Efficacy of New 5-Nitroimidazoles against Metronidazole-Susceptible and -Resistant Giardia,Trichomonas, and Entamoeba spp

ABSTRACT The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via SRN1 and subsequent reactions, were assayed against the protozoan parasites Giardia duodenalis, Trichomonas vaginalis, andEntamoeba histolytica. Two metronidazole-sensitive lines and two metronidazole-resistant lines of Giardia and one line each of metronidazole-sensitive and -resistantTrichomonas were tested. All except one of the compounds were as effective or more effective than metronidazole againstGiardia and Trichomonas, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against Entamoeba. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of Giardia andTrichomonas. However, some drugs were lethal to metronidazole-resistant Giardia and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.

The introduction of nitroheterocyclic drugs in the late 1950s and the 1960s heralded a new era in the treatment of infections caused by gram-negative and -positive bacteria and a range of pathogenic protozoan parasites. The antibiotic azomycin (a 2nitroimidazole), isolated in Japan from a streptomycete, was the first active nitroimidazole to be discovered (15) and acted as the main impetus for the systematic search for drugs with activity against anaerobic protozoa. This led to the synthesis of the 5-nitroimidazole metronidazole (1-␤-hydroxyethyl-2methyl-5-nitroimidazole) and the demonstration of its activity against Trichomonas vaginalis by Cosar and Julou (6). Subsequently, metronidazole was shown to cure giardiasis (21), amoebiasis (18), and Balantidium infections (7). Metronidazole is the drug now most widely used in the treatment of anaerobic protozoan parasitic infections caused by T. vaginalis, Giardia duodenalis, and Entamoeba histolytica (22). It is remarkably safe compared with the toxic amoebicide emetine (12) and is the recommended alternative for the treatment of amoebiasis. Although failures of the treatment of liver abscess and dysentery with metronidazole have been reported (12), no clinical resistance has been reported in E. histolytica. Metronidazole and the related nitroimidazole tinidazole (which is not available in some countries) are also the only drugs effective for the treatment of trichomoniasis and are the drugs of choice for the treatment of giardiasis (26). In the latter cases clinical resistance to these drugs has been documented (9,26,31).
In the event of overt clinical resistance to metronidazole in the anaerobic protozoa, there is no alternative treatment for either trichomoniasis or invasive amoebiasis, keeping in mind the documented cross-resistance between currently used nitroimidazole drugs (22) and worldwide availability. On the positive side, a great deal of flexibility is offered by the side chains attached to the imidazole ring structure bearing the all-impor-tant nitro group. Since the discovery in 1966 (13, 13a, 19) that alkylation of ambident anions by p-nitrobenzyl chloride is an electron-transfer chain process termed S RN 1 (11), the extensions at the sp3 carbons attached to heterocyclic systems have been studied extensively (3). A most attractive feature of S RN 1 reactions is that they proceed under very mild conditions and produce excellent yields of pure products. As a consequence, they are especially valuable for the synthesis of a large number of complex and highly branched compounds. Recently, 5-nitroimidazole derivatives including the lactam-substituted nitroimidazole have been shown to be significantly more effective antiprotozoal agents than metronidazole (8,30). In the study described here we examined the activities of other new 5-nitroimidazole compounds against metronidazole-sensitive and -resistant G. duodenalis (synonymous with Giardia lamblia and Giardia intestinalis) and T. vaginalis and against E. histolytica, the three most medically important anaerobic protozoa.

