CN113372335B - 一种含有1,2,4-三唑硫醚的苯丙氨酸衍生物及其制备方法与应用 - Google Patents
一种含有1,2,4-三唑硫醚的苯丙氨酸衍生物及其制备方法与应用 Download PDFInfo
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- CN113372335B CN113372335B CN202110759172.7A CN202110759172A CN113372335B CN 113372335 B CN113372335 B CN 113372335B CN 202110759172 A CN202110759172 A CN 202110759172A CN 113372335 B CN113372335 B CN 113372335B
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- 150000002993 phenylalanine derivatives Chemical class 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- -1 1,2, 4-triazole thioether Chemical class 0.000 title abstract description 48
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- 239000003814 drug Substances 0.000 claims description 14
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- FSIOTXJNQYVARL-UHFFFAOYSA-N [N+]=1(NC=NC=1)[S-] Chemical compound [N+]=1(NC=NC=1)[S-] FSIOTXJNQYVARL-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 5
- VRPQCVLBOZOYCG-UHFFFAOYSA-N 1-isothiocyanato-4-methoxybenzene Chemical compound COC1=CC=C(N=C=S)C=C1 VRPQCVLBOZOYCG-UHFFFAOYSA-N 0.000 claims description 5
- QJNNHJVSQUUHHE-UHFFFAOYSA-N 2-Methylindole-3-acetic acid Chemical compound C1=CC=C2C(CC(O)=O)=C(C)NC2=C1 QJNNHJVSQUUHHE-UHFFFAOYSA-N 0.000 claims description 5
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- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 5
- 229940125782 compound 2 Drugs 0.000 claims description 4
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- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 3
- SWVMLNPDTIFDDY-FVGYRXGTSA-N methyl (2s)-2-amino-3-phenylpropanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CC1=CC=CC=C1 SWVMLNPDTIFDDY-FVGYRXGTSA-N 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 23
- 239000011734 sodium Substances 0.000 description 22
- ACDFWSNAQWFRRF-VWLOTQADSA-N (2s)-n-methyl-2-[[2-(2-methyl-1h-indol-3-yl)acetyl]amino]-n,3-diphenylpropanamide Chemical compound C([C@@H](C(=O)N(C)C=1C=CC=CC=1)NC(=O)CC=1C2=CC=CC=C2NC=1C)C1=CC=CC=C1 ACDFWSNAQWFRRF-VWLOTQADSA-N 0.000 description 18
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- JTLAIKFGRHDNQM-UHFFFAOYSA-N 1-bromo-2-fluoroethane Chemical compound FCCBr JTLAIKFGRHDNQM-UHFFFAOYSA-N 0.000 description 1
- NIEGKADUBXVLHF-UHFFFAOYSA-N 1-bromo-2-methylsulfonylethane Chemical compound CS(=O)(=O)CCBr NIEGKADUBXVLHF-UHFFFAOYSA-N 0.000 description 1
- WMDHQEHPOVOEOG-UHFFFAOYSA-N 2-(2-bromoethyl)-1,3-dioxane Chemical compound BrCCC1OCCCO1 WMDHQEHPOVOEOG-UHFFFAOYSA-N 0.000 description 1
- OAVCWZUKQIEFGG-UHFFFAOYSA-O 2-(5-methyl-2H-tetrazol-1-ium-1-yl)-1,3-thiazole Chemical compound CC1=NN=N[NH+]1C1=NC=CS1 OAVCWZUKQIEFGG-UHFFFAOYSA-O 0.000 description 1
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- UZGLOGCJCWBBIV-UHFFFAOYSA-N 3-(chloromethyl)pyridin-1-ium;chloride Chemical compound Cl.ClCC1=CC=CN=C1 UZGLOGCJCWBBIV-UHFFFAOYSA-N 0.000 description 1
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- ZDHKVKPZQKYREU-UHFFFAOYSA-N 4-(chloromethyl)pyridine;hydron;chloride Chemical compound Cl.ClCC1=CC=NC=C1 ZDHKVKPZQKYREU-UHFFFAOYSA-N 0.000 description 1
