CN114940694B - 一种甾体类化合物及其制备方法、及在治疗与p53突变有关的肿瘤中的应用 - Google Patents
一种甾体类化合物及其制备方法、及在治疗与p53突变有关的肿瘤中的应用 Download PDFInfo
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Abstract
本发明公开了一种甾体类化合物及其制备方法、及在治疗与p53突变有关的肿瘤中的应用,通过药效团嵌入等结构整合的方式,设计并合成了一系列靶向突变p53的甾体类化合物。本发明甾体类化合物具有高活性、靶向性、低毒性,对多种人肿瘤细胞株显示了良好的p53靶向抗肿瘤活性。
Description
技术领域
本发明涉及化学和医药领域,特别是一种甾体类化合物及其制备方法、及在治疗与p53突变有关的肿瘤中的应用。
背景技术
恶性肿瘤是严重威胁人类健康的常见病、多发病,肿瘤疾病导致的死亡是仅次于心血管疾病的第二大死因。癌症已经成为危害人类健康与生命的大敌,防控癌症已经成为我国的国家战略。目前对恶性肿瘤的三大主要治疗手段为外科手术、化学治疗和放射治疗。其中化学治疗简称化疗,指的是运用抗肿瘤药治疗的方法。化疗药物在治疗过程中引起的不同程度的毒副作用和不良反应仍然是当今世界面临的肿瘤化疗急需解决的难题。迄今为止所用的抗肿瘤药物,几乎都具有不同程度的毒副作用和不良反应。这是由于药物自身的局限性致使药物不能选择性杀灭肿瘤细胞,而且在灭杀癌细胞的同时,对正常的组织细胞也造成损害。因此,研究开发具有高活性、靶向性、低毒性的抗肿瘤药物迫在眉睫。
发明内容
本发明的目的在于克服化疗药物在治疗过程中引起毒副作用和不良反应较严重的问题,提供具有高活性、靶向性、低毒性的抗肿瘤药物,从而提供一种甾体类化合物及其制备方法、及在治疗与p53突变有关的肿瘤中的应用。
本发明的目的通过以下技术方案来实现:
一种式I甾体类化合物或其药用盐,
其中,R1为烷基,环烷基,芳基取代的烷基或环烷基,杂芳基取代的烷基或环烷基,芳基,杂芳基,三氟甲基、卤素、甲氧基、乙氧基、氨基、甲氨基、二甲氨基、乙酰氨基取代的芳基或杂芳基,烯基,或烷基取代的烯基;R2为氢、烷基,环烷基,芳基取代的烷基或环烷基,杂芳基取代的烷基或环烷基,芳基,杂芳基,三氟甲基、卤素、甲氧基、乙氧基、氨基、甲氨基、二甲氨基、乙酰氨基取代的芳基或杂芳基,烯基,或烷基、芳基取代的烯基。
作为优选的,所述式(I)的R1和/或R2中,烷基为1至12个碳原子的直链或支链烷基,环状烷基为3至8个碳原子的环状烷基,芳基为6至14个碳原子的芳香单环或多环系,所述的杂芳基为含有杂原子氮或氧或硫的6至14个原子的芳香单环或多环系,烯基为包含2至10个碳原子的烯基。
其中,烯基可以包括但不限于乙烯基、烯丙基、1-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、3-甲基-2-丁烯基、苯乙烯基甲基;芳基或杂芳基可以被任选一个或多个取代基取代,取代基可以相同或不同,取代基可以包括但不限于1至8个碳原子的直链或支链烷基、环烷基、卤素、甲氧基、乙氧基、氨基、甲氨基、二甲氨基、乙酰氨基;杂芳基可以包括但不限于吡啶基、吲哚基、呋喃基、噻吩基。
作为优选的,式(I)化合物为:
16α-丁氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-异丁氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-正己氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-正辛氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-十二烷氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-环己氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(4-氟苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(3-溴苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(4-甲氧基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(4-叔丁基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(2-甲基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(3-甲基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(4-甲基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(2-噻吩甲氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-(2-呋喃甲氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇、16α-异丁氨基-17β-[(R)-1-羟基-1-烯丙基-乙基]雄甾-5-烯-3β-醇、16α-正丁氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-正己氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-正辛氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-十二烷氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-环丙氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-环戊氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-环己氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-苄氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-氟苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(3-溴苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-甲基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-叔丁基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-叔丁基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(2-吡啶甲氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-色氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(2-呋喃甲氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(2-噻吩甲氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-正己氨基-17β-[(R)-2-羟基-2,3-二甲基-丁基)]雄甾-5-烯-3β-醇、16α-(2-甲基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(3-甲基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-氯苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-氯苄氨)基-17β-(1-羟基-1-甲基-苯乙基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-2-烯戊基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1,4-二甲基-戊烯基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-丙烯基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-4-戊烯基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-苯丁烯基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-4-苯基-丁基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-戊基)雄甾-5-烯-3β-醇、16α-(4-溴苄氨)基-17β-(1-羟基-1,4-二甲基-戊基)雄甾-5-烯-3β-醇、16α-苯氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-溴苯氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-苯乙基氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(4-溴苯乙氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇、16α-(5-己烯-1-氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇,
上述化合物对应的化学结构分别为:
作为优选的,药用盐包括硫酸盐、甲磺酸盐、铵盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、磷酸盐、一氢磷酸盐、二氢磷酸盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物、乙酸盐、丙酸盐、癸酸盐、辛酸盐、丙烯酸盐、甲酸盐、异丁酸盐、酒石酸盐、富马酸盐、草酸盐、马来酸盐、柠檬酸盐、乳酸盐。
本发明还提供了一种甾体类化合物的制备方法,将16α-取代氨基-3β-羟基-孕甾-5-烯-20-酮类化合物与Grinard试剂置于溶剂中反应,得到式(I)化合物。其中16α-取代氨基-3β-羟基-孕甾-5-烯-20-酮类化合物可以以商品化易得的3β-羟基-孕甾-5,16-二烯-20-酮为原料,与氨发生Michael加成反应得到,式(I)化合物合成路线如下式所示:
作为优选的,所述溶剂为四氢呋喃或乙醚,所述反应温度为0℃至所用溶剂的回流温度。
其中,16α-取代氨基-3β-羟基-孕甾-5-烯-20-酮类化合物与Grinard试剂的摩尔比可以为1:1-1:20,优选的为1:12;加入反应液中的Grinard试剂的浓度为0.5-1.5M,加入溶剂的体积与Grinard试剂体积比为3-5:1。
本发明还提供了所述甾体类化合物在治疗与p53突变有关肿瘤药物中的应用。
本发明还提供了一种抗肿瘤药物,包括一种或两种以上所述式(I)所示甾体类化合物或其药用盐和药学上可接受的载体。
本发明的甾体类化合物用作药物时,可以直接使用,或者以药物组合物的形式使用。也可以与其他药物组成复方的形式使用,该药物组合物含有0.1~99%,优选为0.5~90%的本发明化合物,其余为药物学上可接受的,对人和动物无毒和惰性的药物制剂常用的药用载体和/或赋形剂。将本发明的药物组合物以单位体重服用量的形式使用。可以使用不同的药用辅料,制成固体制剂(片剂、胶囊剂、颗粒剂、散剂)、擦剂、软膏剂或针剂。
本发明所述的药物,可以包含式(I)所示甾体类化合物或其药用盐以及一种或多种可药用的稀释剂或载体。可药用载体包括但不限于淀粉、糊精、乳糖、微晶纤维素、一些无机钙盐(如硫酸钙、磷酸氢钙及碳酸钙)、山梨醇、甘露醇或其他药物制剂用的表面活性剂、氧化铝、硬脂酸铝、离子交换材料、缓冲物质如磷酸盐、山梨酸、聚乙烯吡咯酮、纤维素物质、聚乙烯醇、羧甲基纤维素钠、羊毛脂、羟丙基甲基纤维素、乙基纤维素等可用于促进本发明所述化合物、其药用盐或前药药物传递的载体。
本发明所述的药物,可以包含甾体类化合物或其药学上可接受的盐以及其他可药用辅料。可药用辅料包括但不限于:崩解剂如干淀粉、羧甲基淀粉钠、交联羧甲基纤维素钠、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、海藻酸钠等,粘合剂如聚维酮K30、微晶纤维素、褐藻酸钠等,填充剂如无水乳糖、淀粉、葡萄糖、乳糖珠粒等,润滑剂如十二烷基硫酸镁、硬脂酸镁等,以及其他赋形剂、增溶剂、香味剂、着色剂等。
作为优选的,与p53突变有关的肿瘤包括结肠癌、肺癌、胃癌、乳腺癌、卵巢癌、黑色素瘤、血液肿瘤、前列腺癌。
本发明具有以下优点:
甾体类化合物的分子形状、分子量、脂水分配系数等符合成药性要求,且具有良好的生物膜穿透能力,其结构改造与生物及药理活性研究一直是药物研发的热点领域。本发明通过药效团嵌入等结构整合的方式,设计并合成了一系列靶向突变p53的甾体类化合物。
本发明甾体类化合物具有高活性、靶向性、低毒性,对多种人肿瘤细胞株显示了良好的p53靶向抗肿瘤活性。
附图说明
图1靶向突变p53的甾体类化合物具有重激活突变p53的作用。
图2以化合物GAPS-30为例的甾体类化合物增加野生型p53表达。
图3以化合物GAPS-30为例的甾体类化合物增加野生型p53的表达。
图4以化合物GAPS-30为例的甾体类化合物上调p53下游靶蛋白的表达。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚,下面将结合本发明实施方式中的附图,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式是本发明一部分实施方式,而不是全部的实施方式。通常在此处附图中描述和示出的本发明实施方式的组件可以以各种不同的配置来布置和设计。
因此,以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。
需要说明的是,在不冲突的情况下,本发明中的实施方式及实施方式中的特征可以相互组合。
实施例1:
16α-丁氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-1)的制备
16α-丁氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.520mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(6.24mL,1.0M in THF,6.24mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~90:1~60:1)得白色泡沫状固体化合物139mg,收率:66.7%。
1H NMR(400MHz,CDCl3)δ5.33(d,J=5.0Hz,1H),3.50(dq,J=11.2,5.4,4.6Hz,1H),3.37–3.23(m,1H),2.74(dt,J=11.5,7.0Hz,1H),2.49(dt,J=11.4,7.1Hz,1H),2.33–2.14(m,2H),1.93(dtd,J=12.8,5.0,2.2Hz,2H),1.82(d,J=9.7Hz,2H),1.60–1.37(m,9H),1.33(q,J=7.3Hz,2H),1.30–1.16(m,9H),1.11–1.00(m,1H),0.99(s,3H),0.97–0.92(m,1H),0.88(t,J=7.2Hz,3H),0.72(s,3H).
13C NMR(101MHz,CDCl3)δ141.08,121.34,73.11,71.72,67.54,58.99,54.75,50.18,47.66,42.33,42.15,39.76,36.63,32.07,31.82,31.73,31.70,31.30,31.10,29.81,27.44,20.64,20.55,19.48,14.73,14.01.
HRMS(ESI):m/z[m+H]+calcd for C26H45NO2:404.3529;found:404.3523.
本实施例中还对反应物用量、溶剂用量、甲基溴化镁浓度等进行了研究,16α-丁氨基-孕甾-5-烯-3β-羟基-20-酮与甲基溴化镁摩尔比分别调整为1:1、1:5、1:10、1:15、和1:20,为1:1-1:20,加入反应液中的Grinard试剂的浓度为0.5M、1.0M、1.5M,加入溶剂的体积与Grinard试剂体积比为3:1、4:1、5:1,通过调整反应物的用量,也是可以得到16α-丁氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇,但产率稍有降低。
实施例2:
16α-异丁氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-2)的制备
16α-异丁氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.520mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(6.24mL,1.0M in THF,6.24mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1)得白色泡沫状固体化合物145mg,收率:69.0%。
1H NMR(400MHz,CDCl3)δ5.32(d,J=3.0Hz,1H),3.57–3.43(m,1H),3.38–3.25(m,1H),2.64(dd,J=11.6,6.3Hz,1H),2.38–2.12(m,3H),1.99–1.86(m,2H),1.86–1.66(m,3H),1.52(dt,J=26.0,11.3Hz,10H),1.26(s,6H),0.98(s,5H),0.91(t,J=6.0Hz,6H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ141.05,121.31,73.22,71.65,67.09,59.53,55.96,54.59,50.10,42.31,42.19,39.66,37.23,36.60,31.66,31.31,31.27,31.09,28.22,27.23,20.75,20.62,19.47,14.74.
HRMS(ESI):m/z[m+H]+calcd for C26H45NO2:404.3529;found:404.3527.
