CN115073481B - 一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物 - Google Patents
一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物 Download PDFInfo
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- CN115073481B CN115073481B CN202110270901.2A CN202110270901A CN115073481B CN 115073481 B CN115073481 B CN 115073481B CN 202110270901 A CN202110270901 A CN 202110270901A CN 115073481 B CN115073481 B CN 115073481B
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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Abstract
本发明涉及医药技术领域,尤其涉及一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物。本发明提供的呋喃白坚木碱二聚体或其药学上可接受的盐,所述呋喃白坚木碱二聚体或其药学上可接受的盐可激活免疫反应,通过提高T淋巴细胞比例,并增强其功能。
Description
技术领域
本发明涉及医药技术领域,尤其涉及一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物。
背景技术
免疫缺陷或不足导致的疾病如肿瘤、艾滋病或其它病原微生物引起感染性疾病是世界难题,在中国每年有数百万病患者因此死亡。目前临床上使用的化疗、抗病毒和抗生素等药物虽然有一定的疗效,可以使药物直接作用于病灶。但因其毒性大、耐药性强给病人带来巨大的肉体痛苦和精神压力。这些疾病大都会因为免疫下降、或是免疫***缺陷,导致肌体的保护屏障被打破。提高和维持机体免疫力在很大程度上能治疗和预防这些疾病,这也是机体天然的抗病本能。免疫细胞是机体免疫***的主要武器,而T淋巴细胞又是免疫细胞的主要成分,其主要功能是吞噬外来侵袭物,因此,提高机体T淋巴细胞比例、增强其功能是治疗和预防上述免疫疾病的关键点。
发明内容
本发明的目的在于提供一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物,所述呋喃白坚木碱二聚体或其药学上可接受的盐可激活免疫反应,通过提高T淋巴细胞比例,并增强其功能。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种呋喃白坚木碱二聚体或其药学上可接受的盐,具有式Ⅰ所示结构:
其中,所述R1和R1'独立的为氢、羟基或C1~6烷氧基;所述n为0~4的正整数;
所述R2、R2'和R2”独立的为氢或C1~6烷基;所述R3和R3'独立的为氢、羟基或=O;
R4为氢或羟基;R5为无取代基或=O;
所述A--B为-CH2-CH2-、-CH=CH-、-HOCH-CHOH-、-CH2-CHOH-或
所述其药学上可接受的盐为R2'和R2”位取代的有机盐或无机盐。
优选的,当所述A--B为时,所述呋喃白坚木碱二聚体或其药学上可接受的盐具有式Ⅰ-1~式Ⅰ-4所示结构:
式Ⅰ-1;所述式Ⅰ-1中R1为H,R3'为H;或R1为甲氧基,R3'为H;或R1为氢,R3'为=O;
式Ⅰ-2;所述式Ⅰ-2中R1为羟基;或R1为甲氧基;
式Ⅰ-3;所述式Ⅰ-3中R1为氢,R5为无取代基;或
R1为甲氧基,R5为无取代基;或R1为甲氧基,R5为=O;
式Ⅰ-4;所述式Ⅰ-4中R1为氢;或R1为羟基;或
R1为甲氧基。
优选的,当所述A--B为-CH2-CHOH-时,所述-CH2-CHOH-中的羟基位于B位置上,且所述羟基为β-OH,所述呋喃白坚木碱二聚体或其药学上可接受的盐具有式Ⅰ-5所示结构:
式Ⅰ-5;所述式Ⅰ-5中R3'为=O;
当所述A--B为-HOCH-CHOH-时,所述-HOCH-CHOH-中位于A位置上的羟基为α-OH,位于B位置上的羟基为β-OH,所述呋喃白坚木碱二聚体或其药学上可接受的盐具有式Ⅰ-5所示结构:
式Ⅰ-5;所述式Ⅰ-5中R3'为氢。
优选的,当所述A--B为-CH=CH-时,所述呋喃白坚木碱二聚体或其药学上可接受的盐具有式Ⅰ-6~式Ⅰ-11所示结构:
式Ⅰ-6;所述式Ⅰ-6中R1为H,R2为H;或R1为甲氧基,R2为H;或R1为甲氧基,R2甲基;
式Ⅰ-7;所述式Ⅰ-7中R1'为H,R3'为H;或R1'为H,R3'为H;或R1'为H,R3'为=O;
式Ⅰ-8;所述式Ⅰ-8中R3为H,R3'为=O,R4为H;或R3为H,R3'为=O,R4为羟基或R3为=O,R3'为H,R4为氢;
式Ⅰ-9;所述式Ⅰ-9中R1为H,R11为H,R3'为氢;或R1为H,R11为羟基,R3'为氢;或R1为H,R11为甲氧基,R3'为氢;或R1为羟基,R11为氢,R3'为氢;或R1为甲氧基,R11为氢,R3'为氢;或R1为羟基,R11为甲氧基,R3'为=O。
优选的,所述无机酸盐为盐酸盐、氢溴酸盐、硝酸盐、硫酸盐或磷酸盐。
优选的,所述有机酸盐为酒石酸盐、柠檬酸盐、甲酸盐、乙酸盐或乙二酸盐。
本发明还提供了上述技术方案所述的呋喃白坚木碱二聚体的制备方法,包括以下步骤:
将狗牙花属植物或山橙属植物依次进行浸提、萃取和梯度洗脱,得到所述呋喃白坚木碱二聚体。
本发明还提供了上述技术方案所述的呋喃白坚木碱二聚体或其药学上可接受的盐在制备治疗免疫疾病药物中的应用。
本发明还提供了一种药物组合物,包括上述技术方案所述的呋喃白坚木碱二聚体或其药学上可接受的盐和药学上可接受的载体。
优选的,所述呋喃白坚木碱二聚体或其药学上可接受的盐在所述药物组合物中的质量百分含量≥10%。
本发明提供了一种呋喃白坚木碱二聚体或其药学上可接受的盐,具有式Ⅰ所示结构:其中,所述R1和R1'独立的为氢、羟基或C1~6烷氧基;所述n为0~4的正整数;所述R2、R2'和R2”独立的为氢或C1~6烷基;所述R3和R3'独立的为氢、羟基或=O;R4为氢或羟基;R5为无取代基或=O;所述A--B为-CH2-CH2-、-CH=CH-、-HOCH-CHOH-、-CH2-CHOH-或所述其药学上可接受的盐为R2'和R2”位取代的有机盐或无机盐。本发明所述式I是由两个白坚木单体通过呋喃环连接形成的二聚体,和呋喃环打开但没有活性的白坚木二聚体tabernaemontines A–L相比,说明呋喃环是活性的关键基团。另外,呋喃环白坚木——柯南因、呋喃环白坚木——依波加双吲哚都是没有抑制巨噬细胞M2极化的活性,这支持了白坚木单体也是活性的必须单元,即白坚木单体及呋喃环刚性连接方式是活性的基本骨架。
附图说明
图1为CPLD、CPLL、TBYA、MDK和HMD的激活免疫测试数据图;
图2为化合物1-3和5-7激活免疫测试数据图
图3为化合物1体内激活免疫活性数据图;
图4为化合物1提高并增强CD8+T细胞功能的数据图;
