CN114436972B - 帕苯达唑衍生物及其制备方法与应用 - Google Patents
帕苯达唑衍生物及其制备方法与应用 Download PDFInfo
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- CN114436972B CN114436972B CN202210088377.1A CN202210088377A CN114436972B CN 114436972 B CN114436972 B CN 114436972B CN 202210088377 A CN202210088377 A CN 202210088377A CN 114436972 B CN114436972 B CN 114436972B
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
- C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
- C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
- C07D235/30—Nitrogen atoms not forming part of a nitro radical
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Abstract
本发明涉及医药领域,具体涉及帕苯达唑衍生物及其制备方法与应用,帕苯达唑衍生物的结构如式X所示:其中,R1选自烷氧基或烷胺基;R2选自氟,氰基,甲基,三氟甲基,正丁基或正戊基;R3选自氢,甲基或氟,本发明中制备的帕苯达唑衍生物体外对头颈部鳞状细胞癌HNSCC细胞株的增殖具有很好的抑制作用,其能抑制微管蛋白聚合,抑制细胞侵袭,细胞迁移和阻断细胞周期;体内能明显控制HN6异种移植瘤裸鼠模型肿瘤的生长,起到了良好的治疗效果,通过对相关肿瘤标志物的检测也证明了其疗效明显。
Description
技术领域
本发明涉及医药领域,具体涉及帕苯达唑衍生物及其制备方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)是一种口腔、咽和喉部被鳞状上皮覆盖的恶性肿瘤,虽然它起源于黏膜上皮细胞,但具有明显的异质性。除手术切除外,大多数患者需要进行保守治疗。基于细胞毒性的西妥昔单抗、顺铂和5-氟尿嘧啶联合治疗是一种常用的治疗方案,免疫疗法也是一种有前途的治疗方法,虽然对于HNSCC的治疗取得了一些进展,但最佳的治疗方法仍不明确,新药的开发仍是必要和紧迫的。
发明内容
针对现有技术中存在的问题,本发明的目的是提供一种帕苯达唑衍生物及其制备方法与应用,本发明中制备的帕苯达唑衍生物体外对头颈部鳞状细胞癌HNSCC细胞株的增殖具有很好的抑制作用,其能抑制微管蛋白聚合,抑制细胞侵袭,细胞迁移和阻断细胞周期;体内能明显控制HN6异种移植瘤裸鼠模型肿瘤的生长,起到了良好的治疗效果,通过对相关肿瘤标志物的检测也证明了其疗效明显。
为了实现上述目的,本发明的技术方案如下所述:
在本发明的第一方面,提供一种帕苯达唑衍生物,该衍生物的结构如式X所示:
其中,R1选自烷氧基或烷胺基;R2选自氟,氰基,甲基,三氟甲基,正丁基或正戊基;R3选自氢,甲基或氟。
在一种或多种实施方式中,所述帕苯达唑衍生物具有式(I)或式(II)所示结构:
优选地,所述R1选自烷氧基或烷胺基;R2选自氟,氰基,甲基,三氟甲基,正丁基或正戊基;R3选自氢,甲基或氟。
具体地,所述帕苯达唑衍生物选自下列化合物:
化合物6a:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-乙基脲;
化合物6b:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-环己基脲;
化合物6c:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-异丙基脲;
化合物6d:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-丙基脲;
化合物6e:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-己基脲;
化合物6f:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-苯乙基脲;
化合物6g:1-(叔丁基)-3-(6-丁基-1H-苯并[d]咪唑-2-基)脲;
化合物6h:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-环戊基脲;
化合物6i:1-烯丙基-3-(6-丁基-1H-苯并[d]咪唑-2-基)脲;:
化合物6j:2-(3-(6-丁基-1H-苯并[d]咪唑-2-基)脲基)甲基丙烯酸乙酯;
化合物6k:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸异丁酯;
化合物6l:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸苄酯;
化合物6m:2-氯乙基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯;
化合物6n:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸丙酯;
化合物6o:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸己酯;
化合物6p:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸丁酯;
化合物6q:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6r:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸庚酯;
化合物6s:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸环戊酯;
化合物6t:3-氯丙基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯;
化合物6u:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸仲丁酯;
化合物6v:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸烯丙酯;
化合物6w:2-乙基己基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯;
化合物6x:N-(6-丁基-1H-苯并[d]咪唑-2-基)呋喃-2-甲酰胺;
化合物6y:N-(6-丁基-1H-苯并[d]咪唑-2-基)-2-苯基乙酰胺;
化合物6z:N-(6-丁基-1H-苯并[d]咪唑-2-基)四氢-2H-吡喃-4-甲酰胺;
化合物6aa:N-(6-丁基-1H-苯并[d]咪唑-2-基)-2-(噻吩-2-基)乙酰胺;
化合物6ab:(6-氰基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6ac:(6-(三氟甲基)-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6ad:(6-甲基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6ae:(6-氟-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6af:(6-戊基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6ag:(5,6-二氟-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6ah:(5,6-二甲基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
