Design, synthesis, and X-ray crystal structure of a potent dual inhibitor of thymidylate synthase and dihydrofolate reductase as an antitumor agent
Design, synthesis, and X-ray crystal structure of a potent dual inhibitor of thymidylate synthase and dihydrofolate reductase as an antitumor agent
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DOI：
10.1021/jm000200l
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发表时间：
2000-10-19
影响因子：
7.3
通讯作者：
Queener, SF
中科院分区：
文献类型：
--
作者：
Gangjee, A;Yu, JM;Queener, SF
设计并合成了一种新的N-{2-氨基-4-甲基[（吡咯并[2，3-d]嘧啶-5-基）乙基]苯甲酰基}-L-谷氨酸（3a），它是胸苷酸合成酶（TS）和二氢叶酸还原酶（DHFR）的双重抑制剂，也是一种抗肿瘤药物。化合物3b，3a的N7-苄基化类似物，也被合成为抗肿瘤剂。3a的合成是通过12步序列完成的，该序列涉及从2-乙酰基丁内酯经5步合成2-氨基-4-甲基吡咯并-[2，3-d]嘧啶（10）。保护10的2-氨基，在5-位进行区域选择性碘化，然后进行钯催化偶联，得到中间体14，通过还原和皂化将其转化为3a。类似的合成方法用于3b。3a、DHFR和NADPH的三元复合物的X射线晶体结构显示，吡咯并[2，3-d]嘧啶环以“2，4-二氨基模式”结合，其中吡咯氮模拟2，4-二氨基嘧啶的4-氨基部分。这是经典吡咯并[2，3-d]嘧啶抗叶酸剂显示具有这种与DHFR结合的交替模式的第一个例子。化合物3a和3b比LY 231514对干酪乳杆菌和大肠杆菌TS的抑制作用更强。Anglyca 3a对来自人、弓形虫和卡氏肺孢子虫的DHFR也具有更强的抑制作用。3a对甲氨蝶呤（MTX）耐药细胞系的评价表明，3a的交叉耐药性远低于MTX。代谢物保护研究和叶酰聚-γ-谷氨酸合成酶研究表明，3a对培养物中肿瘤细胞生长的抗肿瘤活性是TS和DHFR双重抑制的结果。化合物3a抑制CCRF-CEM和FaDu细胞在培养物中的生长，ED 50值分别为12.5和7.0 nM，并且比MTX对FaDu细胞更有活性。相比之下，化合物3b对两种细胞系均无活性。化合物3a在国家癌症研究所体外临床前抗肿瘤筛选程序中进行了评估，并针对许多肿瘤细胞系提供了纳摩尔范围内的IG（50）值。
A novel N-{2-amino-4-methyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid (3a) was designed and synthesized as a potent dual inhibitor of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as an antitumor agent. Compound 3b, the N7-benzylated analogue of 3a, was also synthesized as an antitumor agent. The synthesis of 3a was accomplished via a 12-step sequence which involved the synthesis of 2-amino-4-methylpyrrolo-[2,3-d]pyrimidine (10) in 5 steps from 2-acetylbutyrolactone. Protection of the 2-amino group of 10 and regioselective iodination at the 5-position followed by palladium-catalyzed coupling afforded intermediate 14 which was converted to 3a by reduction and saponification. Similar synthetic methodology was used for 3b. X-ray crystal structure of the ternary complex of 3a, DHFR, and NADPH showed that the pyrrolo[2,3-d]pyrimidine ring binds in a "2,4-diamino mode" in which the pyrrole nitrogen mimics the 4-amino moiety of 2,4-diaminopyrimidines. This is the first example of a classical pyrrolo[2,3-d]pyrimidine antifolate shown to have this alternate mode of binding to DHFR. Compounds 3a and 3b were more inhibitory than LY231514 against TS from Lactobacillus casei and Escherichia coli. Analogue 3a was also more inhibitory against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii. Evaluation of 3a against methotrexate (MTX)-resistant cell lines with defined mechanisms indicated that cross-resistance of 3a was much lower than that of MTX. Metabolite protection studies and folylpoly-gamma -glutamate synthetase studies suggest that the antitumor activity of 3a against the growth of tumor cells in culture is a result of dual inhibition of TS and DHFR. Compound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED50 values of 12.5 and 7.0 nM, respectively, and was more active against FaDu cells than MTX. In contrast, compound 3b was inactive against both cell lines. Compound 3a was evaluated in the National Cancer Institute in vitro preclinical antitumor screening program and afforded IG(50) values in the nanomolar range against a number of tumor cell lines.