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Antimicrobial Agents and Chemotherapy, September 2003, p. 3015-3017, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.3015-3017.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Antitrypanosomal Activities of Fluoroquinolones with Pyrrolidinyl Substitutions

Elizabeth Nenortas, Tomasz Kulikowicz, Christian Burri, and Theresa A. Shapiro^*

Division of Clinical Pharmacology, Departments of Medicine and of Pharmacology and Molecular Sciences, The Johns Hopkins University, Baltimore, Maryland 21205

Received 11 March 2003/ Returned for modification 16 May 2003/ Accepted 4 June 2003

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Fluoroquinolones with pyrrolidinyl substitutions were tested against Trypanosoma brucei and mammalian cells. Bulky substituents at C-7 or a 1-2-bridging thiazolidine ring increased antitrypanosomal activity and selective toxicity. These compounds trap protein-DNA complexes and inhibit nucleic acid biosynthesis in trypanosomes, characteristics of topoisomerase II inhibition.

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African trypanosomiasis is fatal if untreated (11, 12), and current therapies are limited by toxicity and the emergence of drug-resistant parasites (6). Trypanosomes have abundant topoisomerase II activity in both the nuclear and mitochondrial compartments (4), and classic topoisomerase inhibitors promote the intracellular formation of protein-DNA complexes and are cytotoxic (8). Fluoroquinolones target topoisomerase II in bacteria. We previously found that fluoroquinolones in clinical use have weak antitrypanosomal activity, but some experimental tetracyclic fluoroquinolones exhibit significant cytotoxicity against Trypanosoma brucei in vitro (5, 7). We now report the activities of a series of bicyclic and tricyclic fluoroquinolones.

Topoisomerase inhibitors. Ofloxacin was from R. W. Johnson Pharmaceutical Research Institute (Raritan, N.J.), sparfloxacin was from Rhone-Poulenc Rorer (Vitry sur Seine, France), ciprofloxacin was from Miles, Inc. (West Haven, Conn.), and all PD compounds were from Parke-Davis, Pharmaceutical Research Division, Warner-Lambert Company (Ann Arbor, Mich.). Stock solutions of ciprofloxacin, clinafloxacin, and PD134914 were prepared in sterile water, VM26 was prepared in dimethyl sulfoxide (Aldrich), and all others were prepared in sterile 100 mM NaOH.

Assays. Bloodstream-form T. brucei (MiTat 1.2, strain 427) cells and L1210 mouse leukemia cells were maintained in medium (7). Ten concentrations of each fluoroquinolone were evaluated in quadruplicate (1). Exponentially growing cells were treated with solvent or inhibitor for 20 h, lysed, and incubated with p-nitrophenyl phosphate. Acid phosphatase activity was determined, and 50% effective concentrations of inhibitor (EC₅₀) were obtained (SigmaPlot 4.0; SPSS Science) (1, 3). All EC₅₀ comparisons made were considered statistically significant on the basis of nonoverlapping 95% confidence intervals. Covalent protein-DNA complexes were precipitated from sodium dodecyl sulfate lysates of cells treated with solvent, ciprofloxacin, PD124979, PD120978, PD124816, PD120755, PD143891, or VM-26 (2, 9). Results are expressed as the percentage of total radiolabeled DNA (mean ± standard deviation) captured with a 100 µM concentration of inhibitor. To monitor nucleic acid biosynthesis, log-phase T. brucei cells were harvested and resuspended in [methyl-³H]thymidine (20 Ci/mmol; 250 mCi/ml, final concentration) with or without fluoroquinolone. Samples taken at timed intervals were processed as described previously (2).

Antitrypanosomal activity. We tested 11 fluoroquinolones with pyrrolidine substitutions and three standards with piperazine substitutions (Table 1). Each of these was cytotoxic, not cytostatic, in the assay. The bicyclic pyrrolidinyl fluoroquinolones (nucleus A) form a series which is structurally related to ciprofloxacin and sparfloxacin. Replacement of the piperazinyl group at C-7 of ciprofloxacin or sparfloxacin with 3-aminopyrrolidine (PD124979 and PD124816) resulted in a fivefold improvement in antitrypanosomal activity, and the bulky pyrrolidinyl substituent of PD143891 yielded a greater than 10-fold decrease in the EC₅₀ relative to that of sparfloxacin. Halogenation at C-8 decreased potency up to 12-fold. An amino group at position C-5 roughly doubled the activities of PD134914 and PD124816 compared with those of their 5-H congeners. For PD124816, the detrimental effect of fluorine at C-8 was offset by 5-amination: the EC₅₀ of doubly substituted PD124816 and parent PD124979 were comparable.

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TABLE 1. Antitrypanosomal activities of tested fluoroquinolones

The unusual tricyclic nucleus of PD120978 and PD120755 (nucleus B in Table 1) imparted a 2.5-fold increase in antitrypanosomal activity. An extended 3-[(ethylamino)methyl]-1-pyrrolidinyl group at position 8 tripled the activity, making PD120755 the most potent fluoroquinolone reported here. PD114980 has the same extended pyrrolidinyl group but a different tricyclic backbone (nucleus C in Table 1). The resulting 15-fold loss of activity suggests that this ofloxacin-like nucleus lacks the intrinsic antitrypanosomal activity of nucleus A or B.

