CN110845319B - 一种新黄酮类化合物的制备方法以及在制备治疗骨质疏松药物中的应用 - Google Patents
一种新黄酮类化合物的制备方法以及在制备治疗骨质疏松药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种新黄酮类化合物的制备方法以及在制备治疗骨质疏松药物中的应用,包括化合物1、化合物2、化合物3、化合物4中的至少一种;化合物1为4‑甲氧基黄檀醌;化合物2的R1=OH,R2=OMe,化合物2为3'‑羟基‑4,4'‑二甲氧基黄檀醌;化合物3的R1=H,R2=OH,化合物3为4'‑羟基‑4‑甲氧基黄檀醌;化合物4为S(+)‑3'‑hydroxy‑4',2,4,5‑Tetramethoxydalbergiquinol。本发明该类成分在治疗骨质疏松相关药理学研究方面尚未见国内外文献报道;另外本发明具有成分富集效率高、方法明晰简单等特点。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种新黄酮类化合物的制备方法以及在制备治疗骨质疏松药物中的应用。
背景技术
据统计,全球骨质疏松患者超过2亿,其中,绝经后骨质疏松占骨质疏松的绝大多数。体内雌激素水平快速下降是绝经后女性极易患骨质疏松症的根本原因。目前,临床通常服用甾体类雌激素药物进行治疗,虽可减轻女性绝经期症状,防治骨质疏松,但也增加女性患乳腺癌、子宫内膜癌等恶性肿瘤疾病的风险。为此,寻求更为安全有效的治疗绝经后骨质疏松药物一直是世界医药研究的热点。现代研究表明:非甾体类植物雌激素生物活性约为雌激素的1/500~1/1000,且对乳腺和子宫等雌激素受体阳性的组织器官不会产生刺激增生及其他副作用,显示出良好的应用前景。据文献报道,以降香黄檀为代表的黄檀属植物是非甾体类雌激素主要植物来源之一。新黄酮(neoflavonoids)类成分则是该属植物的特征性成分。本课题组前期从黄檀属植物中分离得到若干新黄酮(neoflavonoids)类成分,并证明这些成分具有不同程度的抗骨质疏松活性。该研究结果尚未见国内外文献报道。本发明可为今后开发安全有效的抗骨质疏松药物提供参考。
发明内容
针对现有技术的不足,本发明旨在提供一种新黄酮类化合物的制备方法以及在制备治疗骨质疏松药物中的应用。
本发明的通过如下技术方案实现。
一、化合物结构式
化合物1:4-甲氧基黄檀醌
化合物2:3'-羟基-4,4'-二甲氧基黄檀醌
化合物3:4'-羟基-4-甲氧基黄檀醌
化合物4:S(+)-3'-hydroxy-4',2,4,5-Tetramethoxydalbergiquinol
(二)制备方法:
东非黑黄檀心材50kg,经粉碎,过40目筛,用70%乙醇加热回流提取3次,合并提取液,回收溶剂并浓缩至无醇味,得总浸膏13.9kg,加适量水制成混悬液,依次用二氯甲烷、乙酸乙酯、正丁醇萃取。
取二氯甲烷部位浸膏6.5kg,经100-200目硅胶柱色谱分离(30cm×130cm),以石油醚-乙酸乙酯(50:1→1:2),乙酸乙酯为洗脱剂进行梯度洗脱,合并TLC检视相同部分,得到16个流分(Frs.1-16)。
流分Fr.2(83.8g)经硅胶柱色谱分离(9cm×45cm),以石油醚-丙酮(13:1→2:1)为流动相进行梯度洗脱,得到10个流分(Fr.2.A-J)。流分Fr.2.D经重结晶得到化合物1,4-甲氧基黄檀醌(5.8g)。
流分Fr.3(101.4g)经200-300目硅胶柱色谱分离(9×45cm),以石油醚-二氯甲烷(8:1→1:20)为洗脱剂进行梯度洗脱,得到11个流分(Frs.3.A-K)。流分Fr.3H(24.0g)经SephadexLH-20柱色谱(25×2500mm),以二氯甲烷-甲醇(1:1)进行洗脱,得到4个(Frs.3H1-4)。流分Fr.3H3(12.2g)经200-300目硅胶柱色谱分离(9×45cm),以石油醚-二氯甲烷(5:1→1:2)为洗脱剂进行梯度洗脱,得到化合物4,S(+)-3'-hydroxy-4',2,4,5-tetramethoxydalbergiquinol(17.3mg)。
