CN106243026B - 烟酸或异烟酸类化合物及其用途 - Google Patents

烟酸或异烟酸类化合物及其用途 Download PDF

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CN106243026B
CN106243026B CN201610546453.3A CN201610546453A CN106243026B CN 106243026 B CN106243026 B CN 106243026B CN 201610546453 A CN201610546453 A CN 201610546453A CN 106243026 B CN106243026 B CN 106243026B
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王慧
张万年
李涛
缪震元
卢晓雪
庄春林
王建新
徐西国
黄洁
吴岳林
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Abstract

本发明公开了一种烟酸或异烟酸类化合物及其用途。本发明提供的化合物，为式I所示化合物或其药学上可接受的盐、酯、溶剂合物。药效试验证明，本发明提供的巯基烟酸类化合物在体外能抑制肉毒毒素内肽酶活性，同时对肉毒毒素中毒小鼠具有明显的保护作用。基于此，本发明提供的化合物可用于制备预防和/或治疗肉毒毒素暴露和/或中毒的药物。

Description

烟酸或异烟酸类化合物及其用途
技术领域
本发明属于药物领域，涉及一种烟酸或异烟酸类化合物及其用途。
背景技术
梭菌属细菌毒素包括肉毒神经毒素和破伤风毒素。破伤风毒素由梭状芽孢杆菌属破伤风梭状杆菌在厌氧环境中产生，在临床上可引起破伤风。肉毒神经毒素(ClostridiumBotulinum Neurotoxins，简称肉毒毒素)是一组已知毒力最强的蛋白质(包括A-G型)，主要是由梭状芽孢杆菌属肉毒杆菌在厌氧环境中产生，其静脉注射半数致死量约为1ng/kg，吸入半数致死剂量约为3ng/kg。常见肉毒毒素中毒主要是由于食用污染的食物、伤口感染产生毒素进入体内及婴儿肠道中的毒素前体通过消化产生活性毒素而发生。此外，A型肉毒毒素已经广泛用于胆碱能神经和肌肉功能障碍的临床治疗，以及应用于美容去皱，治疗使用中有可能因用药过量、误用滥用和(或)不良反应而出现全身肉毒中毒症状。肉毒中毒潜伏期较短，病程发展快，病情较严重，病死率高。其中引起人类中毒的主要是A、B、E、F型，而A型对人的神经毒性最强。目前用于肉毒中毒和破伤风预防和治疗的药物是(型)特异性抗毒素马血清，能对80％以上的中毒病人有效，但此类药物存在明显的副作用，临床报道约9％病例出现血清病和过敏性反应(Black RE,et al.Am J Med,1980,69:567-570)，严重限制了马血清抗毒素的应用，迫切需要寻找更加安全、有效的新型药物。
破伤风和肉毒中毒具有相似的致病过程，是由毒素的重链C端与神经细胞膜上的神经节苷脂结合，酸性环境使得其结构重排，并促使重链N端进入膜内，同时轻链去折叠，二硫键被还原后作为锌离子金属酶转入细胞，催化裂解一类胞内底物蛋白(A、E型肉毒神经毒素作用于突触小体相关蛋白SNAP-25，B、D、F、G型肉毒神经毒素及破伤风毒素作用于突触小泡相关膜蛋白VAMP)，从而影响乙酰胆碱的转移，干预神经冲动的传导，而引起运动神经麻痹或兴奋。如果对结合、转入和催化3个环节进行一个或几个环节的抑制或阻遏，可有效抑制毒素的神经毒性。而以具有酶活性的毒素轻链为靶点设计和发展催化类抑制剂成为近些年的研究热点。Harry B等体外高通量筛选植物、海洋组织、真菌天然提取物对梭菌属毒素产生抑制的物质，发现了30种非亚型抑制剂，其中5种提取物可同时抑制B型、E型肉毒毒素。Smith LA等在2009年发现了喹啉醇衍生物CB7969312是一种潜在抑制剂，通过结合到A型肉毒毒素轻链活性位点的大疏水区口袋的Zn催化区域，有效地中和A型肉毒毒素对N2a细胞的毒性(Roxas-Duncan,V,et al.Agents Chemother.2009,53:3478-3486；Pang,Y.-P.etal.PLos One 2009,4,e7730.)。