CN116715659A - 一种新颖骨架类型细胞松弛素及其在制备具有抗肿瘤活性的药物中的应用 - Google Patents
一种新颖骨架类型细胞松弛素及其在制备具有抗肿瘤活性的药物中的应用 Download PDFInfo
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- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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Abstract
本发明涉及医药化学技术领域,具体公开了从湖北贝母内生真菌Boeremia exigua中分离得到的一个5/5/13三环体系的新颖骨架类型的细胞松弛素类化合物。采用MTT法检测细胞活力,实验结果显示,该化合物对MCF‑7(人乳腺癌细胞)具有明显的抑制活性,IC50值为11.81mM。
Description
技术领域
本发明涉及医药和化学技术领域,特别涉及一种来源于湖北贝母内生真菌Boeremia exigua的细胞松弛素类化合物及其在制备具有抗肿瘤活性的药物中的应用。
背景技术
由于基因的突变、生活习惯的改变以及外部生存环境的不稳定,人类体内的正常细胞突变为癌细胞的风险上升,癌症也成为人类高发疾病之一。细胞凋亡是细胞死亡的天然机制,由于癌细胞中抗凋亡蛋白的过表达以及促凋亡蛋白的低表达,其多个凋亡途径被抑制,这导致癌细胞的耐药性增强,传统的抗癌药物已经难以对抗日益进化的癌细胞。在治疗癌症过程中,通常使用多种手段来激活癌细胞的凋亡途径,使其自然凋亡,达到控制病情恶化以及治愈的治疗目的。目前真菌来源的化合物通过激活不同的凋亡途径,表现出显著的抗癌效果,成为对抗耐药性癌细胞的新希望。
湖北贝母为百合科多年生草本植物,具有化痰止咳、解毒散结的功效。目前在湖北建始、宣恩广泛栽培,为治疗气管炎和慢性支气管炎的常用中药。内生真菌生存于健康植物组织和器官中,与植物处于互惠共生的平衡关系。内生真菌生物自身的合成途径丰富多样且千变万化,它们提供了通过人工化学合成途径难以得到、结构复杂的一系列新型化合物,是新颖结构活性天然产物和临床药物的重要来源,许多农药、抗癌药物以及抗菌药物均直接或间接来源于内生真菌。基于内生真菌具有资源丰富、种类繁多、培养周期短暂、基因操作可行性高等优势,且细胞松弛素类化合物具有多种生物活性,如抗肿瘤、抗菌、抗炎、抗氧化等。申请人对从湖北贝母的内生真菌中分离得到的一个骨架新颖细胞松弛素的抗肿瘤活性进行探究。
发明内容
针对现有技术中存在的不足,本发明的目的在于提供了一种细胞松弛素及其在制备具有抗肿瘤活性的药物中的应用。
一种具有抗肿瘤活性的细胞松弛素,其结构式如下:
分子式:C29H37NO4
名称为:boerelasin A
所述细胞松弛素在制备可抑制MCF-7(人乳腺癌细胞)生长的药物中的应用也属于本发明的保护范围。
本发明与现有技术相比,具有以下优点和有益效果:
1、本发明揭示了一种来源于湖北贝母内生真菌B.exigua的5/5/13三环体系新颖骨架类型的细胞松弛素。
2、本发明提供了一种具有抗肿瘤活性的细胞松弛素,可剂量依赖性地抑制MCF-7细胞的增殖,具有可用于制备新型抗肿瘤药物的潜在用途。
3、本发明拟保护其用途的细胞松弛素,可通过从植物内生真菌中提取纯化来获取,具有培养周期短暂、操作可行性高、无化学污染、绿色环保等优势。
附图说明
图1为具体实施方式中实施例1制备得到的化合物的氢谱图(600MHz,MeOD);
图2为具体实施方式中实施例1制备得到的化合物的碳谱图(150MHz,MeOD)和DEPT图;
图3为具体实施方式中实施例1制备得到的化合物的HMQC图;
图4为具体实施方式中实施例1制备得到的化合物的HMBC图;
