2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 10. 2,4-Diamino-5-(6-quinolylmethyl)- and -[(tetrahydro-6-quinolyl)methyl]pyrimidine derivatives. Further specificity studies.

A series of 18 2,4-diamino-5-[(1,2,3,4-tetrahydro-6-quinolyl)methyl]pyrimidines has been prepared by the condensation of 2,4-diamino-5-(hydroxymethyl)pyrimidine with 1,2,3,4-tetrahydroquinolines in acidic medium. Several derivatives were catalytically aromatized; others were synthesized from these by routine aromatic substitution or by condensations of (anilinomethyl)pyrimidines to give quinolinylmethyl analogues. Compounds with 4-methyl-8-methoxy substitution are closely related to trimethoprim (1a) in structure and are excellent inhibitors of bacterial dihydrofolate reductase, with activity at least equivalent to that of 1a. The highest degree of inhibition was achieved with the rigid aromatic series, but greater specificity was accomplished among the tetrahydroquinoline derivatives. This was directly related to N-1 substitution of 4-methyl-8-methoxy derivatives. The spatial relationships around N-1 and protonation at this site may both affect selectivity. Such compounds also had excellent broad-spectrum in vitro antibacterial activity.

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 11. Quinolylmethyl analogues with basic substituents conveying specificity.

A series of nine 2,4-diamino-5-[6-( or 7-)quinolylmethyl]pyrimidines has been prepared by condensations of quinolinecarboxaldehydes with beta-anilinopropionitriles, followed by treatment with guanidine. All compounds has basic or methoxy substituents at the 2- or 4-positions of the quinoline ring. All of the 6-quinolylmethyl derivatives were highly inhibitory against Escherichia coli dihydrofolate reductase (DHFR), provided that an 8-substituent was present in the quinoline ring. Those compounds that had basic substituents in the 2-position of the quinoline ring were also highly specific for bacterial dihydrofolate DHFR, relative to a vertebrate counterpart. Protonation on the quinoline ring nitrogen is a possible cause of specificity.

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 9. Lipophilic trimethoprim analogues as antigonococcal agents.

Lipophilic analogues of trimethoprim (1) bearing 3,5-dialkyl-4-hydroxy substituents in the benzene ring are much more active in vitro against Neisseria gonorrhoeae than is 1. The 3,5-diisopropyl-4-hydroxy derivative (2) was selected as a candidate for clinical evaluation as an antigonococcal agent, and as part of the preliminary evaluation it was submitted to extended pharmacokinetic and metabolism studies in dogs. Although the compound was not extensively conjugated by metabolic enzymes, one of the methyl groups was metabolized to produce a 3-isopropyl-4-hydroxy-5-(alpha-carboxyethyl)benzyl derivative (43), which was rapidly excreted. Related analogues were likewise extensively metabolized.

2,4-Diamino-5-benzylpyrimidines as antibacterial agents. 7. Analysis of the effect of 3,5-dialkyl substituent size and shape on binding to four different dihydrofolate reductase enzymes.

A group of trimethoprim (TMP) analogues containing 3,5-dialkyl(or halo)-4-alkoxy, -hydroxy, or -amino substitution were analyzed in terms of their inhibitory activities against four dihydrofolate reductase (DHFR) isozymes. Although selectivities were lower than with TMP, the activities against vertebrate DHFR were usually at least 2 orders of magnitude less than against enzymes from microbial sources. However, the profiles of activity were remarkably similar for rat, Neisseria gonorrhoeae, and Plasmodium berghei enzymes in all three series, although somewhat different for Escherichia coli DHFR, leading to the conclusion that the hydrophobic pockets are similar for the first three isozymes. Optimal substitution was reached with 3,5-di-n-propyl or 3-ethyl-5-n-propyl groups. Branching of chains at the alpha-carbon, which resulted in increased substituent thickness, was detrimental to E. coli DHFR inhibition in particular. MR is an inadequate parameter for use in correlating such substituent effects. Conformational changes of the more bulky inhibitors can be invoked to explain some differences in inhibitory pattern. Although log P explains simple substituent effects with the vertebrate DHFRs very well, it is insufficient in the more complex cases described here, where shape is clearly involved as well. Solvent-accessible surface areas were measured for TMP in E. coli and chicken DHFRs, where the coordinates are now known. The environment is more hydrophobic in the latter case; this can also be postulated for rat DHFR, which has a very similar activity profile. As with the mammalian isozymes, N. gonorrhoeae DHFR contains an active site phenylalanine replacing Leu-28 of E. coli DHFR, thus creating a more hydrophobic pocket. A similar replacement may also occur in the P.berghei isozyme. Selectivity for bacterial DHFR is dependent on the nature of the 4-substituent, being low for polar 4-hydroxy compounds but high for polar 4-amino analogues, possibly as a result of solvation differences. With complex substituents, the environment of each atom in the active site must be taken into account to adequately explain structure-activity relationships.

