Classical and Nonclassical Furo[2,3-d]pyrimidines as Novel Antifolates: Synthesis and Biological Activities

Abstract

Classical antifolate analogues containing a novel furo[2,3-d]pyrimidine ring system which include N-[4-[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5- yl)methyl]amino]benzoyl]-L-glutamic acid (1) and its N-9 methyl analogue 2 were synthesized as potential dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Four nonclassical antifolates, 2,4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines 3-6 with 3,4,5-trimethoxy, 3,4,5-trichloro, 3,4-dichloro, and 2,5-dimethoxy substituents, respectively, in the phenyl ring, were also synthesized as potential inhibitors of DHFRs including those from Pneumocystis carinii and Toxoplasma gondii, which are organisms responsible for opportunistic infections in AIDS patients. The classical and nonclassical analogues were obtained via nucleophilic displacements of the key intermediate 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with the appropriate (p-aminobenzoyl)-L-glutamate or substituted aniline. The key intermediate was in turn synthesized from 2,4-diamino-6-hydroxypyrimidine and 1,3-dichloroacetone. The final compounds were tested in vitro against rat liver, (recombinant) human, P. carinii, T. gondii, and Lactobacillus casei DHFRs. The classical analogues showed moderate to good DHFR inhibitory activity (IC50 10(-6)-10(-8) M) with the N-CH3 analogue 2 about twice as potent as 1. The nonclassical analogues were inactive with IC50S > 3 x 10(-5) M. The classical analogues were also evaluated as inhibitors of TS (L. casei, (recombinant) human and human CCRF-CEM), glycinamide ribonucleotide formyltransferase, and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and were found to be inactive against these enzymes. The classical analogues (particularly 2) were significantly cytotoxic toward a variety of tumor cell lines in culture. The nonclassical analogues were marginally active. Both classical compounds were good substrates for human folylpolyglutamate synthetase. Further evaluation of the cytotoxicity of 1 and 2 in CCRF-CEM cells and its sublines, having defined mechanisms of methotrexate (MTX) resistance, demonstrated that the analogues utilize the reduced folate/MTX-transport system and primarily inhibit DHFR and that poly-gamma-glutamylation was crucial to their mechanism of action. Protection studies in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin or the combination of thymidine plus hypoxanthine. Furthermore, for compounds 1 and 2, in contrast to MTX, the FaDu cells were better protected by thymidine alone than hypoxanthine alone, suggesting a predominantly antithymidylate effect.

Novel non-classical C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines as potential inhibitors of dihydrofolate reductase and as anti-opportunistic agents

“…Gangjee et al reported [7][8][9][10] the synthesis of two-carbon atom bridged furo [2,3-d] which on hydrogenation would give the desired target compounds. However the Wittig reaction of I and (1-chloro-ethyl)-benzene under a variety of different conditions of time, base, temperature and solvent failed to afford the desired product III.…”

“…Gangjee et al [5][6][7][8][9][10] reported two-atom bridged classical and non-classical, 6-5 bicyclic analogs, as potential DHFR inhibitors (Fig. 2).…”

Synthesis and evaluation of a classical 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine as antifolates

“…Recently, some 6-5 bicyclic pyrrolo[2, 3-d]pyrimidines such as compounds 3 and 4 TNP351 8,9 were also reported as potent antifolates. Gangjee et al [10][11][12] reported a series of novel furo[2,3-d]pyrimidines 5-9 as potent antifolates. A 2,4-diamino-substituted pyrimidine ring is considered important for potent DHFR inhibition, while a 2-substituted-4-oxopyrimidine ring is considered important for TS inhibition.…”

“…A 2,4-diamino-substituted pyrimidine ring is considered important for potent DHFR inhibition, while a 2-substituted-4-oxopyrimidine ring is considered important for TS inhibition. 2,13,14 Gangjee et al 10,11 suggested a dual DHFR-TS inhibitor model and proposed two binding modes for the 2-amino-4-oxo-pyrrolo[2, 3-d]pyrimidine system as well as the 2,4-diamino-furo[2, 3-d]pyrimidine (Fig. 2), Gangjee et al 15 demonstrated that compound 10, a 4-methyl analog of compound 3, binds in the alternate mode to DHFR.…”

“…Thus, it was of interest to develop additional dual DHFR-TS inhibitors as antitumor agents. Gangjee et al [10][11][12] reported the synthesis of novel, classical 2,4-diamino-5-substitutedfuro[2,3-d]pyrimidines 5-9 as antifolates which have a two-, or three-atom bridge connecting the furo[2, 3-d]pyrimidine ring system to the benzoyl-L-glutamic acid (L-Glu). Compounds 5-8 showed moderate to high potency against DHFR (IC 50 = 1.0 × 10 −6 to 1.0 × 10 −8 M), whereas compound 9 was less active against DHFR (IC 50 = 1.0 × 10 −5 M).…”