CN109627243A - 一种喷昔洛韦-铜配合物、合成方法及其应用 - Google Patents
一种喷昔洛韦-铜配合物、合成方法及其应用 Download PDFInfo
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- 238000010189 synthetic method Methods 0.000 title claims abstract description 15
- 229960001179 penciclovir Drugs 0.000 claims abstract description 36
- JNTOCHDNEULJHD-UHFFFAOYSA-N Penciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(CCC(CO)CO)C=N2 JNTOCHDNEULJHD-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 10
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims abstract description 6
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims abstract description 6
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- DILRJUIACXKSQE-UHFFFAOYSA-N n',n'-dimethylethane-1,2-diamine Chemical compound CN(C)CCN DILRJUIACXKSQE-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic Table
- C07F1/005—Compounds containing elements of Groups 1 or 11 of the Periodic Table without C-Metal linkages
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- C07—ORGANIC CHEMISTRY
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Abstract
本发明公开了一种喷昔洛韦‑铜配合物、合成方法及其应用,所述喷昔洛韦‑铜配合物的合成方法,包括以下步骤:(1)称取物质的量之比为1:1‑2:1的喷昔洛韦和五水硫酸铜,溶于溶剂中,得到混合溶液;(2)将步骤1制得的混合溶液置于反应容器中,反应混合物经液氮冷冻后,抽真空,熔封,反应,即得喷昔洛韦‑铜(II)配合物。本发明的喷昔洛韦‑铜(II)配合物对于多种肿瘤细胞的增殖抑制率高于喷昔洛韦,喷昔洛韦‑铜(II)配合物比喷昔洛韦能够更有效地抑制两种单纯疱疹病毒标准株,喷昔洛韦‑铜(II)配合物对HSV‑I和HSV‑II的抑制作用分别较喷昔洛韦提高了35.8%和30.4%,铜配合物的抗病毒活性增强明显。
Description
【技术领域】
本发明属于抗病毒药物合成技术领域,具体涉及一种喷昔洛韦-铜配合物、合成方法及其应用。
【背景技术】
喷昔洛韦(Penciclovir)是鸟嘌呤类似物类抗病毒药物,用于治疗多种疱疹病毒感染。具有毒性低,病毒敏感性高等特点。喷昔洛韦口服吸收低,常用于局部给药。核苷类抗病毒药,体外对I型和II型单纯疱疹病毒有抑制作用,在病毒感染细胞中,病毒胸腺嘧啶脱氧核苷激酶将该品磷酸化为喷昔洛韦单磷酸盐,然后细胞激酶将喷昔洛韦单磷酸盐转化为喷昔洛韦三磷酸盐。体外实验表明,喷昔洛韦三磷酸盐与脱氧鸟嘌呤核苷三磷酸盐竞争性抑制单纯疱疹病毒多聚酶,从而选择性抑制单纯疱疹病毒DNA的合成和抑制。通过引入铜与喷昔洛韦生成喷昔洛韦-铜配合物增强其在人体和水产品中抗病毒作用。
