CN117486897A - 海洋来源γ-吡喃酮类化合物Asperpyrone A及其制备方法与用途 - Google Patents
海洋来源γ-吡喃酮类化合物Asperpyrone A及其制备方法与用途 Download PDFInfo
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- CN117486897A CN117486897A CN202311320903.3A CN202311320903A CN117486897A CN 117486897 A CN117486897 A CN 117486897A CN 202311320903 A CN202311320903 A CN 202311320903A CN 117486897 A CN117486897 A CN 117486897A
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Abstract
本发明属于天然药物领域，公开了从真菌发酵产物中获得的结构如式I所示的γ‑吡喃酮类化合物或其药用盐。本发明以3株血液肿瘤细胞株(Namalwa,NALM‑6,Raji)为模型，测定所述的γ‑吡喃酮类化合物对肿瘤细胞增殖的抑制作用。实验结果表明，化合物具有较强的细胞毒性，表明γ‑吡喃酮类化合物或其药用盐对多种人血液肿瘤细胞具有强抑制活性。本发明还公开了所述的γ‑吡喃酮类化合物或其药用盐在制备预防和/或治疗血液肿瘤疾病的药物中的用途。
Description
技术领域
本发明属于天然药物领域，涉及γ-吡喃酮类化合物Asperpyrone A及其制备方法与用途，具体明涉及从微生物中，特别是从真菌发酵产物中获得的全新骨架γ-吡喃酮类化合物Asperpyrone A或其药用盐类及其在制备预防和/或治疗血液肿瘤疾病的药物中的用途。
背景技术
恶性肿瘤作为全球较大的公共卫生问题之一，极大地危害人类的健康，并将成为新世纪人类的第一杀手。血液肿瘤是起源于淋巴造血系统的恶性肿瘤，是一类高度异质性的疾病，其包括但不限于白血病、淋巴瘤和多发性骨髓瘤等。由于造血系统和免疫系统遍布全身，一旦发病即属于全身系统性疾病，没有可切除病灶，局部治疗无效，导致治疗手段非常有限，既往的治疗方法以放化疗、分子靶向治疗与骨髓移植为主。除少数成功骨髓移植患者可能治愈外，大部分血液肿瘤患者预后不佳，总体生存率较低，并且复发率高。
化学药物治疗作为治疗血液肿瘤的重要手段之一，在近三十年已经有了巨大的发展和进步，比如多发性骨髓瘤治疗药物蛋白酶体抑制剂硼替佐米、淋巴瘤治疗药物环磷酰胺、阿霉素、长春新碱等。但是这些抗肿瘤药也存在许多不良反应，比如脱发、呕吐、快速产生耐药性等等，导致化学药物无法达到预期的治疗效果。因此，新的抗肿瘤药物的研究与开发是目前药学领域的热点和难点问题之一。
海洋来源微生物的次级代谢产物是新药的重要来源，近年来，人们已从海绵共附生真菌中发现了大量具有新颖结构的化合物，包括生物碱、聚酮、萜类及多肽类等，其中一些化合物具有显著的抗肿瘤、抗疟原虫、抗菌及神经保护等生物活性。并且微生物次级代谢产物具有资源可持续性、不破坏生态环境等特点，因此拥有巨大的开发利用价值。
发明内容
