Binding of the epoxide cryptophycin analog, LY355703 to albumin and its effect on in vitro antiproliferative activity.

Cryptophycin, isolated from the cyanobacterium Nostoc, is a cytotoxic dioxadiazacyclohexadecenetetrone which causes rapid depletion of microtubules in intact cells. In the present report, the effect of protein binding of a new synthetic cryptophycin analog, LY355703 (cryptophycin 52), is discussed. In handling the compound, it was found to bind extensively to surfaces, and a high degree of plasma protein binding was also observed (about 99% in human plasma). Similarly, while LY355703 displays potent antiproliferative activity against several human tumor cell lines in vitro (IC50s ranging from 12 to 40 pM), the addition of human or bovine serum albumin (BSA) to CCRF-CEM cells adapted to serum-free (UltraCHO) medium markedly reduced its anti-proliferative activity. For example, the IC50s for LY355703 in BSA at 0, 4 and 40 mg/ml were 2, 19 and 34 pM, respectively. In comparison, the IC50 only increased 2-fold (4210-8530 pM) for taxol over the same BSA concentration range. When log phase CCRF-CEM cells were exposed to 1 microM [3H]LY355703, there was a rapid accumulation of drug, so that LY355703 reached steady state within 10 min. The rate of LY355703 uptake in log-phase CCRF-CEM human leukemia cells was a linear function of concentration over a wide range (0.25-50 microM), although the cytotoxicity IC50 was 19 pM. Drug accumulation was not inhibited by sodium azide. Although cryptophycin was observed to bind extensively to albumin, binding did not markedly modulate cryptophycin uptake by CCRF-CEM cells. Overall, these results demonstrate that attention must be given to the binding properties of LY355703 and similar cryptophycins while handling these compounds, and that binding to albumin (and probably other cellular components as well) is a significant factor for interpretation of results both in vitro and in vivo.

Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.

Metabolism and disposition of the antifolate LY231514 in mice and dogs.

The metabolism and disposition of LY231514 was studied in mice and dogs. LY231514 is a novel pyrrotopyrimidine-based multi-target antifolate (MTA) showing broad in vivo antitumor activity in mouse models and is currently in phase II human clinical trials. Doses (iv) of the compound showed high plasma levels, resulting in AUC values of 30-33 micrograms-hr/ml for mice and dogs after 20 and 7.5 mg/kg doses, respectively. The compound was eliminated rapidly. Half-life values for mice and dogs were about 7 and 2 hr, respectively. In vitro plasma binding measured 56% in mice, 46% in dogs, and 81% in humans. Fecal elimination was the major excretion pathway in mice after single iv doses of [14C]LY231514. Urine constituted the major route of excretion in dogs. Parent LY231514 accounted for the majority of urinary radiocarbon in mice (90%) and dogs (68%). Minor metabolites were found in urine, but the amounts were too small to isolate or identify. Based on an earlier observation that LY231514 photodegraded to produce reaction products having similar retention times as these minor urinary isolates, a photo-oxidation system was developed which in fact produced these metabolites. Subsequently, these photolytically-produced materials were used as standards to identify two novel in vivo metabolites formed by oxidation of the pyrrolo-pyrimidine ring system of LY231514. The oxidative transformations are similar to those observed for tryptophan and other indoles in that the pyrrole ring is oxidized to give an amide; further oxidation cleaves this ring, one ring carbon is lost, and a ketone is formed.

Disposition and metabolism of the sulfonylurea oncolytic agent LY295501 in mouse, rat, and monkey.

The disposition and metabolism of LY295501 was studied in mice, rats, and monkeys. This novel diaryl sulfonylurea oncolytic agent is structurally related to sulofenur and shows excellent activity in a broad range of mouse antitumor models. The compound is well absorbed, giving plasma concentrations greater than 200 micrograms/ml after oral doses of 30-100 mg/kg, where it appears to be completely bound (> 99.9%) to plasma proteins. The high degree of protein binding may be a factor in its relatively long half-life, which ranges from about 8 hr in rats and 15 hr in mice to 50 hr in monkeys. While more material was excreted in feces than in urine from mice and rats given single oral doses of [14C]LY295501, urine was the major route of elimination in monkeys. Three major metabolites-all formed via oxidation of the saturated part of the benzodihydrofuran moiety-were characterized in the urine of mice, rats, and monkeys. It is interesting that two of these metabolites are derived from opening of this saturated ring, an unusual metabolic process which represents a significant part of the metabolism of LY295501. As with sulofenur, metabolites of 3,4-dichloroaniline formed after metabolic cleavage of the sulfonylurea linkage were also found in urine. Unlike sulofenur, these do not seem to have major toxicological significance, but their formation does explain the minor methemoglobinemia observed in toxicology studies of LY295501. Even though only trace amounts of LY295501 were found in urine, LY295501 is the predominant drug-related material in plasma, along with small amounts of other, relatively nonpolar, metabolites.

