CN102618448B - 补身烷型倍半萜环己烯酮衍生物、其制备方法及用途 - Google Patents
补身烷型倍半萜环己烯酮衍生物、其制备方法及用途 Download PDFInfo
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Abstract
本发明属于医药化工领域,涉及补身烷(drimane)型倍半萜环己烯酮衍生物、其制备方法及用途。具体地,本发明涉及式I化合物,其中,阿拉伯数字表示标位,R和S分别表示相应标位碳原子的绝对构型。本发明还涉及该化合物的制备方法、含有该化合物的组合物、以及抗肿瘤用途。本发明还涉及能够制备该化合物的产紫青霉BD-1-6。本发明的式I化合物能够有效地杀伤肿瘤细胞或者抑制肿瘤细胞的增殖,具有良好的抗肿瘤活性,具有作为抗肿瘤药物的潜力。
Description
技术领域
本发明属于医药化工领域,涉及补身烷(drimane)型倍半萜环己烯酮衍生物、其制备方法及用途。
背景技术
褶叶苔醇(albicanol,也译作“折叶苔醇”)是一种结构比较简单的补身烷(drimane)型倍半萜类化合物,具有式A或式B所示反式六元双环倍半萜骨架结构。
式A 式B
阿拉伯数字表示标位,R和S分别表示相应碳原子的绝对构型
其中,(+)-褶叶苔醇即(+)-albicanol具有上述式A所示5S,9S,10S的绝对构型(Anilkumar AT,et al,Studies in lipase catalyzedtransesterifications:Synthesis of(+)-albicanol,(+)-albicanol acetateand chiral intermediates useful in the synthesis of drimanes andlabdanes,Tetrahedron,2000,56(12):1899-1903;Toshima H,et al,Total synthesis of(+)-albicanol and(+)-albicanol acetate,Biosci.Biotechnol.Biochem.,2001,65(5):1244-1247),而(-)-褶叶苔醇即(-)-albicanol则具有式B所示的5R,9R,10R绝对构型(J,et al,Total synthesis of the marine sesquiterpene hydroquinones zonaroland isozonarol and the sesquiterpene quinones zonarone andisozonarone,Tetrahed.Lett.,2000,41(29):5469-5473;Laube T,et al,Total synthesis of yahazunol,zonarone and isozonarone,Tetrahedron,2000,58(21):4299-4309)。
褶叶苔醇的C-11位羟基由对苯醌(或其氢化物对苯二酚)骨架基团所取代的倍半萜对苯醌(或对苯二酚)衍生物已有报道。这些化合物中既有(+)-褶叶苔醇(式A)的衍生物(Kawashima K,et al,Structureof tauranin and a note on the“C16-acids”obtained from di-andtriterpenoids,Chem.Pharm Bull.,1964,12(7):796-803;KawashimaK,et al,Structure of tauranin,Tetrahed.Lett.,1964,5(20):1227-1231;Ishi S,et al,First syntheses of(-)-tauranin and antibiotic(-)-BE-40644based on lipase-catalyzed optical resolution of albicanol,Chem.Pharm.Bull.,2009,57(10):1103-1106;Wijeratne EMK,et al,Sesquiterpenequinones and related metabolites from Phyllosticta spinarum,a fungalstrain endophytic in Platycladus orientalis of the sonoran desert,J.Nat.Prod.,71(2):218-222;Li C,et al,A new cytotoxic metabolite from adeep sea derived fungus,Phialocephala sp.,Acta Pharmaceut.Sin.,45(10):1275-1278;Kono K,et al,F-12509A,a new sphingosine kinaseinhibitor,produced by a discomycete,J.Antibiot.,2000,53(5):459-466;Maezawa N,et al,Synthesis of a novel sphingosine kinaseinhibitor(-)-F-12509A and determination of its absolute configuration,Tetrahed.Lett.,2007,48(28):4865-4867),也有(-)-褶叶苔醇(式B)的衍生物(Akita H,et al,Synthesis of decalin type chiral synthonsbased on enzymatic functionalization and their