- ZIESYFUVRJDNNW-UHFFFAOYSA-N 5-(chloromethyl)-1,3-thiazole;hydrochloride Chemical compound Cl.ClCC1=CN=CS1 ZIESYFUVRJDNNW-UHFFFAOYSA-N 0.000 description 1
- 125000004070 6 membered heterocyclic group Chemical group 0.000 description 1
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- PQUSVJVVRXWKDG-UHFFFAOYSA-N methyl 2-bromo-2-methylpropanoate Chemical compound COC(=O)C(C)(C)Br PQUSVJVVRXWKDG-UHFFFAOYSA-N 0.000 description 1
- YDCHPLOFQATIDS-UHFFFAOYSA-N methyl 2-bromoacetate Chemical compound COC(=O)CBr YDCHPLOFQATIDS-UHFFFAOYSA-N 0.000 description 1
- ACEONLNNWKIPTM-UHFFFAOYSA-N methyl 2-bromopropanoate Chemical compound COC(=O)C(C)Br ACEONLNNWKIPTM-UHFFFAOYSA-N 0.000 description 1
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- NYAZXHASVIWIRJ-UHFFFAOYSA-N nitridosulfidocarbon(.) Chemical compound [S]C#N NYAZXHASVIWIRJ-UHFFFAOYSA-N 0.000 description 1
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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Abstract
本发明公开了一种含有1,2,4‑三唑硫醚的苯丙氨酸衍生物及其制备方法和应用。所述化合物具有通式I所示的结构。本发明还涉及含有式I结构化合物的药物组合物。活性筛选实验显示本发明化合物具有很好的抗HIV‑1/2活性，因此本发明还提供上述化合物在制备抗艾滋病药物中的应用。
Description
技术领域
本发明属于有机化合物合成与医药应用技术领域，具体涉及一种含有1,2,4-三唑硫醚的苯丙氨酸衍生物及其制备方法与应用。
背景技术
艾滋病(Acquired Immune Deficiency Syndrome，AIDS)是由人免疫缺陷病毒(Human Immunodeficiency Virus，HIV)引起的破坏人体免疫系统的传染性疾病。HIV属于RNA逆转录病毒，根据基因差异分为HIV-1和HIV-2两种亚型，全球流行的主要亚型为HIV-1；HIV-2主要在西非和西欧流行，但随着全球化的不断发展，HIV-2感染的风险在不断增加，现已在全球其他地区也发现有HIV-2感染病例，应引起足够的重视。当前，抗HIV药物仍然是防治艾滋病的有效武器，依据作用靶标的不同，主要分为逆转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂、融合抑制剂以及CCR5协同受体抑制剂。“高效抗逆转录疗法”(Highly ActiveAntiretroviral Therapy，HAART)在很大程度上延长了患者的生存时间，改善了患者的生存质量，但是耐药问题、药物毒副作用、潜伏感染以及长期服用药物的高额费用等问题大大降低了该疗法的功效，限制了该疗法的应用，进而迫使研究者研发新靶点、新机制、新结构的抗艾滋病药物。
HIV-1衣壳是由Gag前体蛋白的一部分剪切得到衣壳蛋白单元以后组装形成的。HIV-1衣壳蛋白在病毒复制的早期阶段和晚期阶段均发挥重要作用，已成为设计新型抗HIV-1药物热点。Pfizer公司通过高通量筛选发现能显著抑制HIV-1复制的小分子化合物PF-74，机制研究表明PF-74通过HIV-1衣壳蛋白结合干扰病毒正常的复制过程。尽管PF-74结构新颖、机制独特、靶点明确，但是，PF-74相对于目前已上市的抗HIV-1的药物疗效较低，活性在微摩尔级别。因此，研发更为高效的衣壳蛋白抑制剂成为近年来抗艾滋病药物研发领域的重要方向。
发明内容
本发明提供了一种含有1,2,4-三唑硫醚的苯丙氨酸衍生物及其制备方法与应用，本发明还提供了上述化合物的部分活性筛选结果及其用途。
本发明的技术方案如下：
一、含有1,2,4-三唑硫醚的苯丙氨酸衍生物
本发明的含有1,2,4-三唑硫醚的苯丙氨酸衍生物，具有如下通式Ⅰ所示的结构：
其中，
R1为卤素、氰基、酯基、甲氧基、氮甲基、烃基；
R2为卤素、氰基、酯基、甲氧基、氮甲基、烃基、硝基、氨基、酰胺基、磺酰胺基；
R3为C1-C6烷基、OC1-C6烷基、C2-C6烯基、C3-C6环烷基、OC3-C6环烷基、炔丙基、吡啶基、苯基、取代苯基、取代苄基、取代萘环、各种取代的六元杂环或各种取代的五元杂环；所述的取代基选自卤素、甲基、甲氧基、三氟甲基、乙酰基、三氟甲氧基、羟基、氰基、硝基、氨基、硼酸、甲砜基、甲磺酰基、N-叔丁氧羰基。
根据本发明优选的，
R1为H、F、Cl、Br、CN、OCH3、NCH3、CH3；
R2为OCH3、H、F、Cl、Br、I、CN、NCH3、CH3、CF3、NH2、NO2；
R3为
根据本发明进一步优选的，含有1,2,4-三唑硫醚的苯丙氨酸衍生物，是下列之一：
二、含有1,2,4-三唑硫醚的苯丙氨酸衍生物的制备方法
含有1,2,4-三唑硫醚的苯丙氨酸衍生物的制备方法，步骤包括：2-甲基吲哚-3-乙酸与L-苯丙氨酸甲酯盐酸盐1在二氯甲烷中发生酰胺缩合反应得到化合物2，化合物2与水合肼在乙醇中回流反应得到中间体3；随后化合物3与4-甲氧基苯基异硫氰酸酯在N,N-二异丙基乙胺的条件下反应得到化合物4，最后化合物4与不同的卤代物发生亲核取代反应得到终产物4(a-x)。
试剂及条件：(i)2-甲基吲哚-3-乙酸，O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸盐，N,N-二异丙基乙胺，二氯甲烷，0℃转至室温；(ii)水合肼，乙醇，80℃；(iii)4-甲氧基苯基异硫氰酸酯，N,N-二异丙基乙胺，N,N-二甲基甲酰胺，50℃；(iv)相应的卤代物，碳酸铯，N,N-二甲基甲酰胺，50℃。
其中，R3为：
三、含有1,2,4-三唑硫醚的苯丙氨酸衍生物的应用