Janda KD等在2010年发现lomofungin能抑制A型肉毒毒素轻链(Ki值6.7±0.7uM)，显示出典型的非竞争性动力学(Eubanks,L.M.,et al.ACSMed.Chem.Lett.2010,1:268-272.)。这些报道的天然产物或化合物对毒素轻链具有体外抑制作用，有些天然产物或化合物在整体毒素和动物模型水平也具有抑毒效应，但活性仍有提升空间。另外，目前报道的大多数化合物具有毒素型特异抑制活性，而交叉抑制作用还有待提高。因此有必要研究发展新的天然产物或活性化合物，可以对梭菌属神经毒素(多型肉毒毒素和破伤风毒素)具有更广范围的体外抑制和拮抗作用，而且可以在整体动物水平体现出较高抗毒活性，从中研发新的治疗药物。
发明内容
本发明的目的是提供一种烟酸或异烟酸类化合物及其用途。
本发明提供的新烟酸或异烟酸类化合物，包括接受的盐、酯、溶剂合物，该化合物的结构通式如式I所示：
Figure GDA0002926961140000021
所述式I中，
R1、R2独立地表示下述基团中的任意一种：氢或-(CH2)mCOOH；
R3、R4、R5、R6、R7独立地表示下述基团中的任意一种：氢、羟基、氨基、卤素、低级烷基、低级烷氧基和低级环烷基；
所述低级烷基是指含1至6个碳原子的直链或支链饱和脂肪烃基；
所述低级烷氧基是指含1至6个碳原子的直链或支链烷氧基；
所述低级环烷基是指含3至7个碳的环基；
X独立地表示下述基团中的任意一种：O、-S＝O和-SO2；
m是0或1或2或3。
本文中，所述低级烷基具体可为甲基、乙基、丙基、异丙基、丁基或叔丁基；
所述低级烷氧基具体可为甲氧基或乙氧基；
所述低级环烷基具体可为环丙基、环丁基、环戊基、环己基或环庚基；
所述卤素具体可为氯、溴、碘或氟；
上述式I所示化合物药学上可接受的盐为吡啶环上取代羧基与金属离子，或吡啶环氮原子与有机酸或无机酸形成的各类盐。
更优的，式I所示化合物选自以下化合物：
2-(2-((5-氯-2-甲氧基苯胺基)乙酰基)磺酰基)异烟酸，其结构式如2a所示；
2-(2-((5-氯-2–苄氧基苯胺基)乙酰基)磺酰基)烟酸，其结构式如2b所示；
2-(2-((5-氯-2-甲氧基苯胺基)乙酰基)亚磺酰基)烟酸，其结构式如2c所示；
2-(2-(5-氯-2-甲氧基苯胺基)乙酰基氧代)异烟酸，其结构式如6a所示；
2-(2-(5-氯-2-甲氧基苯胺基)乙酰基氧代)烟酸，其结构式如6b所示。
Figure GDA0002926961140000031
本发明的第二方面，是提供了上述的烟酸类或异烟酸类化合物及其药学上可接受的盐、酯、溶剂合物的制备方法，为如下方法A或方法B：
所述方法A包括如下步骤：使式II所示化合物进行氧化反应，反应完毕得到式I所示化合物；
Figure GDA0002926961140000032
式II中R1、R2、R3、R4、R5、R6和R7的定义同式I；
所述方法B包括如下步骤：
1)使式III所示化合物与氯乙酰氯或溴乙酰氯反应，得到式IV所示化合物；
Figure GDA0002926961140000033
式III、式IV中R3、R4、R5、R6和R7的定义同式I；
2)使式IV所示化合物与式V所示化合物进行醚化反应，得到所述式I所示化合物；
Figure GDA0002926961140000041
式V中R1和R2的定义同式I。
另外，上述本发明提供的式I所示化合物或其药学上可接受的盐、酯、溶剂合物以及它们的混合物在制备下述产品中的应用，也属于本发明的保护范围：
1)预防和/或治疗肉毒毒素暴露和/或中毒的药物；
2)肉毒毒素内肽酶抑制剂。
本发明还提供了一种肉毒毒素内肽酶抑制剂，其活性成分为前述本发明提供的式I所示化合物或其药学上可接受的盐、酯、溶剂合物以及它们的混合物。
本发明还提供了预防和/或治疗肉毒毒素暴露和/或中毒的药物，其活性成分为前述本发明提供的式I所示化合物或其药学上可接受的盐、酯、溶剂合物以及它们的混合物。
本发明还提供了预防和/或治疗肉毒毒素暴露和/或中毒的药物制剂，其活性成分为前述本发明提供的式I所示化合物或其药学上可接受的盐、酯、溶剂合物以及它们的混合物。
上述应用或肉毒毒素内肽酶抑制剂或药物或药物制剂中，所述肉毒毒素具体可包括A型肉毒毒素和B型肉毒毒素。