图5为具体实施方式中实施例1制备得到的化合物的1H-1H COSY图;
图6为具体实施方式中实施例1制备得到的化合物的ROESY图;
图7为根据图1-6推测实施例1制得的化合物四种可能的构型;
图8为具体实施方式中实施例1制备得到的化合物实测碳谱和计算碳谱比较;
图9为具体实施方式中实施例1制备得到的化合物实测CD图和计算CD图比较;
图10为具体实施方式中实施例1制备得到的化合物对MCF-7细胞活力的影响。
具体实施方式
为使本申请的发明目的、发明内容呈现得更加清楚,下面申请人将结合具体实施例对本发明的技术方案进行清楚、完整的描述。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到实施例1:
内生真菌Boeremia exigua是从中国湖北恩施采集的贝母新鲜植株根部中分离得到,经ITS测序将其鉴定为Boeremia exigua,该菌株保藏于中南民族大学药学院微生物菌种库(已经公开,见:吕晓,叶可,马旭军等.湖北贝母内生真菌Boeremia exigua化学成分及其抗炎活性研究[J].中南民族大学学报(自然科学版),2022,41(2):174-179)。湖北贝母内生真菌Boeremia exigua采用大米固体发酵进行扩大培养:将保存菌株的试管从冰箱取出,在无菌常温环境中放置1小时后,在无菌操作台下取直径为5mm的菌块接种至平板PDA培养基上,常温遮光条件下生长约8天后,从平板PDA培养基上挑取直径为5mm的菌块接种到大米培养基上,共340瓶,培养条件:在25℃下暗培养30d。大米培养基配比:大米50g/瓶,蒸馏水50mL/瓶,置于500mL培养瓶中,经120℃高温灭菌20分钟。
权利要求书和说明书发明内容中所述化合物细胞松弛素的分离纯化过程:将长满菌丝体的大米固体培养基(共36kg)捣碎,用25L有机溶剂(二氯甲烷-甲醇=1:1,v/v)浸泡2天后,离心,将提取液减压浓缩,重复3次浸提操作;将3次浓缩后的提取液合并,减压浓缩蒸干溶剂后,加入少量水溶解,再用乙酸乙酯重复萃取3次(每次萃取的乙酸乙酯用量为4L),将乙酸乙酯部分合并后减压浓缩得到粗提浸膏660g。将浸膏用80-100目正相硅胶柱层析,二氯甲烷-甲醇(二氯甲烷:甲醇=100:0,50:1,20:1,10:1,5:1,v/v)梯度洗脱,并用薄层色谱(展开剂为二氯甲烷:甲醇=10:1,v/v)检测并显色,粗划段得到A–E五个组分。
将D组分(二氯甲烷/甲醇的体积比为10:1时洗脱出的组分,56.8g)经过高效中压液相制备色谱(Biotage SP1,反相填料材料:RP-18,20-45μm,Fuji Silysia ChemicalLtd.,Japan)甲醇水体系(甲醇/水=50:50,60:40,70:30,80:20,90:10,v/v),洗脱流速为20mL/min,五个比例洗脱时间均为60min,并用薄层色谱(展开剂为二氯甲烷:甲醇=10:1,v/v)检测并显色,合并相同或类似的组分,得到六个亚组分,按照极性由小到大依次标记为D1-D6。D3组分(27.3g)为甲醇-水的体积比为60:40时洗脱出来的组分,经Sephadex LH-20凝胶柱色谱(Pharmacia Fine Chemical Co.,Ltd.,Sweden)用甲醇洗脱(甲醇用量为10个柱体积)后,用薄层色谱(展开剂为二氯甲烷:甲醇=10:1,v/v)检测并显色,合并相同或类似的组分,得到8个亚组分,按照极性由小到大依次标记为D3-1~D3-8。第4个柱体积洗脱所得组分D3-4(2.6g)再经正相硅胶柱色谱石油醚-丙酮体系(石油醚:丙酮=10:1-1:1,v/v)梯度洗脱,用薄层色谱(展开剂为石油醚:丙酮=4:1,v/v;薄层层析硅胶板,青岛海洋化工厂)检测并显色,得到5个组分,按洗脱先后顺序将该5个组分依次编号为D3-4-1~D3-4-5。其中D3-4-3组分(13mg,石油醚:丙酮=4:1时洗脱下来)再经高效液相制备色谱(Agilent1260;色谱柱:Agilent Zorbax SB-C18,规格:9.4mm×150mm,5μm;乙腈-水=60:40-75:25,v/v;流速:4mL/min)梯度洗脱35min,制备得2.5mg本发明的化合物(保留时间为18min)。