本发明公开了含有1,2,4-三唑硫醚的苯丙氨酸衍生物抗HIV-1/2活性筛选结果及其作为HIV-1/2抑制剂的首次应用。通过实验证明本发明的含有1,2,4-三唑硫醚的苯丙氨酸衍生物可作为HIV-1/2抑制剂用于制备抗艾滋病药物。本发明还提供上述化合物在制备抗HIV-1/2药物中的应用。
目标化合物的抗HIV-1/2活性和毒性实验
对按照上述方法合成的一类含有1,2,4-三唑硫醚的苯丙氨酸衍生物进行了细胞水平的抗HIV-1/2活性和毒性测试，它们的抗HIV-1和HIV-2活性和毒性数据列于表1中，以文献报道的衣壳蛋白抑制剂PF-74为阳性对照。
由表1可以看出，本发明所提供的含有1,2,4-三唑硫醚的苯丙氨酸衍生物是一系列结构新颖的HIV抑制剂，绝大部分化合物表现出优秀的抑制HIV-1/2野生株的活性。其中，化合物4l(EC50＝0.59±0.08μM)和4r(EC50＝0.59±0.06μM)的对HIV-1野生株活性尤为突出，优于阳性对照PF-74(EC50＝0.79±0.09μM)；此外，化合物4i、4j、4o以及4v也表现出了与PF-74相当的抗HIV-1活性。对于HIV-2野生株，化合物4r(EC50＝2.69±0.17μM)的抗病毒活性也要优于PF-74(EC50＝3.79±0.39μM)；此外，4c、4d、4g、4j、4q、4s、4v等七个化合物表现出了与PF-74相当的抗HIV-2活性。因此该类化合物具有较大的价值，可作为制备抗HIV的候选药物加以利用开发。
因此，本发明所提供的含有1,2,4-三唑硫醚的苯丙氨酸衍生物可作为HIV-1/2抑制剂用于制备抗艾滋病药物。
一种抗HIV-1/2的药物组合物，含有上述的含有1,2,4-三唑硫醚的苯丙氨酸衍生物及其药学上可接受的盐与药用辅料，制成不同剂型的药物。
具体实施方式
通过下述实施例有助于理解本发明，但是不能限制本发明的内容。
实施例1：甲基(2-(2-甲基-1H-吲哚-3-基)乙酰基)-L-苯丙氨酸(2)的制备
将起始原料2-甲基吲哚-3-乙酸(1.06mmol，0.2g)加入到10mL的二氯甲烷中，然后向此溶液中加入O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸盐(1.59mmol，0.60g)。冰浴条件下搅拌0.5h，然后加入N,N-二异丙基乙胺(2.12mmol，0.34mL)和L-苯丙氨酸甲酯盐酸盐(1.16mmol，0.25g)，撤去冰浴，室温搅拌8h。待反应完毕，减压蒸除溶剂，然后向瓶内残留物中加入20mL饱和碳酸氢钠溶液，乙酸乙酯萃取(3×8mL)；分取有机层，加入1NHCl溶液20mL，乙酸乙酯萃取(3×8mL)；合并有机层，加饱和食盐水洗涤(3×25mL)，有机相用无水硫酸钠干燥；过滤，减压蒸干溶剂得到中间体化合物甲基(2-(2-甲基-1H-吲哚-3-基)乙酰基)-L-苯丙氨酸(2)的粗品0.23g，白色固体，产率61.3％，1H NMR(400MHz,DMSO-d6)δ10.74(s,1H,NH),8.24(d,J＝7.7Hz,1H,NH),7.33(d,J＝7.8Hz,1H,PhH),7.27–7.19(m,4H,PhH),7.16(d,J＝6.9Hz,2H,PhH),6.97(t,J＝7.4Hz,1H,PhH),6.88(t,J＝7.4Hz,1H,PhH),4.52–4.37(m,1H,CH),3.59(s,3H,CH3),3.45(s,2H,CH2),3.03(dd,J＝13.7,5.2Hz,1H,CH2),2.91(dd,J＝13.8,9.2Hz,1H,CH2),2.25(s,3H,CH3).EI-MS:m/z 351.4[M+H]+,373.2[M+Na]+,C21H22N2O3(350.16).
实施例2：(S)-N-(1-肼基-1-氧代-3-苯基丙烷-2-基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(3)的制备
将甲基(2-(2-甲基-1H-吲哚-3-基)乙酰基)-L-苯丙氨酸2(0.43mmol，0.15g)溶解于20mL无水乙醇中，然后添加水合肼(4.29mmol，0.13mL)，85℃回流反应10h，TLC监测反应完成，减压蒸除溶剂，用甲醇:二氯甲烷＝1:1溶解残余物，硅胶柱层析(甲醇:二氯甲烷＝1:30)，减压浓缩，真空干燥，最终得到(S)-N-(1-肼基-1-氧代-3-苯基丙烷-2-基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(3)。白色固体，产率：82.3％。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),9.17(s,1H,NH),7.96(d,J＝8.3Hz,1H,NH),7.27(d,J＝7.7Hz,1H,PhH),7.18(d,J＝8.1Hz,6H,PhH),6.94(t,J＝7.2Hz,1H,PhH),6.85(t,J＝7.2Hz,1H,PhH),4.43(t,J＝7.1Hz,1H,CH),4.20(s,2H,NH2),3.42(s,2H,CH2),2.89(dd,J＝13.6,4.9Hz,1H,CH2),2.77(dd,J＝13.7,9.3Hz,1H,CH2),2.22(s,3H,CH3).EI-MS:m/z 351.4[M+H]+,C20H22N4O2(350.17).
实施例3：(S)-N-(1-(5-巯基-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4)
将(S)-N-(1-肼基-1-氧代-3-苯基丙烷-2-基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺3(0.19mmol，0.065g)、4-甲氧基苯基异硫氰酸酯(0.19mmol，0.028mL)和N,N-二异丙基乙胺(0.37mmol，0.062mL)溶于N,N-二甲基甲酰胺(10mL)中，50℃反应8h；减压蒸干溶剂，加入2N氢氧化钠溶液(10mL)，100℃反应8h。过滤，所得固体用乙酸乙酯和石油醚重结晶得到(S)-N-(1-(5-巯基-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4)。白色固体，产率：76.5％，熔点：272–274℃。1H NMR(400MHz,DMSO-d6)δ13.85(s,1H,SH),10.72(s,1H,NH),8.65(d,J＝7.4Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.23–7.15(m,4H,PhH),7.12–6.73(m,8H,PhH),4.61(q,J＝7.4Hz,1H,CH),3.76(s,3H,OCH3),3.05(dd,J＝13.5,6.8Hz,1H,CH2),2.95(dd,J＝13.5,8.0Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 498.2[M+H]+,C20H22N4O2(487.19).
