CN113444135B - 一种牛樟芝四环三萜糖苷及其酶催化制备方法和用途 - Google Patents
一种牛樟芝四环三萜糖苷及其酶催化制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种牛樟芝四环三萜糖苷及其酶催化制备方法和用途。为解决牛樟芝四环三萜存在的水溶性差、生物利用度低等问题，本发明采用绿色、高效、环保的糖基转移酶YjiC1对牛樟芝三萜进行糖基化修饰，获得了一系列结构新颖的牛樟芝四环三萜糖苷，具有良好的抑制肿瘤增殖的活性，生物利用度显著提高。这类牛樟芝四环三萜糖苷或其药学上可接受的盐、酯、溶剂化物、立体异构体、互变异构体、前药以及其混合物可用于制备治疗/预防肿瘤药物及保健品。
Description
技术领域
本发明涉及四环三萜糖苷，具体涉及一种牛樟芝四环三萜糖苷及其酶催化制备方法和药物应用。
背景技术
恶性肿瘤是严重危害人类健康的疾病之一，具有发病率高和致死率高等特点。现代临床治疗以化疗为主，会产生较大的毒副作用，降低患者的生存质量。天然产物在抗肿瘤、降低不良反应等方面具有独特的优势，是新药发现的重要来源。
牛樟芝(Antrodia camphorata或A.cinnamomea)又称樟芝、血灵芝，是中国台湾地区特有的珍贵药用真菌。在中国台湾地区有数百年药用历史，民间普遍用于抗癌、保肝、解酒、增强免疫。牛樟芝含有丰富的四环三萜类化合物，其结构特异，主要分为麦角甾烷型和羊毛甾烷型。其中麦角甾烷型三萜骨架仅29个碳，C4位有特异的α-甲基取代，支链在C24与C28位形成双键，存在C25立体异构现象，此类结构仅存在于多孔菌科真菌；羊毛甾烷型三萜骨架大多由31个碳组成，在C24位有特异的亚甲基取代，存在Δ7和Δ7(9)的成对现象，氧化程度明显低于灵芝三萜。
三萜类化合物被认为是牛樟芝的主要活性成分，具有抗癌、保肝、抗炎、免疫调节等多种药理活性，其中抗肿瘤作用备受关注。去氢齿孔酸(Dehydroeburicoic acid)是牛樟芝中含量最高的三萜类成分之一，属于羊毛甾烷型，经小鼠和细胞实验证实有一定的抗肿瘤活性(Du YC等,Phytomedicine 2012,19,788-796)。虽然牛樟芝三萜的骨架独特、活性明确，但该类成分存在水溶性差、生物利用度低等问题，限制了其药物开发。我们的药代动力学研究也表明，部分牛樟芝三萜，特别是低极性的羊毛甾烷，在药材提取物中含量较高，服用牛樟芝后血药浓度却很低(Qiao X等,RSC Advance 2015,5,47040-47052)。因此，对牛樟芝三萜进行结构修饰具有重要的现实意义和良好的应用前景。
发明内容
本发明的目的在于克服现有牛樟芝四环三萜水溶性、生物利用率低的问题，提供一系列结构新颖的经过酶催化修饰得到的牛樟芝四环三萜糖苷，并提供了其制备方法和药物应用。本发明的发明人尤其出乎预料地发现，经糖基化修饰的牛樟芝三萜皂苷可以显著提高药理活性、溶解性，并改善吸收与生物利用率，由此完成了本发明。
本发明的第一个方面涉及式(I)或式(II)化合物：
其中，R1选自-OH、＝O、O-糖基；R2和R3各自独立选自H、C1-6烷基、-OH；R4、R5和R6各自独立选自C1-6烷基、-COOH；R7选自H、C1-6烷基；R8选自H、-OH、O-糖基；R9选自H、-OH、O-糖基；R10选自H和-OH；条件是，所述R1、R8、R9中至少一者是O-糖基。
本发明的式(I)、式(II)化合物或其药学上可接受的盐、酯、溶剂化物、立体异构体、互变异构体、前药以及它们的混合物为有效成分制备的药物也属于本发明的保护范围。
上述O-糖基中，所述糖基优选为六碳糖基，更优选为吡喃葡萄糖基，最优选为吡喃葡萄糖-1-基。
上述C1-6烷基例如甲基、乙基、丙基、异丙基、丁基等。
本发明的式(I)、式(II)化合物可以分别是如下式(I’)、式(II’)所示立体构型的化合物：
其中R1至R10如前所定义。
在本发明的一些具体实施方案中，本发明的式(I)、式(II)化合物分别是如下式(IA)、式(IIA)化合物：
其中R1、R2、R5、R6、R8、R10如前所定义，且式(IA)中R1和R8至少一者是O-糖基。
上述式(IA)、式(IIA)化合物可以为式(IA’)、式(IIA’)所示立体构型的化合物：
其中R1、R2、R5、R6、R8、R10如前所定义。
在本发明的一些具体实施方案中，本发明提供的牛樟芝四环三萜糖苷选自下面所示的化合物：
本发明的另一个方面涉及合成本发明化合物或其药学上可接受的盐、酯、溶剂化物、立体异构体、互变异构体的方法，其包括以下步骤：在糖基转移酶的作用下将来源于牛樟芝的四环三萜化合物与糖供体反应，，生物催化合成式(I)或式(II)结构的化合物。这些牛樟芝四环三萜化合物的结构与式(I)、式(II)化合物相比，是在相应的R1、R8、R9当中至少有一个取代基为-OH。
其中，所述糖基转移酶优选为来自枯草芽孢杆菌的糖基转移酶YjiC1(GenBankAccession Number JX982974)，这是一种特异性的葡萄糖基转移酶，可以根据文献(Li K等,Adv.Synth. Catal.2017,359,3765-3772)公开的方法制备得到。糖基转移酶YjiC1选择性地在牛樟芝四环三萜化合物的R1、R8、R9中至少一处位点上催化O-糖基化，合成式(I)或式(II)结构的牛樟芝四环三萜糖苷。所述糖供体优选为核苷酸糖，更优选为UDP-糖基供体，例如UDP-葡萄糖(UDP-Glc)、UDP-半乳糖(UDP-Gal)等。
