Thymidylate synthetase inhibitors. Synthesis of N-substituted 5-aminomethyl-2'-deoxyuridine 5'-phosphates

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Abstract
As part of a program to design selective cancer cell thymidylate synthetase inhibitors, a series of substituted 5-aminomethyl-2''-deoxyuridines with variations in the amine substituent was synthesized in order to elucidate structural criteria that provided strong binding of a 5-substituted 2''-deoxyuridine to the enzyme. A series of substituted 5-aminomethyl-2''-deoxyuridines was synthesized as analogues of 5-thymidylyltetrahydrofolic acid, a proposed intermediate in the thymidylate synthetase catalyzed reaction. 1-(3,5-Di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosyl)-5-chloromethyluracil was treated with the appropriate amine to give the ester protected 5-aminomethyl nucleoside. Removal of the ester groups was accomplished with anhydrous potassium carbonate in methanol to afford the free .beta.-nucleoside. In this way 5-(2-dimethylaminoethylaminomethyl)-2''-deoxyuridine (5a), 5-dimethylaminomethyl-2''-deoxyuridine (5b), 5-N-methylpiperazinylmethyl-2''-deoxyuridine (5c) and 5-pyrrolidinylmethyl-2''-deoxyuridine (5d) were prepared. Compounds 5a,b,d were converted to the respective 5''-phosphates 6a,b,d. All 3 compounds were substrate competitive inhibitors of thymidylate synthetase purified from Escherichia coli, calf thymus and [mouse] Ehrlich ascites tumor cells. The most active compound was 6a with KI of 6, 3.1 and 14 .mu.M observed for the respective enzymes.