CN116715718A - 乌苏烷型三萜类化合物、其微生物转化制备方法及应用 - Google Patents
乌苏烷型三萜类化合物、其微生物转化制备方法及应用 Download PDFInfo
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- CN116715718A CN116715718A CN202310669587.4A CN202310669587A CN116715718A CN 116715718 A CN116715718 A CN 116715718A CN 202310669587 A CN202310669587 A CN 202310669587A CN 116715718 A CN116715718 A CN 116715718A
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- ursane
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
- C07J71/001—Oxiranes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- C12P33/00—Preparation of steroids
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
本发明属于医药技术领域,公开了一种乌苏烷型三萜类化合物、其微生物转化制备方法及应用,该乌苏烷型三萜类化合物同时具有11β,12β‑三元氧环和28,13β‑内酯。本发明利用微生物的酶体系,以熊果酮酸为底物,特异性的同时在乌苏烷型三萜母核11β,12β位引入三元氧环,在28,13β位引入内酯。此外,对乌苏烷型三萜母核非化学反应活性位点1位、7位和21位7成功地引入了羟基或双键,获得了七个结构新颖的乌苏烷型三萜化合物,并通过体外抗肿瘤细胞试验证实,七个化合物均具有显著的抗肿瘤活性,可以作为抗肿瘤药物的活性成分,具有广泛的医药用途。
Description
技术领域
本发明涉及医药技术领域,涉及一种乌苏烷型三萜类化合物、其微生物转化制备方法及应用,具体涉及一种同时具有11β,12β-三元氧环和28,13β-内酯的新型乌苏烷型三萜的制备方法及其在制备抗肿瘤药物中的应用。
背景技术
占全球药物市场约25%的药物来自植物次生代谢产物,其中数量和种类最多的是萜烯烃类化合物及其含氧衍生物(萜烯类),根据其结构中异戊二烯单元(C5H8)的数量,可分为单萜(C10)、倍半萜(C15)、二萜(C20)、三萜(C30)等。恶性肿瘤是当代最常见的疾病之一,随着人们生活节奏的加快及压力的加大,恶性肿瘤的发病率正逐年上升。因此,积极寻找天然药物在抗肿瘤治疗中具有重要的临床价值。
天然的三萜类化合物有100多种不同的骨架类型。近年来,越来越多的研究者开始关注乌苏烷型三萜在抗菌、抗炎、抗肿瘤、细胞毒性、保护肝脏等方面生物活性的研究。但是,由于三萜分子的高度疏水性大大限制了它们作为药物在临床的有效使用。目前,提高三萜类化合物生物利用度的最常见方法之一是通过化学修饰。现有技术中,由于五环三萜类化合物结构的特殊性,母核缺乏化学反应活性基团。利用有机化学方法对乌苏烷型三萜进行结构修饰的位点主要为结构中原有的两种常见的化学反应活性基团——A环上3位的羟基以及D/E环上28位的羧基。乌苏烷型三萜母核上的大多数C-H键为非化学反应活性位点,采用常规化学反应方法难以对母核结构进行修饰,从而获得母核上具有羟基、羰基等化学活性基团修饰的衍生物。因此,严重限制了乌苏烷型三萜化学结构修饰的反应位点与衍生物的多样性。另一种方法是生物转化,应用条件温和,利用具有高区域和立体选择性的微生物的催化活性来进行。因而成为了复杂天然产物结构修饰与改造的有力工具。本课题组长期从事天然活性成分的微生物转化研究,尤其是天然活性三萜的微生物转化研究。利用多种微生物对天然来源的常见三萜(包括达玛烷型、环阿尔廷烷型、乌苏烷型、齐墩果烷型三萜和羽扇豆烷型等)进行转化研究,发现微生物酶体系能选择性催化三萜母核上的多个非化学反应活性位点,从而获得母核上具有羟基、羰基等化学活性基团的衍生物(Journalofnaturalproducts2021,84:2664-2674;Phytochemistry2021,182:112608;NaturalProductResearch2021,35(16):2685-2690;Phytochemistry2019,166:112076;PlantaMedica2019,85(1):56-61)。微生物转化产物一方面可以获得更强生物活性的衍生物直接用于药物开发,另一方面经微生物转化后母核上新引进的化学活性基团增加了进行化学修饰与改造的位点,解决了三萜类化合物有机化学制备衍生物反应位点少的难题。