在本发明的一个具体实施方案中，采用的牛樟芝四环三萜化合物如下所示：去氢齿孔酸、去氢硫色多孔菌酸、25R-antcin K、25S-antcin K、25R-camphoratin A、25S-camphoratin A、 antcamphin E、25S-antcin C。
上述酶催化制备牛樟芝四环三萜糖苷的方法具体可以是：将所述牛樟芝四环三萜化合物溶解于溶媒(如缓冲液，例如NaH2PO4-Na2HPO4缓冲液)中，加入糖供体和糖基转移酶YjiC1，使其反应一段时间(例如在37℃、200rpm的摇床中反应8h)，终止反应(例如通过加入甲醇终止反应)，萃取(例如用乙酸乙酯萃取)，然后进行分离纯化(例如采用半制备液相色谱法)，得到所述牛樟芝四环三萜糖苷。选择性地在R1、R8、R9中至少一处位点上催化O-糖基化，合成式(I)或式(II)结构的牛樟芝四环三萜糖苷。
本发明的另一个方面涉及一种药物组合物或保健食品组合物，其中含有本发明的牛樟芝四环三萜糖苷化合物或其药学上可接受的盐、酯、溶剂化物、立体异构体、互变异构体。
在该方面的一个具体实施方案中，所述药物组合物或保健食品组合物可采用各种形式，如片剂、散剂、丸剂、注射剂、胶囊剂、膜剂、栓剂、膏剂、冲剂等剂型；其在形状方面可采用各种形状。上述各种形式均可以按照药学领域或食品领域的常规方法制备。
在该方面的一个具体实施方案中，所述药物组合物或保健食品组合物中还可以加入一种或多种药学上可接受的载体或辅料。所述载体或辅料包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
本发明的另一个方面涉及本发明的牛樟芝四环三萜糖苷化合物或其药学上可接受的盐、酯、溶剂化物、立体异构体、互变异构体在制备抗肿瘤药物或保健食品中的应用。
本发明对前述本发明化合物的体外抗肿瘤活性进行了初步测试和评价，选取了4种人源肿瘤细胞(人早幼粒白血病HL60细胞、人脑胶质瘤U251细胞、人结肠癌S480细胞、人乳腺癌MCF7细胞)，结果显示本发明具体实施方案中的化合物1对于HL60细胞、U251细胞、SW480细胞增殖活性的抑制作用显著增强，优于对照化合物(化合物1的苷元，去氢齿孔酸，dehydroeburicoic acid)。提示本发明提供的四环三萜糖苷可用于制备治疗和/或预防肿瘤的药物及保健品。
本发明人进一步对本发明的化合物1与对照化合物(去氢齿孔酸)进行了体内代谢研究， Balb/c小鼠口服给药，检测血药浓度，结果表明本发明化合物的生物利用度较对照化合物明显提高。
与现有技术相比，发明人提供了一系列结构新颖的樟芝四环三萜糖苷及其酶催化合成方法，这些糖苷有良好的抑制肿瘤增殖的活性，生物利用度明显提高，优于催化前的底物苷元，具有较好的潜在要用价值，有望用于抗肿瘤药物的制备。
附图说明
图1为实施例1分离纯化的YjiC1酶的SDS-PAGE检测图，其中M泳道为分子量标记物，Y泳道为YjiC1酶。
图2为实施例2中HPLC检测牛樟芝四环三萜糖苷化合物(化合物1–10)的生物转化图。
图3为化合物1的1H NMR谱。
图4为化合物1的13C NMR谱。
图5为化合物2的1H NMR谱。
图6为化合物2的13C NMR谱。
图7为化合物3的1H NMR谱。
图8为化合物3的13C NMR谱。
图9为化合物4的1H NMR谱。
图10为化合物4的13C NMR谱。
图11为化合物5的1H NMR谱。
图12为化合物5的13C NMR谱。
图13为化合物6的1H NMR谱。
图14为化合物6的13C NMR谱。
图15为化合物7的1H NMR谱。
图16为化合物7的13C NMR谱。
图17为化合物8的1H NMR谱。
图18为化合物8的13C NMR谱。
图19为化合物9的1H NMR谱。
图20为化合物9的13C NMR谱。
图21为化合物10的1H NMR谱。
图22为化合物10的13C NMR谱。
图23为化合物1与对照化合物去氢齿孔酸分别灌胃给药后小鼠血浆的去氢齿孔酸浓度。
具体实施方式
下面结合实施例进一步阐述本发明，应理解，以下实施例仅用于说明本发明而不用于限制本发明的保护范围。
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、YjiC1酶的纯化。
一、实验材料和方法
YjiC1酶(GenBank Accession Number JX982974)来自枯草芽孢杆菌(Bacillussubtilis) (Li K等,Adv.Synth.Catal.2017,359,3765-3772)，取枯草芽孢杆菌菌落接种于含50μg/mL 卡那霉素的LB培养基(3mL)中，置于37℃、200rpm的摇床上过夜培养。将培养好的种子液加入500mLLB液体培养基中(1:100,v:v)，继续于37℃、200rpm的摇床上培养至OD600 值约0.4-0.6；加入终浓度为0.2mM的IPTG，在18℃、180rpm摇床上继续培养24h，诱导蛋白表达。收集诱导表达的培养物，8000rpm、4℃离心20min收集菌体，用50mmol/L NaH2PO4-Na2HPO4缓冲液(10-15mL，含10mM咪唑和300mM NaCl，pH 8.0)溶解成悬浮液，冰上超声破碎(Φ6，35％，3s，3s，15–20min)，10000rpm、4℃下离心30min，收集上清液即为粗酶液。采用Ni-NTA琼脂糖树脂纯化蛋白，将上述40mL粗酶液中加入2mL Ni-NTA树脂，冰浴1h，用50mmol/L的NaH2PO4-Na2HPO4缓冲液(pH 8.0，含10mM咪唑和300mM NaCl)进行纯化，继续用50mM NaH2PO4-Na2HPO4(Wash buffer，20mM咪唑， 300mM NaCl，pH 8.0)洗脱，收集各流份。最后用含有不同咪唑浓度50mM、100mM、150 mM、200mM、250mM和500mM的Elution buffer(洗脱缓冲液：pH 8.0，50mM NaH2PO4-Na2HPO4，300mM NaCl)洗脱，收集各流份。