Sulfonimidamide analogs of oncolytic sulfonylureas.

A series of sulfonimidamide analogs of the oncolytic diarylsulfonylureas was synthesized and evaluated for (1) in vitro cytotoxicity against CEM cells, (2) in vivo antitumor activity against subaxillary implanted 6C3HED lymphosarcoma, and (3) metabolic breakdown to the o-sulfate of p-chloroaniline. The separated enantiomers of one sulfonimidamide analog displayed very different activities in the in vivo screening model. In general, several analogs demonstrated excellent growth inhibitory activity in the 6C3HED model when dosed orally or intraperitoneally. A correlative structure-activity relationship to the oncolytic sulfonylureas was not apparent.

Pharmacological characterization of LY335979: a potent cyclopropyldibenzosuberane modulator of P-glycoprotein.

The above data indicate that LY335979 displays the following characteristics of an 'ideal modulator' of Pgp-mediated multidrug resistance: high affinity binding to Pgp, high potency for in vitro reversal of drug resistance, high therapeutic index (activity was demonstrated at doses ranging from 1-30 mg/kg) observed in in vivo antitumor efficacy experiments, and a lack of pharmacokinetic interactions that alter the plasma concentration of coadministered oncolytic agents. These desirable features strongly suggest that LY335979 is an exciting new clinical agent to test the hypothesis that inhibition of P-glycoprotein activity will result in reversal of multidrug resistance in human tumors.

Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979.

Overexpression of P-glycoprotein (Pgp) by tumors results in multidrug resistance (MDR) to structurally unrelated oncolytics. MDR cells may be sensitized to these oncolytics when treated with a Pgp modulator. The present study evaluates LY335979 as a modulator both in vitro and in vivo. LY335979 (0.1 microM) fully restored sensitivity to vinblastine, doxorubicin (Dox), etoposide, and Taxol in CEM/VLB100 cells. LY335979 modulated Dox cytotoxicity even when LY335979 (0.5 microM) was removed 24 h prior to the cytotoxicity assay. LY335979 blocked [3H]azidopine photoaffinity labeling of the M(r) approximately 170,000 Pgp in CEM/VLB100 plasma membranes and competitively inhibited equilibrium binding of [3H]vinblastine to Pgp (Ki of approximately 0.06 microM). Treatment of mice bearing P388/ADR murine leukemia cells with LY335979 in combination with Dox or etoposide gave a significant increase in life span with no apparent alteration of pharmacokinetics. LY335979 also enhanced the antitumor activity of Taxol in a MDR human non-small cell lung carcinoma nude mouse xenograft model. Thus, LY335979 is an extremely potent, efficacious modulator that apparently lacks pharmacokinetic interactions with coadministered anticancer drugs and is, therefore, an exciting new agent for clinical evaluation for reversal of Pgp-associated MDR.

Novel acid labile COL1 trityl-linked difluoronucleoside immunoconjugates: synthesis, characterization, and biological activity.

LY207702 (1) is a difluorinated purine nucleoside that exhibits impressive antitumor activity in preclinical models. This agent, however, also possesses cardiotoxicity which limits the potential clinical utility of this novel drug candidate. We therefore developed linker chemistry whereby regioselective N6-tritylation of LY207702 (1) allowed this drug to be coupled to epsilon-lysine amino groups of mAb's reactive with human tumor-associated antigens. The resulting immunoconjugates 3 possessed conjugation ratios ranging from 5 to 7 mol of LY207702/mol of mAb, minimal aggregate content (5-10%), and good immunoreactivity. The electronic nature of substituents on the aromatic rings of the trityl group dictated the degree of acid lability of the trityl linker. Increased electronic stabilization of the transient trityl carbocation led to increase in the release rate of free drug, i.e., m-DMT 10a = p-DMT 10b > p-MMT 10d > p-T 10f. Consequently, the more acid labile DMT conjugates 3a and 3b proved to be the most potent cytotoxic agents, and the most stable p-T conjugate 3f exhibited the least antitumor activity when evaluated in vitro and in vivo. p-MeT-linked conjugate 3e, the most stable construct that retained excellent in vivo antitumor activity, was selected for more extensive evaluation. No detectable free drug or metabolite was observed in mouse plasma at a single intravenous dose of p-MeT conjugate 3e, which was consistent with its predicted stability under physiological conditions. This construct did, however, exhibit significant antigen-mediated antitumor activity in vivo. No cardiotoxicity was detected in mice dosed with conjugate 3e (6 mg/kg free drug content per day for 21 days) equivalent to approximately 8 times the total dose required for complete regression of well-established (approximately 1 g) HC1 human colon tumor xenografts in nude mice. Cardiotoxicity was induced in 20% of free drug 1 treated group at the equivalent dose. Cardiomyopathy was, however, observed when the dose of conjugate 3e was increased to 8 mg/kg per day for 21 days. These data suggest that antitumor activity of LY207702 (1) was maintained and its cardiotoxic potential reduced when this agent was administered to human tumor xenograft bearing nude mice as COL1-N6-p-MeT-207702 conjugate 3e.