上述药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体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。
所述药物可以制成注射液、悬浮剂、粉剂、片剂、颗粒剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。
药效试验证明，本发明提供的巯基烟酸类化合物在体外能抑制肉毒毒素内肽酶活性，同时对肉毒毒素中毒小鼠具有明显的保护作用。
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
实施例1、2-(2-((5-氯-2-甲氧基苯胺基)乙酰基)磺酰基)异烟酸的合成
称取2-((2-((5-氯-2-甲氧基苯基)氨基)-2-氧代乙基)硫醚)异烟酸30mg溶于3mL无水乙酸中，缓慢加入双氧水0.2mL，室温反应2h，柱纯化得到白色固体，收率42％。
该产物的结构确证结果如下：
1H NMR(300MHz,DMSO-d6)δ:9.86(s,1H),8.97(d,J＝4.9MHz,1H),8.29(s,1H),8.15(dd,J＝1.2,4.9MHz,1H),7.92(d,J＝2.4MHz,1H),7.04-7.16(m,2H),4.92(s,2H),3.84(s,3H)；ESI-MS(m/z):383.06(M-H+),766.87(2M-H+)。由上可知，该化合物结构正确，目标化合物编号2a。
Figure GDA0002926961140000051
实施例2、2-(2–((5-氯-2-苄氧基苯胺基)乙酰基)磺酰基)烟酸的合成
按照与上相同的步骤，仅将2-((2-((5-氯-2-甲氧基苯基)氨基)-2-氧代乙基)硫醚)异烟酸替换为2-((2-((5-氯-2-苄氧基苯基)氨基)-2-氧代乙基)硫醚)烟酸，得到2-(2–((5-氯-2-苄氧基苯胺基)乙酰基)磺酰基)烟酸。该产物为白色固体，收率53％。
该产物的结构确证结果如下：
1H NMR(300MHz,DMSO-d6)δ:10.03(s,1H),8.42(d,J＝7.4MHz,1H),8.21(s,1H),7.99(s,1H),7.51(d,J＝7.5MHz,2H),7.32-7.44(m,3H),7.11(s,2H),6.75(t,1H),5.29(s,2H),5.13(s,2H)；ESI-MS(m/z):459.01(M-H+)。由上可知，该化合物结构正确，目标化合物编号2b。
Figure GDA0002926961140000052
实施例3、2-(2-((5-氯-2-甲氧基苯胺基)乙酰基)亚磺酰基)烟酸的合成
按照与上相同的步骤，仅将2-((2-((5-氯-2-甲氧基苯基)氨基)-2-氧代乙基)硫醚)异烟酸替换为2-((2-((5-氯-2-甲氧基苯基)氨基)-2-氧代乙基)硫醚)烟酸，得到2-(2-((5-氯-2-甲氧基苯胺基)乙酰基)亚磺酰基)烟酸。该产物为白色固体，收率48％。
该产物的结构确证结果如下：
1H NMR(300MHz,DMSO-d6)δ:7.97(s,1H),8.68(dd,J＝1.6,4.8MHz,1H),8.28(dd,J＝1.6,7.7MHz,1H),8.18(d,J＝2.2MHz,1H),7.35(q,1H),6.98-7.09(m,2H),4.00(s,2H),3.78(s,3H)；ESI-MS(m/z):383.17(M-H+),767.01(2M-H+)。由上可知，该化合物结构正确，目标化合物编号2c。
Figure GDA0002926961140000061
实施例4、2-(2-(5-氯-2-甲氧基苯胺基)乙酰基氧代)异烟酸的合成
称取2-甲氧基-5-氯苯胺0.5g溶于10mL无水二氯甲烷中，加入三乙胺1mL，置于50mL茄形瓶中。精密量取氯乙酰氯0.25mL，稀释于2mL无水二氯甲烷中，缓慢滴加至反应瓶中，室温反应4h。用水淬灭反应，用二氯甲烷萃取三次，合并浓缩有机相，柱层析得到2-氯-N-(5-氯-2-甲氧基苯基)乙酰胺，收率78％。
1H NMR(300MHz,DMSO-d6)δ:9.68(s,1H),8.12(d,J＝2.5MHz,1H),7.19(dd,J＝2.6,8.8MHz,1H),7.12(d,J＝8.8MHz,1H),4.43(s,2H),3.89(s,3H)；ESI-MS(m/z):272(M+K+).