二、实验结果
YjiC1酶产物采用SDS-PAGE检测结果如图1所示，蛋白纯度＞90％。
实施例2、糖基转移酶YjiC1催化合成三萜糖苷(化合物1–10)的分析反应
一、实验材料和方法
本方法中提及的所有化学试剂均购自北京化工厂。各三萜糖苷的合成参见实施例3-8。
反应体系(100μL)：三萜底物(0.2mM)，UDP-葡萄糖(0.5mM)，YjiC1纯酶(50μg)，50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，37℃反应8h；加入200μL甲醇终止反应，涡旋振荡，14,000rpm离心30min，取上清液进行HPLC检测。每个底物进行两个平行反应，转化率的计算通过HPLC谱图中底物和产物的峰面积进行。
色谱条件：
仪器：Waters e2695高效液相色谱仪；
色谱柱：Agilent ZORBAX SB-C18(4.6mm×250mm,5μm)柱，连接Agilent ZORBAXSB-C18预柱(4.6mm×12.5mm,5μm)；
流动相：乙腈(A)-0.1％甲酸水溶液(B)；
洗脱程序：0–20min，20％A–100％A；20–25min，100％A；
流速：1mL/min；
进样量：10μL；
检测波长：254nm。
二、实验结果。
糖基转移酶YjiC1酶对牛樟芝四环三萜类化合物具有较高的催化转化率，其中麦角甾烷型四环三萜(化合物5–10)的催化转化率在92％以上，羊毛甾烷型四环三萜(化合物1–4) 的催化转化率在66％以上，见图2。
实施例3、三萜糖苷化合物1的合成
一、实验材料和方法。
取适量去氢齿孔酸(47.50mg,0.10mM)溶解于250mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP-葡萄糖(122.0mg,0.20mM)和YjiC1纯酶液(450 μg)，于37℃、200rpm的摇床上反应8h，加入甲醇(2倍体积)终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，甲醇复溶。
二、实验结果
采用半制备液相色谱YMC Pack ODS-A column(10mm×250mm,5μm)，色谱条件： 0-30min,45％-100％乙腈水溶液，30-40min,100％乙腈；检测波长：254nm，流速：2mL/min，得到化合物1(37.8mg,60.0％,白色固体)，并采用NMR(见图3和4)和HRESIMS确定其结构。
产率：60.0％,37.8mg。HRESIMS:m/z 629.4036([M-H]-,calcd.for C37H57O8,629.4053).1H NMR(400MHz,pyridine-d5)δ:2.38,2.55(2H,m,H-1),1.87,2.34(2H,m,H-2),3.40(1H,dd,J＝ 11.8,3.9Hz,H-3),1.22(1H,m,H-5),5.59(1H,d,J＝6.2Hz,H-7),2.38(1H,overlapped,H-12a), 2.54(1H,overlapped,H-12b),0.99(3H,overlapped,H-18),0.99(3H,overlapped,H-19),1.03(3H, overlapped,H-26),1.03(3H,overlapped,H-27),4.90(1H,s,H-28a),4.94(1H,s,H-28b),1.32(3H, m,H-29),1.10(1H,s,H-30),1.09(1H,s,H-31),4.93(1H,d,J＝7.8Hz,H-1'),4.05(1H,m,H-2'), 4.01(1H,m,H-3'),4.26(2H,m,H-4',5'),4.43,4.59(2H,m,H-6').13C NMR(100MHz, pyridine-d5)δ:36.3(C-1),27.5(C-2),89.2(C-3),39.8(C-4),50.2(C-5),23.6(C-6),121.5(C-7), 143.2(C-8),146.8(C-9),37.8(C-10),117.0(C-11),36.5(C-12),44.6(C-13),50.9(C-14),32.0 (C-15),27.6(C-16),48.5(C-17),16.6(C-18),23.2(C-19),49.5(C-20),178.9(C-21),33.1(C-22), 32.1(C-23),156.2(C-24),34.6(C-25),22.3(C-26),22.4(C-27),107.4(C-28),28.7(C-29),17.5 (C-30),26.2(C-31),107.4(C-1'),76.2(C-2'),78.7(C-3'),72.2(C-4'),79.1(C-5'),63.4(C-6').
