The synthesis and biological activity are reported for 21 6-substituted 2,4-diaminopyrido[3,2-d]pyrimidine analogues (4-24) of piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) and as antitumor agents. Recombinant DHFR from Pneumocystis carinii (pc) and native DHFR from Toxoplasma gondii (tg) were the target enzymes tested; these organisms are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity for the pathogenic DHFR. The synthesis of S9-bridged compounds 4-6 was achieved by aryl displacement of 2,4-diamino-6-chloropyrido[3, 2-d]pyrimidine (27) with thiol nucleophiles. Oxidation of 4-6 with hydrogen peroxide in glacial acetic acid afforded the corresponding sulfone analogues 7-9. The N9-bridged compounds 10-24 were synthesized from their precursor 3-amino-6-(arylamino)-2-pyridinecarbonitriles via a thermal cyclization with chloroformamidine hydrochloride. Unlike the S9-bridged compounds, the arylamino side chains of the N9-bridged analogues were introduced prior to the formation of the 2, 4-diaminopyrido[3,2-d]pyrimidine nucleus. A reversed two-atom-bridged analogue (25) was also synthesized using a synthetic strategy similar to that utilized for compounds 10-24. The IC50 values of these compounds against pcDHFR ranged from 0.0023 x 10(-6) M for 2,4-diamino-6-(N-methyl-3',4'-dimethoxyanilino)pyrido[3, 2-d]pyrimidine (21), which was the most potent, to 90.4 x 10(-6) M for 2,4-diamino-6-(4'-methoxyanilino)pyrido[3,2-d]pyrimidine (12), which was the least potent. The three S9-bridged compounds tested were more potent than the corresponding sulfone-bridged compounds for all three DHFRs. N9-Methylation increased the potency by as much as 17 000-fold (compounds 15 and 21). None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was again 21 with an IC50 value of 0.00088 x 10(-6) M and the least potent was 12 with an IC50 of 2.8 x 10(-6) M. N9-Methylation afforded an increase in potency of up to 770-fold (compound 15 NH vs 21 N-CH3) compared to the corresponding N9-H analogue. In contrast to pcDHFR, several analogues had a greater selectivity ratio for tgDHFR compared to trimetrexate (TMQ) or PTX, most notably 2, 4-diamino-6-[(3',4'- dimethoxyphenyl)thio]pyrido[3,2-d]pyrimidine (4), 2,4-diamino-6-[(2'-methoxyphenyl)sulfonyl]pyrido[3, 2-d]pyrimidine (7), and 2,4-diamino-6-(2', 5'-dimethoxyanilino)pyrido[3,2-d]pyrimidine (14) which combined relatively high potency at 10(-7)-10(-8) M along with selectivity ratios of 3.97, 6.67, and 4.93, respectively. Several analogues synthesized had better selectivity ratios than TMQ or PTX for both pcDHFR and tgDHFR, and the potencies of the N9-methylated compounds were comparable to or greater than that of TMQ or PTX. Selected compounds were evaluated as inhibitors of the growth of a variety of tumor cells in culture. The N9-CH3 analogues were, in general, highly potent with GI50 values in the nanomolar range. The N9-H and S9 analogues were less potent with GI50 values in the millimolar to micromolar range.

中文翻译：

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吡曲氨辛的6位取代的2,4-二氨基吡啶并[3,2-d]嘧啶类似物，作为大鼠肝脏，卡氏肺孢子虫和弓形虫的二氢叶酸还原酶的抑制剂，并用作抗肿瘤剂。