Drugs.
All compounds used in this study were identified by spectral data, purified by chromatography on silica gel columns, and recrystallized from appropriate solvents. Their purity was checked with appropriate controls by thinlayer chromatography and elemental analysis (C, H, N). The purity was always over 99.6%. The synthesis, structural identification, and purity of compounds 1, 2, 3, 4, 5, 6, 7, 8, and 13 have been reported previously (8,27,28). Data for products 9, 10, 11, and 12 were presented at 33rd International Meeting on Medicinal Chemistry (28a).
Compounds 9 and 10 were synthesized by the reaction of the bis-chloride (compound 1) with p-toluenesulfinic salt in dimethyl sulfoxide. The E and Z isomers (compounds 11 and 12, respectively) were obtained by the LD-S RN 1 mechanism after subjecting compound 9 to 2-nitropropane anion (Fig. 1).
Metronidazole ( Fig. 1) was from Sigma. All drugs were dissolved in chromatography-grade dimethylformamide (DMF) (Sigma) at 100 mM and were diluted into medium as required.
Cultures. All strains used in this work are described in Table 1. Growth Conditions. Giardia and Trichomonas parasites were grown in TYI-S-33 medium supplemented with bile (1 mg/ml; Sigma) (17). The same medium without bile was used for Entamoeba. Parasites were grown at 37°C (35.5°C for Entamoeba) in filled 5-ml plastic tubes and were maintained upright. Giardia and Trichomonas trophozoites were subcultured every 2 to 3 days, and Entamoeba trophozoites were subcultured every 3 to 4 days.
Metronidazole-resistant lines of protozoa were maintained in culture in the presence of metronidazole (Table 1). Drug assays. Trophozoites for drug assays were harvested in the mid-logarithmic phase of growth and were distributed identically among 5-ml tubes; 2 ϫ 10 5 to 9 ϫ 10 5 Giardia trophozoites, 2 ϫ 10 6 to 7 ϫ 10 6 trichomonads, and 3 ϫ 10 4 Entamoeba parasites were seeded per ml. On the following day different concentrations (0.5, 1, 5, 10, 50, 100, and 500 M) of drugs in a maximum volume of 25 l of DMF were added to the tubes, and the parasites were allowed to grow for 3 days in the presence of drug. Twenty-five microliters of DMF alone did not affect the parasites. For assays of antigiardial activity, all the medium including unattached trophozoites was removed after 3 days and was replaced with fresh medium without drug. Medium and unattached Entamoeba parasites were also removed after 3 days. For drug assays with trichomonads, the tubes were centrifuged, the medium containing drug was removed but the trophozoites and debris were left in place, and fresh medium was added. After a further 4 days the assay was terminated.
Metronidazole-resistant Giardia and Trichomonas parasites were allowed to grow without metronidazole for 2 days prior to the commencement of the drug assays.
Parasite viability was evaluated on each day and was finally scored on the basis of a viable or nonviable culture. The viability score did not depend on the numbers of parasites present but depended on the presence of live (motile) parasites. It was important to score the cultures on a daily basis because the organisms in some cultures, particularly those containing the Trichomonas strain that we used, grew very rapidly. In some assays, untreated controls and cultures with low levels of drug reached maximum growth prior to the change of medium or soon afterward and were in decline by the end of the assay. These cultures were scored as viable. The data presented in Table 2 do not indicate whether confluent cultures versus only a few live parasites were obtained at the end of the assay.
The antiprotozoal activities of all of the drugs were compared with that of metronidazole within the same experiment. The minimum lethal concentration (MLC) of metronidazole was estimated as the lowest concentration of drug with which no viable organisms were observed at the termination of the assay.

RESULTS
Drug assays. In the assays described here the MLCs of metronidazole for susceptible strains were 50 to 100 M, which are consistent with those in previous reports (10, 16) but higher than the previously reported doses of metronidazole inhibitory for Giardia which have relied on a variety of criteria (26). The MLC of about 10 M metronidazole for Entamoeba and the MLC of 100 M (16 g/ml) metronidazole for Trichomonas grown under anaerobic conditions are similar to previously reported MLCs (see reference 20 and references therein; 14). The high levels of metronidazole resistance that developed in Giardia lines WB1B-M3 and BRIS/83/HEPU/106-2ID 10 and the Trichomonas line BRIS/92/STDL/F1623-M1 are described in Table 1.
The activities of the 12 new 5-nitroimidazole compounds tested and 1 previously tested compound were compared with that of metronidazole against the test organisms ( Table 2). The aim of this study was to identify those compounds which were consistently and significantly more effective than metronidazole against Giardia, Trichomonas, and Entamoeba. All compounds except compound 3 were as effective or more effective than metronidazole, and none was as effective as compound 13 against all three parasite species tested. Compounds 4, 6, 7, 10, 11, 12, and 13 demonstrated increased activity over that of metronidazole against Giardia and Trichomonas. Compounds 1, 2, and 13 maintained similar levels of activity against metronidazole-sensitive and -resistant Giardia parasites. When we assayed parasites of Giardia sp. strain BRIS/83/HEPU/106 against several of the most effective drugs (compounds 4, 6, 10, 12, and 13) on three occasions, the MLCs ranged between Ͻ1 and 5 M on all occasions.
Metronidazole-resistant Trichomonas was generally resistant to the same concentrations of all of the nitroimidazole compounds tested.