实施例4、三萜糖苷化合物2-4的合成
一、实验材料和方法。
取适量去氢硫色多孔菌酸(Dehydrosulphurenic acid)(20.50mg,0.04mM)溶解于190mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP-葡萄糖(50.3mg,0.08 mM)和YjiC1纯酶液(350μg)，于37℃、200rpm的摇床上反应8h，加入甲醇(2倍体积) 终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，甲醇复溶。
二、实验结果
采用半制备液相色谱YMC Pack ODS-A column(10mm×250mm,5μm)，色谱条件： 0-35min,25％-100％乙腈水溶液，35-45min,100％乙腈；检测波长：254nm，流速：2mL/min，得到化合物2(1.4mg,5％,白色固体)、3(8.7mg,34％,白色固体)、4(13.1mg,41％,白色固体)，并采用NMR(图5-10)和HRESIMS确定他们的结构。
化合物2产率：5％,1.4mg。HRESIMS:m/z 645.4007([M-H]-,calcd.for C37H57O9,645.4003).1H NMR(400MHz,pyridine-d5)δ:2.41,2.73(2H,m,H-1),1.88,2.35(2H,m,H-2),4.69(1H,dd,J＝11.6,2.5Hz,H-3),1.27(1H,m,H-5),6.48(1H,d,J＝6.4Hz,H-7),1.40(1H,overlapped,H-12a),1.83(1H,overlapped,H-12b),1.13(3H,s,H-18),1.03(3H,overlapped,H-19), 1.01(3H,overlapped,H-26),1.01(3H,overlapped,H-27),4.87(1H,s,H-28a),4.91(1H,s,H-28b), 1.30(3H,s,H-29),1.09(1H,s,H-30),1.48(1H,s,H-31),4.93(1H,d,J＝7.7Hz,H-1'),4.05(1H, m,H-2'),4.01(1H,m,H-3'),4.25(2H,m,H-4',H-5'),4.43,4.58(2H,m,H-6').13C NMR(100 MHz,pyridine-d5)δ:37.2(C-1),27.5(C-2),89.2(C-3),39.8(C-4),50.2(C-5),23.6(C-6),122.5 (C-7),142.3(C-8),147.2(C-9),37.9(C-10),116.7(C-11),36.6(C-12),45.3(C-13),52.9(C-14), 74.1(C-15),40.0(C-16),46.9(C-17),17.2(C-18),23.3(C-19),49.3(C-20),179.1(C-21),33.1 (C-22),32.2(C-23),156.2(C-24),34.6(C-25),22.3(C-26),22.4(C-27),107.5(C-28),28.7(C-29),17.5(C-30),18.7(C-31),107.4(C-1'),76.2(C-2'),78.7(C-3'),72.2(C-4'),79.2(C-5'),63.4(C-6').
化合物3产率：34％,8.7mg。HRESIMS:m/z 645.3989([M-H]-,calcd.for C37H57O9,645.4003).1H NMR(400MHz,pyridine-d5)δ:2.30,2.75(2H,m,H-1),1.14,1.91(2H,m,H-2),3.45(1H,t,J＝7.6Hz,H-3),1.27(1H,m,H-5),6.90(1H,d,J＝6.1Hz,H-7),2.40(1H,overlapped,H-12a),2.66(1H,overlapped,H-12b),1.14(3H,s,H-18),1.10(3H,s,H-19),1.00(3H, overlapped,H-26),1.00(3H,overlapped,H-27),4.83(1H,s,H-28a),4.87(1H,s,H-28b),1.06(3H, m,H-29),1.09(1H,s,H-30),1.40(1H,s,H-31),5.08(1H,d,J＝7.8Hz,H-1'),4.10(1H,m,H-2'), 4.03(1H,m,H-3'),4.33(1H,m,H-4'),4.32(1H,m,H-5'),4.42,4.55(2H,m,H-6').13C NMR(100 MHz,pyridine-d5)δ:37.8(C-1),29.0(C-2),78.4(C-3),39.6(C-4),49.9(C-5),24.1(C-6),123.8 (C-7),141.4(C-8),147.4(C-9),38.3(C-10),116.0(C-11),36.7(C-12),44.6(C-13),53.2(C-14), 85.8(C-15),61.6(C-16),46.9(C-17),17.2(C-18),23.4(C-19),49.2(C-20),179.1(C-21),33.0 (C-22),32.1(C-23),155.9(C-24),34.5(C-25),22.2(C-26),22.3(C-27),107.5(C-28),29.1(C-29), 16.9(C-30),19.1(C-31),106.9(C-1'),75.8(C-2'),78.7(C-3'),72.2(C-4'),79.3(C-5'),63.3(C-6').
