Rosowsky A, Freisheim J H, Moran R G, Solan V C, Bader H, Wright J E, Radike-Smith M
J Med Chem. 1986 May;29(5):655-60. doi: 10.1021/jm00155a012.
Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.
合成了抗肿瘤抗叶酸剂甲氨蝶呤（MTX）的类似物，其中谷氨酸（Glu）部分被鸟氨酸（Orn）、2,4-二氨基丁酸（Dab）或2,3-二氨基丙酸（Dap）取代。还合成了用Orn取代Glu的氨蝶呤（AMT）类似物。MTX类似物是通过4-氨基-4-脱氧-N10-甲基蝶酸（mAPA）与Nω-叔丁氧羰基-α,ω-二氨基链烷酸在氰基磷酸二乙酯存在下反应，然后用三氟乙酸（TFA）脱保护，或通过对硝基苯基-mAPA与Nω-叔丁氧羰基-α,ω-二氨基链烷酸反应并随后用TFA处理得到的。AMT类似物（APA-Orn）是通过对硝基苯基4-氨基-4-脱氧-N10-甲酰基蝶酸酯与硅烷化Nδ-叔丁氧羰基-L-鸟氨酸在55℃的N,N-二甲基甲酰胺（DMF）中反应3天（产率45%）、皂化（83%）和TFA裂解（89%）合成的。APA-Orn是L1210小鼠白血病二氢叶酸还原酶（DHFR）（IC50 = 0.072 μM）和小鼠肝脏部分纯化的叶酰聚谷氨酸合成酶（FPGS）（Ki = 0.15±0.06 μM）的有效抑制剂。MTX类似物（mAPA-Orn）对这两种酶同样有活性，对DHFR的IC50为0.160 μM，对FPGS抑制的Ki为20.4±7.7 μM。其他MTX类似物和先前报道的赖氨酸衍生物（mAPA-Lys）显示出与mAPA-Orn相似的DHFR亲和力，但缺乏作为FPGS抑制剂的活性。带正电荷的氨基似乎对细胞摄取不利，与MTX和AMT（IC50 = 0.002 μM）相比，这些化合物对野生型L1210细胞的细胞毒性较低（IC50 = 0.40 - 2.4 μM）证明了这一点。另一方面，mAPA-Orn和APA-Orn对L1210/R81细胞都比相应的Glu衍生物MTX和AMT更有效，这表明在这些MTX耐药细胞中可能发生一个“自我增强”过程，该过程涉及通过干扰还原型叶酸的聚谷氨酸化来增强抗叶酸活性。APA-Orn是迄今为止发现的最有效的DHFR和FPGS双重抑制剂，但其作为治疗剂的有效性可能需要某种形式的前药修饰来中和侧链的末端氨基。