实施例3:
16α-正己氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-3)的制备
16α-正己氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.480mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.80mL,1.0M in THF,5.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物131mg,收率:63.2%。
1H NMR(400MHz,CDCl3)δ5.32(d,J=4.9Hz,1H),3.47(dd,J=7.9,3.3Hz,1H),3.28(td,J=9.3,3.3Hz,1H),2.69(ddd,J=11.3,7.5,6.1Hz,1H),2.45(dt,J=11.3,7.0Hz,1H),2.32–2.14(m,2H),1.98–1.88(m,2H),1.85–1.75(m,2H),1.44(dtd,J=31.6,11.5,10.0,7.0Hz,10H),1.33–1.12(m,15H),1.10–1.00(m,1H),0.98(s,3H),0.94(dd,J=11.4,4.8Hz,1H),0.85(t,J=6.7Hz,3H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ141.15,121.26,73.02,71.64,67.73,58.84,54.80,50.21,48.09,42.31,42.09,39.77,37.23,36.62,32.11,31.85,31.76,31.68,31.31,31.03,30.27,27.54,27.14,22.67,20.62,19.47,14.69,14.18.
HRMS(ESI):m/z[m+H]+calcd for C28H49NO2:432.3842;found:432.3830.
实施例4:
16α-正辛氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-4)的制备
16α-正辛氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.450mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.40mL,1.0M in THF,5.40mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=170:1~150:1~120:1~100:1)得白色泡沫状固体化合物172mg,收率:83.0%。
1H NMR(400MHz,CDCl3)δ5.33(d,J=4.9Hz,1H),3.49(dtd,J=19.6,9.3,7.9,5.8Hz,2H),2.95(ddd,J=11.9,8.4,6.2Hz,1H),2.69–2.55(m,1H),2.34–2.13(m,2H),1.99–1.87(m,2H),1.87–1.77(m,4H),1.76–1.41(m,9H),1.37(d,J=19.4Hz,5H),1.31–1.22(m,11H),1.06(td,J=12.4,10.4,4.3Hz,2H),0.98(s,3H),0.86(t,J=6.8Hz,3H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ140.75,121.51,73.91,71.78,66.18,59.80,54.25,49.88,47.68,42.44,42.33,39.54,37.23,36.55,31.89,31.69,31.41,31.36,31.19,29.96,29.35,29.29,27.15,26.82,22.73,20.64,19.49,14.84,14.19.
HRMS(ESI):m/z[m+H]+calcd for C30H53NO2:460.4155;found:460.4151.
实施例5:
16α-十二烷氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-5)的制备
16α-十二氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=150:1~120:1~90:1)得白色泡沫状固体化合物130mg,收率:63%。
1H NMR(400MHz,CDCl3)δ5.32(d,J=5.0Hz,1H),3.50(tt,J=10.5,4.3Hz,1H),3.28(td,J=9.2,3.1Hz,1H),2.68(dt,J=11.5,6.7Hz,1H),2.45(dt,J=11.3,7.0Hz,1H),2.33–2.12(m,2H),1.94(dd,J=12.1,3.1Hz,2H),1.89–1.74(m,2H),1.60–1.33(m,10H),1.25(d,J=13.9Hz,27H),1.10–0.90(m,6H),0.86(t,J=6.7Hz,3H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ141.16,121.28,73.01,71.67,67.77,58.85,54.82,50.24,48.10,42.33,42.10,39.80,37.25,36.64,32.15,32.03,31.78,31.70,31.33,31.04,30.35,29.77,29.75,29.69,29.66,29.47,27.56,27.50,22.79,20.64,19.48,14.70,14.23.
HRMS(ESI):m/z[m+H]+calcd for C34H61NO2:516.4781;found:516.4772.
实施例6:
16α-环己氨基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-6)的制备
16α-环己氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.480mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.80mL,1.0M in THF,5.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=150:1~120:1~90:1)得白色泡沫状固体化合物136mg,收率:65%。
1H NMR(400MHz,CDCl3)δ5.35–5.27(m,1H),3.63–3.41(m,2H),2.80–2.69(m,1H),2.35–2.13(m,2H),2.00(q,J=5.2Hz,1H),1.97–1.88(m,4H),1.87–1.34(m,15H),1.30(s,5H),1.25(s,6H),1.05(td,J=13.4,12.3,3.8Hz,1H),0.98(s,3H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ141.04,121.26,73.45,71.56,65.88,55.75,55.15,54.30,50.00,42.30,42.22,39.54,37.24,36.59,32.87,31.67,31.54,31.24,31.20,30.75,26.43,25.63,24.55,24.34,20.60,19.48,14.70.
HRMS(ESI):m/z[m+H]+calcd for C28H47NO2:430.3685;found:430.3688.
实施例7:
16α-(4-氟苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-7)的制备
16α-(4-氟苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.450mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.50mL,1.0M in THF,5.50mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=90:1~70:1~50:1)得白色泡沫状固体化合物118mg,收率:57%。
1H NMR(400MHz,CDCl3)δ7.30–7.24(m,2H),6.99(t,J=8.7Hz,2H),5.35(d,J=5.4Hz,1H),3.89(d,J=12.8Hz,1H),3.67(d,J=12.8Hz,1H),3.51(tt,J=11.2,4.6Hz,1H),3.34(td,J=9.4,3.1Hz,1H),3.19(s,3H),2.34–2.17(m,2H),1.97(dtd,J=12.5,5.3,2.9Hz,2H),1.89–1.75(m,2H),1.67–1.35(m,8H),1.25(s,6H),1.16(s,3H),1.06(td,J=13.9,4.2Hz,1H),1.00(s,3H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ163.46,161.02,141.17,134.67,130.20,130.12,121.25,115.52,115.30,73.25,71.71,67.67,57.89,54.77,51.40,50.19,42.34,42.32,39.77,37.24,36.64,31.78,31.70,31.66,31.32,30.96,27.33,20.64,19.48,14.66.
HRMS(ESI):m/z[m+H]+calcd for C29H42FNO2:456.3278;found:456.3276.
实施例8:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-8)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(270mg,0.540mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(6.47mL,1.0M in THF,6.47mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~100:1~80:1)得白色泡沫状固体化合物132mg,收率:48.0%。
1H NMR(400MHz,CDCl3)δ7.42(d,J=8.4Hz,2H),7.18(d,J=8.3Hz,2H),5.37–5.32(m,1H),3.84(d,J=13.0Hz,1H),3.64(d,J=13.0Hz,1H),3.51(tt,J=11.4,9.7,4.3Hz,1H),3.33(td,J=9.5,9.0,3.6Hz,1H),2.34–2.16(m,2H),1.96(dt,J=12.9,3.3Hz,2H),1.89–1.71(m,3H),1.63–1.34(m,9H),1.33–1.13(m,9H),1.11–0.91(m,5H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ141.15,138.33,131.63,130.19,121.26,121.15,77.46,73.20,71.73,67.91,58.04,54.79,51.65,50.18,42.33,42.30,39.78,37.22,36.63,31.86,31.79,31.70,31.31,30.92,27.52,20.63,19.48,14.66.
HRMS(ESI):m/z[m+H]+calcd for C29H42BrNO2:516.2477;found:516.2476.
实施例9:
16α-(3-溴苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-9)的制备
16α-(3-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~90:1~60:1)得白色泡沫状固体化合物128mg,收率:62%。
1H NMR(400MHz,CDCl3)δ7.44(d,J=1.8Hz,1H),7.40–7.34(m,1H),7.26(s,2H),7.18(t,J=7.8Hz,1H),5.35(d,J=5.4Hz,1H),3.87(d,J=13.0Hz,1H),3.68(d,J=13.0Hz,1H),3.52(tt,J=11.2,4.5Hz,1H),3.39–3.28(m,1H),2.35–2.17(m,2H),1.97(dt,J=12.3,3.3Hz,2H),1.88–1.78(m,2H),1.61–1.39(m,8H),1.31(dd,J=12.7,4.2Hz,1H),1.25(d,J=3.4Hz,5H),1.20(s,3H),1.07(td,J=13.9,13.4,4.2Hz,1H),1.00(s,3H),0.72(s,3H).
13C NMR(101MHz,CDCl3)δ141.57,141.15,131.46,130.56,130.23,127.15,122.62,121.32,73.22,71.80,67.90,58.25,54.80,51.80,50.21,42.38,39.82,37.25,36.67,31.85,31.82,31.75,31.35,30.98,27.48,20.67,19.51,14.69.
HRMS(ESI):m/z[m+H]+calcd for C29H42BrNO2:516.2477;found:516.2478.
实施例10:
16α-(4-甲氧基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-10)的制备
16α-(4-甲氧基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.440mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.31mL,1.0M in THF,5.31mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~100:1~80:1)得白色泡沫状固体化合物131mg,收率:63.2%。
1H NMR(400MHz,CDCl3)δ7.28(d,J=8.6Hz,2H),6.88(d,J=8.6Hz,2H),5.35(d,J=5.2Hz,1H),4.11(d,J=13.1Hz,1H),3.79(s,3H),3.75–3.67(m,2H),3.52(tt,J=11.0,4.6Hz,1H),3.46–3.33(m,1H),2.48–2.38(m,1H),2.34–2.16(m,2H),2.02–1.76(m,7H),1.65(dd,J=17.7,8.6Hz,1H),1.57–1.40(m,5H),1.40–1.32(m,1H),1.29(s,3H),1.08(s,3H),1.04–1.00(m,1H),0.98(s,3H),0.67(s,3H).
13C NMR(101MHz,CDCl3)δ159.81,141.01,130.47,126.80,121.32,114.46,73.81,71.72,65.67,63.71,57.44,55.45,54.42,50.23,49.97,42.50,42.34,39.48,37.23,36.60,31.71,31.57,31.33,31.23,29.44,28.48,26.35,20.61,19.49,14.63.
HRMS(ESI):m/z[m+H]+calcd for C30H45NO3:468.3478;found:468.3475.
实施例11:
16α-(4-叔丁基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-11)的制备
16α-(4-叔丁基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.420mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.02mL,1.0M in THF,5.02mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物145mg,收率:70.1%。
1H NMR(400MHz,CDCl3)δ7.33(d,J=8.3Hz,2H),7.24(d,J=8.3Hz,2H),5.35(d,J=4.8Hz,1H),3.88(d,J=12.7Hz,1H),3.69(d,J=12.6Hz,1H),3.57–3.46(m,1H),3.38(td,J=9.4,2.9Hz,1H),2.34–2.16(m,2H),2.02–1.89(m,2H),1.87–1.75(m,2H),1.61(ddd,J=14.5,11.3,6.0Hz,8H),1.29(s,9H),1.25(s,4H),1.16(s,3H),1.08(dd,J=14.3,4.3Hz,1H),1.03-0.96(m,5H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ150.50,141.11,135.71,128.29,125.49,121.30,73.25,71.69,67.61,58.07,54.75,51.76,50.14,42.32,42.27,39.74,37.22,36.62,34.59,31.75,31.68,31.64,31.45,31.30,30.99,27.26,20.63,19.48,14.68.
HRMS(ESI):m/z[m+H]+calcd for C33H51NO2:494.3998;found:494.3991.
实施例12:
16α-(2-甲基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-12)的制备
16α-(2-甲基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.460mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.51mL,1.0M in THF,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物110mg,收率:52.9%。
1H NMR(400MHz,CDCl3)δ7.25–7.20(m,1H),7.17–7.10(m,3H),5.35(d,J=4.5Hz,1H),3.86(d,J=12.6Hz,1H),3.68(d,J=12.6Hz,1H),3.59–3.46(m,1H),3.39(dt,J=9.6,6.1Hz,1H),2.35(s,3H),2.33–2.17(m,2H),1.98(ddt,J=16.1,11.3,3.0Hz,2H),1.82(d,J=11.7Hz,2H),1.68–1.34(m,9H),1.33–1.24(m,2H),1.23(s,3H),1.17(s,3H),1.07(td,J=13.9,4.1Hz,1H),1.00(s,3H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ141.18,137.15,136.69,130.56,129.10,127.52,126.05,121.29,73.01,71.75,67.91,58.25,54.82,50.23,50.07,42.35,42.32,39.81,37.25,36.65,32.14,31.81,31.72,31.35,30.87,27.43,20.66,19.49,19.12,14.67.
HRMS(ESI):m/z[m+H]+calcd for C30H45NO2:452.3529;found:452.3526.
