CN106349342B - 吡啶并吲哚并咪唑酮丁酰-Val-Phe-氨基葡萄糖,其制备,活性和应用 - Google Patents
吡啶并吲哚并咪唑酮丁酰-Val-Phe-氨基葡萄糖,其制备,活性和应用 Download PDFInfo
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Abstract
本发明公开了[(S)‑2,3,4,9‑四氢‑1H‑吡啶并[3,4‑b]吲哚并(2,2‑二甲基‑咪唑‑4‑酮‑3‑基)]‑2‑甲基丁酰‑Val‑Phe‑氨基葡萄糖,公开了它的制备方法,公开了它的抗肿瘤作用和抗炎作用,因而本发明公开了它在制备抗肿瘤药物和抗炎药物中的应用。
Description
技术领域
本发明涉及下式的[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-Phe-氨基葡萄糖,涉及它的制备方法,涉及它的抗肿瘤作用和抗炎作用,因而本发明涉及它在制备抗肿瘤药物和抗炎药物中的应用。本发明属于生物医药领域。
技术背景
肿瘤是局部组织的细胞异常增生而形成的新生物。所有的恶性肿瘤总称为癌症。恶性肿瘤严重威胁着人类健康。在临床肿瘤的手术治疗、放疗和化疗中,化疗应用最广泛,发明疗效好和毒副作用低的抗肿瘤新药一直是药物研究的热点。在抗肿瘤新药研究中,发明人曾公开下面结构的化合物(式中AA为氨基酸残基)按1μmol/kg剂量腹腔单次给药,每天一次,连续给药7天具有抗肿瘤作用。发明人并不满意这利疗效。
经过5年研究,发明人发现上面构中的AA-OBzl用Val-Phe-氨基葡萄糖代替,不仅可以增强抗肿瘤活性,而且可以获得额外的抗炎活性。根据这些研究结果,发明人提出了本发明。
发明内容
本发明的第一个内容是提供下式的[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-Phe-氨基葡萄糖。
本发明的第二个内容是提供[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-Phe-氨基葡萄糖的制备方法,该方法包括以下步骤:(1)将L-色氨酸在硫酸的催化下与甲醛进行Pictet-Spengler缩合得到2,3,4,9-四氢-β-咔啉-3-羧酸;
(2)冰浴下向2,3,4,9-四氢-β-咔啉-3-羧酸与N,N-二甲基甲酰胺,DMF,的溶液中加入二碳酸二叔丁酯,Boc2O,N-甲基吗啉催化得到N-Boc-2,3,4,9-四氢-β-咔啉-3-羧酸;
(3)在二环己基碳二亚胺,DCC,和N-羟基苯并三唑,HOBt,存在下N-Boc-2,3,4,9-四氢-β-咔啉-3-羧酸在无水THF(四氢呋喃)中与L-Leu-OMe缩合为N-Boc-2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe;
(4)在氯化氢-乙酸乙酯溶液中N-Boc-2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe脱去Boc生成2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe;
(5)三乙胺催化下2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe在甲醇和丙酮溶液中避光反应得到2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰甲酯;
(6)在2N NaOH溶液中2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰甲酯在2N NaOH溶液中水解生成2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酸;
(7)在DCC和HOBt存在下2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酸在无水DMF中与Val-OBzl缩合为2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-OBzl;
(8)在Pd/C催化下2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-OBzl氢解为2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val;
(9)在DCC和HOBt存在下,Boc-Phe在无水DMF中与氨基葡萄糖缩合为Boc-Phe-氨基葡萄糖;
(10)在4N氯化氢-乙酸乙酯溶液中Boc-Phe-氨基葡萄糖脱去Boc生成Phe-氨基葡萄糖;
(11)在DCC和HOBt存在下2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基于酰-Val在无水DMF中与Phe-氨基葡萄糖缩合为2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖。
本发明的第三个内容是评价2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖抑制S180肿瘤小鼠肿瘤生长的作用。