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 5. 3',5'-Dimethoxy-4'-substituted-benzyl analogues of trimethoprim.

Forty trimethoprim analogues in which the para substituent in the benzene ring was varied were prepared for antibacterial evaluation. All were very potent inhibitors of Escherichia coli dihydrofolate reductase. The similarity of their inhibitory activities strongly suggested that the side chains beyond the first two atoms were not in contact with the enzyme. However, among 38 ether derivatives which varied widely in their bulk and lipophilicity, very few approached trimethoprim in their broad-spectrum in vitro antibacterial activity. The 4'-methyl and 4'-ethyl analogues and the allyloxy and gamma-chloropropoxy ethers had activities fairly close to that of trimethoprim. The two ethers were chosen for further evaluation in vivo. Neither compound quite matched trimethoprim in efficacy in mice, and their half-lives, as well as that of the beta-methoxyethoxy analogue, were found to be shorter in dogs.

2,4-Diamino-5-benzylpyrimidines as antibacterial agents. 4. 6-Substituted trimethoprim derivatives from phenolic Mannich intermediates. Application to the synthesis of trimethoprim and 3,5-dialkylbenzyl analogues.

The preparation of a wide variety of 6-substituted trimethoprim analogues was readily accomplished by the reaction of 2,4-diamino-6-substituted-pyrimidines with 2,6-dimethoxy-4-[(N,N-dimethylamino)methyl]phenol at 120--160 degrees C. The less reactive 2,6-dialkyl-4-[(N,N-dimethylamino)methyl]phenols reacted successfully with 2,4-diamino-6-(alkylthio)pyrimidines to give 5-(substituted benzyl)pyrimidines. The phenolic groups of the products were alkylated in high yield when a nonreactive 6-substituent was present in the pyrimidine ring. 6-(Alkylthio) groups were easily removed with Raney nickel. Trimethoprim was thus obtained in high yield from its 6-(methylthio) counterpart. The 6-substituted trimethoprim analogues all had low activity as inhibitors of Escherichia coli dihydrofolate reductase and as antibacterial agents.

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 2. C-Alkylation of pyrimidines with Mannich bases and application to the synthesis of trimethoprim and analogues.

A new route to 5-(p-hydroxybenzyl)pyrimidines has been developed which utilizes phenolic Mannich bases plus pyrimidines containing at least two activating groups. The products can be alkylated on the phenolic oxygen or on the pyrimidine N-1 atom, depending on conditions. This method has been used to prepare trimethoprim, a broad-spectrum antibacterial agent, starting from 2,4-diaminopyrimidine and 2,6-dimethoxyphenol.

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 3. C-Benzylation of aminopyridines with phenolic Mannich bases. Synthesis of 1- and 3-deaza analogues of trimethoprim.

Electrophilic substitution of 2,4-diaminopyridine by 2,6-disubstituted -4-[(N,N-dimethylamino)methyl]phenols and by halogens (bromine and fluorine) produces 3-benzyl and 3-halo derivatives, plus a small amount of disubstitution at the 3,5 positions. Treatment of a 2,4-diamino-3-halopyridine with phenolic Mannich bases gives 5- and N-benzylation. 2,4-Diamino-3-bromo-5-(4-hydroxy-3,5-dimethoxybenzyl)pyridine was methylated on the phenolic group in good yield and dehalogenated to produce 3-deazatrimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyridine]. This compound is about 300-fold less active as an inhibitor of Eschericia coli dihydrofolate reductase than is trimethoprim. 2,6-Diaminopyridine is very readily dibenzylated at the 3,5 positions as well as on an amino group, by a phenolic Mannich base; use of a fourfold excess of the pyridine provided a 3-benzylated 2,6-diaminopyridine in 50% yield; this was inactive as an inhibitor of dihydrofolate reductase at 10(-4) M. 2-Amino- and 4-aminopyridines do not produce C-benzylated products under the conditions reported here.