CN115160236B - Corallorazines类化合物及其制备方法和应用 - Google Patents
Corallorazines类化合物及其制备方法和应用 Download PDFInfo
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- CN115160236B CN115160236B CN202210554072.5A CN202210554072A CN115160236B CN 115160236 B CN115160236 B CN 115160236B CN 202210554072 A CN202210554072 A CN 202210554072A CN 115160236 B CN115160236 B CN 115160236B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
- C07D241/06—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members
- C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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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
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/02—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
- C07C233/09—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to carbon atoms of an acyclic unsaturated carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and unsaturated
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/88—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having the nitrogen atom of at least one of the carboxamide groups further acylated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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Abstract
本发明公开了具有如下通式(I)所示结构的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物。本发明公开的十种化合物Corallorazines D~Corallorazines M,均可由同一株放线菌通过发酵培养后提纯后得到。本发明还公开了Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物在制备用于治疗或预防癌症尤其是结肠癌的药物中的应用。
Description
技术领域
本发明涉及微生物药物领域技术领域,具体涉及Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物及其在治疗或预防癌症、炎症、或其它增殖性疾病的药物中的应用,特别是制备用于抗结肠癌类的药物中的应用,并提供了Corallorazines类化合物的制备方法。
背景技术
结肠癌是导致与癌症相关的死亡中最常见的癌症之一,在我国结肠癌死亡率排在所有类型癌症中的第五位,并有其发病率逐年上升及其病患者逐年年轻化的趋势。有调查显示,结肠癌患者中,发生远处转移或复发的几率超过50%,且晚期患者中5年生存率不到5%。因此发现先导化合物并将其开发成药物,对于延长结肠癌患者的生存质量和时间是十分重要的。
目前人们已经很难从现有菌株的二次代谢产物发现结构新颖、活性好的天然产物,而对于少数发现的新菌株,往往其二次代谢产物的生物活性也不太理想。微生物中蕴藏着丰富的活性二次代谢产物,其研究开发,能帮助解决当前陆地药用资源紧张的问题,同时对于开发先导化合物具有十分重要的意义。
Corallorazines类化合物于2014年从C.coralloides B035的发酵产物中首次分离得到,是一种由脱氢丙氨酸和甘氨酸通过半胺键连接而形成哌嗪环的新型二肽,再通过酰胺键进一步连接到一个不寻常的脂肪酰基链,这些氨基酸通过半胺基官能团连接,形成立体异构体。
Corallorazine A在结构上与二酮哌嗪有一些相似之处,并且已经报道了一种密切相关的合成化合物,其结构式如下式(Ⅱ)所示,它成为Phenylahistin合成的副产物。下式(Ⅲ)为Phenylahistin的结构式。目前,Phenylahistin已经开发为上市药物Plinabulin,其结构式如式(Ⅳ)所示。
关于Corallorazines类化合物目前报道的仅有Corallorazines A-C,其结构式如下所示:
Schmitz A等(Schmitz A,Kehraus S,T F,et,al.Corallorazinesfrom the Myxobacterium Corallococcus coralloides.Journal of Natural Products,2014,77(1),159-163.doi:10.1021/np400740u)在琼脂扩散试验测定得到CorallorazinesA对细菌和真菌无效,对大麻素受体无亲和力,对人白细胞弹性蛋白酶也无抑制作用。Corallorazines A-C仍是尚待开发的化合物。