图5为CPLD提高并增强CD8+T细胞功能的数据图。
具体实施方式
本发明提供了一种呋喃白坚木碱二聚体或其药学上可接受的盐,具有式Ⅰ所示结构:
式Ⅰ;
其中,所述R1和R1'独立的为氢、羟基或C1~6烷氧基;所述n为0~4的正整数;
所述R2、R2'和R2”独立的为氢或C1~6烷基;所述R3和R3'独立的为氢、羟基或=O;
R4为氢或羟基;R5为无取代基或=O;
所述A--B为-CH2-CH2-、-CH=CH-、-HOCH-CHOH-、-CH2-CHOH-或
所述其药学上可接受的盐为R2'和R2”位取代的有机盐或无机盐。
在本发明中,当所述A--B为时,所述呋喃白坚木碱二聚体或其药学上可接受的盐优选具有式Ⅰ-1~式Ⅰ-4所示结构:
式Ⅰ-1;所述式Ⅰ-1中R1为H,R3'为H(记为化合物1);或R1为甲氧基,R3'为H(记为taberyunineA或TBYA);或R1为氢,R3'为=O(记为化合物2);
式Ⅰ-2;所述式Ⅰ-2中R1为羟基(记为taberyunine C);或R1为甲氧基(记为10-dehydroxyl-conophylline);
式Ⅰ-3;所述式Ⅰ-3中R1为氢,R5为无取代基(记为化合物3);或R1为甲氧基,R5为无取代基(记为conophylline(CPLL));或R1为甲氧基,R5为=O(记为化合物4);
式Ⅰ-4;所述式Ⅰ-4中R1为氢(记为化合物5);或R1为羟基(记为化合物6);或R1为甲氧基(记为化合物7)。
在本发明中,当所述A--B为-CH2-CHOH-时,所述-CH2-CHOH-中的羟基位于B位置上,且所述羟基为β-OH,所述呋喃白坚木碱二聚体或其药学上可接受的盐优选具有式Ⅰ-5所示结构:
式Ⅰ-5;所述式Ⅰ-5中R3'为=O(记为化合物8);
当所述A--B为-HOCH-CHOH-时,所述-HOCH-CHOH-中位于A位置上的羟基为α-OH,位于B位置上的羟基为β-OH,所述呋喃白坚木碱二聚体或其药学上可接受的盐优选具有式Ⅰ-5所示结构:
式Ⅰ-5;所述式Ⅰ-5中R3'为氢(记为conophyllinine)。
在本发明中,当所述A--B为-CH=CH-时,所述呋喃白坚木碱二聚体或其药学上可接受的盐优选具有式Ⅰ-6~式Ⅰ-11所示结构:
式Ⅰ-6;所述式Ⅰ-6中R1为H,R2为H(记为conophyllidine(CPLD));或R1为甲氧基,R2为H(记为10-O-methyl-conophyllidine);或R1为甲氧基,R2甲基(10,15-dimethyl-conophyllidine);
式Ⅰ-7;所述式Ⅰ-7中R1'为H,R3'为H(记为melodineine K(MD K));或R1'为H,R3'为H(记为19'-hydroxymelodinineK(HMD K));或R1'为H,R3'为=O(记为3'-acetonyl-melodinine K);
式Ⅰ-8;所述式Ⅰ-8中R3为H,R3'为=O,R4为H(记为化合物9);或R3为H,R3'为=O,R4为羟基(记为化合物10);或R3为=O,R3'为H,R4为氢(记为化合物11);
式Ⅰ-9;所述式Ⅰ-9中R1为H,R11为H,R3'为氢(记为taberyunine B);或R1为H,R11为羟基,R3'为氢(记为化合物12);或R1为H,R11为甲氧基,R3'为氢(记为化合物13);或R1为羟基,R11为氢,R3'为氢(记为化合物14);或R1为甲氧基,R11为氢,R3'为氢(记为化合物15);或R1为羟基,R11为甲氧基,R3'为=O(记为化合物16)。
在本发明中,所述无机酸盐为盐酸盐、氢溴酸盐、硝酸盐、硫酸盐或磷酸盐;
所述有机酸盐为酒石酸盐、柠檬酸盐、甲酸盐、乙酸盐或乙二酸盐。
本发明还提供了上述技术方案所述的呋喃白坚木碱二聚体的制备方法,包括以下步骤:
将狗牙花属植物或山橙属植物依次进行进行浸提、萃取和梯度洗脱,得到所述呋喃白坚木碱二聚体。
在本发明中,所述狗牙花属植物优选为药用狗牙花、狗牙花、伞房狗牙花,尖蕾狗牙花和平脉狗牙花;所述山橙属植物优选为山橙或薄叶山橙。
在本发明中,所述浸提优选在有机溶剂中进行;所述有机溶剂优选为醇类溶剂、酮类溶剂、酯类溶剂、醚类溶剂或卤代烷类溶剂,更优选为C1-6醇、C3-6酮、C3-6酯、C2-6醚或C1-6卤代烷;所述C1-6醇优选为甲醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇、叔丁醇、正戊醇、异戊醇、环戊醇、正己醇或环己醇;所述C3-6酮优选为丙酮、甲乙酮或甲基异丁酮;所述C3-6酯优选甲酸乙酯或乙酸乙酯丙酸乙酯;所述C2-6醚优选为甲醚或***;所述C1-6卤代烷优选包括二氯甲烷、氯仿或二氯乙烷。
所述浸提后,本发明还优选包括将得到的浸提液进行减压浓缩得到总提取物。
在本发明中,所述萃取采用的萃取剂优选为乙酸乙酯;具有过程优选为将得到的总提取物的pH值调节至1.0~4.5后,采用乙酸乙酯进行萃取后,将得到的酸水层的pH值调节至7.0~11.0后,采用乙酸乙酯进行二次萃取。
在本发明中,所述梯度洗脱优选采用硅胶柱层析的方式进行,采用的洗脱机优选为氯仿-甲醇***(1:0→0:1,v/v)。
在本发明中,式Ⅰ-1~式Ⅰ-11所示结构的化合物的提取过程具体为:
将75kg药用狗牙花(Tabernaemontanabovina)的干燥叶粉碎后由甲醇浸提3次(3×20L),将得到的提取液减压浓缩后调节pH值至2~3,采用乙酸乙酯萃取2次;将萃取得到的酸水层的pH值调节至7~9,再用等体积乙酸乙酯萃取3次,乙酸乙酯层经合并浓缩后得到生物碱总碱(875g)。总碱进行硅胶柱层析,氯仿-甲醇洗脱(1:0~1:1,v/v),合并馏分后得到组分I-Ⅹ。将246g第Ⅲ组分先通过RP-18中压色谱柱梯度洗脱(30~100%甲醇)划为12小段(Ⅲ-1~Ⅲ-12)。Ⅲ-10(45g)由RP-1中压色谱柱经50~90%甲醇洗脱得7小份(Ⅲ-10-1~Ⅲ-10-7),其中Ⅲ-10-7经两次凝胶(甲醇)纯化后进一步通过HPLC制备(60~75%乙腈)纯化得化合物6。Ⅲ-11(37g)继续由RP-18-中压色谱柱经50~100%甲醇溶液梯度洗脱得8小份(Ⅲ-11-1~Ⅲ-10-8),其中Ⅲ-11-5经凝胶(甲醇)纯化后进一步经HPLC制备(70~85%乙腈)纯化得化合物15;Ⅲ-11-8经凝胶(甲醇)纯化后进一步通过HPLC制备(C18制备柱,60~75%乙腈)纯化得化合物5。Ⅲ-12(20g)继续由RP-18-中压色谱柱经梯度洗脱(60-100%甲醇)得5小份(Ⅲ-12-1~Ⅲ-12-5),其中Ⅲ-12-4经两次凝胶(甲醇)纯化后进一步经HPLC制备(C18制备柱,65-80%乙腈)纯化得化合物16。Ⅲ-12-5经凝胶(甲醇)细分后进一步经HPLC制备(C18制备柱,65-80%乙腈)纯化得10-dehydroxyl-conophylline。第Ⅳ组分(79g)先通过RP-18中压色谱柱经甲醇—水溶液梯度洗脱(15~85%)划为7段(Ⅳ-1~Ⅳ-7)。Ⅳ-5(16g)继续由RP-18中压色谱柱经甲醇—水溶液梯度洗脱(30~80%)得5小份(Ⅳ-5-1~Ⅳ-5-5),其中Ⅳ-5-3经凝胶(甲醇)纯化后进一步经HPLC制备(C18制备柱,45-60%乙腈)纯化得conophyllinine;Ⅳ-5-4经凝胶(甲醇)纯化后进一步经HPLC制备(C18制备柱,45-60%乙腈)纯化得化合物8。
