Piper J R, McCaleb G S, Montgomery J A, Kisliuk R L, Gaumont Y, Thorndike J, Sirotnak F M
Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35255.
J Med Chem. 1988 Nov;31(11):2164-9. doi: 10.1021/jm00119a018.
The title compounds were prepared in extensions of a general synthetic approach used earlier to prepare 5-alkyl-5-deaza analogues of classical antifolates. Wittig condensation of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (2a) and its 5-methyl analogue 2b with [4-(methoxycarbonyl)benzylidene] triphenylphosphorane gave 9,10-ethenyl precursors 3a and 3b. Hydrogenation (DMF, ambient, 5% Pd/C) of the 9,10-ethenyl group of 3b followed by ester hydrolysis led to 4-[2-(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)ethyl]ben zoi c acid (5), which was converted to 5-methyl-5,10-dideazaaminopterin (6) via coupling with dimethyl L-glutamate (mixed-anhydride method using i-BuOCOCl) followed by ester hydrolysis. Standard hydrolytic deamination of 6 gave 5-methyl-5,10-dideazafolic acid (7). Intermediates 3a and 3b were converted through concomitant deamination and ester hydrolysis to 8a and 8b. Peptide coupling of 8a,b (using (EtO)2POCN) with diesters of L-glutamic acid gave intermediate esters 9a and 9b. Hydrogenation of both the 9,10 double bond and the pyrido ring of 9a and 9b (MeOH-0.1 N HCl, 3.5 atm, Pt) was followed by ester hydrolysis to give 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (11a) and the 5-methyl analogue 11b. Biological evaluation of 6, 7, 11a, and 11b for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for growth inhibition and transport characteristics toward L1210 cells revealed 6 to be less potent than methotrexate in the inhibition of DHFR and cell growth. Compounds 6, 11a, and 11b were transported into cells more efficiently than methotrexate. Growth inhibition IC50 values for 11a and 11b were 57 and 490 nM, respectively; the value for 11a is in good agreement with that previously reported (20-50 nM). In tests against other folate-utilizing enzymes, 11a and 11b were found to be inhibitors of glycinamide ribonucleotide formyltransferase (GAR formyltransferase) from one bacterial (Lactobacillus casei) and two mammalian (Manca and L1210) sources with 11a being decidedly more inhibitory than 11b. Neither 11a nor 11b inhibited aminoimidazolecarboxamide ribonucleotide formyltransferase. These results support reported evidence that 11a owes its observed antitumor activity to interference with the purine de novo pathway with the site of action being GAR formyltransferase.
通过扩展先前用于制备经典抗叶酸剂的5-烷基-5-脱氮类似物的通用合成方法，制备了标题化合物。2,4-二氨基吡啶并[2,3-d]嘧啶-6-甲醛（2a）及其5-甲基类似物2b与[4-（甲氧基羰基）亚苄基]三苯基膦进行维蒂希缩合反应，得到9,10-乙烯基前体3a和3b。3b的9,10-乙烯基进行氢化反应（DMF，常温，5% Pd/C），然后进行酯水解反应，得到4-[2-（2,4-二氨基-5-甲基吡啶并[2,3-d]嘧啶-6-基）乙基]苯甲酸（5），通过与L-谷氨酸二甲酯偶联（使用异丁基氯甲酸酯的混合酸酐法），然后进行酯水解反应，将其转化为5-甲基-5,10-二脱氮氨基蝶呤（6）。6进行标准的水解脱氨反应得到5-甲基-5,10-二脱氮叶酸（7）。中间体3a和3b通过同时进行脱氨和酯水解反应转化为8a和8b。8a、b（使用（EtO）2POCN）与L-谷氨酸二酯进行肽偶联反应，得到中间体酯9a和9b。9a和9b的9,10-双键和吡啶环进行氢化反应（甲醇-0.1 N盐酸，3.5 atm，Pt），然后进行酯水解反应，得到5,10-二脱氮-5,6,7,8-四氢叶酸（11a）及其5-甲基类似物11b。对6、7、11a和11b进行生物活性评估，考察其对从L1210细胞中分离的二氢叶酸还原酶（DHFR）的抑制作用、对L1210细胞的生长抑制作用以及转运特性，结果显示6在抑制DHFR和细胞生长方面的效力低于甲氨蝶呤。化合物6、11a和11b比甲氨蝶呤更有效地转运进入细胞。11a和11b的生长抑制IC50值分别为57和490 nM；11a的值与先前报道的值（20 - 50 nM）吻合良好。在针对其他利用叶酸的酶的测试中，发现11a和11b是来自一种细菌（干酪乳杆菌）和两种哺乳动物（Manca和L1210）来源的甘氨酰胺核糖核苷酸甲酰基转移酶（GAR甲酰基转移酶）的抑制剂，11a的抑制作用明显强于11b。11a和11b均不抑制氨基咪唑甲酰胺核糖核苷酸甲酰基转移酶。这些结果支持了已报道的证据，即11a观察到的抗肿瘤活性归因于对嘌呤从头合成途径的干扰，作用位点为GAR甲酰基转移酶。
