CN114920730A - 一种雷帕霉素开环降解杂质及其制备方法和应用 - Google Patents
一种雷帕霉素开环降解杂质及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及医药化工领域,尤其涉及一种雷帕霉素开环降解杂质及其制备方法和应用。通过碱水解开环、酸中和、纯化,得到雷帕霉素开环化合物,结构式如下:
Description
技术领域
本发明涉及医药化工领域,尤其涉及一种雷帕霉素开环降解杂质及其制备方法和应用。
背景技术
雷帕霉素(rapamycin,又称西罗莫司sirolimus),1975年由加拿大Ayerst实验室的Vezina等人从太平洋Easler岛土壤样品中分离的吸水链霉菌(Streptomyeeshygroscopicus)发酵产生得到。1977年Morris等首先发现其具有免疫抑制作用。雷帕霉素通过与体内的FKBP12(FK506结合蛋白12)结合后抑制哺乳动物雷帕霉素靶蛋白mTOR(themammalian target of rapamycin)的激活,抑制蛋白质合成,从而抑制了细胞因子引发的T淋巴细胞增殖。mTOR还是与肿瘤发生、发展和转移密切相关的分子靶位。雷帕霉素不但作为器官移植抗排斥药物和治疗冠状动脉再狭窄的支架涂层在临床上应用,而且已经在实验室中被证实可抑制许多癌细胞的生长,包括横纹肌肉瘤、神经母细胞瘤、肺小细胞癌、骨癌、胰脏癌、白血病癌细胞及B细胞淋巴癌等。雷帕霉素衍生物在治疗癌症方面已经得到临床应用,雷帕霉素具有广泛的应用前景。1999年雷帕霉素通过FDA批准在美国首次上市,至今已在美国、欧盟、日本、中国等国家上市,主要作为新型免疫抑制剂用于肾移植排异,国内已有片剂、口服液、胶囊等制剂上市。
雷帕霉素是36元大环内酯化合物,化学结构复杂,稳定性差,生产、贮存过程中易产生降解产物。雷帕霉素及其制剂的质量是药物品质的一个重要标准,对雷帕霉素的纯度和杂质有严格规定要求,开发杂质对照品,保证雷帕霉素及其制剂的质量十分重要。其中雷帕霉素开环化合物是雷帕霉素主要杂质之一。因此,为提高雷帕霉素及其制剂的质量控制的准确性,本发明提出一种雷帕霉素开环降解杂质的制备方法。
发明内容
本发明要解决的技术问题,在于提供一种雷帕霉素开环降解杂质及其制备方法和作为雷帕霉素及其制剂质量检查的杂质对照品的用途。
本发明是这样实现的:
一种雷帕霉素开环降解杂质的制备方法,包括如下步骤:
(1)开环:将雷帕霉素溶于有机溶剂中,在碱性条件下水解开环;
(2)酸中和:开环后在酸性条件下形成雷帕霉素开环化合物;
(3)纯化:加入萃取溶剂萃取1-3次,浓缩有机层,干燥后进行柱层析。
进一步地,步骤(1)所述有机溶剂包括甲醇、乙醇、丙酮、乙腈、异丙醇、四氢呋喃、二甲基甲酰胺等溶剂中的一种;特别优选乙醇或丙酮。
进一步地,步骤(1)所述碱性条件采用的碱包括氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸氢二钠、醋酸铵、醋酸钠等试剂溶液中的一种;特别优选氢氧化钠。
更进一步地,所述碱的浓度为0.5-1.5mol/L;特别优选1.0mol/L。
更进一步地,在碱性条件下反应0.5-1.5小时;特别优选反应1.0小时。
进一步地,步骤(2)所述酸性条件采用的酸包括盐酸、氢溴酸、硫酸、磷酸、甲酸、乙酸、乙二酸等中的一种;特别优选盐酸或乙酸。
更进一步地,所述酸的浓度为0.8-1.2mol/L;特别优选1.0mol/L。
更进一步地,加入酸后调节pH=5~6,然后反应0.5-1.5小时;特别优选反应1.0小时。
进一步地,步骤(3)所述萃取溶剂包括乙酸乙酯、二氯甲烷、三氯甲烷、甲苯、正丁醇等溶剂中的一种;特别优选乙酸乙酯或二氯甲烷。
进一步地,步骤(3)所述柱层析采用的固定相为C1、C4、C8和C18反相硅胶中的一种;特别优选C18反相硅胶;采用的洗脱剂为甲醇、异丙醇、丙酮、乙腈等含水有机溶剂的一种,含水量为30%-80%(重量分数);特别优选丙酮或乙腈,含水量为40%-60%(重量分数)。
本发明通过在碱性条件下使雷帕霉素中的酯基水解开环,从而生成相应的盐,然后再进行酸反应,生成雷帕霉素开环化合物。如果先采用在酸性条件反应,易产生三烯双键移位和7位甲氧差向异构化等副反应;碱浓度过低,不产生酯基水解开环,碱浓度过高,副反应多等问题。因此,只有合适的碱性条件下进行内酯水解开环,碱浓度以0.5-1.5mol/L为宜。
本发明制备的雷帕霉素开环化合物纯度高于95%,可直拉作为雷帕霉素及其制剂质量检查的杂质对照品使用。
本发明具有如下优点:本发明制备的雷帕霉素开环化合物是雷帕霉素的主要杂质之一,作为雷帕霉素及其制剂质量检查的杂质对照品,用于雷帕霉素及其制剂质量控制。本发明提供的一种雷帕霉素开环降解杂质的制备方法,该方法反应条件温和,操作简单,可以得到高纯度的雷帕霉素开环化合物,提高雷帕霉素及其制剂的质量控制的准确性。
具体实施方式
本发明的合成路线如下:
对本发明得到的雷帕霉素开环化合物结构确证:
雷帕霉素开环化合物主要构象的NMR信号归属见下表。
1H and 13C-NMR chemical shifts of compounda.(inD6-DMSO)
a1H and13CNMR were measured at 500MHz and 125MHz,respectively
结构式如下:
雷帕霉素开环化合物HPLC检测方法:
色谱条件色谱柱Kromasil 100-5C18柱(4.6mmx250mm,5μm);流动相20mmol/L甲酸铵溶液(用甲酸调至pH3.8,A):乙腈(含1%甲基叔丁醚,B),线性梯度洗脱,程序见表1;流速1.5ml/min;柱温35℃,样品室温度4℃;检测波长277nm;进样量20μl。
取雷帕霉素开环化合物样品20mg,精密称定,置25ml量瓶中,加乙腈溶解并稀释至刻度,摇匀,得雷帕霉素开环化合物溶液;精密量取20μl,注入液相色谱仪,记录色谱图。
表1梯度洗脱程序
实施例1
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率88.7%,HPLC纯度83.5%。
实施例2
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL甲醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去甲醇溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率84.3%,HPLC纯度79.7%。