狗牙花(T.divaricata)干燥叶片(7.5kg)粉碎,甲醇常温浸提3次,减压浓缩得到总提取物。总提取物调节pH至2~3,等体积乙酸乙酯萃取3次;酸水层调节pH至7~9,再用等体积乙酸乙酯萃取3次,得到总生物碱(68g)经正相硅胶柱层析,氯仿—甲醇***(1:0→0:1,v/v)洗脱,合并后得到化合物5个组分(Fr.Ⅰ-Ⅴ)。Fr.Ⅱ(25g)经过中压柱层析划段,甲醇—水***(10–100%)梯度洗脱,划为5段(Fr.Ⅱ-1~5)。Fr.Ⅱ-2(2g)进一步经中压划段,甲醇—水***(30~55%)梯度洗脱,划为5段(Fr.II-2-1~5);Fr.II-2-4(0.5g)经过Sephadex LH-20凝胶柱处理,以及HPLC纯化(50-65%乙腈)得到化合物3。Fr.II-3(8.5g)析出白色柱状结晶,母液(5g)经中压分离,10~80%甲醇梯度洗脱为4段(Fr.II-3-1~4)。Fr.II-3-4经HPLC纯化(C18制备柱,45-60%乙腈)得到化合物4。Fr.II-4(3.5g)经中压划段,甲醇—水***(45-65%)梯度洗脱,划为7段(Fr.II-4-1~7)。Fr.II-4-5(2.0g)经过Sephadex LH-20凝胶柱处理得到4段,其中,Fr.II-4-5-3经HPLC纯化(65-80%乙腈)得到化合物7;Fr.II-4-5-4经HPLC纯化(C18制备柱,65~80%乙腈)得到化合物12;Fr.II-5(0.7g)经中压划段,45~65%甲醇梯度洗脱划为8段(Fr.II-5-1~8);Fr.II-5-8(0.1g)经过Sephadex LH-20凝胶柱处理,HPLC纯化(65~75%乙腈)得到化合物13。
伞房狗牙花Tabernaemontana corymbosa干燥茎叶(15kg),粉碎后用甲醇提取液,减压得到总浸膏。总浸膏用0.5%的HCl溶液溶解完全,调pH到2~3,用等体积乙酸乙酯萃取3次;酸水部位用10%的氨水调节pH到9~10,用等体积乙酸乙酯萃取3次,乙酸乙酯层合并浓缩得到生物碱总碱(61.5g)。经硅胶柱层析,氯仿-丙酮梯度洗脱(从1:0到2:1),合并相同份后得到8个部分(Fr.I-VIII)。IV(11.3g)通过C18中亚制备(MeOH—H2O:40%、50%、60%、70%、80%)得到IV-1~IV-5五份。IV-4(1.2g)通过C18中亚制备(MeOH-H2O,40-65%),和高压制备(C18柱)MeOH-H2O(60~70%)梯度洗脱得到Taberyunine C。Fr.VIII(20.3g)通过中压划段(甲醇水:15~65%)得到2个组分VIII-I和VIII-II。VIII-I(8.2g)通过硅胶柱层析(氯仿—丙酮5:1~0:1)得到5个组分。VIII-I-2(1.5g)经过硅胶柱层析(氯仿—甲醇20:1~9:1)得到VIII-I-2-1,再进一步经过HPLC纯化(甲醇—水:40~55%)得到conophyllidine和conophylline。VIII-I-5(0.42g)经过HPLC纯化(甲醇—水:60~75%)得到10-dehydroxyl-conophylline。VIII-II(4.7g)经过硅胶柱层析(氯仿—甲醇5:1~0:1)得到3个组分VIII-II-1~VIII-II-3。VIII-II-1(0.9g)通过硅胶柱层析(氯仿—甲醇:10:1)得到VIII-II-1-1,再经过HPLC纯化(甲醇水:70~85%)得到taberyunineA和taberyunine B。
山橙M.suaveolens干燥的茎(28kg)粉碎后用甲醇浸提4次(4×75L),提取液减压回收得到总浸膏。总浸膏调pH到2~3后用等体积乙酸乙酯萃取3次;酸水部位调节pH到7~9用等体积乙酸乙酯萃取3次,所得到生物碱总碱(250g)进行硅胶柱层析,氯仿—丙酮梯度洗脱得到三段(Fr.Ⅰ-Ⅲ)。Fr.Ⅱ(3.0g)经中压分离(30~80%甲醇)得到九个组分Ⅱ-1~Ⅱ-9。Ⅱ-2经过中压分离(60-80%甲醇)得到化合物melodinine K。Ⅱ-9(1.1g)经中压划段得到5份,Ⅱ-9-1~Ⅱ-9-5,Ⅱ-9-1经过Sephadex LH-20葡聚糖凝胶(100%甲醇)分为两分Ⅱ-9-1-1~Ⅱ-9-1-2。Ⅱ-9-1-1经HPLC纯化(50~65%乙腈)得化合物10;Ⅱ-9-1-2分别经Sephadex LH-20葡聚糖凝胶(甲醇)、再HPLC纯化(50~65%乙腈)得化合物2;Ⅱ-9-2经Sephadex LH-20葡聚糖凝胶(甲醇)洗脱后再经HPLC纯化(45~60%乙腈)得化合物9;Ⅱ-9-3经H P L C纯化(55~70%乙腈)得化合物11。
山橙M.suaveolens干燥茎叶(3.0kg),粉碎后用95%的乙醇浸泡3次,每次24小时,提取液合并,减压蒸馏除去溶剂,粗提物悬浮于水中。加入1%的盐酸水溶液调节pH 2~3并搅拌,用EtOAc萃取,用10%的氨水溶液将水层的酸碱度调节至pH 9~10,再用EtOAc萃取得总生物碱浸膏9.8g。用10g硅胶拌样,100g硅胶柱层析,石油醚—丙酮(40:1~2:1)梯度洗脱,得到IV个组分。第III组分(3.2g)用RP-18反相柱乙腈—水5~40%洗脱得到III-1~III-3,其中III-3再用HPLC纯化得到melodinine K,第IV组分(0.8g)HPLC(70~80%甲醇)纯化得到化合物3'-acetonyl-melodinine K和19'-hydroxy-melodinine K。
薄叶山橙干燥茎叶(14kg)同上处理方法获得总生物碱浸膏17g。硅胶柱层析,氯仿-甲醇梯度洗脱,合并近似组分得到5份。第4馏分(2g))硅胶柱层析,氯仿—甲醇(15:1~8:1)洗脱,此后进行硅胶常压及中压柱层析,以石油醚/丙酮(4:1)、石油醚/乙酸乙酯(3:1)、氯仿/丙酮(9:1)及甲醇/水(60~80%)等洗脱***反复纯化,得到化合物melodinineK。
将得到的melodinine K、三氯甲烷、高氯酸和间氯过氧苯甲酸混合,在0度条件下,发生氧化反应,得到化合物1;反应式如下所示:
将得到的melodinine K、先和间氯过氧苯甲酸、三氯甲烷,再和三氟乙酸酐、丙酮混合,在0度条件下,发生氧化、亲核反应,得到3'-acetonyl-melodinine K;反应式如下所示:
将conophyllidine、碘甲烷和乙腈混合,在室温条件下,发生取代反应,得到10-O-methyl-conophyllidine;反应式如下所示:
将conophyllidine、碘甲烷和乙腈混合,在50度条件下,发生取代反应,得到10,15-O-dimethyl-conophyllidine;反应式如下所示:
本发明还提供了上述技术方案所述的呋喃白坚木碱二聚体或其药学上可接受的盐在制备治疗免疫疾病药物中的应用。
本发明还提供了一种药物组合物,包括上述技术方案所述的呋喃白坚木碱二聚体或其药学上可接受的盐和药学上可接受的载体。
在本发明中,所述呋喃白坚木碱二聚体或其药学上可接受的盐在所述药物组合物中的质量百分含量优选≥10%,更优选≥20%,最优选≥50%。本发明对所述药学上可接受的载体的种类没有任何特殊的限定,采用本领域技术人员熟知的种类即可。
