Synthesis and antitumor activity of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine.

The synthesis of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW301U, 7) by a route that has general applicability to the preparation of many 6-(substituted benzyl)-5-methylpyrido[2,3-d]pyrimidines is described. The key intermediate, 2,4-diamino-7,8-dihydro-6-(2,5-dimethoxybenzyl)-5-methyl-7-oxopyrido[2,3-d]pyrimidine (4), is converted to the 7-chloro compound 5 by treatment with a 1:1 complex of N,N-dimethylformamide--thionyl chloride, and 5 is hydrogenolyzed with palladium on charcoal in the presence of potassium hydroxide to yield 7. BW301U is a potent lipid-soluble inhibitor of mammalian dihydrofolate reductase and has significant activity against the Walker 256 carcinosarcoma in rats.

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 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. 13. Some alkenyl derivatives with high in vitro activity against anaerobic organisms.

A series of 2,4-diamino-5-(3,5-dialkenyl-4-methoxy- or -4-hydroxybenzyl)pyrimidines was prepared from [(allyloxy)benzyl]pyrimidines by Claisen rearrangements, and the resulting allyl phenols were further modified by methylation and rearrangement to 1-propenyl analogues. Analogous 3,4-dimethoxy-5-alkenyl derivatives were prepared by similar techniques. High in vitro antibacterial activity was obtained against certain anaerobic organisms, such as Bacteroides species and Fusobacterium, which was equal to or better than the control, metronidazole, in several cases. The profile was similar against Neisseria gonorrhoeae and Staphylococcus aureus. The 3,5-bis(1-propenyl)-4-methoxy derivative 8 was 1 order of magnitude more active against Escherichia coli dihydrofolate reductase than its saturated counterpart, and it was also more active than trimethoprim, 1. However, it was considerably less active in vitro against the Gram-negative organisms. The 3,4-dimethoxy-5-alkenyl, -5-alkyl, and -5-alkoxy analogues had very high broad-spectrum antibacterial activity. However, pharmacokinetic studies of four of the compounds in dogs and rats and in vivo studies with an abdominal sepsis model in rats showed no advantages over trimethoprim.

Metabolism of a novel antitumor agent, crisnatol, by a human hepatoma cell line, Hep G2, and hepatic microsomes. Characterization of metabolites.

Metabolism of the anticancer agent crisnatol was investigated using a human hepatoma cell line, Hep G2, and human liver microsomes. Crisnatol was metabolized extensively by both systems. The TLC/autoradiographic analysis showed that the crisnatol metabolite profile was similar for both systems and the major metabolites were shown to have structural characteristics similar to those formed by the rat. The Hep G2 cells formed three isomeric dihydrodiols; one of these has been identified by GC/MS and 1H-NMR as the crisnatol 1,2-dihydrodiol. Human liver microsomes also formed two isomeric dihydrodiols with 1,2-dihydrodiol as the major isomer and, in addition, produced 1-hydroxycrisnatol. Crisnatol concentrations of 1.3 micrograms/mL completely inhibited the replication of Hep G2 cells as measured by thymidine incorporation and cell growth kinetics and, at this concentration, cell viability decreased by only 35% as determined by vital staining of cells using neutral red dye.

The role of dietary nitrate and nitrite in the reductive deamination of sulfadiazine by the rat, guinea pig, and neonatal calf.

Previous metabolic depletion studies of 14C-sulfadiazine (SDZ) in the neonatal calf led to identification of two novel metabolites, 2-benzenesulfonamidopyrimidine (desNH2SDZ) and 2-benzenesulfonamido-4-hydroxypyrimidine. The novelty of the biotransformation prompted examination of mechanisms for the reductive deamination of SDZ in vivo. In subsequent work, it was found that neonatal calves did not consistently convert SDZ to desNH2SDZ; however, calves that were treated simultaneously with nitrite did. Further, when SDZ was given orally to guinea pigs, whose diet is high in nitrate and who demonstrate the capacity to reduce nitrate to nitrite in the oral cavity, SDZ was transformed to desNH2SDZ. Rats did not reductively deaminate SDZ even if they consumed a diet high in nitrate for two weeks prior to treatment with SDZ. However, they did so when nitrite was added to their diet. These observations strongly suggest that reductive deamination of sulfonamides is dependent on the ingestion of nitrite or the reduction of dietary nitrate to nitrite. This reduction of nitrate to nitrite proceeds in the oral cavity, presumably via microflora residing there.

Lipid-soluble inhibitors of dihydrofolate reductase. III: Quantitative thin-layer and high-performance liquid chromatographic methods for measuring plasma concentrations of the antifolate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido-[2,3-d]pyrimidine.

Specific methods using high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for the analysis of the potent antifolate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido-[2,3-d]pyrimidine (I) in plasma were developed. The HPLC system employed paired-ion chromatography using a mobile phase of water-acetonitrile (65:35, v/v) in conjunction with a reversed-phase C-1 column and detection by UV absorbance measurement. The TLC system utilized a scanning densitometer operating in the reflectance mode with detection of I by fluorescence measurement. The lower limits of detection for the HPLC and TLC methods were approximately 0.005 micrograms/ml. The coefficients of variation for the measurement of drug concentrations over the range of 0.04-1.0 microgram/ml of plasma were 5 and 6%, respectively.

Competitive protein binding assay for piritrexim.

A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with [125l]methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found. published HPLC method was found.

Specific TLC tissue residue determination of sulfadiazine following fluorescamine derivatization.

