Synthesis of quaternised 2-aminopyrimido[4,5-d]pyrimidin-4(3H)-ones and their biological activity with dihydrofolate reductase.
In a program to design and develop mechanism-based compounds active as substrates and inhibitors of dihydrofolate reductase (DHFR), we report the synthesis and physical properties of the 6-methyl- (7), 8-methyl- (8a), and 8-ethyl- (8b) derivatives of the parent 2-aminopyrimido[4,5-d]pyrimidin-4-(3H)-one (6). These compounds are the first members of a class of heterocycles related to 8-alkylpterins (N8-alkyl-2-aminopteridin-4(8H)-ones) (2a-2c), which have been shown to be novel substrates for DHFR. Three methods were developed for the synthesis of target compounds 7, 8a and 8b; however, the optimum yields (1-8%) could not be improved because the products decomposed by ring opening (e.g. to 2,4-diamino-5-methyliminomethylpyrimidin-6(1H)-one (9)) under the reaction conditions. The marked pi-electron deficiency of compounds 7, 8a and 8b is the likely cause for the susceptibility of the quaternised pyrimidine ring in the related cations 10, 15a and 15b, respectively, to add nucleophiles, thus promoting the opening of the pyrimidopyrimidine ring system. 1H-NMR spectroscopic studies of compounds 7, 8a and 8b revealed a fast and reversible covalent hydration of the associated cations across the C7z.sbnd;N8 bond for the N6-methyl derivative 7 and across the N6z.sbnd;C7 bond for the N8-methyl derivative 8a. UV spectroscopic studies of methyl derivatives 7 and 8a as well as the parent heterocycle 6 showed that protonation of the latter occurred at N1, while methylation with iodomethane proceeded at N6 and N8. The basicities of the N-methyl derivatives 7 and 8a (pK(a) ca. 5.5) are similar to those of 8-alkylpterins 2; this is an essential element of the design to promote binding to DHFR in their protonated form. Enzyme kinetics of 7, 8a and 8b with chicken DHFR confirmed our predictions that they are substrates, with apparent K(m) values of 3.8, 0.08, and 0.65 mM, and apparent V(max) values of 0.47, 2.27, and 0.30 nmol L(-1) min(-1) (for enzyme concentration 0.122 micro M), respectively. The parent compound 6 was not a substrate.
Gebauer
Markus G
MG
Computational Proteomics and Therapy Design Group, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia.
McKinlay
Carolyn
C
Gready
Jill E
JE
D000818:Animals; D002645:Chickens; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000494:pharmacology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D009249:NADP / Q000378:metabolism; D011622:Pterins / Q000138:chemical synthesis* / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D011622:Pterins; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase