用。
为实现本发明目的，本发明分别将胶霉毒素6位上的羟基与N-Boc-L-氨基酸酯化成盐得到系列衍生物，此方法可提高其稳定性，降低其毒副作用，同时增强其对LSD1的抑制活性。
本发明提供的L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物的结构通式如下所示：
Figure BDA0003015475910000021
其中R2为氢或C1-C3的烷基；R1为氢，C1-C10的直链或支链烷基；或R1为含N的5-10元饱和杂环或杂环芳基；或R1为含硫的C1-C10直链或支链烷基；或R1为C1-C5烷基取代的氨基；或R1为苯基或苯基取代的C1-C3烷基；或R1、R2相连接后为-(CH2)n-，n＝2-7，且R2与氮原子相连。
优选：R2为氢，甲基；R1为氢，C1-C5的直链或支链烷基；或R1为含N的5-10元杂环芳基；或R1为含硫的C1-C5直链或支链烷基；或R1为C1-C5烷基取代的氨基；或R1为苯基或苯基取代的C1-C3烷基；或R1、R2相连接后为-(CH2)n-，n＝3-5，且R2与氮原子相连。
更优选：R2为氢，甲基；R1为如下基团之一：
Figure BDA0003015475910000031
或R1、R2为-(CH2)4-，即通式I为下式：
Figure BDA0003015475910000032
为了制剂需要，本发明还需要进一步保护通式I化合物可药用的盐，可接受的盐为有机酸盐、无机酸盐、有机碱盐或无机碱盐，其中有机酸包括乙酸、甲磺酸、柠檬酸、富马酸、马来酸、乙醇酸、乳酸、水杨酸、琥珀酸、对甲苯磺酸、酒石酸、甲磺酸、丙二酸、硫辛酸；无机酸包括，盐酸、氢溴酸、硝酸、硫酸、磷酸；有机碱包括葡甲胺、氨基葡萄糖；无机碱包括碱金属钠、钾、钡、钙、镁、锌的碱性化合物。
本发明所述的L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物通过如下合成路线得到：
Figure BDA0003015475910000033
a、将胶霉毒素(Gliotoxin，1)溶解于二氯甲烷中，以DCC(二环己基碳二亚胺和DMAP(4-二甲氨基吡啶)作为催化剂，与各种N-Boc-氨基酸发生酯化反应，柱色谱纯化得到中间产物2a-2p；
b、将上步所得中间产物2a-2p溶解于二氯甲烷中，加入过量的三氟乙酸使其脱掉Boc保护基并成盐，将反应体系在旋转蒸发仪上进行浓缩后加入异丙醚使其析出得到产物3a-3p(通式I)。
Figure BDA0003015475910000041
R1、R2同上。
本发明创新点及优点：以LSD1为靶点设计合成了系列胶霉毒素衍生物，体外酶活性测定结果表明：所合成的衍生物对LSD1的抑制活性均明显高于母体胶霉毒素。可将其用于抗肿瘤药物的开发，应用于临床治疗人食道癌、胃癌、肺癌、结直肠癌以及乳腺癌等疾病，具有很好的应用前景。不仅扩大了ETP类化合物的结构类型，还为进一步研究其抗肿瘤作用机制打下基础，对开发新型抗肿瘤药物具有重要的理论意义和实际应用价值。
具体实施方式
通过以下具体实例进一步说明本发明，但应注意本发明的范围并不接受这些实施例的任何限制。
实施例1：
Figure BDA0003015475910000042
称取200mg胶霉毒素溶解于4mL二氯甲烷中，在常温下搅拌并加入1.1eq的N-Boc-L-肌氨酸、1eq的DCC和0.1eq的DMAP,反应过程中每30min进行TLC监测，1.5-2h后反应完全。向反应体系中加入50mL二氯甲烷稀释，将反应体系用饱和的NH4Cl溶液洗涤三次，用二氯甲烷对水相反萃一次。合并有机相，并用饱和的NaCl溶液洗涤三次，用无水硫酸镁干燥12h，浓缩有机相得粗提取物，使用柱色谱分离纯化得淡黄色固体。用二氯甲烷溶解上述产物，冰浴下加入300μL三氟乙酸搅拌，反应过程中每30min进行TLC监测，2-3h后反应完全，用旋转蒸发仪除去体系中的溶剂，常温条件下加入异丙醚搅拌过夜，经真空泵抽滤，收集终产物并用异丙醚洗涤，得淡黄色固体3a，产率61％，m.p.:120.0-121.8℃；IR(KBr,cm-1):3432,1765,1682,1384,1203,1136,799,721,668.1H NMR(400MHz,DMSO-d6)δ8.54(s,2H),6.05(dt,J＝5.8,2.9Hz,1H),5.97(ddd,J＝8.5,5.0,2.8Hz,1H),5.65(d,J＝9.7Hz,1H),5.49(s,1H),5.17(d,J＝12.7Hz,1H),5.06(d,J＝12.7Hz,1H),4.86(d,J＝13.2Hz,1H),4.56(d,J＝13.1Hz,1H),4.23(d,J＝7.6Hz,1H),3.65(d,J＝17.8Hz,1H),3.23(s,1H),3.18(d,J＝2.7Hz,1H),3.10(s,3H),1.44(d,J＝7.2Hz,3H).13C NMR(100MHz,DMSO-d6)δ168.84,164.92,163.06,158.40,132.29,129.48,123.62,119.03,116.17,75.82,75.39,72.62,69.49,60.93,47.76,35.73,28.02,15.57.ESI-HRMS:m/z cacld.For C16H20N3O5S2[M-CF3COO-]+:398.0839,found 398.0846。