A spectrodensitometric method for the direct determination of sulfadiazine at the tissue residue level (0.1 ppm) is based upon the measurement of the fluorescence of a sulfadiazine-fluorescamine derivative formed directly on a TLC plate by dipping it into a fluorescamine solution. The linear dynamic range for the assay is about 150 from 200 to 0.2 ng, the lower limit of sensitivity. Recoveries from various spiked tissues including milk, eggs, liver, kidneys, muscle, skin, and fat varied with the tissue type but were reproducible. The assay technique has also been used for the assay of sulfamethoxazole and has been explored for use in specifically assaying sulfonamide mixtures.

Baquiloprim, a new antifolate antibacterial: in vitro activity and pharmacokinetic properties in cattle.

During examination of the half-lives in cattle of a series of 5-substituted diaminobenzyl-pyrimidines, it was found that replacement of the phenyl ring of trimethoprim (TMP) by bicyclic structures, particularly a quinolyl group, led to increases in half-life. The presence of a dimethylamino group on the quinolyl ring of the compound baquiloprim (BQP) conferred a half-life of about 10 hours. In contrast to TMP (half-life about one hour), BQP was well absorbed from the gastrointestinal tract in all ages of cattle, plasma concentrations reaching a plateau on the day after dosing followed by a slow decline. BQP showed the same high broad spectrum antibacterial activity as TMP, with marked synergy with sulphonamides. Its differential binding of the dihydrofolate reductases of Escherichia coli and rat liver predicted a margin of safety similar to that of TMP. The results of efficacy studies in mice in comparison with TMP showed that the longer half-life of BQP was associated with greater efficacy, and therapeutic properties superior to those of TMP in cattle were therefore predicted for BQP.

Experimental induction of Proteus mirabilis cystitis in the pony and evaluation of therapy with trimethoprim-sulfadiazine.

Proteus mirabilis cystitis was induced in 9 ponies by chemically eroding the bladder mucosa before the organism was inoculated. Comparisons were made in the treatment of P mirabilis cystitis between ponies treated daily for 13 days with a trimethoprim-sulfadiazine (TMP-SDZ) paste and both positive and negative controls. Urine cultures from ponies treated with TMP-SDZ became negative for P mirabilis between days 3 and 9 after the start of the treatment, whereas positive controls remained infected until day 13. Urine cultures from all ponies were negative for P mirabilis on day 28. Urine concentrations of TMP and SDZ were relatively high after day 1 of therapy.

Pharmacokinetics of trimethoprim and sulfadiazine in the dog: urine concentrations after oral administration.

Serial blood samples were obtained from 12 healthy adult dogs given equivalent subcutaneous and oral doses of the antibacterial combination, trimethoprim-sulfadiazine (1:5). By using a 1-compartment open model, pharmacokinetic parameters for both drugs were estimated from the mean serum concentration data after oral administration. Trimethoprim and sulfadiazine were rapidly absorbed, reaching maximum concentrations in 1 and 4 hours with serum elimination half-lives of 2.5 and 9.9 hours, respectively. After a single oral dose (30 mg/kg, combined ingredients) was given, both drugs were present in urine for up to 24 hours at concentrations exceeding the minimum inhibitory concentrations for common pathogenic bacteria.

Serum concentrations of trimethoprim and sulfadiazine following oral paste administration to the horse.

Two fasted and 2 fed horses were dosed orally with a combined trimethoprim and sulfadiazine paste formulation at a dose of 35 mg (1:5 combined active ingredients)/kg. Serum concentrations of each drug were determined periodically for 3 consecutive days for the 4 horses. The extent and rate of absorption for trimethoprim were variable, but peak serum concentrations occurred generally within 3 hours; sulfadiazine absorption was slower, reaching peak concentrations by 6 hours. Fasting did not have a consistent effect on the serum concentration profiles for either drug. Both drugs achieved serum concentrations that equaled or exceeded the minimum inhibitory concentrations necessary to inhibit the growth of certain pathogens common to the horse. Thus, the paste formulation provides an effective means of dose administration of horses.

Metabolism and disposition by the rat of 35S-sulfadiazine alone and in the presence of trimethoprim.

The tissue distribution and metabolism of 35S-sulfadiazine (I, SDZ) alone and in the presence of trimethoprim (TMP) was studied in the male rat. In the 72-hr period following a single oral dose (30 mg/kg) of 35S-SDZ/TMP (5/1, w/w), 87% of the radioactivity was recovered in the urine and 15% of the radioactivity was recovered in the feces. The concentrations of drug-related material in the plasma or tissues after 72 hr were less than 0.1 ppm with the exception of the liver (0.13 ppm). Aside from intact drug, the two major urinary metabolites (greater than 5% of the radioactivity in urine) were N4-acetylsulfadiazine (II) and sulfadiazine N4-glucuronide (VI). Three minor urinary metabolites (less than 5%) were identified as N4-acetyl-2-sulfanilamido-4-hydroxypyrimidine (IV), 2-sulfanilamido-4-hydroxypyrimidine (III) and 2-sulfanilamido-5-hydroxypyrimidine (V). Metabolites IV and V are novel metabolites of SDZ and have not been reported previously for any species. The relative amounts of sulfadiazine and its metabolites excreted in the urine and feces as well as the distribution of intact drug and 35S in rat tissues were determined. The metabolites were screened for antibacterial activity; the N4-acetylated metabolites II and IV were inactive, whereas the hydroxypyrimidine metabolites III and V were active against a few organisms but in general much less active than I.