发明人利用单菌株多产物策略(OSMAC)，对来自南海蜂海绵来源真菌榴红曲霉Aspergillus puniceus(菌株保藏编号：MCCC 3A00856)在不同培养基(包括PDB培养基，YPD培养基及大米培养基)下进行培养，借助HPLC-DAD-MS分析，以及Antibase数据库分析排重，发现该菌株中可能含有一类新的化学成分。以UV和LC-MS导向，综合使用各种色谱手段对可能新结构实现了定向分离。采用各种波谱学方法，以及计算13C NMR和单晶X-ray实验确定其为一类全新骨架、具有独特的6/6/6/6/6环系的γ-吡喃酮类化合物Asperpyrone A，并发现化合物Asperpyrone A对多种血液肿瘤具有较强的抑制作用，因此，该化合物在治疗人血液肿瘤相关疾病方面具有较好的应用前景。同时，本发明化合物Asperpyrone A的制备方法工艺简单，用时短，大大降低药品的成本，适合大规模生产。
本发明的目的是提供结构如式I所示的用作医药用途的γ-吡喃酮类化合物或其药用盐：
化合物Asperpyrone A的分子式为：C22H18O11，相对分子量为：458。
所述的γ-吡喃酮类化合物的药用盐为γ-吡喃酮类化合物与无机酸、有机酸、氨基酸或磺酸形成的盐；所述的无机酸为盐酸或硫酸；所述的有机酸为乙酸、三氟乙酸、柠檬酸、马来酸、草酸、琥珀酸、苯甲酸、酒石酸、富马酸、扁桃酸、抗坏血酸或苹果酸；所述的氨基酸为丙氨酸、天冬氨酸或赖氨酸；所述的磺酸为甲磺酸或对甲苯磺酸。
本发明所述的γ-吡喃酮类化合物是从南海蜂海绵来源真菌Aspergilluspuniceus(菌株保藏编号：MCCC 3A00856)大米发酵产物中首次分离得到的。
本发明的另一个目的是提供一种所述的γ-吡喃酮类化合物的制备方法，包括以下步骤：
真菌Aspergillus puniceus经大米发酵后得到大米发酵产物，大米发酵产物用95％乙醇超声提取2次，再用50％乙醇超声提取1次，将提取物进行过滤，合并滤液，滤液减压浓缩除去乙醇，得到混悬液，混悬液依次使用石油醚、乙酸乙酯萃取，将萃取液进行减压浓缩，分别得到石油醚部位萃取物、乙酸乙酯部位萃取物；乙酸乙酯部位萃取物进行MCI柱层析，以EtOH-H2O系统为洗脱剂进行梯度洗脱，得到45个组分，分别记为组分M1-组分M45；组分M16进行Sephadex LH-20柱层析，以甲醇为洗脱剂进行洗脱，得到8个亚组分，分别记为亚组分M16-1-亚组分M16-8；亚组分M16-4经半制备RP-HPLC(半制备反相高效液相色谱)纯化得到式I所示的γ-吡喃酮类化合物。
优选的，所述的大米发酵产物的制备：在温度28℃下，把菌株Aspergilluspuniceus(菌株保藏编号：MCCC 3A00856)接种在PDA培养基平皿上，28℃培养7天；将复苏的菌株接种到100mL灭菌后的PDB种子培养基中，在温度28℃下，以转速180rpm培养3天，得到种子培养液；将大米和去离子水按照质量比1:1混合得到大米培养基，按照种子培养液和大米培养基的体积质量比为1:20mL/g，将种子培养液接种到大米培养基中，在温度28℃下培养30天，得到大米发酵物。
优选的，每次超声提取的时间为40min。
优选的，用95％乙醇超声提取时，95％乙醇和大米发酵物的体积比为3:1。用50％乙醇超声提取时，50％乙醇和大米发酵物的体积比为3:1。
优选的，混悬液使用石油醚萃取3次，每次萃取时，石油醚和混悬液的体积比为3:1；混悬液使用乙酸乙酯萃取3次，每次萃取时，乙酸乙酯和混悬液的体积比为3:1。
优选的，进行MCI柱层析时，以MCI GEL CHP20/P120为填料，所述的EtOH-H2O系统依次为H2O、10％EtOH、20％EtOH、30％EtOH、45％EtOH、60％EtOH、80％EtOH和100％EtOH，利用HPLC对各流分进行检测，并对相似组分进行合并，最终得到45个组分(组分M1-M45)。
