Highly Active Anti- Pneumocystis carinii Compounds in a Library of Novel Piperazine-Linked Bisbenzamidines and Related Compounds

Abstract

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 ...

The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis

“…AR-12 showed moderate activity against both species, with 72-h 50% inhibitory concentrations (IC 50 s) of 4.8 g/ml for P. carinii and 1.8 g/ml for P. murina. IC 50 s for pentamidine, an agent used for treatment and prophylaxis, are typically less than 1 g/ml (30). AR-12 has a remarkably broad spectrum of antifungal activity at concentrations that are similar to those obtained in humans during a phase I anticancer clinical trial.…”

In Vitro Selection and In Vivo Efficacy of Piperazine- and Alkanediamide-Linked Bisbenzamidines against Pneumocystis Pneumonia in Mice

“…The selection criteria for the compounds evaluated in these studies were based on their activities in our in vitro drug screening system and their lack of or low toxicities in one or more mammalian cell lines (12). For each compound, Table 1 shows the structure in relation to the parent compound, the in vitro efficacy against P. carinii, expressed as the IC 50 (the concentration of compound required to reduce the ATP content of treated cells by 50% versus that of untreated cells), the in vitro activity rank, and the IC 50 for the A549 cell line, a human lung cell line (toxicity).…”

“…Bisbenzamidines were synthesized in the laboratory of Tien Huang, College of Pharmacy, Xavier University of Louisiana, according to previously described procedures (12,13,28,29,33,37,38). The structures and purity of the compounds were confirmed by spectroscopic methods (infrared and highresolution proton nuclear magnetic resonance) and elemental analyses.…”

“…A series of compounds in which pentamidine was modified by a 1,4-piperazinediyl, alkanediamide, or phenylenediamide moiety as the central linker to modulate conformational flexibility was developed in our laboratories (12,13,28,29,37). Forty-six of these agents were evaluated in the ATP assay, and some were found to be more active than pentamidine against P. carinii, with minimal toxicity to mammalian cells (12,37). For this study, we have extended these observations to examine the activities and toxicities of selected bisbenzamidine de- …”

“…This system is semiautomated and permits the screening of hundreds of compounds per year. Promising agents are selected on the basis of anti-Pneumocystis activity and a lack of toxicity or low toxicity in one to three mammalian cell lines for further study in an immunosuppressed rodent model of Pneumocystis pneumonia (12,43,45). The relationship of this system to human pneumocystosis has become more complicated by the increasing evidence of genetic diversity and host specificity among Pneumocystis populations from different mammalian species (3,18,25,32).…”