然而,与用化学合成的方法对有机化合物进行结构修饰与改造不同。微生物转化的方法是不能从理论上或者发酵方法本身预测或确定哪个微生物具有转化的能力,也不能预测或确定转化产物的化学结构,只有获得转化产物并经结构鉴定后才能确认。已有研究表明,即使是结构相似的底物,经过微生物酶体系进行选择性催化后所获得的转化产物结构差异亦会非常显著。例如白桦脂酸和白桦脂酮酸,这两个化合物的结构差异只在于C-3位的羟基和羰基,但是它们经微生物转化后所获得的衍生物却有显著的差异(Journalofnaturalproducts2021,84:2664-2674;Phytochemistry2021,182:112608)。同时,即使是相同的先导化合物,经不同的微生物酶催化后所获得的衍生物结构也会有显著差异,而这也是微生物转化的多样性特色。因而,微生物转化法作为一种有机化合物结构修饰的方法,其产物结构具有多样性和不确定性的特征,只有制备获得并经结构鉴定后才能最终确定。因此,微生物转化法并不能像化学合成方法那样先设计好化合物,然后再去反向合成。同时,自然界中微生物的种类繁多,挑选到合适的微生物菌株是采用微生物转化法对化合物进行结构修饰与改造的关键。
发明内容
有鉴于此,本发明的目的是提供一种同时具有11β,12β-三元氧环和28,13β-内酯的新型乌苏烷型三萜类化合物或其药学上可成的盐及其制备方法,该新型乌苏烷型三萜类化合物可用于制备抗肿瘤的药物。
本发明提供的新型乌苏烷型三萜类化合物为结构式为式Ⅰ、式Ⅱ、式Ⅲ、式Ⅳ、式Ⅴ、式Ⅵ和式Ⅶ的化合物:
结构式为式Ⅰ—式Ⅶ的化合物均为本发明首次公开的新型乌苏烷型三萜类化合物。
本发明还提供了上述新型乌苏烷型三萜类化合物的制备方法,包括如下步骤:
1)发酵培养微生物,向培养基中加入熊果酮酸,接着进行转化培养,除去菌丝体后得到发酵液,所述微生物为曲霉属的菌株;
2)将所述发酵液经萃取后,蒸干萃取液,得到转化粗提物;
3)转化粗提物经硅胶柱色谱,以二氯甲烷:甲醇作为流动相进行梯度洗脱,收集流份后经HPLC分析合并得5个组分。
4)将所述组分用反相高效液相色谱纯化,得到新型乌苏烷型三萜类化合物。
上述制备方法的合成路线如下:
优选的,步骤1)中,转化培养温度为26℃,摇床转速为160转/分,培养时间为168小时。
优选的,步骤2)中,所述萃取的萃取溶剂为乙酸乙酯。
优选的,步骤3)中,优选梯度洗脱条件为二氯甲烷:甲醇100:1-80:1-60:1-50:1-20:1。
本发明还提供了上述乌苏烷型三萜类化合物在制备抗肿瘤药物中的应用,所述肿瘤为宫颈癌、白血病、神经母细胞瘤、前列腺癌细胞、肝癌、乳腺癌和结肠癌中的一种或几种。
与现有技术相比,本发明利用微生物转化的方法,能特异性的同时在乌苏烷型三萜母核11β,12β位引入三元氧环,在28,13β位引入内酯。所引入的上述基团均具有立体的选择性。此外,还能对乌苏烷型三萜母核非化学反应活性位点1位、7位和21位成功地进行结构修饰,引入了化学反应活性基团羟基或双键,从而获得了结构新颖的乌苏烷型三萜化合物式Ⅰ—式Ⅶ,通过体外抗肿瘤细胞试验证实,化合物Ⅰ—式Ⅶ具有显著的抗肿瘤活性,可以作为抗肿瘤药物的活性成分,也可以作为化学合成的中间体,具有广泛的用途。
具体实施方式
为了进一步说明本发明,下面结合实施例对本发明提供的新型乌苏烷型三萜及其制备方法进行详细描述。
实施例1:结构式为式Ⅰ—式Ⅶ的化合物的制备
本发明采用微生物转化方法,以熊果酮酸为原料,经过发酵、提取、分离等步骤制备本发明化合物。曲霉属(Aspergillus)的菌株可以购自中国科学院微生物菌种保藏管理中心(CGMCC),选用马铃薯培养基,于固体斜面培养基上置4℃冰箱内保存。
以赭曲霉AspergillusochraceusCGMCC3.5324为例,制备结构式为式Ⅰ—式Ⅶ的化合物的过程如下:
1)发酵、转化以及萃取
将赭曲霉AspergillusochraceusCGMCC3.5324接入2个250mL三角瓶(装有100mL马铃薯培养基)中,作为种子液。于摇床上160rpm、26℃下振荡培养12小时后,待菌丝生长处于旺盛期,用无菌移液管吸取1mL的种子液,加入到20个1000mL摇瓶(装有400mL马铃薯培养基)中。振荡培养24小时后,每个摇瓶中加入20mg熊果酮酸(0.2mL,100mg/mL乙醇溶液),共用400mg底物。相同条件下继续转化3天,将发酵液过滤,滤除菌丝体,滤液用等体积的乙酸乙酯萃取3次,萃取液减压浓缩至干,得到转化物粗提物约0.85g。
2)硅胶柱色谱分离
转化粗提物经硅胶柱色谱进行分离。二氯甲烷:甲醇梯度洗脱(100:1-80:1-60:1-50:1-20:1)。收集流份,经HPLC分析后合并,得合并组分A-E。
3)反相高效液相色谱纯化
合并组分B用反相高效液相色谱纯化。制备条件为半制备用色谱柱YMCODSA-5μm,10.0×250mm,乙腈-水(45:55,V/V),流速3.0mL/min,检测波长203nm。得到结构式为式Ⅰ、式Ⅱ和式Ⅲ的转化产物。合并组分C用反相高效液相色谱纯化。制备条件为半制备用色谱柱YMCODSA-5μm,10.0×250mm,乙腈-水(60:40,V/V),流速3.0mL/min,检测波长203nm。得到结构式为式Ⅳ和式Ⅴ的转化产物。合并组分D用反相高效液相色谱纯化。制备条件为半制备用色谱柱YMCODSA-5μm,10.0×250mm,乙腈-水(66:34,V/V),流速3.0mL/min,检测波长203nm。得到结构式为式Ⅵ和式Ⅶ的转化产物。化合物式Ⅰ—式Ⅶ的质谱和波谱学数据如下所示。