化合物4产率：41％,13.1mg。HRESIMS:m/z 807.4523([M-H]-,calcd.forC43H67O14, 807.4531).1H NMR(400MHz,pyridine-d5)δ:1.02,2.12(2H,m,H-1),1.03(2H,m,H-2),3.39 (1H,dd,J＝11.8,3.9Hz,H-3),1.23(1H,m,H-5),6.87(1H,d,J＝5.8Hz,H-7),2.40(1H, overlapped,H-12a),2.65(1H,overlapped,H-12b),1.12(3H,s,H-18),1.02(3H,overlapped,H-19), 1.01(3H,overlapped,H-26),1.01(3H,overlapped,H-27),4.83(1H,s,H-28a),4.87(1H,s,H-28b), 1.17(3H,s,H-29),1.05(1H,s,H-30),1.40(1H,s,H-31),4.92(1H,d,J＝7.7Hz,H-1'),4.04(1H, m,H-2'),4.01(1H,m,H-3'),4.25(2H,m,H-4',5'),4.43,4.56(2H,m,H-6'),5.06(1H,d,J＝7.8 Hz,H-1”),4.10(1H,m,H-2”),4.02(1H,m,H-3”),4.32(1H,m,H-4”),4.33(1H,m,H-5”),4.37, 4.56(2H,m,H-6”).13C NMR(100MHz,pyridine-d5)δ:37.7(C-1),27.5(C-2),89.3(C-3),39.8 (C-4),49.9(C-5),23.8(C-6),123.4(C-7),141.4(C-8),147.1(C-9),37.8(C-10),116.1(C-11),36.7 (C-12),44.6(C-13),53.2(C-14),85.9(C-15),62.3(C-16),46.9(C-17),17.2(C-18),23.2(C-19), 49.2(C-20),179.1(C-21),33.0(C-22),32.1(C-23),155.9(C-24),34.5(C-25),22.2(C-26),22.4 (C-27),107.5(C-28),28.6(C-29),17.4(C-30),19.1(C-31),107.4(C-1'),76.1(C-2'),78.7(C-3'), 72.2(C-4'),79.1(C-5'),63.4(C-6'),107.0(C-1”),75.8(C-2”),73.7(C-3”),72.2(C-4”),79.3(C-5”), 63.3(C-6”).
实施例5、三萜糖苷化合物5、6的合成
一、实验材料和方法。
分别称取适量25R-antcin K(9.5mg,0.02mM),25S-antcin K(9.9mg,0.02mM)溶解于 120mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP-葡萄糖(25.5mg,0.04mM)和YjiC1纯酶液(240μg)，于37℃、200rpm的摇床中反应8h，加入甲醇(2 倍体积)终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，甲醇复溶。
二、实验结果
采用半制备液相色谱YMC Pack ODS-Acolumn(10mm×250mm,5μm)，色谱条件： 0-35min,15％-80％乙腈水溶液，35-45min,80％-100％乙腈水溶液；检测波长：254nm，流速：2mL/min，分别得到化合物5(11.7mg,90％,白色固体)、6(11.3mg,92％,白色固体)，并采用NMR(图11-14)和HRESIMS确定他们的结构。
化合物5产率：90％,11.7mg。HRESIMS:m/z 649.3582([M-H]-,calcd.forC35H53O11, 649.3588).1H NMR(400MHz,pyridine-d5)δ:1.25,3.13(2H,m,H-1),1.97,2.78(2H,m,H-2), 4.09(1H,brs,H-3),2.22(1H,m,H-5),4.63(1H,t,J＝8.4Hz,H-7),2.44(1H,d,J＝13.2Hz, H-12a),2.92(1H,d,J＝13.3Hz,H-12b),2.72(1H,m,H-14),0.82(3H,s,H-18),2.08(3H,s, H-19),0.85(3H,d,J＝6.0Hz,H-21),3.47(3H,q,J＝6.9Hz,H-25),1.51(3H,d,J＝7.0Hz, H-27),5.06(1H,overlapped,H-28a),5.23(1H,s,H-28b),1.84(3H,s,H-29),5.05(1H,overlapped, H-1'),3.99(1H,m,H-2'),4.04(1H,m,H-3'),4.19(1H,m,H-4'),4.26(1H,m,H-5'),4.30,4.55(2H, m,H-6').13C NMR(100MHz,pyridine-d5)δ:30.0(C-1),27.0(C-2),74.9(C-3),74.4(C-4),43.7 (C-5),29.6(C-6),79.7(C-7),151.4(C-8),146.7(C-9),38.6(C-10),201.9(C-11),59.0(C-12),48.5 (C-13),54.3(C-14),25.1(C-15),28.5(C-16),55.3(C-17),12.7(C-18),21.2(C-19),36.6(C-20), 18.9(C-21),34.9(C-22),32.1(C-23),150.8(C-24),47.1(C-25),177.2(C-26),17.5(C-27),110.8 (C-28),28.3(C-29),105.6(C-1'),76.0(C-2'),78.5(C-3'),72.8(C-4'),79.0(C-5'),63.7(C-6').