application to thesynthesis of(-)-ambrox and(+)-zonarol,Tetrahed.Asymmetr.,1998,9(10):1789-1799;J,et al,Total synthesis of the marinesesquiterpene hydroquinones zonarol and isozonarol and thesesquiterpene quinones zonarone and isozonarone,Tetrahed.Lett.,2000,41(29):5469-5473;Laube T,et al,Total synthesis of yahazunol,zonarone and isozonarone,Tetrahedron,2000,58(21):4299-4309;Laube T,et al,Total synthesis of two 12-nordrimanes and thepharmacological activie sesquiterpene hydroquinone yhazunol,Tetrahedron,2005,61(5):1141-1148)。
在褶叶苔醇的C-11位羟基由对苯醌骨架基团所取代的倍半萜对苯醌衍生物中,对苯醌环上的一个双键被氧化成环氧三元环、同时一个羰基被还原成羟基的环己烯酮衍生物也已有报道(Fujimoto H,et al,Immunomodulatory constituents from an ascomycete,Eupenicilliumcrustaceum,and revised absolute structure of macrophorin D,J.Nat.Prod.,64(9):1234-1237;Sassa T,et al,New terpene-linkedcyclohexenone epoxides,macrophorin A,B,and C,produced by thefungus caused Macrophoma fruit rot of apple,Agric.Biol.Chem.,1983,47(1):187-189;Ayer WA,et al,Three piperazinediones and adrimane diterpenoid from Penicillium brevi-compactum,Phytochem.,1990,29(5):1661-1665;Sassa T,et al,Macrophorin D,a newself-growth inhibitor of the causal fungus of Macrophoma fruit rot ofapple,Agric.Biol.Chem.,1984,48(7):1923-1925)。但与本发明的补身烷型倍半萜环己烯酮衍生物直接相关的、在环己烯酮环上进一步连接六元内酯环形成桥联双环结构的化合物尚未见文献报道。
另外,在褶叶苔醇的C-11位羟基由对苯醌骨架基团所取代的倍半萜对苯醌衍生物中,对苯醌环上的一个双键被氧化成环氧三元环、结构与本发明的化合物相近的环己烯二酮类衍生物也有报道(FujimotoH,et al,Immunomodulatory constituents from an ascomycete,Eupenicillium crustaceum,and revised absolute structure ofmacrophorin D,J.Nat.Prod.,64(9):1234-1237;Wang H,et al,Aflavinines and other antiinsectan metabolites from the ascostromataof Eupenicillium crustaceum and related species,Appl.Environ.Microbiol.,1995,61(12):4429-4435)。但所有该类化合物的结构均不同于本发明的补身烷型倍半萜环己烯酮衍生物。譬如,在已知的该类化合物中Macrophorin F(Sassa T,et al,Isolation and identification ofnew antifungal macrophorines E,F,and G as malonyl meroterpenesfrom Botryosphaeria berengeriana,Biosci.Biotechnol,Biochem.,1998,62(11):2260-2262)具有与本发明的一个补身烷型倍半萜环己烯酮衍生物相同的倍半萜环己烯酮碳骨架,但其立体结构未确切测定,而且整个分子为丙二酸酯衍生物,化学结构与本发明的化合物不同。又如,文献(Lin X,et al,A new cytotoxic sesquiterpene quinone produced byPenicillium sp.F00120 isolated from a deep sea sediment sample,Mar.Drugs,2012,10(1):106-115)最近报道了命名为penicilliumin A的化合物。根据该文献记载,该化合物的平面结构与本发明的一个补身烷型倍半萜环己烯酮衍生物相同,但光学活性却与本发明的化合物不同。该文献仅测定了penicilliumin A中补身烷型倍半萜骨架的相对构型,而环己烯酮环上的不对称碳原子的相对构型以及整个分子的绝对构型却并未能予以测定。所述补身烷型倍半萜骨架即使是相对构型相同也仍有光学活性不同的两种完全相反的绝对构型,而环己烯酮环上的不对称碳原子也同样有完全相反的两种绝对构型。因此,相对构型相同的补身烷型倍半萜骨架与具有不对称碳原子的环己烯酮片段组合后,可形成4种具有不同绝对构型和光学活性的分属于2对对映异构体的不同化合物分子结构。因此,penicilliumin A实际上是分子真实结构不明确的化合物。本发明的化合物不仅通过CD谱测定和基于量子化学的ECD谱计算,确切测定了整个分子的绝对构型,而且还显示出与penicilliumin A不同的旋光活性,因此是与penicilliumin A不同的补身烷型倍半萜环己烯酮衍生物。