本发明对所述药物组合物的给药剂型没有任何特殊的限定,采用本领域技术人员熟知的给药剂型即可。
在本发明中,所述药物组合物的给药途径优选为经口给药或非经口给药。每天的给药量优选为1~1000mg。当所述给药途径为经口给药时,所述药物组合物还优选包括药用辅剂;所述药用辅剂优选包括赋形剂、崩解剂、粘合剂、润滑剂、抗氧剂、包衣剂、着色剂、芳香剂和表面活性剂中的一种或几种;所述药用辅剂在所述药物组合物中的质量百分含量优选≥10%,更优选≥20%,最优选≥50%;所述药物组合物的给药剂型优选为颗粒剂、胶囊或片剂。当所述给药途径为非经口给药时,所述药物组合物的给药剂型优选为注射液、输液剂或栓剂。
下面结合实施例对本发明提供的呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
将75kg药用狗牙花(Tabernaemontanabovina)的干燥叶粉碎后由甲醇浸提3次(3×20L),将得到的提取液减压浓缩后调节pH值至2~3,采用乙酸乙酯萃取2次;将萃取得到的酸水层的pH值调节至7~9,再用等体积乙酸乙酯萃取3次,乙酸乙酯层经合并浓缩后得到生物碱总碱(875g)。总碱进行硅胶柱层析,氯仿-甲醇洗脱(1:0~1:1,v/v),合并馏分后得到组分I-Ⅹ。将246g第Ⅲ组分先通过RP-18中压色谱柱梯度洗脱(30~100%甲醇)划为12小段(Ⅲ-1~Ⅲ-12)。Ⅲ-10(45g)由RP-1中压色谱柱经50~90%甲醇洗脱得7小份(Ⅲ-10-1~Ⅲ-10-7),其中Ⅲ-10-6经凝胶(甲醇洗脱)分离后进一步经HPLC制备(C18制备柱,70~85%乙腈)纯化得化合物14;Ⅲ-10-7经两次凝胶(甲醇)纯化后进一步通过HPLC制备(60~75%乙腈)纯化得化合物6。Ⅲ-11(37g)继续由RP-18-中压色谱柱经50~100%甲醇溶液梯度洗脱得8小份(Ⅲ-11-1~Ⅲ-10-8),其中Ⅲ-11-5经凝胶(甲醇)纯化后进一步经HPLC制备(70~85%乙腈)纯化得化合物15;Ⅲ-11-8经凝胶(甲醇)纯化后进一步通过HPLC制备(C18制备柱,60~75%乙腈)纯化得化合物5。Ⅲ-12(20g)继续由RP-18-中压色谱柱经梯度洗脱(60-100%甲醇)得5小份(Ⅲ-12-1~Ⅲ-12-5),其中Ⅲ-12-4经两次凝胶(甲醇)纯化后进一步经HPLC制备(C18制备柱,65-80%乙腈)纯化得化合物16。Ⅲ-12-5经凝胶(甲醇)细分后进一步经HPLC制备(C18制备柱,65-80%乙腈)纯化得10-dehydroxyl-conophylline。第Ⅳ组分(79g)先通过RP-18中压色谱柱经甲醇—水溶液梯度洗脱(15~85%)划为7段(Ⅳ-1~Ⅳ-7)。Ⅳ-5(16g)继续由RP-18中压色谱柱经甲醇—水溶液梯度洗脱(30~80%)得5小份(Ⅳ-5-1~Ⅳ-5-5),其中Ⅳ-5-3经凝胶(甲醇)纯化后进一步经HPLC制备(C18制备柱,45-60%乙腈)纯化得conophyllinine;Ⅳ-5-4经凝胶(甲醇)纯化后进一步经HPLC制备(C18制备柱,45-60%乙腈)纯化得化合物8。
狗牙花(T.divaricata)干燥叶片(7.5kg)粉碎,甲醇常温浸提3次,减压浓缩得到总提取物。总提取物调节pH至2~3,等体积乙酸乙酯萃取3次;酸水层调节pH至7~9,再用等体积乙酸乙酯萃取3次,得到总生物碱(68g)经正相硅胶柱层析,氯仿—甲醇***(1:0→0:1,v/v)洗脱,合并后得到化合物5个组分(Fr.Ⅰ-Ⅴ)。Fr.Ⅱ(25g)经过中压柱层析划段,甲醇—水***(10–100%)梯度洗脱,划为5段(Fr.Ⅱ-1~5)。Fr.Ⅱ-2(2g)进一步经中压划段,甲醇—水***(30~55%)梯度洗脱,划为5段(Fr.II-2-1~5);Fr.II-2-4(0.5g)经过Sephadex LH-20凝胶柱处理,以及HPLC纯化(50-65%乙腈)得到化合物3。Fr.II-3(8.5g)析出白色柱状结晶,母液(5g)经中压分离,10~80%甲醇梯度洗脱为4段(Fr.II-3-1~4)。Fr.II-3-4经HPLC纯化(C18制备柱,45-60%乙腈)得到化合物4。Fr.II-4(3.5g)经中压划段,甲醇—水***(45-65%)梯度洗脱,划为7段(Fr.II-4-1~7)。Fr.II-4-5(2.0g)经过Sephadex LH-20凝胶柱处理得到4段,其中,Fr.II-4-5-3经HPLC纯化(65-80%乙腈)得到化合物7;Fr.II-4-5-4经HPLC纯化(C18制备柱,65~80%乙腈)得到化合物12;Fr.II-5(0.7g)经中压划段,45~65%甲醇梯度洗脱划为8段(Fr.II-5-1~8);Fr.II-5-8(0.1g)经过Sephadex LH-20凝胶柱处理,HPLC纯化(65~75%乙腈)得到化合物13。
伞房狗牙花Tabernaemontana corymbosa干燥茎叶(15kg),粉碎后用甲醇提取液,减压得到总浸膏。总浸膏用0.5%的HCl溶液溶解完全,调pH到2~3,用等体积乙酸乙酯萃取3次;酸水部位用10%的氨水调节pH到9~10,用等体积乙酸乙酯萃取3次,乙酸乙酯层合并浓缩得到生物碱总碱(61.5g)。经硅胶柱层析,氯仿-丙酮梯度洗脱(从1:0到2:1),合并相同份后得到8个部分(Fr.I-VIII)。IV(11.3g)通过C18中亚制备(MeOH—H2O:40%、50%、60%、70%、80%)得到IV-1~IV-5五份。IV-4(1.2g)通过C18中亚制备(MeOH-H2O,40-65%),和高压制备(C18柱)MeOH-H2O(60~70%)梯度洗脱得到Taberyunine C。Fr.VIII(20.3g)通过中压划段(甲醇水:15~65%)得到2个组分VIII-I和VIII-II。VIII-I(8.2g)通过硅胶柱层析(氯仿—丙酮5:1~0:1)得到5个组分。VIII-I-2(1.5g)经过硅胶柱层析(氯仿—甲醇20:1~9:1)得到VIII-I-2-1,再进一步经过HPLC纯化(甲醇—水:40~55%)得到conophyllidine和conophylline。VIII-I-5(0.42g)经过HPLC纯化(甲醇—水:60~75%)得到10-dehydroxyl-conophylline。VIII-II(4.7g)经过硅胶柱层析(氯仿—甲醇5:1~0:1)得到3个组分VIII-II-1~VIII-II-3。VIII-II-1(0.9g)通过硅胶柱层析(氯仿—甲醇:10:1)得到VIII-II-1-1,再经过HPLC纯化(甲醇水:70~85%)得到taberyunineA和taberyunine B。