【发明内容】
本发明提供一种喷昔洛韦-铜配合物的合成方法,由以下方程式合成,
具体包括以下步骤:
(1)称取物质的量之比为1:1-2:1的喷昔洛韦和五水硫酸铜,溶解于极性溶剂中,得到混合溶液;
(2)将步骤1制得的混合溶液置于反应容器中,反应混合物经液氮冷冻后,将容器抽至真空,熔封,然后于80-130℃条件下反应至完全,即得喷昔洛韦-铜(II)配合物。
优选地,步骤1中所述的极性溶剂可以是水或者乙醇中的一种或它们两者以任意配比的混合溶剂。
更优选地,所述的乙醇体积浓度为50-75%。
所述极性溶剂的用量可根据需要确定,通常情况下,1mmol的喷昔洛韦(PCV)和1mmol的五水硫酸铜(CuSO4·5H2O)一共用5-30mL的极性溶剂来溶解,优选为10~20mL。在具体的溶解步骤中,可将喷昔洛韦(PCV)和五水硫酸铜(CuSO4·5H2O)分别用极性溶剂溶解,再混合在一起反应;也可将喷昔洛韦(PCV)和五水硫酸铜(CuSO4·5H2O)混合后,再加极性溶剂。
优选地,步骤2中所述的反应容器通为耐压耐温的厚壁玻璃管。
优选地,步骤2中所述于80-130℃条件下反应至完全需要1-48小时。
优选地,反应温度为90-110℃,反应时间为8-12小时。
本发明还提供一种喷昔洛韦-铜配合物的应用,是对人和水产品具有抗病毒药物的应用。
本发明具有下述效果:
本发明的喷昔洛韦的金属配合物在国内外均未见报道,本发明所报道的喷昔洛韦-铜配合物是首次报道。喷昔洛韦-铜(II)配合物的增殖抑制率高于喷昔洛韦,且在20μM的浓度下,抑制率高于50%,表现出了明显高于喷昔洛韦配体的体外抗肿瘤活性。喷昔洛韦-铜(II)配合物比喷昔洛韦更有效地抑制HSV-I(sm44)和HSV-II(333)感染vero细胞所致的细胞病变效应CPE。喷昔洛韦-铜(II)配合物对HSV-I和HSV-II的抑制作用分别较喷昔洛韦提高了35.8%和30.4%。上述结果均与喷昔洛韦与铜(II)的配位结合作用引起潜在的正协同效应直接相关。
【附图说明】
图1为喷昔洛韦-铜(II)配合物的晶体结构图。
图2为喷昔洛韦-铜(II)配合物的红外光谱图。
【具体实施方式】
为便于更好地理解本发明,通过以下实施例加以说明,这些实施例属于本发明的保护范围,但不限制本发明的保护范围。
实施例1
一种喷昔洛韦-铜配合物的合成方法,是将喷昔洛韦(PCV)(1.5mmol,0.377g)和五水硫酸铜(CuSO4·5H2O)(1.0mmol,0.250g)一起放到一根一端封口的厚壁玻璃管中,加入10mL混合溶剂(乙醇/水=2:1)。用液氮将混合反应液冷冻后,在抽真空的条件下,将玻璃管另一端熔封。将玻璃管避光放置到100℃的烘箱中反应12小时。之后,将玻璃管缓慢降温至室温,管内生成绿色的配合物晶体产物。经结构表征和单晶X射线衍射分析,确定该产物即为喷昔洛韦-铜(II)配合物(图1为喷昔洛韦-铜(II)配合物的晶体结构图),产率75%。
红外光谱(KBr,cm--1):3400,2931,2375,1666,1633,1487,1413,1384,1120,1033,859,776,689,619,510(附图2).
元素分析结果:Anal.Calcd.for C20H42CuN10O16S(%):C,31.00;H,5.42;N,18.08.Found:C 29.77,H 5.68,N 17.93;
分子量:M.W.=774.26.
实施例2
一种喷昔洛韦-铜配合物的合成方法,是将喷昔洛韦(PCV)(1.0mmol,0.250g)和五水硫酸铜(CuSO4·5H2O)(1.0mmol,0.250g)一起放到一根一端封口的厚壁玻璃管中,加入5mL混合溶剂(乙醇/水=3:1)。用液氮将混合反应液冷冻后,在抽真空的条件下,将玻璃管另一端熔封。将玻璃管避光放置到120℃的烘箱中反应2小时。之后,将玻璃管缓慢降温至室温,管内生成绿色的配合物晶体产物。经结构表征和单晶X射线衍射分析,确定该产物即为喷昔洛韦-铜(II)配合物(图1为喷昔洛韦-铜(II)配合物的晶体结构图),产率48%。