目前已知,与本发明的补身烷型倍半萜环己烯酮衍生物结构相近或相关的有些化合物具有一定生物活性。譬如,macrophorin F对部分植物病原真菌显示有抗真菌活性(Sassa T,et al,Isolation andidentification of new antifungal macrophorines E,F,and G as malonylmeroterpenes from Botryosphaeria berengeriana,Biosci.Biotechnol,Biochem.,1998,62(11):2260-2262);penicilliumin A对肿瘤细胞A375、B16、Hela等有一定的细胞毒活性(Lin X,et al,A new cytotoxicsesquiterpene quinone produced by Penicillium sp.F00120 isolatedfrom a deep sea sediment sample,Mar.Drugs,2012,10(1):106-115)。又如,具有环己烯二酮结构的有些macrophorin类化合物有免疫抑制活性(Fujimoto H,et al,Immunomodulatory constituents from anascomycete,Eupenicillium crustaceum,and revised absolute structureof macrophorin D,J.Nat.Prod.,64(9):1234-1237)。
发明内容
本发明人通过创造性的劳动和不懈的努力,得到了产紫青霉(Penicillium purpurogenum)BD-1-6,并提取分离得到了补身烷型倍半萜环己烯酮衍生物(式I化合物)。本发明人惊奇地发现,式I化合物能够有效地杀伤肿瘤细胞或者抑制肿瘤细胞的增殖,具有良好的抗肿瘤活性,故而具有作为抗肿瘤药物的潜力。由此提供了下述发明:
本发明的一个方面涉及产紫青霉BD-1-6,其保藏编号为CGMCCNo.5525,保藏日期为2011年12月6日,保藏地点为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)。
该菌株是将从天津塘沽驴驹河渤海湾潮间带海泥样品中分离并经分类学研究鉴定为产紫青霉(Penicillium purpurogenum)的青霉菌属的真菌野生株产紫青霉G59经硫酸二乙酯诱变所获得的突变株BD-1-6。其具有如下微生物菌学特征:
菌落在查氏培养基上25℃培养12天直径达17-30mm,平坦、近于平坦或有几道同心环纹;质地绒状或兼絮状;分生孢子面暗灰绿色或暗绿色,近于橄榄柠檬色、褐橄榄色、或微暗橄榄绿色;菌丝体橘黄色、黄色或橙红色;反面暗红色、橘红色或紫红色;分生孢子梗发生于基质,发生气生菌丝者少,孢梗茎(70-)100-250(-300)×2.5-3.2(-3.5)μm,壁平滑,顶端通常膨大;帚状枝双轮生,偶有三轮生或单轮生,彼此紧贴而近于平行;梗基每轮4-8个,9.0-13(-14)×2.5-3.0μm;瓶梗每轮4-6个,9.5-13×(1.8-)2.0-2.4μm,披针形,梗茎明显;分生孢子呈椭圆形,充分成熟时部分呈近球形,2.8-3.5(-4.0)×2.2-3.0μm,壁平滑或稍粗糙;分生孢子链较疏松,叉开或近于圆柱形。
本发明的另一方面涉及式I化合物,
式I
其中,
阿拉伯数字表示标位,R和S分别表示相应标位碳原子的绝对构型;其结构特征为:分子结构中具有绝对构型为5S9S10S的与(+)-褶叶苔醇相同的补身烷型倍半萜碳骨架,并在该碳骨架的碳11位通过碳-碳单键连接了由环己烯二酮衍生的取代基团。
本发明的再一方面涉及式I化合物的制备方法,包括如下步骤:
将本发明的产紫青霉BD-1-6进行发酵培养,获得含有式I化合物的发酵物,将发酵物进行分离纯化,得到式I化合物;
具体地,所述分离纯化包括液液萃取、柱层析、薄层层析、以及高效液相层析。
根据本发明所述的制备方法,包括如下步骤:
1)将本发明的产紫青霉BD-1-6进行发酵培养,获得发酵液;
2)将发酵液过滤,滤取菌体并悬浮于50%-90%(v/v)的丙酮水溶液中,超声破碎菌体细胞,室温浸提过滤,滤液经减压浓缩至不含丙酮后用乙酸乙酯萃取,得乙酸乙酯萃取物;
3)将乙酸乙酯萃取物通过硅胶柱层析(二氯甲烷-丙酮体积比1∶0→0∶1洗脱)分为粗组分,再经对所得粗组分的第二次硅胶柱层析(环己烷-丙酮体积比100∶0→60∶40洗脱)或Sephadex LH-20柱层析(二氯甲烷-甲醇体积比1∶1洗脱)分离,制得含有所述化合物的柱层析组分;
4)将含有所述化合物的柱层析组分用HPLC分离(C-18柱,甲醇-水体积分数80∶20或77∶23洗脱),制得所述化合物。
在本发明的一个实施方案中,所述的制备方法,其中,步骤2)中的丙酮水溶液为丙酮占70%-90%(v/v)的丙酮水溶液;具体地,为丙酮占75%-85%(v/v)的丙酮水溶液,例如丙酮占75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、或85%(v/v)的丙酮水溶液。