实施例13:
16α-(3-甲基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-13)的制备
16α-(3-甲基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.460mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.51mL,1.0M in THF,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物127mg,收率:61.1%。
1H NMR(400MHz,CDCl3)δ7.25–7.20(m,1H),7.17–7.10(m,3H),5.35(d,J=4.5Hz,1H),3.86(d,J=12.6Hz,1H),3.68(d,J=12.6Hz,1H),3.59–3.46(m,1H),3.39(dt,J=9.6,6.1Hz,1H),2.35(s,3H),2.33–2.17(m,2H),1.98(ddt,J=16.1,11.3,3.0Hz,2H),1.82(d,J=11.7Hz,2H),1.68–1.34(m,9H),1.33–1.24(m,2H),1.23(s,3H),1.17(s,3H),1.07(td,J=13.9,4.1Hz,1H),1.00(s,3H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ141.18,137.15,136.69,130.56,129.10,127.52,126.05,121.29,73.01,71.75,67.91,58.25,54.82,50.23,50.07,42.35,42.32,39.81,37.25,36.65,32.14,31.81,31.72,31.35,30.87,27.43,20.66,19.49,19.12,14.67.
HRMS(ESI):m/z[m+H]+calcd for C30H45NO2:452.3529;found:452.3531.
实施例14:
16α-(4-甲基苄氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-14)的制备
16α-(4-甲基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.460mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.51mL,1.0M in THF,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物123mg,收率:59.2%。
1H NMR(400MHz,CDCl3)δ7.19(d,J=7.8Hz,2H),7.10(d,J=7.8Hz,2H),5.35(d,J=4.8Hz,1H),3.84(d,J=12.6Hz,1H),3.66(d,J=12.6Hz,1H),3.51(tt,J=10.7,4.4Hz,1H),3.35(td,J=9.0,3.7Hz,1H),2.31(s,3H),2.30–2.17(m,2H),2.02–1.91(m,2H),1.87–1.79(m,2H),1.63–1.41(m,8H),1.38(dd,J=13.0,3.5Hz,1H),1.30(dd,J=12.5,4.1Hz,1H),1.24(s,3H),1.22(s,1H),1.18(s,3H),1.06(td,J=13.6,3.9Hz,1H),1.00(s,3H),0.96(dd,J=11.3,4.8Hz,1H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ141.15,136.90,136.30,129.19,128.41,121.30,73.11,71.71,67.89,57.96,54.82,52.00,50.21,42.34,42.24,39.79,37.23,36.64,31.93,31.80,31.70,31.33,30.94,27.50,21.20,20.64,19.48,14.67.
HRMS(ESI):m/z[m+H]+calcd for C30H45NO2:452.3529;found:452.3527.
实施例15:
16α-(2-噻吩甲氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-15)的制备
16α-(2-噻吩甲氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.470mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.61mL,1.0M in THF,5.61mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物126mg,收率:59.0%。
1H NMR(400MHz,CDCl3)δ7.20(dd,J=4.8,1.5Hz,1H),6.93(p,J=3.4Hz,2H),5.34(d,J=5.1Hz,1H),4.12(d,J=13.7Hz,1H),3.89(d,J=13.7Hz,1H),3.58–3.47(m,1H),3.42(td,J=9.2,3.3Hz,1H),2.98(br,3H),2.35–2.16(m,2H),2.01–1.89(m,2H),1.87–1.75(m,2H),1.66–1.35(m,8H),1.34–1.14(m,9H),1.13–1.01(m,1H),0.99(s,4H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ142.63,141.13,126.74,125.44,124.89,121.29,73.23,71.72,67.78,57.86,54.72,50.16,46.71,42.38,42.33,39.77,37.22,36.63,31.76,31.69,31.66,31.30,30.87,27.44,20.65,19.49,14.64.
HRMS(ESI):m/z[m+H]+calcd for C27H41NO2S:444.2936;found:444.2939.
实施例16:
16α-(2-呋喃甲氨)基-17β-(1-羟基-1-甲基-乙基)雄甾-5-烯-3β-醇(GAPS-16)的制备
16α-(2-呋喃甲氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.490mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加甲基溴化镁(5.83mL,1.0M in THF,5.83mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~60:1)得白色泡沫状固体化合物124mg,收率:59.3%。
1H NMR(400MHz,CDCl3)δ7.37–7.29(m,1H),6.29(dd,J=3.2,1.9Hz,1H),6.20(d,J=3.2Hz,1H),5.33(d,J=5.2Hz,1H),3.88(d,J=14.2Hz,1H),3.72(d,J=14.2Hz,1H),3.50(tq,J=10.5,5.7,5.0Hz,1H),3.35(td,J=9.2,3.3Hz,1H),2.33–2.16(m,2H),1.99–1.88(m,2H),1.87–1.76(m,2H),1.60–1.34(m,8H),1.34–1.25(m,1H),1.24(s,4H),1.19(s,3H),1.06(td,J=13.8,13.2,4.0Hz,1H),0.99(s,3H),0.97–0.92(m,1H),0.71(s,3H).
13C NMR(101MHz,CDCl3)δ153.02,141.97,141.11,121.31,110.41,107.40,71.73,71.71,67.72,58.08,54.71,50.18,44.68,42.35,42.32,39.80,37.25,36.64,31.89,31.72,31.30,30.95,27.40,20.66,19.48,14.63.
HRMS(ESI):m/z[m+H]+calcd for C27H41NO3:428.3165;found:428.3159.
实施例17:
16α-异丁氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-17)的制备和
16α-异丁氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.520mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(6.19mL,1.0M in Et2O,6.19mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=50:1~30:1)得白色泡沫状固体化合物111mg,收率:50.1%。
1H NMR(400MHz,CDCl3)δ6.05(ddt,J=17.4,10.8,7.1Hz,1H),5.34(d,J=5.0Hz,1H),5.15–4.96(m,2H),3.51(tt,J=10.4,4.4Hz,1H),3.32(td,J=9.2,3.3Hz,1H),2.55(dd,J=11.4,6.4Hz,1H),2.38–2.14(m,5H),1.95(dd,J=12.6,4.0Hz,2H),1.88–1.76(m,3H),1.64(p,J=6.6Hz,2H),1.60–1.38(m,8H),1.38–1.30(m,1H),1.25(s,4H),1.19(d,J=15.7Hz,1H),1.07(dt,J=14.0,6.7Hz,1H),0.99(s,4H),0.89(t,J=6.5Hz,6H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ141.10,135.29,121.34,117.25,74.62,71.75,66.33,59.14,56.39,54.76,50.15,47.78,42.32,39.86,37.23,36.63,32.07,31.77,31.70,31.34,28.76,25.45,20.98,20.86,20.62,19.47,15.01.
HRMS(ESI):m/z[m+H]+calcd for C28H47NO2:430.3685;found:430.3684.
实施例18:
16α-正丁氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-18)的制备
16α-正丁氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.520mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(6.19mL,1.0M in Et2O,6.19mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=50:1~30:1)得白色泡沫状固体化合物182mg,收率:85.2%。
1H NMR(400MHz,CDCl3)δ6.03(ddt,J=17.0,9.8,7.0Hz,1H),5.38–5.26(m,1H),5.14–4.94(m,2H),3.61–3.43(m,1H),3.32(td,J=9.2,3.1Hz,1H),2.69(dt,J=11.2,6.6Hz,1H),2.45(dt,J=10.9,6.9Hz,1H),2.36–2.14(m,4H),1.93(ddd,J=11.3,5.4,2.7Hz,2H),1.88–1.75(m,3H),1.60–1.28(m,13H),1.25(d,J=2.0Hz,3H),1.21–1.12(m,1H),1.11–0.92(m,5H),0.87(t,J=7.1Hz,3H),0.74(d,J=1.9Hz,3H).
13C NMR(101MHz,CDCl3)δ141.11,135.27,121.30,117.21,66.32,58.74,54.78,50.15,47.81,42.32,42.30,39.85,37.23,36.63,32.37,32.15,31.77,31.69,31.34,25.47,20.63,19.47,15.02,14.06.
HRMS(ESI):m/z[m+H]+calcd for C28H47NO2:430.3685;found:430.3681.
实施例19:
16α-正己氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-19)的制备
16α-正己氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.480mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.77mL,1.0M in Et2O,5.77mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=60:1~40:1)得白色泡沫状固体化合物138mg,收率:62.8%。
1H NMR(400MHz,CDCl3)δ6.02(ddt,J=17.2,10.3,6.9Hz,1H),5.39–5.24(m,1H),5.17–4.92(m,2H),3.49(tt,J=11.3,4.5Hz,1H),3.33(dt,J=9.8,6.0Hz,1H),2.69(ddd,J=11.3,7.7,6.1Hz,1H),2.45(ddd,J=11.4,8.0,6.3Hz,1H),2.31–2.13(m,4H),2.01–1.88(m,2H),1.88–1.72(m,2H),1.63–1.33(m,10H),1.24(d,J=7.0Hz,10H),0.97(s,5H),0.91–0.79(m,3H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ141.10,135.12,121.25,117.34,74.68,71.63,66.16,58.81,54.71,50.12,48.12,47.76,42.32,39.83,37.24,36.61,31.95,31.81,31.78,31.73,31.68,31.32,30.08,27.12,25.37,22.63,20.61,19.44,15.00,14.14.
HRMS(ESI):m/z[m+H]+calcd for C30H51NO2:458.3998;found:458.3995.
实施例20:
16α-正辛氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-20)的制备
16α-正辛氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.450mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.41mL,1.0M in Et2O,5.41mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=70:1~50:1)得白色泡沫状固体化合物145mg,收率:66.2%。
1H NMR(400MHz,CDCl3)δ6.03(ddt,J=17.3,10.5,6.9Hz,1H),5.33(dd,J=5.2,2.7Hz,1H),5.13–5.01(m,2H),3.50(tt,J=10.6,4.5Hz,1H),3.39–3.28(m,1H),2.70(ddd,J=11.5,7.8,6.1Hz,1H),2.45(ddd,J=11.3,7.9,6.3Hz,1H),2.32–2.16(m,4H),1.99–1.72(m,6H),1.47(dtt,J=17.5,13.1,8.9Hz,10H),1.33–1.20(m,16H),1.12–1.01(m,1H),0.99(s,3H),0.86(t,J=6.8Hz,4H),0.75(d,J=7.1Hz,3H).
13C NMR(101MHz,CDCl3)δ141.08,135.17,121.32,117.36,74.67,71.72,66.22,58.80,54.75,50.13,48.17,47.79,42.32,39.84,37.24,36.62,32.02,31.95,31.75,31.69,31.33,30.20,29.60,29.31,27.48,27.03,25.43,22.75,20.62,19.47,15.02,14.20.
HRMS(ESI):m/z[m+H]+calcd for C32H55NO2:486.4311;found:486.4312.
实施例21:
16α-十二烷氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-21)的制备
16α-十二烷氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(4.80mL,1.0M in Et2O,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~60:1)得白色泡沫状固体化合物172mg,收率:79.3%。
1H NMR(400MHz,CDCl3)δ6.00(ddt,J=17.2,10.2,7.0Hz,1H),5.32(dt,J=5.0,2.1Hz,1H),5.15–5.01(m,2H),3.49(ddd,J=11.1,6.5,4.7Hz,1H),3.38(td,J=9.3,2.4Hz,1H),2.80(dt,J=11.5,7.1Hz,1H),2.52(dt,J=11.5,7.3Hz,1H),2.24(dddd,J=33.9,15.8,8.9,2.9Hz,4H),2.00–1.85(m,2H),1.80(dt,J=11.9,3.8Hz,2H),1.63(dd,J=11.0,5.4Hz,2H),1.56–1.43(m,5H),1.40–1.32(m,2H),1.23(d,J=4.0Hz,20H),1.04(td,J=13.8,12.9,4.0Hz,1H),0.97(s,3H),0.86(t,J=6.8Hz,3H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ140.89,134.55,121.35,117.98,74.87,71.64,65.82,59.17,54.48,49.96,47.98,47.67,42.41,42.29,39.77,37.20,36.55,32.01,31.65,31.57,31.22,29.73,29.62,29.51,29.44,27.31,24.95,22.78,20.60,19.45,15.04,14.22.
HRMS(ESI):m/z[m+H]+calcd for C36H63NO2:541.4859;found:541.4860.
实施例22:
16α-环丙氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-22)的制备
16α-环丙氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.540mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(6.46mL,1.0M in Et2O,6.46mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=90:1~70:1)得白色泡沫状固体化合物135mg,收率:60.5%。
1H NMR(400MHz,CDCl3)δ6.11–5.94(m,1H),5.77(td,J=17.2,7.0Hz,1H),5.33(d,J=3.5Hz,1H),5.13–4.96(m,4H),3.60–3.41(m,2H),2.56(td,J=7.0,3.5Hz,1H),2.37–2.08(m,6H),1.94(dt,J=14.8,5.3Hz,2H),1.88–1.75(m,2H),1.59–1.39(m,8H),1.38–1.28(m,2H),1.25(d,J=3.7Hz,3H),1.11–1.01(m,1H),1.00–0.97(m,3H),0.87(t,J=7.4Hz,3H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ141.10,135.32,135.20,121.28,117.28,117.21,74.68,71.73,66.90,57.00,55.58,54.65,50.14,47.74,42.35,42.32,39.91,37.23,36.62,32.52,31.73,31.69,31.31,27.25,25.36,20.63,19.47,15.00,10.22,10.19.
HRMS(ESI):m/z[m+H]+calcd for C27H43NO2:414.3372;found:414.3368.
实施例23:
16α-环戊氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-23)的制备
16α-环戊氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.500mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(6.01mL,1.0M in Et2O,6.01mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~50:1)得白色泡沫状固体化合物156mg,收率:70.9%。
1H NMR(400MHz,CDCl3)δ6.04(ddt,J=17.2,10.4,7.0Hz,1H),5.34(d,J=5.4Hz,1H),5.13–5.01(m,2H),3.58–3.46(m,1H),3.41(td,J=9.4,3.0Hz,1H),3.13(p,J=5.8Hz,1H),2.26(ddd,J=19.6,8.8,3.5Hz,4H),1.94(tt,J=9.9,4.1Hz,2H),1.82(tq,J=12.3,7.1,5.4Hz,3H),1.75–1.61(m,4H),1.56–1.41(m,10H),1.33(qd,J=8.8,4.0Hz,2H),1.27–1.23(m,6H),1.06(d,J=4.1Hz,1H),1.00(s,3H),0.95(dd,J=11.3,4.9Hz,1H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ141.09,135.24,121.36,117.26,74.70,71.78,66.81,57.63,56.68,54.75,50.17,47.83,42.34,42.28,39.92,37.24,36.65,34.54,32.40,31.93,31.79,31.72,31.37,29.82,25.34,23.99,20.64,19.49,14.98.
HRMS(ESI):m/z[m+H]+calcd for C29H47NO2:442.3685;found:442.3682.
实施例24:
16α-环己氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-24)的制备
16α-环己氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.480mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.80mL,1.0M in Et2O,5.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~50:1)得白色泡沫状固体化合物146mg,收率:66.5%。
1H NMR(400MHz,CDCl3)δ6.13–5.94(m,1H),5.33(d,J=2.8Hz,1H),5.12–4.94(m,2H),3.58–3.42(m,2H),2.54–2.42(m,1H),2.34–2.15(m,4H),2.02–1.88(m,4H),1.87–1.76(m,3H),1.66(td,J=13.3,12.7,8.0Hz,3H),1.60–1.26(m,10H),1.26–1.01(m,11H),0.99(s3H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ141.11,135.24,121.30,117.18,74.68,71.69,66.77,54.97,54.91,54.72,50.16,47.80,42.31,42.17,39.90,37.22,36.62,35.03,33.12,32.32,31.76,31.68,31.34,26.14,25.23,25.01,24.59,20.61,19.46,14.93.
HRMS(ESI):m/z[m+H]+calcd for C30H49NO2:456.3842;found:456.3834.
实施例25:
16α-苄氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-25)的制备
16α-苄氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.470mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.69mL,1.0M in Et2O,5.69mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=30:1~25:1~20:1)得白色泡沫状固体化合物158mg,收率:71.7%。
1H NMR(400MHz,CDCl3)δ7.34–7.28(m,4H),7.26(m,1H),6.03(ddt,J=17.2,10.3,7.0Hz,1H),5.36(dt,J=5.3,2.0Hz,1H),5.16–5.00(m,2H),3.92(d,J=12.7Hz,1H),3.71(d,J=12.7Hz,1H),3.52(t,J=4.6Hz,1H),3.43(td,J=9.5,2.6Hz,1H),2.38–2.17(m,4H),2.04–1.89(m,2H),1.83(dp,J=9.8,3.3,2.9Hz,2H),1.74–1.21(m,12H),1.16(s,3H),1.09(dd,J=14.3,4.3Hz,1H),1.00(s,4H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ141.06,134.92,128.70,128.65,127.58,121.36,117.76,74.83,71.78,66.30,58.02,54.71,52.28,50.09,47.62,42.54,42.36,39.86,37.24,36.64,31.76,31.73,31.57,31.36,25.25,20.65,19.49,14.99.
HRMS(ESI):m/z[m+H]+calcd for C31H45NO2:464.3529;found:464.3527.
实施例26:
16α-(4-氟苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-26)的制备
16α-(4-氟苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.450mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.46mL,1.0M in Et2O,5.46mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=50:1~30:1~20:1)得白色泡沫状固体化合物182mg,收率:85.2%。
1H NMR(400MHz,CDCl3)δ7.26–7.20(m,2H),7.01–6.91(m,2H),6.01(ddt,J=17.4,10.6,7.0Hz,1H),5.37–5.26(m,1H),5.15–4.98(m,2H),3.82(d,J=12.7Hz,1H),3.63(d,J=12.6Hz,1H),3.55–3.41(m,1H),3.36(td,J=9.1,3.3Hz,1H),2.32–2.17(m,4H),1.94(dt,J=11.8,3.3Hz,2H),1.86–1.73(m,2H),1.59–1.19(m,10H),1.15(s,3H),1.06(dd,J=14.0,4.0Hz,1H),0.98(s,4H),0.72(s,3H).
13C NMR(101MHz,CDCl3)δ163.31,160.88,141.14,134.97,130.13,130.05,121.13,117.46,115.39,115.18,74.76,71.55,66.35,57.78,54.68,51.58,50.06,47.54,42.42,42.26,39.81,37.17,36.57,31.82,31.72,31.62,31.27,25.29,20.56,19.42,14.91.
HRMS(ESI):m/z[m+H]+calcd for C31H44FNO2:482.3434;found:482.3431.
实施例27:
16α-(4-溴苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-27)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(4.80mL,1.0M in Et2O,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~60:1~50:1)得白色泡沫状固体化合物134mg,收率:61.7%。
1H NMR(400MHz,CDCl3)δ7.41(d,J=8.3Hz,2H),7.18(d,J=8.3Hz,2H),6.00(ddt,J=17.2,10.4,7.0Hz,1H),5.32(d,J=5.7Hz,1H),5.14–4.99(m,2H),3.84(t,J=12.0Hz,1H),3.64(d,J=13.0Hz,1H),3.58–3.45(m,1H),3.38(td,J=9.4,2.7Hz,1H),2.39–2.13(m,4H),2.02–1.89(m,2H),1.89–1.73(m,2H),1.68–1.40(m,8H),1.40–1.20(m,5H),1.15(s,3H),1.12–1.01(m,1H),0.98(s,4H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ141.10,137.65,134.80,131.69,130.32,121.34,121.22,117.75,74.87,71.66,66.27,57.96,54.65,51.57,50.04,47.54,42.51,42.28,39.81,37.19,36.59,31.72,31.65,31.52,31.28,25.21,20.58,19.45,14.95.
HRMS(ESI):m/z[m+H]+calcd for C31H44BrNO2:452.4539;found:452.4546.
实施例28:
16α-(3-溴苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-28)的制备
16α-(3-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(4.80mL,1.0M in Et2O,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物151mg,收率:69.5%。
1H NMR(400MHz,CDCl3)δ7.44–7.34(m,2H),7.28–7.23(m,1H),7.20–7.13(m,1H),6.02(ddt,J=13.9,10.3,6.9Hz,1H),5.38–5.30(m,1H),5.14–5.01(m,2H),3.83(d,J=12.9Hz,1H),3.66(d,J=12.9Hz,1H),3.51(tt,J=12.1,4.6Hz,1H),3.39(td,J=8.9,3.5Hz,1H),2.38–2.15(m,4H),2.06–1.91(m,3H),1.87–1.74(m,3H),1.62–1.38(m,8H),1.39–1.16(m,6H),1.12–0.91(m,5H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ141.58,141.11,134.98,131.43,130.49,130.21,127.19,122.54,121.26,117.58,74.79,71.71,66.48,58.14,54.72,51.93,50.10,47.60,42.54,42.32,39.88,37.22,36.62,31.91,31.78,31.69,31.32,25.38,20.62,19.47,14.97.
HRMS(ESI):m/z[m+H]+calcd for C31H44BrNO2:452.4539;found:452.2626.
实施例29:
16α-(4-甲基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-29)的制备
16α-(4-甲基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.460mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.51mL,1.0M in Et2O,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~60:1~50:1)得白色泡沫状固体化合物140mg,收率:63.7%。
1H NMR(400MHz,CDCl3)δ7.19(d,J=8.0Hz,2H),7.10(d,J=7.8Hz,2H),6.04(ddt,J=17.3,10.4,7.0Hz,1H),5.38–5.32(m,1H),5.13–4.99(m,2H),3.82(d,J=12.5Hz,1H),3.65(d,J=12.5Hz,1H),3.51(tt,J=11.2,4.5Hz,1H),3.40(td,J=9.1,3.1Hz,1H),2.31(s,4H),2.30–2.20(m,3H),2.04–1.91(m,2H),1.88–1.76(m,2H),1.62–1.38(m,8H),1.38–1.19(m,3H),1.18(s,3H),1.10–1.02(m,1H),0.99(s,3H),0.98–0.91(m,1H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ141.11,136.92,136.10,135.16,129.19,128.48,121.27,117.37,74.73,71.68,66.38,57.87,54.75,52.08,50.10,47.63,42.43,42.30,39.84,37.20,36.61,31.86,31.77,31.67,31.32,25.36,21.20,20.61,19.46,14.97.
HRMS(ESI):m/z[m+H]+calcd for C32H47NO2:478.3685;found:478.3686.
实施例30:
16α-(4-叔丁基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-30)的制备
16α-(4-叔丁基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.420mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.02mL,1.0M in Et2O,5.02mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=80:1~60:1~50:1)得白色泡沫状固体化合物134mg,收率:61.8%。
1H NMR(400MHz,CDCl3)δ7.32(d,J=8.2Hz,2H),7.24(d,J=8.1Hz,2H),6.03(ddt,J=17.2,10.3,7.0Hz,1H),5.35(d,J=5.0Hz,1H),5.15–5.00(m,2H),3.87(d,J=12.5Hz,1H),3.68(d,J=12.5Hz,1H),3.57–3.47(m,1H),3.43(td,J=9.3,2.6Hz,1H),2.35–2.18(m,4H),2.01–1.89(m,2H),1.89–1.76(m,3H),1.71–1.31(m,9H),1.29(s,9H),1.25(s,1H),1.17(s,3H),1.13–1.03(m,1H),1.00(s,3H),0.98–0.92(m,1H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ150.50,141.07,135.01,128.37,125.49,121.32,117.60,74.79,71.71,66.26,58.05,54.71,51.91,50.06,47.63,42.47,42.31,39.82,37.21,36.60,34.58,31.74,31.68,31.44,31.31,25.21,20.61,19.47,14.99.
HRMS(ESI):m/z[m+H]+calcd for C35H53NO2:520.4155;found:520.4154.
实施例31:
16α-(4-甲氧基苄氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-31)的制备
16α-(4-叔丁基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.440mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.31mL,1.0M in Et2O,5.31mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物158mg,收率:72.3%。
1H NMR(400MHz,CDCl3)δ7.22(d,J=8.6Hz,2H),6.83(d,J=8.6Hz,2H),6.04(ddt,J=17.2,10.3,7.0Hz,1H),5.36(dt,J=4.1,1.9Hz,1H),5.15–4.98(m,2H),3.78(s,3H),3.64(d,J=12.4Hz,1H),3.53(dt,J=11.2,6.3Hz,1H),3.40(td,J=9.3,3.1Hz,1H),2.27(ddd,J=18.7,9.4,3.8Hz,4H),2.05–1.92(m,2H),1.89–1.76(m,2H),1.52(tdt,J=25.1,14.8,4.4Hz,9H),1.41–1.31(m,2H),1.25(s,7H),1.17(s,3H),1.00(s,3H),0.91–0.81(m,2H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ158.98,141.08,135.17,129.80,121.39,117.47,113.95,74.76,71.83,66.43,57.75,55.43,54.80,51.74,50.14,47.68,42.48,42.36,39.89,37.24,36.66,32.07,31.88,31.81,31.73,31.37,29.84,25.38,22.84,20.65,19.50,15.01,14.27.
HRMS(ESI):m/z[m+H]+calcd for C32H47N2O3:494.3634;found:494.3627.
实施例32:
16α-(2-吡啶甲氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-32)的制备
16α-(2-吡啶甲氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.470mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.68mL,1.0M in Et2O,5.68mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=20:1~10:1)得白色泡沫状固体化合物126mg,收率:57.2%。
1H NMR(400MHz,CDCl3)δ8.55–8.41(m,1H),7.69–7.57(m,1H),7.30(dd,J=7.9,3.1Hz,1H),7.15(t,J=4.1Hz,1H),6.02(ddt,J=17.2,9.9,7.0Hz,1H),5.32(d,J=4.9Hz,1H),5.11–4.91(m,2H),4.02(dd,J=14.1,2.5Hz,1H),3.82(dd,J=14.1,4.9Hz,1H),3.50(tt,J=12.4,5.2Hz,1H),3.40(td,J=9.3,2.3Hz,1H),2.42–2.12(m,4H),2.01–1.87(m,2H),1.80(d,J=11.5Hz,2H),1.68–1.25(m,10H),1.19(d,J=13.0Hz,4H),1.09–0.90(m,6H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ159.13,148.93,141.00,136.92,135.97,135.11,122.84,122.77,122.30,121.36,117.34,116.77,74.77,74.54,71.54,68.91,66.69,58.27,57.51,54.55,53.60,53.43,50.11,50.07,47.59,44.08,42.56,42.32,40.03,39.92,37.23,36.60,32.03,31.66,31.27,31.21,28.21,25.40,20.63,19.46,15.25,15.11.
HRMS(ESI):m/z[m+H]+calcd for C30H44N2O2:465.3481;found:465.3471.
实施例33:
16α-色氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-33)的制备
16α-色氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.420mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.06mL,1.0M in Et2O,5.06mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(乙酸乙酯/甲醇=50:1)得白色固体化合物117mg,收率:53.7%。
1H NMR(400MHz,CDCl3)δ8.31(s,1H),7.58(d,J=7.8Hz,1H),7.33(d,J=8.1Hz,1H),7.18(t,J=7.5Hz,1H),7.10(t,J=7.4Hz,1H),6.98(s,1H),6.05(td,J=17.1,6.9Hz,1H),5.31(d,J=5.0Hz,1H),5.17–4.99(m,2H),3.51(tt,J=10.4,4.6Hz,1H),3.40(td,J=9.4,2.7Hz,1H),3.14–3.03(m,1H),2.99(dt,J=15.7,7.2Hz,1H),2.86(td,J=11.9,10.2,6.1Hz,2H),2.32–2.20(m,3H),1.92(dt,J=12.5,3.2Hz,1H),1.89–1.73(m,3H),1.44(tq,J=14.0,7.7,5.4Hz,8H),1.27(s,3H),1.14–1.02(m,2H),0.98(s,3H),0.96–0.83(m,1H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ140.98,136.54,135.20,127.44,122.27,122.04,121.37,119.33,118.87,117.37,113.60,111.36,74.83,71.75,65.98,58.87,54.63,50.06,48.03,47.77,42.31,39.79,37.23,36.59,31.85,31.69,31.63,31.27,26.24,25.49,20.60,19.45,15.01.
HRMS(ESI):m/z[m+H]+calcd for C34H48N2O2:517.3794;found:517.3789.
实施例34:
16α-(2-呋喃甲氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-34)的制备
16α-(2-呋喃甲氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.490mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.83mL,1.0M in Et2O,5.83mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=60:1~40:1~30:1)得白色粉末状固体化合物148mg,收率:67.2%。
1H NMR(400MHz,CDCl3)δ7.32(d,J=1.0Hz,1H),6.28(dd,J=3.2,1.9Hz,1H),6.18(d,J=3.2Hz,1H),6.02(ddt,J=17.1,10.2,7.0Hz,1H),5.33(dt,J=5.3,2.0Hz,1H),5.18–4.98(m,2H),3.84(d,J=14.1Hz,1H),3.69(d,J=14.1Hz,1H),3.51(tt,J=11.2,4.6Hz,1H),3.38(td,J=9.1,3.1Hz,1H),2.35–2.16(m,4H),2.00–1.87(m,2H),1.86–1.77(m,2H),1.60–1.21(m,10H),1.19(s,3H),1.06(td,J=13.8,13.3,4.0Hz,1H),0.99(s,3H),0.95(dd,J=11.5,4.9Hz,1H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ153.23,141.86,141.08,135.09,121.31,117.51,110.36,107.22,74.66,71.70,66.43,57.95,54.66,50.10,47.61,44.80,42.48,42.32,39.89,37.22,36.62,32.04,31.72,31.69,31.30,25.32,20.63,19.47,14.97.
HRMS(ESI):m/z[m+H]+calcd for C29H35NO3:454.3321;found:454.3323.
实施例35:
16α-(2-噻吩甲氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-35)的制备
16α-(2-噻吩甲氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.470mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.61mL,1.0M in Et2O,5.61mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=50:1~30:1~20:1)得白色粉末状固体化合物158mg,收率:71.5%。
1H NMR(400MHz,CDCl3)δ7.18(dd,J=4.8,1.5Hz,1H),6.97–6.83(m,2H),6.04(ddt,J=17.2,10.3,7.0Hz,1H),5.40–5.26(m,1H),5.14–5.02(m,2H),4.08(d,J=13.6Hz,1H),3.87(d,J=13.6Hz,1H),3.49(dtt,J=21.7,9.2,4.2Hz,2H),2.39–2.17(m,4H),2.03–1.90(m,2H),1.86–1.78(m,2H),1.63–1.38(m,8H),1.38–1.22(m,2H),1.20(s,3H),1.06(td,J=13.7,13.3,4.0Hz,1H),0.99(s,4H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ142.83,141.12,135.14,126.71,125.36,124.74,121.27,117.45,74.77,71.71,66.49,57.77,54.70,50.11,47.56,46.80,42.57,42.32,39.90,37.23,36.63,31.83,31.78,31.69,31.32,25.40,20.64,19.47,14.97.
HRMS(ESI):m/z[m+H]+calcd for C29H35NO2S:470.3093;found:470.3088.
实施例36:
16α-正己氨基-17β-[(R)-2-羟基-2,3-二甲基-丁基)]雄甾-5-烯-3β-醇(GAPS-36)的制备
16α-正己氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.480mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加异丙基溴化镁(5.80mL,1.0M in THF,5.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色粉末状固体化合物121mg,收率:55.0%。
1H NMR(400MHz,CDCl3)δ5.33(d,J=5.1Hz,1H),3.50(dt,J=11.5,6.3Hz,1H),3.31(dt,J=9.0,5.8Hz,1H),2.68(ddd,J=11.2,8.1,5.9Hz,1H),2.46(ddd,J=11.2,8.3,6.1Hz,1H),2.33–2.11(m,2H),2.02–1.87(m,2H),1.87–1.76(m,2H),1.76–1.61(m,2H),1.60–1.32(m,10H),1.32–1.15(m,10H),1.13–1.03(m,1H),1.02–0.77(m,13H),0.72(s,3H).
13C NMR(101MHz,CDCl3)δ141.11,121.33,76.43,71.70,62.05,59.07,54.76,50.18,48.15,42.38,42.33,39.16,37.26,36.65,36.13,32.02,31.87,31.85,31.70,31.43,30.15,27.18,25.15,22.66,20.63,19.46,17.64,17.50,15.17,14.18.
HRMS(ESI):m/z[m+H]+calcd for C30H53NO2:460.4155;found:460.4150.
实施例37:
16α-(2-甲基苄氨)基-17β-[(R)-2-羟基-2-甲基-1-丁烯基)]雄甾-5-烯-3β-醇(GAPS-37)的制备
16α-(2-甲基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.460mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.51mL,1.0M in THF,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=150:1~120:1~100:1~80:1)得白色粉末状固体化合物139mg,收率:64.0%。
1H NMR(400MHz,CDCl3)δ7.25–7.19(m,1H),7.14(s,3H),6.02(td,J=17.1,6.9Hz,1H),5.36(s,1H),5.07(t,J=13.7Hz,2H),3.83(d,J=12.4Hz,1H),3.67(d,J=12.5Hz,1H),3.57–3.37(m,2H),2.34(s,3H),2.32–2.12(m,4H),1.98(t,J=14.1Hz,3H),1.82(d,J=11.1Hz,2H),1.67–1.21(m,12H),1.19(s,3H),1.12–0.90(m,5H),0.76(s,3H).
13C NMR(101MHz,CDCl3)δ141.12,137.18,136.55,135.13,130.44,129.03,127.42,126.03,121.21,117.36,74.60,71.61,66.27,58.22,54.71,50.07,50.04,47.51,42.45,42.26,39.82,37.18,36.58,32.10,31.75,31.63,31.29,25.43,20.58,19.44,19.18,14.97.
HRMS(ESI):m/z[m+H]+calcd for C32H47NO2:478.3685;found:478.3681.
实施例38:
16α-(3-甲基苄氨)基-17β-[(R)-2-羟基-2-甲基-1-丁烯基)]雄甾-5-烯-3β-醇(GAPS-38)的制备
16α-(3-甲基苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.460mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.51mL,1.0M in THF,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=150:1~120:1~100:1~80:1)得白色粉末状固体化合物143mg,收率:65.0%。
1H NMR(400MHz,CDCl3)δ7.18(t,J=7.3Hz,1H),7.10(d,J=9.0Hz,2H),7.05(d,J=7.6Hz,1H),6.04(td,J=16.8,8.0Hz,1H),5.35(s,1H),5.08(t,J=13.9Hz,2H),3.82(d,J=12.3Hz,1H),3.66(d,J=12.4Hz,1H),3.52(d,J=11.3Hz,1H),3.41(t,J=8.8Hz,1H),2.31(d,J=8.0Hz,7H),2.07–1.90(m,2H),1.82(d,J=11.0Hz,2H),1.66–1.39(m,8H),1.20(s,6H),0.99(s,5H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ141.09,139.07,138.14,135.13,129.20,128.43,128.06,125.56,121.26,117.40,71.66,66.38,58.03,54.73,52.42,50.07,47.61,42.42,42.27,39.83,37.18,36.59,31.90,31.75,31.64,31.30,25.33,21.46,20.59,19.46,14.96.
HRMS(ESI):m/z[m+H]+calcd for C32H47NO2:478.3685;found:478.3687.
实施例39:
16α-(4-氯苄氨)基-17β-[(R)-2-羟基-2-甲基-1-丁烯基)]雄甾-5-烯-3β-醇(GAPS-39)的制备
16α-(4-氯苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.440mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.26mL,1.0M in THF,5.26mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~100:1~70:1)得白色粉末状固体化合物156mg,收率:72.0%。
1H NMR(400MHz,CDCl3)δ6.04(td,J=17.0,7.0Hz,1H),5.36(s,1H),5.20–5.02(m,2H),3.85(d,J=12.9Hz,1H),3.66(d,J=12.9Hz,1H),3.52(dt,J=11.5,6.3Hz,1H),3.39(t,J=9.0Hz,1H),2.26(d,J=23.9Hz,5H),2.12–1.91(m,3H),1.83(d,J=12.1Hz,2H),1.72–1.39(m,9H),1.39–1.13(m,6H),1.00(s,6H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ141.12,137.64,134.96,133.02,129.87,128.63,121.18,117.53,71.60,66.41,57.88,54.68,51.65,50.04,47.55,42.45,42.26,39.81,31.79,31.73,31.63,31.27,25.33,20.56,19.44,14.94.
HRMS(ESI):m/z[m+H]+calcd for C31H44ClNO2:498.3139;found:498.3141.
实施例40:
16α-苄氨基-17β-(1-羟基-1-甲基-苯乙基)雄甾-5-烯-3β-醇(GAPS-40)的制备
16α-苄氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.470mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加苄基溴化镁(5.69mL,1.0M in THF,5.69mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物189mg,收率:77.6%。
1H NMR(400MHz,CDCl3)δ7.48-7.40(m,2H),7.38(ddd,J=7.9,6.8,1.2Hz,2H),7.28–7.18(m,2H),7.36–7.31(m,3H),6.11(dt,J=17.2,10.3,7.0Hz,1H),,3.93(d,J=11.8Hz,1H),3.71(d,J=11.8Hz,1H),3.54(t,J=4.8Hz,1H),3.44(td,J=9.6,2.8Hz,1H),2.39–2.18(m,4H),2.06–1.92(m,2H),1.84(dt,J=9.8,3.3,2.8Hz,2H),1.72–1.24(m,14H),1.19(s,3H),1.11(dd,J=14.6,4.8Hz,2H),1.08(s,3H),0.76(s,3H).