流分Fr.6(2.1kg)经硅胶柱色谱分离(30cm×110cm),以石油醚:二氯甲烷(150:1-1:10)为流动相进行梯度洗脱,得到11个流分(Fr.6.A-K)。流分Fr.6.B(42.4g)经SephadexLH-20柱色谱得到6个流分(Fr.6.B(1-6))。Fr.6.B4(32.2g)经重结晶得到化合物2,3'-羟基-4,4'-二甲氧基黄檀醌(19.5g)。Fr.6.I(71.8g)经Sephadex LH-20柱色谱及重结晶得到化合物3,4'-羟基-4-甲氧基黄檀醌(1g)。
与现有技术相比,本发明的优点是:该类成分在治疗骨质疏松相关药理学研究方面尚未见国内外文献报道;另外本发明具有成分富集效率高、方法明晰简单等特点。
附图说明
图1为本发明的化合物1,4-甲氧基黄檀醌的1H-NMR图谱;
图2为本发明的化合物1,4-甲氧基黄檀醌的13C-NMR图谱;
图3为本发明的化合物2,3′-羟基-4,4′-二甲氧基黄檀醌的1H-NMR图谱;
图4为本发明的化合物2,3′-羟基-4,4′-二甲氧基黄檀醌的13C-NMR图谱;
图5为本发明的化合物3,4′-羟基-4甲氧基黄檀醌的1H-NMR图谱;
图6为本发明的化合物3,4′-羟基-4甲氧基黄檀醌的13C-NMR图谱;
图7为本发明的化合物4,S(+)-3'-hydroxy-4',2,4,5-tetramethoxydalbergiquinol的1H-NMR图谱;
图8为本发明的化合物4,S(+)-3'-hydroxy-4',2,4,5-tetramethoxydalbergiquinol的13C-NMR图谱;
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,但不作为本发明的限定。
一、化合物结构式
化合物1:4-甲氧基黄檀醌
化合物2:3'-羟基-4,4'-二甲氧基黄檀醌
化合物3:4'-羟基-4-甲氧基黄檀醌
化合物4:S(+)-3'-hydroxy-4',2,4,5-Tetramethoxydalbergiquinol
(二)制备方法:
东非黑黄檀心材50kg,经粉碎,过40目筛,用70%乙醇加热回流提取3次,合并提取液,回收溶剂并浓缩至无醇味,得总浸膏13.9kg,加适量水制成混悬液,依次用二氯甲烷、乙酸乙酯、正丁醇萃取。
取二氯甲烷部位浸膏6.5kg,经100-200目硅胶柱色谱分离(30×130cm),以石油醚-乙酸乙酯(50:1→1:2),乙酸乙酯为洗脱剂进行梯度洗脱,合并TLC检视相同部分,得到16个流分(Frs.1-16)。
流分Fr.2(83.8g)经硅胶柱色谱分离(9cm×45cm),以石油醚-丙酮(13:1→2:1)为流动相进行梯度洗脱,得到10个流分(Fr.2.A-J)。流分Fr.2.D经重结晶得到化合物1,4-甲氧基黄檀醌。
流分Fr.3(101.4g)经200-300目硅胶柱色谱分离(9×45cm),以石油醚-二氯甲烷(8:1→1:20)为洗脱剂进行梯度洗脱,得到11个流分(Frs.3.A-K)。流分Fr.3H(24.0g)经SephadexLH-20柱色谱(25×2500mm),以二氯甲烷-甲醇(1:1)进行洗脱,得到4个(Frs.3H1-4)。流分Fr.3H3(12.2g)经200-300目硅胶柱色谱分离(9×45cm),以石油醚-二氯甲烷(5:1→1:2)为洗脱剂进行梯度洗脱,得到化合物4,S(+)-3'-hydroxy-4',2,4,5-tetramethoxydalbergiquinol。