本发明的再一方面涉及一种组合物,其含有本发明的式I化合物;可选地,所述组合物还含有一种或多种药用载体或赋形剂。具体地,所述组合物为药物组合物。所述组合物或药物组合物能够用于抗肿瘤或抑制肿瘤细胞增殖或杀伤肿瘤细胞。
本发明的式I化合物可与各种药物上可接受的载体、赋形剂或辅料配伍制成抗肿瘤药物,用于肿瘤的治疗。
本发明化合物可单独或以药物组合物的形式给药。给药途径可以是口服、非肠道或局部给药。药物组合物可根据给药途径配成各种适宜的剂型。
本发明化合物的药物组合物可以以下面的任意方式施用:口服,喷雾吸入,直肠用药,鼻腔用药,颊部用药,局部用药,非肠道用药,如皮下,静脉,肌内,腹膜内,鞘内,心室内,胸骨内和颅内注射或输入,或借助一种外植储器用药。其中优选口服、腹膜内或静脉内给药方式。
当口服用药时,本发明化合物可制成任意口服可接受的制剂形式,包括但不限于片剂、胶囊、水溶液或水悬浮液。其中,片剂使用的载体一般包括乳糖和玉米淀粉,另外也可加入润滑剂如硬脂酸镁。胶囊制剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。水悬浮液制剂则通常是将活性成分与适宜的乳化剂和悬浮剂混合使用。任选地,以上口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
当皮肤局部施用时,本发明化合物可制成适当的软膏、洗剂或霜剂制剂形式,其中将活性成分悬浮或溶解于一种或多种载体中。软膏制剂可使用的载体包括但不限于:矿物油、液体凡士林、白凡士林、丙二醇、聚氧化乙烯、聚氧化丙烯、乳化蜡和水;洗剂或霜剂可使用的载体包括但不限于:矿物油、脱水山梨糖醇单硬脂酸酯、吐温60、十六烷酯蜡、十六碳烯芳醇、2-辛基十二烷醇、苄醇和水。
本发明化合物还可以无菌注射制剂形式用药,包括无菌注射水或油悬浮液或无菌注射溶液。其中,可使用的载体和溶剂包括水、林格氏溶液和等渗氯化钠溶液。另外,灭菌的非挥发油也可用作溶剂或悬浮介质,如单甘油酯或二甘油酯。
本发明的再一方面涉及本发明的式I化合物或者本发明任一项所述的组合物在制备杀伤肿瘤细胞或抑制肿瘤细胞增殖的药物或试剂中的用途;具体地,所述肿瘤细胞为白血病细胞、宫颈癌细胞、胃癌细胞、乳腺癌细胞、肺癌细胞、肝癌细胞、或肠癌细胞;具体地,所述白血病细胞为慢性髓细胞性白血病细胞或急性早幼粒细胞白血病细胞;具体地,所述肿瘤细胞为人慢性髓细胞性白血病K562细胞、人急性早幼粒细胞白血病HL-60细胞、人子宫颈癌HeLa细胞、人胃癌BGC-823细胞、或人乳腺癌MCF-7细胞。
本发明的再一方面涉及一种在体内或体外杀伤肿瘤细胞或者抑制肿瘤细胞增殖的方法,包括使用有效量的式I化合物或者本发明任一项所述的组合物的步骤;具体地,所述肿瘤细胞为白血病细胞、宫颈癌细胞、胃癌细胞、乳腺癌细胞、肺癌细胞、肝癌细胞、或肠癌细胞;具体地,所述白血病细胞为慢性髓细胞性白血病细胞或急性早幼粒细胞白血病细胞;具体地,所述肿瘤细胞为人慢性髓细胞性白血病K562细胞、人急性早幼粒细胞白血病HL-60细胞、人子宫颈癌HeLa细胞、人胃癌BGC-823细胞、或人乳腺癌MCF-7细胞。
本发明采用MTT法,测试了式I化合物对人慢性髓细胞性白血病K562细胞、人急性早幼粒细胞白血病HL-60细胞、人子宫颈癌HeLa细胞、人胃癌BGC-823细胞、人乳腺癌MCF-7细胞增殖的抑制作用。经实验证实,式I化合物可显著抑制上述人癌细胞的(体外)增殖,因而具有抗肿瘤作用。
本发明的再一方面涉及本发明的式I化合物或者本发明任一项所述的组合物在制备抗肿瘤药物中的用途;具体地,所述肿瘤为白血病、宫颈癌、胃癌、乳腺癌、肺癌、肝癌、或肠癌;具体地,所述白血病为慢性髓细胞性白血病、急性早幼粒细胞白血病。
本发明的再一方面涉及一种治疗和/或预防和/或辅助治疗癌症的方法,包括给予受试者有效量的本发明的式I化合物或者本发明任一项所述的组合物的步骤;所述癌症为白血病、宫颈癌、胃癌、乳腺癌、肺癌、肝癌、或肠癌;具体地,所述白血病为慢性髓细胞性白血病、急性早幼粒细胞白血病。
需要指出的是,本发明化合物使用剂量和使用方法取决于诸多因素,包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医师的主观判断。优选的使用剂量介于0.01-100mg/kg体重/天。
本发明的再一方面涉及本发明的产紫青霉BD-1-6在制备本发明的式I化合物中的用途。
本发明中,
术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
术语“受试者”可以指患者或者其它接受式I化合物或者本发明任一项所述的药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物,特别是哺乳动物,例如人、狗、猴、牛、马等。
术语“疾病和/或病症”是指所述受试者的一种身体状态,该身体状态与本发明所述疾病和/或病症有关。
在本发明中,如果没有特别说明,所述有机溶液例如丙酮溶液(水溶液)或者乙醇溶液(水溶液)或者乙酸乙酯溶液(水溶液)的浓度均指体积浓度(v/v)。
发明的有益效果
本发明的式I化合物能够有效地杀伤肿瘤细胞或者抑制肿瘤细胞的增殖,具有良好的抗肿瘤活性,具有作为抗肿瘤药物的潜力。
涉及保藏的生物材料
产紫青霉BD-1-6已于2011年12月6日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101;保藏编号为CGMCC No.5525;分类命名为Penicillium purpurogenum。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
在下面的实施例中,