13C NMR(101MHz,CDCl3)δ141.06,140.22,135.92,128.70,128.65,128.40,127.58,126.01,125.88,121.36,74.88,68.42,66.32,58.02,54.71,52.31,50.10,47.06,42.56,42.38,39.88,37.15,36.60,31.77,31.71,31.54,31.33,25.84,20.63,19.51,14.97.
HRMS(ESI):m/z[m+H]+calcd for C35H47NO2:514.3685;found:514.3682.
实施例41:
16α-(4-氯苄氨)基-17β-(1-羟基-1-甲基-苯乙基)雄甾-5-烯-3β-醇(GAPS-41)的制备
16α-(4-氯苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.440mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加苄基溴化镁(5.26mL,1.0M in THF,5.26mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=110:1~90:1~60:1)得白色泡沫状固体化合物192mg,收率:79.9%。
1H NMR(400MHz,CDCl3)δδ7.55–7.48(m,2H),7.41(dd,J=7.6,6.4,1.4Hz,2H),7.05–6.89(m,1H),6.04(td,J=17.0,7.0Hz,1H),5.20–5.02(m,2H),3.66(d,J=12.9Hz,1H),3.52(dt,J=11.5,6.3Hz,1H),3.39(t,J=9.0Hz,1H),2.26(d,J=23.9Hz,5H),1.83(d,J=12.1Hz,2H),1.79-1.74(m,6H),1.72–1.54(m,6H),1.335–1.23(m,6H),1.10(s,6H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ141.12,140.23,137.55,133.02,132.70,131.21,129.87,129.65,128.72,128.63,121.18,74.77,69.11,66.41,57.88,54.54,51.85,50.23,47.59,44.23,42.37,42.31,39.28,31.67,31.59,31.42,31.21,27.73,20.61,19.43,14.96.
HRMS(ESI):m/z[m+H]+calcd for C35H46ClNO2:548.3295;found:548.3293.
实施例42:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-2-烯戊基)雄甾-5-烯-3β-醇(GAPS-42)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加2-丁烯基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~80:1)得白色泡沫状固体化合物168mg,收率:71.0%。
1H NMR(400MHz,CDCl3)δ7.43(d,J=7.4Hz,2H),7.20(d,J=6.4Hz,2H),6.21(ddt,J=16.0,12.4,4.0Hz,1H),5.31(d,J=6.4Hz,1H),5.20–4.97(m,2H),3.62(d,J=14.0Hz,1H),3.55–3.49(m,1H),3.39(td,J=8.4,2.4Hz,1H),2.38–2.19(m,4H),2.08–1.90(m,2H),1.88–1.70(m,2H),1.71(m,3H),1.69–1.44(m,8H),1.38–1.21(m,5H),1.14(s,3H),1.11–0.99(m,1H),0.97(s,4H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ141.25,137.77,134.84,131.88,129.99,126.33,121.57,121.86,74.46,71.56,66.65,57.15,54.58,51.15,50.58,47.65,42.31,42.20,39.18,37.37,36.95,31.25,31.21,31.28,31.33,25.24,20.59,19.85,18.25,14.92.
HRMS(ESI):m/z[m+H]+calcd for C32H46BrNO2:556.2790;found:556.2801.
实施例43:
16α-(4-溴苄氨)基-17β-(1-羟基-1,4-二甲基-戊烯基)雄甾-5-烯-3β-醇(GAPS-43)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.440mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加2-甲基-2-丁烯基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物172mg,收率:75.4%。
1H NMR(400MHz,CDCl3)δ7.40(d,J=7.4Hz,2H),7.18(d,J=7.2Hz,2H),6.28(m,1H),5.25-5.20(m,1H),5.18–4.85(m,2H),3.70(d,J=14.0Hz,1H),3.49–3.44(m,1H),3.40(td,J=7.8,2.6Hz,1H),2.40–2.20(m,4H),2.15–1.88(m,2H),1.85–1.79(m,2H),1.72–1.68(m,6H),1.65–1.40(m,8H),1.34–1.19(m,4H),1.21(s,3H),1.19–1.10(m,1H),0.98(s,4H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ140.59,137.68,134.74,131.65,129.20,126.01,121.24,121.16,75.98,71.20,66.45,57.05,54.17,49.58,49.10,47.32,42.28,42.65,39.52,37.75,36.90,31.92,31.84,31.25,31.01,24.96,20.45,19.88,19.06,18.74,14.93.
HRMS(ESI):m/z[m+H]+calcd for C33H48BrNO2:570.2947;found:570.2949.
实施例44:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-丙烯基)雄甾-5-烯-3β-醇(GAPS-44)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加乙烯基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~60:1)得白色泡沫状固体化合物165mg,收率:78.1%。
1H NMR(400MHz,CDCl3)δ7.55(d,J=8.0Hz,2H),7.37(d,J=7.6Hz,2H),5.88(dd,J=17.2,10.6Hz,1H),5.28(m,1H),5.19(dd,J=17.2,1.7Hz,1H),5.15(dd,J=10.6,1.7Hz,1H),3.61(d,J=14.0Hz,1H),3.44–3.40(m,1H),3.37(m,1H),2.35–2.14(m,4H),2.10-1.98(m,2H),1.85-1.77(m,2H),1.66–1.48(m,8H),1.38–1.21(m,5H),1.18(s,3H),0.95(s,3H),0.76(s,3H).
13C NMR(101MHz,CDCl3)δ144.47,139.99,139.01,132.28,130.67,121.35,120.22,110.01,73.61,70.66,66.32,57.25,55.34,51.02,50.91,47.33,43.84,39.21,37.83,36.75,31.85,31.28,31.84,31.71,27.84,21.34,19.82,14.96.
HRMS(ESI):m/z[m+H]+calcd for C30H42BrNO2:528.2477;found:528.2473.
实施例45:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-4-戊烯基)雄甾-5-烯-3β-醇(GAPS-45)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加1-丁烯基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~60:1)得白色泡沫状固体化合物174mg,收率:78.2%。
1H NMR(400MHz,CDCl3)δ7.42(d,J=8.4Hz,2H),7.21(d,J=8.4Hz,2H),5.80(m,1H),5.34(d,J=5.6Hz,1H),5.24–5.18(m,2H),3.82(t,J=12.0Hz,1H),3.70(d,J=14.0Hz,1H),3.61–3.48(m,1H),3.42(m,1H),2.38–2.15(m,4H),2.14–1.90(m,2H),1.86–1.74(m,2H),1.69–1.43(m,10H),1.42–1.18(m,6H),1.18(s,3H),1.20(m,1H),0.99(s,4H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ142.02,139.40,134.79,131.92,130.27,121.65,121.22,116.99,74.69,71.62,66.14,57.47,54.45,51.53,50.48,47.18,42.75,42.56,40.12,37.15,36.60,31.75,31.86,31.62,31.47,27.81,25.28,20.52,19.55,14.92.
HRMS(ESI):m/z[m+H]+calcd for C32H46BrNO2:556.2790;found:556.2788.
实施例46:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-苯丁烯基)雄甾-5-烯-3β-醇(G APS-46)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加1-肉桂基溴化镁(4.80mL,1.0M in Et2O,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应约2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到无色的油状物,硅胶柱层析(二氯甲烷/甲醇=100:1~80:1~50:1)得白色泡沫状固体化合物183mg,收率:74.0%。
1H NMR(400MHz,CDCl3)δ7.42(d,J=8.4Hz,2H),7.32–7.26(m,2H),7.11(m,4H),6.32(d,J=8.4Hz,1H),6.05(m,1H),5.37(m,1H),5.28(d,J=5.4Hz,1H),3.84(m,1H),3.69(m,1H),3.57–3.23(m,1H),3.43(m,1H),2.34–2.25(m,4H),2.22–1.95(m,2H),1.87–1.79(m,3H),1.68–1.47(m,6H),1.42–1.18(m,6H),1.16(s,3H),1.23(m,1H),0.98(s,4H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ142.35,139.75,136.76,134.33,131.25,130.12,127.91,128.85,128.26,127.66,125.91,121.85,121.79,76.12,71.07,66.23,57.27,54.42,51.85,50.21,47.85,42.45,40.80,37.15,36.38,31.81,31.51,31.20,31.74,27.68,25.34,20.49,19.82,14.92.
HRMS(ESI):m/z[m+H]+calcd for C37H48BrNO2:618.2947;found:618.2946.
实施例47:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-4-苯基-丁基)雄甾-5-烯-3β-醇的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(25mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加苯丙基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=110:1~90:1~60:1)得白色泡沫状固体化合物175mg,收率:70.6%。
1H NMR(400MHz,CDCl3)δ7.39(d,J=8.4Hz,2H),7.34–7.27(m,2H),7.09(m,4H),5.37(m,1H),5.28(d,J=5.4Hz,1H),3.86(m,1H),3.71(m,1H),3.59–3.27(m,2H),3.44(m,1H),2.35–2.27(m,4H),2.27–1.98(m,2H),1.88–1.81(m,3H),1.67–1.49(m,7H),1.45–1.20(m,6H),1.17(s,3H),1.25(m,1H),0.99(s,4H),0.76(s,3H).
13C NMR(101MHz,CDCl3)δ142.47,139.79,136.81,134.34,131.85,130.74,127.89,128.93,128.68,121.81,121.79,76.37,71.28,66.51,57.72,54.47,51.88,50.58,47.45,42.47,40.76,37.26,36.38,36.32,31.85,31.57,31.74,31.54,27.68,25.84,20.44,19.27,14.89.
HRMS(ESI):m/z[m+H]+calcd for C37H50BrNO2:620.3103;found:620.3105.
实施例48:
16α-(4-溴苄氨)基-17β-(1-羟基-1-甲基-戊基)雄甾-5-烯-3β-醇(GAPS-48)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加丁基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=120:1~90:1~70:1)得白色泡沫状固体化合物159mg,收率:71.2%。
1H NMR(400MHz,CDCl3)δ7.41(d,J=8.4Hz,2H),7.21(d,J=8.4Hz,2H),5.26(m,1H),3.81(m,1H),3.72(d,J=14.0Hz,1H),3.59(m,1H),3.44(m,1H),2.33-2.19(m,4H),2.14–1.90(m,3H),1.88–1.72(m,2H),1.70–1.46(m,10H),1.44–1.21(m,6H),1.19(s,3H),1.21(m,1H),0.98(s,3H),0.89(s,3H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ142.07,134.81,131.89,130.31,121.74,121.23,74.18,71.82,66.72,57.38,54.54,51.28,50.38,47.26,42.71,42.45,40.98,40.21,37.16,36.57,31.13,31.89,31.84,31.42,27.87,25.42,25.23,23.33,20.24,19.45,14.89,14.13.
HRMS(ESI):m/z[m+H]+calcd for C32H48BrNO2:558.2947;found:558.2948.
实施例49:
16α-(4-溴苄氨)基-17β-(1-羟基-1,4-二甲基-戊基)雄甾-5-烯-3β-醇(GAPS-49)的制备
16α-(4-溴苄氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.400mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加2-甲基丁基溴化镁(4.80mL,1.0M in THF,4.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=110:1~90:1~50:1)得白色泡沫状固体化合物168mg,收率:73.4%。
1H NMR(400MHz,CDCl3)δ7.40(d,J=7.4Hz,2H),7.21(d,J=7.4Hz,2H),5.27(m,1H),3.71(m,1H),3.45–3.41(m,1H),3.39(m,1H),2.39–2.25(m,4H),2.22–1.91(m,2H),1.83–1.80(m,2H),1.64–1.43(m,8H),1.33–1.21(m,4H),1.20(s,3H),1.19–1.09(m,5H),0.99(s,4H),0.92(m,6H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ140.79,137.82,134.75,129.29,121.28,121.19,75.34,71.31,66.33,57.09,54.28,49.74,49.18,47.12,42.86,42.70,39.55,37.72,36.58,33.16,31.85,31.35,31.63,24.13,23.21,20.34,19.16,19.50,18.28,14.73.
HRMS(ESI):m/z[m+H]+calcd for C33H50BrNO2:572.3103;found:572.3105.
实施例50:
16α-苯氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-50)的制备
16α-苯氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.491mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.89mL,1.0M in Et2O,5.89mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=50:1~40:1~30:1)得白色泡沫状固体化合物167mg,收率:76.0%。
1H NMR(400MHz,CDCl3)δ7.08–6.67(m,4H),6.59(m,2H),5.36(t,J=5.4Hz,1H),5.16–5.00(m,2H),3.57(t,J=4.6Hz,1H),3.46(m,2H),2.32–2.18(m,4H),2.09–1.91(m,4H),1.84(m,2H),1.75–1.23(m,12H),1.17(s,3H),1.06(m,1H),1.01(s,3H),0.73(s,3H).