实施例2：
Figure BDA0003015475910000051
用N-Boc-甲基-L-丙氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3b，产率65％，m.p.:125.9-126.8℃；IR(KBr,cm-1):3429,1761,1685,1420,1383,1202,1062,798,722,703,672.1H NMR(400MHz,DMSO-d6)δ9.33(s,2H),6.06(dt,J＝5.8,3.1Hz,1H),5.98(ddd,J＝8.1,4.8,2.7Hz,1H),5.65(d,J＝9.8Hz,1H),5.18(d,J＝12.7Hz,1H),5.08(d,J＝12.7Hz,1H),4.86(d,J＝13.1Hz,1H),4.56(d,J＝13.1Hz,1H),4.25(q,J＝7.1Hz,1H),3.23(s,1H),3.18(s,1H),3.10(s,3H),2.62(s,3H),1.67(ddq,J＝39.4,8.2,3.9Hz,1H),1.48(d,J＝7.2Hz,3H).13C NMR(100MHz,DMSO-d6)δ168.17,164.92,163.06,158.13,132.21,129.42,123.66,119.08,118.09,75.79,75.32,72.63,69.45,61.04,54.92,35.71,30.43,28.03,13.63.ESI-HRMS:m/z cacld.For C17H22N3O5S2[M-CF3COO-]+:412.0995,found 412.1100。
实施例3：
Figure BDA0003015475910000061
用N-Boc-L-2-氨基丁酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3c，产率48％，m.p.:121.6-122.5℃；IR(KBr,cm-1):3433,2928,1762,1685,1414,1379,1356,1203,1137,1061,835,799,722.1H NMR(400MHz,DMSO-d6)δ8.52(s,3H),5.98(dd,J＝5.1,2.8Hz,1H),5.90(ddd,J＝9.7,5.0,2.8Hz,1H),5.58(d,J＝9.8Hz,1H),5.41(d,J＝18.3Hz,1H),5.12(d,J＝12.8Hz,1H),4.99(d,J＝12.7Hz,1H),4.79(d,J＝13.1Hz,1H),4.49(dd,J＝13.0,2.8Hz,1H),3.61-3.54(m,1H),3.16(d,J＝1.7Hz,1H),3.11(d,J＝1.6Hz,1H),3.03(s,3H),1.84–1.76(m,2H),0.91(t,J＝7.5Hz,3H).13C NMR(100MHz,DMSO-d6)δ168.32,164.91,163.05,158.12,132.27,129.46,123.63,119.04,114.97,75.81,75.31,72.62,69.48,61.01,52.89,35.73,28.04,23.33,8.99.ESI-HRMS:m/z cacld.ForC17H22N3O5S2[M-CF3COO-]+:412.0995,found 412.1001。
实施例4：
Figure BDA0003015475910000071
用N-Boc-L-正缬氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3d，产率45％，m.p.:102.2-103.2℃；IR(KBr,cm-1):3430,2966,1762,1684,1419,1381,1203,1135,1062,799,722.1H NMR(400MHz,DMSO-d6)δ8.49(s,3H),5.96(d,J＝4.4Hz,1H),5.92-5.86(m,1H),5.57(d,J＝9.8Hz,1H),5.42(s,1H),5.10(d,J＝12.7Hz,1H),4.97(d,J＝12.7Hz,1H),4.77(d,J＝13.2Hz,1H),4.47(d,J＝13.1Hz,1H),4.08(t,J＝6.1Hz,1H),3.57(d,J＝17.9Hz,1H),3.12(d,J＝18.6Hz,1H),3.01(s,3H),1.74-1.65(m,2H),1.38-1.25(m,2H),0.80(t,J＝7.4Hz,3H).13C NMR(100MHz,DMSO-d6)δ168.42,164.91,163.05,157.99,132.29,129.46,123.63,119.03,118.39,75.78,75.30,72.62,69.50,61.07,51.69,35.74,31.96,28.00,17.53,13.42.ESI-HRMS:m/z cacld.For C18H24N3O5S2[M-CF3COO-]+:426.1152,found 426.1156。