Characterization of an O-glucuronide metabolite of the CCK-B antagonist LY288513 and its plasma levels in mouse, rat, and dog.

LY288513 [(4S,5R)-trans-N-(4-bromophenyl)-3-oxo-4,5-diphenyl-1- pyrazolidinecarboxamide] has been developed as a cholecystokinin-B antagonist for anxiolytic indications. In the drug disposition studies reported herein, we found LY288513 was metabolized rapidly in mice and rats to form a glucuronide adduct. This metabolite was isolated from rodent urine and further characterized to determine whether a ring-nitrogen or the oxygen atom in the pyrazolidinone nucleus of LY288513 was bonded to the glucuronide. Using 13C NMR plus two-dimensional rotating frame Overhauser enhancement spectroscopy analysis and IR analysis of model compounds, we determined the metabolite was an O-glucuronide. The major elimination pathway for [14C]LY288513 was fecal, with > 75% of total radiocarbon excreted within 24 hr. Plasma levels for parent and metabolite in the three species varied substantially. The plasma half-life was significantly longer in dogs (approximately 12 hr) than mice (approximately 24 hr) or rats (2.6-3.6 hr). Consistent with this, in dogs relatively little O-glucuronide was observed in plasma, but in rats and mice the AUCs for glucuronide were greater than the parent AUC. A dose-ranging study in rats revealed oral absorption of LY288513 is nonsaturated, even after 800 mg/kg oral doses. A plot of AUC vs. dose revealed a consistent overproportional increase in AUC for both parent and glucuronide metabolite with increasing dose.

Pharmacokinetics of the anticancer agent sulofenur in mice, rats, monkeys, and dogs.

The absorption and pharmacokinetics of sulofenur [N-(indan-5-sulfonyl)-N'-(4-chlorophenyl)urea, LY186641] and its major metabolites were examined in mice, rats, monkeys, and dogs. The compound is a diarylsulfonylurea currently being evaluated as an oncolytic agent in phase I and II trials. In all species, sulofenur was well absorbed after an oral dose, but over a prolonged period, and sulofenur exhibited a fairly long half-life of elimination from plasma. These values ranged from 6 h in rats up to 30, 110, and 200 h in mice, monkeys, and dogs, respectively, at doses (240-1000 mg/m2) within the range of those used in clinical trials. Experiments describing the high degree of binding of sulofenur to plasma proteins (consistently > 99%) help to explain these relatively long half-lives. There is, however, a large difference between these plasma half-lives in the species studied. Sulofenur was previously found to be extensively metabolized to products that are excreted primarily into the urine. In this study, its major metabolites, which are found mainly in the urine, were also minor components of the drug-related material (< 10% of the sulofenur concentrations) in the plasma of rats treated with sulofenur. The absorption, binding characteristics, and elimination of these major metabolites after their administration to rats were also compared with sulofenur.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation of the antiviral agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and characteristics of a mesoionic ribose metabolite.