山橙M.suaveolens干燥的茎(28kg)粉碎后用甲醇浸提4次(4×75L),提取液减压回收得到总浸膏。总浸膏调pH到2~3后用等体积乙酸乙酯萃取3次;酸水部位调节pH到7~9用等体积乙酸乙酯萃取3次,所得到生物碱总碱(250g)进行硅胶柱层析,氯仿—丙酮梯度洗脱得到三段(Fr.Ⅰ-Ⅲ)。Fr.Ⅱ(3.0g)经中压分离(30~80%甲醇)得到九个组分Ⅱ-1~Ⅱ-9。Ⅱ-2经过中压分离(60-80%甲醇)得到化合物melodinine K。Ⅱ-9(1.1g)经中压划段得到5份,Ⅱ-9-1~Ⅱ-9-5,Ⅱ-9-1经过Sephadex LH-20葡聚糖凝胶(100%甲醇)分为两分Ⅱ-9-1-1~Ⅱ-9-1-2。Ⅱ-9-1-1经HPLC纯化(50~65%乙腈)得化合物10;Ⅱ-9-1-2分别经Sephadex LH-20葡聚糖凝胶(甲醇)、再HPLC纯化(50~65%乙腈)得化合物2;Ⅱ-9-2经Sephadex LH-20葡聚糖凝胶(甲醇)洗脱后再经HPLC纯化(45~60%乙腈)得化合物9;Ⅱ-9-3经H P L C纯化(55~70%乙腈)得化合物11。
山橙M.suaveolens干燥茎叶(3.0kg),粉碎后用95%的乙醇浸泡3次,每次24小时,提取液合并,减压蒸馏除去溶剂,粗提物悬浮于水中。加入1%的盐酸水溶液调节pH 2~3并搅拌,用EtOAc萃取,用10%的氨水溶液将水层的酸碱度调节至pH 9~10,再用EtOAc萃取得总生物碱浸膏9.8g。用10g硅胶拌样,100g硅胶柱层析,石油醚—丙酮(40:1~2:1)梯度洗脱,得到IV个组分。第III组分(3.2g)用RP-18反相柱乙腈—水5~40%洗脱得到III-1~III-3,其中III-3再用HPLC纯化得到melodinine K,第IV组分(0.8g)HPLC(70~80%甲醇)纯化得到化合物3'-acetonyl-melodinine K和19'-hydroxy-melodinine K。
薄叶山橙干燥茎叶(14kg)同上处理方法获得总生物碱浸膏17g。硅胶柱层析,氯仿-甲醇梯度洗脱,合并近似组分得到5份。第4馏分(2g))硅胶柱层析,氯仿—甲醇(15:1~8:1)洗脱,此后进行硅胶常压及中压柱层析,以石油醚/丙酮(4:1)、石油醚/乙酸乙酯(3:1)、氯仿/丙酮(9:1)及甲醇/水(60~80%)等洗脱***反复纯化,得到化合物melodinineK。
将得到的melodinine K、三氯甲烷、高氯酸和间氯过氧苯甲酸混合,在0度条件下,发生氧化反应,得到化合物1;反应式如下所示:
将得到的melodinine K、先和间氯过氧苯甲酸、三氯甲烷,再和三氟乙酸酐和丙酮混合,在0度条件下,发生氧化、亲核反应,得到3'-acetonyl-melodinine K;反应式如下所示:
将conophyllidine、碘甲烷和乙腈混合,在室温条件下,发生取代反应,得到10-O-methyl-conophyllidine;反应式如下所示:
将conophyllidine、碘甲烷和乙腈混合,在50度条件下,发生取代反应,得到10,15-O-dimethyl-conophyllidine;反应式如下所示:
实施例1提取得到的一系列的呋喃白坚木碱二聚体的光谱数据为:
化合物1:淡黄色粉末,紫外光谱UV(MeOH)λ(max):196,245,330nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表-1和2;质谱ESI-MS m/z:719([M+H]+),分子式C42H46N4O7。
化合物2:淡黄色粉末,紫外光谱UV(MeOH)λ(max):191,254,329nm;核磁1H(800MHz)和13C(200MHz)NMR(acetone-d6)数据见表-1和2;质谱ESI-MS m/z:733([M+H]+),分子式C42H44N4O8。
化合物3:淡黄色粉末:紫外光谱UV(MeOH)λmax:196,254,328nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表-1和2;质谱ESIMS m/z:787[M+Na]+,分子式为C43H48N4O9。
化合物4:淡黄色粉末;紫外光谱UV(MeOH)λ(max):196,254,332nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表-1和2;质谱ESIMS m/z:809.3405[M–H],分子式C44H50N4O11。
化合物5:黄色粉末:紫外光谱UV(MeOH)λ(max):196,248,330nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表1和2;质谱ESIMS m/z:765[M+H]+,分子式C43H48N4O9。
化合物6:黄色粉末:紫外光谱UV(MeOH)λ(max):196,248,332nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表1和2;质谱ESIMS m/z:765[M+H]+,分子式C43H48N4O9。
化合物7:黄色粉末;紫外光谱UV(MeOH)λmax:196,254,332nm.核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表1和2;质谱ESIMS m/z 795.3601[M+H]+,分子式C44H50N4O10。
化合物8:黄色粉末:紫外光谱UV(MeOH)λ(max):196,240,320,340nm;核磁1H(600MHz)和13C(150MHz)NMR data(CD3OD)数据见表1和2;质谱ESIMS m/z:810[M+H]+;分子式C44H50N4O11。
化合物9:0淡黄色粉末,紫外光谱UV(MeOH)λ(max):198,254,328nm;核磁1H(800MHz)和13C(200MHz)NMR(acetone-d6)数据见表-1和2;质谱ESIMS m/z:717[M+H]+,分子式为C42H44N4O7。
化合物10:淡黄色粉末,紫外光谱UV(MeOH)λ(max):196,254,332nm;核磁1H(600MHz)和13C NMR(150MHz)(acetone-d6)数据见表-1和2;质谱ESIMS m/z:733[M+H]+,分子式C42H44N4O8。
化合物11:淡黄色粉末,紫外光谱UV(MeOH)λ(max):195,254,329nm;核磁1H(800MHz)和13C(200MHz)NMR(acetone-d6)数据见表-3和4;质谱ESIMS m/z:717[M+H]+,分子式C42H44N4O7。
化合物12:淡黄色粉末;紫外光谱UV(MeOH)λmax:196,243,331nm;核磁1H(500MHz)和13C(125MHz)NMR(acetone-d6)数据见表3和4;ESIMS m/z:749[M+H]+;分子式为C43H48N4O8。
化合物13:淡黄色粉末;紫外光谱UV(MeOH)λmax:196,242,328nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6),数据见表3和4;质谱ESIMS m/z 785.3526[M+Na]+,分子式为C44H50N4O8。
化合物14:黄色粉末:紫外光谱UV(MeOH)λ(max):198,250,326nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表3和4;质谱ESIMS m/z:749[M+H]+,分子式C43H48N4O8。