实施例4：化合物4(a-x)的制备
将(S)-N-(1-(5-巯基-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺4(0.30mmol，0.15g)、相应的卤代物(0.39mmol)和碳酸铯(0.90mmol，0.20g)溶于N,N-二甲基甲酰胺(10mL)中，50℃反应8h。反应毕，加水25mL淬灭反应，乙酸乙酯萃取(3×10mL)，合并有机相，饱和氯化钠溶液洗涤(3×25mL)，用无水硫酸钠干燥，过滤并减压浓缩，最后使用乙酸乙酯和石油醚重结晶得目标化合物4(a-x)。
选用溴乙酸甲酯与化合物4反应得到甲基(S)-2-((4-(4-甲氧基苯基)-5-(1-(2-(2-甲基-1H-吲哚-3-基)乙酰胺基)-2-苯乙基)-4H-1,2,4-三唑-3-基)硫代)乙酸酯(4a)。
白色固体，产率：62.3％，熔点：80-82℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.71(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.23–7.13(m,4H,PhH),7.14–6.79(m,8H,PhH),4.73(q,J＝7.5Hz,1H,CH),4.03(s,2H,CH2),3.77(s,3H,OCH3),3.65(s,3H,OCH3),3.38(s,2H,CH2),3.13(dd,J＝13.3,7.2Hz,1H,CH2),3.01(dd,J＝13.3,7.7Hz,1H,CH2),2.23(s,3H,CH3).EI-MS:m/z 570.26[M+H]+,592.33[M+Na]+,C31H31N5O4S(569.21).
选用溴丙酸甲酯与化合物4反应得到甲基(S)-3-((4-(4-甲氧基苯基)-5-(1-(2-(2-甲基-1H-吲哚-3-基)乙酰胺基)-2-苯乙基)-4H-1,2,4-三唑-3-基)硫代)丙酸酯(4b)。
白色固体，产率：62.3％，熔点：80-82℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.71(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.23–7.13(m,4H,PhH),7.14–6.79(m,8H,PhH),4.73(q,J＝7.5Hz,1H,CH),4.03(s,2H,CH2),3.77(s,3H,OCH3),3.65(s,3H,OCH3),3.38(s,2H,CH2),3.13(dd,J＝13.3,7.2Hz,1H,CH2),3.01(dd,J＝13.3,7.7Hz,1H,CH2),2.23(s,3H,CH3).EI-MS:m/z 584.10[M+H]+,606.20[M+Na]+,C32H33N5O4S(583.23).
选用4-氯甲基吡啶盐酸盐与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((吡啶-4-基甲基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4c)。
白色固体，产率：70.3％，熔点：82-84℃。1H NMR(400MHz,DMSO-d6)δ10.73(s,1H,NH),8.75(d,J＝7.2Hz,1H,NH),7.31(d,J＝7.7Hz,1H,PhH),7.28–7.08(m,5H,PhH),7.03–6.82(m,7H,PhH),4.59(q,J＝7.3Hz,1H,CH),4.46(dt,J＝14.0,7.1Hz,1H,CH2),4.37–4.25(m,1H,CH2),3.75(s,3H,OCH3),3.68(s,3H,OCH3),3.38(s,2H,CH2),3.00(dd,J＝11.7,7.5Hz,2H,CH2),2.93–2.85(m,2H,CH2),2.24(s,3H,CH3).EI-MS:m/z 584.10[M+H]+,606.20[M+Na]+,C32H33N5O4S(583.23).
选用3-氯甲基吡啶盐酸盐与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((吡啶-3-基甲基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4d)。
白色固体，产率：67.2％，熔点：82-84℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.68(d,J＝7.6Hz,1H,NH),8.53(d,J＝1.9Hz,1H,pyridinyl-H),8.45(dd,J＝4.8,1.5Hz,1H,pyridinyl-H),7.69(dt,J＝7.8,1.8Hz,1H,PhH),7.29(dd,J＝7.8,4.2Hz,2H,PhH),7.19(d,J＝8.0Hz,1H,PhH),7.17–7.11(m,3H,pyridinyl-H+PhH),6.93(ddd,J＝7.6,3.8,1.7Hz,3H,PhH),6.89–6.80(m,3H,PhH),4.70(t,J＝7.5Hz,1H,CH),4.32(s,2H,CH2),3.74(s,3H,OCH3),3.36(d,J＝7.6Hz,2H,CH2),3.13(dd,J＝13.2,7.5Hz,1H,CH2),3.00(dd,J＝13.3,7.5Hz,1H,CH2),2.23(s,3H,CH3).EI-MS:m/z 589.5[M+H]+,611.4[M+Na]+,C32H33N5O4S(588.23).
选用2-氯甲基吡啶盐酸盐与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((吡啶-2-基甲基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4e)。
白色固体，产率：72.3％，熔点：82-84℃。1H NMR(400MHz,DMSO-d6)δ10.72(s,1H,NH),8.73(d,J＝7.7Hz,1H,NH),8.45(d,J＝4.8Hz,1H,pyridinyl-H),7.72(d,J＝1.9Hz,1H,pyridinyl-H),7.42–7.37(m,1H,PhH),7.37–7.22(m,3H,PhH),7.22–7.14(m,4H,pyridinyl-H+PhH),6.95(td,J＝7.5,6.3,2.8Hz,4H,PhH),6.90–6.83(m,3H,PhH),4.70(q,J＝7.5Hz,1H,CH),4.43(s,2H,CH2),3.75(s,3H,OCH3),3.38(s,2H,CH2),3.13(dd,J＝13.3,7.3Hz,1H,CH2),3.01(dd,J＝13.3,7.6Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 589.20[M+H]+,611.41[M+Na]+,C32H33N5O4S(588.23).
选用1-溴-2-(甲基磺酰基)乙烷与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((2-(甲磺酰基)乙基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4f)。
白色固体，产率：64.3％，熔点：92-94℃。1H NMR(400MHz,DMSO-d6)δ10.72(s,1H,NH),8.73(d,J＝7.2Hz,1H,NH),7.30(d,J＝7.7Hz,1H,PhH),7.27–7.06(m,5H,PhH),7.03–6.83(m,7H,PhH),4.71–4.58(m,2H,CH2),4.52(ddd,J＝14.2,7.9,6.2Hz,1H,CH),3.76(s,3H,OCH3),3.67(dq,J＝14.3,7.1Hz,2H,CH2),3.38(d,J＝4.0Hz,2H,CH2),3.09(s,3H,CH3),3.07–2.89(m,2H,CH2),2.24(s,3H,CH3).EI-MS:m/z 604.4[M+H]+,C31H33N5O4S2(603.20).