以下称为化合物1的本发明的化合物,经光学活性相关比旋光度([α]D)和圆二色散(CD)谱测定,质谱(MS)、高分辨质谱(HR-MS)、紫外(UV)光谱、红外(IR)光谱、核磁共振(NMR)谱解析,计算ECD与实测CD谱对比分析等,鉴定其具有如下式I所示碳11位取代基为R1的化学结构,其绝对构型为5S9S10S6′S;
以下称为化合物2的本发明的化合物,经光学活性相关比旋光度([α]D)和圆二色散(CD)谱测定,质谱(MS)、高分辨质谱(HR-MS)、紫外(UV)光谱、红外(IR)光谱、核磁共振(NMR)谱解析,计算ECD与实测CD谱对比分析等,鉴定其具有如下式I所示碳11位取代基为R2的化学结构,其绝对构型为5S9S10S4′R5′R6′S。
式I
阿拉伯数字表示标位,R和S表示相应碳原子的绝对构型
在下面实施例的结构研究中,熔点(mp)用北京天地宇科技有限责任公司X-4型精密显微熔点测定仪,温度未校正。比旋光度([α]D)用美国Rudolph Research公司Autopol II型旋光仪测定。电喷雾离子化质谱(ESI-MS)用美国AB公司API 3000型液相色谱-质谱联用仪、高分辨电喷雾离子化质谱(HR-ESI-MS)用美国Agilent公司6520Q-TOF液相色谱-质谱联用仪测定。紫外(UV)光谱用澳大利亚GBC公司Cintra 20型紫外可见分光光度仪、红外(IR)光谱用美国Bruker公司Tensor-27型红外光谱仪测定。核磁共振(NMR)用日本JEOL公司JNM-ECA-400型超导核磁共振仪(400MHz1H-NMR,100MHz13C-NMR)测定。圆二色(CD)谱用法国Biologic Science公司MOS 450型圆二色谱仪测定。电子圆二色(ECD)谱的理论计算工作全部采用Gaussian 09软件完成。计算采用密度泛函理论(DFT)和含时密度泛函理论(TD-DFT)方法进行。首先采用B3LYP/6-31G方法对目标化合物的所有可能构型的构象进行了优化,再对优化所得的结构进行了ECD谱计算(TDDFT,B3LYP/6-311++G(2d,p)),溶剂化效应采用PCM模型。
实施例1:微生物发酵培养与化合物1和化合物2的制备
1.发酵培养与发酵物的提取处理
1.1)生产菌株
本实施例中用于发酵生产化合物1和2的产素菌是保藏在中国微生物菌种保藏管理委员会普通微生物中心的产紫青霉(Penicilliumpurpurogenum)BD-1-6株,保藏编号为5525即CGMCC No.5525。
1.2)发酵培养
从4℃冰箱保存的产紫青霉(Penicillium purpurogenum)BD-1-6的PDA培养基(组成:葡萄糖2%、琼脂2%、NaCl 1.5%,用20%土豆的水煮液配制)试管斜面,用接种环在无菌条件下刮取适量孢子,划线接种于新配制的PDA固体培养基平板上,28℃培养箱中活化培养4天。从活化培养4天的试管斜面,用接种环刮取孢子适量,接种于装有200ml液体发酵培养基(组成:葡萄糖2%、麦芽糖1%、甘露醇2%、谷氨酸1%、蛋白胨0.5%、酵母浸粉0.3%)的1个500ml三角烧瓶中,28℃、200rpm摇床培养48h,获得一级种子培养液约200ml。将该一级种子培养液按5%接种量接种到7个分别装有200ml液体发酵培养基(组成:葡萄糖2%、麦芽糖1%、甘露醇2%、谷氨酸1%、蛋白胨0.5%、酵母浸粉0.3%)的500ml三角烧瓶中,28℃、200rpm摇床培养48h,获得二级种子培养液约1400ml。将所得二级种子培养液再按5%接种量接种到100个分别装有200ml液体发酵培养基(组成:葡萄糖2%、麦芽糖1%、甘露醇2%、谷氨酸1%、蛋白胨0.5%、酵母浸粉0.3%)的500ml三角烧瓶中,28℃、200rpm摇床发酵12天,获得发酵液共计约20L。
2.提取处理与乙酸乙酯浸膏的制备
将全部发酵液约20L用4层纱布过滤,分为滤液和菌体。菌体用体积分数为80%的丙酮水溶液约10L混悬,超声2h,室温浸提12h后用4层纱布过滤,重复操作3次,合并滤液,减压浓缩至不含丙酮后,残余水混悬液约6L用等体积乙酸乙酯萃取3次,得菌体提取物的乙酸乙酯萃取浸膏17g。
3.乙酸乙酯浸膏的柱层析分离与含有化合物1和化合物2的柱层析组分的制备
将上述乙酸乙酯萃取物浸膏17g用适量氯仿-甲醇(体积比1∶1)溶解,加40g硅胶(100~200目)拌样,干燥后研磨均匀并添加到装有100~200目硅胶300g的常压玻璃柱上(柱床:4.5cm×40cm),以二氯甲烷-丙酮(体积比1∶0→0∶1)为洗脱剂,通过逐步增加洗脱剂中丙酮的体积分数增大洗脱剂极性来进行梯度洗脱柱层析分离,各接约300ml为每个流份,根据薄层层析检测结果,合并相应洗脱流份,浓缩获得6个组分:Fr-1(4.6g,二氯甲烷洗脱部分),Fr-2(2.6g,二氯甲烷洗脱部分)、Fr-3(1.1g,二氯甲烷-丙酮体积比95∶5洗脱部分)、Fr-4(3.1g,二氯甲烷-丙酮体积比95∶5洗脱部分)、Fr-5(1.7g,二氯甲烷-丙酮体积比95∶5→80∶20洗脱部分)、Fr-6(2g,丙酮洗脱部分)。
Fr-3(1.1g)用适量氯仿-甲醇(体积比1∶1)溶解并加2g硅胶(100~200目)拌样,干燥后研磨均匀并添加到装有100~200目硅胶25g的常压玻璃柱上(柱床:1.5cm×35cm),以环己烷-丙酮(体积比100∶0→60∶40)为洗脱剂,通过逐步增加洗脱剂中丙酮的体积分数增大洗脱剂极性来进行梯度洗脱柱层析分离,根据薄层层析检测结果,收集合并相应洗脱液浓缩,得3个组分:Fr-3-1(480mg,环己烷洗脱部分)、Fr-3-2(121mg,环己烷-丙酮体积比95∶5→85∶15洗脱部分)、Fr-3-3(310mg,环己烷-丙酮体积比80∶20→75∶25→60∶40洗脱部分)。其中,Fr-3-2(121mg)为含有化合物1的柱层析组分。