流分Fr.6(2.1kg)经硅胶柱色谱分离(30×110cm),以石油醚:二氯甲烷(150:1-1:10)为流动相进行梯度洗脱,得到11个流分(Fr.6.A-K)。流分Fr.6.B(42.4g)经SephadexLH-20柱色谱得到6个流分(Fr.6.B(1-6))。Fr.6.B4(32.2g)经重结晶得到化合物2,3'-羟基-4,4'-二甲氧基黄檀醌(19.5g)。Fr.6.I(71.8g)经Sephadex LH-20柱色谱及重结晶得到化合物3,4'-羟基-4-甲氧基黄檀醌。
(三)化学成分结构鉴定
化合物1,4-甲氧基黄檀醌:黄色针状结晶,氢谱数据显示存在一个单取代苯环结构(7.30(2H,q,J=5.8,5.4Hz,H-2′,6′),7.23(1H,t,J=7.3Hz,H-4′),7.19(2H,d,J=7.1Hz,H-3′,5′),),一个端烯结构(6.10(1H,ddd,J=17.2,10.2,6.7Hz,H-2),5.27(1H,d,J=10.2Hz,H-3,5.00(1H,d,J=17.2Hz,H-3),4.93(1H,dd,J=6.7Hz,H-1)),一个甲氧基(3.81(3H,s,OCH3-4″)),碳谱数据显示有两个羰基(186.27(C-2″),182.36(C-5″)),一个端烯(137.25(C-2),118.21(C-3),),一个单取代苯环(128.77(C-3′,5′),128.56(C-2′,6′),127.20(C-4′)),鉴定该化合物为4-甲氧基黄檀醌。其波谱数据如下:1H-NMR(600MHz,CDCl3)δ7.30(2H,q,J=5.8,5.4Hz,H-2′,6′),7.23(1H,t,J=7.3Hz,H-4′),7.19(2H,d,J=7.1Hz,H-3′,5′),6.49(1H,s,H-3),6.10(1H,ddd,J=17.2,10.2,6.7Hz,H-2),5.91(1H,s,H-6″),5.27(1H,d,J=10.2Hz,H-3),5.00(1H,d,J=17.2Hz,H-3),4.93(1H,dd,J=6.7Hz,H-1),3.81(3H,s,OCH3-4″).13C-NMR(150MHz,CDCl3)δ47.04(C-1),137.25(C-2),118.21(C-3),150.99(C-1″),186.27(C-2″),107.90(C-3″),158.48(C-4″),182.36(C-5″),131.56(C-6″),139.34(C-1′),128.56(C-2′,6′),128.77(C-3′,5′),127.20(C-4′),56.31(OCH3-4″)。
化合物2,3'-羟基-4,4'-二甲氧基黄檀醌:红棕色块状结晶,HR-ESI-MS数据显示其分子质量为m/z 301.1071[M+H+],理论值为301.1076[M+H+],故分子式为C17H16O5。氢谱数据中可以观察到两组耦合系统,分别为6.05(1H,ddd,J=17.0,10.2,6.7Hz,2′),5.24(1H,dt,J=10.3,1.2Hz,=CH2cis),4.98(1H,dt,J=17.1,1.4Hz,=CH2trans),4.82(1H,dd,J=6.7,1.2Hz,H-3′),和6.78(1H,d,J=8.3Hz,H-5″),6.73(1H,d,J=2.2Hz,H-2″),6.67(1H,dd,J=8.3,2.1Hz,H-6″),以及两个单峰质子、两组甲氧基质子和一个宽锋质子。碳谱数据中可以观察到两个羰基,一个端烯。结合核磁共振二维谱(HMBC、HSQC),6.46(1H,d,J=1.2Hz,3)与δ151.19(C-1),186.48(C-2),158.57(C-4)相关,在3位;5.90(1H,s,6)与δ151.19(C-1),186.48(C-2),158.57(C-4),182.52(C-5)相关,在6位;5.63(1H,s,3″-OH)与114.79(C-2″),145.83(C-3″),145.79(C-4″)相关,在3″位;3.85(3H,s,4″-OCH3)和3.79(3H,s,4′-OCH3)分别与145.79(C-4″),158.57(C-4)相关,故分别在4″、4位。