本发明的第四个内容是评价2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖对ICR小鼠炎症的抑制作用。
附图说明
图1[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-Phe-氨基葡萄糖的合成路线.i)稀硫酸,40%甲醛;ii)Boc2O,DMF,三乙胺;iii)DCC,HOBt,NMM,无水THF;iv)4N氯化氢,甲醇,丙酮,三乙胺;v)2N NaOH;vi)Pd/C,氢气。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1制备(S)-2,3,4,9-四氢-β-咔啉-3-羧酸(1)
将2500mL蒸馏水置于2500mL反应瓶中,缓慢加入1.3mL浓硫酸,搅拌2min,再向得到的稀硫酸中加入32.6g(159mmol)L-色氨酸,超声至完全溶解,再加入65mL 40%甲醛溶液,室温搅拌7h TLC(CH2Cl2∶CH3OH,5∶1)监测终止反应。向反应液中缓慢滴加浓氨水,调pH至6,静置半小时,过滤,滤饼分别用蒸馏水,丙酮洗涤3次,收集滤饼转移至表面皿中晾干,最后得到31.5g(91%)标题化合物,为无色固体。ESI-MS(m/e)215[M-H]-。
实施例2制备N-Boc-(S)-2,3,4,9-四氢-β-咔啉-3-羧酸(2)
将41.9g(193.9mmol)(S)-2,3,4,9-四氢-β-咔啉-3-羧酸加到300mL DMF中,冰浴搅拌下向悬浮液中加50.8g(233mmol)(Boc)2O,然后滴加三乙胺调反应液pH至10,室温反应48h,每8h水泵抽气1次,TLC(CH2Cl2∶CH3OH,5∶1)显示原料点消失,终止反应。减压浓缩,残留物用300mL乙酸乙酯溶解,用5%KHSO4溶液洗3次,乙酸乙酯层用无水硫酸钠于燥12h,过滤,滤液减压浓缩。残留物加入少量CH2Cl2超声为悬浮液后过滤,收集滤饼。滤液浓缩后重复上述步骤,直至溶液内无悬浮物终止。收集滤饼得33.94g(55%)标题化合物,为无色固体。ESI-MS(m/e)315[M-H]-。
实施例3制备N-Boc-(S)-2,3,4,9-四氢-β-咔啉-3-甲酰-L-亮氨酸甲酯(3)
冰浴下将31.35g(99.7mmol)N-Boc-(S)-2,3,4,9-四氢-β-咔啉-3-羧酸溶解于250mL无水THF并加13.39g(99.7mmol)HOBt,然后加24.51g(119.6mmol)DCC与150mL无水THF的溶液,反应30min;再加19.87g(99.7mmol)L-亮氨酸甲酯,用NMM调反应液pH至9,室温反应24h。TLC(石油醚∶丙酮,3∶1)显示原料点消失,终止反应。过滤,滤液减压浓缩。残留物用250mL乙酸乙酯溶解,依次用饱和 NaHCO3溶液洗3次,饱和NaCl溶液洗3次,5%KHSO4溶液洗3次,饱和NaCl溶液洗3次,5%NaHCO3溶液洗3次,饱和NaCl溶液洗3次。合并的乙酸乙酯层用无水硫酸钠干燥12h,过滤,滤液减压浓缩得43.42g(99%)标题化合物,为黄色固体。ESI-MS(m/e)444[M+H]+。
实施例4制备[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酸甲酯(4)
冰浴搅拌下向21.0g(48.4mmol)N-Boc-(S)-2,3,4,9-四氢-β-咔啉-3-甲酰-L-亮氨酸甲酯滴入220mL氯化氢-乙酸乙酯溶液(4N),冰浴下反应4h TLC(石油醚∶丙酮,3∶1)监测原料点消失。抽干溶剂,残留物用110mL CH3OH溶解,加入110mL丙酮,用三乙胺调反应液pH至9,室温避光反应2周。TLC(石油醚∶丙酮,3∶1)显示原料点消失,终止反应。反应液过滤,滤液减压浓缩有沉淀析出。再过滤,滤液减压浓缩又有沉淀析出。重复此步骤,直至无沉淀析出。收集滤饼。滤液浓缩后用400mL乙酸乙酯溶解,用饱和NaCl溶液洗8次,合并的乙酸乙酯层用无水硫酸钠干燥12h后过滤,滤液减压浓缩,残留物用少量CH3OH悬浮,过滤,收集滤饼。滤液重复此步骤,直至无沉淀析出。合并收集的所有滤饼,晾干得11.75g(65%)标题化合物,为无色固体。ESI-MS(m/e)384[M+H]+。
实施例5制备[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酸(5)
向10.32g(26.9mmol)[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酸甲酯加入300mL THF和100mL CH3OH。冰浴搅拌下用2NNaOH调反应液的pH至12。冰浴反应48h TLC(石油醚∶丙酮,3∶1)监测原料点消失,终止反应。冰浴下用饱和KHSO4溶液将反应液pH调至7。减压浓缩去除THF和CH3OH后,冰浴下用5%KHSO4溶液将残留物液的pH调至2,使析出大量无色沉淀。过滤,滤饼用少量蒸馏水洗涤,将滤饼晾干得9.45g(95%)标题化合物,为浅黄色固体。ESI-MS(m/e)368[M-H]-。
实施例6制备[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-OBzl(6)