DISCUSSION
The observations that important antibacterial and antiprotozoal activities of nitroimidazoles are associated with reductive metabolism have led to considerable interest in nitroimidazole reduction chemistry and the synthesis of new, highly effective drugs. Recently, we demonstrated that 1-methyl-2chloromethyl-5-nitroimidazole reacted by the S RN 1 mechanism with various aliphatic, cyclic, or heterocyclic nitronate anions led to a new class of 5-nitroimidazoles bearing a trisubstituted double bond at the 2 position (8,29). The subsequent structure-activity relationships revealed that the most antimicrobial and antiparasitic compounds showed a greater resonance conjugation in the molecular structure. In order to increase the conjugate system and in connection with mechanistic studies, we have synthesized new mono-and bis-alkylating agents and explored their reactivities with nitronate anions. The structures and antiprotozoal activities of these compounds are described here.
A great deal of variation in the antiprotozoal efficacies of the 13 compounds tested was revealed. Only one compound was less effective than metronidazole against all three species of protozoa examined. All other compounds were as effective a The metronidazole (MZ) concentration was maintained in the medium for metronidazole-resistant lines. Metronidazole at 1 M is equal to 0.17 g/ml.
b The MLC is the lowest concentration of metronidazole at which no viable organisms were observed following exposure to drug for 3 days and growth for a further 4 days in fresh drug-free medium.
c Metronidazole-resistant lines. or more effective than metronidazole against some or all organisms tested. The lactam-substituted compound (compound 13) was significantly more effective than metronidazole against Giardia (50 to 100 times more effective) and Trichomonas (50 times more effective), but against the Entamoeba strain that we used the compound was not as effective as it was previously (30). Compound 3 was uniformly less effective than metronidazole on every occasion in which it was tested, showing that the substitution on the imidazole ring by an acyl group resulted in a loss of antiprotozoal activity. The influence of the substitution by one or two alkylnitromethyl groups (compounds 4 and 6) was also noted, and the highly conjugated imidazoles (compounds 7 and 8) were generally less active than the corresponding bis-C-alkylation product (compound 4) or the monounsaturated product (compound 6). However, only compound 13 remained very effective against the metronidazole-resistant Giardia lines BRIS/83/HEPU/106-2ID 10 and WB1B-M3.
The new compounds tested have side chains that are more hydrophobic than those of metronidazole, and in some cases this may reflect the increased activities of the new compounds because the site of metronidazole activation in the anaerobic protozoa is the membrane-localized electron transport pathway (4,22,24). Whether the increased hydrophobicity would be detrimental or otherwise to the activities of these drugs in vivo remains to be tested.
With the clinical nitroimidazole resistance in Trichomonas being well documented, there is clearly a great need for the development of new antiparasitic drugs. However, cross-resistance among the nitroimidazole drugs in Giardia and Trichomonas has been documented previously (2,26) and was again demonstrated in this study. Except for compound 13 (which was effective against metronidazole-resistant Giardia), no drug is likely to be uniformly more effective than metronidazole in vivo against highly metronidazole-resistant parasites.
The mechanism of action of metronidazole in anaerobes requires reduction of the critical nitro group to toxic radicals by ferredoxin, which itself is reduced by the membrane-localized enzyme pyruvate:ferredoxin oxidoreductase (PFOR) (22). In highly metronidazole-resistant Trichomonas, which we have used in these studies, there is no PFOR or ferredoxin (4), and metronidazole and the other 5-nitroimidazoles tested here were not activated in these organisms. In metronidazole-resistant Giardia, although PFOR activity is decreased (24), it is still detectable and is thus able to activate 5-nitroimidazole drugs, as we have seen in this study. In both these parasites we have reported alternative oxoacid oxidoreductases which do not apparently reduce the characterized ferredoxins and which are at least as active or more active in metronidazole-resistant lines than in their drug-sensitive parent strain (4,24). These alternative pathways in the anaerobic protozoa are poorly understood and may well be the targets of highly active 5-nitroimidazoles or related compounds. For this reason and with the encouraging results obtained with compounds such as the lactam-substituted nitroimidazole (compound 13), continued assessment of new 5-nitroimidazole drugs is extremely worthwhile.