在本发明的第二方面,提供一种第一方面所述帕苯达唑衍生物的制备方法,包括如下步骤:
(1)化合物1的胺基在浓硫酸的催化下被乙酸酐中乙酰化,然后乙酰基的邻位被乙酸酐和硝酸硝基化得到化合物2;随后,化合物2在甲醇中被氢氧化钾溶液水解成化合物3,进而硝基在甲醇中被H2/Pd还原得到化合物4;
(2)2-甲基-2-硫代异脲硫酸盐在碱性条件下与异氰酸酯、氯甲酸酯或酰氯发生取代反应,得到5a-5aa;
(3)化合物4和5a-5aa在酸性条件下发生环化反应生成帕苯达唑衍生物(6a-6ah);
其中步骤(1)和(2)顺序不分先后;
试剂及条件:(Ⅰ)苯胺,乙酸酐,浓硫酸,45~55℃,0.5~1.5h;硝酸,0℃,30~50min。(Ⅱ)KOH,60~70℃,0.5~1.5h。(Ⅲ)H2,Pd/C,室温,23~25h。(Ⅳ)异氰酸酯、氯甲酸酯或酰氯,N,N-二异丙基乙胺,室温,46~50h。(Ⅴ)CH3COOH:H2O=3:10,90~110℃,2.5~3.5h。
具体地,所述制备方法如下:
(1)将醋酸和少量浓硫酸冷却至0℃,加入化合物1;45~55℃搅拌0.5~1.5h后,将混合物冷却至室温,再加入乙酸酐;之后,再次混合物冷却0℃,添加浓硝酸,在0℃搅拌30~50min;将反应液加入到冰水中,得到化合物2;
将化合物2溶于甲醇,加入40%KOH溶液,在60~70℃下搅拌0.5~1.5h;反应溶液用干燥后过滤,除去溶剂得到化合物3;
将化合物3,Pd/C和甲醇混合,使用氢气进行还原;将反应溶液在室温下搅拌23~25h,Pd/C过滤,得到化合物4;
(2)在2-甲基-2-硫脲硫酸酯、DIPEA和乙腈的混合物中,滴加异氰酸酯、氯甲酸酯或酰氯;室温下搅拌46~50h,去除溶剂;加入乙酸乙酯和水,充分搅拌后分液,将有机相洗涤、干燥,产物纯化得到化合物5a-5aa;
(3)将化合物4、5a-5aa与溶剂在90~110℃下搅拌2.5~3.5h,将反应溶液冷却至室温,调节PH至碱性;然后加入二氯甲烷,充分搅拌后分液,有机相干燥、过滤、浓缩、纯化得到所述帕苯达唑衍生物。
优选地,步骤(3)中所述溶剂为乙酸水溶液,其中VCH3COOH:VH2O=3:10;
优选地,所述异氰酸酯选自异氰酸乙酯、异氰酸环己酯、异氰酸异丙酯、异氰酸丙酯、异氰酸己酯、异氰酸苯乙酯、异氰酸叔丁酯、异氰酸环戊酯、异氰酸烯丙酯或甲基丙烯酸异氰基乙酯;
优选地,所述氯甲酸酯选自氯甲酸异丁酯、氯甲酸苄酯、氯甲酸氯乙酯、氯甲酸丙酯、氯甲酸己酯、氯甲酸丁酯、氯甲酸异丙酯、氯甲酸庚酯、氯甲酸环戊酯、氯甲酸-2-乙基己酯、氯甲酸氯丙酯、氯甲酸-1-甲基丙酯或氯甲酸烯丙酯;
优选地,所述酰氯选自呋喃甲酰氯、苯乙酰氯、四氢吡喃-4-甲酰氯或2-噻吩乙酰氯。
在本发明的第三方面,提供了一种药物组合物,其包含上述帕苯达唑衍生物。
在本发明的第四方面,提供了一种药物制剂,其包含上述帕苯达唑衍生物或上述药物组合物,以及药学上可接受的载体和/或辅料。所述制剂选自口服制剂和肠胃外给药制剂,可以为片剂、丸剂、胶囊或注射剂。
在本发明的第五方面,提供上所述帕苯达唑衍生物在制备治疗头颈部鳞状细胞癌HNSCC的药物中的应用;
在一种或多种实施方式中,所述头颈部鳞状细胞癌包括人舌鳞癌和人咽鳞癌。
体外活性研究表明,本发明公开的帕苯达唑衍生物对于测试的三种HNSCC细胞株的增殖具有很好的抑制作用,其能抑制微管蛋白聚合,抑制细胞侵袭,细胞迁移和阻断细胞周期。
体内活性研究表明,本发明公开的帕苯达唑衍生物在口服80mg/kg的剂量能明显控制HN6异种移植瘤裸鼠模型肿瘤的生长,起到了良好的治疗效果,通过对相关肿瘤标志物的检测也证明了其疗效明显。
体内外活性表明这是本发明公开的帕苯达唑衍生物是一种有潜力的治疗头颈部鳞状细胞癌HNSCC的分子,为药物的研发提供一定的基础。
如本文所述的,术语“药物组合物”,其还可以是指“组合物”,其可用于在受试者特别是哺乳动物中实现治疗或预防本发明所述疾病或病症。
本发明化合物的药物组合物,可以以以下的任意方式施与:口服、喷雾吸入、直肠给药、鼻腔给药、***给药、局部给药、非肠道给药如皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内或颅内注射或输入,或借助一种外植的储器用药,其中优选口服、肌注、腹膜内或静脉内用药方式。
本发明化合物或含有它的药物组合物可以单位剂量形式给药。给药剂型可以是液体剂型、固体剂型。液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混旋剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂、包合物、填埋剂、贴剂、擦剂等。
本发明的药物组合物中还可以含有常用的载体,这里所述可药用载体包括但不局限于:离子交换剂,氧化铝,硬脂酸铝,卵磷脂,血清蛋白如人血清蛋白,缓冲物质如磷酸盐,甘油,山梨酯,山梨酸钾,饱和植物脂肪酸的部分甘油酯混合物,水,盐或电解质,如硫酸鱼精蛋白,磷酸氢二钠,磷酸氢钾,氯化钠,锌盐,胶态氧化硅,三硅酸镁,聚乙烯吡咯烷酮,纤维素物质,聚乙二醇,羧甲基纤维素钠,聚丙烯酸酯,蜂蜡,羊毛酯等。载体在药物组合物中的含量可以是1重量%-98重量%,通常大约占到80重量%。为方便起见,局部麻醉剂,防腐剂,缓冲剂等可直接溶于载体中。
口服片剂和胶囊可以含有赋形剂如粘合剂,如糖浆,***胶,山梨醇,黄芪胶,或聚乙烯吡咯烷酮,填充剂,如乳糖,蔗糖,玉米淀粉,磷酸钙,山梨醇,氨基乙酸,润滑剂,如硬脂酸镁,滑石,聚乙二醇,硅土,崩解剂,如马铃薯淀粉,或可接受的增润剂,如月桂醇钠硫酸盐。片剂可以用制药学上公知的方法包衣。
口服液可以制成水和油的悬浮液,溶液,乳浊液,糖浆,也可以制成干品,用前补充水或其它合适的媒质。这种液体制剂可以包含常规的添加剂,如悬浮剂,山梨醇,纤维素甲醚,葡萄糖糖浆,凝胶,羟乙基纤维素,羧甲基纤维素,硬脂酸铝凝胶,氢化的食用油脂,乳化剂,如卵磷脂,山梨聚糖单油酸盐,***树胶;或非水载体(可能包含可食用油),如杏仁油,油脂如甘油,乙二醇,或乙醇;防腐剂,如对羟基苯甲酸甲酯或丙酯,山梨酸。如需要可添加调味剂或着色剂。
栓剂可包含常规的栓剂基质,如可可黄油或其它甘油酯。
对于胃肠外投药,液态剂型通常由化合物和一种消毒的载体制成。载体首选水。依照所选载体和药物浓度的不同,化合物既可溶于载体中也可制成悬浮溶液,在制成注射用溶液时先将化合物溶于水中,过滤消毒后装入封口瓶或安瓿中。
必须认识到,通式X、通式I~II化合物的最佳给药剂量和间隔是由化合物性质和诸如给药的形式、路径以及所治疗的特定哺乳动物等外部条件决定的,而这一最佳给药剂量可用常规的技术确定。同时也必须认识到,最佳的疗程,即同时X化合物在额定的时间内每日的剂量,可用本领域内公知的方法确定。
本发明的具体实施方式具有以下有益效果:
对本发明所公开的帕苯达唑衍生物的体外活性研究表明,其对于测试的三种HNSCC细胞株的增殖具有很好的抑制作用,其能抑制微管蛋白聚合,抑制细胞侵袭,细胞迁移和阻断细胞周期;
对本发明所公开的帕苯达唑衍生物的体内活性研究表明,其在口服80mg/kg的剂量能明显控制HN6异种移植瘤裸鼠模型肿瘤的生长,起到了良好的治疗效果,通过对相关肿瘤标志物的检测也证明了其疗效明显;
体内外活性表明本发明所公开的帕苯达唑衍生物是一种有潜力的治疗HNSCC的分子,为治疗HNSCC的药物的研发提供一定的基础。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为:HN6细胞中微管蛋白的免疫染色。用化合物(100nmol/L)处理HN6细胞24h,免疫荧光测定显示微管结构。红色(Alexa555)用于标记微管蛋白,蓝色(DAPI)用于标记细胞核;比例尺为20μm;A为用DMSO处理的HN6细胞;B为用6q处理的HN6细胞;C为用帕苯达唑处理的HN6细胞;D为用6v处理的HN6细胞。
图2为:6q和6v能有效抑制肿瘤细胞的迁移和侵袭。A为化合物处理24h后,分别于0h和24h拍摄6孔板中生长的细胞,计算划痕愈合率;用200μL的吸头制造划痕,用倒置荧光显微镜拍照;B为帕苯达唑、6q、6v处理后细胞划痕愈合率;数据表示为平均值±SEM(n=3,*P<0.05,***P<0.001);C为用或不用化合物处理24h后,通过基质的细胞用结晶紫染色;D为帕苯达唑、6q、6v对细胞的侵袭率;数据表示为均值±SEM(n=3,***P<0.001)。