优选的，半制备RP-HPLC的色谱柱为Capcell Pak PFP，规格为：5μm，10×250mm；流动相为：15％ACN-0.1％TFA水溶液，等度洗脱，流速：1.5mL/min。
本发明以3株血液肿瘤细胞株(Namalwa,NALM-6,Raji)为模型，测定所述的γ-吡喃酮类化合物对肿瘤细胞增殖的抑制作用。实验结果表明，化合物具有较强的细胞毒性，对三株细胞的IC50分别为5.28、0.38和3.59μmol/L，表明所述的γ-吡喃酮类化合物或其药用盐对多种人血液肿瘤细胞具有强抑制活性，可用作制备抗血液肿瘤相关疾病的药物。与现有的同类抗肿瘤药物相比，所述的γ-吡喃酮类化合物具有更加优越的治疗效果。
本发明的另一个目的是提供所述的γ-吡喃酮类化合物或其药用盐在制备预防和/或治疗血液肿瘤疾病的药物中的用途。
所述的血液肿瘤疾病为白血病或淋巴瘤。
本发明的另一个目的是提供一种药物组合物，所述的药物组合物以所述的γ-吡喃酮类化合物或其药用盐作为活性成分或主要活性成分，与一种或多种药学上可接受的载体制成的抗血液肿瘤的制剂。
所述的药物组合物可用于抗血液肿瘤的临床治疗。
所述γ-吡喃酮类化合物或其药用盐也可与已知药物组成复方制剂用于相关癌症疾病的治疗。
所述的药物组合物中，所述的γ-吡喃酮类化合物或其药用盐的重量比为0.1～99.9％，药物可接受的载体的重量比为0.1～99.9％。
所述的药物组合物以适合药用的制剂形式存在。
所述的药物组合物的制剂为片剂、胶囊剂、颗粒剂、丸剂、散剂、膏剂、混悬剂、注射剂、粉针剂、栓剂、霜剂、滴剂或贴剂等。其中，所述的片剂为糖衣片剂、薄膜衣片剂、肠溶衣片剂或缓释片剂；所述的胶囊剂为硬胶囊剂、软胶囊剂、缓释胶囊剂；所述的粉针剂为冻干粉针剂。
本发明所述的药物组合物，作为制剂形式，每剂中含有的γ-吡喃酮类化合物或其药用盐的有效量为0.1～1000mg，所述每剂指的是，每一制剂单位，如片剂的每片，胶囊的每粒，也可指每次服用剂量，如每次服用100mg。
本发明的药物组合物在制备成粉剂、片剂、可分散粉剂、胶囊、扁囊剂、栓剂和软膏形式的固体或半固体药物制剂时，可使用固体载体。可使用的固体载体优选为选自稀释剂、调味剂、增溶剂、润滑剂、悬浮剂、粘合剂、膨胀剂等中的一种或多种物质，或可为包封物质。在粉状制剂中，在载体中含有5～70％的微粒化活性成分。适宜的固体载体包括碳酸镁、硬脂酸镁、滑石粉、蔗糖、乳糖、果胶、糊精、淀粉、明胶、甲基纤维素、羧甲基纤维素钠、低沸点蜡、可可脂等。由于它们易于给药，片剂，粉剂、扁囊剂和胶囊等代表最有利的口服固体制剂。
本发明的液体制剂包括溶液剂、悬液和乳液。例如，非胃肠道给药的注射制剂可为水或水-丙二醇溶液形式，调节其等渗度，pH等使适于活体的生理条件。液体制剂还可制成在聚乙二醇、水溶液中的溶液形式。可通过将活性成分溶解在水中，再加入适量的着色剂、调味剂、稳定剂和增稠剂，来制备口服水溶液。可将微粒化的活性成分分散在粘性物质如天然和合成胶、甲基纤维素、羧甲基纤维素钠和其它已知悬浮剂中制备适于口服的水悬液。
为了易于给药及剂量均一，将上述药物制剂配制成剂量单位形式是特别有利的。制剂的剂量单位形式指适于作为单一剂量的物理分离单位，每个单位含有产生所期望的治疗效果的计算好的预定量的活性成分。这种剂量单位形式可为包装形式，如片剂、胶囊或装在小管或小瓶中的粉剂，或装在管或瓶中的软膏、凝胶或霜剂。