实施例5：
Figure BDA0003015475910000072
用N-Boc-L-缬氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3e，产率68％，m.p.:102.6-103.5℃；IR(KBr,cm-1):3432,1685,668.1H NMR(400MHz,DMSO-d6)δ8.59-8.51(m,3H),6.05(dd,J＝5.2,2.8Hz,1H),5.97(ddd,J＝9.8,5.0,2.8Hz,1H),5.65(d,J＝10.0Hz,1H),5.46(s,1H),5.21(d,J＝12.8Hz,1H),5.06(d,J＝12.8Hz,1H),4.86(d,J＝13.4Hz,1H),4.56(d,J＝13.3Hz,1H),4.08(d,J＝4.4Hz,1H),3.65(d,J＝17.7Hz,1H),3.23(d,J＝3.3Hz,1H),3.18(d,J＝1.6Hz,1H),3.10(s,3H),1.02(dd,J＝6.9,4.7Hz,6H).13C NMR(100MHz,DMSO-d6)δ167.82,164.92,163.03,158.09,132.29,129.47,123.63,119.03,114.93,75.80,75.26,72.62,69.50,61.18,57.20,35.74,29.43,28.11,17.89,17.61.ESI-HRMS:m/z cacld.For C18H24N3O5S2[M-CF3COO-]+:426.1152,found 426.1156。
实施例6：
Figure BDA0003015475910000081
用N-Boc-甲基-L-缬氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3f，产率64％，m.p.:122.7-123.5℃；IR(KBr,cm-1):3429,2973,1758,1684,1467,1419,1382,1355,1202,1137,1061,799,721.1H NMR(400MHz,DMSO-d6)δ9.09(s,2H),5.82(dt,J＝5.7,2.9Hz,1H),5.74(ddd,J＝8.2,4.9,2.7Hz,1H),5.42(d,J＝9.8Hz,1H),5.23(s,1H),5.04(d,J＝12.8Hz,1H),4.89(d,J＝12.8Hz,1H),4.62(d,J＝13.1Hz,1H),4.32(d,J＝13.1Hz,1H),3.88(d,J＝4.0Hz,1H),3.42(d,J＝17.8Hz,1H),3.00(s,1H),2.95(d,J＝2.7Hz,1H),2.87(s,3H),2.27(p,J＝1.8Hz,2H),2.05(tt,J＝7.1,3.6Hz,1H),0.84(d,J＝7.0Hz,3H),0.76(d,J＝6.8Hz,3H).13C NMR(100MHz,DMSO-d6)δ166.87,164.92,163.09,158.02,132.28,129.48,123.64,119.04,118.18,75.74,75.17,72.63,69.48,65.29,61.24,35.73,32.12,28.82,28.13,18.77,16.79.ESI-HRMS:m/z cacld.For C19H26N3O5S2[M-CF3COO-]+:440.1308,found 440.1313。
实施例7：
Figure BDA0003015475910000091
