CN115340560B - 一种西罗莫司类似物及其制备方法和应用 - Google Patents
一种西罗莫司类似物及其制备方法和应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
本发明属于医药领域,尤其涉及一种西罗莫司类似物及其制备方法和应用,所述西罗莫司类似物如式1所示。所述制备方法包含:从发酵液中提取,获得含西罗莫司类似物的粗品,对粗品进行正相层析,得到含西罗莫司类似物的一次层析物,随后对获得的一次层析物进行反相层析,得到西罗莫司类似物的纯品。该西罗莫司类似物具有抗真菌及抗肿瘤作用。
Description
技术领域
本发明属于医药领域,尤其涉及一种西罗莫司类似物及其制备方法和应用。
背景技术
西罗莫司(sirolimus)又称雷帕霉素(rapamycin),是一种亲脂性含氮36元大环内酯类抗生素,分子式C51H79NO13,分子量914.2,其结构式如下:
西罗莫司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;西罗莫司在延长哺乳动物生命周期方面具有潜在作用,研究表明,西罗莫司能恢复阿尔茨海默病(AD)实验小鼠的相应缺陷症状,恢复认知和记忆水平,降低大脑组织的损伤。西罗莫司对AD病具有缓解作用。
目前通过化学半合成的方法对西罗莫司进行结构改造获得一些衍生物,如依维莫司(everolimus,RADO01)、biolimus A9、zotarolimus、替西罗莫司(temsirolimus,CCI-779)已作为药物支架的涂料或抗肿瘤靶向药物应用于临床。
本申请人在吸水链霉菌FC904(Streptomyces hygroscopicus FC904)[该菌株的培养和发酵条件见:程元荣等,中国抗生素杂志,2002,27(12):709-712]发酵液中已分离到:脯氨酰雷帕霉素(prolyrapamycin)[杨国新等.海峡药学,2008,20(3):72-74]、28-差向雷帕霉素(28-epirapamycin)[杨国新等,中国抗生素杂志,2008,33(2):80-83]、7-羟基-雷帕霉素(7-O-demethyl-rapamycin)[陈夏琴等.中国抗生素杂志,2016,41(4):267-270]、7-O-乙基雷帕霉素(7-O-ethyl-rapamycin)[陈夏琴等.中国抗生素杂志,2012,37(11):69-73]、雷帕霉素二醛(rapamycin dialdehyde)[杨国新等,中国抗生素杂志,2018,43(4):450-456]、29-O-去甲基雷帕霉素(29-O-demethyl-rapamycin)[杨国新等,中国抗生素杂志,2019,44(2):208-213],申请人从该发酵液中获得一种新的西罗莫司类似物31-去甲基雷帕霉素,并验证了其具有抗肿瘤和抗真菌活性作用。
发明内容
本发明所要解决的技术问题在于提供一种西罗莫司类似物及其制备方法和应用。
本发明是这样实现的:
本发明首先提供了一种西罗莫司类似物,其特征在于:为31-差向雷帕霉素,结构如式1所示:
本发明还提供了所述西罗莫司类似物的制备方法,包括以下步骤:
(1)将西罗莫司发酵液进行固液分离,获得菌丝体,用有机溶剂浸提菌丝体,得到浸提液后,将浸提液减压浓缩,得到浓缩物。
(2)用与水不相溶的有机溶剂萃取浓缩物,除去水层以获得有机相,有机相浓缩除去有机溶剂后获得粗提物。对粗提物进行硅胶层析,洗脱后得初步纯化的31-差向雷帕霉素。
(3)将初步纯化的31-差向雷帕霉素进行1-2次的C18反相硅胶层析,以甲醇水溶液洗脱,得到31-差向雷帕霉素纯品。
进一步地:
步骤(1)所述有机溶剂为低级醇、低级酮、羧酸酯、卤代烷烃中的一种或几种。
步骤(2)所述的有机溶剂为羧酸酯、卤代烷烃、甲苯中的一种或几种。