鉴定该化合物为3′-羟基-4,4′-二甲氧基黄檀醌。其波谱数据如下,1H NMR(600MHz,CDCl3)δ6.78(1H,d,J=8.3Hz,H-5″),6.73(1H,d,J=2.2Hz,H-2″),6.67(1H,dd,J=8.3,2.1Hz,H-6″),,6.05(1H,ddd,J=17.0,10.2,6.7Hz,2′),5.90(1H,s,6),5.63(1H,s,3″-OH),5.24(1H,dt,J=10.3,1.2Hz,=CH2cis),4.98(1H,dt,J=17.1,1.4Hz,=CH2trans),4.82(1H,dd,J=6.7,1.2Hz,H-3′),3.85(3H,s,4″-OCH3),3.79(3H,s,4-OCH3).13C NMR(151MHz,CDCl3)δ151.19(C-1),186.48(C-2),107.97(C-3),158.57(C-4),182.52(C-5),131.52(C-6),132.55(C-1″),114.79(C-2″),145.83(C-3″),145.79(C-4″),110.90(C-5″),120.26(C-6″),46.46(C-1′),137.39(C-2′),118.05(C-3′),56.38(4″-OCH3),56.08(4-OCH3)。
化合物3,4'-羟基-4-甲氧基黄檀醌:红棕色块状结晶,氢谱数据显示该化合物具有一个对位取代的苯环(7.08(2H,d,J=8.4HZ,H-2',6'),6.80(2H,d,J=8.4HZ,H-3',5')),一个端烯结构(6.10(1H,ddd,J=17.0,10.2,6.6HZ,H-8),5.93(1H,s,H-3),5.28(1H,d,J=10.2HZ,H-9a),5.00(2H,J=17.2HZ,H-9b),4.89(1H,d,J=6.4HZ,H-7)),一个甲氧基(3.80(3H,s,4-OCH3)),一个羟基。碳谱数据显示两个羰基(186.4(C-2),182.4(C-5),),一个对位取代的苯环,一个端烯结构。鉴定该化合物为4′-羟基-4-甲氧基黄檀醌。其波谱数据如下:1H-NMR(600MHz,CDCl3)δ:7.08(2H,d,J=8.4HZ,H-2',6'),6.80(2H,d,J=8.4HZ,H-3',5'),6.49(1H,s,H-6),6.10(1H,ddd,J=17.0,10.2,6.6HZ,H-8),5.93(1H,s,H-3),5.28(1H,d,J=10.2HZ,H-9a),5.00(2H,J=17.2HZ,H-9b),4.89(1H,d,J=6.4HZ,H-7),3.80(3H,s,4-OCH3);13C-NMR(151MHz,CDCl3)δ:186.4(C-2),107.8(C-3),158.5(C-4),182.4(C-5),131.3(C-6),46.2(C-7),137.4(C-8),118.0(C-9),151.2(C-1'),129.8(C-2',6'),115.6(C-3',5'),154.7(C-4'),56.3(4-OCH3)。