化合物15:黄色粉末;紫外光谱UV(MeOH)λ(max):198,250,330nm;核磁1H(600MHz)和13C(150Hz)NMR(acetone-d6)数据见表3和4;质谱ESIMS m/z:763[M+H]+,分子式C44H50N4O8。
化合物16:黄色粉末;紫外光谱UV(MeOH)λ(max):198,245,335nm;核磁1H(600MHz)和13C(150MHz)NMR(acetone-d6)数据见表3和4;质谱ESIMS m/z:793[M+H]+,分子式C44H48N4O10。
TaberyunineA:淡黄色粉末;紫外光谱UV(MeOH)λ(max):242,330nm;核磁1HNMR(600MHz,acetone-d6)δ:7.40(1H,s,H-9′),6.58(1H,s,H-12),6.56(1H,s,H-12′),5.99(1H,d,J=8.1Hz,H-9),5.92(1H,d,J=8.1Hz,H-10),4.97(1H,dd,J=8.0,4.8Hz,H-14),4.88(1H,d,J=8.0Hz,H-3),4.08(1H,brd,J=4.8Hz,H-15),3.71(3H,s,CO2Me′),3.69(3H,s,CO2Me),3.65(3H,s,11-OMe),0.90(3H,t,J=7.2Hz,H-18′),0.66(3H,t,J=7.2Hz,H-18);13C(150MHz)NMR(acetone-d6)数据见表-5;质谱HR-ESI-MS m/z:749.3552[M+H]+,分子式为C43H48N4O8。
Taberyunine C:白色粉末;紫外光谱UV(MeOH)λ(max):241,328nm;核磁1HNMR(600MHz,acetone-d6)δ:8.88(1H,s,NH),9.39(1H,s,NH′),7.42(1H,s,H-9′),6.57(1H,s,H-12′),5.99(1H,d,J=8.4Hz,H-10),5.64(1H,d,J=8.4Hz,H-9),4.97(1H,dd,J=7.8,4.8Hz,H-14),4.89(1H,d,J=7.8Hz,H-3),4.08(1H,brd,J=4.8Hz,H-15),3.72(6H,s,C′O2Me和CO2Me),3.73(3H,s,11-OMe),0.92(3H,t,J=7.8Hz,H-18′),0.68(3H,t,J=7.2Hz,H-18);13C(150MHz)NMR(acetone-d6)数据见表-5。质谱HRESIMS m/z765.3480[M+H]+,分子式C43H49N4O9。
10-Dehydroxyl-conophylline:白色粉末;紫外光谱UV(MeOH)λ(max):248,329nm;核磁1H(600MHz)NMR(acetone-d6)δ:8.88(1H,s,NH),9.39(1H,s,NH′),7.42(1H,s,H-9′),6.57(1H,s,H-12′),5.99(1H,d,J=8.4Hz,H-10),5.64(1H,d,J=8.4Hz,H-9),4.97(1H,dd,J=7.8,4.8Hz,H-14),4.89(1H,d,J=7.8Hz,H-3),4.08(1H,brd,J=4.8Hz,H-15),3.72(6H,s,CO2Me′和CO2Me),3.65(3H,s,11-OMe),0.92(3H,t,J=7.2Hz,H-18′),0.66(3H,t,J=7.2Hz,H-18);质谱ESI-MS m/z 779.3623[M+H]+,分子式为C44H50N4O9。
Conophylline:白色粉末;紫外光谱UV(MeOH)λ(max):243,330nm;核磁1H NMR(acetone-d6,400MHz):δ8.78(1H,s,NH),4.84(1H,d,J=7.7Hz,H-3),5.65(1H,s,H-9),4.93(1H,dd,J=7.7,4.3Hz,H-14),4.09(1H,m,H-15),0.70(3H,t,J=7.1Hz,H-18),2.56(1H,s,H-21),3.70(3H,s,16-COOCH3),9.37(1H,s,NH'),7.19(1H,s,H-9'),6.58(1H,s,H-12'),3.16(1H,m,H-14'),0.76(3H,t,J=7.2Hz,H-18'),2.66(1H,s,H-21'),3.71(3H,s,16'-COOCH3),3.72(3H,s,11'-OCH3),3.86(3H,s,12'-OCH3);13C(100MHz)NMR(acetone-d6),数据见表-5。
Conophyllinine黄色粉末;核磁1H NMR(500MHz acetone-d6,):δ8.77(1H,s,NH),5.75(1H,s,H-9),4.91(1H,dd,J=7.6,3.5Hz,H-14),4.83(1H,d,J=7.6Hz,H-3),3.83(3H,s,12-OCH3),3.72(3H,s,11-OCH3),3.68(3H,s,16-COOCH3),0.68(3H,t,J=7.5Hz,H-18),9.26(1H,s,NH'),7.38(1H,s,H-9'),6.58(1H,s,H-12'),3.70(3H,s,16'-COOCH3),0.82(3H,t,J=7.5Hz,H-18');13C(125MHz)NMR(acetone-d6)数据见表-5。
Conophyllidine:淡黄色粉末,紫外光谱UV(MeOH)λ(max):242,330nm;核磁1HNMR(acetone-d6,400MHz):δ:9.37(1H,s,NH'),8.77(1H,s,NH),7.40(1H,s,H-9'),6.59(1H,s,H-12'),5.62(1H,s,H-9),4.93(1H,dd,J=7.6,3.6Hz,H-14),4.82(1H,d,J=7.6Hz,H-3),4.05(1H,d,J=3.6Hz,H-15),3.86(3H,s,12'-OCH3),3.72(3H,s,11'-OCH3),3.71(3H,s,16'-COOCH3),3.70(3H,s,16-COOCH3),0.70(3H,t,J=7.4Hz,H-18),0.69(3H,t,J=7.4Hz,H-18');13C(acetone-d6)NMR(100MHz)数据见表-5。
10-O-methyl-conophyllidine:淡黄色粉末,紫外光谱UV(MeOH)λ(max):242,330nm;核磁1H(400MHz)NMR(acetone-d6)δ:9.29(1H,s,NH),6.20(1H,d,J=7.2,H-9),6.46(1H,d,J=7.2,H-9),7.01(1H,d,J=7.2,H-11),7.00(1H,d,J=7.2,H-12),5.01(1H,dd,J=7.8,3.6Hz,H-14),4.07(1H,dd,J=3.6Hz,H-15),4.83(1H,s,H-3),7.46(1H,s,H-9'),6.56(1H,s,H-12'),5.87(2H,overlap,H-14'/15'),3.77(3H,s,COOCH3),3.66(3H,s,C'OOC'H3),2.08(3H,s,CH2COCH3),0.84(3H,t,J=7.4Hz,H-18'),0.67(3H,t,J=7.4Hz,H-18)。质谱HRESIMS m/z:849.4075[M+H]+,分子式为C48H56N4O10。