用N-Boc-L-正亮氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3g，产率53％，m.p.:163.4-164.3℃；IR(KBr,cm-1):3429,2929,1761,1684,1420,1379,1203,1136,722,668.1H NMR(400MHz,DMSO-d6)δ8.57(s,3H),6.03(s,1H),5.98(dt,J＝8.1,3.5Hz,1H),5.66(d,J＝9.8Hz,1H),5.18(d,J＝12.7Hz,1H),5.06(d,J＝12.7Hz,1H),4.85(d,J＝13.1Hz,1H),4.56(d,J＝13.0Hz,1H),4.16(t,J＝6.0Hz,1H),3.23(s,1H),3.18(s,1H),3.10(s,3H),1.82(dt,J＝10.6,5.5Hz,2H),1.41(ddd,J＝13.7,9.1,5.9Hz,1H),1.36-1.20(m,4H),0.86(t,J＝7.0Hz,3H).13C NMR(100MHz,DMSO-d6)δ168.36,164.91,163.05,158.13,132.18,129.39,123.65,119.07,117.93,75.77,75.30,72.61,69.47,61.03,51.84,35.72,29.57,28.00,26.09,21.63,13.48.ESI-HRMS:m/z cacld.For C19H26N3O5S2[M-CF3COO-]+:440.1308,found 440.1311。
实施例8：
Figure BDA0003015475910000092
用N-Boc-L-叔亮氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3h，产率47％，m.p.:112.8-113.7℃；IR(KBr,cm-1):329,2919,1757,1686,1383,1178.1HNMR(400MHz,DMSO-d6)δ8.54(s,3H),6.05(dt,J＝5.9,3.0Hz,1H),5.97(ddd,J＝8.2,5.0,2.8Hz,1H),5.65(d,J＝9.8Hz,1H),5.19(d,J＝12.9Hz,1H),5.05(d,J＝12.8Hz,1H),4.85(d,J＝13.2Hz,1H),4.58-4.52(m,1H),3.94(s,1H),3.62(d,J＝5.8Hz,1H),3.22(s,1H),3.18(d,J＝1.6Hz,1H),3.10(s,3H),1.06(s,9H).13C NMR(100MHz,DMSO-d6)δ167.56,164.89,163.03,158.04,132.29,129.48,123.63,119.03,118.33,75.76,75.22,72.62,69.51,61.41,60.68,35.76,33.42,28.19,26.12.ESI-HRMS:m/z cacld.For C19H26N3O5S2[M-CF3COO-]+:440.1308,found 440.1312。
实施例9：
Figure BDA0003015475910000101
用N-Boc-L-亮氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3i，产率64％，m.p.:105.8-106.7℃；IR(KBr,cm-1):3732,3703,3434,2963,1763,1685,1420,1380,1202,1136,1062,799,721,668,649.1H NMR(400MHz,DMSO-d6)δ8.48(s,3H),5.98(dt,J＝5.8,3.0Hz,1H),5.90(ddd,J＝8.2,4.9,2.7Hz,1H),5.58(d,J＝9.7Hz,1H),5.43(s,1H),5.12(d,J＝12.7Hz,1H),4.97(d,J＝12.7Hz,1H),4.78(d,J＝13.1Hz,1H),4.49(d,J＝13.1Hz,1H),3.16(s,1H),3.11(d,J＝2.5Hz,1H),3.03(s,3H),1.63(dddd,J＝48.7,28.2,13.7,7.0Hz,4H),0.84(t,J＝6.2Hz,6H).13C NMR(100MHz,DMSO-d6)δ168.61,164.90,163.06,158.36,132.25,129.43,123.64,119.04,114.94,75.75,75.27,72.62,69.48,61.08,50.47,35.74,27.99,23.73,22.04,21.90.ESI-HRMS:m/z cacld.For C19H26N3O5S2[M-CF3COO-]+:440.1308,found 440.1312。
实施例10：
Figure BDA0003015475910000111