Fr-5(1.7g)用适量二氯甲烷-甲醇(体积比1∶1)溶解,湿法上样,添加到用二氯甲烷-甲醇(体积比1∶1)预装的Sephadex LH-20柱上(柱床:2.0cm×130cm),以二氯甲烷-甲醇(体积比1∶1)为洗脱剂进行层析分离,各接5ml为每个层析流份,根据薄层检测结果收集合并和浓缩,依次按洗脱先后顺序得4个组分:Fr-5-1(590mg)、Fr-5-2(760mg)、Fr-5-3(113mg)、Fr-5-4(230mg)。其中,Fr-5-3(113mg)为含有化合物2的柱层析组分。
4.化合物1和化合物2的分离制备
4.1)化合物1的HPLC分离精制
将含有化合物1的组分Fr-3-2(121mg)用适量甲醇溶解并经0.22μm滤膜过滤后,用Waters 600型HPLC系统(Waters 600控制器、Waters 600泵、Waters 2414 RI检测器、Waters 2996 PDA检测器、Empower色谱工作站),利用Senshu pak C-18半制备柱(8mm×250mm)进行室温HPLC分离(以甲醇-水体积分数80∶20为流动相,流速2.0ml/min,样品甲醇溶液体积1.6ml,浓度62.5mg/ml,每次进样100μl,检测波长为254nm),制得化合物1(6.2mg,保留时间tR=20.1min)。
4.2)化合物2的HPLC分离精制
将含有化合物2的组分Fr-5-3(113mg)用适量甲醇溶解并经0.22μm滤膜过滤后,用Waters 600型HPLC系统(Waters 600控制器、Waters 600泵、Waters 2414 RI检测器、Waters 2996 PDA检测器、Empower色谱工作站),利用Senshu pak C-18半制备柱(8mm×250mm)进行室温HPLC分离(以甲醇-水体积分数77∶23为流动相,流速2.0ml/min,样品甲醇溶液体积约1.5ml,样品浓度75.3mg/ml,每次进样100μl,检测波长为254nm),制得化合物2(21mg,保留时间tR=21.0min)。
5.化合物1和化合物2的理化常数与波谱数据
化合物1为无色油状物,易溶于甲醇,可溶于丙酮、氯仿,不溶于水,[α]D 20-13.7(c 0.1,CHCl3),[α]D 20-9.3(c 0.5,MeOH)。正离子ESI-MSm/z:383[M+Na]+;负离子ESI-MS m/z:359[M-H]-,395[M+Cl]-;正离子HR-ESI-MS m/z:实测值383.2206[M+Na]+,计算值383.2198(C22H32O4Na[M+Na]+)。UVλmax nm(logε)in MeOH:236(4.0)。IR vmaxcm-1:3392,2941,1682,1457,1443,1388,1368,1243,1208,1092,1036,898。CD(0.27mM,MeOH)Δε(nm):0(456),-0.54(361),0(320),+1.14(260),0(247.6),-1.18(238),0(226),+3.73(209)。CDλmax nm(mdeg)inMeOH at 100μg/ml:456(0),361(-0.5001),320(0),260(+1.0413),247.6(0),238(-1.0774),226(0),209(+3.4168)。1H-NMR(400MHz,CDCl3)δ:6.82(1H,br s,H-2′),4.75(1H,br s,Ha-12),4.54(1H,br d,J=17.2Hz,Ha-7′),4.44(1H,br d,J=17.2Hz,Hb-7′),4.25(1H,br s,Hb-12),3.12(1H,d,J=16.0Hz,Ha-5′),2.97(1H,d,J=16.0Hz,Hb-5′),2.28(1H,ddd,J=12.8,4.1,2.4Hz,He-7),1.90(1H,dd,J=12.8,5.4Hz,Ha-11),1.89(1H,td,J=12.8,4.8Hz,Ha-7),1.86(1H,dd,J=12.8,4.7Hz,Hb-11),1.77(1H,dd,J=5.4,4.7Hz,H-9),1.74(1H,dm,J=12.8Hz,He-6),1.63(1H,dt,J=12.6,2.5Hz,He-1),1.45-1.54(2H,m,H2-2),1.37(br d,J=12.4Hz,He-3),1.25(1H,qd,J=12.8,4.1Hz,Ha-6),1.18(1H,td,J=12.4,4.8Hz,Ha-3),1.13(1H,dd,J=12.8,2.4Hz,H-5),1.07(1H,td,J=12.6,4.8Hz,Ha-1),0.85(3H,s,H3-13),0.75(3H,s,H3-14),0.57(3H,s,H3-15)。13C-NMR(100MHz,CDCl3)δ:201.2(C-1′),196.6(C-4′),150.9(C-3′),148.9(C-8),134.4(C-2′),107.1(C-12),77.4(C-6′),59.6(C-7′),55.6(C-5),53.1(C-5′),50.5(C-9),42.0(C-3),39.9(C-10),38.7(C-1),38.1(C-7),34.8(C-11),33.7(C-4),33.5(C-13),24.6(C-6),21.6(C-14),19.3(C-2),15.0(C-15)。
对符合上述MS、UV、IR、NMR数据的绝对构型为5S9S10S6′S、5S9S10S6′R、5R9R10R6′S、5R9R10R6′R的四种不同结构化合物进行了ECD计算,结果表明,只有绝对构型为5S9S10S6′S的本发明化合物1的计算ECD图谱与上述实测CD谱吻合,因而其绝对构型得以确定。