冰浴和搅拌下向6.0g(16.29mmol)[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酸与350mL无水THF的溶液中加入2.2g(16.29mmol)HOBt及4.02g(19.6mmol)DCC,反应30min。之后,加入7.39g(16. 29mmol)L-Val-OBzl并用NMM将反应液的pH调至9,反应13h TLC(石油醚∶丙酮,3∶1)监测原料点消失,终止反应。反应液过滤,滤液减压浓缩,残留物用乙酸乙酯溶解,得到的溶液依次用饱和NaHCO3溶液洗3次,饱和NaCl溶液洗3次,5%KHS O4溶液洗3次,饱和NaCl溶液洗3次,5%NaHCO3溶液洗3次,饱和NaCl溶液洗3次。乙酸乙酯层用无水硫酸钠干燥12h,过滤,滤液减压浓缩,残留物用硅胶柱层析纯化得6.62g(72.3%)标题化合物,为无色固体。ESI-MS(m/e)559[M+H]+。
实施例7制备[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val(7)
向2.3g(4.12mmol)[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-OBzl和100mL CH3OH的溶液中加230mg Pd/C,搅拌均匀,通入氢气,反应48h,TLC(石油醚∶丙酮,3∶1)监测原料点消失,终止反应。反应液过滤,滤液减压浓缩,得1.73g(90%)标题化合物,为黄色固体。ESI-MS(m/e)467[M-H]-。
实施例8制备Boc-Phe-氨基葡萄糖
冰浴和搅拌下向10.0g(37.7mmol)Boc-Phe与300mL无水DMF的溶液中加入5.0g(37.7mmol)HOBt及9.3g(45.2mmol)DCC,反应30min。之后,加入12.0g(56.6mmol)盐酸氨基葡萄糖并用NMM将反应液的pH调至9,室温反应36h,TLC(CH2Cl2∶CH3OH∶CH3CO2H,15∶1∶1.5)监测原料点消失,终止反应。反应液过滤,滤液减压浓缩得6.0g(38%)标题化合物,为无色固体。ESI-MS(m/e)425[M-H]-。
实施例9制备Phe-氨基葡萄糖
冰浴和搅拌下6.0g(14.08mmol)Boc-Phe-氨基葡萄糖与120mL 4N氯化氢-乙酸乙酯溶液反应4h,TLC(CH2Cl2∶CH3OH∶CH3CO2H,15∶1∶1.5)监测原料点消失,终止反应。反应液减压浓缩,残留物加无水乙醚减压浓缩除去游离氯化氢,得5.7g(100%)标题化合物,为无色固体。ESI-MS(m/e)326[M+H]+。
实施例10制备[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val-Phe-氨基葡萄糖(8)
冰浴和搅拌下向200mg(0.43mmol)[(S)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚并(2,2-二甲基-咪唑-4-酮-3-基)]-2-甲基丁酰-Val与40mL无水DMF的溶液中加58mg(0.43mmol)HOBt及185mg(0.51mmol)DCC,反应30min。之后,加185mg(0. 51mmol)Phe-氨基葡萄糖并用NMM将反应液的pH调至9,室温避光反应24h,T LC(CH2Cl2∶CH3OH∶CH3CO2H,15∶1∶1.5)监测原料点消失,终止反应。反应液过滤,滤液减压浓缩,残留物用10mL CH3OH和10mL蒸馏水的混合溶剂溶解,用MILL EX GP 0.22μm滤膜过滤,用HPLC纯化,得37mg(11%)标题化合物,为淡黄色固体。ESI-MS(m/e)775[M-H]-;Mp 173-175℃;[α]=17.0(c=0.04,CH3OH); 1HNMR(300MHz,DMSO-d6)δ/ppm:10.868(s,1H),8.125(d,J=6.9Hz,1H),7.725(d,J=8.4Hz,1H),7.440(d,J=7.2Hz,1H),7.161(m,9H),6.446(d,J=4.2Hz,1H),4.877(t,J=3.9Hz,2H),4.581(m,3H),4.172(m,1H),3.794(m,3H),3.486(m,5H),2.904(m,4H),1.970(m,3H),1.625(m,3H),1.306(s,3H),1.275(s,1H),0.925(d,J=6.3Hz,6H),0.822(d,J=6.6Hz,3H),0.772(dd,J1=6.6Hz,J2=23.4Hz,6H)。
实验例1评价化合物8抑制S180小鼠肿瘤生长的活性
测定前将本发明衍生物用生理盐水将样品溶解;将阿霉素溶于生理盐水。无菌条件下取接种于ICR小鼠7-10天的S180肉瘤,加入适量生理盐水配制成瘤细胞悬液,细胞数为1×107个/mL,接种于健康雄性ICR小鼠前肢腋皮下,每只小鼠注射0.2mL。肿瘤接种24h后,治疗组小鼠每日腹腔注射0.2mL本发明衍生物的水溶液,连续给药9天,剂量为0.01μmol/kg。空白组小鼠每日腹腔注射0.2mL生理盐水。以阿霉素(剂量为2μmol/kg)作阳性对照。实验进行至第10天,称小鼠体重,并剖取各组小鼠的肿瘤称重,结果列入表1。数据表明,化合物8治疗小鼠的瘤重明显小于生理盐水治疗小鼠的瘤重。说明化合物8具有好的抗肿瘤活性。与发明背景中提到的已经公开的化合物相比,有效剂量降低了100倍,获得了意想不到的技术效果。