图3为:为6q和6v诱导HN6细胞凋亡,阻滞细胞周期在G2/M期。A为细胞用化合物处理24h;选择Annexin-FITC和PI对凋亡细胞进行染色,具有代表性的流式细胞仪点图显示;B为各组凋亡细胞百分比;数据表示为平均值±SEM(n=3,*P<0.05,**P<0.01,***P<0.001);C为用化合物处理细胞24h,选择PI染液染色DNA,用流式细胞仪检测DNA含量;HN6细胞具有代表性的DNA分布直方图;D为细胞周期分布,数据表示为平均值±SEM(n=3)。
图4为:6q和6v对异种移植瘤模型有较好的治疗作用。A-D为小鼠肿瘤体积和重量;移植瘤小鼠被给予帕苯达唑80mg/kg、6q、6v或溶剂,每日1次灌胃。测量肿瘤体积,记录体重;结果用标准差表示(n=5);B为实体瘤被手术切除;E-G为裸鼠血清中CYFRA21-1、SCCAg、TPS水平;空白组为正常无瘤裸鼠,对照组为未处理异种移植裸鼠;结果用标准差表示(n=5);*P<0.05,**P<0.01。
图5为:化合物对HFF-1和昆明小鼠的毒性;A-C用一系列浓度的帕苯达唑、6q和6v处理HFF-1细胞72h的细胞存活率;CCK8法检测细胞活力;数据以平均SEM表示,n=3。D和E帕苯达唑、6q、6v的生存曲线;M表示男性,F表示女性;实验的设计与实施按照GB 15193.3-2014中Korbor法和限度法试验的规定进行。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本申请使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
下面结合具体的实施例对本发明作进一步的解释和说明。
实施例1:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-乙基脲(6a)
试剂及条件:(Ⅰ)4-正丁基苯胺,乙酸酐,浓硫酸,50℃,1h;硝酸,0℃,4min;(Ⅱ)KOH,65℃,1h;(Ⅲ)H2,Pd/C,室温,24h;(Ⅳ)异氰酸乙酯,N,N-二异丙基乙胺,室温,48h;(Ⅴ)CH3COOH:H2O=3:10,100℃,3h。
步骤1:N-(4-丁基-2-硝基苯基)乙酰胺(2)的合成
将醋酸和少量浓硫酸加到250mL瓶中,冷却至0℃,逐滴加化合物1。50℃搅拌1h后,将混合物冷却至室温,再加入乙酸酐。之后,再次混合物冷却0℃,慢慢添加浓硝酸,在0℃搅拌40min。将反应液加入到大量的冰水中,将产生的黄色沉淀过滤出来,干燥得到粗产品。粗产物经硅胶层析进一步纯化,得到黄色固体产物。1H NMR(400MHz,DMSO)δ10.17(s,1H),7.74(d,J=1.1Hz,1H),7.51(d,2H),2.67–2.60(m,2H),2.05(s,3H),1.60–1.52(m,2H),1.35–1.25(m,2H),0.90(t,J=7.3Hz,3H).ESI-MS:m/z[M+H]+calcd for C12H17N2O3 +237.12,found 237.49.Mp 72.8–73.6℃.
步骤2:4-丁基-2-硝基苯胺(3)的合成
在250mL的瓶子中,将化合物2溶于甲醇;加入40%KOH溶液(7.4mL,50.78mmol),在65℃下搅拌1h。反应溶液用无水Na2SO4干燥后过滤,减压除去溶剂。经硅胶层析纯化得到产物为黄色油状物。1H NMR(400MHz,DMSO)δ7.74(s,1H),7.30(s,2H),7.27(d,1H),6.95(d,J=8.6Hz,1H),2.47(d,J=7.7Hz,2H),1.54–1.44(m,2H),1.34–1.23(m,2H),0.89(t,J=7.3Hz,3H).ESI-MS:m/z[M-H]-calcd for C10H13N2O2 -193.10,found 193.23.
步骤3:4-丁基苯-1,2-二胺(4)的合成
在250mL三颈烧瓶中,加入化合物3,Pd/C和甲醇。将整个***密封,用氮气换空气,再用氢气置换氮气。将反应溶液在室温下搅拌24h,Pd/C过滤,浓缩得到油状产物。1H NMR(400MHz,DMSO)δ6.39(d,J=7.7Hz,1H),6.33(d,J=1.8Hz,1H),6.19(dd,J=7.7,1.8Hz,1H),4.26(s,4H),2.32(t,J=7.6Hz,2H),1.48–1.40(m,2H),1.31–1.22(m,2H),0.87(t,J=7.3Hz,3H).ESI-MS:m/z[M+H]+calcd for C10H17N2 +165.14,found 165.14.
步骤4:1,3-双(乙基氨基羰基)-2-甲基-2-硫代异脲(5a)的合成
在2-甲基-2-硫脲硫酸酯、DIPEA和乙腈的混合物中,滴加异氰酸乙酯。室温下搅拌48h,然后减压去除溶剂。加入乙酸乙酯和水,充分搅拌后分液,用饱和氯化钠溶液洗涤有机相2次,然后用无水Na2SO4干燥,产物经硅胶层析纯化后得到白色油状物5a。1H NMR(400MHz,CDCl3)δ12.59(s,1H),5.40(d,J=76.0Hz,2H),3.27(dq,J=13.8,7.0Hz,4H),2.31(s,3H),1.16(dt,J=15.4,7.6Hz,6H).ESI-MS:m/z[M+H]+calcd for C8H17N8O2S+233.11,found232.79.
步骤5:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-乙基脲(6a)的合成
将化合物4、5与溶剂(CH3COOH:H2O=3:10)在100℃下搅拌3h,将反应溶液冷却至室温,用饱和NaHCO3水溶液调节PH至碱性。然后加入二氯甲烷,充分搅拌后分液,有机相用无水Na2SO4干燥后过滤,浓缩后,经硅胶层析纯化得到产物白色固体产物。白色固体(48mg,37.9%yield).1HNMR(400MHz,DMSO)δ11.31(s,1H),9.80(s,1H),7.37(s,1H),7.23(d,J=8.0Hz,1H),7.16(s,1H),6.85(d,J=8.0Hz,1H),3.21(dt,J=14.0,7.1Hz,2H),2.59(dd,J=16.8,9.1Hz,2H),1.56(dt,J=15.1,7.5Hz,2H),1.30(dq,J=14.6,7.3Hz,2H),1.11(t,J=7.2Hz,3H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ154.69,148.73,135.05,121.50,35.57,34.53,34.39,22.20,15.77,14.30.ESI-HRMS:m/z[M+H]+calcd forC14H21N4O+261.1715,found 267.1712.HPLC purity 99.1%,tR=10.558min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 253.6–254.9℃.
按照与实施例1相同的方法,采用不同的原料,合成以下化合物。
实施例2:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-环己基脲(6b)
白色固体(36mg,37.6%yield).1H NMR(400MHz,DMSO)δ11.31(s,1H),9.66(s,1H),7.50(s,1H),7.23(d,J=8.0Hz,1H),7.16(s,1H),6.85(d,J=8.0Hz,1H),3.63–3.54(m,1H),2.60(t,J=7.6Hz,2H),1.89–1.81(m,2H),1.72–1.64(m,2H),1.60–1.51(m,3H),1.37–1.21(m,7H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ153.88,148.70,135.08,121.52,48.29,35.57,34.38,33.12,25.66,24.64,22.20,14.29.ESI-HRMS:m/z[M+H]+calcd for C18H27N4O+315.2185,found 315.2178.HPLC purity 97.0%,tR=6.551min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 119.9–120.6℃.