用N-Boc-L-蛋氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3j，产率57％，m.p.:129.2-130.1℃；IR(KBr)νmax:3726,3433,2920,1762,1682,1427,1382,1201,1136,799,720,668,649cm-1.1H NMR(400MHz,DMSO-d6)δ(ppm):8.39(s,3H),5.80(d,J＝4.0Hz,1H),5.73(dt,J＝8.2,3.4Hz,1H),5.41(d,J＝9.6Hz,1H),4.96(d,J＝12.7Hz,1H),4.81(d,J＝12.7Hz,1H),4.60(d,J＝13.1Hz,1H),4.31(d,J＝12.9Hz,1H),4.02(t,J＝6.3Hz,1H),3.40(d,J＝17.7Hz,1H),2.98(s,1H),2.93(d,J＝3.3Hz,1H),2.85(s,3H),2.26(p,J＝1.7Hz,2H),1.88-1.74(m,5H).13C NMR(100MHz,DMSO-d6)δ(ppm):168.02,164.88,163.08,158.38,132.26,129.46,123.62,119.04,117.99,75.75,75.29,72.62,69.50,61.19,50.87,35.74,29.48,28.14,28.05,14.17.ESI-HRMS:m/z cacld.For C18H24N3O5S3[M-CF3COO-]+:458.0873,found 458.0877。
实施例11：
Figure BDA0003015475910000112
用N-Boc-L-2-苯基甘氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3k，产率61％，m.p.:142.8-143.6℃；IR(KBr)νmax:3726,3703,3623,3431,2923,1762,1681,1187,1137,1062,720,679,668,650,617cm-1.1H NMR(400MHz,DMSO-d6)δ(ppm):9.04(s,3H),7.52-7.34(m,6H),5.96(dt,J＝5.6,3.1Hz,1H),5.89(ddd,J＝8.3,4.8,2.8Hz,1H),5.58(d,J＝9.5Hz,1H),5.40(d,J＝5.9Hz,1H),5.09(d,J＝12.6Hz,1H),4.91(dd,J＝12.7,3.6Hz,1H),4.78(d,J＝13.2Hz,1H),3.55(d,J＝17.8Hz,1H),3.08(d,J＝17.7Hz,1H),2.81(s,1H),2.62(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):167.33,164.81,162.86,158.34,132.28,131.95,129.74,129.44,128.96,128.38,123.62,118.97,115.11,75.82,75.21,72.53,69.56,61.29,55.20,35.67,27.48.ESI-HRMS:m/z cacld.For C21H22N3O5S2[M-CF3COO-]+:460.0995,found 460.1001。
实施例12：
Figure BDA0003015475910000121
用N-Boc-L-苯丙氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3l，产率65％，m.p.:130.6-131.4℃；IR(KBr)νmax:3439,2920,1762,1685,1383,1199,427cm-1.1H NMR(400MHz,DMSO-d6)δ(ppm):8.70(s,3H),7.35-7.25(m,5H),6.05(dt,J＝5.6,2.9Hz,1H),5.97(ddd,J＝7.9,4.8,2.7Hz,1H),5.65(d,J＝9.7Hz,1H),5.59–5.33(m,1H),5.17(d,J＝12.7Hz,1H),4.84(dd,J＝12.4,9.7Hz,2H),4.55(d,J＝13.0Hz,1H),4.47-4.42(m,1H),3.67-3.58(m,1H),3.21(d,J＝5.5Hz,1H),3.17(d,J＝4.6Hz,1H),3.13-3.08(m,1H),2.83(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):167.87,164.77,162.92,158.11,134.46,132.22,129.48,129.27,128.65,127.27,123.62,119.06,114.93,75.85,75.13,72.57,69.50,60.80,52.98,36.02,35.69,27.70.ESI-HRMS:m/z cacld.For C22H24N3O5S2[M-CF3COO-]+:474.1152,found 474.1155。
实施例13：
Figure BDA0003015475910000131