称取2-氯-N-(5-氯-2-甲氧基苯基)乙酰胺0.1g，2-羟基异烟酸0.56g，碳酸钾0.19g置于20mL茄形瓶中，加入5mL N,N-二甲基甲酰胺，室温进行醚化反应过夜。用大量水与乙酸乙酯萃取，合并浓缩有机相，柱层析得到2-(2-(5-氯-2-甲氧基苯胺基)乙酰基氧代)异烟酸。白色固体，收率57％。
该产物的结构确证结果如下：
1H NMR(300MHz,DMSO-d6)δ:10.02(s,1H),8.42(d,J＝7.2MHz,1H),8.22(d,J＝6.7MHz,1H),8.04(s,1H),7.10(m,2H),6.75(t,1H),5.11(s,2H),3.87(s,3H)；ESI-MS(m/z):335.31(M-H+)。由上可知，该化合物结构正确，目标化合物编号6a。
Figure GDA0002926961140000062
实施例5、2-(2-(5-氯-2-甲氧基苯胺基)乙酰基氧代)烟酸的合成
按照与上相同的步骤，仅将2-羟基异烟酸替换为2-羟基烟酸，得到白色固体，收率62％。
该产物的结构确证结果如下：
1H NMR(300MHz,DMSO-d6)δ:9.82(s,1H),8.08(d,J＝2.0MHz,1H),7.63(d,J＝6.8MHz,1H),7.33(d,J＝6.7MHz,1H),7.09-7.12(m,1H),6.80(d,J＝9.8MHz,1H),6.54(dd,J＝1.5,6.8MHz,1H),4.85(s,2H),3.87(s,3H)；ESI-MS(m/z):335.15(M-H+)。由上可知，该化合物结构正确，目标化合物编号6b。
Figure GDA0002926961140000071
实施例6、新的巯基烟酸类化合物对A型肉毒毒素内肽酶活性的体外抑制作用。
采用底物切割法检测化合物活性。A型肉毒毒素内肽酶(BoNT/A-LC)的表达和纯化参见文献(L.Li,B.R.Singh,High-Level expression,purification,andcharacterization of recombinant type A botulinum neurotoxin light chain,Protein Expr Purif.1999,17:339–344.)。A型肉毒毒素底物CYA的构建和表达纯化参见文献(D.R.Ruge,F.M.Dunning,T.M.Piazza,B.E.Molles,M.Adler,F.N.Zeytin,W.C.Tucker,Detection of six serotypes of botulinum neurotoxin using fluorogenicreporters,J.Anal.Biochem.2011,411:200–209.)。在50μl反应液(50mM Hepes–NaOH pH7.4,10mM NaCl,0.1％Tween20,5mM dithiothreitol,10μM ZnCl2)中，加入终浓度10nM的BoNT/A-LC和100μg的化合物，37℃共孵育15min，再加入终浓度4μM的底物CYA(针对A型肉毒毒素)，体外切割30min。SDS-PAGE检测和分析通过凝胶成像系统(Bio-Rad公司MolecularImager chemiDocTM XRS Imaging System)完成，凝胶图像拍摄通过凝胶采集和图像分析系统(ChampGel 5000Plus公司SAGECREATION)完成。数据通过Gel-Pro analyzer软件分析处理，进行蛋白片段灰度分析，与对照组进行比较，计算出化合物的抑制率。
底物切割法检测化合物体外抑制活性结果如表1所示，化合物对A型肉毒毒素内肽酶活性具有不同的抑制作用，其抑制率范围0-60.0％，其中实施例1所得2c所示化合物的抑制效果显著，抑制率可达60.0％。
表1.底物切割法检测化合物对A型肉毒毒素的抑制效果