化合物Ⅰ:3-羰基-21β-羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯(3-oxo-21β-hydroxyl-11β,12β-epoxyl-urs-28,13β-olide):熔点257–263℃;旋光度 红外光谱主要的吸收峰(KBr):νmax3612,2943,1765,1718,1377,1226,1048cm-1;高分辨质谱m/z529.3173[M+COOH]-(calcd.forC31H45O7,529.3165);核磁共振氢谱和碳谱数据见表1。
化合物Ⅱ:3-羰基-1β,21β-二羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯(3-oxo-1β,21β-dihydroxyl-11β,12β-epoxyl-urs-28,13β-olide):熔点278–280℃;旋光度 红外光谱主要的吸收峰(KBr):νmax3668,3527,2961,1768,1722,1362,1239,1054cm-1;高分辨质谱m/z545.3156[M+COOH]-(calcd.forC31H45O8,545.3114);核磁共振氢谱和碳谱数据见表1。
化合物Ⅲ:3-羰基-21β-羟基-11β,12β-环氧基-1(2)-双键-乌苏烷-28,13β-内酯(3-oxo-21β-hydroxyl-11β,12β-epoxyl-urs-1-ene-28,13β-olide):熔点242–245℃;旋光度 红外光谱主要的吸收峰(KBr):νmax3622,3031,2955,1765,1689,1332,1267,1091cm-1;高分辨质谱m/z527.2993[M+COOH]-(calcd.forC31H43O7,527.3009);核磁共振氢谱和碳谱数据见表1。
化合物Ⅳ:3-羰基-7α,21β-二羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯(3-oxo-7α,21β-dihydroxyl-11β,12β-epoxyl-urs-28,13β-olide):熔点270–274℃;旋光度 红外光谱主要的吸收峰(KBr):νmax3685,3618,2956,1767,1717,1322,1247,1053cm-1;高分辨质谱m/z545.3130[M+COOH]-(calcd.forC31H45O8,545.3114);核磁共振氢谱和碳谱数据见表2。
化合物Ⅴ:3-羰基-7β,21β-二羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯(3-oxo-7β,21β-dihydroxyl-11β,12β-epoxyl-urs-28,13β-olide):熔点268–273℃;旋光度 红外光谱主要的吸收峰(KBr):νmax3647,3586,2959,1765,1713,1351,1232,1055cm-1;高分辨质谱m/z545.3123[M+COOH]-(calcd.forC31H45O8,545.3114);核磁共振氢谱和碳谱数据见表2。
化合物Ⅵ:3,21-二羰基-7β-羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯(3,21-dioxo-7β-hydroxyl-11β,12β-epoxyl-urs-28,13β-olide):熔点247–249℃;旋光度红外光谱主要的吸收峰(KBr):νmax3674,2956,1765,1721,1714,1366,1267,1068cm-1;高分辨质谱m/z543.2968[M+COOH]-(calcd.forC31H43O8,543.2958);核磁共振氢谱和碳谱数据见表2。
化合物Ⅶ:3-羰基-21β-羟基-7-甲基-11β,12β-环氧基-7-双键-26-无甲基乌苏烷-28,13β-内酯(3-oxo-21β-hydroxyl-7-methyl-11β,12β-epoxyl-7-ene-26-norurs-28,13β-olide):熔点282–284℃;旋光度 红外光谱主要的吸收峰(KBr):νmax3598,2971,1767,1716,1380,1233,1048cm-1;高分辨质谱m/z527.3017[M+COOH]-(calcd.forC31H43O7,527.3009);核磁共振氢谱和碳谱数据见表3。
表1.化合物Ⅰ—Ⅲ的核磁氢谱和碳谱数据
表2.化合物Ⅳ—Ⅵ的核磁氢谱和碳谱数据
表3.化合物Ⅶ的核磁氢谱和碳谱数据
以上结果表明,所得化合物式Ⅰ—式Ⅶ结构正确。
实施例2:化合物式Ⅰ—式Ⅶ的抗肿瘤活性
1)实验材料