报道了吡咯特辛（PTX）的21个6-取代的2,4-二氨基吡啶并[3,2-d]嘧啶类似物（4-24）作为二氢叶酸还原酶（DHFR）的抑制剂和抗肿瘤剂的合成和生物活性。来自卡氏肺孢子虫（PC）的重组DHFR和来自弓形虫（tg）的天然DHFR是测试的目标酶。这些生物体是艾滋病患者致命的机会性感染的原因。大鼠肝脏（rl）DHFR用作哺乳动物参考酶，以确定对病原性DHFR的选择性。S9桥联化合物4-6的合成是通过将2，4-二氨基-6-氯吡啶并[3,2-d]嘧啶（27）用硫醇亲核试剂进行芳基置换而实现的。在冰醋酸中用过氧化氢氧化4-6，得到相应的砜类似物7-9。通过其前体3-氨基-6-（芳基氨基）-2-吡啶腈通过与氯甲am盐酸盐的热环化合成N9-桥连的化合物10-24。与S9桥联的化合物不同，N9桥联的类似物的芳基氨基侧链是在形成2，4-二氨基吡啶并[3,2-d]嘧啶核之前引入的。还使用类似于用于化合物10-24的合成策略合成了反向的两个原子桥接的类似物（25）。这些化合物对pcDHFR的IC50值范围为0.0023 x 10（-6）M，对于2,4-二氨基-6-（N-甲基-3'，4'-二甲氧基苯胺基）吡啶基[3，2-d]嘧啶（ 21）最有效，对于2,4-二氨基-6-（4'-甲氧基苯胺基）吡啶并[3,2-d]嘧啶（12）最小，为90.4 x 10（-6）M有力的。对于所有三种DHFR，测试的三种S9桥连化合物比相应的砜桥连化合物更有效。N9-甲基化将效力提高了多达17000倍（化合物15和21）。没有类似物对pcDHFR具有选择性。针对tgDHFR，最有效的类似物再次为21，IC50值为0.00088 x 10（-6）M，最低的类似物为12，IC50为2.8 x 10（-6）M。与相应的N9-H类似物相比，最多可折叠770倍（化合物15 NH对21 N-CH3）。与pcDHFR相比，与曲美曲塞（TMQ）或PTX相比，几种类似物对tgDHFR的选择性更高，最著名的是2，4-二氨基-6-[（3'，4'-二甲氧基苯基）硫代]吡啶基[3,2 -d]嘧啶（4），2,4-二氨基-6-[（（2'-甲氧基苯基）磺酰基]吡啶基[3、2-d]嘧啶（7）和2 4-二氨基-6-（2'，5'-二甲氧基苯胺基）吡啶基[3,2-d]嘧啶（14），在10（-7）-10（-8）M时具有较高的效能，且选择性为分别为3.97、6.67和4.93。合成的几种类似物对pcDHFR和tgDHFR的选择性比TMQ或PTX好，并且N9-甲基化化合物的效能与TMQ或PTX相当或更高。评价所选化合物作为培养物中多种肿瘤细胞生长的抑制剂。N9-CH3类似物通常具有很高的效价，其GI50值在纳摩尔范围内。N9-H和S9类似物的效力较低，其GI50值在毫摩尔至微摩尔范围内。2-d]嘧啶（14）在10（-7）-10（-8）M时具有较高的效能，选择性比分别为3.97、6.67和4.93。合成的几种类似物对pcDHFR和tgDHFR的选择性比TMQ或PTX好，并且N9-甲基化化合物的效能与TMQ或PTX相当或更高。评价所选化合物作为培养物中多种肿瘤细胞生长的抑制剂。N9-CH3类似物通常具有很高的效价，其GI50值在纳摩尔范围内。N9-H和S9类似物的效力较低，其GI50值在毫摩尔至微摩尔范围内。2-d]嘧啶（14）在10（-7）-10（-8）M时具有较高的效能，选择性比分别为3.97、6.67和4.93。合成的几种类似物对pcDHFR和tgDHFR的选择性比TMQ或PTX好，并且N9-甲基化化合物的效能与TMQ或PTX相当或更高。评价所选化合物作为培养物中多种肿瘤细胞生长的抑制剂。N9-CH3类似物通常具有很高的效价，其GI50值在纳摩尔范围内。N9-H和S9类似物的效力较低，其GI50值在毫摩尔至微摩尔范围内。N9甲基化化合物的效能与TMQ或PTX相当或更高。评价所选化合物作为培养物中多种肿瘤细胞生长的抑制剂。N9-CH3类似物通常具有很高的效价，其GI50值在纳摩尔范围内。N9-H和S9类似物的效力较低，其GI50值在毫摩尔至微摩尔范围内。N9甲基化化合物的效能与TMQ或PTX相当或更高。评价所选化合物作为培养物中多种肿瘤细胞生长的抑制剂。N9-CH3类似物通常具有很高的效价，其GI50值在纳摩尔范围内。N9-H和S9类似物的效力较低，其GI50值在毫摩尔至微摩尔范围内。