Figure GDA0002926961140000072
Figure GDA0002926961140000081
实施例7、新的巯基烟酸类化合物对B型肉毒毒素内肽酶活性的体外抑制作用。
采用底物切割法检测化合物活性。B型肉毒毒素内肽酶(BoNT/B-LC)的表达和纯化参见文献(J.Gilsdorf,N.Gul,L.A.Smith,Expression,purification,andcharacterization of Clostridium botulinum type B light chain,Protein ExprPurif 46(2006)256–267.)。B型肉毒毒素底物CYB的构建和表达纯化参见文献(D.R.Ruge,F.M.Dunning,T.M.Piazza,B.E.Molles,M.Adler,F.N.Zeytin,W.C.Tucker,Detection ofsix serotypes of botulinum neurotoxin using fluorogenic reporters,J.Anal.Biochem.2011,411:200–209.)。在50μl反应液(50mM Hepes–NaOH pH 7.4,10mMNaCl,0.1％Tween20,5mM dithiothreitol,10μM ZnCl2)中，加入终浓度10nM的BoNT/B-LC和100μg的化合物，37℃共孵育15min，再加入终浓度4μM的底物CYB(针对B型肉毒毒素)，体外切割30min。SDS-PAGE检测和分析通过凝胶成像系统(Bio-Rad公司Molecular ImagerchemiDocTM XRS Imaging System)完成，凝胶图像拍摄通过凝胶采集和图像分析系统(ChampGel 5000Plus公司SAGECREATION)完成。数据通过Gel-Pro analyzer软件分析处理，进行蛋白片段灰度分析，与对照组进行比较，计算出化合物的抑制率。
底物切割法检测化合物体外抑制活性结果如表1所示，化合物对B型肉毒毒素内肽酶活性具有不同的抑制作用，其抑制率范围0-85.0％，其中，实施例2所得6b所示化合物的抑制效果显著，抑制率可达85.0％，其次为实施例2所得6a所示化合物，抑制率为80％。
表2.底物切割法检测化合物对B型肉毒毒素的抑制效果
Figure GDA0002926961140000082
实施例8.新的巯基烟酸类化合物对动物A型肉毒毒素中毒的保护效果
A型肉毒毒素(BoNT/A)的提取和鉴定方法参见文献(C.J.Malizio,M.C.Goodnough,E.A.Johnson,Purification of Clostridium botulinum type Aneurotoxin,Methods Mol Biol.2000,145:27-39.)，Balb/c小鼠16-18g购自军事医学科学院实验动物中心。肉毒毒素中毒的保护性实验参照文献描述(C.H.Hatheway,J.D.Snyder,J.E.Seals,T.A.Edell,G.E.Lewis,Jr.Antitoxin levels in botulism patientstreated with trivalent equine botulism antitoxin to toxin types A,B,andE.Infect Dis 1984,150:145-151.)的小鼠肉毒中毒模型方法，简要方法如下：以Balb/C小鼠为测试动物，进行随机分组，10只/组，1mg的不同化合物为待检样品，先给小鼠尾静脉注射5倍半数致死剂量(5LD50)的A型肉毒毒素，再给小鼠尾静脉注射1mg的化合物(20％1,3-丙二醇溶解的化合物)溶液，同时设置空白组和对照组。持续观察超过5天，观察小鼠肉毒中毒体征(如出现蜂腰，皮毛倒立，呼吸微弱，四肢麻痹直至死亡)，记录小鼠生存时间，计算存活率。