实施例3:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-异丙基脲(6c)
白色固体(42mg,41.9%yield).1H NMR(400MHz,DMSO)δ11.32(s,1H),9.55(s,1H),7.30(d,J=32.2Hz,1H),7.22(d,J=7.8Hz,1H),7.15(s,1H),6.84(d,J=7.8Hz,1H),3.90–3.78(m,1H),2.65–2.57(m,2H),1.55(dt,J=15.1,7.5Hz,2H),1.30(dd,J=14.7,7.4Hz,2H),1.15(d,J=6.5Hz,6H),0.89(t,J=7.3Hz,3H).ESI-HRMS:m/z[M+H]+calcd forC15H23N4O+275.1872,found 275.1871.HPLC purity 95.8%,tR=9.114min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 144.5–147.4℃.
实施例4:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-丙基脲(6d)
白色固体(39mg,46.7%yield).1H NMR(400MHz,DMSO)δ11.27(s,1H),9.78(s,1H),7.48(s,1H),7.23(d,J=8.0Hz,1H),7.15(s,1H),6.84(d,J=8.0Hz,1H),3.15(dd,J=12.8,6.5Hz,2H),2.60(t,J=7.5Hz,2H),1.56(dd,J=14.8,7.4Hz,2H),1.48(dd,J=14.3,7.2Hz,2H),1.30(dd,J=14.7,7.4Hz,2H),1.18(t,J=11.8Hz,2H),0.90(dt,J=12.0,6.1Hz,6H).ESI-HRMS:m/z[M+H]+calcd for C15H23N4O+275.1872,found 275.1871.HPLCpurity 99.6%,tR=9.261min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 171.8–172.9℃.
实施例·:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-己基脲(6e)
白色固体(30mg,31.1%yield).1H NMR(400MHz,DMSO)δ11.26(s,1H),9.71(s,1H),7.43(s,1H),7.23(d,J=8.0Hz,1H),7.15(s,1H),6.84(d,J=8.1Hz,1H),3.18(dd,J=12.8,6.6Hz,2H),2.60(t,J=7.6Hz,2H),1.61–1.52(m,2H),1.51–1.44(m,2H),1.35–1.26(m,8H),0.88(q,J=7.1Hz,6H).13C NMR(101MHz,DMSO)δ154.74,148.73,135.07,121.51,39.61,35.57,34.38,31.41,29.97,26.48,22.53,22.19,14.36,14.29.ESI-HRMS:m/z[M+H]+calcd for C18H29N4O+317.2341,found 317.2336.HPLC purity 96.7%,tR=7.152min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 202.5–203.9℃.
实施例6:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-苯乙基脲(6f)
白色固体(52mg,37.0%yield).1H NMR(400MHz,DMSO)δ11.30(s,1H),9.73(s,1H),7.47(s,1H),7.33–7.26(m,4H),7.24–7.20(m,2H),7.14(s,1H),6.84(d,J=8.1Hz,1H),3.43(q,J=13.2,6.8Hz,2H),2.80(t,J=14.0,6.9Hz,2H),2.60(t,J=7.6Hz,2H),1.60–1.50(m,2H),1.37–1.25(m,2H),0.90(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ139.81,139.51,135.10,129.20,129.12,128.86,126.69,126.64,121.52,41.27,36.13,35.56,34.37,22.19,14.30.ESI-HRMS:m/z[M+H]+calcd for C20H25N4O+337.2028,found337.2023.HPLC purity 98.9%,tR=11.688min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 180.2–181.7℃.
实施例7:1-(叔丁基)-3-(6-丁基-1H-苯并[d]咪唑-2-基)脲(6g)
无色透明油状物(62mg,35.3%yield).1H NMR(400MHz,DMSO)δ11.37(s,1H),9.46(s,1H),7.23(d,J=8.0Hz,2H),7.15(s,1H),6.84(d,J=8.0Hz,1H),2.60(t,J=7.6Hz,2H),1.59–1.51(m,2H),1.35(s,9H),1.28(d,J=4.6Hz,2H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ153.63,148.59,134.95,121.40,50.27,35.60,34.43,29.29,22.22,14.30.ESI-HRMS:m/z[M+H]+calcd for C16H25N4O+289.2089,found 289.2022.HPLC purity98.2%,tR=11.820min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 84.4–86.9℃.
实施例8:1-(6-丁基-1H-苯并[d]咪唑-2-基)-3-环戊基脲(6h)
白色固体(104mg,55.0%yield).1H NMR(400MHz,DMSO)δ11.36(s,1H),9.60(s,1H),7.46(s,1H),7.23(d,J=8.0Hz,1H),7.15(s,1H),6.85(d,J=8.0Hz,1H),4.07–3.98(m,1H),2.61(t,J=7.0Hz,2H),1.92–1.85(m,2H),1.70–1.65(m,2H),1.60–1.53(m,4H),1.43(dd,J=12.2,6.2Hz,2H),1.31(dd,J=14.8,7.4Hz,2H),0.90(t,J=7.3Hz,3H).13CNMR(101MHz,DMSO)δ154.26,148.68,135.09,121.52,51.52,35.57,34.39,33.23,23.65,23.60,22.21,14.30.ESI-HRMS:m/z[M+H]+calcd for C17H25N4O+301.2028,found301.2022.HPLC purity 95.6%,tR=18.058min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 180.5–182.9℃.
实施例9:1-烯丙基-3-(6-丁基-1H-苯并[d]咪唑-2-基)脲(6i)
白色固体(50mg,60.2%yield).1H NMR(400MHz,DMSO)δ11.32(s,1H),9.84(s,1H),7.61(s,1H),7.23(d,J=8.0Hz,1H),7.16(s,1H),6.85(d,J=8.0Hz,1H),5.97–5.85(m,1H),5.20(d,J=17.2Hz,1H),5.11(d,J=10.3Hz,1H),3.91–3.81(m,2H),2.63–2.58(m,2H),1.63–1.51(m,2H),1.37–1.25(m,2H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ154.71,148.66,136.19,135.42,135.15,121.59,115.45,41.97,35.56,34.38,22.20,14.30.ESI-HRMS:m/z[M+H]+calcd for C15H21N4O+273.1715,found 273.1710.HPLC purity99.7%,tR=15.687min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 223.4–225.6℃.
实施例10:2-(3-(6-丁基-1H-苯并[d]咪唑-2-基)脲基)甲基丙烯酸乙酯(6j)
白色固体(135mg,64.4%yield).1H NMR(400MHz,DMSO)δ11.18(s,1H),10.18(dd,J=221.4,53.7Hz,1H),7.77(s,1H),7.23(d,J=8.0Hz,1H),7.15(s,1H),6.86(d,J=8.1Hz,1H),6.12(s,1H),5.71(s,1H),4.20(t,J=5.3Hz,2H),3.51(q,J=5.4Hz,2H),2.61(t,J=7.6Hz,2H),1.90(s,3H),1.61–1.51(m,2H),1.35–1.25(m,2H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ166.94,154.95,148.64,136.29,135.18,126.43,121.63,64.07,38.68,35.55,34.36,22.18,18.46,14.29.ESI-HRMS:m/z[M+H]+calcd forC18H25N4O3 +345.1927,found 345.1919.HPLC purity 99.6%,tR=8.850min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 221.4–223.9℃.