13C NMR(101MHz,CDCl3)δ145.66,140.82,134.85,129.67,121.86,120.37,118.42,113.51,74.25,71.88,66.58,58.27,54.68,50.36,47.85,42.73,42.64,39.68,37.36,36.48,31.89,31.61,31.50,31.41,25.67,20.51,19.13,14.93.
HRMS(ESI):m/z[m+H]+calcd for C30H44NO2:450.3372;found:450.3374.
实施例51:
16α-(4-溴苯氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-51)的制备
16α-(4-溴苯氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.411mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(4.93mL,1.0M in Et2O,4.93mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=90:1~70:1~50:1)得白色泡沫状固体化合物185mg,收率:85.1%。
1H NMR(400MHz,CDCl3)δ7.39(d,J=8.4Hz,2H),7.22(d,J=8.4Hz,2H),6.57(m,1H),5.32(d,J=5.7Hz,1H),5.23–4.97(m,2H),3.60–3.45(m,1H),3.41(m,1H),2.37–2.11(m,4H),2.01–1.90(m,2H),1.88–1.71(m,2H),1.67–1.41(m,8H),1.39–1.23(m,5H),1.17(s,3H),1.14–1.00(m,1H),0.99(s,4H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ141.32,137.52,134.45,131.78,130.64,121.15,121.27,117.87,74.87,71.62,66.23,57.75,54.47,50.45,47.38,42.55,42.86,39.84,37.79,36.45,31.38,31.41,31.98,31.86,25.24,20.52,19.47,14.93.
HRMS(ESI):m/z[m+H]+calcd for C30H42BrNO2:528.2477;found:528.2476.
实施例52:
16α-苯乙基氨基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-52)的制备
16α-苯乙基氨基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.459mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.51mL,1.0M in Et2O,5.51mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=90:1~70:1~50:1)得白色泡沫状固体化合物167mg,收率:76.5%。
1H NMR(400MHz,CDCl3)δ7.29–7.26(m,4H),7.19(m,1H),6.03(m,1H),5.23(dt,J=5.3,2.0Hz,1H),5.16–5.00(m,2H),3.52(t,J=4.6Hz,1H),3.43(m,1H),2.88(m,2H),2.62(m,2H),2.37–2.18(m,4H),2.05–1.90(m,2H),1.78(m,2H),1.69–1.22(m,12H),1.17(s,3H),1.11(m,1H),1.01(s,4H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ140.89,139.45,134.91,128.05,127.99,127.51,121.47,117.25,74.38,71.63,66.54,58.21,54.58,50.12,49.75,47.47,42.18,42.35,39.42,37.65,36.35,35.29,31.98,31.52,31.41,31.05,25.35,20.19,19.28,14.97.
HRMS(ESI):m/z[m+H]+calcd for C32H47NO2:478.3685;found:478.3687.
实施例53:
16α-(4-溴苯乙氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-53)的制备
16α-(4-溴苯乙氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.389mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(4.66mL,1.0M in THF,4.66mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到棕黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=70:1~50:1~30:1)得白色泡沫状固体化合物159mg,收率:73.5%。
1H NMR(400MHz,CDCl3)δ7.76(d,J=8.4Hz,2H),7.11(d,J=8.4Hz,2H),5.33(d,J=5.4Hz,1H),5.22–4.99(m,2H),3.61–3.47(m,1H),3.39(m,1H),2.89(m,2H),2.67(m,2H),2.38–2.14(m,4H),2.32–1.99(m,2H),1.86–1.73(m,2H),1.68–1.40(m,8H),1.39–1.25(m,4H),1.19(s,3H),1.15–1.03(m,1H),0.98(s,4H),0.74(s,3H).
13C NMR(101MHz,CDCl3)δ140.81,138.01,134.74,131.54,131.08,121.83,120.57,118.04,74.32,71.46,66.22,57.38,54.28,50.85,49.52,47.34,42.51,42.26,39.57,37.03,36.31,35.59,31.75,31.45,31.21,31.07,25.65,20.36,19.75,14.89.
HRMS(ESI):m/z[m+H]+calcd for C32H46BrNO2:556.2790;found:556.2789.
实施例54:
16α-(5-己烯-1-氨)基-17β-[(R)-1-羟基-1-烯丙基-乙基)]雄甾-5-烯-3β-醇(GAPS-54)的制备
16α-(5-己烯-1-氨)基-孕甾-5-烯-3β-羟基-20-酮(200mg,0.484mmol)溶于无水四氢呋喃(20mL)中,氩气保护,搅拌充分溶解并置于冰浴条件下,然后缓慢滴加烯丙基溴化镁(5.80mL,1.0M in Et2O,5.80mmol,12eq.),之后恢复室温,在室温条件下继续反应2小时。经TLC(薄层层析)检测反应完毕后,在冰浴条件下用饱和氯化铵溶液淬灭反应。四氢呋喃萃取,收集有机相,用无水硫酸钠干燥,过滤之后减压浓缩得到米黄色稠油状物,硅胶柱层析(二氯甲烷/甲醇=110:1~90:1~70:1)得白色泡沫状固体化合物158mg,收率:71.7%。
1H NMR(400MHz,CDCl3)δ5.89(m,2H),5.31(d,J=5.4Hz,1H),5.12-5.05(m,2H),4.88-4.84(m,2H),3.34(m,1H),3.34(dt,J=9.8,6.0Hz,1H),2.68(m,1H),2.42(m,1H),2.37–2.11(m,4H),2.04–1.86(m,2H),1.85–1.73(m,2H),1.66–1.34(m,8H),1.23(m,11H),0.99(s,5H),0.97(m,3H),0.75(s,3H).
13C NMR(101MHz,CDCl3)δ140.87,139.11,134.52,121.80,117.83,116.27,75.14,71.08,66.02,58.17,54.68,50.45,48.52,47.48,42.34,39.75,37.70,36.26,33.44,31.85,31.73,31.68,31.57,31.24,30.55,27.33,25.52,20.84,19.41,15.54.
HRMS(ESI):m/z[m+H]+calcd for C30H49NO2:456.3842;found:456.3845.
药效学实验部分
一、相对荧光素酶活实验
1.实验方法
将突变p53(mutp53)(R273H和R175H)转入到H1299(p53 null)细胞中,同时将PUMA启动子荧光素酶报告基因载体构建至细胞中,构建了H1299-p53 R273H-WT PUMApromoter,H1299-p53R175H-WT PUMA promoter BS2稳定细胞株。取生长状态良好的H1299(p53R273H)-WT PUMA promoter BS2、H1299(p53R175H)-WT PUMA promoter BS2细胞,用0.25%的胰蛋白酶消化后制成细胞悬液,血球计数板计数,按照一定的细胞密度接种到24孔板中(不同的细胞设置不同的接种密度),37℃培养箱中培养过夜;加入10μM甾体类化合物、50μM PRIMA-1作为正对照(已经被证实作为mut-p53的重激活剂)处理24h,吸取细胞上清进行后通过Secrete-Pair TM Dual Luminescence Assay Kit进行相对荧光酶活检测。
具体步骤如下:
i.取生长良好,汇合度80-90%的H1299 p53R175H/p53R273H-PUMA promoter细胞(稳定表达PUMA启动子荧光素酶报告基因),按1.5×105个细胞/孔接种至6孔板中,晃匀,放入37℃培养箱孵育24h至细胞贴璧完全。
ii.将贴璧完全的H1299 p53R175H/p53R273H-PUMA promoter细胞从培养箱取出,吸净旧培养基,加入2mL含相应工作浓度化合物的新鲜RPMI 1640培养基,晃匀,放入37℃培养箱孵育15h。
iii.GLuc、SEAP都属于分泌型的蛋白,因此化合物处理完毕后,可轻轻收集细胞培养上清,立即检测GLuc、SEAP的活性。
1)检测GLuc的活性
(1)收集化合物处理完毕的100μL细胞培养上清至1.5mL离心管,然后置于室温。
(2)将10×的GL-S缓冲液取出并在常温下解冻,充分混匀后取适量10×的GL-S缓冲液用超纯水稀释为1×GL-S缓冲液。1×GL-S缓冲液用量为100μL/反应。
(3)避光环境下,往1×GL-S缓冲液加入1/100总体积的Substrate GL(100×),充分混匀,配得GLuc工作液。
(4)将待检测上清及GLuc工作液分别移入25℃杂交炉中温育25分钟。
(5)取一个干净的不透光96孔酶标板,将温育完毕的上清及工作液取出分别移取10,100μL至孔中,用移液枪温和混匀。
(6)混匀完毕后,96孔板放入25℃杂交炉孵育1分钟,然后将酶标仪的读板模式设置为“化学发光”,检测96孔板中GLuc氧化荧光素所发生的生物萤光。(室温孵育后应争取在5分钟内读板)。
2)检测SEAP的活性
(1)吸取50μL细胞培养上清至新的1.5mL离心管,然后在65℃杂交炉中加热15min,取下置于冰浴备用。
(2)将10×的AP缓冲液取出并在常温下自然解冻,充分混匀后取适量的10×的AP缓冲液用超纯水稀释为1×AP缓冲液。1×GL-S缓冲液的用量为100μL/反应。
(3)避光环境下,往1×AP缓冲液加入1/100总体积的Substrate AP(100×),充分混匀,配得SEAP工作液。
(4)将待检测上清及SEAP工作液分别移入25℃杂交炉中温育10分钟。
(5)取一个干净的不透光96孔酶标板,将温育完毕的上清及SEAP工作液取出分别移取10、100μL至孔中,用移液枪温和混匀。
(6)混匀完毕后,96孔板放入25℃杂交炉孵育10分钟,然后通过酶标仪检测96孔板中SEAP与底物反应的发光强度。
2.实验结果:
靶向突变p53的甾体类化合物合物筛选结果见图1
本实验对GAPS-1-GAPS-46进行了研究,靶向突变p53的甾体类化合物的相对荧光素酶活结果显示上述46种化合物均具有重激活突变p53的作用,且其中GAPS-4、GAPS-8、GAPS-11、GAPS-12、GAPS-14、GAPS-24、GAPS-26、、GAPS-27、GAPS-28、GAPS-29、GAPS-30、GAPS-33、GAPS-38、GAPS-46、GAPS-53(见灰框)对重激活突变p53的效果比较突出,图中以PRIMA-1为阳性对照,以5-FU及顺铂为阴性对照。
二、细胞增殖抑制实验
1.实验方法
取生长状态良好的细胞,用0.25%的胰蛋白酶消化后制成细胞悬液,血球计数板计数,按照一定的细胞密度接种到96孔板中(不同的细胞设置不同的接种密度),37℃培养箱中培养过夜;甾体类化合物设置5个浓度梯度(0.01、0.1、1、10、50μM)3个复孔,37℃培养箱中培养72h后终止培养;每孔加入5mg/mL的MTT溶液25μL,放置于培养箱中4h;去除细胞悬液加入150μl DMSO,置于摇床上振荡10min;多功能酶标仪(美国Bio-TeK)仪器490nm波长下测定OD值.根据测定的OD值计算出IC50值和细胞的相对存活率.