表1化合物对8对S180荷瘤小鼠肿瘤生长的影响
n=15;a)与生理盐水比p<0.01。
实验例2评价化合物8的抗炎活性
20±2g ICR雄性小鼠随机分为空白对照组、阳性用药组及给药组,小鼠使用前静息1天,操作间保持室内温度22℃,每组小鼠10只。一次口服生理盐水(剂量为0.2mL/20g)或阿司匹林的生理盐水溶液(剂量为1.11mmol/kg)或化合物8的生理盐水溶 液给(剂量为1μmol/kg)30分钟后,往小白鼠的左耳外廓涂二甲苯(0.045mL),1小时后将小鼠麻醉,颈椎脱臼处死。将小鼠的左和右耳剪下,用直径7mm的打孔器在两耳的相同位置,取圆形耳片,分别称重,求出两圆耳片的重量差作为肿胀度,以表示。本实验数据统计均采用t检验和方差分析。结果见表2。可以看出化合物8治疗组的鼠耳肿胀度与生理盐水组相比具有显著性差异,表明化合物8在发挥体内抗肿瘤作用下,还具有抗炎作用。与发明背景中提到的已经公开的化合物相比,抗炎症活性是意想不到的技术效果。
表2化合物8的抗炎活性
n=10;a)与生理盐水比p<0.01。
Claims (4)
1.下式的2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖
2.权利要求1的2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖的制备方法,该方法包括:
(1)将L-色氨酸在硫酸的催化下与甲醛进行Pictet-Spengler缩合得到2,3,4,9-四氢-β-咔啉-3-羧酸;
(2)冰浴下向2,3,4,9-四氢-β-咔啉-3-羧酸与N,N-二甲基甲酰胺,DMF,的溶液中加入二碳酸二叔丁酯,Boc2O,N-甲基吗啉催化得到N-Boc-2,3,4,9-四氢-β-咔啉-3-羧酸;
(3)在二环己基碳二亚胺,DCC,和N-羟基苯并三唑,HOBt,存在下N-Boc-2,3,4,9-四氢-β-咔啉-3-羧酸在无水THF(四氢呋喃)中与L-Leu-OMe缩合为N-Boc-2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe;
(4)在氯化氢-乙酸乙酯溶液中N-Boc-2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe脱去Boc生成2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe;
(5)三乙胺催化下2,3,4,9-四氢-β-咔啉-3-甲酰-Leu-OMe在甲醇和丙酮溶液中避光反应得到2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰甲酯;
(6)在甲醇溶液中2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰甲酯在2N NaOH溶液中水解生成2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酸;
(7)在DCC和HOBt存在下2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酸在无水DMF中与Val-OBzl缩合为2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-OBzl;
(8)在在Pd/C催化下2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-OBzl氢解为2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val;
(9)在DCC和HOBt存在下,Boc-Phe在无水DMF中与氨基葡萄糖缩合为Boc-Phe-氨基葡萄糖;
(10)在氯化氢-乙酸乙酯溶液中Boc-Phe-氨基葡萄糖脱去Boc生成Phe-氨基葡萄糖;
(11)在DCC和HOBt存在下2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val在无水DMF中与Phe-氨基葡萄糖缩合为2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖。
3.权利要求1的2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖在制备抗肿瘤药物中的应用。
4.权利要求1的2,3,4,9-四氢-1H-吡啶并吲哚并-2,2-二甲基-咪唑-4-酮-3-基-2-甲基丁酰-Val-Phe-氨基葡萄糖在制备抗炎药物中的应用。
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氨基葡萄糖拟肽类衍生物的合成;刘俊朋;《中国优秀硕士学位论文全文数据库(电子期刊)_工程科技I辑》;20131215(第12期);第1.1.2节,1.2.2节 |
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