化合物2为白色结晶型粉末(甲醇),mp 122-123℃,易溶于甲醇,可溶于丙酮、氯仿,不溶于水,[α]D 20+21.0(c 1.0,MeOH)。正离子ESI-MSm/z:419[M+H]+,441[M+Na]+;负离子ESI-MS m/z:531[M+CF3CO2]-。正离子HR-ESI-MS m/z:实测值419.2429[M+H]+,计算值419.2434(C24H35O6[M+H]+);UVλmax nm(logε)in MeOH:234(3.87)。IR vmaxcm-1:3405,2934,1733,1694,1459,1442,1388,1365,1277,1246,1229,1114,1067,1044,1021,889。CD(0.12mM,MeOH)Δε(nm):0(401),+1.41(338),0(295),-9.06(247),0(228.7)。CDλmax nm(mdeg)in MeOHat 50μg/ml:401(0),338(+0.5560),295(0),247(-3.5745),228.7(0)。1H-NMR(400MHz,acetone-d6)δ:6.12(1H,br s,H-2′),4.90(1H,br s,Ha-12),4.79(1H,br s,Hb-12),4.41(2H,br s,H2-7′),3.96(1H,s,H-5′),3.05(1H,d,J=17.2Hz,Ha-8′),2.91(1H,d,J=17.2Hz,Hb-8′),2.36(1H,ddd,J=12.9,3.9,2.5Hz,He-7),2.15-2.23(1H,AB type,Ha-11),2.11(1H,td,J=12.9,4.8Hz,Ha-7),1.95-2.03(2H,AB type,H-9,Hb-11),1.73(1H,br dm,J=13.7Hz,He-1),1.73(1H,dm,J=12.9Hz,He-6),1.58(1H,qt,J=13.7,3.4Hz,Ha-2),1.45(1H,dquint,J=13.7,3.4Hz,He-2),1.36(1H,dt,J=13.7,3.4,He-3),1.31(1H,qd,J=12.9,3.9Hz,Ha-6),1.17(1H,dd,J=12.9,2.5Hz,H-5),1.14(1H,td,J=13.7,3.4Hz,Ha-3),1.12(1H,td,J=13.7,3.4Hz,Ha-1),0.84(3H,s,H3-13),0.78(3H,s,H3-14),0.70(3H,s,H3-15)。13C-NMR(100MHz,acetone-d6)δ:192.1(C-1′),167.6(C-9′),164.3(C-3′),149.9(C-8),120.2(C-2′),107.8(C-12),85.1(C-6′),74.5(C-5′),71.6(C-4′),60.5(C-7′),56.0(C-5),50.1(C-9),43.1(C-8′),42.7(C-3),40.8(C-10),39.2(C-1),38.6(C-7),34.0(C-4),33.6(C-13),25.0(C-6),22.6(C-11),21.8(C-14),19.7(C-2),15.0(C-15)。
对符合上述MS、UV、IR、NMR数据绝对构型为5S9S10S4′R5′R6′S、5S9S10S4′S5′S6′R、5R9R10R4′R5′R6′S、5R9R10R4′S5′S6′R的四种不同结构化合物进行了ECD计算,结果表明,只有5S9S10S4′R5′R6′S绝对构型的本发明化合物2的计算ECD图谱与上述实测CD谱吻合,因而其绝对构型得以确定。
实施例2:化合物1和化合物2的抗肿瘤活性测试
1.实验材料
1)被测样品溶液的配制
受试样品为上述实施例1中分离精制的纯品化合物1和化合物2。5-氟尿嘧啶(阿拉丁试剂有限公司,批号5402)和三水多烯紫杉醇(北京奇米沃科技有限公司,批号20110326)用作阳性对照样品。精密称取适量样品,分别用DMSO配成所需浓度的溶液,供测试活性。
2)细胞系及细胞的继代培养
活性测试采用人慢性髓细胞性白血病K562细胞系、人急性早幼粒细胞白血病HL-60细胞系、人宫颈癌HeLa细胞系、人胃癌BGC-823系、人乳腺癌MCF-7系(上述细胞均可以商购获得,例如从上海雅吉生物科技有限公司、上海锐聪实验室设备有限公司、上海麦莎生物科技有限公司等购买)。
K562、HL-60、HeLa、BGC-823、MCF-7细胞分别用含10%胎牛血清以及青霉素和链霉素各100μg/ml的RPMI-1640培养基常规传代,并于37℃通入5%二氧化碳的细胞培养箱中培养维护。
2.活性测试方法
样品的抗肿瘤活性采用MTT法结合细胞形态学检测的方法进行测试。分别取对数生长期的K562、HL-60、HeLa、BGC-823、MCF-7细胞,用新鲜RPMI-1640培养基配制成细胞密度为2×104个/ml的细胞悬液,接种于96孔板中,每孔200μl。接种后,悬浮细胞K562和HL-60于37℃培养2h,而贴壁细胞HeLa、BGC-823、MCF-7则于37℃培养12h。之后,样品组每孔加样品液各2μl,空白对照组则每孔加DMSO各2μl,继续于37℃培养48h。培养结束后在光学显微镜下观察细胞形态变化,判断有无细胞凋亡或细胞坏死的形态特征,必要时拍照。每孔加预冷的5mg/ml的MTT溶液(用PBS溶液配制)各20μl,37℃孵育4h后,于4℃、2000rpm离心10min,吸去上清液,每孔各加150μl DMSO,置于酶标仪上充分振荡使MTT紫色产物完全溶解,测量每孔570nm处的OD值。实验中样品和空白对照组均分别设三个平行孔,取OD平均值,按IR%=(OD空白-OD样品)/OD空白×100%公式,计算样品对受试癌细胞的抑制率(IR%)。样品对受试癌细胞的半数抑制浓度(IC50)则由不同浓度下的抑制率求算。