实施例3
一种喷昔洛韦-铜配合物的合成方法,是将喷昔洛韦(PCV)(2.0mmol,0.506g)和五水硫酸铜(CuSO4·5H2O)(1.0mmol,0.250g)一起放到一根一端封口的厚壁玻璃管中,加入20mL混合溶剂(乙醇/水=1:1)。用液氮将混合反应液冷冻后,在抽真空的条件下,将玻璃管另一端熔封。将玻璃管避光放置到80℃的烘箱中反应48小时。之后,将玻璃管缓慢降温至室温,管内生成绿色的配合物晶体产物。经结构表征和单晶X射线衍射分析,确定该产物即为喷昔洛韦-铜(II)配合物(图1为喷昔洛韦-铜(II)配合物的晶体结构图),产率70%
实施例4
体外抗肿瘤活性实验
具体步骤如下:
所需试剂与原料:PBS晶体购于北京中杉公司;干粉RPMI1640培养基、胰蛋白酶粉末和小牛血清购于Hydone公司;MTT和CCK-8购于南京凯基生物科技发展有限公司。RPMI1640培养液,干粉RPMI1640培养基,DMEN培养基,四甲基偶氮唑盐[MTT,3-(4,5-dimethylthbm)1-2-yl-2,5-diphenyl-tetrazoliumbromide]。实验所用细胞株均购于中国科学院上海生命科学研究院。
试剂及细胞使用方法:
PBS缓冲液:将袋装的PBS晶体溶解于二次亚沸水中,转移到1L容量瓶进行定容,高温灭菌后于4℃保存备用;
RPMI1640培养液:将袋装的培养基溶于二次亚沸水中,用HCl调节PH为7.2-7.4,转移到1L容量瓶进行定容后4℃下过夜,用已消毒的过滤器过滤后于-20℃保存备用;
胰蛋白酶溶液:称取适量的胰蛋白酶,用一定体积的PBS缓冲液进行溶解,使溶液浓度为0.25%,4℃下过夜,用已消毒的过滤器过滤后于-20℃保存备用;
所用细胞株:人胃癌细胞SGC-7901、人肝癌细胞BEL-7404、人大细胞肺癌细胞A549、人乳腺癌细胞MCF-7、人宫颈癌细胞HeLa。
药物溶液浓度:将喷昔洛韦及其铜(II)配合物用DMSO溶解,配制成2×10-3M的储液备用。
测试方法:采用MTT法,即四甲基偶氮唑盐微量酶反应比色法,是一种检测细胞存活和生长的方法。MTT是一种能够接受氢原子而被还原的燃料,其检测原理为活细胞线粒体中的烟酰胺腺嘌呤二核苷酸磷酸(NADP)转氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒并沉积在细胞中,而死细胞无此功能。DMSO能溶解细胞中的甲瓒,用酶标仪在一定波长处测定其光密度值,在一定细胞数范围内,MTT结晶形成的量与细胞数成正比,因此可根据测得的光密度(OD)值来判定活细胞的数量,根据如下公式计算出药物对肿瘤细胞生长的抑制率:
抑制率(%)=(1-实验组平均OD值/对照组平均OD值)×100%
实验步骤:
细胞株的培养:将所选的细胞株置于10%小牛血清、100U/mL青霉素、100U/mL链霉菌的RPMI1640培养液中,在37℃下含5%CO2的培养箱中培养。每天在倒置的显微镜下观察细胞生长的情况。
(1)取对数生长期细胞,经0.25%胰蛋白酶消化后,用10%小牛血清的培养液调整细胞悬液浓度,每孔190μL接种于96孔培养板中,使待测细胞密度至1000-10000孔,(边缘孔用无菌PBS填充);
(2)5%CO2,37℃孵育,至细胞单层铺满,孔底加入浓度梯度的药物10μL,其中DMSO终浓度≤1%,同时设相应的阴性对照组(培养液中只有细胞和等量DMSO,无药物)和空白对照组(培养液中只有等量的药物,无细胞),在实验过程中,每个浓度梯度平行设有4个复孔以保证实验的真实性;
(3)5%CO2,37℃孵育48小时,倒置显微镜下观察。
(4)每孔加入10μL MTT溶液(5mg/mL BPS缓冲液,即0.5%MTT),继续培养4h,在呈色后中止培养尽量吸尽孔内残余培养液;
