CN116730884A - 一种含硫角环素二聚体结构化合物、其制备方法和用途 - Google Patents
一种含硫角环素二聚体结构化合物、其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种含硫角环素二聚体结构化合物、其制备方法和用途,属于医药技术领域,其结构式如式I所示,由放线菌发酵制备得到,本发明具有抑制炎症性肠道氧化应激反应,提取制备方法简单,原料可持续利用。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种含硫角环素二聚体结构化合物,同时还涉及该含硫角环素二聚体结构化合物的制备方法,及该含硫角环素二聚体结构化合物在制备抗炎症性肠病药物中的应用。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD)是一类由遗传、免疫、感染、环境及饮食习惯等多种因素所致的特异性自身免疫反应,包括溃疡性结肠炎(ulcerativecolitis,UC)和克罗恩病(Crohn’s disease,CD)。目前市面上治疗IBD的治疗药物包括常规治疗(氨基水杨酸盐、糖皮质激素、免疫抑制剂、抗生素)和生物制剂(抗TNF-α单克隆抗体)。从疗效上看,常规治疗虽能改善症状,但复发率高且对重症患者疗效不佳,而生物制剂安全性和有效性仍需扩大规模临床试验加以评估。如,抗TNF-α使用者中,约30%无应答。因此,研发疗效显著、副作用小、成本低廉的新型小分子IBD治疗药物,具有重要临床需求。
角环素(Angucyclines)类化合物属于II型聚酮(T2PKS)类天然产物,其多样独特的化学结构使其成为小分子天然药物研究领域关注的热点。天然来源的含硫化合物在药物研发中占据重要的地位。据报道,2019年全球销售额前200名药品中,包含41种含硫药物,占20.5%。常见的磺胺类药物、青霉素、头孢类抗生素等也都属于经典的含硫药物。但是,含硫元素的角环素(Angucyclines)类化合物比较罕见。迄今为止,只有12个含硫角环素化合物被发现。其中,含有硫醚二聚这一特殊结构特征的角环素(Angucyclines)类化合物更少(目前仅报道了7个),导致对该类化合物的药用活性及开发利用严重不足。至今未见具有缓解肠道炎症活性的含硫角环素二聚体结构的报道。
葡聚糖硫酸钠(Dextran Sulfate Sodium,DSS)诱导的果蝇肠道炎症模型是研究溃疡性结肠炎疾病的经典模型。DSS喂食果蝇后会破坏其肠上皮中肠道表皮细胞之间的连接,从而造成肠道损伤。
发明内容
本发明的目的在于克服上述缺点而提供的一种具有抑制炎症性肠道氧化应激反应,提取制备方法简单,原料可持续利用的含硫角环素二聚体结构化合物。
本发明的另一目的在于提供该含硫角环素二聚体结构化合物的制备方法。
本发明的再一目的在于提供该含硫角环素二聚体结构化合物在制备抗炎症性肠病药物中的应用。
本发明目的及解决其主要技术问题是采用以下技术方案来实现的:
本发明的一种含硫角环素二聚体结构化合物,其结构式如式I所示:
本发明的一种含硫角环素二聚体结构化合物的制备方法,包括如下步骤:
(1)将放线菌Spongiactinospora rosea活化后,转移至100mL含种子培养基(包含3g/100mL色氨酸大豆汤,0.5g/100mL酵母提取物,10g/100mL蔗糖,pH调至7.2-7.4)的锥形瓶中,随后置于摇床200rpm,28℃振荡培养72小时,得种子液;
(2)将种子液以10%体积比(mL)比例的接种量接到发酵培养基(包含3g/100mL色氨酸大豆汤,0.5g/100mL酵母提取物,10g/100mL蔗糖,pH调至7.2-7.4)中,置于摇床200rpm,28℃振荡培养14天,获得菌株的发酵培养物;
(3)收集发酵液,用等体积乙酸乙酯(EtOAc)萃取发酵菌液3次,合并萃取液,用旋转蒸发仪蒸干乙酸乙酯溶液后,得到乙酸乙酯萃取部位;
(4)上述乙酸乙酯萃取部位用体积比90%(v/v)甲醇水溶解后,用等体积石油醚脱脂,脱脂后,调整溶液比例为60%(v/v)甲醇水,用等体积二氯甲烷萃取,得二氯甲烷层。