化合物6产率：92％,11.3mg。HRESIMS:m/z 649.3594([M-H]-,calcd.forC35H53O11, 649.3588).1H NMR(400MHz,pyridine-d5)δ:1.25,3.13(2H,m,H-1),1.97,2.78(2H,m,H-2), 4.09(1H,brs,H-3),2.22(1H,m,H-5),4.63(1H,t,J＝8.4Hz,H-7),2.44(1H,d,J＝13.2Hz, H-12a),2.92(1H,d,J＝13.3Hz,H-12b),2.72(1H,m,H-14),0.82(3H,s,H-18),2.08(3H,s, H-19),0.85(3H,d,J＝6.0Hz,H-21),3.47(3H,q,J＝6.9Hz,H-25),1.51(3H,d,J＝7.0Hz, H-27),5.06(1H,overlapped,H-28a),5.23(1H,s,H-28b),1.84(3H,s,H-29),5.05(1H,overlapped, H-1'),3.99(1H,m,H-2'),4.04(1H,m,H-3'),4.19(1H,m,H-4'),4.26(1H,m,H-5'),4.30,4.55(2H, m,H-6').13C NMR(100MHz,pyridine-d5)δ:30.0(C-1),27.0(C-2),74.9(C-3),74.4(C-4),43.7 (C-5),29.6(C-6),79.7(C-7),151.4(C-8),146.7(C-9),38.6(C-10),201.9(C-11),59.0(C-12),48.5 (C-13),54.3(C-14),25.1(C-15),28.5(C-16),55.3(C-17),12.7(C-18),21.2(C-19),36.6(C-20), 18.9(C-21),34.9(C-22),32.1(C-23),150.8(C-24),47.1(C-25),177.2(C-26),17.5(C-27),110.8 (C-28),28.3(C-29),105.6(C-1'),76.0(C-2'),78.5(C-3'),72.8(C-4'),79.0(C-5'),63.7(C-6').
实施例6、三萜糖苷化合物7、8的合成
一、实验材料和方法。
分别称取适量25R-camphoratin A(10.0mg,0.02mM)和25S-camphoratin A(10.4mg,0.02 mM)溶解于210mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP- 葡萄糖(25.9mg,0.04mM)和YjiC1纯酶液(250μg)，于37℃、200rpm的摇床上反应8h，加入甲醇(2倍体积)终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，甲醇复溶。
二、实验结果
采用半制备液相色谱YMC Pack ODS-A column(10mm×250mm,5μm)，色谱条件： 0-35min,15％-80％乙腈水溶液，35-45min,80％-100％乙腈水溶液；检测波长：254nm，流速：2mL/min，得到化合物7(11.6mg,89％,白色固体)、8(11.0mg,85％,白色固体)，并采用 NMR(图15-18)和HRESIMS确定他们的结构。
化合物7产率：89％,11.6mg。HRESIMS:m/z 649.3579([M-H]-,calcd.forC35H53O11, 649.3588).1H NMR(400MHz,pyridine-d5)δ:1.97,2.79(2H,m,H-1),1.86(2H,m,H-2),3.89 (1H,overlapped,H-3),1.56(2H,m,H-4),2.09(1H,m,H-5),4.45(1H,overlapped,H-7),4.41(1H, s,H-12),3.56(1H,m,H-14),0.90(3H,s,H-18),1.56(3H,s,H-19),1.06(3H,d,J＝6.3Hz,H-21), 3.45(3H,q,J＝7.0Hz,H-25),1.51(3H,d,J＝7.0Hz,H-27),5.04(1H,overlapped,H-28a),5.23 (1H,s,H-28b),1.26(3H,d,J＝6.7Hz,H-29),5.00(1H,d,J＝7.7Hz,H-1'),4.06(1H,m,H-2'), 4.00(1H,m,H-3'),4.29(1H,m,H-4'),4.46(1H,m,H-5'),4.45,4.56(2H,m,H-6').13C NMR(100 MHz,pyridine-d5)δ:29.9(C-1),30.9(C-2),70.5(C-3),36.2(C-4),40.5(C-5),32.5(C-6),79.3 (C-7),151.5(C-8),144.1(C-9),37.5(C-10),203.2(C-11),81.9(C-12),51.2(C-13),47.7(C-14), 24.9(C-15),27.9(C-16),46.3(C-17),12.6(C-18),18.5(C-19),36.8(C-20),18.4(C-21),35.3 (C-22),32.4(C-23),151.3(C-24),47.7(C-25),178.0(C-26),17.8(C-27),110.4(C-28),17.4 (C-29),105.9(C-1'),76.1(C-2'),78.6(C-3'),72.4(C-4'),79.2(C-5'),63.5(C-6').
化合物8产率：85％,11.0mg。HRESIMS:m/z 649.3589([M-H]-,calcd.forC35H53O11, 649.3588).1H NMR(400MHz,pyridine-d5)δ:1.97,2.79(2H,m,H-1),1.86(2H,m,H-2),3.89 (1H,overlapped,H-3),1.56(2H,m,H-4),2.09(1H,m,H-5),4.45(1H,overlapped,H-7),4.41(1H, s,H-12),3.56(1H,m,H-14),0.90(3H,s,H-18),1.56(3H,s,H-19),1.06(3H,d,J＝6.3Hz,H-21), 3.45(3H,q,J＝7.0Hz,H-25),1.51(3H,d,J＝7.0Hz,H-27),5.04(1H,overlapped,H-28a),5.23 (1H,s,H-28b),1.26(3H,d,J＝6.7Hz,H-29),5.00(1H,d,J＝7.7Hz,H-1'),4.06(1H,m,H-2'), 4.00(1H,m,H-3'),4.29(1H,m,H-4'),4.46(1H,m,H-5'),4.45,4.56(2H,m,H-6').13C NMR(100 MHz,pyridine-d5)δ:29.9(C-1),30.9(C-2),70.5(C-3),36.2(C-4),40.5(C-5),32.5(C-6),79.3 (C-7),151.5(C-8),144.1(C-9),37.5(C-10),203.2(C-11),81.9(C-12),51.2(C-13),47.7(C-14), 24.9(C-15),27.9(C-16),46.3(C-17),12.6(C-18),18.5(C-19),36.8(C-20),18.4(C-21),35.3 (C-22),32.4(C-23),151.3(C-24),47.7(C-25),178.0(C-26),17.8(C-27),110.4(C-28),17.4 (C-29),105.9(C-1'),76.1(C-2'),78.6(C-3'),72.4(C-4'),79.2(C-5'),63.5(C-6').