用N-Boc-甲基-L-苯丙氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3m，产率52％，m.p.:118.6-119.4℃；IR(KBr)νmax:3432,1681,1191,720,668cm-1.1HNMR(400MHz,DMSO-d6)δ(ppm):9.57(s,1H),7.47–7.14(m,6H),6.05(t,J＝3.8Hz,1H),6.01–5.92(m,1H),5.65(d,J＝9.8Hz,1H),5.19(d,J＝12.7Hz,1H),4.79(dd,J＝20.3,12.9Hz,2H),4.54(d,J＝13.0Hz,1H),4.46(dd,J＝9.6,4.7Hz,1H),3.35(d,J＝4.7Hz,1H),3.32(d,J＝4.8Hz,1H),3.20(s,1H),3.16(s,1H),3.07(dd,J＝13.9,9.6Hz,1H),2.74(s,3H),2.66(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):167.19,164.77,162.92,158.22,134.11,132.12,129.41,129.20,128.71,127.38,123.65,119.11,114.98,75.80,74.93,72.55,69.46,60.78,60.71,35.64,34.78,31.34,27.55.ESI-HRMS:m/z cacld.ForC23H26N3O5S2[M-CF3COO-]+:488.1308,found 488.1300。
实施例14：
Figure BDA0003015475910000132
用N-Boc-L-高苯丙氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3n，产率51％，m.p.:113.5-114.4℃；IR(KBr)νmax:3426,2924,1763,1685,1428,1379,1354,1201,1063,799,721,701,425cm-1.1H NMR(400MHz,DMSO-d6)δ(ppm):8.68(s,3H),7.31(t,J＝7.5Hz,2H),7.21(dd,J＝7.7,5.5Hz,3H),6.05(dt,J＝5.9,3.0Hz,1H),5.97(ddd,J＝8.0,4.9,2.8Hz,1H),5.65(d,J＝9.6Hz,1H),5.22(d,J＝12.7Hz,1H),5.07(d,J＝12.7Hz,1H),4.85(dd,J＝12.8,3.0Hz,1H),4.55(d,J＝12.8Hz,1H),4.20(d,J＝6.2Hz,1H),3.65(d,J＝17.8Hz,1H),3.21(d,J＝18.1Hz,1H),3.10(s,3H),2.82-2.60(m,3H),2.11(td,J＝12.1,11.2,5.9Hz,2H).13C NMR(100MHz,DMSO-d6)δ(ppm):168.16,164.91,163.09,140.12,132.24,129.45,128.49,128.15,126.24,123.61,119.04,75.76,75.31,72.60,69.51,61.15,51.60,35.74,31.83,30.14,28.08.ESI-HRMS:m/z cacld.For C25H26F3N3O7S2[M-CF3COO-]+:488.1308,found 488.1313。
实施例15：
Figure BDA0003015475910000141
用N-Boc-L-色氨酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3o，产率63％，m.p.:158.8-159.7℃；IR(KBr)νmax:3429,2922,1762,1676,1457,1431,1356,1194,799,747,721cm-1.1H NMR(400MHz,DMSO-d6)δ(ppm):11.01(d,J＝2.6Hz,1H),8.58(s,3H),7.45(d,J＝7.9Hz,1H),7.29(d,J＝8.0Hz,1H),7.18(d,J＝2.4Hz,1H),7.02(t,J＝7.5Hz,1H),6.94(t,J＝7.4Hz,1H),5.95(q,J＝3.5Hz,1H),5.88(dt,J＝8.3,3.8Hz,1H),5.56(d,J＝9.7Hz,1H),5.43(s,1H),5.08(d,J＝12.7Hz,1H),4.73(d,J＝13.1Hz,1H),4.65(d,J＝12.7Hz,1H),4.27(t,J＝7.1Hz,1H),3.57-3.49(m,1H),3.21(d,J＝7.1Hz,2H),3.08(d,J＝17.8Hz,1H),2.64(s,3H),0.95(d,J＝6.0Hz,1H).13C NMR(100MHz,DMSO-d6)δ(ppm):168.31,164.76,162.92,158.43,136.30,132.27,129.48,126.75,124.77,123.61,121.26,119.02,118.71,117.85,115.21,111.62,106.43,75.82,75.13,72.59,69.48,60.85,52.63,35.69,27.41,26.60.ESI-HRMS:m/z cacld.For C24H25N4O5S2[M-2CF3COO--H+]+:513.1255,found513.1263。