将二氯甲烷层经Sephadex LH-20凝胶柱色谱分离,以CH2Cl2/MeOH(1:1,v/v)作为溶剂进行洗脱,得到组分Fr.A-Fr.I,组分Fr.C进一步以CH3CN/H2O为洗脱剂(10-100%,v/v),流速10mL/min,经反相中压柱色谱(ODS)洗脱,得Fr.C.1-Fr.C.14,组分Fr.C.7进一步以CH2Cl2/MeOH(50:1,v/v)为洗脱剂,经正相硅胶柱色谱分离,得Fr.C.7.1和Fr.C.7.2,组分Fr.C.7.1经反相半制备高效液相分离,CH3OH/H2O(70%,v/v)洗脱,流速2.5mL/min,制备得式I所述硫角环素二聚体结构化合物。
本发明的一种含硫角环素二聚体结构化合物在制备抗炎症性肠病药物中的应用。
本发明一种含硫角环素二聚体结构化合物或其药用盐在作为抗炎性肠病药物应用。
所述的药物还包括药学上可接受的载体。
本发明同现有技术相比,具有明显的优点和有益效果,由以上技术方案可知,本发明采用的原料为放线菌发酵产物,制备流程简单,资源可持续利用。本发明如式I的化合物在5nM化合物浓度下,能够有效缓解5%DSS处理诱导的果蝇寿命缩短表型,通过改善果蝇肠道功能来维持成体果蝇生存。本发明通过构建DSS诱导的果蝇肠道炎症损伤模型体内试验进一步发现,5nM浓度下,该化合物能够有效缓解5%DSS处理诱导的果蝇肠道氧化应激反应,具有潜在的抗gstd1介导的氧化应激的作用。本发明为研究和开发新的抗炎症性肠病药物提供了新的候选化合物,也为开发利用微生物药用资源提供了科学依据。
附图说明
图1为本发明化合物的核磁共振氢谱;
图2为本发明化合物的核磁共振碳谱;
图3为本发明化合物的单晶结构;
图4为本发明化合物实测ECD和计算ECD曲线;
图5为本发明化合物对DSS诱导果蝇生存率影响结果;
图6为本发明化合物抑制果蝇肠道氧化应激活性结果。
具体实施方式
实施例1
一种含硫角环素二聚体结构化合物的制备方法,包括如下步骤:
(1)将放线菌Spongiactinospora rosea活化后,转移至100mL含种子培养基(包含3g/100mL色氨酸大豆汤,0.5g/100mL酵母提取物,10g/100mL蔗糖,pH调至7.2-7.4)的锥形瓶中,随后置于摇床200rpm,28℃振荡培养72小时,得种子液。
(2)将种子液以体积比10%(v/v)比例的接种量分别接到1L的含有发酵培养基(包含3g/100mL色氨酸大豆汤,0.5g/100mL酵母提取物,10g/100mL蔗糖,pH调至7.2-7.4)的锥形瓶中,总计30L,置于摇床200rpm,28℃振荡培养14天,获得菌株的发酵培养物。
(3)收集发酵液,离心除菌丝体,滤液吸入真空反应器,加入等体积乙酸乙酯萃取3次,合并萃取液,用旋转蒸发仪蒸干乙酸乙酯溶液后得到乙酸乙酯萃取部位66.7g
(4)上述乙酸乙酯萃取部位用90%(v/v)甲醇水溶解后,用等体积石油醚脱脂3次,脱脂后,调整溶液比例为60%(v/v)甲醇水,用等体积二氯甲烷萃取,得二氯甲烷层6.2g。将二氯甲烷层经Sephadex LH-20凝胶柱色谱分离,以CH2Cl2/MeOH(1:1,v/v)作为溶剂进行洗脱,得到9个组分Fr.A-Fr.I。组分Fr.C(258.1mg)进一步以CH3CN/H2O为洗脱剂(10-100%,v/v),流速10mL/min,经反相中压柱色谱(ODS)洗脱,得到14个组分Fr.C.1-Fr.C.14.组分Fr.C.7(56.8mg)进一步以CH2Cl2/MeOH(50:1,v/v)为洗脱剂,经正相硅胶柱色谱分离,得Fr.C.7.1和Fr.C.7.2。最后,组分Fr.C.7.1(22.6mg)经反相半制备高效液相分离,CH3OH/H2O(70%,v/v)洗脱,流速2.5mL/min,制备得式I所述硫角环素二聚体结构化合物10.8mg。
(二)化合物的结构鉴定
化合物通过高分辨质谱、核磁共振谱(1H-NMR,13C-NMR,DEPT,HSQC、HMBC、COSY)、紫外、旋光、X-单晶衍射综合分析,确定其结构式为
化合物为橙黄色针状晶体,分子式为C40H31O10S。m/z 703.1607[M+H]+(calcd forC40H31O10S,703.1632).(c0.1,MeOH);UV(MeOH)λmax(logε),234(3.63),261(3.71),375(3.13)nm.核磁数据如表1所示。
表1,化合物的1H(600MHz)和13C(150MHz)-NMR(DMSO)数据
ɑ峰重叠信号.