实施例7、三萜糖苷化合物9的合成
一、实验材料和方法。
取适量antcamphin E(9.8mg,0.02mM)溶解于200mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP-葡萄糖(24.8mg,0.04mM)和YjiC1纯酶液(250 μg)，于37℃、200rpm的摇床上反应8h，加入甲醇(2倍体积)终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，甲醇复溶。
二、实验结果
采用半制备液相色谱YMC Pack ODS-A column(10mm×250mm,5μm)，色谱条件： 0-35min,25％-80％乙腈水溶液，35-45min,80％-100％乙腈水溶液；检测波长：254nm，流速：2mL/min，得到化合物9(11.7mg,90％,白色固体)，并采用NMR(图19和20)和HRESIMS 确定其结构。
化合物9产率：90％,11.7mg。HRESIMS:m/z 647.3433([M-H]-,calcd.forC35H51O11, 647.3431).1H NMR(400MHz,pyridine-d5)δ:1.56,3.35(2H,m,H-1),1.74,2.46(2H,m,H-2), 4.69(1H,t,J＝8.3Hz,H-7),2.49(1H,overlapped,H-12a),2.96(1H,overlapped,H-12b),2.69 (1H,m,H-14),0.82(3H,s,H-18),2.00(3H,s,H-19),0.88(3H,d,J＝5.8Hz,H-21),3.46(3H,q,J ＝6.8Hz,H-25),1.52(3H,d,J＝7.0Hz,H-27),5.07(1H,overlapped,H-28a),5.24(1H,s,H-28b), 1.68(3H,s,H-29),5.09(1H,d,J＝7.8Hz,H-1'),4.02(1H,m,H-2'),4.06(1H,m,H-3'),4.18(1H, m,H-4'),4.27(1H,m,H-5'),4.30,4.58(2H,m,H-6').13C NMR(100MHz,pyridine-d5)δ:37.3 (C-1),35.1(C-2),213.9(C-3),77.0(C-4),51.4(C-5),29.9(C-6),79.4(C-7),152.7(C-8),144.7 (C-9),37.9(C-10),201.7(C-11),58.9(C-12),48.4(C-13),54.2(C-14),25.3(C-15),28.6(C-16), 55.2(C-17),12.8(C-18),20.9(C-19),36.6(C-20),19.0(C-21),34.9(C-22),32.2(C-23),150.8 (C-24),47.2(C-25),177.2(C-26),17.6(C-27),110.9(C-28),24.1(C-29),105.6(C-1'),76.0(C-2'), 78.8(C-3'),72.8(C-4'),79.2(C-5'),63.8(C-6').
实施例8、三萜糖苷化合物10的合成
一、实验材料和方法。
取适量25S-antcin C(9.6mg,0.02mM)溶解于210mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP-葡萄糖(24.8mg,0.04mM)和YjiC1纯酶液(250μg)，于37℃、200rpm的摇床上反应8h，加入甲醇(2倍体积)终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，甲醇复溶。
二、实验结果
采用半制备液相色谱YMC Pack ODS-A column(10mm×250mm,5μm)，色谱条件： 0-35min,25％-80％乙腈水溶液，35-45min,80％-100％乙腈水溶液；检测波长：254nm，流速：2mL/min，得到化合物10(11.1mg,88％,白色固体)，并采用NMR(图21和22)和HRESIMS 确定其的结构。
化合物10产率：88％,11.1mg。HRESIMS:m/z 631.3483([M-H]-,calcd.forC35H51O10, 631.3482).1H NMR(400MHz,pyridine-d5)δ:2.43,4.33(2H,m,H-1),3.47,3.62(2H,m,H-2), 3.35(1H,m,H-4),2.51(1H,m,H-5),4.48(1H,t,J＝7.9Hz,H-7),2.46(1H,d,J＝13.4Hz, H-12a),2.95(1H,d,J＝13.7Hz,H-12b),0.84(3H,s,H-18),1.56(3H,s,H-19),0.87(3H,d,J＝ 5.3Hz,H-21),1.52(3H,d,J＝6.7Hz,H-27),5.07(1H,s,H-28a),5.23(1H,s,H-28b),1.23(3H,d, J＝6.5Hz,H-29),5.01(1H,d,J＝7.8Hz,H-1'),5.38(1H,m,H-2'),5.12(1H,m,H-3'),5.13(1H, m,H-4'),5.38(1H,m,H-5'),5.52,5.69(2H,m,H-6').13C NMR(100MHz,pyridine-d5)δ:36.6 (C-1),38.5(C-2),211.8(C-3),44.8(C-4),49.1(C-5),34.9(C-6),78.6(C-7),153.1(C-8),143.7 (C-9),37.3(C-10),201.8(C-11),58.5(C-12),48.6(C-13),54.2(C-14),25.0(C-15),28.6(C-16), 55.2(C-17),12.8(C-18),18.0(C-19),36.5(C-20),19.0(C-21),33.4(C-22),32.3(C-23),150.9 (C-24),47.0(C-25),177.5(C-26),17.5(C-27),110.8(C-28),12.3(C-29),106.0(C-1'),76.0(C-2'), 78.9(C-3'),72.3(C-4'),79.3(C-5'),63.4(C-6').
