CN113388002A - 一种环孢菌素相关化合物的制备方法 - Google Patents
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Abstract
本发明公开了一种环孢菌素相关化合物的制备方法,本发明方法操作简单,后处理方便,收率高,制备所得化合物对环孢菌素有很高的参考价值。并且本申请通过对环孢菌素相关化合物的制备,对环孢菌素进行效能科学评价、药理研究、药代动力学等提供了重要依据,便于其在人体内的代谢过程以及药物动力学进行更深入的研究,使其具有更好的药用前景。
Description
技术领域
本发明涉及环肽类免疫抑制剂的制备方法,具体涉及一种环孢菌素相关化合物的制备方法。
技术背景
环孢菌素是一种从丝状真菌培养液中分离出的由11个氨基酸组成的环肽。该药发现于1969年,1976年瑞士Sandoz公司首次报道了由半知菌属多孔木霉(后更名为雪白白僵菌)和光泽柱孢菌产生的环孢菌素。此后各国的科学家又陆续报道了由13种其他产生菌,如茄病镰刀菌和侵菅新赤壳菌等产生环孢菌素。
环孢菌素在器官移植治疗中发挥了重大作用,奠定并推动了器官移植的发展。环孢菌素作为免疫抑制剂它还被应用于一些自身免疫性疾病的治疗,如对类风湿关节炎及白塞(贝赫切特综合征),取得了较为满意的疗效,对I型糖尿病、牛皮癣和寄生虫病如疟疾、血吸虫等有一定疗效。此外,环孢菌素还具有广泛的其它生物学活性如抗真菌、抗寄生虫、抗HIV、抗炎、逆转肿瘤细胞多药耐药等作用。
由于环孢菌素稳定性较差,现有的环孢菌素的合成方法是采用发酵的方法制备得到。本发明报道一种环孢菌素开环后的化合物的制备方法,暂未有文献报道,制备所得化合物是环孢菌素的开环产物,属于代谢产物。
发明内容
发明目的:针对上述现有技术,本申请提供了一种环孢菌素相关化合物的制备方法。
技术方案:本申请所述的一种环孢菌素相关化合物的制备方法,包括以下步骤:
(1)固相合成:
1)以CTC-Resin为固相载体,首先偶合Fmoc-Leu-OH,得到Fmoc-Leu-Resin,脱去Fmoc保护得到H-Leu-Resin;
2)由Fmoc-N-Me-Leu-OH与步骤1)得到的H-Leu-Resin偶联,得到Fmoc-N-Me-Leu-Leu-Resin,然后依次缩合Fmoc-D-Ala-OH,Fmoc-Ala-OH,Fmoc-N-Me-Leu-OH,Fmoc-Val-OH,Fmoc-N-Me-Leu-OH,Fmoc-N-Me-Gly-OH,Fmoc-Abu-OH;
3)最后偶合(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic acid(81135-41-3),裂解除去树脂,得到片段一;
(2)羧基液相偶联甲氨:将步骤(1)得到的片段一偶联得到片段二;
(3)将步骤(2)得到的片段二,纯化得到环孢菌素相关化合物。
进一步的,上述制备方法步骤(1)中:
步骤(1)中,步骤1)固相载体偶联氨基酸的偶联剂为DIEA。
步骤(1)中,步骤2)偶联剂为DIC+A或DIEA+B,A为HOBT或HOAT,B为HBTU、HATU、PyBOP或TBTU。
步骤(1)中,步骤3)偶联剂为DIC+A或者B+A+C,其中A为HOBT或HOAT,B为HBTU、HATU、TBTU或PyBOP,C为DIEA或NMM。
步骤(1)中,步骤3)裂解树脂所用试剂为体积比为1:1~1:4的三氟乙醇和二氯甲烷,优选体积比为1:2的三氟乙醇和二氯甲烷。裂解试剂的用量为按照CTC-Resin重量的10倍(w:v=1:10)加入相应体积的量,除去树脂。
进一步的,步骤(2)中,甲氨偶联溶剂为N,N-二甲基甲酰胺,偶联剂为DIC+A或DIEA+B,其中A为HOBT,B为HBTU、HATU、PyBop或TBTU。反应温度为20~30℃,反应时间为12~24小时。其中优选DIC+HOBT,其他偶联试剂产物较杂。缩合完成将反应液加入到50倍体积的水中析出,抽滤后用油泵旋干。
进一步的,步骤(3)中,裂解序列中的保护所用试剂为三氟乙酸、水和三异丙基硅烷溶液,其中,三氟乙酸的体积比为80%-90%,水的体积比为5%-10%,三异丙基硅烷的体积比为5%-10%。三氟乙酸、水、三异丙基硅烷的优选体积比为90:10:10。反应温度为10~20℃,反应时间为1~2小时。
步骤(3)反应完成后,旋蒸旋干,提纯目标物采用色谱柱纯化:流动相A相:0.1%TFA水溶液,流动相B相:0.1%TFA乙腈溶液,梯度:40%-80%。
有益效果:本申请环孢菌素相关化合物的制备方法,首先通过固相合成片段一,最后通过HOBT+DIC液相中偶联甲氨,有效的提高了收率,并降低了成本。并且本申请通过对环孢菌素相关化合物的研究,可以对环孢菌素在人体内的代谢过程以及药物动力学进行更深入的研究,使其具有更好的药用前景。
附图说明
图1是本申请环孢菌素相关化合物的合成流程图;
图2是本申请环孢菌素相关化合物质谱及HPLC检测图。
具体实施方式