试验例1:化合物对DSS诱导果蝇生存率的影响
1.试验材料
果蝇培养基、滤纸片、果蝇培养管、塞子、托盘、酒精、二氧化碳、二氧化碳管道、智能果蝇培养箱、细毛刷,移液枪、蔗糖、甲基亚枫(DMSO)、体式显微镜、Gstd1-GFP转基因果蝇品系。
2.试验过程
a.果蝇的准备
果蝇在装有果蝇培养基的培养管里产卵后灭活,等待孵化;分别收集3-5日龄内雌性Gstd1-GFP基因型果蝇,取孵化后同一批果蝇,每管30只;
b.溶液及其他材料准备
分别配置0.1%(v/v)DMSO溶液、5%蔗糖溶液(5g/100mL)、5%(v/v)DSS溶液、1nM、5nM、10nM化合物不同浓度的溶液(溶于0.1%(v/v)DMSO);圆形滤纸片数张。
c.试验设计
试验一共分成六个处理组,每组重复三次。阳性对照组(5%DSS(v/v)溶液)、阴性对照组1(5%蔗糖溶液(5g/100mL))、阴性对照组2(0.1%DMSO(v/v)溶液)、试验组1(1nM化合物溶液)、试验组2(5nm化合物溶液)、试验组3(10nM化合物溶液),依次做好标记和日期。
d.试验步骤
首先,在智能果蝇培养箱(29℃)中对六个处理组的果蝇提前进行2小时饥饿处理。其次,准备六个新的果蝇培养管,并在其底部放入两层滤纸片;然后,按照如下体系用移液枪将配置好的溶液分别对应加入六个处理组中的滤纸片上:阴性对照组1和阳性对照组加入200ul5%蔗糖溶液(5g/100mL)、阴性对照组2加入200ul0.1%DMSO溶液(v/v)、试验组1加入200ul1nM化合物溶液、试验组2加入200ul5nM化合物溶液、试验组3加入200ul10nM化合物溶液。并做好标记备用,静置或轻轻扭动培养管,避免滤纸表明上形成大量积液;最后,将饥饿处理后的果蝇,用细毛刷在显微镜和二氧化碳工作台上分别对应转入处理好的培养管中,并置于智能果蝇培养箱(26℃)中培养。如上操作连续处理3天,每天更换新的滤纸和加入对应溶液,期间观察果蝇死亡情况,及时更新试验果蝇数目。
在试验第4天,进行5%DSS溶液(v/v)处理。首先,对果蝇进行饥饿处理2小时,饥饿期间,分别更换新的滤纸和溶液;加液方法如下:试验组1,2,3分别加入200ul5%DSS溶液(v/v),阳性对照组加入200ul5%DSS溶液(v/v);阴性对照组1继续加入200ul5%蔗糖溶液,阴性对照组2继续加入200ul0.1%DMSO(v/v);最后,将饥饿处理后的果蝇分别对应转入对应果蝇管中。每天更换新的滤纸和加入对应溶液,期间每隔几小时观察并记录果蝇的死亡情况,最后根据数据做出生长曲线图。
3.试验结果
试验结果显示(图5),阴性对照组的果蝇均呈现出正常的生存曲线,与阴性对照组相比,5%DSS(v/v)处理显著缩短果蝇的正常寿命,其中5nM化合物预处理组能够有效缓解5%DSS(v/v)处理诱导的果蝇寿命缩短表型,提示该化合物可能通过改善果蝇肠道功能来维持成体果蝇生存。
试验例2:化合物对DSS诱导果蝇肠道氧化应激水平的影响
1.试验材料
果蝇培养基、滤纸片、果蝇培养管、塞子、托盘、酒精、二氧化碳、二氧化碳管道、智能果蝇培养箱、细毛刷,移液枪、蔗糖、甲基亚枫(DMSO)、体式显微镜、Gstd1-GFP转基因果蝇品系,解剖镊子,倒置荧光显微镜,显微镜玻片。
2.试验过程