选用1-溴-2-氟乙烷与化合物4反应得到(S)-N-(1-(5-((2-氟乙基)硫代)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4g)。
白色固体，产率：68.5％，熔点：178-180℃。1H NMR(400MHz,DMSO-d6)δ10.72(s,1H,NH),8.74(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.7Hz,1H,PhH),7.24–7.14(m,4H,PhH),7.08–6.80(m,8H,PhH),4.80–4.65(m,2H,CH2),4.58(t,J＝5.9Hz,1H,CH),3.76(s,3H,OCH3),3.48–3.41(m,2H,CH2),3.38(s,2H,CH2),3.14(dd,J＝13.3,7.2Hz,1H,CH2),3.02(dd,J＝13.3,7.8Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 544.5[M+H]+,566.6[M+Na]+,C30H30FN5O2S(543.21).
选用1-溴-2,2-二氟乙烷与化合物4反应得到(S)-N-(1-(5-((2,2-二氟乙基)硫代)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4h)。
白色固体，产率：72.3％，熔点：202-204℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.73(d,J＝7.7Hz,1H,NH),7.29(d,J＝7.7Hz,1H,PhH),7.26–7.09(m,5H,PhH),7.03–6.80(m,7H,PhH),6.26(tt,J＝56.3,4.1Hz,1H,CH),4.71(q,J＝7.5Hz,1H,CH),3.76(s,3H,OCH3),3.62(td,J＝15.9,4.0Hz,2H,CH2),3.36(d,J＝10.9Hz,2H,CH2),3.12(dd,J＝13.3,7.1Hz,1H,CH2),3.01(dd,J＝13.3,7.8Hz,1H,CH2),2.23(s,3H,CH3).EI-MS:m/z 562.17[M+H]+,584.26[M+Na]+,C30H29F2N5O2S(561.20).
选用2-(4-吗啉)乙基溴与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((2-吗啉乙基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4i)。
白色固体，产率：65.6％，熔点：98-100℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.72(d,J＝7.7Hz,1H,NH),7.31(d,J＝7.7Hz,1H,PhH),7.27–7.09(m,5H,PhH),7.01–6.83(m,7H,PhH),4.70(q,J＝7.5Hz,1H,CH),3.76(s,3H,OCH3),3.56–3.48(m,4H,morpholinyl-H),3.39(s,2H,CH2),3.23(t,J＝6.8Hz,2H,CH2),3.13(dd,J＝13.2,7.3Hz,1H,CH2),3.01(dd,J＝13.3,7.7Hz,1H,CH2),2.56(t,J＝6.9Hz,2H,CH2),2.40–2.31(m,4H,morpholinyl-H),2.24(s,3H,CH3).EI-MS:m/z 611.36[M+H]+,633.40[M+Na]+,C34H38N6O3S(610.27).
选用溴乙酰胺与化合物4反应得到(S)-N-(1-(5-((2-氨基-2-氧乙基)硫代)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4j)。
白色固体，产率：72.5％，熔点：120-122℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.72(d,J＝7.7Hz,1H,NH),7.65(s,1H,PhH),7.30(d,J＝7.8Hz,1H,PhH),7.26–7.13(m,5H,PhH+NH2),7.11–6.79(m,8H,PhH),4.72(q,J＝7.5Hz,1H,CH),3.88(s,2H,CH2),3.77(s,3H,OCH3),3.38(s,2H,CH2),3.13(dd,J＝13.3,7.2Hz,1H,CH2),3.01(dd,J＝13.3,7.8Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 555.28[M+H]+,577.27[M+Na]+,C30H30N6O3S(544.21).
选用3-溴-1,1,1-三氟丙烷与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((3,3,3-三氟丙基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4k)。
白色固体，产率：63.7％，熔点：90-92℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.70(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.23–7.13(m,4H,PhH),7.09–6.79(m,8H,PhH),4.71(q,J＝7.5Hz,1H,CH),3.75(s,3H,OCH3),3.37(d,J＝9.3Hz,2H,CH2),3.25(dd,J＝8.6,6.6Hz,2H,CH2),3.14(dd,J＝13.3,7.3Hz,1H,CH2),3.02(dd,J＝13.3,7.7Hz,1H,CH2),2.71(ddd,J＝19.2,9.6,4.7Hz,2H,CH2),2.23(s,3H,CH3).EI-MS:m/z594.20[M+H]+,616.30[M+Na]+,C31H30F3N5O2S(593.21).
选用4-(3-溴丙基)吗啉与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((3-吗啉丙基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4l)。
白色固体，产率：61.7％，熔点：88-90℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.71(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.25–7.11(m,4H,PhH),7.01–6.77(m,7H,PhH),4.70(q,J＝7.5Hz,1H,CH),3.75(s,3H,OCH3),3.54(t,J＝4.7Hz,4H,morpholinyl-H),3.37(d,J＝9.7Hz,2H,CH2),3.13(dd,J＝13.3,7.4Hz,1H,CH2),3.04(dd,J＝8.3,6.0Hz,2H,CH2),3.02–2.97(m,1H,CH2),2.29(d,J＝6.3Hz,4H,morpholinyl-H),2.23(s,3H,CH3),1.76(q,J＝7.0Hz,2H,CH2),0.84(tt,J＝6.6,4.2Hz,2H,CH2).EI-MS:m/z625.30[M+H]+,C35H40N6O3S(624.29).
选用2-环己基溴乙烷与化合物4反应得到(S)-N-(1-(5-((2-环己基乙基)硫代)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4m)。
淡黄色固体，产率：70.3％，熔点：90-92℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.70(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.23–7.11(m,4H,PhH),7.06–6.91(m,4H,PhH),6.91–6.60(m,4H,PhH),4.70(q,J＝7.5Hz,1H,CH),3.75(s,3H,OCH3),3.37(d,J＝8.9Hz,2H,CH2),3.13(dd,J＝13.2,7.5Hz,1H,CH2),3.01(q,J＝7.1,6.7Hz,3H,CH2),2.23(s,3H,CH3),1.63(d,J＝12.0Hz,5H CH2×2+CH),1.46(q,J＝7.1Hz,2H,CH2),1.31–1.12(m,4H,CH2×2),0.89–0.81(m,2H,CH2).EI-MS:m/z 608.26[M+H]+,630.40[M+Na]+,C36H41N5O2S(607.30).