化合物在小鼠肉毒中毒模型中的抗毒作用如表3所示，化合物可以不同程度抑制A型肉毒毒素对受试动物的致死效应，其中，实施例1所得2c所示化合物的保护率最高(100％)，其次实施例2所得6a所示化合物的保护率为60％。
表3.化合物在小鼠A型肉毒中毒模型中的抗毒作用
Figure GDA0002926961140000091
注：表3中安慰剂为溶媒(体积百分浓度20％的1,3-丙二醇水溶液)；抗毒素为A型马血清抗毒素(购自中国食品药品检定研究院)
实施例9.巯基烟酸类化合物对动物B型肉毒毒素中毒的保护效果
B型肉毒毒素(BoNT/B)的提取和鉴定方法参见文献(H.Arimitsu,K.Inoue,Y.Sakaguchi,J.Lee,Y.Fujinaga,T.Watanabe,T.Ohyama,R.Hirst,K.Oguma,Purificationof fully activated Clostridium botulinum serotype B toxin for treatment ofpatients with dystonia,Infect Immun.71(2003)1599-1603.)，Balb/c小鼠14-16g购自军事医学科学院实验动物中心。
肉毒毒素中毒的保护性实验参照文献描述(C.H.Hatheway,J.D.Snyder,J.E.Seals,T.A.Edell,G.E.Lewis,Jr.Antitoxin levels in botulism patientstreated with trivalent equine botulism antitoxin to toxin types A,B,andE.Infect Dis 1984,150:145-151.)的小鼠肉毒中毒模型方法，简要方法如下：以Balb/C小鼠为测试动物，进行随机分组，10只/组，1mg的不同化合物为待检样品，先给小鼠尾静脉注射3倍半数致死剂量(3LD50)的B型肉毒毒素，再给小鼠尾静脉注射1mg的化合物(20％1,3-丙二醇溶解的化合物)溶液，同时设置空白组和对照组。持续观察超过5天，观察小鼠肉毒中毒体征(如出现蜂腰，皮毛倒立，呼吸微弱，四肢麻痹直至死亡)，记录小鼠生存时间，计算存活率。
化合物在小鼠肉毒中毒模型中的抗毒作用如表4所示，化合物可以不同程度抑制B型肉毒毒素对受试动物的致死效应，其中，实施例2所得6b所示化合物的保护率最高，为80％；其次为实施例1所得2c所示化合物和实施例2所得6a所示化合物，其保护率均为20％。
表4.化合物在小鼠B型肉毒中毒模型中的抗毒作用
Figure GDA0002926961140000101
注：表4中安慰剂为溶媒(体积百分浓度20％的1,3-丙二醇水溶液)；抗毒素为B型马血清抗毒素(购自中国食品药品检定研究院)。

Claims (7)

1.化合物或其药学上可接受的盐；
所述化合物为
Figure FDA0002906754240000011
Figure FDA0002906754240000012
2.权利要求1所述化合物或其药学上可接受的盐在制备下述产品中的应用：
1)预防和/或治疗肉毒毒素暴露和/或中毒的药物；
2)肉毒毒素内肽酶抑制剂。
3.一种肉毒毒素内肽酶抑制剂，其活性成分为权利要求1所述化合物或其药学上可接受的盐。
4.一种预防和/或治疗肉毒毒素暴露和/或中毒的药物，其活性成分为权利要求1所述化合物或其药学上可接受的盐。
5.一种预防和/或治疗肉毒毒素暴露和/或中毒的药物制剂，其活性成分为权利要求1所述化合物或其药学上可接受的盐。
6.根据权利要求2所述的应用，其特征在于：所述肉毒毒素包括A型肉毒毒素和B型肉毒毒素。
7.权利要求3所述的肉毒毒素内肽酶抑制剂或权利要求4所述的药物或权利要求5所述的药物制剂，其特征在于：所述肉毒毒素包括A型肉毒毒素和B型肉毒毒素。
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