步骤(2)所述硅胶层析,以丙酮体积百分数为20%的丙酮-石油醚混合溶剂洗脱。
步骤(3)所述甲醇水溶液中,甲醇与水的体积比为40:60~90:10,更进一步体积比为75:25~85:15。
最后,本发明提供了所述西罗莫司类似物在制备抗肿瘤的药物中的应用。
进一步地,所述肿瘤包括直肠癌、肺癌、肾癌、食管癌、胃癌、乳腺癌。
以及,所述西罗莫司类似物在制备抗真菌的药物中的应用。
进一步地,所述真菌包括白色念珠菌。
本发明具有如下优点:本发明通过对西罗莫司发酵液分离、纯化获得了一种新的西罗莫司类似物--31-差向雷帕霉素,且该化合物具有抗肿瘤和抗真菌活性作用。
附图说明
下面参照附图结合实施例对本发明作进一步的说明。
图1为粗提物的HPLC液相色谱图。
图2为半制备上柱样的HPLC液相色谱图。
图3为纯品的HPLC液相色谱图。
图4为化合物31-差向雷帕霉素的紫外谱图。
图5为化合物31-差向雷帕霉素的质谱图。
图6为化合物31-差向雷帕霉素的1H-NMR谱图。
图7为化合物31-差向雷帕霉素的13C-NMR图。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。
实施例1
西罗莫司发酵液(该菌株的培养和发酵条件见:程元荣等,中国抗生素杂志,2002,27(12):709-712)10000ml,离心收集菌丝体,分别用2000mL95%乙醇浸泡菌丝体两次,每次搅拌60分钟后,离心收集酒精浸泡液,减压浓缩浸泡液去除乙醇,剩余部分用2000mL乙酸乙酯萃取,分出的水层用1000mL乙酸乙酯再萃取一次,合并乙酸乙酯层,减压浓缩有机相层除去乙酸乙酯,得粗提物约16.19g,其中,31-差向雷帕霉素的保留时间为19.040min,如图1所示(色谱条件如下所述)。
称取200-300目硅胶800g装填玻璃层析柱,粗提物用适量丙酮溶解后上样,样品吸附后,用以丙酮体积百分数为20%(v/v)的丙酮-石油醚混合溶剂洗脱,分部收集,HPLC跟踪检测,合并所需部分,减压浓缩除去溶剂,得到初步纯化的浅黄色泡沫状固体524mg,其中,31-差向雷帕霉素的保留时间为18.930min,如图2所示。将此浅黄色泡沫状固体分成两份。
取一份浅黄色泡沫状固体溶解于适量甲醇中,制备液相色谱仪分离,分段收集,HPLC检测。合并所需部分,减压浓缩去除甲醇后用等体积的二氯甲烷分别萃取两次,合并二氯甲烷层,减压浓缩除去二氯甲烷获得31-差向雷帕霉素粗产物。
将两份浅黄色泡沫状固体经制备液相分离得到的31-差向雷帕霉素粗产物合并,共得47mg。
将粗产物47mg再进行一次制备液相色谱仪分离,获得31-差向雷帕霉素纯品3.4mg,如图3所示。
制备色谱分离条件:岛津液相色谱仪LC-8A,色谱柱:UniSil 10-100C18(10μm,21.2mm×250mm),检测波长:UV 277nm,柱温:室温,流速:8mL/min,流动相为甲醇︰水=80︰20。
HPLC检测条件:色谱柱:Hypersil BDS C18柱(5μm,3mm×250mm),检测波长:UV277nm,柱温:50℃,流速:1.1mL/min,流动相A:0.27g/L磷酸二氢钾溶液,流动相B:乙腈,以表1所述比例运行。
表1 流动相配比运行表
化合物31-差向雷帕霉素的理化性质见表2。
表2 31-差向雷帕霉素的理化性质
31-差向雷帕霉素在DMSO中,存在两种构象,主要构象与次要构象比值接近2.5:1,表3列出31-差向雷帕霉素主要构象的13C-NMR和1H-NMR化学位移归属。
表3 31-差向雷帕霉素的13C-NMR和1H-NMR化学位移归属a,b(D6-DMSO)
a 1H和13C NMR分别在500MHz与125MHz测试
b主要构象的化学位移值
实施例2
实验细胞:人结直肠腺癌细胞HCT-15、人食管癌细胞ECA-109、人非小细胞肺癌细胞A549、人腺胃癌细胞SGC7901、人肾透明细胞腺癌细胞786-0、人乳腺癌细胞T47D,上述细胞均来自上海细胞库。
