The family of nitric oxide synthases (NOS) catalyzes the conversion of l-arginine to l-citrulline and nitric oxide (NO), an important cellular messenger molecule which has been implicated in the pathophysiology of septic shock and inflammatory and neurodegenerative disease states. NOS can be maximally activated by the ubiquitous cofactor, (6R)-5,6,7,8-tetrahydrobiopterin (H4Bip), and antagonists of H4Bip may be of therapeutic importance to inhibit pathologically high NO formation. The 4-amino substituted analogue of H4Bip was reported to be a potent NOS inhibitor. Therefore, we developed a series of novel 4-amino pteridine derivatives, anti-pterins, to pharmacologically target the neuronal isoform of nitric oxide synthase (NOS-I). To functionally characterize the pterin/anti-pterin interaction and establish a structure−activity relationship (SAR), we systematically altered the substituents in the 2-, 4-, 5-, 6-, and 7-position of the pteridine nucleus. Varying the substitution pattern in the 2-, 5-, and 7-position resulted in no significant inhibitory effect on enzyme activity. In contrast, bulky substituents in the 6-position, such as phenyl, markedly increased the inhibitory potency of the reduced 4-amino-5,6,7,8-tetrahydropteridines, possibly as a consequence of hydrophobic interactions within NOS-I. However, this was not the case for the aromatic 4-amino pteridines. Interestingly, chemical modification of the 4-amino substituent by dialkyl/diaralkylation together with 6-arylation of the aromatic 2,4-diamino pteridine resulted in potent and efficacious inhibitors of NOS-I, suggesting possible hydrophilic and hydrophobic interactions within NOS-I. This SAR agrees with (a) the recently published crystal structure of the oxygenase domain of the inducible NOS isoform (NOS-II) and (b) the comparative molecular field analysis of selected NOS-I inhibitors, which resulted in a 3D-QSAR model of the pterin binding site interactions. Further optimization should be possible when the full length structure of NOS-I becomes available.

SummaryA series of new 6-substituted 2,4-diaminopteridines (3-23) and pterins (26-43) have been synthesized by nucleophilic displacement reactions at the side chain of 6-bromomethyl-2,4-diaminopteridine (2) and 6-bromomethylpterin (25) using various types of 0-, N-and S-nucleophiles. Acid and base hydrolyses allow easy conversion of the 2,4-diaminopteridines (3-5, 7, 9, 10, 12, 19-21 and 23) into the corresponding pterins (26-32, 39-42). Acylations take place at the amino as well as at the functional groups in the side chain (44-46). A versatile protection of the amino group in pterins is its transformation into the N ,N -dimethylaminomethylcnimino function by amide-acetals (47, 50) allowing subsequently selective acylations at the side chain (48, 49).

Summary The syntheses of the title compounds have been achieved starting from 4,5-diamino-2-methylpyrimidin-6( 1 H)one. Sufficient amounts of the quinonoid 7,8-dihydropteridin-4(6H)-ones (12-14) were formed upon oxidation of the corresponding 5,6,7 ,8-tetrahydro derivatives (15 -17) to show that they are substrates of dihydroptcridine reductase (human). The apparent Km and Kcal values of (12-14) were determined. These provided evidence that the tautomeric form of the natural cofactor 7,8-dihydro(6H)biopterin has the endocyclic C2- N3 double bond structure (29).

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