鉴于此,本发明人对Corallorazines的拟诺卡氏菌属Nocardiopsis metallicusDSM44598进行研究,从高氏一号液体培养基和大米培养基中共分离得到10个结构新颖Corallorazines类化合物(Corallorazines D~Corallorazines M),其中部分具有良好抗结肠癌活性,为先导化合物的开发提供支持。
发明内容
本发明的目的在于提供具有如通式(I)所示结构的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物及其在治疗或预防癌症、炎症、或其它增殖性疾病的药物中的应用,特别是制备用于抗结肠癌类的药物中的应用,并提供了Corallorazines类化合物的制备方法。
本发明的第一方面,提供了具有如下通式(I)所示结构的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物:
其中,
R3=-OH或H或Me
R4=Me或H。
所述Corallorazines类化合物为实施例1~10所制得的化合物,其结构与命名如下:
。
本发明的第二方面,提供了一种如本发明第一方面所述的Corallorazines类化合物的制备方法,包括以下步骤:
(1)将放线菌接种于高氏一号培养基中,摇床培养,获得种子液;所述放线菌为德国微生物菌种保藏中心保藏号为DSM44598的拟诺卡氏菌;
(2)将步骤(1)所得的种子液接种于大米固体培养基中,静置培养,经有机溶剂多次萃取后获得发酵产物粗提物;
(3)将步骤(2)所得的发酵产物粗提物进行分离纯化,得到所述Corallorazines类化合物。
优选地,步骤(1)中,所述高氏一号培养基的配制包括:相对于1L所述的高氏一号培养基,将可溶性淀粉20g、硝酸钾1g、磷酸氢二钾0.5g、硫酸镁0.5g、硫酸亚铁0.01g、琼脂20g、海盐25g混合后加水至1L,再调节pH值至7.0~7.2。
优选地,步骤(1)中,所述摇床培养条件为:培养温度为23~33℃,震荡速度为100~260rpm,培养时间为2~6天。
优选地,步骤(2)中,所述大米固体培养基由大米和海水组成,其中大米质量与海水体积比为35g~45g:50ml~70ml。
优选地,步骤(2)中,所述静置培养条件为:培养温度为26~30℃,静置培养55~65天。
优选地,步骤(2)中,所述有机溶剂为乙酸乙酯。
优选地,步骤(3)中,所述分离纯化,具体为:
(a)使用填料为硅胶的硅胶柱色谱纯化所述发酵产物粗提物,用体积比为6:1、3:1、2:1、1:1的石油醚/乙酸乙酯体系或体积比为70:1、50:1、30:1、10:1、1:1、0:1的二氯甲烷/甲醇体系梯度洗脱,收集含有目标化合物的馏分并合并;
(b)使用填料为十八烷基键合硅胶的反向色谱进一步分离纯化,采用的流动相为体积比为15~70:70~100甲醇/水体系或体积比为15~40:80~100乙腈/水体系,得到所述Corallorazines类化合物。
本发明制备方法中,将硅胶柱层析中所得馏分用反相高效液相色谱分离,收集不同流动相下不同洗脱时间的产物即可分别得到实施例1~10所制得的化合物。
优选地,检测波长为210nm,流动相为乙腈/水体系,以10mL/min进行梯度洗脱;
流动相为体积浓度为15~100%的乙腈/水体系时,收集21~22min的洗脱液得到Corallorazines D;
流动相为体积浓度为30~100%的乙腈/水体系时,收集19.7~20.7min的洗脱液得到Corallorazines E;
流动相为体积浓度为20~100%的乙腈/水体系时,收集19.5~20.5min的洗脱液得到Corallorazines F;
优选地,检测波长为210nm,流动相为甲醇/水体系,以10mL/min进行梯度洗脱;
流动相为体积浓度为20~100%的甲醇/水体系时,收集23.5~24.5min的洗脱液得到Corallorazines G;
流动相为体积浓度为20~100%的甲醇/水体系时,收集34~35min的洗脱液得到Corallorazines H;
流动相为体积浓度为30~100%的甲醇/水体系时,收集29.5~30.5min的洗脱液得到Corallorazines I;
流动相为体积浓度为15~100%的甲醇/水体系时,收集18~19min的洗脱液得到Corallorazines J;
流动相为体积浓度为20~100%的甲醇/水体系时,收集29.5~30.5min的洗脱液得到Corallorazines K;
流动相为体积浓度为15~100%的甲醇/水体系时,收集19.5~20.5min的洗脱液得到Corallorazines L;
流动相为体积浓度为20~100%的甲醇/水体系时,收集29~30min的洗脱液得到Corallorazines M;
采用上述的制备工艺可以制备高产率、高纯度的Corallorazines D~Corallorazines M化合物。
为进一步测试本发明制备得的Corallorazines类化合物的生物活性,采用HCT116结肠癌细胞进行化合物的抗肿瘤活性评价试验。
试验表明,本发明分离得到的Corallorazines D与Corallorazines F具有较好的抗HCT116结肠癌细胞的活性,因此具有用于制备用于治疗或预防癌症尤其是结肠癌的药物的潜力。
在上述试验的基础上,本发明的第三方面,提供了所述的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物,在制备用于治疗或预防癌症、炎症、或其它增殖性疾病的药物中的应用,优选在制备用于抗结肠癌类的药物中的应用;
所述癌症包括结直肠癌、肝癌、结肠癌、胰腺癌、卵巢癌、乳腺癌、膀胱癌、前列腺癌、白血病、骨髓瘤、胶质瘤。
本发明的第四方面,提供了一种用于治疗或预防癌症、炎症、或其它增殖性疾病的药物,所述药物含有治疗有效量的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物,以及一种或多种可药用的载体、赋形剂、佐剂、辅料和/或稀释剂;