选用2-氯甲基嘧啶盐酸盐与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((嘧啶-2-基甲基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4n)。
白色固体，产率：72.3％，熔点：90-92℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.71(t,J＝5.8Hz,3H,NH+pyrimidinyl-H),7.40(t,J＝4.9Hz,1H,pyrimidinyl-H),7.31(d,J＝7.8Hz,1H,PhH),7.28–7.05(m,5H,PhH),6.96(qd,J＝4.9,2.5Hz,4H,PhH),6.92–6.82(m,3H,PhH),4.73(q,J＝7.5Hz,1H,CH),4.52(s,2H,CH2),3.76(s,3H,OCH3),3.39(s,2H,CH2),3.13(dd,J＝13.3,7.3Hz,1H,CH2),3.01(dd,J＝13.3,7.6Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 590.33[M+H]+,612.34[M+Na]+,C33H31N7O2S(589.23).
选用(2-溴甲基)二甲胺与化合物4反应得到(S)-N-(1-(5-((2-(二甲氨基)乙基)硫代)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4o)。
白色固体，产率：67.3％，熔点：84-86℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.69(d,J＝7.6Hz,1H,NH),7.31(d,J＝7.8Hz,1H,PhH),7.22–7.13(m,4H,PhH),7.07–6.92(m,4H,PhH),6.93–6.71(m,4H,PhH),4.71(q,J＝7.5Hz,1H,CH),3.76(s,3H,OCH3),3.39(s,2H,CH2),3.21(t,J＝6.8Hz,2H,CH2),3.14(dd,J＝13.3,7.4Hz,1H,CH2),3.02(dd,J＝13.3,7.6Hz,1H,CH2),2.24(s,3H,CH3),2.12(s,6H,NCH3×2).EI-MS:m/z 569.29[M+H]+,591.37[M+Na]+,C32H36N6O2S(568.26).
选用4-(甲磺酰基)苄溴与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((4-(甲磺酰基)苄基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4p)。
白色固体，产率：72.7％，熔点：96-98℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.71(d,J＝7.6Hz,1H,NH),7.81(d,J＝8.3Hz,2H,PhH),7.53(d,J＝8.3Hz,2H,PhH),7.30(d,J＝7.8Hz,1H,PhH),7.19(d,J＝8.0Hz,1H,PhH),7.14(dd,J＝5.1,1.8Hz,3H,PhH),7.02–6.89(m,4H,PhH),6.85(t,J＝7.4Hz,4H,PhH),4.67(q,J＝7.5Hz,1H,CH),4.39(s,2H,CH2),3.74(s,3H,OCH3),3.36(d,J＝9.0Hz,2H,CH2),3.19(s,3H,CH3),3.12(dd,J＝13.2,7.4Hz,1H,CH2),3.00(dd,J＝13.3,7.6Hz,1H,CH2),2.23(s,3H,CH3).EI-MS:m/z 666.35[M+H]+,688.34[M+Na]+,C36H35N5O4S2(665.21).
选用4-(溴甲基)苯磺酰胺与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((4-氨磺酰苄基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4q)。
白色固体，产率：69.2％，熔点：120-122℃。1H NMR(400MHz,DMSO-d6)δ10.70(s,1H,NH),8.69(d,J＝7.6Hz,1H,NH),7.73(d,J＝8.3Hz,2H,PhH),7.47(d,J＝8.3Hz,2H,PhH),7.34(s,2H,NH2),7.30(d,J＝7.7Hz,1H,PhH),7.24–7.10(m,4H,PhH),6.94(t,J＝7.0Hz,4H,PhH),6.86(d,J＝7.6Hz,4H,PhH),4.69(q,J＝7.5Hz,1H,CH),4.36(s,2H,CH2),3.74(s,3H,OCH3),3.36(d,J＝11.1Hz,2H,CH2),3.12(dd,J＝13.3,7.3Hz,1H,CH2),3.00(dd,J＝13.3,7.6Hz,1H,CH2),2.23(s,3H,CH3).EI-MS:m/z 667.37[M+H]+,689.42[M+Na]+,C35H34N6O4S2(666.21).
选用溴乙腈与化合物4反应得到(S)-N-(1-(5-((氰基)硫)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯基乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4r)。
白色固体，产率：62.5％，熔点：80-82℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.77(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.23–7.13(m,4H,PhH),7.07–6.82(m,8H,PhH),4.74(q,J＝7.5Hz,1H,CH),4.24(s,2H,CH2),3.77(s,3H,OCH3),3.38(d,J＝10.4Hz,2H,CH2),3.16(dd,J＝13.3,7.0Hz,1H,CH2),3.04(dd,J＝13.3,7.9Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 537.28[M+H]+,559.34[M+Na]+,C30H28N6O2S(536.20).
选用溴乙炔与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-(丙-2-炔基-1-基硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4s)。
白色固体，产率：73.2％，熔点：78-80℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.74(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.24–7.14(m,4H,PhH),7.07–6.60(m,8H,PhH),4.73(q,J＝7.6Hz,1H,CH),3.88(d,J＝2.6Hz,2H,CH2),3.76(s,3H,OCH3),3.39(s,2H,CH2),3.20(t,J＝2.6Hz,1H,CH2),3.14(dd,J＝13.2,7.2Hz,1H,CH2),3.02(dd,J＝13.3,7.7Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 536.32[M+H]+,558.36[M+Na]+,C31H29N5O2S(535.20).