具体步骤如下:
(1)取生长良好,汇合度80-90%的肿瘤细胞,按5×103个细胞/孔接种至96孔板中,晃匀,放入37℃培养箱孵育至细胞贴璧完全。
(2)加入甾体类化合物处理细胞,使用培养基梯度稀释化合物母液,稀释完毕后涡旋混匀,按照设置的浓度梯度,以20μL/孔分别加入96孔板中,每个浓度3个复孔。晃匀,放入37℃培养箱孵育72h。
(3)孵育至相应时间点后将96孔板拿出,在超净工作台中避光加入MTT溶液(终浓度为5μg/mL)。然后放回培养箱继续孵育4h。
(4)孵育完毕,使用负压吸引泵吸除96孔板中液体并加入二甲基亚砜(150μL/孔),放在37℃摇床上摇荡15min,使紫蓝色甲瓒晶体充分溶解,最后用酶联免疫检测仪测定波长490nm处的OD值。
2.实验结果:
靶向突变p53的甾体类化合物对肿瘤细胞株的体外抗肿瘤增殖作用见表1。
表1靶向突变p53的甾体类化合物抗肿瘤增殖作用(IC50(μM))
本发明靶向突变p53的甾体类化合物对HT29、H1299 p53 null、H1299 p53 R175H、H1299 p53 R273H、HCT116p53 null共5个肿瘤细胞株进行了体外细胞增殖抑制实验,以顺铂(MW30)作为阳性对照药物,结果表明所制备的化合物对上述肿瘤细胞有良好的抑制作用,数据见上表1。
三、免疫荧光
1.实验方法
取生长状态良好的HT29、SK-BR-3细胞种于带有盖玻片的六孔板中,待细胞贴壁后,用甾体类化合物(5或10μM)处理48h后.1×PBS洗涤细胞,固定液(5%多聚甲醛:1×PBS:20%蔗糖=6:3:1)固定10min,5×BSA室温封闭2h,加一抗PAb240抗体(识别突变型p53)、PAb1620抗体(识别野生型p53)孵育过夜,加Goat-anti-mouse 488(绿光)荧光二抗,避光室温孵育2h,1×PBS洗涤,染DAPI,室温孵育15min,然后封片,拍片,观察甾体类化合物处理HT29、SK-BR-3细胞后PAb240(识别突变型p53)与PAb1620(识别野生型p53)表达情况。
具体步骤如下:
(1)细胞接种:先用1×PBS清洗灭菌的盖玻片,之后将盖玻片放到六孔板中。取生长良好,汇合度80-90%的HT29、SK-BR-3细胞,按2×105个细胞/孔接种至六孔板中,晃匀,放入37℃培养箱孵育至细胞贴璧完全。
(2)化合物处理:将贴璧完全的HT29、SK-BR-3细胞从培养箱取出,吸净旧培养基,加入10mL新鲜1640培养基(实验组均含10μM的化合物),晃匀,放入37℃培养箱孵育48h。
(3)细胞固定:药物处理结束后,取出6孔板,吸掉培养基,用移液管沿着孔壁加入1×PBS,每孔加入2mL,缓缓摇晃清洗2次,吸去PBS,然后在每孔加入1mL固定液(5%多聚甲醛:1×PBS:20%蔗糖=6:3:1),固定10min。
(4)细胞清洗:吸除固定液,用1×PBS洗3次,清洗时用移液管沿着孔壁加入1×PBS,轻轻摇晃六孔板,放置2min。
(5)细胞打孔:每孔加入800μL的1%NP-40,处理5min,其目的是为了在细胞膜上打孔,提高细胞膜的通透性。随后重复步骤4。
(6)BAS封闭:每孔加入800μL的5%BSA,室温静置,封闭2h。封闭结束后重复步骤4。
(7)一抗孵育:p53抗体PAb1620以及PAb240稀释比例参考抗体说明书配制(1:500,用1%BSA稀释),加一抗时,直接将抗体轻轻滴加到孔内的盖玻片上,每个玻片滴加80μL,将抗体铺满整个盖玻片,一抗室温孵育1h或者4℃过夜。一抗孵育结束后重复步骤4。
(8)二抗孵育:二抗孵育步骤与一抗相似,用对应的荧光二抗(1:1500,用1%BSA稀释)覆盖盖玻片,室温避光孵育1h(注:荧光二抗IgG568荧光显微镜下显红色,IgG488显绿色,根据需要选取不同的荧光二抗)。二抗孵育结束后重复步骤4。
(9)染核:每孔加入80μL DAPI(1:500,用1×PBS稀释)染细胞核,室温20min,操作同步骤7,该过程也需要避光操作。随后重复步骤4。
(10)封片和爬片:向载玻片中央滴加一滴防猝灭剂,用小镊子小心夹取盖玻片,将盖玻片有细胞的一面朝下放于滴有防淬灭剂处的载玻片上。放盖玻片时,先将盖玻片与防淬灭剂接触,然后再将另一侧缓缓放下,避免气泡产生。随即放入暗盒中,防止荧光淬灭,可立即拍照保存也可以放在4℃保存较长一段时间。
(11)拍片观察:用倒置荧光显微镜观察制好的片子,并进行拍照保存。
2.实验结果
本发明靶向突变p53的甾体类化合物中,以化合物GAPS-30为例,其能够增加突变p53对识别野生型p53抗体Pab1620的反应性,同时降低突变p53对识别突变型p53抗体Pab240的反应性,如图2所示,为甾体类化合物恢复突变p53野生型功能提供了证据。
四、免疫沉淀
1.实验方法
免疫沉淀是一种分离特定蛋白并分析的方法。基于抗原抗体免疫反应及proteinA/G可与抗体Fc片段特异结合的特性,可富集并定量分析特定蛋白及潜在互作的因子。用甾体类化合物(10μM)处理HT29和SK-BR-3细胞48h。收集细胞,用细胞裂解缓冲液(50mM Tris-HCl pH7.4,150mM NaCl,1%NP-40,1mM EDTA,0.25%C24H39NaO4)和蛋白酶抑制剂和磷酸酶抑制剂(Roche)冰冻裂解细胞。采用抗p53一抗(PAb1620(Millipore,OP33)、PAb240(SANTA,SC-99)、IgG(Millipore,AP124P)或蛋白A/G琼脂糖进行免疫沉淀实验。洗珠5次,用抗p53(Santa,SC-126)进行western blotting分析。
具体步骤如下:
(1)细胞接种:取生长良好,汇合度80-90%的HT29、SK-BR-3细胞,按2×106个细胞/皿接种至10cm培养皿(大皿),晃匀,放入37℃培养箱孵育24h至细胞贴璧完全。
(2)化合物处理:将贴璧完全的HT29、SK-BR-3细胞从培养箱取出,吸净旧培养基,加入10mL新鲜1640培养基(实验组均含5μM的化合物),晃匀,放入37℃培养箱孵育48h。
(3)收集细胞:用细胞刮收集细胞并将细胞悬液移入15mL的离心管中,4℃、1000g离心5mins,弃去上清,以1mL预冷1×PBS重悬细胞后将悬液转至1.5mL的离心管,4℃,以1000g离心5mins,弃去上清并将所留沉淀存于-80℃待用。
(4)蛋白提取:以适量RIPA裂解液重悬细胞沉淀后,在冰浴中对样品进行超声破碎,功率设为25%,超声10s,间隔6s,超声10次。然后,样品置于4℃的360°静音混匀器裂解2-3h。4℃、10000g离心30mins,小心转移上清至新的1.5mL离心管。
(5)预洗:取20μL Protein A+G琼脂糖珠至新1.5mLEP管并以1mL 1×PBS重悬清洗琼脂糖珠后,4℃,8000rpm离心1min,弃上清,重复两次。将(2)蛋白样品及1μg的IgG抗体移入EP管中置于360°静音混匀仪4℃预洗2h从而去除与珠子非特异性结合的蛋白,4℃下,5000rpm,离心30mins后,将上清转入新的已预冷1.5mL EP管备用。
(6)免疫沉淀反应:根据蛋白浓度测定结果,取100-500μg的蛋白样品至新的1.5mLEP管,加入1μg目的抗体(或IgG),移至4℃的360°静音混匀仪中免疫反应过夜。次日,将免疫反应样品完全转移到20μL已用PBS充分清洗的Protein A+G琼脂糖珠中,补加600μL预冷1×PBS,在4℃的360°静音混匀仪继续孵育过夜。
(7)清洗:样品从4℃中取出,冰浴静置2mins后,6000g、4℃,离心1min,弃上清。沿壁加入1mL预冷1×PBS,轻轻清洗珠子后,4℃、6000g,离心1min,弃上清,清洗8-10次。
(8)变性及蛋白质印迹:往已清洗样品管中加入16μL 1×PBS及4μL 5×Loadingdye,短暂低速离心后,放入沸水浴煮沸7-10mins使结合于琼脂糖珠的免疫复合物完全解离。已变性样品冷凝后,离心并进行SDS-PAGE电泳。
(9)本实验所需试剂配制
(1)0.5M EDTA(pH7.4)溶液的配制:
(2)RIPA裂解液的配制:
(3)抗体的配制:
2.实验结果
如图3所示,靶向突变p53的甾体类化合物中,以化合物GAPS-30为例,GAPS-30降低了突变p53的表达,同时增加了野生型p53的表达。
五、蛋白质印迹法
1.实验方法
蛋白质印迹法(免疫印迹试验)即Western Blot。它是分子生物学、生物化学和免疫遗传学中常用的一种实验方法。其基本原理是通过PAGE(聚丙烯酰氨凝胶电泳)分离的蛋白质样品,转移到固相载体(例如PVDF膜)上,固相载体以非共价键形式吸附蛋白质,且能保持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋白质或多肽作为抗原,与对应的抗体起免疫反应,再与酶或同位素标记的第二抗体起反应,经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。
具体步骤如下:
(1)取对数生长期的HT29、SK-BR-3细胞,采用化合物(5/10μM)处理6h、12h、24h,用细胞刮将细胞刮下,4℃离心,用1×PBS清洗一遍,移至1.5mL离心管中得到细胞沉淀。
(2)加入适宜的细胞裂解液,对细胞沉淀进行超声处理,条件25-30Hz,超10s停5s,共10次,如果沉淀未被完全超声彻底可以适当增加超声次数。提取出的蛋白利用考蓝或BCA方法进行定量。
(3)配置蛋白上样体系:15μL、20μg,蛋白上样体系中加溴酚蓝(已加β-巯基乙醇)终浓度为1×上样体系;体系配好后,沸水煮10min左右,冷却离心。
(4)电泳:配置12%的分离胶和5%的浓缩胶,配置1×的Running Buffer(含1%的SDS),准备上样。跑胶条件:恒压80V跑30min后切换恒压120V跑1小时30min。
(5)转膜:配置转膜液,200mL甲醇+100mL的10×Running Buffer纯水定容到1L,PVDF膜用之前要用甲醇活化;按照板子黑面-海绵-三层滤纸-胶-PVDF膜-三层滤纸-海绵-板子白面顺序制备转膜装置。转膜条件:恒流200mA、2h。
(6)5%牛奶封闭,先用1×PBST清洗PVDF膜上的转膜液,然后用5%的牛奶常温封闭2小时或是37℃封闭1h或是4℃摇床封闭过夜。
(7)标一抗:用1×PBST清洗封闭牛奶,然后用配好的p53(Do-1)(1:500)(SC-126,Santa)、PUMA(1:100)(12450S,CST)、p21(1:1000)(2947S,CST)、Bax(1:500)(5023,CST)抗体4℃摇床过夜。
(8)标二抗:用1×PBST清洗标过一抗的PVDF膜,按照1:10000比例稀释相应种属的HRP标记二抗,室温2h。
(9)显影:用1×PBST清洗标过二抗的PVDF膜,按照1:1比例配置显影液,进行显影。
2.实验结果
靶向突变p53的甾体类化合物中,以化合物GAPS-30为例,GAPS-30上调了p53下游蛋白的表达水平,如图4所示。
实施例55:
片剂的制备:
按实施例GAPS-27的方法制得本发明化合物,取其中一种化合物或几种化合物的混合物,按其与赋形剂重量比为1:5-1:10的比例加入赋形剂,制粒压片。
实施例56:
口服液制剂的制备:
按实施例GAPS-30的方法制得本发明化合物,取其中一种化合物或几种化合物的混合,按常规口服液制法制成口服液。
实施例57:
胶囊剂、颗粒剂、或冲剂的制备:
按实施例GAPS-33的方法制得本发明化合物,取其中一种化合物或几种化合物的混合,按其与赋形剂重量比为5:1的比例加入赋形剂,制成胶囊或颗粒剂或冲剂。
尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
3.据权利要求1 所述甾体类化合物或其药用盐,其特征在于:药用盐包括硫酸盐、铵盐、甲磺酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、磷酸盐、一氢磷酸盐、二氢磷酸盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物、乙酸盐、丙酸盐、癸酸盐、辛酸盐、丙烯酸盐、甲酸盐、异丁酸盐、酒石酸盐、富马酸盐、草酸盐、马来酸盐、柠檬酸盐、以及乳酸盐中的任一种。
4.一种权利要求1所述甾体类化合物的制备方法,其特征在于:将16α-取代氨基-3β-羟基-孕甾-5-烯-20-酮类化合物与Grinard 试剂置于溶剂中反应,得到式(I)化合物。
5.根据权利要求4 所述甾体类化合物的制备方法,其特征在于:所述溶剂为四氢呋喃或乙醚,16α-取代氨基-3β-羟基-孕甾-5-烯-20-酮类化合物与Grinard 试剂反应的温度为0℃至所用溶剂的回流温度。
6.权利要求1-3任意一项所述甾体类化合物或其药用盐在制备用于治疗与 p53突变有关肿瘤药物中的应用。
7.根据权利要求6所述的应用,其特征在于:与p53突变有关的肿瘤包括结肠癌、肺癌、乳腺癌、卵巢癌、黑色素瘤、血液肿瘤、以及前列腺癌中的任一种。
8.一种抗肿瘤药物,其特征在于:包括一种或两种以上权利要求1-3任意一项所述甾体类化合物或其药用盐和药学可接受的载体。
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