3.实验结果
1)MTT法测试结果
在MTT法测试中,化合物1和化合物2对受试K562、HL-60、HeLa、BGC-823、MCF-7细胞均显示出抑制细胞增殖的抗肿瘤活性,测试结果如下面的表1和表2中所示。
表1:化合物1和化合物2对人癌细胞的抑制效果
表2:化合物1和化合物2对人癌细胞的抑制效果
2)细胞形态学检测结果
在光学倒置显微镜下观察到,上述受试癌细胞分别经100μg/ml化合物1和化合物2处理48h后,视野中部分细胞呈胞体膨大、细胞质凝集等坏死性细胞形态,表明化合物1和化合物2主要通过对受试癌细胞的杀伤性细胞毒活性发挥其抑制癌细胞增殖的抗肿瘤作用。
4.结论
化合物1和化合物2对人慢性髓细胞性白血病K562细胞、人急性早幼粒细胞白血病HL-60细胞、人子宫颈癌HeLa细胞、人胃癌BGC-823细胞、人乳腺癌MCF-7细胞均具有抗肿瘤活性,且主要通过杀伤性细胞毒作用发挥其抑制癌细胞增殖的抗肿瘤作用,因此化合物1和化合物2可用作为肿瘤细胞增殖抑制剂或抗肿瘤剂。
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解,根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
Claims (22)
1.产紫青霉BD-1-6,其保藏编号为CGMCC No.5525,保藏日期为2011年12月6日,保藏地点为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)。
3.权利要求2所述的式I化合物的制备方法,包括如下步骤:
将权利要求1所述的产紫青霉BD-1-6进行发酵培养,获得含有式I化合物的发酵物,将发酵物进行分离纯化,得到式I化合物。
4.根据权利要求3所述的制备方法,其中,所述分离纯化包括液液萃取、柱层析、薄层层析以及高效液相层析。
5.根据权利要求3所述的制备方法,包括如下步骤:
1)将权利要求1所述的产紫青霉BD-1-6进行发酵培养,获得发酵液;
2)将发酵液过滤,滤取菌体并悬浮于体积浓度为50%-90%的丙酮水溶液中,超声破碎菌体细胞,室温浸提过滤,滤液经减压浓缩至不含丙酮后用乙酸乙酯萃取,得乙酸乙酯萃取物;
3)将乙酸乙酯萃取物通过硅胶柱层析分为粗组分,再经对所得粗组分的第二次硅胶柱层析或Sephadex LH-20柱层析分离,制得含有所述化合物的柱层析组分;
4)将含有所述化合物的柱层析组分用HPLC分离,制得所述化合物。
6.根据权利要求5所述的制备方法,其中,步骤2)中所用丙酮水溶液为体积浓度70%-90%的丙酮水溶液。
7.根据权利要求5所述的制备方法,其中,步骤2)中所用丙酮水溶液为体积浓度75%-85%的丙酮水溶液。
8.根据权利要求5所述的制备方法,其中,步骤3)中所述硅胶柱层析用二氯甲烷–丙酮体积比1:0→0:1洗脱。
9.根据权利要求5所述的制备方法,其中,步骤3)中所述第二次硅胶柱层析用环己烷–丙酮体积比100:0→60:40洗脱。
10.根据权利要求5所述的制备方法,其中,步骤3)中所述Sephadex LH-20柱层析用二氯甲烷–甲醇体积比1:1洗脱。
11.根据权利要求5所述的制备方法,其中,步骤4)中所述HPLC分离用C-18柱,甲醇–水体积分数80:20或77:23洗脱。
12.一种组合物,其含有权利要求2所述的式I化合物。
13.根据权利要求12所述的组合物,其还含有一种或多种药用载体或赋形剂。
14.权利要求2所述的式I化合物或者权利要求12或13所述的组合物在制备杀伤肿瘤细胞或抑制肿瘤细胞增殖的药物或试剂中的用途,其中,所述肿瘤细胞为白血病细胞、宫颈癌细胞、胃癌细胞或乳腺癌细胞。
15.根据权利要求14所述的用途,其中,所述白血病细胞为慢性髓细胞性白血病细胞或急性早幼粒细胞白血病细胞。
16.根据权利要求14所述的用途,其中,所述肿瘤细胞为人慢性髓细胞性白血病K562细胞、人急性早幼粒细胞白血病HL-60细胞、人子宫颈癌HeLa细胞、人胃癌BGC-823细胞或人乳腺癌MCF-7细胞。
17.一种在体外杀伤肿瘤细胞或者抑制肿瘤细胞增殖的方法,包括使用有效量的权利要求2所述的式I化合物或者权利要求12或13所述的组合物的步骤,其中,所述肿瘤细胞为白血病细胞、宫颈癌细胞、胃癌细胞或乳腺癌细胞。
18.根据权利要求17所述的方法,其中,白血病细胞为慢性髓细胞性白血病细胞或急性早幼粒细胞白血病细胞。
19.根据权利要求17所述的方法,其中,所述肿瘤细胞为人慢性髓细胞性白血病K562细胞、人急性早幼粒细胞白血病HL-60细胞、人子宫颈癌HeLa细胞、人胃癌BGC-823细胞或人乳腺癌MCF-7细胞。
20.权利要求2所述的式I化合物或者权利要求12或13所述的组合物在制备抗肿瘤药物中的用途,其中,所述肿瘤为白血病、宫颈癌、胃癌或乳腺癌。
21.根据权利要求20所述的用途,其中,所述白血病为慢性髓细胞性白血病或急性早幼粒细胞白血病。
22.权利要求1所述的产紫青霉BD-1-6在制备权利要求2所述的式I化合物中的用途。
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产紫青霉G59的庆大霉素抗性突变株新产抗肿瘤活性产物研究;柴云晶等;《国际药学研究杂志》;20110630;第38卷(第3期);全文 * |
柴云晶等.产紫青霉G59的庆大霉素抗性突变株新产抗肿瘤活性产物研究.《国际药学研究杂志》.2011,第38卷(第3期),全文. |
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