实施例11:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸异丁酯(6k)
白色固体(51mg,51.8%yield).1H NMR(400MHz,DMSO)δ11.57(s,2H),7.28(d,J=7.7Hz,1H),7.20(s,1H),6.89(d,J=8.0Hz,1H),3.92(t,J=8.3Hz,2H),2.61(t,J=7.5Hz,2H),2.01–1.90(m,1H),1.59–1.51(m,2H),1.35–1.26(m,2H),0.93(d,J=6.7Hz,6H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ135.61,121.97,71.33,35.58,34.39,28.04,22.22,19.35,14.29.ESI-HRMS:m/z[M+H]+calcd for C16H24N3O2 +290.1869,found190.1863.HPLC purity 97.2%,tR=16.446min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 135.9–137.4℃.
实施例12:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸苄酯(6l)
白色固体(50mg,50.8%).1H NMR(400MHz,DMSO)δ11.77(s,2H),7.49–7.34(m,5H),7.26(d,J=8.1Hz,1H),7.19(s,1H),6.89(d,J=8.0Hz,1H),5.24(s,2H),2.60(t,J=7.5Hz,2H),1.59–1.50(m,2H),1.29(dt,J=14.5,7.3Hz,2H),0.89(t,J=7.3Hz,3H).13CNMR(101MHz,DMSO)δ155.89,148.61,137.00,135.78,128.93,128.47,122.14,113.46,113.04,66.87,35.54,34.35,22.20,14.29.ESI-HRMS:m/z[M+H]+calcd for C19H21N3O2 +324.1712,found 324.1704.HPLC purity 99.8%,tR=15.432min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 199.2–201.7℃.
实施例13:2-氯乙基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯(6m)
白色固体(41mg,44.6%yield).1H NMR(400MHz,DMSO)δ11.88(s,1H),7.35(d,J=8.1Hz,1H),7.27(s,1H),6.93(d,J=8.1Hz,1H),4.57(t,J=8.0Hz,2H),4.20(t,J=8.0Hz,2H),2.63(t,J=7.6Hz,2H),1.62–1.53(m,2H),1.37–1.26(m,2H),0.90(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ155.05,145.48,135.79,122.28,117.20,111.37,63.97,44.38,35.59,34.35,22.20,14.29.ESI-HRMS:m/z[M+H]+calcd for C14H19ClN3O2 +296.1166,found315.2178.HPLC purity 99.8%,tR=8.219min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 199.2–201.7℃.Mp 182.8–184.7℃.
实施例14:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸丙酯(6n)
白色固体(38mg,45.8%yield).1H NMR(400MHz,DMSO)δ11.48(s,2H),7.26(s,1H),7.18(s,1H),6.90(s,1H),4.11(s,2H),2.61(s,2H),1.61(d,J=39.7Hz,4H),1.31(s,2H),0.91(d,J=6.5Hz,6H).13C NMR(101MHz,DMSO)δ121.96,66.91,35.57,34.38,22.31,22.21,14.30,10.70.ESI-HRMS:m/z[M+H]+calcd for C15H22N3O2+276.1712,found276.1705.HPLC purity 99.3%,tR=9.068min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 204.5–206.8℃.
实施例15:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸己酯(6o)
白色固体(56mg,60.0%yield).1H NMR(400MHz,DMSO)δ11.50(s,2H),7.26(d,J=8.1Hz,1H),7.18(s,1H),6.89(d,J=8.1Hz,1H),4.14(t,J=6.6Hz,2H),2.65–2.58(m,2H),1.65(dd,J=13.7,6.9Hz,2H),1.55(dd,J=15.1,7.7Hz,2H),1.38–1.26(m,8H),0.93–0.86(m,6H).13C NMR(101MHz,DMSO)δ135.49,121.90,65.39,35.57,34.38,31.37,28.90,25.44,22.49,22.21,14.35,14.30.ESI-HRMS:m/z[M+H]+calcd for C18H28N3O2 +318.2182,found318.2173.HPLC purity 99.4%,tR=12.993min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 201.2–203.5℃.
实施例16:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸丁酯(6p)
白色固体(53mg,50.1%yield).1H NMR(400MHz,DMSO)δ11.58(s,2H),7.28(d,J=7.8Hz,1H),7.20(s,1H),6.89(d,J=7.7Hz,1H),4.15(t,J=6.1Hz,2H),2.61(t,J=6.8Hz,2H),1.67–1.51(m,4H),1.43–1.27(m,4H),0.96–0.85(m,6H).13C NMR(101MHz,DMSO)δ148.23,135.55,121.94,113.61,65.15,35.58,34.39,31.00,22.22,19.03,14.30,14.08.ESI-HRMS:m/z[M+H]+calcd for C16H24N3O2 +290.1869,found 290.1860.HPLC purity99.3%,tR=10.557min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 211.7–213.2℃
实施例17:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6q)
白色固体(49mg,58.5%yield).1H NMR(400MHz,DMSO)δ11.65(s,2H),7.30(d,1H),7.23(s,1H),6.91(d,1H),5.09–4.90(m,1H),2.62(t,2H),1.67–1.49(m,2H),1.43–1.20(m,8H),0.90(t,3H).13C NMR(101MHz,DMSO)δ154.72,147.98,135.56,121.94,113.54,69.21,35.60,34.44,22.32,22.24,14.29.ESI-HRMS:m/z[M+H]+calcd forC15H22N3O2+276.1712,found 276.1706.HPLC purity 99.6%,tR=8.792min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 122.4–125.1℃.
实施例18:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸庚酯(6r)
白色固体(91mg,75.2%yield).1H NMR(400MHz,DMSO)δ11.45(s,2H),7.26(d,J=7.5Hz,1H),7.18(s,1H),6.89(d,J=8.3Hz,1H),4.14(s,2H),2.62(s,2H),1.60(d,J=32.7Hz,4H),1.28(s,10H),0.88(s,6H).13C NMR(101MHz,DMSO)δ135.56,121.97,65.43,35.56,34.36,31.66,28.92,28.80,25.73,22.48,22.20,14.40,14.29.ESI-HRMS:m/z[M+H]+calcd for C19H30N3O2 +332.2338,found 332.2328.HPLC purity 97.8%,tR=12.155min,250mm×4.6mm,CH3OH:H2O=85:15,0.8mL/min.Mp 194.1–195.2℃.
实施例19:环戊基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯(6s)
白色固体(45mg,49.0%yield).1H NMR(400MHz,DMSO)δ11.56(s,2H),7.30–7.26(m,1H),7.20(s,1H),6.89(d,J=8.1Hz,1H),5.16(t,J=5.7Hz,1H),2.62(t,J=7.6Hz,2H),1.92(dd,J=12.5,6.5Hz,2H),1.76–1.66(m,4H),1.63–1.52(m,4H),1.36–1.27(m,2H).13C NMR(101MHz,DMSO)δ155.12,148.05,135.54,121.92,78.24,35.59,34.42,32.74,23.79,22.24,14.30.ESI-HRMS:m/z[M+H]+calcd for C17H24N3O2 +302.1869,found302.1860.HPLC purity 99.6%,tR=11.135min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 171.8–174.8℃.
实施例20:3-氯丙基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯(6t)
白色固体(40mg,42.4%yield).1H NMR(400MHz,DMSO)δ11.58(s,2H),7.27(d,J=8.1Hz,1H),7.19(s,1H),6.91(d,J=8.1Hz,1H),4.26(t,J=6.2Hz,2H),3.75(t,J=6.4Hz,2H),2.62(t,J=7.6Hz,2H),2.16–2.05(m,2H),1.61–1.51(m,2H),1.35–1.25(m,2H),0.90(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ155.01,151.30,148.54,147.31,136.80,123.02,113.40,112.97,62.82,42.23,35.47,34.24,31.84,22.17,14.27.ESI-HRMS:m/z[M+H]+calcd for C16H21ClN3O2 +310.1322,found 310.1314.HPLC purity 99.6%,tR=8.599min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 184.2–185.9℃.