(5)每孔加入150μL DMSO,平板震荡器振荡5min,使结晶物充分溶解,最后比色以空白凋零,用酶标仪570nm/630nm双波长测定去除本底光吸收值后的吸光度(A)值;
由上述体外抗肿瘤活性测试结果可以看出,对于上述受测的5种典型的人源肿瘤细胞株,喷昔洛韦-铜(II)配合物的增殖抑制率均高于喷昔洛韦,且在20μM的浓度下,抑制率均高于50%,表现出了明显高于喷昔洛韦配体的体外抗肿瘤活性,这与喷昔洛韦与铜(II)的配位结合作用引起潜在的正协同效应直接相关。
实施例5
病毒增殖和效价滴定:两种单纯疱疹病毒标准株:HSV-I(sm44)和HSV-II(333)均在成纤维肾细胞vero细胞中增殖和制备。vero细胞接种96孔微量培养板,待细胞长成单层后弃去生长液(含10%小牛血清,GIBCO公司产品),PBS洗1次,接种10倍稀释(用MEM)的各稀释度病毒液,每滴度4孔。37℃吸附1小时,PBS洗1次。加维持液(含2%小牛血清)置37℃培养。镜检观察细胞病变效应(CPE)。记录实验结果,按Reed—muench法计算HSV-I(sm44)和HSV-II(333)原液的感染性滴度。
CPE抑制实验:vero细胞传代接种96孔板,待细胞长成单层后,弃去生长液,PBS洗l次,接种HSV-I(sm44)和HSV-II(333)。37℃吸咐1小时,弃去病毒液,PBS洗1次。药物从62.5μg/mL处用细胞维持液作倍比稀释,加入不同稀释度的药物―细胞维持液,置37℃培养。实验设药物对照、病毒对照和正常细胞对照。待病毒对照CPE达到4+,记录实验结果,按Reed—muench法计算半数有效浓度(IC50),即使50%细胞培养孔发生CPE的药物浓度。
实验结果:
PCV-Cu比PCV能够更为有效地抑制HSV-I(sm44)和HSV-II(333)感染vero细胞所致的细胞病变效应CPE。用Reed—Muench法计算得:PCV-Cu对HSV-I和HSV-II的半数有效浓度(IC50)分别为1.22μg/mL和2.85μg/mL。相对来看,PCV对HSV-I和HSV-II的半数有效浓度(IC50)分别为1.90μg/mL和4.10μg/mL。因此,PCV-Cu对HSV-I和HSV-II两种单纯疱疹病毒的抑制作用分别较PCV提高了35.8%和30.4%,铜配合物的抗病毒活性增强明显。
以上内容是对本发明所作的进一步详细说明,不能认定本发明的只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。
Claims (8)
1.一种喷昔洛韦-铜配合物的合成方法,其特征在于,由以下方程式合成,
具体包括以下步骤:
(1)称取物质的量之比为1:1-2:1的喷昔洛韦和五水硫酸铜,溶解于极性溶剂中,得到混合溶液;
(2)将步骤1制得的混合溶液置于反应容器中,反应混合物经液氮冷冻后,将容器抽至真空,熔封,然后于80-130℃条件下反应至完全,即得喷昔洛韦-铜(II)配合物。
2.根据权利要求1所述的喷昔洛韦-铜配合物的合成方法,其特征在于,步骤1中所述的极性溶剂可以是水或者乙醇中的一种或它们两者以任意配比的混合溶剂。
3.根据权利要求2所述的喷昔洛韦-铜配合物的合成方法,其特征在于,所述的乙醇体积浓度为50-75%。
4.根据权利要求1所述的喷昔洛韦-铜配合物的合成方法,其特征在于,步骤2中所述的反应容器通为耐压耐温的玻璃管。
5.根据权利要求1所述的喷昔洛韦-铜配合物的合成方法,其特征在于,步骤2中所述于80-130℃条件下反应至完全需要1-48小时。
6.根据权利要求5所述的喷昔洛韦-铜配合物的合成方法,其特征在于,所述于90-110℃条件下反应至完全需要8-12小时。
7.一种根据权利要求1-6任一项所述合成方法合成的喷昔洛韦-铜配合物。
8.一种根据权利要求7所述的喷昔洛韦-铜配合物的应用,其特征在于,是对人和水产品具有抗肿瘤、抗病毒药物的应用。
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