The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compound selectivity. By those means, small aromatic molecules of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, symmetrical bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (K(i) = 2-4 μM). The putative mode of inhibition is discussed according to molecular modeling supported by in vitro tests.

Conformationally restricted bisbenzamidines and related congeners have been synthesized and evaluated for activity against two Plasmodium falciparum strains. The most active compounds, bisbenzamidines linked by a 1,4-piperazinediyl core, had IC(50) values between 3 and 18 nM against both chloroquine-susceptible and -resistant parasites and IC(50) values for cytotoxicity greater than 5 microM, using the A549 human lung epithelial cell line. DNA binding affinity, as estimated by DeltaT(m), did not correlate with either antiparasite effects or cytotoxicity. Each of the active bisbenzamidines interfered with the formation of hemozoin in cell-free systems.

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Trimethoprim-sulfamethoxazole and pentamidine isethionate have been used extensively for the prophylaxis and therapy of pneumonia caused by Pneumocystis jirovecii. Problems associated with toxicity and potential emerging resistance for both therapies necessitate the development of safe and effective analogs or new treatment strategies. In the present study, a library of 36 compounds was synthesized by using the pentamidine molecule as the parent compound modified by a 1,4-piperazinediyl moiety as the central linker to restrict conformation flexibility. The compounds were evaluated for anti-Pneumocystis carinii activity in a bioluminescent ATP-driven assay. Four of the compounds were highly active, with 50% inhibitory concentration (IC 50 ) values of <0.01 g/ml; four had very marked activity (IC 50 < 0.10 g/ml); ten had marked activity (IC 50 < 1.0 g/ml); nine had moderate activity (IC 50 < 10 g/ml); one had slight activity (IC 50 ‫؍‬ 34.1 g/ml); and the remaining eight did not demonstrate activity in this assay system. The high level of activity was specifically associated with an alkyl chain length of five to six carbons attached to one of the nitrogens of the bisamidinium groups. None of the highly active compounds and only one of the very marked compounds exhibited any toxicity when evaluated in three mammalian cell lines. The strategy of substitution of 1,4-piperazine-linked bisbenzamidines produced compounds with the highest level of activity observed in the ATP assay and holds great promise for the development of efficacious anti-P. carinii therapy.Despite advances in the treatment of human immunodeficiency virus infection, Pneumocystis jirovecii pneumonia remains a leading cause of opportunistic infection and mortality in human immunodeficiency virus-infected patients. Currently available anti-Pneumocystis drugs are limited by significant problems of efficacy, toxicity, and emerging resistance (14,21,37,38). No member of the genus Pneumocystis can be maintained continuously outside the mammalian lung. Thus, drug development, as well as other aspects of investigation of this organism family, has been hindered.The effective use of pentamidine isethionate for the treatment of human Pneumocystis pneumonia was first reported in 1958 (18), and the early experience with the drug was summarized in 1967 (19). Trimethoprim-sulfamethoxazole (TMP-SMZ) later became the therapy of choice for this pneumonia due to increased efficacy and reduced toxicity (16). Despite concerted efforts focusing on modifications of the dihydrofolate reductase and dihydropteroate inhibitor portions of TMP-SMZ and the diamidine structure of pentamidine, no compound with increased anti-Pneumocystis carinii properties without toxicity has emerged as a clinical drug (11). With the potential problem of emerging resistance to the sulfa component of TMP-SMZ (1, 21, 26), the significant failure rate of prophylactic pentamidine, and its limited spectrum (17) and associated toxicity (2), it is necessary to identify new therapies or modifications of ...

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A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystis carinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibitors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were the most potent compounds against P. carinii. N,N′-bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4 displayed potent inhibition (IC50 = 2–3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the amidine groups in 4 were moved from the para to the meta positions or replaced with amides.

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