虽然剂量单位形式中所含活性成分的量可以变化，但一般根据所选择活性成分的效力，调节在1～800mg范围内。
本领域技术人员可按常规方法确定适于某种情况的优选剂量。一般，开始治疗的量低于活性成分的最佳剂量，然后逐渐增加给药剂量，直到达到最佳治疗效果。为治疗需要，总的日剂量可一次给药或分数次给药。
附图说明
图1为化合物Asperpyrone A杀伤人Burkitt's淋巴瘤细胞(Namalwa)的浓度-生存率曲线。
图2为化合物Asperpyrone A杀伤B细胞急性淋巴细胞白血病细胞系(NALM-6)的浓度-生存率曲线。
图3为化合物Asperpyrone A杀伤人淋巴瘤细胞细胞系(Raji)的浓度-生存率曲线。
具体实施方式
下面通过实施例对本发明的技术方案进行详细说明，以下实施例是为了帮助本领域技术人员更好地理解本发明，但不以任何方式限制本发明。
本领域技术人员基于本发明的技术方案，适用于从任何微生物，而不限于从真菌Aspergillus puniceus的大米发酵物制备γ-吡喃酮类化合物。
实施例1
菌株来源：榴红曲霉Aspergillus puniceus，由中国海洋微生物菌种保藏管理中心共享菌株得到，菌株保藏编号：MCCC 3A00856的菌株。
PDB种子培养基：葡萄糖15g/L，马铃薯浸粉5g/L，氯化钠5g/L，蛋白胨10g/L,NaCl30g/L。
真菌Aspergillus puniceus的发酵：
在温度28℃下，把菌株Aspergillus puniceus(菌株保藏编号：MCCC 3A00856)接种在PDA(马铃薯葡萄糖琼脂)培养基平皿上，28℃培养7天；然后，将复苏的菌株接种到500mL三角瓶内，三角瓶中事先加入100mL灭菌后的PDB种子培养基，在28℃的摇床中，以转速180rpm培养3天，得到种子培养液。进行放大发酵：准备160个菌种发酵袋，每袋加入50g大米和50mL去离子水作为大米培养基，封口，用高压灭菌锅进行灭菌(121℃，15min)，冷却；最后，于超净台中，在每个发酵袋中加入5mL种子培养液，在28℃恒温培养箱中培养30天，得到大米发酵物。
实施例2
取实施例1获得的大米发酵物，用95％乙醇超声提取(95％乙醇和大米发酵物的体积比3:1)，提取2次，每次40min，再用50％乙醇超声提取(50％乙醇和大米发酵物的体积比3:1)40min，每次超声提取后进行过滤并合并滤液，然后减压浓缩至无醇味将乙醇除去，得到混悬液约2L，再依次使用石油醚、乙酸乙酯对该混悬液进行萃取，石油醚、乙酸乙酯分别萃取3次，每次萃取，有机溶剂和混悬液的体积比3:1，将萃取液进行减压浓缩，分别得到石油醚部位萃取物、乙酸乙酯部位萃取物。
实施例3
化合物1的分离、制备：
取实施例2乙酸乙酯部位萃取物进行MCI柱层析，以MCI GEL CHP20/P120为填料，平均粒径120μm，以EtOH-H2O进行梯度洗脱(H2O、10％EtOH、20％EtOH、30％EtOH、45％EtOH、60％EtOH、80％EtOH和100％EtOH)，利用HPLC对各流分进行检测，并对相似组分进行合并，得到45个组分(记为组分M1-组分M45)；
组分M16进行Sephadex LH-20柱层析，以甲醇为洗脱剂进行洗脱，得到8个亚组分(记为亚组分M16-1-亚组分M16-8)。
亚组分M16-4经半制备RP-HPLC(Capcell Pak PFP,粒径5μm,内径10mm×柱长250mm)，流动相：15％ACN-含0.1％TFA的H2O(即体积分数15％的乙腈和体积分数0.1％三氟乙酸的混合水溶液)，等度洗脱，流速：1.5mL/min，制备得化合物1，将化合物1命名为Asperpyrone A。