Antitrypanosomal activity in the A ring system is thus favored by (i) substitution at C-7 with extended pyrrolidinyl > pyrrolidinyl > piperidinyl, (ii) placement of -NH₂ at C-5, (iii) no substitutions at C-8, and (iv) introduction of the thiazolidine ring that bridges the N-1 and C-2 positions (nucleus B). Single-step substitutions involving any of these features produced compounds with increased activity, and the combination of two such motifs resulted in PD143891 and PD120755, which were the most potent compounds tested.

Selective toxicity. When tested against mammalian L1210 cells, selectivities of ciprofloxacin and sparfloxacin were worsened by a 3-aminopyrrolidinyl substituent at C-7. Interestingly, however, the bulky aromatic extension at C-7 in PD143891 restored relative toxicity to trypanosomes. A similar trend was seen in the tricyclic series, where PD120978 was equally toxic to both cell types but PD120755 was twofold more active against trypanosomes. Although a C-5 amino group enhanced antitrypanosomal activity, this was offset by disproportionately greater toxicity to L1210 cells (PD124979 versus PD134914). Selectivity is not large between mammalian cells and trypanosomes, which are also eukaryotes. Nevertheless, some motifs did provide a modest but statistically significant difference in selective toxicity that was reproducible in pairwise comparisons.

Formation of protein-DNA complexes. Intracellular cleavable complex formation was concentration dependent for each inhibitor, and with PD143891 it reached levels comparable to those obtained with VM-26, a potent antitumor agent (58% ± 8% and 56% ± 8%, respectively) (9, 10). Complexes were increased by substituting a 3-aminopyrrolidine for piperazine (as in PD124979, 5% ± 1%) or increasing the bulk of the pyrrolidine substituent in PD120755 and PD143891 (35% ± 10% and 58% ± 8%, respectively).

Inhibition of nucleic acid biosynthesis. To determine whether fluoroquinolones affect nucleic acid biosynthesis, cells were labeled with [methyl-³H]thymidine in the presence or absence of inhibitor (Fig. 1). In treated cells there was a time- and concentration-dependent inhibition of incorporation. The order of inhibitor potency was ciprofloxacin < PD143891 < PD120755. For both pyrrolidinyl compounds, DNA synthesis was halted by 2 h of exposure. To our knowledge this is the first report of fluoroquinolone-mediated inhibition of DNA synthesis in trypanosomes.

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FIG. 1. Fluoroquinolone inhibition of nucleic acid biosynthesis in trypanosomes. T. brucei cells were suspended in labeling medium with or without fluoroquinolone, and duplicate samples were taken at the indicated times. Relative to controls (diamonds, bold line), incorporation was reduced in cells treated with ciprofloxacin (triangles), PD143891 (circles), or PD 120755 (squares) at 10 µM (open symbols, dashed lines) or 100 µM (closed symbols, solid lines). The background of 21,552 dpm has been subtracted from all values.

The structure-antitrypanosomal activity relationships (Table 1) correlate well with the ability to inhibit nucleic acid biosynthesis (Fig. 1) and to promote intracellular cleavable complex formation. These findings indicate that fluoroquinolone-mediated inhibition of type II DNA topoisomerase activity may be a useful approach for development of much-needed new antitrypanosomal agents.

 
We thank John Domagala of Parke-Davis Pharmaceutical Research for providing investigational fluoroquinolones; Rhone-Poulenc Rorer for pefloxacin; Annette Bodley and Suji Xie for technical assistance; and Rahul Bakshi, Jane Scocca, and John Domagala for thoughtfully reading the manuscript.

This work was supported by National Institutes of Health grant AI28855, the Burroughs Wellcome Fund, the Swiss National Research Foundation (C.B.), and the Janggen-Poehn Foundation (C.B.).

 
* Corresponding author. Mailing address: The Johns Hopkins University School of Medicine, 301 Hunterian Building, 725 North Wolfe St., Baltimore, MD 21205-2185. Phone: (410) 955-1888. Fax: (410) 955-2634. E-mail: tshapiro{at}jhmi.edu.

Present address: Swiss Tropical Institute, Swiss Center for International Health, CH-4002 Basel, Switzerland.

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8. Shapiro, T. A. 1993. Inhibition of topoisomerases in African trypanosomes. Acta Trop. 54:251-260.[CrossRef][Medline]
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9. Shapiro, T. A., and P. T. Englund. 1990. Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. Proc. Natl. Acad. Sci. USA 87:950-954.[Abstract/Free Full Text]
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10. Shapiro, T. A., V. A. Klein, and P. T. Englund. 1989. Drug-promoted cleavage of kinetoplast DNA minicircles. Evidence for type II topoisomerase activity in trypanosome mitochondria. J. Biol. Chem. 264:4173-4178.[Abstract/Free Full Text]
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Antimicrobial Agents and Chemotherapy, September 2003, p. 3015-3017, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.3015-3017.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nenortas, E. Articles by Shapiro, T. A. Search for Related Content PubMed PubMed Citation Articles by Nenortas, E. Articles by Shapiro, T. A.