实施例16：
Figure BDA0003015475910000151
用N-Boc-L-2-哌啶甲酸代替N-Boc-L-肌氨酸，其他操作同实施例1，得淡黄色固体3p，产率51％，m.p.:142.6-143.5℃；IR(KBr)νmax:3435,1754,1684,1267,1202,1129,720cm-1.1H NMR(400MHz,DMSO-d6)δ(ppm):9.44(s,2H),6.05(dd,J＝5.1,2.8Hz,1H),5.97(ddd,J＝8.1,4.9,2.8Hz,1H),5.65(d,J＝9.8Hz,1H),5.51(s,1H),5.19(d,J＝12.7Hz,1H),5.08(d,J＝12.8Hz,1H),4.86(d,J＝13.1Hz,1H),4.56(d,J＝13.2Hz,1H),4.25(dd,J＝11.5,3.4Hz,1H),3.30(d,J＝12.3Hz,1H),3.24-3.18(m,1H),3.09(s,3H),3.01-2.94(m,1H),2.10(d,J＝13.3Hz,1H),1.75(dd,J＝19.2,9.0Hz,3H),1.59(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):167.70,164.93,163.02,158.37,132.30,129.48,123.63,119.03,115.52,75.85,75.35,72.62,69.50,60.93,55.35,43.34,35.72,28.03,25.51,21.11,21.03.ESI-HRMS:m/z cacld.For C19H24N3O5S2[M-CF3COO-]+:438.1152,found 438.1156。
实施例17：本发明合成的新型L-氨基酸-6-胶霉毒素酯三氟乙酸盐系列衍生物
实验方法
1.LSD1重组蛋白的纯化及浓度测定
将LSD1蛋白用Ni-NTA亲和填料纯化后，利用超滤管反复离心使蛋白除盐，再用离子交换柱进一步纯化后低温离心浓缩蛋白。对于纯化后的蛋白，用Braford蛋白定量法测定LSD1蛋白的浓度。
2.LSD1抑制活性的测定过程
(1)蛋白与底物进行孵育：实验对每板设定空白对照孔和100％孔，空白孔中只有蛋白，不加入化合物样品底物及H3K4me2，分别用1.25μLDMSO与2.5μL缓冲液代替。100％孔中加入蛋白和H3K4me2底物反应，不加入化合物样品，用1.25μL DMSO进行替代。先将终浓度0.25μM重组蛋白与不同浓度的化合物(包括阳性对照ORY-1001)及HEPES缓冲溶液加入96孔板中室温孵育10 min，再加入2.5μL多肽底物H3K4me2于HEPES缓冲溶液中37℃恒温孵育30min，摇床转速160 rpm。
(2)加入检测相关试剂：在孔中加入0.1μL Amplex Red溶液及1μL辣根过氧化酶溶液的混合溶液室温孵育5 min。
(3)酶标仪读数：以吸收光535 nm，发射光595 nm检测生成的过氧化氢量。
(4)化合物对LSD1蛋白抑制率的计算公式：
抑制率(％)＝(百分百组-实验组)/(百分百组-空白组)×100％。
表1本发明化合物3a-3p的抑制活性
Figure BDA0003015475910000161

Claims (3)

1.L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物，其特征在于，具有通式Ⅰ所示结构：
Figure FDA0003408680230000011
其中，R2为氢，甲基；R1为如下基团之一：
Figure FDA0003408680230000012
或R1、R2相连后为-(CH2)4-，即化学式为：
Figure FDA0003408680230000013
2.如权利要求1所述的L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物，其特征在于，选如下化合物：
Figure FDA0003408680230000014
Figure FDA0003408680230000021
3.如权利要求1或2所述的L-氨基酸-6-胶霉毒素酯三氟乙酸盐类化合物在制备抗癌药物中的应用，其特征在于，以其为活性成份，制备用于治疗人食道癌、胃癌、肺癌、结直肠癌或乳腺癌药物。
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