选用2-(2-溴乙基)-1,3-二氧杂环己烷与化合物4反应得到(S)-N-(1-(5-((2-(1,3-二噁烷-2-基)乙基)硫代)-4-(4-甲氧基苯基)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4t)。
白色固体，产率：72.5％，熔点：78-80℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.72(d,J＝7.7Hz,1H,NH),7.30(d,J＝7.8Hz,1H,PhH),7.26–7.11(m,4H,PhH),7.10–6.61(m,8H,PhH),4.70(q,J＝7.5Hz,1H,CH),4.57(t,J＝5.0Hz,1H,CH),3.98(dd,J＝11.4,4.2Hz,2H,CH2),3.76(s,3H,OCH3),3.66(t,J＝12.0Hz,2H,CH2),3.39(s,2H,CH2),3.14(dd,J＝13.2,7.4Hz,1H,CH2),3.02(dt,J＝13.6,7.5Hz,3H,CH2+CH2),2.24(s,3H,CH3),1.85(ddt,J＝12.4,8.1,4.1Hz,3H,CH2+CH2),1.32(d,J＝13.3Hz,1H,CH2).EI-MS:m/z 612.46[M+H]+,634.56[M+Na]+,C34H37N5O4S(611.26).
选用4-(溴甲基)苯硼酸与化合物4反应得到(S)-(4-((4-(4-甲氧基苯基)-5-(1-(2-(2-甲基-1H-吲哚-3-基)乙酰胺基)-2-苯乙基)-4H-1,2,4-三唑-3-基)硫代)甲基)苯基)硼酸(4u)。
白色固体，产率：62.7％，熔点：144-146℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.70(d,J＝7.6Hz,1H,NH),8.02(s,2H,B(OH)2),7.71(d,J＝7.9Hz,2H,PhH),7.31(d,J＝7.8Hz,1H,PhH),7.25(d,J＝7.9Hz,2H,PhH),7.22–7.10(m,4H,PhH),6.95(t,J＝7.5Hz,4H,PhH),6.86(t,J＝7.6Hz,4H,PhH),4.70(q,J＝7.5Hz,1H,CH),4.37–4.24(m,2H,CH2),3.74(s,3H,OCH3),3.38(d,J＝8.8Hz,2H,CH2),3.13(dd,J＝13.3,7.4Hz,1H,CH2),3.01(dd,J＝13.3,7.6Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 630.6[M-H]-,C35H34BN5O4S(631.24).
选用5-(氯甲基)噻唑盐酸盐与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((噻唑-5-基甲基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4v)。
白色固体，产率：73.1％，熔点：88-90℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.95(s,1H,thiazolyl-H),8.73(d,J＝7.7Hz,1H,NH),7.77(s,1H,thiazolyl-H),7.32(d,J＝7.8Hz,1H,PhH),7.23–7.12(m,4H,PhH),7.07–6.91(m,4H,PhH),6.91–6.72(m,4H,PhH),4.71(q,J＝7.5Hz,1H,CH),4.62(d,J＝2.7Hz,2H,CH2),3.74(s,3H,OCH3),3.38(d,J＝6.4Hz,2H,CH2),3.15(dd,J＝13.2,7.6Hz,1H,CH2),3.03(dd,J＝13.2,7.4Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z595.12[M+H]+,617.24[M+Na]+,C32H30N6O2S2(594.19).
选用4-(氯甲基)噻唑盐酸盐与化合物4反应得到(S)-N-(1-(4-(4-甲氧基苯基)-5-((噻唑-4-基甲基)硫代)-4H-1,2,4-三唑-3-基)-2-苯乙基)-2-(2-甲基-1H-吲哚-3-基)乙酰胺(4w)。
白色固体，产率：63.9％，熔点：87-89℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),9.01(s,1H,thiazolyl-H),8.71(d,J＝7.7Hz,1H,NH),7.50(d,J＝2.0Hz,1H,thiazolyl-H),7.31(d,J＝7.8Hz,1H,PhH),7.26–7.11(m,4H,PhH),6.96(td,J＝7.3,6.3,2.7Hz,4H,PhH),6.91–6.66(m,4H,PhH),4.71(q,J＝7.5Hz,1H,CH),4.46(s,2H,CH2),3.75(s,3H,OCH3),3.38(d,J＝8.1Hz,2H,CH2),3.14(dd,J＝13.2,7.4Hz,1H,CH2),3.02(dd,J＝13.3,7.5Hz,1H,CH2),2.24(s,3H,CH3).EI-MS:m/z 595.38[M+H]+,617.39[M+Na]+,C32H30N6O2S2(594.19).
选用2-溴代异丁酸甲酯与化合物4反应得到甲基(S)-2-((4-(4-甲氧基苯基)-5-(1-(2-(2-甲基-1H-吲哚-3-基)乙酰胺基)-2-苯乙基)-4H-1,2,4-三唑-3-基)硫代)-2-甲基丙酸酯(4x)。
白色固体，产率：64.7％，熔点：82-84℃。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H,NH),8.76(d,J＝7.7Hz,1H,NH),7.31(d,J＝7.8Hz,1H,PhH),7.23–7.10(m,4H,PhH),6.94(dt,J＝7.5,5.6Hz,4H,PhH),6.86(t,J＝7.5Hz,4H,PhH),4.71(q,J＝7.5Hz,1H,CH),3.75(s,3H,OCH3),3.53(s,3H,OCH3),3.39(d,J＝5.7Hz,2H,CH2),3.14(dd,J＝13.1,7.6Hz,1H,CH2),3.03(dd,J＝13.2,7.5Hz,1H,CH2),2.24(s,3H,CH3),1.42(d,J＝7.1Hz,6H,CH3×2).EI-MS:m/z 598.43[M+H]+,620.43[M+Na]+,C33H35N5O4S(597.24).
实施例5：抗HIV活性实验(MT-4细胞模型)
参见①Pauwels R,et al.J.Virol.Methods.1988,20,309.②Pannecouque C,etal.Nat Protocols2008,3,427.