化合物的结构鉴定:将本实施例1制备所得的化合物细胞松弛素,加0.5mL的氘代甲醇溶解,用200μL移液枪转移至核磁管中,在核磁共振仪(德国布鲁克Avance III600MHz)上检测氢谱、碳谱以及二维谱图(如图1-6)。综合各理化数据解得该化合物的结构并将其命名为boerelasin A。
所得细胞松弛素的核磁共振数据:
该化合物的绝对构型通过量子化学确定。其中C-3、C-4、C-5、C-16和C-20是骨架上的碳,因此其构型应该与细胞松弛素基本骨架一致,即3S4R5S16R20R。剩余C-8和C-9有四种可能的构型(1a-8R9S、2a-8R9R、3a-8S9S、4a-8S9R,如图7所示),我们通过在mPW1PW91-SCRF/6-31+G(d,p)//M06-2X/def2SVP(PCM溶剂模型)理论水平上计算NMR,结果显示3a-3S4R5S8S9S16R20R的计算值与实验值最吻合(R2=0.9986)(图8),因此我们可以确定boerelasin A绝对构型为3S4R5S8S9S16R20R。另外,我们对四种构型的ECD图谱也进行了计算,结果显示8S9S构型的ECD图谱和实测值更吻合(图9),与碳谱计算结果一致。
其他理化数据:
外观:浅黄色无定型粉末,根据高分辨质谱HRESIMS([M+H]+,m/z 464.2797)根据上述检测的结果,确认本实施例所得化合物的结构式为:
分子式:C29H37NO4
实施例2:MTT检测细胞活力
原理:MTT法即MTT比色法,是一种操作简便、灵敏度高、经济实惠、结果直观的测试细胞活力的方法。MTT是一种四甲基偶氮唑盐,全称为3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,性状为黄色粉末。活细胞线粒体中的琥珀酸脱氢酶具有还原外源性MTT的作用,可将MTT还原为不溶于水的蓝色(或蓝紫色)晶体甲臜(Formazan),而死细胞无此作用。DMSO(二甲亚砜)可以溶解沉积在细胞中的甲臜,通过酶标仪在570nm波长下进行吸光值测定,从而可以测出甲臜的含量。一般情况下,由于甲臜生成量与活细胞数量成正比,则可以根据光密度OD值计算得出活细胞的相对数量(将空白对照组活细胞设定为100%)
实验材料与仪器:
细胞株:人乳腺癌细胞系(MCF-7)(中国科学院昆明植物研究所);DMEM培养基和无菌PBS(pH=7.4,0.01M)(Hyclone公司),胎牛血清(浙江天杭生物科技股份有限公司)、MTT(Sigma),DMSO(Biosharp);细胞96孔板、细胞刮刀、细胞培养瓶(Corning);
多功能酶标仪(德国TECAN),二氧化碳培养箱(Thermo),倒置显微镜(Nikon),高压灭菌锅(上海博迅医疗生物仪器股份有限公司),制冰机(Panasonic),电子天平(赛多利斯)。
实验步骤:
用含有10%胎牛血清的DMEM培养基在恒温培养箱中(5% CO2,37℃)传代培养MCF-7细胞。取对数生长期生长状态良好的MCF-7细胞,用含有10%胎牛血清的DMEM培养基配制成细胞悬液,调整浓度为1×105个/mL,接种到96孔板中,每孔100μL,为避免边缘效应,只在中间区域6×10孔种上细胞,在细胞孔外围孔中加入100μL无菌PBS,5% CO2、37℃条件下培养8h。用不含胎牛血清的DMEM培养基配制六个浓度梯度的实施例1所得化合物,分别为40,30,20,10,5,1μM。待细胞贴壁后,弃去孔中的DMEM培养基,药物组加入不同浓度的化合物,100μL/孔,空白组每孔加入100μL无菌PBS,阳性对照组加入顺铂(溶液配制同实施例1所得化合物),然后将96孔板置于恒温培养箱中继续培养12h。接着在每个细胞孔中加入100μL浓度为0.5mg/mL的MTT溶液,5%CO2、37℃条件下培养4h后终止培养,弃去上清液,每个细胞孔中加入100μL DMSO,置于摇床震荡摇匀,使沉积在细胞中的甲臜充分溶解。使用酶标仪在572nm波长处测量各孔的吸光值,记录OD值并计算各待测化合物的存活率(存活率%=药物组/空白组×100%),再计算得出IC50值。
实验结果:
结果表明化合物boerelasin A对MCF-7(人乳腺癌细胞)具有明显的抑制活性。随着化合物浓度增加,抑制效果也相应增加,存在统计学意义,因此认为该化合物对MCF-7细胞生长抑制作用呈剂量依赖性,其IC50值为11.81μM,优于阳性对照药顺铂(21.69μM)。
Claims (10)
1.一种新颖骨架类型的细胞松弛素类化合物,其结构式为:
2.根据权利要求1所述的细胞松弛素类化合物,其特征在于,所述化合物的结构式为:
3.一种权利要求1或2所述的化合物的制备方法,其特征在于,包括以下步骤:
1)将湖北贝母内生真菌B.exigua大米固体发酵产物用有机混合溶剂浸泡提取,将提取液浓缩至干,再加水溶解,用乙酸乙酯进行萃取;
2)对乙酸乙酯萃取液进行硅胶柱分离,得到A-E五个组分;
3)对D组分进行中压液相制备色谱分离,得到D1~D6六个亚组分;
4)D3组分经凝胶柱色谱分离,得到D3-1~D3-8八个亚组分;
5)对D3-4组分进行硅胶柱分离,得到D3-4-1~D3-4-5五个亚组分;
6)对D3-4-3组分进行高效液相制备色谱纯化,得到所述化合物。
4.根据权利要求3所述的制备方法,其特征在于,步骤1)中的有机混合试剂是体积比为1:1的二氯甲烷-甲醇混合溶剂。
5.根据权利要求3所述的制备方法,其特征在于,步骤2)中硅胶柱分离的条件包括:以二氯甲烷-甲醇为洗脱液,按体积比为100:0、50:1、20:1、10:1、5:1的比例依次进行洗脱,TLC检测,得五个洗脱组分,其中展开剂为二氯甲烷:甲醇=10:1,v/v。
6.根据权利要求3所述的制备方法,其特征在于,步骤3)中的中压液相制备色谱分离的条件包括:RP-18色谱柱,以甲醇-水为洗脱液,按体积比为50:50、60:40、70:30、80:20、90:10的比例依次进行洗脱,流速为20mL/min,每个比例洗脱时间均为60min,TLC检测,得六个洗脱亚组分,其中展开剂为二氯甲烷:甲醇=10:1,v/v。
7.根据权利要求3所述的制备方法,其特征在于,步骤4)中的凝胶柱色谱分离,以甲醇为洗脱剂,TLC检测,得八个洗脱亚组分,其中展开剂为二氯甲烷:甲醇=10:1,v/v。
8.根据权利要求3所述的制备方法,其特征在于,步骤5)中的硅胶柱分离的条件包括:以石油醚-丙酮为洗脱液,按体积比10:1-1:1的比例梯度洗脱,TLC检测,得5个洗脱组分,其中展开剂为石油醚:丙酮=4:1,v/v。
9.根据权利要求3所述的制备方法,其特征在于,步骤6)中的高效液相制备色谱的条件包括:C18色谱柱,以乙腈-水为洗脱液,按所述乙腈和水的初始体积比为60:40,最终体积比为75:25进行梯度洗脱35min,流速为4mL/min。
10.权利要求1或2所述的化合物在制备抗肿瘤药物中的应用;
进一步,权利要求1或2所述的化合物在制备抑制人乳腺癌细胞MCF-7生长的药物中的应用。
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