The biotransformation of the antiinfluenza agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896, I) was studied. In addition to a urea metabolite (II) formed by transformation of the cyanamide functionality, another highly polar metabolite was found in mouse urine and in BSC-1, MDCK, and other cell culture incubations of [14C]LY217896. Using 13C-labeled LY217896 together with NMR and MS techniques, this highly polar metabolite was identified as a ribose derivative (III), which apparently exists in a mesoionic form (i.e. positive and negative charges within the same ring system). It was also found that this ribose is formed from LY217896 and ribose-1-phosphate in a reaction catalyzed by the enzyme purine nucleoside phosphorylase, but that the reverse reaction (cleavage of the ribose) is not observed under the conditions used. When tested in vitro using the same assay as that used to measure the antiviral activity of LY217896, this ribose and the urea metabolite exhibit essentially no activity. The presence of a ribose has been implicated in the activity of antiviral compounds such as ribavirin and anticancer agents like 2-aminothiadiazole and tiazofurin, which are structurally similar to LY217896. These activities have been postulated to involve either mono- or triphosphorylated forms, or NAD-type analogs. Possible implications of the formation of this mesoionic ribose metabolite for the mechanism of antiviral activity of LY217896 are discussed.

Comparison of metabolism and toxicity to the structure of the anticancer agent sulofenur and related sulfonylureas.

The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.

Metabolism and disposition of the anticancer agent sulofenur in mouse, rat, monkey, and human.

The elimination and metabolism of sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641] was examined in mice, rats, monkeys, and humans. The compound, which is efficacious in a wide number of solid-tumor in vivo models, is currently being developed as an anticancer agent. Its diarylsulfonylurea structure is unique among such agents, and the basis of its activity is unknown but apparently novel. A major goal of these studies was to determine whether p-chloroaniline is formed in significant quantities during the course of sulofenur metabolism. p-Chloroaniline, capable of being formed by hydrolysis of this diarylsulfonylurea, is known to induce methemoglobinemia and/or hemolytic anemia. In animal studies using rats and monkeys, as well as in clinical trials of sulofenur, elevated levels of methemoglobin have been noted. The metabolism was thus compared to the known metabolism of p-chloroaniline. Sulofenur (I) is well absorbed in both monkey and human; practically all of the excreted radiolabel from an oral dose is in the urine. Metabolism is extensive; the major excretion products are the 1-hydroxyindanyl (II) and 1-ketoindanyl (III) derivatives in all species, along with significant amounts of the 3-hydroxyindanyl (IV) and 3-ketoindanyl (V) metabolites in the mouse and rat. Dihydroxyindanyl secondary metabolites also are present, but no sulofenur is observed in the urine samples. Known metabolites account for over 95% of the radiocarbon present in urine samples from a patient given [14C-p-chlorophenyl]sulofenur.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism and disposition of p-chloroaniline in rat, mouse, and monkey.

The metabolism and disposition of [14C]p-chloroaniline was studied in the male Fisher 344 rat, female C3H mouse, and male rhesus monkey. Greater than 90% of the radiocarbon is eliminated through the urine in all species. A major route of metabolism is orthohydroxylation, whereby 2-amino-5-chlorophenyl sulfate is the major excretion product in all species. Excretion of p-chloro-oxanilic acid and p-chloroglycolanilide, oxidation products of p-chloroacetanilide, is significant in the rat. The appearance of p-chloroacetanilide along with p-chloroaniline in plasma after an oral dose of [14C]p-chloroaniline also was studied in rhesus monkey. In addition to p-chloroacetanilide, 2-amino-5-chlorophenyl sulfate is found to be a major circulating metabolite.

Thiol-mediated incorporation of radiolabel from 1-[14C]-methyl-4-phenyl-5-nitrosoimidazole into DNA. A model for the biological activity of 5-nitroimidazoles.

1-Methyl-4-phenyl-5-nitrosoimidazole (5NO), which has properties consistent with the biologically active form of a 5-nitroimidazole, was radiolabeled (1-[14C]-methyl) and shown to bind to DNA, but at a rate too slow to account for its bactericidal effect. In the presence of physiological intracellular concentrations of such thiols as glutathione, however, binding was enhanced by 2-3 orders of magnitude, which is quantitatively sufficient to account for the bactericidal effect of 5NO. That 5NO binding was greater for poly[d(G-C).d(G-C)] than for poly[d(A-T).d(A-T)] suggests that the reactive species binds to nucleophilic bases on DNA, a suggestion which is also supported by our finding of a thiol-dependent reaction to form an adduct between 5NO and aniline.

Mammalian cell toxicity and bacterial mutagenicity of nitrosoimidazoles.