下面结合具体实施例对本申请作出详细说明。
本申请中所用试剂列表:
实施例1
一种环孢菌素相关化合物的制备方法,合成流程如图1所示,具体包括以下步骤:
步骤一:
Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-Resin的合成:
1)将10g取代度0.4~0.6mmol/g的CTC-Resin加入多肽反应器中,加入2gFmoc-Leu-OH和10ml DIEA反应2小时后,抽去反应液,DMF洗涤两次;脱去Fmoc保护后,DMF洗涤两次,DCM洗涤一次,DMF洗涤两次;
2)先偶联Fmoc-N-Me-Leu-OH,然后依次缩合Fmoc-D-Ala-OH,Fmoc-Ala-OH,Fmoc-N-Me-Leu-OH,Fmoc-Val-OH,Fmoc-N-Me-Leu-OH,Fmoc-N-Me-Gly-OH,Fmoc-Abu-OH反应时间均为2小时,脱保护时间为0.5小时,反应终点以茚三酮法检测为准;反应结束后,DMF洗涤两次,DCM洗涤一次,DMF洗涤两次;
3)将洗涤后的树脂再用甲醇洗涤两次后,用油泵抽干得到:Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-Resin;
步骤二:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acid溶解到20ml DMF溶液中去,加入0.5g HOBT和0.5ml DIC,混合均匀后加入到步骤一的树脂中,室温搅拌反应2小时;反应终点以茚三酮法检测为准;反应结束后,DMF洗涤两次,DCM洗涤一次。用100mL TFE/DCM=1:4裂解试剂切割两遍,每次1小时。过滤树脂收集裂解液,用油泵拉干得到2.1g黄色固体;
步骤三:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-NH-CH3的合成:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu2.1g溶解到100ml DMF溶液中,NH2-CH3.HCl 0.5g加入到DMF中,加入0.5g HOBT和0.2mlDIC,搅拌12小时后用油泵拉干,得到固体1.9g;
步骤四:
环孢菌素相关化合物的提纯:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-NH-CH3 1.9g用80%乙腈和水溶解,过滤;
纯化设备:汉邦UV3000/NP7000,色谱柱:DAC-50,C18,10um,波长:220.流动相A相:0.1%TFA水溶液,流动相B相:0.1%TFA乙腈溶液,梯度:40%-80%,时间为60分钟,得到0.6g环孢菌素相关化合物;
结构确认后确认序列为:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-NH-CH3。(图2为化合物质谱及HPLC检测图)
实施例2
一种环孢菌素相关化合物的制备方法,合成流程如图1所示,具体包括以下步骤:
步骤一:
Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-Resin的合成:
1)将10g取代度0.4~0.6mmol/g的CTC-Resin加入多肽反应器中,加入2gFmoc-Leu-OH和10ml DIEA反应2小时后,抽去反应液,DMF洗涤两次;脱去Fmoc保护后,DMF洗涤两次,DCM洗涤一次,DMF洗涤两次;
2)先偶联Fmoc-N-Me-Leu-OH,然后依次缩合Fmoc-D-Ala-OH,Fmoc-Ala-OH,Fmoc-N-Me-Leu-OH,Fmoc-Val-OH,Fmoc-N-Me-Leu-OH,Fmoc-N-Me-Gly-OH,Fmoc-Abu-OH反应时间均为2小时,脱保护时间为0.5小时,反应终点以茚三酮法检测为准;反应结束后,DMF洗涤两次,DCM洗涤一次,DMF洗涤两次;
3)将洗涤后的树脂再用甲醇洗涤两次后,用油泵抽干得到:Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-Resin;
步骤二:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-Methyl Ester溶解到20ml DMF溶液中去,加入4ml Pip溶液,混合均匀后加入到步骤一的树脂中,室温搅拌反应4小时;反应终点以茚三酮法检测为准;反应结束后,DMF洗涤两次,DCM洗涤一次。用100mL TFE/DCM=1:4裂解试剂切割两遍,每次1小时。过滤树脂收集裂解液,用油泵拉干得到0.8g黄色固体;
步骤三:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-NH-CH3的合成:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu0.8g溶解到100ml DMF溶液中,NH2-CH3.HCl 0.5g加入到DMF中,加入0.5g HOBT和0.2mlDIC,搅拌12小时后用油泵拉干,得到固体0.9g;