实施例21:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸仲丁酯(6u)
白色固体(46mg,52.2%yield).1H NMR(400MHz,CDCl3)δ10.68(s,1H),7.56(s,1H),7.25(s,1H),7.02(d,J=7.9Hz,1H),5.08–4.98(m,1H),2.71(t,J=7.7Hz,2H),1.95–1.83(m,1H),1.80–1.69(m,1H),1.69–1.60(m,2H),1.44(d,J=6.3Hz,3H),1.38(dt,J=12.9,6.5Hz,2H),1.02(t,J=7.4Hz,3H),0.94(t,J=7.3Hz,3H).ESI-HRMS:m/z[M+H]+calcd for C16H24N3O2 +290.1869,found 290.1862.HPLC purity 99.4%,tR=10.562min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.
实施例22:(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸烯丙酯(6v)
白色固体(36mg,43.3%yield).1H NMR(400MHz,DMSO)δ11.58(s,2H),7.26(d,J=7.9Hz,1H),7.18(s,1H),6.90(d,J=7.8Hz,1H),6.05–5.94(m,1H),5.38(d,J=17.2Hz,1H),5.25(d,J=10.2Hz,1H),4.67(d,J=4.2Hz,2H),2.62(t,J=7.2Hz,2H),1.60–1.50(m,2H),1.31(q,J=14.2,7.0Hz,2H),0.90(t,J=7.1Hz,3H).13C NMR(101MHz,DMSO)δ135.69,133.61,122.05,118.14,65.79,35.54,34.34,22.20,14.29.ESI-HRMS:m/z[M+H]+calcdfor C15H20N3O2 +274.1556,found 274.1550.HPLC purity 98.8%,tR=8.042min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 211.2–212.9℃.
实施例23:2-乙基己基(6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸酯(6w)
无色油状物(39mg,38.9%yield).1H NMR(400MHz,DMSO)δ11.58(d,J=32.0Hz,2H),7.28(dd,J=8.0,3.1Hz,1H),7.20(d,J=2.6Hz,1H),6.90(d,J=8.1Hz,1H),4.07(dd,J=11.0,7.8Hz,2H),2.62(t,J=7.6Hz,2H),1.64–1.52(m,3H),1.38–1.26(m,10H),0.91–0.86(m,9H).13C NMR(101MHz,DMSO)δ155.72,148.22,135.58,121.97,113.54,67.59,38.97,35.58,34.39,30.20,28.85,23.61,22.92,22.22,14.38,14.29,11.29.ESI-HRMS:m/z[M+H]+calcd for C20H32N3O2 +346.2495,found 346.2488.HPLC purity 97.1%,tR=8.608min,250mm×4.6mm,CH3OH:H2O=90:10,0.8mL/min.Mp 61.3–63.4℃.
实施例24:N-(6-丁基-1H-苯并[d]咪唑-2-基)呋喃-2-甲酰胺(6x)
白色固体(39mg,45.2%yield).1H NMR(400MHz,DMSO)δ12.10(s,2H),7.89(s,1H),7.37(s,1H),7.32(d,J=7.6Hz,1H),7.23(s,1H),6.97(d,J=7.6Hz,1H),6.66(s,1H),2.67–2.59(m,2H),1.61–1.51(m,2H),1.36–1.27(m,2H),0.90(t,J=7.3Hz,3H).ESI-HRMS:m/z[M+H]+calcd for C16H18N3O2 +284.1399,found 284.1393.HPLC purity 98.2%,tR=7.137min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 149.2–152.3℃.
实施例25:N-(6-丁基-1H-苯并[d]咪唑-2-基)-2-苯基乙酰胺(6y)
白色固体(45mg,48.1%yield).1H NMR(400MHz,DMSO)δ11.99(s,2H),7.38–7.25(m,7H),6.92(d,J=11.5Hz,1H),3.72(s,2H),2.63(t,J=15.1,7.6Hz,2H),1.61–1.51(m,2H),1.34–1.27(m,2H),0.89(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ170.76,146.84,135.73,129.67,128.84,127.23,42.78,35.60,34.38,22.22,14.30.ESI-HRMS:m/z[M+H]+calcd for C19H22N3O+308.1763,found 315.2178.HPLC purity 98.3%,tR=9.064min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 171.0–173.0℃.
实施例26:N-(6-丁基-1H-苯并[d]咪唑-2-基)四氢-2H-吡喃-4-甲酰胺(6z)
白色固体(48mg,52.3%yield).1H NMR(400MHz,DMSO)δ11.92(s,1H),11.41(s,1H),7.31(d,J=8.1Hz,1H),7.23(s,1H),6.91(d,J=8.1Hz,1H),3.91(d,J=11.3Hz,2H),3.35(dd,J=8.5,6.1Hz,2H),2.79–2.72(m,1H),2.62(t,J=7.6Hz,2H),1.78–1.66(m,4H),1.59–1.53(m,2H),1.35–1.28(m,2H),0.90(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO)δ174.60,147.01,135.57,122.10,66.71,41.19,35.61,34.39,29.02,22.23,14.30.ESI-HRMS:m/z[M+H]+calcd for C17H24N3O2 +302.1869,found 302.1861.HPLC purity 99.0%,tR=7.284min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp 158.0–159.2℃.
实施例27:N-(6-丁基-1H-苯并[d]咪唑-2-基)-2-(噻吩-2-基)乙酰胺(6aa)
棕色油状物(23mg,22.9%yield).1H NMR(400MHz,CDCl3)δ7.44(d,J=8.2Hz,1H),7.31(s,1H),7.25–7.21(m,1H),7.06(d,J=8.2Hz,1H),6.98(d,J=3.3Hz,2H),4.48(s,2H),2.70(t,J=7.7Hz,2H),1.65–1.58(m,2H),1.39–1.32(m,2H),0.92(t,J=7.3Hz,3H).13C NMR(101MHz,CDCl3)δ151.88,137.82,137.75,127.40,126.94,125.32,123.55,35.81,34.24,30.04,22.31,13.97.ESI-HRMS:m/z[M+H]+calcd for C17H20N3OS+314.1327,found315.2178.HPLC purity 98.7%,tR=8.935min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.
实施例28:(6-氰基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6ab)
灰色固体(60mg,65.4%yield).1H NMR(400MHz,DMSO)δ12.26(s,1H),11.59(s,1H),7.84(s,1H),7.56(d,J=8.2Hz,1H),7.48(d,J=8.2Hz,1H),5.08–4.96(m,1H),1.32(d,J=6.3Hz,6H).13C NMR(101MHz,DMSO)δ153.81,150.04,125.29,120.73,69.98,22.20.ESI-HRMS:m/z[M+H]+calcd for C12H13N4O2 +245.1039,found 245.1031.HPLC purity99.7%,tR=4.666min,250mm×4.6mm,CH3OH:H2O=80:20,0.8mL/min.Mp>300℃.
实施例29:(6-(三氟甲基)-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6ac)
灰色固体(56mg,68.7%yield).1H NMR(400MHz,DMSO)δ11.96(s,2H),7.76(s,1H),7.60(d,J=8.3Hz,1H),7.41(d,J=8.3Hz,1H),5.09–4.97(m,1H),1.34(s,3H),1.32(s,3H).13C NMR(101MHz,DMSO)δ153.91,149.68,127.05,124.36,118.13,69.87,22.19.ESI-HRMS:m/z[M+H]+calcd for C12H13F3N3O2 +288.0960,found 288.0952.HPLCpurity 99.8%,tR=7.905min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 202.2–204.9℃.