It is currently believed that the biological activity of such therapeutic 5-nitroimidazoles as metronidazole is mediated by a short-lived, highly toxic species that arises from nitro group reduction. We found that the 5-nitroimidazole, 1-methyl-4-phenyl-5-nitroimidazole (5-NO2), is at least 1000-fold less cytotoxic for CHO cells and mutagenic for Ames tester strain TA100 than its homologous nitroso compound, 1-methyl-4-phenyl-5-nitrosoimidazole (5-NO). Such evidence, along with previous work showing a similar relative bactericidal potency of these compounds, is consistent with the labile nitrosoimidazole being a biologically active species of the nitroimidazole, and indicates that mammalian cells are very susceptible to such an active form. The high potency of both 5-NO and 1-methyl-4-nitroso-5-phenylimidazole (4-NO), in contrast to the lack of potency of 1-methyl-4-nitro-5-phenylimidazole (4-NO2) relative to 5-NO2, is additional evidence to support the suggestion that the activity of a nitroimidazole is determined mainly by the ease with which it is reduced.

Nitrosoimidazoles: highly bactericidal analogues of 5-nitroimidazole drugs.

It is believed that metronidazole and related 5-nitroimidazoles are activated by reduction of the nitro group and that the active species has a nitrogen functionality of intermediate oxidation state. However, the preparation and isolation of the active forms of the 5-nitroimidazoles used therapeutically have proven elusive. To pursue this problem we have prepared both 1-methyl-4-phenyl-5-nitrosoimidazole (3) and 1-methyl-4-nitroso-5-phenylimidazole (5) from 4(5)-nitroso-5(4)-phenylimidazole (1). We have also prepared the homologous nitroimidazoles. Escherichia coli mutants with defects in DNA repair were found to be sensitive to both 1-methyl-4-phenyl-5-nitroimidazole (4) and metronidazole, but fairly resistant to 1-methyl-4-nitro-5-phenylimidazole (6), a finding in accord with the relative biological activity of 4- and 5-nitroimidazoles examined previously. In contrast, all three nitroso compounds are considerably more bactericidal than their analogous nitro compounds under both aerobic and anaerobic conditions, a finding that provides direct evidence that reduction of the nitro group is responsible for activation of the nitroimidazoles. Further evidence is also consistent with the possibility that the nitrosoimidazoles are themselves biologically active species derived from nitroimidazoles, although a more conservative interpretation is simply that they are more facilely converted to such active species.

Chemical and biological properties of acetyl derivatives of the hydroxylamino reduction products of metronidazole and dimetridazole.

Metronidazole and related 5-nitroimidazoles undergo reduction of their nitro group apparently to produce such reactive species as 5-hydroxylaminoimidazoles. To define the role of these species we have sought ways to prepare them by the catalytic reduction of metronidazole, dimetridazole and flunidazole. Although their respective 5-hydroxylaminoimidazoles were too unstable to be isolated directly, their O,N-diacetyl derivatives were isolable. Of these, the diacetyl derivative of the hydroxylamine derived from dimetridazole, O,N-diacetyl-1,2-dimethyl-5-hydroxylaminoimidazole (DiacDMH), was used for further study. DiacDMH was converted to its monoacetyl derivative, N-acetyl-1,2-dimethyl-5-hydroxylaminoimidazole (AcDMH), by enzymatic deacylation. Both DiacDMH and AcDMH were examined for bactericidal activity against such strains as Bacteroides fragilis, Clostridium perfringens, and Escherichia coli strain SR58, which are known to be sensitive to dimetridazole, as well as a variety of other bacteria. No bactericidal activity was detected, even in the presence of deacetylating enzymes. As the 5-hydroxylaminoimidazole itself could not be shown to form in these bacterial incubations, it remains uncertain whether or not the hydroxylamino functionality of a 5-nitroimidazole has bactericidal activity.

Formation of an amino reduction product of metronidazole in bacterial cultures: lack of bactericidal activity.

To investigate whether the amino reduction product of metronidazole has antibacterial activity, 5-amino-1-beta-hydroxyethyl-2-methylimidazole (AMN) was synthesized and tested against Bacteroides fragilis and Escherichia coli strain SR58, both of which are known to be sensitive to metronidazole. Neither of these strains was found to be sensitive either to AMN or to the equivalent amine derived from dimetridazole, 5-amino-1,2-dimethylimidazole. Both of these amines are relatively stable in the presence of bacteria, making it possible to examine the bacterial reduction of radiolabeled metronidazole in the presence of AMN. This experiment indicated that at least 17% of the metronidazole that disappeared under the reducing conditions of the bacterial medium was converted to AMN. We conclude, therefore, that AMN forms during the activation of metronidazole by bacterial reduction but is not a bactericidal form of metronidazole.