化合物1的结构鉴定：
化合物1为橙红色针状结晶(甲醇)，HR-ESI-MS测出准分子离子峰m/z 459.0905[M+H]+提示其分子组成为C22H18O11(calcd for C22H19O11,459.0922)，不饱和度为14。化合物1的1HNMR谱(600MHz,DMSO-d6)显示出2个三取代的双键[δH 6.54(1H,brt,J＝1.2Hz)和6.50(1H,brt,J＝1.2Hz)]；2个连氧亚甲基[δH 4.46(4H,overlap)]；2个孤立甲基[1.30(3H,s)和1.81(3H,s)]；1个连氧甲基[δH 3.30(3H,s)]；以及3个活泼[δH 5.46(1H,s),5.87(1H,t,J＝6Hz),5.83(1H,t,J＝6Hz)]质子的共振信号。化合物1的13C NMR谱显示出22个碳信号，并结合DEPT谱分析，表明除了上述氢谱中基团相对应的碳信号之外，还有13个季碳信号，包括3个共轭羰基碳(δC 195.5,170.2和168.8)；3个烯碳(δC 109.9,119.1,122.4)和5个连氧烯碳(δC154.7,142.4,141.4,140.4和140.2)；以及2个连氧的sp3季碳(δC 98.5和79.4)。
γ-吡喃酮类化合物Asperpyrone A经1D NMR、2D NMR、HRESIMS、IR、UV、ECD、单晶X衍射(铜靶)分析等多种现代波谱技术，确定了化合物Asperpyrone A的化学结构如式I所示。
表1.化合物1的NMR数据(600MHz,DMSO-d6)
实施例4
化合物1杀伤肿瘤细胞能力测试
Cell Counting Kit是一种基于水溶性四唑盐(WST)的细胞增殖和细胞毒性检测方法。它的原理是在电子耦合试剂存在的情况下，WST可以被线粒体内的一些脱氢酶还原为可溶性的橙黄色甲臜(formazan)。甲臜的数量与活细胞的数量成正比。细胞增殖越快、细胞毒性越小、细胞数量越多，则颜色越深，颜色的深浅与细胞数量呈现良好的线性关系。
药物溶液的配制：取2mg化合物Asperpyrone A，用100μL DMSO配成浓度为20mg/mL(43.7mM)的化合物Asperpyrone A储液，用完全培养基将化合物Asperpyrone A储液稀释104倍至4.37μM，之后依次倍比稀释得到系列浓度的药物溶液。
细胞：Namalwa细胞(人Burkitt’s淋巴瘤细胞)、NALM-6细胞(B细胞急性淋巴细胞白血病细胞系)、Raji细胞(人淋巴瘤细胞系)，都采用RPMI-1640完全培养基培养。
操作步骤：将对数生长期的Namalwa细胞(人Burkitt’s淋巴瘤细胞)、NALM-6细胞(B细胞急性淋巴细胞白血病细胞系)、Raji细胞(人淋巴瘤细胞系)，用0.25％胰酶消化，调整细胞浓度为2.5×104个/mL。铺板，将调整浓度后的细胞培养液加入96孔板中，每孔200μL，周边以无血清的培养基封闭，将细胞置于培养箱中培养至对数期。弃去原培养液，给药组每孔加入200μL不同浓度的药物溶液。每个浓度设立3个复孔，同时设立空白对照组和溶剂对照组，空白对照组继完全培养基培养，溶剂对照组使用含DMSO(200μL体系含有0.02μLDMSO)的完全培养基培养。在细胞培养箱中继续培养48h。取出96孔板，弃上清液，每孔加入100μL含有10％的CCK溶液的完全培养基，在细胞培养箱中继续培养1-4小时，用酶标仪测定450nm处的吸光值。
