Studies are described examining a new class of 4-aminofolate analogues modified by an N to C conversion and alkyl substitution at the N-5 position of aminopterin and methotrexate. All of these analogues were equivalent to aminopterin and methotrexate as inhibitors of tumor cell dihydrofolate reductase (K1 = 3.49-5.16 pm). N to C conversion at the N-5 position of aminopterin reduced its influx (inferred from the change in K1) 3-fold, but the same modification increased influx of methotrexate 2–3-fold in Sarcoma 180 cells. Alkylation (methyl or ethyl) of this position on 5-deazaaminopterin increased influx 3-fold, while a similar alteration of 5-deazamethotrexate increased influx 4–5-fold. Influx of the methotrexate analogues was increased a total of 14-fold as a result of these modifications. Similar differences among these analogues were observed for inhibition of Sarcoma 180 cell growth in culture. Inhibitory potency was in the ascending order methotrexate < 5-deazamethotrexate < 5-deazaaminopterin < aminopterin < 5-alkyl (methyl or ethyl) analogues of 5-deazaaminopterin and 5-deazamethotrexate (the ethyl analogues were 2-fold more inhibitory than the methyl analogues). All of the analogues examined were equivalent in regard to efflux from Sarcoma 180 cells. Differences in transport alone did not account for all of the increased inhibitory potency (up to 33-fold) of the 5-alkyl-5-deaza analogues compared to the parent compounds. The extent of polyglutamylation of 5-deazaaminopterin and 5-deazamethotrexate and their 5-alkyl derivatives in Sarcoma 180 cells was substantially less compared to aminopterin and equivalent to methotrexate. Transport inward of 5-deazaaminopterin in isolated crypt cell epithelium from mouse small intestine was 2-fold lower than aminopterin (influx Km = 14.2 ± 2 µm), while influx of 5-deazamethotrexate was 2-fold greater than methotrexate (influx Km = 98.6 ± 23). However, transport inward of all of 5-alkyl derivatives of these 5-deaza analogues was intermediate [influx Km = 44.4 ± 11 (SEM) to 49.8 ± 12 µm] between values for aminopterin and methotrexate. These differences accounted, to some extent, for the reduced toxicity of the 5-alkyl-5-deazaaminopterin analogues compared to aminopterin and the increased toxicity of 5-methyl-5-deazamethotrexate compared to methotrexate. All of the 5-alkyl derivatives of aminopterin and methotrexate were more active in vivo than methotrexate against four murine tumor models. Modest improvement in activity over methotrexate was observed in the Lewis lung tumor, but substantially greater improvement (3–6-fold greater reduction in tumor growth) was observed for some of these analogues against Sarcoma 180 and T241 fibrosarcoma. Greatest improvement in activity (10–20-fold greater reduction in tumor growth compared to methotrexate) was seen with the EO771 mammary adenocarcinoma. On a schedule of once per day for 5 days, a number of animals (10–60%) treated with these new analogues were tumor free shortly after cessation of therapy.

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Supported in part by Grants CA-08748, CA-18856, CA-22764, and CA-25236 from the National Cancer Institute, Department of Health and Human Services and the Elsa U. Pardee Foundation.

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