选取5nM化合物浓度作为检测其对DSS诱导果蝇肠道氧化应激水平的影响。一共分为三个处理组,阴性对照组(5%蔗糖溶液(g/100mL)),阳性对照组(5%DSS溶液(v/v))、,试验组(5%DSS溶液+5nM化合物)。试验前期果蝇收集和处理步骤与试验一的试验步骤一致。当阳性对照组在5%DSS处理后,果蝇死亡数目到达一半时,分别对阴性对照组,阳性对照组,试验组中死亡的果蝇肠道在显微镜下进行解剖,收集,每组10根完整肠道,在荧光显微镜下观察Gstd1-GFP荧光强度,用以分析化合物对DSS诱导果蝇肠道gstd1介导的氧化应激强弱的影响。
3.试验结果
试验结果表明(图6),与对照组(A-A’)相比,5%DSS处理(B-B’)能够显著诱导果蝇肠道gstd1介导的氧化应激反应。果蝇在处理前连续饲喂5nM的化合物四天后(C-C’),能够有效缓解5%DSS处理诱导的肠道氧化应激反应。体内果蝇模型结果说明,该化合物具有潜在的抗gstd1介导的氧化应激的作用。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,任何未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、变换材质、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (7)
1.一种含硫角环素二聚体结构化合物,其结构式如式I所示:
2.一种含硫角环素二聚体结构化合物的制备方法,包括如下步骤:
(1)将放线菌Spongiactinospora rosea活化后,转移至100mL含种子培养基的锥形瓶中,随后置于摇床200rpm,28℃振荡培养72小时,得种子液;
(2)将种子液以体积比10%的接种量接到发酵培养基中,置于摇床200rpm,28℃振荡培养14天,获得菌株的发酵培养物;
(3)收集发酵液,用等体积乙酸乙酯(EtOAc)萃取发酵菌液3次,合并萃取液,用旋转蒸发仪蒸干乙酸乙酯溶液后,得到乙酸乙酯萃取部位;
(4)上述乙酸乙酯萃取部位用体积比90%(v/v)甲醇水溶解后,用等体积石油醚脱脂,脱脂后,调整溶液比例为60%(v/v)甲醇水,用等体积二氯甲烷萃取,得二氯甲烷层。
将二氯甲烷层经Sephadex LH-20凝胶柱色谱分离,以CH2Cl2/MeOH(1:1,v/v)作为溶剂进行洗脱,得到组分Fr.A-Fr.I,组分Fr.C进一步以CH3CN/H2O为洗脱剂(10-100%,v/v),流速10mL/min,经反相中压柱色谱(ODS)洗脱,得Fr.C.1-Fr.C.14,组分Fr.C.7进一步以CH2Cl2/MeOH(50:1,v/v)为洗脱剂,经正相硅胶柱色谱分离,得Fr.C.7.1和Fr.C.7.2,组分Fr.C.7.1经反相半制备高效液相分离,CH3OH/H2O(70%,v/v)洗脱,流速2.5mL/min,制备得式I所述硫角环素二聚体结构化合物。
3.如权利要求2所述的一种含硫角环素二聚体结构化合物的制备方法,其中:种子培养基为包含3g/100mL色氨酸大豆汤,0.5g/100mL酵母提取物,10g/100mL蔗糖,pH调至7.2-7.4。
4.如权利要求2所述的一种含硫角环素二聚体结构化合物的制备方法,其中:发酵培养基为包含3g/100mL色氨酸大豆汤,0.5g/100mL酵母提取物,10g/100mL蔗糖,pH调至7.2-7.4。
5.一种含硫角环素二聚体结构化合物在制备抗炎症性肠病药物中的应用。
6.一种含硫角环素二聚体结构化合物或其药用盐在作为抗炎性肠病药物应用。
7.如权利要求5或6的抗炎性肠病药物,包括药学上可接受的载体。
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