肿瘤细胞生存率％＝(给药孔荧光吸收值-空白孔的荧光吸收值)/(溶剂对照孔的荧光吸收值-空白孔的荧光吸收值)×100％
测定每个浓度下的肿瘤细胞的生存率，使用SigmaPlot软件计算得到待测样品的IC50值。
实验结果：化合物Asperpyrone A杀伤3株肿瘤细胞的IC50值以及浓度-生存率曲线分别如表2和图1所示。表明化合物Asperpyrone A对人血液肿瘤细胞具有强抑制活性，可用作制备抗血液肿瘤相关疾病的药物。
表2.化合物Asperpyrone A杀伤3株肿瘤细胞的IC50值
| 肿瘤细胞 | IC50(μmol/L) |
| Namalwa细胞 | 5.28 |
| NALM-6细胞 | 0.38 |
| Raji细胞 | 3.59 |
Claims (10)
1.结构如式I所示的γ-吡喃酮类化合物或其药用盐：
2.根据权利要求1所述的γ-吡喃酮类化合物或其药用盐，其特征在于：所述的γ-吡喃酮类化合物的药用盐为γ-吡喃酮类化合物与无机酸、有机酸、氨基酸或磺酸形成的盐。
3.根据权利要求2所述的γ-吡喃酮类化合物或其药用盐，其特征在于：所述的无机酸为盐酸或硫酸；所述的有机酸为乙酸、三氟乙酸、柠檬酸、马来酸、草酸、琥珀酸、苯甲酸、酒石酸、富马酸、扁桃酸、抗坏血酸或苹果酸；所述的氨基酸为丙氨酸、天冬氨酸或赖氨酸；所述的磺酸为甲磺酸或对甲苯磺酸。
4.一种权利要求1所述的γ-吡喃酮类化合物的制备方法，其特征在于：包括以下步骤：真菌Aspergillus puniceus经大米发酵后得到大米发酵产物，大米发酵产物用95％乙醇超声提取2次，再用50％乙醇超声提取1次，将提取物进行过滤，合并滤液，滤液减压浓缩除去乙醇，得到混悬液，混悬液依次使用石油醚、乙酸乙酯萃取，将萃取液进行减压浓缩，分别得到石油醚部位萃取物、乙酸乙酯部位萃取物；乙酸乙酯部位萃取物进行MCI柱层析，以EtOH-H2O系统为洗脱剂进行梯度洗脱，得到45个组分，分别记为组分M1-组分M45；组分M16进行Sephadex LH-20柱层析，以甲醇为洗脱剂进行洗脱，得到8个亚组分，分别记为亚组分M16-1-亚组分M16-8；亚组分M16-4经半制备RP-HPLC纯化得到式I所示的γ-吡喃酮类化合物。
5.根据权利要求4所述的γ-吡喃酮类化合物的制备方法，其特征在于：进行MCI柱层析时，以MCI GEL CHP20/P120为填料。
6.根据权利要求4所述的γ-吡喃酮类化合物的制备方法，其特征在于：半制备RP-HPLC的色谱柱为Capcell Pak PFP，规格为：5μm，10×250mm；流动相为：15％乙腈-0.1％三氟乙酸水溶液，等度洗脱，流速：1.5mL/min。
7.权利要求1所述的γ-吡喃酮类化合物或其药用盐在制备预防和/或治疗血液肿瘤疾病的药物中的用途。
8.根据权利要求7所述的用途，其特征在于：所述的血液肿瘤疾病为白血病或淋巴瘤。
9.一种药物组合物，其特征在于：所述的药物组合物以权利要求1所述的γ-吡喃酮类化合物或其药用盐作为活性成分或主要活性成分，与药学上可接受的载体制成的制剂。
10.根据权利要求9所述的药物组合物，其特征在于：所述的制剂为片剂、胶囊剂、颗粒剂、丸剂、散剂、膏剂、混悬剂、注射剂、粉针剂、栓剂、霜剂、滴剂或贴剂。
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