步骤四:
环孢菌素相关化合物的提纯:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-NH-CH3 1.9g用80%乙腈和水溶解,过滤;
纯化设备:汉邦UV3000/NP7000,色谱柱:DAC-50,C18,10um,波长:220。流动相A相:0.1%TFA水溶液,流动相B相:0.1%TFA乙腈溶液,梯度:40%-80%,时间为60分钟,得到0.1g环孢菌素相关化合物。
结构确认后确认序列为:
(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic-acyl-Abu-N-Me-Gly-N-Me-Leu-Val-N-Me-Leu-Ala-D-Ala-N-Me-Leu-Leu-NH-CH3。
Claims (10)
1.一种环孢菌素相关化合物的制备方法,其特征在于,包括以下步骤:
(1)固相合成:
1)以CTC-Resin为固相载体,首先偶合Fmoc-Leu-OH,得到Fmoc-Leu-Resin,脱去Fmoc保护得到H-Leu-Resin;
2)由Fmoc-N-Me-Leu-OH与步骤1)得到的H-Leu-Resin偶联,得到Fmoc-N-Me-Leu-Leu-Resin,然后依次缩合Fmoc-D-Ala-OH,Fmoc-Ala-OH,Fmoc-N-Me-Leu-OH,Fmoc-Val-OH,Fmoc-N-Me-Leu-OH,Fmoc-N-Me-Gly-OH,Fmoc-Abu-OH;
3)最后偶合(4S,5R)-5-((R,E)-hex-4-en-2-yl)-3-methyl-2-oxooxazolidine-4-carboxylic acid(81135-41-3),裂解除去树脂,得到片段一;
(2)羧基液相偶联甲氨:将步骤(1)得到的片段一偶联得到片段二;
(3)将步骤(2)得到的片段二,纯化得到环孢菌素相关化合物。
2.根据权利要求1中所述的制备方法,其特征在于,步骤(1)中,步骤1)固相载体偶联氨基酸的偶联剂为DIEA。
3.根据权利要求1中所述的制备方法,其特征在于,步骤(1)中,步骤2)偶联剂为DIC+A或DIEA+B,其中A为HOBT或HOAT,B为HBTU、HATU、PyBOP或TBTU。
4.根据权利要求1中所述的制备方法,其特征在于,步骤(1)中,步骤3)偶联剂为DIC+A或者B+A+C,其中A为HOBT或HOAT,B为HBTU、HATU、TBTU或PyBOP,C为DIEA或NMM。
5.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,步骤3)裂解树脂所用试剂为体积比为1:1~4的三氟乙醇和二氯甲烷。
6.根据权利要求1中所述的制备方法,其特征在于,步骤(1)中,步骤3)裂解树脂所用试剂为体积比为1:2的三氟乙醇和二氯甲烷。
7.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,成环溶剂为N,N-二甲基甲酰胺。
8.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,偶联剂为DIC+A或DIEA+B,其中A为HOBT,B为HBTU、HATU、PyBop或TBTU。
9.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,反应温度为20~30℃,反应时间为12~24小时。
10.根据权利要求1所述的制备方法,其特征在于,步骤(3)中,提纯目标物采用色谱柱纯化:流动相A相:0.1%TFA水溶液,流动相B相:0.1%TFA乙腈溶液,梯度:40%-80%。
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CN101120012A (zh) * | 2004-12-17 | 2008-02-06 | 伊素制药公司 | 环孢菌素类似物的代谢物 |
CN104744570A (zh) * | 2013-12-31 | 2015-07-01 | 深圳先进技术研究院 | 一种环孢菌素的合成方法 |
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CN101120012A (zh) * | 2004-12-17 | 2008-02-06 | 伊素制药公司 | 环孢菌素类似物的代谢物 |
CN104744570A (zh) * | 2013-12-31 | 2015-07-01 | 深圳先进技术研究院 | 一种环孢菌素的合成方法 |
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