10,15-O-dimethyl-conophyllidine:淡黄色粉末,紫外光谱UV(MeOH)λ(max):242,330nm;质谱HRESIMS m/z:863.4231[M+H]+,分子式为C49H58N4O10。
Melodinine K:白色粉末;紫外光谱UV(MeOH)λmax:242,330nm;核磁1HNMR(600MHz,acetone-d6)δ:9.27(1H,s,NH),9.39(1H,s,NH′),7.46(1H,s,H-9′),6.99(1H,d,J=7.2Hz,H-11),6.94(1H,d,J=7.2Hz,H-12),6.58(1H,s,H-12′),6.40(1H,d,J=7.2Hz,H-10),6.11(1H,d,J=7.2Hz,H-9),5.89(2H,overlap,H-14'/15'),5.00(1H,dd,J=7.8,3.6Hz,H-14),4.89(1H,d,J=7.6Hz,H-3),4.07(1H,dd,J=3.6Hz,H-15),3.72(3H,s,C′O2Me′),3.71(3H,s,CO2Me),0.82(3H,t,J=7.2Hz,H-18'),0.67(3H,t,J=7.2Hz,H-18);13C(150MHz)NMR(acetone-d6)数据见表-6;质谱ESI-MS m/z703[M+H]+,分子式为C42H46N4O6。
19'-Hydroxy-melodinine K:白色粉末;紫外光谱UV(MeOH)λmax:242,330nm;核磁1HNMR(acetone-d6,400MHz)δ:9.24(1H,s,NH),9.33(1H,s,N'H),7.43(1H,s,H-9'),6.96(1H,t,J=7.3Hz,H-11),6.91(1H,d,J=7.3Hz,H-12),6.56(1H,s,H-12'),6.54(1H,t,J=7.3Hz,H-10),6.05(1H,d,J=7.3Hz,H-9),5.85(2H,overlap,H-14'/15'),4.93(1H,dd,J=7.8,3.6Hz,H-14),4.83(1H,d,J=7.8Hz,H-3),4.05(1H,d,J=3.6Hz,H-15),3.77(3H,s,COOCH3),3.69(3H,s,C'OOC'H3),3.55(1H,q,J=6.5Hz),1.00(3H,d,J=6.6Hz,H-18'),0.65(3H,t,J=7.8Hz,H-18);13C(150MHz)NMR(acetone-d6)数据见表-6;质谱HR-ESI-MS m/z:719.3443[M+H]+,分子式为C42H46N4O7。
3'-acetonyl-melodinine K:白色粉末;紫外光谱UV(MeOH)λmax 330,247nm;核磁1H(600MHz)NMR(acetone-d6)δ:9.49(1H,s,NH),9.29(1H,s,NH),7.46(1H,s,H-9'),7.01(1H,t,J=7.2,H-11),7.00(1H,d,J=7.2,H-12),6.56(1H,s,H-12'),6.46(1H,t,J=7.2,H-10),6.20(1H,d,J=7.2,H-9),5.87(2H,overlap,H-14'/15'),4.98(1H,dd,J=7.8,3.6Hz,H-14),4.83(1H,s,H-3),4.07(1H,dd,J=3.6Hz,H-15),3.77(3H,s,COOCH3),3.66(3H,s,C'OOC'H3),2.08(3H,s,CH2COCH3),0.84(3H,t,J=7.4Hz,H-18'),0.67(3H,t,J=7.4Hz,H-18);13C(150MHz)NMR(acetone-d6)数据见表-6。质谱HRESIMS m/z759.3761[M+H]+,分子式为C45H50N4O7。
Taberyunine B:淡黄色粉末:紫外光谱UV(MeOH)λmax:204,242,330nm;核磁1H(600MHz)NMRδ:9.23(1H,s,NH),9.39(1H,s,NH′),7.44(1H,s,H-9′),6.57(2H,s,H-12/12′),5.99(1H,d,J=8.0Hz,H-9),5.91(1H,d,J=8.0Hz,H-10),5.80(2H,overlap,H-14′/15′),4.95(1H,dd,J=7.8,4.8Hz,H-14),4.89(1H,d,J=7.8Hz,H-3),4.06(1H,d,J=4.8Hz,H-15),3.70(3H,s,C'OOC'H3),3.69(3H,s,COOCH3),3.63(3H,s,11-OMe),0.84(3H,t,J=7.2Hz,H-18′),0.66(3H,t,J=7.2Hz,H-18);13C(150MHz)NMR(acetone-d6)数据见表-6;HRESIMS m/z 733.3612[M+H]+,分子式C43H49N4O7。
表1~6为实施例1提取得到的一系列的呋喃白坚木碱二聚体的核磁数据归属表:
表1化合物1~10的1HNMR数据归属(acetone-d6)。arecorded at 600MHz;bat800MHz
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表2化合物1~10的13C NMR数据归属(acetone-d6)。arecorded at 150MHz,bat200MHz.
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表3化合物11-16的1H NMR数据归属(acetone-d6)。arecorded at 800MHz;bat500MHz,c at 600MHz.
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表4化合物11-16的13C NMR数据归属(acetone-d6).arecorded at 200MHz;bat125MHz;cat 150MHz.
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表5 taberyunineA、taberyunine C、conophylline、conophyllinine、conophyllidine的13C NMR数据归属(acetone-d6)
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arecordedat 150MHz;bat 100MHz;cat 125MHz
表6 Melodinine K、19'-hydroxy-melodinine K、3'-acetonyl-melodinine K、taberyunine B的13C NMR数据归属(acetone-d6)
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arecordedon 150MHz;brecordedon 100MHz.
