Modification of the potent thymidylate synthase (TS) inhibitor 1-[[N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- prop-2-ynylamino]benzoyl]amino]methyl]-3-nitrobenzene (4a) has led to the synthesis of quinazolinone antifolates bearing functionalized alkyl substituents at C2. A general synthetic route was developed which involved coupling the appropriate 1-[[N-[4-(alkylamino)benzoyl)amino]methyl]-3-nitrobenzene 20-22 with a 6-(bromomethyl)-2-(acetoxymethyl)-3,4-dihydro-4-oxoquinazoline 9 or 10. Replacement of the 2-acetoxy group by a chlorine atom followed by the displacement of the halogen of 25a-c by various nucleophiles led to compounds 26-40. Good TS (IC50 < 1 microM) and growth inhibition (IC50 0.1-1 microM) were found with most of these new antifolates. TS inhibitors in this series do not apparently require the reduced folate carrier (RFC) for cell entry (they most likely penetrate the cell membrane by passive diffusion) and are not polyglutamated. N, O, S, Cl, and CN as well as large amino and mercapto substituents were tolerated by the enzyme. The simultaneous incorporation of 7-methyl and 2'-F substituents gave a series of highly potent agents inhibiting cell growth at concentrations < 1 microM (24, 27bc; 30-32b, 35b). The incorporation of suitable C2 substituents has overcome the decrease in aqueous solubility observed with lipophilic quinazoline antifolates. This is best illustrated by compound 31a, where up to a 54-fold increase in solubility has been achieved by the incorporation of an N-methylpiperazine nucleus into the C2-methyl group of 4a.