The paper describes the design, synthesis, and testing of inhibitors of folate-synthesizing enzymes and of whole cell cultures of Candida albicans. The target enzymes used were dihydropteroic acid synthase (SYN) and dihydrofolate reductase (DHFR). Several series of new 2,4-diaminopyrimidines were synthesized and tested as inhibitors of DHFR and compared with their activity against DHFR derived from mycobacteria and Escherichia coli. To test for selectivity, also rat DHFR was used. A series of substituted 4-aminodiphenyl sulfones was tested for inhibitory activity against SYN and the I(50) values compared to those obtained previously against Plasmodium berghei- and E. coli-derived SYN. Surprisingly, QSAR equations show very similar structural dependencies. To find an explanation for the large difference in the I(50) values observed for enzyme inhibition (SYN, DHFR) and for inhibition of cell cultures of Candida, mutant strains with overexpressed efflux pumps and strains in which such pumps are deleted were included in the study and the MICs compared. Efflux pumps were responsible for the low activity of some of the tested derivatives, others showed no increase in activity after pumps were knocked out. In this case it may be speculated that these derivatives are not able to enter the cells.

Quantitative structure-activity studies have been performed for a series of 2-substituted isonicotinic acid hydrazides by correlating electronic, steric, and lipophilic properties of the substituents with the biological activity date (MIC) from serial dilution tests with Mycobacterium tuberculosis (strain H 37 Rv). The reaction rates for the quaternization of 2-substituted pyridines with methyl iodide were also determined. The rate constants show a similar dependence on the steric and electronic effects of the substituents as the antibacterial activities of the corresponding pyridine-4-carboxylic acid hydrazides. The obtained correlations give evidence that the reactivity of the pyridine nitrogen atom is essential for the biological activity of 2-substituted isonicotinic acid hydrazides and seem to support the hypothesis that isonicotinic acid derivatives are incorporated into an NAD analogue.

The steady state growth of E. coli cultures is inhibited by either sulfonamide (SA) or trimethoprim (TMP) with a rate constant, k_app1The new steady state is obtained immediately after the addition of TMP whereas a lag phase of about 5 generations is observed for SA. In TMP-treated cultures the slope of the logarithmic growth curve decreases after an initial inhibited growth and a second steady state is established, k_app2 The appearance of the second ‘phase’is dependent on the number of germs but not on the number of generations. Using prewashed cell cultures it was shown that the presence of the first phase is due to an antagonist excreted into the culture medium. The inhibitory effect of TMP is also partly antagonized by thymidine. The inhibitory effect of TMP is about 5 times larger than the effect of sulfamethoxazole (Gantanol®, GT) as evaluated from the plot of 1/k₀––k_app vs. 1/C or C k₀/k₀––k_app vs. C respectively. In cultures where the growth was totally stopped by high concentration of SA no inhibitory effect of TMP is observable. The inhibitory effect of TMP is found to be reversible if the drug containing culture is diluted. The antibacterial effects of TMP alone were seen to be considerably more complex than those of sulfonamides despite their presumable similar sites of action. Even at very high concentrations SA act only bacteriostatically, whereas TMP alone can cause bactericidal effects if a certain threshold of concentration (≥1 µmol/l) is passed. (A decrease in numbers of cells of about 90% can be obtained within 8 h.) Combinations of TMP and SA at concentrations where both drugs are acting merely bacteriostatically lead to effects considerably greater than would be expected from simple additivity. This study of generation rate constants in inhibited cultures indicates that the claim for synergism and bactericidal effect as usually defined is indeed valid for this combination. It seems possible that both the effects in inhibited cultures by TMP alone and its combination with sulfonamides might be explained if one were to assume that TMP has an additional mode of action separate from its known inhibition of dihydrofolate reductase.

Bacterial growth kinetic experiments have been performed to study the potentiating effect of trimethoprim (TMP) and TMP derivatives on the antibacterial action of sulfonamides (SA). The TMP derivatives showed different inhibitory effects when used alone. Derivatives with decreased inhibitory activity excerted ‘bactericidal’ effects only at high concentrations. A rather fast development of bacteria with lower sensitivity against these derivatives has also been observed. In the combined action with SA, however, the same maximal ‘kill’ rate was observed for TMP derivatives as tetroxoprim or derivative IV (table I) at low concentrations of these inhibitors. Under the experimental conditions there is no significant difference between the studied TMP derivatives and various SA in the combined action.

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