实验方法:
1.取细胞以1×104/mL的细胞密度接种于96孔培养板中,每孔100μL,置37℃5%CO2及饱和湿度的条件下培养24h。
2.待其贴壁后,加入31-差向雷帕霉素纯品,其终浓度达到0.0006μM、0.0024μM、0.0098μM、0.039μM、0.156μM、0.625μM、2.5μM、10μM,每组4个复孔,每孔终体积为200μL。置上述条件下培养72h。
3.培养结束后,每孔加入50μL4℃预冷的TCA溶液(50%,w/v),放置于4℃中固定细胞1h后,用去离子水冲洗3-5遍,室温晾干。
4.每孔加入0.4%(w/v)的SRB染液100μL,染色15min后倒掉,用1%(v/v)乙酸冲洗3-5遍,室温晾干。
5.用100μL Tris-base碱液(10mM,pH=10.5)溶解染料,水平摇床上振荡20min,采用酶标仪540nm处测定光吸收值。
6.计算增殖抑制率=[(对照组—实验组)/对照组D570]×100%。
7.使用Graphpad prism软件,输入药物浓度和抑制率,计算IC50。
实验结果如下:
细胞 | 31-差向雷帕霉素相对IC50(μM) |
T47D | 0.03 |
HCT15 | 0.13 |
A549 | 0.04 |
786-0 | 0.11 |
SGC7901 | 0.42 |
ECA109 | 0.55 |
结果表明,31-差向雷帕霉素具有抗肿瘤活性。
实施例3
1、待测菌悬液的制备:将2株白色念珠菌(CPCC360003/CMCC(F)98001)分别接种于沙保罗琼脂培养基上,置28℃培养48h后,各取1接种环新培养物分别混悬于2ml无菌生理盐水中震荡15秒,使之充分混合均匀后,用血球计数板计数,用沙保罗液体培养基稀释至1×105~5×105CFU/ml(终浓度)。
2、待测药物配置:DMSO溶解待测药物(31-差向雷帕霉素纯品),配成200ug/ml溶液。
3、检测步骤:取无菌圆底96孔板,先在各排孔中加入100ul空白沙保罗液体培养液,第1行第1孔加入100ul待测药物200ug/ml,混合均匀后,从第1孔混合液中吸取100ul移至第2孔混合,依次至12号孔进行倍比稀释,最后每孔添加100ul终浓度1×105~5×105CFU/ml白色念珠菌悬液,实验设置3行平行实验,2行不加药物作为阴性对照实验。28℃培养48h后,观察结果。
4、结果判断:检查对照管的受试菌生长情况是否良好,肉眼观察,药物最低浓度管无菌生长者,即为受试菌的MIC。
5、结果如下:
检测结果表明白色念珠菌CPCC360003、白色念珠菌CMCC(F)98001对31-差向雷帕霉素敏感,具有较强的抑菌能力。
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 (1)
1.一种西罗莫司类似物的制备方法,其特征在于,所述西罗莫司类似物为31-差向雷帕霉素,结构如式1所示:
所述制备方法包括下述步骤:
(1)取西罗莫司发酵液,将发酵液进行固液分离,获得菌丝体,用有机溶剂浸提菌丝体,得到浸提液,将浸提液减压浓缩,得到浓缩物;
(2)将浓缩物进行萃取,除去水层以获得有机相;对有机相分别进行碱洗、水洗、浓缩后获得粗提物,溶解所述粗提物进行硅胶层析,洗脱后得初步纯化的31-差向雷帕霉素;
(3)将初步纯化的31-差向雷帕霉素进行2次的C18反相硅胶层析,以甲醇水溶液洗脱,得到31-差向雷帕霉素纯品;所述甲醇水溶液中,甲醇与水的体积比为40:60~90:10;
步骤(1)所述有机溶剂为95%乙醇;
步骤(2)所述硅胶层析,以丙酮体积百分数为20%的丙酮-石油醚混合溶剂洗脱;
步骤(2)所述萃取采用的有机溶剂为乙酸乙酯。
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