所述可药用的盐包括盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
可将活性化合物制成适合于通过任何适当途径给药的形式,通过常规方法使用一种或多种药学上可接受的载体来配制本公开的组合物。因此,本公开的活性化合物可以配制成用于口服给药、注射(例如静脉内、肌肉内或皮下)给药,吸入等给药的各种剂型。本公开的化合物也可以配制成持续释放剂型,例如片剂、硬或软胶囊、水性或油性混悬液、乳剂、注射液、可分散性粉末或颗粒、栓剂、或糖浆等。
本发明与现有技术相比,其有益效果至少有:
本发明提供了具有如通式(I)所示结构的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物;
本发明提供了十种化合物Corallorazines D~Corallorazines M的制备方法;
本发明提供了所述的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物,在制备用于治疗或预防癌症、炎症、或其它增殖性疾病的药物中的应用,尤其在制备用于抗结肠癌类的药物中的应用;
本发明提供了一种用于治疗或预防癌症、炎症、或其它增殖性疾病的药物;
本发明公开的十种化合物Corallorazines D~Corallorazines M,均可由同一株放线菌通过发酵培养后提纯后得到,其结构为天然产物中首次发现具有结构新颖性;
经生物活性评价试验发现,本发明制备得的Corallorazines D与CorallorazinesF具有较好的抗HCT116结肠癌细胞的活性,因此具有用于制备用于治疗或预防癌症尤其是结肠癌的药物的潜力。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的操作方法,通常按照常规条件,或按照制造厂商所建议的条件。
实验方法与实验设备
化合物的结构确定采用核磁共振(NMR)和/或质谱(MS)。NMR化学位移(δ)以10-6(ppm)为单位给出。NMR的测定采用Bruker AVⅢ600MHz NMR核磁共振波谱仪,测定使用的常规氘代溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)和氘代甲醇(Methanol-d4),以四甲基硅烷(TMS)为内标。质谱(MS)的测定采用液相色谱-质谱联用仪(LC-MS),电离源:电喷雾电离(ESI)。生产商为:Agilent,型号为:Agilent 1260,色谱柱为Eclipse Plus 3.6μm100×4.6mm。
薄层层析硅胶板由青岛海洋化工有限公司生产,薄层色谱(TLC)使用的硅胶板厚度0.2-0.25mm;规格50×200mm。薄层制备色谱(prep-TLC)使用的硅胶板厚度0.4-0.5mm;规格200×200mm。
柱层析硅胶通常使用由青岛海洋化工有限公司生产的硅胶,规格100-200目或300-400目。
高效液相制备色谱采用Agilent Pursuit C-18(10μm,21.2×250mm)色谱柱。
菌种来源
本发明放线菌为拟诺卡氏菌Nocardiopsis metallicus DSM44598,订购网址:https://www.dsmz.de/collection/catalogue/details/culture/DSM-44598。
培养基
所述高氏一号培养基:以1L高氏一号培养基计,将可溶性淀粉20g、硝酸钾1g、磷酸氢二钾0.5g、硫酸镁0.5g、硫酸亚铁0.01g、琼脂20g、海盐25g混合后加水至1L,再调节pH值至7.0~7.2。
大米固体培养基:由大米和海水组成,其中大米质量与海水体积比为35g~45g:50ml~70ml。
实施例1:Corallorazines D的制备与表征
1)菌种种子液
将放线菌接种于500mL锥形瓶中,每瓶含250mL高氏一号培养基,在28℃、180rpm下摇床培养4天,得到接种有放线菌的种子液。
2)化合物的发酵
将步骤1)得到的接种有放线菌的接种液至大米培养基(该大米培养基由以下组分制成:大米40g;海水60mL),接种体积为8mL,在28℃下静置培养55天,用等体积乙酸乙酯萃取3次,得到发酵产物粗提物。
3)化合物的收集
将步骤2)得到的发酵产物粗提物进行硅胶柱层析,用体积比为6:1、3:1、2:1、1:1的石油醚/乙酸乙酯体系梯度洗脱,收集含有新化合物的馏分并合并。
再将所得硅胶柱层析所得馏分用反相高效液相色谱分离(Agilent Pursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为15~100%乙腈/水体系,以10mL/min梯度洗脱,收集21~22min的色谱峰,回收溶剂,即可得Corallorazines D。
制备得的Corallorazines D分子式根据高分辨质谱HR-ESI-MS推测为C20H32N2O5([M+Na]+403.2202,calculated 403.2203)。Corallorazines D的核磁共振数据及信号归属如表1所示:
表1(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
实施例2:Corallorazines E的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为30~100%的乙腈/水体系,以10mL/min梯度洗脱,收集19.7~20.7min的色谱峰,回收溶剂,即得Corallorazines E。
制备得的Corallorazines E分子式根据高分辨质谱HR-ESI-MS推测为C20H32N2O5([M+Na]+403.2202,calculated 403.2203)。Corallorazines E的核磁共振数据及信号归属如表2所示:
表2(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例3:Corallorazines F的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为20~100%的乙腈/水体系,以10mL/min梯度洗脱,收集19.5~20.5min的色谱峰,回收溶剂,即得Corallorazines F。
制备得的Corallorazines F分子式根据高分辨质谱HR-ESI-MS推测为C20H32N2O4([M+Na]+387.2252,calculated 387.2254)。Corallorazines F的核磁共振数据及信号归属如表3所示:
表3(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例4:Corallorazines G的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为20~100%的甲醇/水体系,以10mL/min梯度洗脱,收集23.5~24.5min的色谱峰,回收溶剂,即得Corallorazines G。
制备得的Corallorazines G分子式根据高分辨质谱HR-ESI-MS推测为C20H34N2O5([M+Na]+405.2362,calculated 405.2360)。Corallorazines G的核磁共振数据及信号归属如表4所示:
表4(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
实施例5:Corallorazines H的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为20~100%的甲醇/水体系,以10mL/min梯度洗脱,收集34~35min的色谱峰,回收溶剂,即得Corallorazines H。
制备得的Corallorazines H分子式根据高分辨质谱HR-ESI-MS推测为C20H32N2O4([M+H]+367.2592,calculated 367.2594)。Corallorazines H的核磁共振数据及信号归属如表5所示:
表5(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
实施例6:Corallorazines I的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为30~100%的甲醇/水体系,以10mL/min梯度洗脱,收集29.5~30.5min的色谱峰,回收溶剂,即得Corallorazines I。
制备得的Corallorazines I分子式根据高分辨质谱HR-ESI-MS推测为C20H34N2O4([M+H]+367.2591,calculated 367.2591)。Corallorazines I的核磁共振数据及信号归属如表6所示,:
表6(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例7:Corallorazines J的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积浓度为15~100%的甲醇/水体系,以10mL/min梯度洗脱,收集18~19min的色谱峰,回收溶剂,即得Corallorazines J。
制备得的Corallorazines J分子式根据高分辨质谱HR-ESI-MS推测为C13H23NO([M+H]+210.1854,calculated 210.1852)。Corallorazines J的核磁共振数据及信号归属如表7所示,
表7(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例8:Corallorazines K的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积分数为20~100%的甲醇/水体系,以10mL/min梯度洗脱,收集29.5~30.5min的色谱峰,回收溶剂,即得Corallorazines K。
制备得的Corallorazines K分子式根据高分辨质谱HR-ESI-MS推测为C13H23NO2([M+Na]+248.1620,calculated 248.1620)。Corallorazines K的核磁共振数据及信号归属如表8所示:
表8(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例9:Corallorazines L的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积分数为15~100%的甲醇/水体系,以10mL/min梯度洗脱,收集19.5~20.5min的色谱峰,回收溶剂,即得Corallorazines L。
制备得的Corallorazines L分子式根据高分辨质谱HR-ESI-MS推测为C13H23NO3([M+Na]+264.1575,calculated 264.1570)。Corallorazines L的核磁共振数据及信号归属如表9所示:
表9(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例10:Corallorazines M的制备与表征
将实施例1中硅胶柱层析中所得馏分,用反相高效液相色谱分离(AgilentPursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为体积分数为20~100%的甲醇/水体系,以10mL/min梯度洗脱,收集29~30min的色谱峰,回收溶剂,即得Corallorazines M.