实施例30:(6-甲基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6ad)
白色固体(33mg,34.6%yield).1H NMR(400MHz,DMSO)δ11.67(s,2H),7.29(d,J=8.0Hz,1H),7.23(s,1H),6.89(d,J=8.1Hz,1H),5.06–4.92(m,1H),2.36(s,3H),1.31(d,J=6.2Hz,6H).13C NMR(101MHz,DMSO)δ154.69,147.92,130.28,122.54,69.23,22.32,21.72.ESI-HRMS:m/z[M+H]+calcd for C12H16N3O2 +234.1243,found 234.1236.HPLC purity99.9%,tR=6.709min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 195.0–197.0℃.
实施例31:(6-氟-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6ae)
白色固体(39mg,41.5%yield).1H NMR(400MHz,DMSO)δ11.70(s,2H),7.39(dd,J=8.6,5.0Hz,1H),7.20(dd,J=9.6,2.2Hz,1H),6.94–6.87(m,1H),5.06–4.94(m,1H),1.31(d,J=6.3Hz,6H).ESI-HRMS:m/z[M+H]+calcd for C11H13FN3O2 +238.0992,found238.0985.HPLC purity 99.9%,tR=6.020min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp>300℃.
实施例32:(6-戊基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6af)
白色固体(61mg,755.2%yield).1H NMR(400MHz,DMSO)δ7.27(dd,J=8.0,3.7Hz,1H),7.19(s,1H),6.89(d,J=8.1Hz,1H),5.03–4.91(m,1H),2.61(t,J=7.6Hz,2H),1.63–1.51(m,2H),1.37–1.22(m,10H),0.86(t,J=6.7Hz,3H).13C NMR(101MHz,DMSO)δ154.79,147.96,135.56,121.92,69.16,35.87,31.87,31.38,22.46,22.32,14.40.ESI-HRMS:m/z[M+H]+calcd for C16H24N3O2 +290.1869,found 290.1864.HPLC purity 99.8%,tR=17.844min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp 128.0–129.0℃.
实施例33:(5,6-二氟-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6ag)
淡黄色固体(51mg,51.6%yield).1H NMR(400MHz,DMSO)δ11.98(s,1H),11.37(s,1H),7.39(t,J=9.2Hz,2H),5.06–4.93(m,1H),1.31(d,J=6.2Hz,6H).13C NMR(101MHz,DMSO)δ153.83,148.83,69.72,22.21.ESI-HRMS:m/z[M+H]+calcd for C11H12F2N3O2 +256.0898,found 256.0893.HPLC purity 99.7%,tR=6.511min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp>300℃.
实施例34:(5,6-二甲基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯(6ah)
白色固体(42mg,47.4%yield).1H NMR(400MHz,DMSO)δ11.52(s,2H),7.18(s,2H),5.04–4.90(m,1H),2.25(s,6H),1.30(d,J=6.1Hz,6H).13C NMR(101MHz,DMSO)δ155.02,147.56,129.30,114.38,69.04,22.35,20.33.ESI-HRMS:m/z[M+H]+calcd forC13H18N3O2 +248.1399,found 248.1393.HPLC purity 99.6%,tR=7.806min,250mm×4.6mm,CH3OH:H2O=75:25,0.8mL/min.Mp>300℃.
实施例35:化合物抑制CAL-27,HN6和Fadu细胞增殖活性测定
本实施例用于测定本发明帕苯达唑衍生物对HNSCC细胞系CAL-27,HN6和Fadu细胞增殖的抑制活性。试验方案如下:将细胞以5000个/孔的浓度接种于96孔透明板中,每孔100μL。在5%CO2,37℃条件下培养12h。然后,将无血清培养基中制备的一系列化合物溶液(100μL)加入孔中,继续培养72h,采用CCK8法评价细胞存活率,用BMG公司的CLARIOstar酶标仪记录450nm处的吸光度值,最后用GraphPad prism 7.00计算IC50值。本发明受试化合物抑制肿瘤细胞增殖的IC50值见下表。
表1帕苯达唑衍生物抑制HN6细胞增殖活性和抑制微管蛋白聚合活性
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表2帕苯达唑衍生物抑制HN6,CAL-27,Fadu细胞增殖活性
结论:本发明中的化合物体外细胞增殖表明,化合物6q和6v在活性上优于帕苯达唑,为设计新型的药物提供了思路。
实施例36:体外微管蛋白聚合实验
本实施例用于测定本发明帕苯达唑衍生物对体外微管蛋白聚合的抑制活性。试验方案如下:根据制造商的方案,使用微管蛋白聚合试剂盒进行测试。微管蛋白、化合物(20μM)和GTP在37℃下混合入孔中。随着微管的聚合,报告基团被加入到微管中,荧光逐渐增强。1h内每分钟用BMG CLARIOstar酶标仪记录荧光信号,以微管蛋白聚合速率计算化合物抑制微管聚合的IC50。试验结果见表1。
结论:6n、6q、6u、6v、6z、11e、11g等许多化合物单独抑制微管蛋白聚合的活性都优于帕苯达唑,为设计新型的化合物提供了思路。
实施例37:衍生物6q,6v对细胞微管蛋白结构的影响测定
本实施例用于测定衍生物6q,6v对细胞微管蛋白结构的影响。试验方案如下:将HN6细胞接种于激光共聚焦培养皿(20000个/培养皿)中,12h后加入100nmol/L化合物溶液。培养24h后,丢弃培养基,加入4%多聚甲醛,室温固定15min。PBS洗3次,细胞在封闭液(5%正常山羊血清和0.3%Triton X-100PBS)中封闭1h。弃去封闭液后,加入α-微管蛋白抗体,在4℃下孵育细胞过夜。PBS洗碗3次,加入Anti-Rabbit IgG(H+L),F(ab’)2Fragment(Alexa555Conjugate),室温避光孵育1h。用DAPI(10μg/mL)对细胞核进行染色。最后,利用高速共聚焦平台采集图像。
结论:从图1可以看到DMSO处理后的细胞(图1A)形态良好,微管蛋白形成了规则的网络结构;而100nmol/L的帕苯达唑(图1C)、6q(图1B)或6v(图1D)处理后,细胞失去了规则的微管蛋白网络,大部分微管蛋白聚集在细胞核周围。
实施例38:衍生物6q,6v对细胞侵袭和细胞迁移的影响测定