测试例
激活免疫测试:BMMs是从骨髓中分离并诱导培养的原代细胞,能够很好地反映巨噬细胞的功能,是研究巨噬细胞功能常用的细胞。将小鼠股骨骨髓分离后在含有mCSF因子的培养基中培养6-7天获得成熟的BMMs,用IL-4刺激12h可诱导其极化为免疫抑制型的M2型,高表达ARG1,将蛋白提取后,在体外进行精氨酸水解,并进一步使用α-isonitrosopropiophenone与水解产物尿素反应显色,在540nm波长下测定吸光度,作为ARG1活性的标志。具体过程如下:细胞经药物(CPLD、CPLL、TBYA、MDK、HMD、化合物1-3和化合物5-7)预处理后(1μM),IL-4刺激12h,经PBS清洗,使用含蛋白酶抑制剂的裂解液裂解细胞,取细胞总蛋白,加入0.2–0.4mM的Mn2+后,55℃加热10分钟以充分活化精氨酸酶ARG1。加入0.5mM的精氨酸,37℃孵育1~2小时以充分水解精氨酸。加入含有硫酸、磷酸的酸溶液终止反应,加入9%的α-isonitrosopropiophenone乙醇溶液,95℃加热15~30分钟即可显色。实验结果如图1所示,由图1可知,化合物CPLD、CPLL、TBYA、MDK和HMD能显著抑制巨噬细胞中IL-4诱导的arginase表达,抑制巨噬细胞M2极化;由图1可知(其中A为抑制巨噬细胞M2极化,B为CPLD、CPLL、TBYA、MDK和HMD结构式,C为化合物浓度依赖关系),CPLD、CPLL、TBYA、MDK和HMD的能显著抑制巨噬细胞中IL-4诱导的arginase表达,能够浓度依赖地抑制巨噬细胞M2的极化。由图2可知化合物1-3、5-7也能够浓抑制巨噬细胞M2的极化。
依赖免疫的活性验证:在小鼠黑色素瘤模型上,化合物1在2mg/kg剂量下连续腹腔给药4次,测试结果如图3所示,化合物1具有明显的抑瘤作用,且其抑瘤活性依赖于免疫***,在正常小鼠黑色素瘤模型(A)与乳腺癌模型(B)中,化合物1显著抑制肿瘤的成长,且抑制乳腺癌的自发性肺转移(C和D);使用Balb/c裸鼠构建的黑色素瘤模型(E)与乳腺癌细胞模型(F)中,化合物1的抗肿瘤活性消失;
提高并增强CD8+T细胞功能的验证:通过FACS分析,化合物1的测试结果如图3所示,化合物1能够促进肿瘤内免疫细胞的浸润,上调CD8+T细胞的比例、下调髓系细胞的比例,而对CD4+T细胞、Treg细胞、NK细胞、B细胞等无明显影响。进一步研究发现,化合物1能够促进CD8+T细胞的功能,增强IFNγ、Granzyme B、TNFα、上调PD1、LAG3、TIM3。此外,化合物1能够下调CD206+巨噬细胞的比例(图-4)。其中,(A)说明化合物1能激活肿瘤免疫反应;(B)调节微环境中免疫细胞类群的比例;(C-E)上调T细胞、尤其是CD8+T细胞的比例;(F-H)对Treg比例无显著影响;(I,J)促进CD8+T细胞的功能,上调IFNγ、Granzyme B、TNFα的表达;(K,L)同时上调PD1、LAG3、TIM3的表达;(M,N)下调肿瘤微环境中髓系细胞(CD11b+)比例;(O,P)尤其是M2型TAMs(CD206hi)的比例.*P<0.05;**P<0.01;
COLD的测试结果如图5所示,发现CPLD处理能够显著提高瘤内CD8+T细胞比例,而对CD4+T细胞无显著影响(图-5A~E),同时对Treg比例无影响(F-G)。胞内染色发现,CPLD能够促进CD8+T细胞的功能,增强IFNγ、Granzyme B、TNFα的表达(H~K)。进一步的免疫荧光染色实验同样发现CPLD能够增加CD8+T细胞比例并促进Granzyme B的表达(L)。更近一步地,我们用anti-CD8抗体连续注射小鼠,删除其体内的CD8+T细胞,以此构建黑色素瘤模型,再考察化合物的抗肿瘤活性。结果显示,在CD8+T细胞删除的肿瘤模型与裸鼠肿瘤模型上,CPLD的抗肿瘤作用显著降低,证实CPLD能够通过调节TAMs功能进一步促进CD8+T细胞功能从而发挥抗肿瘤活性(M~N)。
实施例2
硫酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的硫酸乙醇溶液至pH=4,过滤,干燥,得到硫酸盐。
实施例3
盐酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的盐酸乙醇溶液至pH=4,过滤,干燥,得到盐酸盐。
实施例4
磷酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的磷酸溶液至pH=4,过滤,干燥,得到磷酸盐。
实施例5
酒石酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的酒石酸溶液至pH=4,过滤,干燥,得到酒石酸盐。
实施例6
柠檬酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的柠檬酸溶液至pH=4,过滤,干燥,得到柠檬酸盐。
实施例7
甲酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的甲酸溶液至pH=4,过滤,干燥,得到甲酸盐。
实施例8
乙二酸盐的制备:
将实施例1制备得到的28个物质中,分别加入质量浓度为4%的乙二酸溶液至pH=4,过滤,干燥,得到乙二酸盐。
实施例9
注射液的制备:
将实施例2~8制备得到的盐加注射用水,精滤、灌封灭菌,得到注射液。
实施例10
粉针剂的制备:
将实施例2~8制备得到的盐溶于无菌注射用水中,搅拌至溶解,用无菌抽滤漏斗过滤,再无菌精滤,封装于安瓿中,低温冷冻干燥后无菌熔封,得到粉针剂。
实施例11
粉剂的制备:
以9:1的质量比,将实施例2~8制备得到的盐与赋形剂混合,得到粉剂。
实施例12
片剂的制备:
以1:(5~10)的质量比,将实施例2~8制备得到的盐与赋形剂混合,压片,得到片剂。
实施例13
以5:1的质量比,将实施例2~8制备得到的盐与赋形剂混合制成胶囊、颗粒剂或冲剂。
实施例14
以3:1的质量比,将实施例2~8制备得到的盐与赋形剂混合制成胶囊、颗粒剂或冲剂。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.呋喃白坚木碱二聚体或其药学上可接受的盐在制备通过激活免疫治疗黑色素瘤的药物中的应用,其特征在于,所述呋喃白坚木碱二聚体结构为如下所示的任意一种:
所述式Ⅰ-1中R1为H,R3'为H;或R1为氢,R3'为=O;
所述式Ⅰ-3中R1为氢,R5为无取代基;
所述式Ⅰ-4中R1为氢;或R1为羟基。
2.如权利要求1所述的应用,其特征在于,所述的呋喃白坚木碱二聚体的药学上可接受的盐为无机酸盐或有机酸盐;所述无机酸盐为盐酸盐、氢溴酸盐、硝酸盐、硫酸盐或磷酸盐;所述有机酸盐为酒石酸盐、柠檬酸盐、甲酸盐、乙酸盐或乙二酸盐。
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