化合物4,S(+)-3'-hydroxy-4',2,4,5-tetramethoxydalbergiquinol:白色粉末,用HR-TOF-ESI-MS(m/z 329.1376[m-H])推导出其分子式为C19H22O5,不饱和度为9。1H-NMR显示B环中有ABX系统:δH6.77(1H,d,J=2.0Hz,H-2′),6.75(1H,d,J=8.4Hz,H-5′),6.66(1H,dd,J=8.4,2.0Hz,H-6′),两个单重芳香峰:δH 6.68(1H,s,H-6)and 6.53(1H,s,H-3),四个单重甲氧基峰:δH3.87(3H,s,4-OCH3),3.84(3H,s,4′-OCH3),3.78(3H,s,5-OCH3)and 3.73(3H,s,2-OCH3),一个羟基峰:δH5.65(1H,br,3′-OH)。在δH 6.23(1H,ddd,J=16.9,10.2,6.6Hz,H-8),5.17(1H,dt,J=10.2,1.6Hz,H-9a),5.02(1H,d,J=6.6Hz,H-7),4.92(1H,dt,J=16.9,1.6Hz,H-9b)的信号。13C-NMR显示化合物1有19个信号峰,其中15个与黄檀酚骨的架信号一致,。HMBC显示H-3(δH 6.53)与C-1(δC 123.7)和C-5(δC 143.1)相关,H-6与C-2(δC151.3)和C-4(δC 148.2)相关,说明δH 6.53和6.68在A环上。HMBC显示δH 5.65与C-2′(δC115.0),C-3′(δC145.3),C-4′(δC145.0)相关证明3′-OH位于B环的C-3′上。根据HMBC和HSQC的相关性可以看出,4-OCH3(δH3.87)与C-4(δC148.2)相关,4′-OCH3(δH3.84)与C-4′(δC145.0)相关,5-OCH3(δH3.78)与C-5(δC143.1)相关,2-OCH3(δH3.73)与C-2(δC151.3)相关.该化合物的旋光度为+21.5°,因此C-7为S构型。
(四)药效作用
MTT法测定小鼠成骨细胞MC3T3-E1(subclone 14)细胞增殖率:将培养皿中的MC3T3-E1细胞放入37℃,5%CO2培养箱进行培养。细胞培养液包括DMEM培养基、10%热灭活胎牛血清(FBS)、100U/mL的青霉素和100μg/mL的链霉素,2~3d换液1次,3~4d传代1次。
取MC3T3-E1细胞2×104个/孔接种于96孔板中进行细胞增殖实验,每孔放入200μL培养液。实验组加入不同终浓度的待测化合物,阳性对照组为10-7mol mL-1浓度的雌二醇,空白对照组加入等体积的DMEM,每组6个复孔。继续培养24、48、72、96h,在培养结束前4h每孔加入5mg/mL(0.5%)的MTT 20μL。培养箱继续培养4h,取出,吸弃培养液,PBS漂洗一次,每孔各加入DMSO 150μL,Minisaker震荡溶解10~30min后,室温避光孵育15min,待结晶充分溶解后,用酶标仪读取各孔在570nm波长下的光吸收值,计算细胞增值率。
碱性磷酸酶(ALP)活性检测:不同浓度待测化合物作用细胞24、48、72、96h后,弃去细胞培养液后用PBS轻轻地冲洗2次,用0.2%的TritonX-100裂解液100μL处理后,冰上裂解细胞30min。将处理后的样品4℃、12 000×g离心条件下离心15min。按ALP检测试剂盒及BCA法分别测定ALP值和蛋白含量,计算细胞内ALP的活性,以U/(g·port)表示。取上清液测定ALP活性与蛋白浓度。取蛋白上清液100μL加入含1.0mg·mL-1pNPP、1mol·L-1二乙醇胺及0.5mmol·L-1MgCl2的缓冲液100μL,37℃避光孵育30min,每孔加入3mol·L-1NaOH 50μL终止反应,在405nm波长下测定光吸收。BCA法测定样品蛋白浓度,用于校正ALP活性。
以上所述仅为本发明较佳的实施例,并非因此限制本发明的实施方式及保护范围,对于本领域技术人员而言,应当能够意识到凡运用本发明说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案,均应当包含在本发明的保护范围内。
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