本实施例用于测定衍生物6q,6v对细胞侵袭和细胞迁移的影响。试验方案如下:细胞迁移测定:HN6细胞接种在6孔板中,每孔1×106个细胞。培养12h后,200μL吸头创造划痕。用无血清培养基洗孔3次,用倒置荧光显微镜(IX73+DP80,物镜:10×)对划痕进行拍照。然后加入0.1μmol/L无血清培养基制备的化合物溶液,培养24h后再次拍摄划痕愈合情况。计算划痕愈合百分比。细胞侵袭测定:基底膜基质(Corning,cat#356234)用无血清培养基稀释20倍后,加入100μL于上室细胞培养***物(24孔板,孔径8.0μm,Corning,cat#353097)。然后,将小室置于37℃下放置5h,然后将液体吸出。小室用无血清培养基洗涤1次,加入100μL不含血清的培养基制备的化合物溶液。将HN6细胞(100μL,105个/孔)加入上室细胞培养皿中,下室加入600μL含血清培养基。培养24h后,将小室从24孔板上取下,轻轻擦拭上层细胞。用PBS洗涤一次,在室温下用4%多聚甲醛溶液固定10min。PBS洗涤2次后,用0.1%结晶紫溶液染色10min后洗涤、倒置、风干并拍照。最后用33%的乙酸溶解结晶紫,在570nm波长下测定吸光度。
结论:用帕苯达唑、6q或6v处理24h后,测试了划痕愈合率。从图2可以看出,与对照组(44.0%)相比,帕苯达唑组(12.8%)、6q组(6.27%)和6v组(8.1%)划痕愈合率显著降低。此外,与帕苯达唑相比,6q对细胞迁移的抑制更显著。在细胞侵袭实验中,化合物也显著抑制了细胞跨越基质的侵袭过程,浓度为200nmol/L。当浓度增加到500nmol/L时,在帕苯达唑组中只有少数细胞能穿过基质,而在6q和6q组中几乎没有细胞能成功侵入下层。总之,6q和6v可以有效抑制肿瘤细胞的迁移和侵袭。
实施例39:衍生物6q,6v对细胞凋亡和细胞周期的影响测定
本实施例用于测定衍生物6q,6v对细胞凋亡和细胞周期的影响。试验方案如下:细胞凋亡测定:将HN6细胞接种于6孔板,每孔30万个细胞。12h后,用0.1μmol/L或1μmol/L化合物溶液处理细胞24h后收集,PBS洗1次,结合缓冲液洗1次。然后加入100μL结合缓冲液悬浮细胞,加入5μL Annexin FITC和5μL PI染色液,室温避光孵育15min。流式细胞术(CytoFLEXS)分析染色结果。细胞周期测定:将HN6细胞接种于6孔板,每孔30万个细胞。12h后,用0.1μmol/L无血清培养基配制的化合物溶液处理细胞24h,收集细胞,PBS冲洗,70%乙醇固定过夜。PBS洗1次,100μL RNase A溶液重悬,37℃水浴30min后染色。加入400μL PI染色液,4℃暗孵育30min,流式细胞术(CytoFLEX S)分析染色结果。
结论:从图3可以看出,与对照组相比,0.1μmol/L和1μmol/L的化合物处理细胞24h后均能有效诱导细胞凋亡。随着浓度的增加,凋亡细胞的比例也增加。考虑到微管蛋白在细胞有丝***中的重要作用,还评价了化合物对细胞周期的影响。显然,0.1μmol/L 6q或6v均能成功将细胞周期阻滞在G2/M期。综上所述,6q和6v可有效诱导HN6细胞凋亡,并将细胞周期阻滞在G2/M期,从而抑制肿瘤的病理过程。
实施例40:衍生物6q,6v对异种移植瘤模型裸鼠的治疗效果测定
本实施例用于测定衍生物6q,6v对异种移植瘤模型裸鼠的治疗效果。试验方案如下:4周龄雄性裸鼠,适应性喂养5天后,皮下注射100万个HN6肿瘤细胞,使其生长至适当大小。小鼠随机分为4组,每组5只。将帕苯达唑、6q、6v粉末与0.5%羧甲基纤维素钠溶液配制成混悬液,每日灌胃1次,连续2周。用电子卡尺测量肿瘤大小,并用ELISA的方法测量血清中SCCAg,TPS和CYFRA21-1的含量。所有程序均按照实验动物研究机构的实验动物护理和使用指南进行。
从图4可以看出6q和6v对异种移植瘤模型有较好的治疗作用。结论:从图4可以看出,化合物在裸鼠体内能有效控制肿瘤体积的增长,且体重没有明显下降,显示出其在治疗剂量下的良好疗效和生物相容性。移植肿瘤的裸鼠血清中三种肿瘤标志物水平均显著升高。治疗两周后,肿瘤生长得到有效控制,三种肿瘤标志物血清水平均明显降低。
实施例41:衍生物6q,6v对SPF昆明鼠急性毒性及对人皮肤成纤维细胞(HFF-1)毒性测定
本实施例用于测定衍生物6q,6v对SPF昆明鼠急性毒性及对人皮肤成纤维细胞(HFF-1)的毒性。适应性喂养后,将小鼠随机分为雄、雌两组,每组10只。化合物用0.5%羧甲基纤维素钠溶液配制成混悬液,每次0.4mL灌胃给药。灌胃前禁食6h,自由饮水;灌胃后禁食2h。对小鼠进行观察,并记录其体重和生存情况。衍生物6q,6v对人皮肤成纤维细胞(HFF-1)的毒性测定操作见实施例35。
结论:从图5可以看出,给予1g/kg帕苯达唑或6v的小鼠均无不良反应,且状态良好,无死亡。给予150mg/kg或250mg/kg 6q的小鼠没有表现出任何不良反应,直到实验结束都保持活跃,没有死亡。给予630mg/kg 6q的小鼠中,一只雌鼠和一只雄鼠存活。给予1g/kg6q的小鼠在3h后出现虚弱、嗜睡和体温过低的症状,给予630mg/kg 6q的小鼠在12h后出现上述情况。经计算,帕苯达唑和6v的LD50值均大于1g/kg,6q的LD50值为341mg/kg。6q的LD50远高于其有效治疗剂量,且在治疗剂量下小鼠未见异常。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种帕苯达唑衍生物在制备治疗头颈部鳞状细胞癌HNSCC的药物中的应用,其特征在于,所述帕苯达唑衍生物选自下列化合物:
化合物6q: (6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸异丙酯;
化合物6v: (6-丁基-1H-苯并[d]咪唑-2-基)氨基甲酸烯丙酯;
所述头颈部鳞状细胞癌选自人舌鳞癌和人咽鳞癌。
2.如权利要求1所述的应用,其特征在于,所述帕苯达唑衍生物的制备方法包括如下步骤:
(1)化合物1的胺基在浓硫酸的催化下被乙酸酐乙酰化,然后乙酰基的邻位被乙酸酐和硝酸硝基化得到化合物2;随后,化合物2在甲醇中被氢氧化钾溶液水解成化合物3,进而硝基在甲醇中被H2/Pd还原得到化合物4;
(2)2-甲基-2-硫代异脲硫酸盐在碱性条件下与氯甲酸酯发生取代反应,得到5a-5aa,所述的氯甲酸酯选自氯甲酸异丙酯或氯甲酸烯丙酯;
(3)化合物4和5a-5aa在酸性条件下发生环化反应生成帕苯达唑衍生物6q或6v;
其中步骤(1)和(2)顺序不分先后;
;其中R2选自正丁基;R3选自氢。
3.如权利要求2所述的应用,其特征在于,所述制备方法如下:
(1)将醋酸和少量浓硫酸冷却至0 oC,加入化合物1;45~55 oC搅拌0.5~1.5 h后,将混合物冷却至室温,再加入乙酸酐;之后,再次混合物冷却0 oC,添加浓硝酸,在0 oC搅拌30~50min;将反应液加入到冰水中,得到化合物2;
将化合物2溶于甲醇,加入35~45% KOH溶液,在60~70 oC下搅拌0.5~1.5 h;反应溶液用干燥后过滤,除去溶剂得到化合物3;
将化合物3,Pd/C和甲醇混合,使用氢气进行还原;将反应溶液在室温下搅拌23~25 h,Pd/C过滤,得到化合物4;
(2)在2-甲基-2-硫代异脲硫酸盐、DIPEA和乙腈的混合物中,滴加氯甲酸酯;室温下搅拌46~50 h,去除溶剂;加入乙酸乙酯和水,充分搅拌后分液,将有机相洗涤、干燥,产物纯化得到化合物5a-5aa;
(3)将化合物4、5a-5aa与溶剂在90~110 oC下搅拌2.5~3.5 h,将反应溶液冷却至室温,调节PH至碱性;然后加入二氯甲烷,充分搅拌后分液,有机相干燥、过滤、浓缩、纯化得到所述帕苯达唑衍生物。
4.如权利要求3所述的应用,其特征在于,步骤(3)中所述溶剂为乙酸水溶液,其中VCH3COOH: VH2O=3:10。
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