制备得的Corallorazines M分子式根据高分辨质谱HR-ESI-MS推测为C15H25NO4([M+Na]+306.1674,calculated 306.1676)。Corallorazines M的核磁共振数据及信号归属如表10所示:
表10(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
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实施例11:化合物的抗肿瘤活性测定
1、测试目的
本实验通过检测化合物对HCT116结肠癌细胞的生长抑制作用,根据IC50评价本发明制得的化合物对抗HCT116结肠癌细胞的活性。
2、实验方法
采用磺酰罗丹明B(Sulforhodamine B,SRB)比色法检测化合物对人结肠癌细胞株HCT116细胞的增殖抑制作用。取对数生长期的细胞,配置成5×104个/mL,以100μL/孔铺于96孔培养板,CO2培养箱中培养24h,取出培养板后于每孔中加入不同浓度的待测样品,每个浓度设3个复孔,加药完成后,置于CO2培养箱中继续培养72h后取出培养板,弃去培养液,每孔加入100μL 4℃冰箱预冷的质量百分数10%的三氯醋酸(TCA)固定,静置5min后,再将培养板移至4℃冰箱过夜。倒掉固定液,每孔用去离子水洗涤5遍,甩干,空气干燥。每孔加入70μL SRB溶液,室温25℃放置20min,去上清液,用质量百分数1%醋酸洗涤5次,空气干燥。结合的SRB用100μL/孔10mmoL/L Tris碱液(pH=10.5)振荡溶解。置于酶标仪中测定各孔光吸收,测定波长为515nm。根据各孔OD值计算药物对细胞增殖抑制率:抑制率=[1-(OD515给药孔/OD515对照孔)]×100%,根据各浓度抑制率计算半数抑制浓度IC50。
其中待测样品是由实验组Corallorazines D~CorallorazinesM、阳性对照组阿霉素以及空白对照组细胞培养液组成。
3、实验结果
抗结肠癌细胞抑制率测定结果表明,Corallorazines D和Corallorazines F对HCT116细胞都具有很好的抑制作用,其他实验组HCT116细胞抑制效果不明显,IC50均大于10μM。其中Corallorazines D和Corallorazines F对HCT116细胞的IC50如表11所示。
表11 Corallorazines D、F和阿霉素的抑制HCT116细胞增值的效果
化合物 | Corallorazines D | Corallorazines F | 阿霉素 |
IC50(μM) | 6.2±0.05 | 4.4±0.1 | 1.2±0.07 |
Claims (4)
1.Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物,其特征在于,所述Corallorazines类化合物选自如下化合物:
。
2.根据权利要求1所述的Corallorazines类化合物的制备方法,其特征在于,包括以下步骤:
(1)将放线菌接种于高氏一号培养基中,摇床培养,获得种子液;所述放线菌为德国微生物菌种保藏中心保藏号为DSM44598的拟诺卡氏菌;
所述高氏一号培养基的配制包括:相对于1L所述的高氏一号培养基,将可溶性淀粉20g、硝酸钾1g、磷酸氢二钾0 .5g、硫酸镁0 .5g、硫酸亚铁0 .01g、琼脂20g、海盐25g混合后加水至1L,再调节pH值至7.0~7.2;
所述摇床培养条件为:培养温度为23~33℃,震荡速度为100~260 rpm,培养时间为2~6天;
(2)将步骤(1)所得的种子液接种于大米固体培养基中,静置培养,经有机溶剂乙酸乙酯多次萃取后获得发酵产物粗提物;
所述大米固体培养基由大米和海水组成,其中大米质量与海水体积比为35g~45g:50ml~70ml;
所述静置培养条件为:培养温度为26~30℃,静置培养55~65天;
(3)将步骤(2)所得的发酵产物粗提物进行分离纯化,得到所述Corallorazines类化合物;
所述分离纯化,具体为:
(a)使用填料为硅胶的硅胶柱色谱纯化所述发酵产物粗提物,用体积比为6:1、3:1、2:1、1:1的石油醚/乙酸乙酯体系或体积比为70:1、50:1、30:1、10:1、1:1、0:1的二氯甲烷/甲醇体系梯度洗脱,收集含有目标化合物的馏分并合并;
(b)使用填料为十八烷基键合硅胶的反向色谱进一步分离纯化,采用的流动相为体积比为15~70:70~100甲醇/水体系或体积比为15~40:80~100乙腈/水体系,得到所述Corallorazines类化合物。
3.根据权利要求1所述的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物,在制备用于治疗或预防癌症的药物中的应用;
所述癌症为结肠癌。
4.一种用于治疗或预防癌症的药物,其特征在于,所述药物含有治疗有效量的根据权利要求1所述的Corallorazines类化合物,或其互变异构体、外消旋体、对映异构体、非对映异构体、可药用的盐或它们的混合物,以及一种或多种可药用的载体、赋形剂、佐剂和/或稀释剂;
所述可药用的盐选自盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
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