仪器与试剂:CO2培养箱(JouanIGO150);酶标仪(Bio-TEKELx800);荧光倒置显微镜(OlympusIX51);MTT细胞增殖及细胞毒性检测试剂盒(碧云天生物技术研究所)、RPMI1640培养基(GibcolBRL),Rnase A、胎牛血清、二甲基亚砜(DMSO)、胰蛋白酶(上海生物工程有限公司)。
测试用肿瘤细胞株:Hela细胞(人宫颈癌细胞)、K562细胞(人白血病细胞)、K562/ADR细胞(人白血病耐药细胞)、SH-SY5Y细胞(人神经母细胞瘤细胞)、Du-145(人前列腺癌细胞)、HePG2细胞(人肝癌细胞)、MCF-7细胞(人乳腺癌细胞)、CT26细胞(结肠癌细胞),购于中国医学科学院肿瘤研究所。
测试样品:熊果酮酸及实施例1所合成得到的化合物式Ⅰ—式Ⅶ,纯度在95%以上;同时,选取顺铂为阳性对照药物,各化合物均以DMSO溶解后稀释。
2)实验方法
采用MTT法测定各受试化合物对肿瘤细胞株的半数抑制率IC50值:取对数生长期的肿瘤细胞,用含10%小牛血清的RPMI1640培养液调整细胞浓度为5×105/mL,接种于96孔培养板,药物处理组和细胞对照组加入每孔100μL细胞悬液,每组设3个复孔,空白对照组只加入RPMI1640全培养基,每孔100μL,设3个复孔。将96孔培养板置于37℃、5%CO2培养箱培养24h后,加入不同浓度的受试样品,使终浓度为0.1-100μM,继续培养72h。按MTT法于酶标仪,测定570nm的吸光度(A)值,计算抑制率[抑制率=(1-实验组A值/对照组A值)×100%]。实验重复3次。应用SPSS11.5软件作回归方程,计算各受试样品对肿瘤细胞作用72h的半数抑制浓度(IC50)。
3)实验结果
根据MTT法测试结果,计算测试样品对上述细胞的IC50值,结果如表2所示。
表2.测试样品体外细胞毒活性筛选结果
结果表明,本发明的化合物式Ⅰ—式Ⅶ具有良好的抗肿瘤活性,可以作为抗肿瘤药物的活性成分,应用于抗肿瘤药物的制备。
本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (10)
1.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3-羰基-21β-羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅰ结构:
2.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3-羰基-1β,21β-二羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅱ结构:
3.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3-羰基-21β-羟基-11β,12β-环氧基-1(2)-双键-乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅲ结构:
4.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3-羰基-7α,21β-二羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅳ结构:
5.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3-羰基-7β,21β-二羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅴ结构:
6.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3,21-二羰基-7β-羟基-11β,12β-环氧基-乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅵ结构:
7.一种乌苏烷型三萜类化合物,其特征在于,所述乌苏烷型三萜类化合物为3-羰基-21β-羟基-7-甲基-11β,12β-环氧基-7-双键-26-无甲基乌苏烷-28,13β-内酯,所述乌苏烷型三萜类化合物具有式Ⅶ结构:
8.一种乌苏烷型三萜类化合物的微生物转化制备方法,其特征在于,包括如下步骤:
1)发酵培养微生物,向培养基中加入熊果酮酸,接着进行转化培养,除去菌丝体后得到发酵液,所述微生物为赭曲霉Aspergillus ochraceus CGMCC 3.5324;
2)将所述发酵液经萃取,得到转化物;
3)将所述转化物经过硅胶柱色谱纯化,所述硅胶柱纯化采用二氯甲烷-甲醇两相系统梯度洗脱,收集合并组分;
4)将所述组分用反相高效液相色谱纯化,得到结构式为式Ⅰ—式Ⅶ的乌苏烷型三萜类化合物;
9.根据权利要求8所述的制备方法,其特征在于,所述萃取的萃取溶剂为乙酸乙酯。
10.权利要求1-7任一项所述的乌苏烷型三萜类化合物在制备抗肿瘤药物中的应用,所述肿瘤为宫颈癌、白血病、神经母细胞瘤、前列腺癌细胞、肝癌、乳腺癌和结肠癌中的一种或几种。
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