术语解释：MT-4细胞：人急性淋巴母细胞白血病细胞；MTT分析法：MTT即为3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐；商品名:噻唑蓝；DMSO：二甲基亚砜。
测试原理
由于HIV感染的MT-4细胞在一定时间内(5-7天)会发生病变，因此向HIV感染的MT-4细胞悬浊液中加入适当浓度的待检测化合物溶液，经过一段时间(5-7天)的培养后，用MTT分析法测定MT-4细胞活力，得到保护50％细胞免于细胞病变的药物浓度(EC50)即可得出目标化合物的抗HIV的活性。同时得到目标化合物使50％未感染HIV的细胞发生病变的浓度(CC50)。MTT分析法原理：MTT即溴化-3-(4,5-二甲基-2-噻唑基)-2,5-二苯基四唑氮,可与活的细胞内琥珀酸脱氢酶相结合，而不与死亡细胞发生反应。目前MTT法是一种快捷反映细胞活力的酶分析方法。
测试材料和方法
(1)HIV-1野生株、HIV-2野生株：由比利时Leuven大学Rega研究院微生物与免疫学研究所提供。
(2)MT-4细胞：由比利时Leuven大学Rega研究院微生物与免疫学研究所提供。
(3)MTT：购自美国Sigma公司。
(4)样品处理：样品临用前溶于DMSO配成适当浓度，并用双蒸水作5倍稀释，各5个稀释度。
(5)阳性对照：PF-74。
(6)测试方法：样品稀释后加入到HIV感染MT-4细胞悬浊液中，经过一段时间后用MTT比色法测定细胞活力，于酶标仪中，在590nm下记录吸光度(A)值，计算出EC50、CC50以及SI。
(7)MTT染色法：加入样品培养一段时间后，再向每孔加入MTT溶液(5mg/mL)20μL，继续培养若干小时，弃染色液，并向每孔加入150μL的DMSO，充分混合，于酶标仪中，在590nm下记录吸光度。
具体操作如下：将化合物用DMSO或水溶解后用磷酸盐缓冲液稀释，将3×105MT-4细胞与100μL不同浓度的化合物溶液在37℃共同预孵育1h。然后向该混合物中加入100μL适当浓度的病毒稀释液，将细胞于37℃孵育1h。洗涤三次后，将细胞再次分别悬浮于含有或不含化合物的培养基质中。接着将细胞在5％CO2环境中，于37℃下再孵育7天，并于感染后的第三天用含有或不含化合物的培养基质补充原培养液。每种培养条件都重复操作两次。对病毒的细胞病变作用每天都用反向光学显微镜监控。一般来讲，本实验中所用的病毒稀释液常常会在病毒感染后第五天发生细胞病变。药物抑制浓度以药物对病毒致细胞病变作用产生50％抑制作用而同时对细胞无直接毒性的浓度(EC50)表示。值得强调的是，当化合物水溶性较差，需要用DMSO才能溶解时，DMSO体积比浓度相对于水来讲，一般会低于10％(DMSO在MT-4细胞培养介质中最终浓度小于2％)。因为DMSO能影响测试化合物抗病毒活性，对含有相同浓度DMSO溶液的抗病毒活性对比空白实验也应该平行操作进行。另外，DMSO最终浓度(1/1000)远远低于影响HIV-1/2在MT-4细胞中复制所需浓度。
目标化合物的体外抗HIV-1/2野生株活性筛选数据由比利时Leuven大学Rega研究院微生物与免疫学研究所提供，所有的活性数据都经过至少两次独立、平行的实验测得，结果见表1。
表1化合物抗HIV-1/2野生株活性、细胞毒性以及选择指数
注：a EC50：保护50％感染HIV的MT-4细胞免于细胞病变的化合物浓度；b CC50：目标化合物使50％未感染HIV的细胞发生病变的浓度；cSI：选择性系数，CC50/EC50的比值；dNA：无法计算；eND：无法测定；
四、结论
本发明所提供的含有1,2,4-三唑硫醚的苯丙氨酸衍生物是一系列结构新颖的HIV抑制剂，由表1可以看出，绝大部分化合物表现出优秀的抑制HIV-1/2野生株的活性。其中，化合物4l(EC50＝0.59±0.08μM)和4r(EC50＝0.59±0.06μM)的对HIV-1野生株活性尤为突出，稍优于阳性对照PF-74(EC50＝0.79±0.09μM)；此外，化合物4i、4j、4o以及4v也表现出了与PF-74相当的抗HIV-1活性。对于HIV-2野生株，化合物4r(EC50＝2.69±0.17μM)的抗病毒活性优于PF-74(EC50＝3.79±0.39μM)；此外，4c、4d、4g、4j、4q、4s、4v等七个化合物表现出了与PF-74相当的抗HIV-2活性。该类化合物由于其新颖的结构骨架，具有非常大的研发价值，可作为制备抗HIV的先导化合物加以研究。
Claims (4)
1.含有1,2,4-三唑硫醚的苯丙氨酸衍生物或其药学上可接受的盐，其特征在于，具有如下所示的结构：
2.如权利要求1所述的含有1,2,4-三唑硫醚的苯丙氨酸衍生物的制备方法，其特征在于步骤包括：2-甲基吲哚-3-乙酸与L-苯丙氨酸甲酯盐酸盐在二氯甲烷中发生酰胺缩合反应得到化合物2，化合物2与水合肼在乙醇中回流反应得到中间体3；随后化合物3与4-甲氧基苯基异硫氰酸酯在N,N-二异丙基乙胺的条件下反应得到化合物4，最后化合物4与不同的卤代物发生亲核取代反应得到终产物4(a-x)；反应路线如下：
试剂及条件：(i)2-甲基吲哚-3-乙酸，O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸盐，N,N-二异丙基乙胺，二氯甲烷，0℃转至室温；(ii)水合肼，乙醇，80℃；(iii)4-甲氧基苯基异硫氰酸酯，N,N-二异丙基乙胺，N,N-二甲基甲酰胺，50℃；(iv)相应的卤代物，碳酸铯，N,N-二甲基甲酰胺，50℃。
3.一种如权利要求1任一所述的含有1,2,4-三唑硫醚的苯丙氨酸衍生物在制备抗艾滋病毒药物中的应用。
4.一种药物组合物，包含权利要求1任一所述的含有1,2,4-三唑硫醚的苯丙氨酸衍生物和一种或多种药学上可接受载体或赋形剂。
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