实施例3
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL丙酮溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去丙酮溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率83.1%,HPLC纯度77.2%。
实施例4
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL四氢呋喃溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去四氢呋喃溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率76.6%,HPLC纯度75.4%。
实施例5
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钾溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率86.2%,HPLC纯度81.6%。
实施例6
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加0.5mol/L碳酸钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率84.3%,HPLC纯度78.5%。
实施例7
室温下,将雷帕(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L氢溴酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率82.3%,HPLC纯度76.7%。
实施例8
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L乙酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加乙酸乙酯溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率81.2%,HPLC纯度75.3%。
实施例9
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加石油醚溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率81.7%,HPLC纯度78.4%。
实施例10
室温下,将雷帕霉素(9.15g,0.01mol)加入100mL乙醇溶剂中搅拌溶解,溶解后滴加1mol/L氢氧化钠溶液11.0mL,滴毕搅拌反应1小时,反应完毕后,浓缩除去乙醇溶剂,加水100mL溶解,滴加1mol/L盐酸溶液,调节pH=5~6,搅拌反应1小时,反应完毕后,加二氯甲烷溶剂100mL,震荡摇匀,萃取二次,合并后浓缩有机层,干燥后得雷帕霉素开环化合物固体物。收率84.2%,HPLC纯度81.6%。
实施例11
室温下,将实施例1的雷帕霉素开环化合物固体物1.0g,经层析柱纯化,C18反相硅胶为固定相,洗脱剂开始用丙酮:水(6:4),雷帕霉素开环化合物组分洗脱完毕后,改用丙酮洗脱,得雷帕霉素开环化合物纯品,收率81.7%,HPLC纯度97.6%。
实施例12
室温下,将实施例1的雷帕霉素开环化合物固体物1.0g,经层析柱纯化,C18反相硅胶为固定相,洗脱剂开始用甲醇:水(6:4),雷帕霉素开环化合物组分洗脱完毕后,改用甲醇洗脱,得雷帕霉素开环化合物纯品,收率74.6%,HPLC纯度95.8%。
实施例13
室温下,将实施例1的雷帕霉素开环化合物固体物1.0g,经层析柱纯化,C18反相硅胶为固定相,洗脱剂开始用乙腈:水(6:4),雷帕霉素开环化合物组分洗脱完毕后,改用乙腈洗脱,得雷帕霉素开环化合物纯品,收率78.1%,HPLC纯度96.5%。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
Claims (10)
1.一种雷帕霉素开环降解杂质的制备方法,其特征在于:包括如下步骤:
(1)开环:将雷帕霉素溶于有机溶剂中,在碱性条件下水解开环;
(2)酸中和:开环后在酸性条件下形成雷帕霉素开环化合物;
(3)纯化:加入萃取溶剂萃取1-3次,浓缩有机层,干燥后进行柱层析。
2.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:步骤(1)所述有机溶剂包括甲醇、乙醇、丙酮、乙腈、异丙醇、四氢呋喃、二甲基甲酰胺。
3.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:步骤(1)所述碱性条件采用的碱包括氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸氢二钠、醋酸铵、醋酸钠。
4.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:所述碱的浓度为0.5-1.5mol/L。
5.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:步骤(2)所述酸性条件采用的酸包括盐酸、氢溴酸、硫酸、磷酸、甲酸、乙酸、乙二酸。
6.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:所述酸的浓度为0.8-1.2mol/L。
7.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:步骤(3)所述萃取溶剂包括乙酸乙酯、二氯甲烷、三氯甲烷、甲苯、正丁醇。
8.根据权利要求1所述的雷帕霉素开环降解杂质的制备方法,其特征在于:步骤(3)所述柱层析采用的固定相为C1、C4、C8和C18反相硅胶中的一种;采用的洗脱剂包括甲醇、异丙醇、丙酮、乙腈,含水量为30%-80%。
10.如权利要求9所述雷帕霉素开环化合物作为雷帕霉素及其制剂质量检查的杂质对照品的用途。
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