实施例9、化合物1与对照化合物(去氢齿孔酸)的体外抗肿瘤活性测试
一、实验材料和方法。
HL60、U251、SW480、MCF7细胞购自美国菌种保存中心(ATCC)。以U251细胞为例，细胞用10％胎牛血清的DMEM培养基(HL60培养于IMDM培养基)培养，维持温度37℃， 5％CO2饱和湿度，使用0.25％胰蛋白酶-EDTA消化传代，按1×104/孔接种于96孔板12小时后，吸去培养基。分别加入含20μM化合物1和对照化合物的DMEM培养基100μL，继续培养24小时，向每孔加入10μL MTS溶液(美国Promega公司)，继续培养2～4小时，酶标仪测定490nm下各孔吸光度。
以空白组为0％，对照组为100％，计算化合物1与对照化合物的HL60、U251、SW480、MCF7的细胞毒性。
二、实验结果
化合物1对HL60、U251、SW480细胞显示出显著的细胞毒性，其IC50值分别为12.8、9.3、15.9μM，是对照化合物去氢齿孔酸的5倍以上，如表1所示。以上数据说明，牛樟芝四环三萜加糖之后活性明显增强。
表1.化合物1与对照化合物(去氢齿孔酸)的细胞毒活性(IC50，μM)
a阳性药。IC50表示为平均值±SD(n＝3)。N.T.：未检测。
实施例10、化合物1及其对照化合物去氢齿孔酸的小鼠体内代谢研究
一、实验材料和方法。
4-6周雄性Balb/c鼠购自北京大学医学部实验动物部，给予12h光照12h黑暗，恒温，正常饲料及水饲养，于无菌环境中适应1天后进行实验。
动物随机分为3组(n＝6)，空白对照组灌胃等量0.3％羧甲基纤维素钠溶液，去氢齿孔酸给药组灌胃去氢齿孔酸(32mg/kg)，三萜糖苷化合物1给药组灌胃等摩尔量的化合物1(43 mg/kg)。分别在给药后1h、4h眼眶取血，血液样品立即4℃ 8000g离心10min取血清，37℃真空浓缩，甲醇复溶，过0.22μm的微孔滤膜，进行Q-Exactive MS检测。
二、实验结果。
检测各组小鼠体内去氢齿孔酸的血药浓度。小鼠口服去氢齿孔酸1h后，去氢齿孔酸的血药浓度达到607ng/mL，4h后血药浓度明显降低至40ng/mL，如图23。对比之下，化合物1在体内水解为去氢齿孔酸，1h后，血药浓度相对较低(116ng/mL)，但4h后，血药浓度显著高于对照化合物给药组，高达999ng/mL，是其苷元的25倍。结果表明，化合物1与其苷元去氢齿孔酸相比，生物利用度明显提高，即催化合成的新化合物具有更好的生物利用度。
Claims (14)
1.一种四环三萜糖苷，为式(IA)所示的化合物：
其中，R1选自-OH、＝O、O-糖基；R8选自H、-OH、O-糖基；且R1和R8中至少一者是O-糖基。
2.如权利要求1所述的四环三萜糖苷，其特征在于，所述O-糖基中的糖基为六碳糖基。
3.如权利要求2所述的四环三萜糖苷，其特征在于，所述六碳糖基为吡喃葡萄糖基。
4.如权利要求3所述的四环三萜糖苷，其特征在于，所述吡喃葡萄糖基为吡喃葡萄糖-1-基。
5.如权利要求1所述的四环三萜糖苷，其特征在于，所述四环三萜糖苷是式(IA’)所示立体构型的化合物：
其中R1和R8如权利要求1所定义，且式(IA’)中R1和R8至少一者是O-糖基。
6.如权利要求1所述的四环三萜糖苷，其特征在于，所述四环三萜糖苷选自下列化合物1～4：
7.权利要求1～6任意一项所述四环三萜糖苷的制备方法，在糖基转移酶的作用下将如下底物I所示的四环三萜化合物与糖供体反应，生物催化合成所述四环三萜糖苷；
在底物I的结构式中，R1选自-OH、＝O、O-糖基；R2和R3为甲基；R4为-COOH；R5和R6为甲基；R7为甲基；R8选自H、-OH、O-糖基；R9和R10为H；条件是，所述R1和R8中至少一者是-OH。
8.如权利要求7所述的制备方法，其特征在于，所述糖基转移酶是来自枯草芽孢杆菌的糖基转移酶YjiC1，GenBank登录号为JX982974。
9.如权利要求7所述的制备方法，其特征在于，所述糖供体为核苷酸糖。
10.如权利要求9所述的制备方法，其特征在于，所述糖供体为UDP-糖基供体。
11.如权利要求10所述的制备方法，其特征在于，所述糖供体为UDP-葡萄糖。
12.如权利要求7所述的制备方法，其特征在于，所述底物I选自来源于牛樟芝的下述四环三萜化合物：去氢齿孔酸、去氢硫色多孔菌酸。
13.权利要求1～6任意一项所述四环三萜糖苷或其药学上可接受的盐、立体异构体、互变异构体在制备抗肿瘤药物中的应用。
14.一种药物组合物，其中含有权利要求1～6任意一项所述四环三萜糖苷或其药学上可接受的盐、立体异构体、互变异构体。
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