Metabolic fate of the antipsychotic agent, mazapertine, in man--API-MS and MS/MS identification of urinary metabolites.

The in vivo metabolism of the antipsychotic agent mazapertine was studied after oral administration of mazapertine succinate (40 mg/subject) to two healthy volunteers, and urine (0-24 hours) was obtained for metabolite identification using API-ionspray LC/MS and MS/MS analysis. Unchanged mazapertine (12% of the sample) plus 10 metabolites were profiled, quantified, and tentatively identified on the basis of MS data, Glusulase-hydrolysis, and by comparison to synthetic samples. The formation of mazapertine metabolites are via seven metabolic pathways: (1) phenylhydroxylation, (2) piperidyl oxidation, (3) O-dealkylation, (4) N-dephenylation, (5) oxidative N-debenzylation, (6). depiperidylation, and (7) glucuronidation. Pathways 1, 2, 5 and 7 formed 4-OH-phenyl-mazapertine (M1, 18%) and 4-OH-piperidyl (M2, 14%)-mazapertine, carboxybenzoylpiperidine (M8, 10%) and its glucuronide (M9, 14%) as four major metabolites. Six moderate and minor metabolites (M3-M7 & M10; each < or =10%) formed via a combination of pathways 1-6. Mazapertine is extensively metabolized in humans.

Distribution and metabolism of the antipsychotic agent mazapertine succinate in rats.

The pharmacokinetics and drug disposition of 14C 1-[3-[[4-[2-(1-methylethoxy)phenyl]-1-piperazinyl]methyl]benzoy]piperidine succinate (RWJ-37796, mazapertine, Mz) have been investigated in male and female Sprague-Dawley rats. Approximately 93% of the orally administered radioactive dose (30 mg/kg) was recovered after 7 days. Fecal elimination accounted for approximately 63% of the dose while urine accounted for 30%. The rate of elimination of 14C Mz was rapid with 81% of the total fecal and 94% of the total urinary radioactivity being excreted within 24 h. There were no significant gender differences in the overall excretion pattern. The maximal plasma concentration of Mz and total radioactivity occurred at 0.5h after dosing and plasma concentrations were consistently higher in female rats. The Mz concentration declined rapidly in plasma with a terminal half-life<2 h. The total radioactive dose in plasma displayed a considerably longer terminal half-life of 9-13 h. Mz and a total of 15 metabolites were isolated and identified in these samples. Unchanged Mz accounted for <5% of the radioactive dose in excreta samples and <8% of the sample in plasma (0-24 h). Metabolites were formed by phenyl hydroxylation, piperidyl oxidation, O-dealkylation, N-dephenylation, oxidative N-debenzylation and glucuronide conjugation.

Metabolism of the new anxiolytic agent, a pyrido[1,2-]benzimidazole (PBI) analog (RWJ-53050), in rat and human hepatic S9 fractions, and in dog; identification of cytochrome p450 isoforms mediated in the human microsomal metabolism.

The in vitro and in vivo metabolism of RWJ-53050, an anxiolytic agent, was investigated after incubation with rat and human hepatic S9 fractions, and human microsomes and 7 microsomes containing individual human CYP isoforms, CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in the presence of NADPH-generating system, and a single oral dose administration to dogs (30 mg/kg). Unchanged RWJ-53050 (> or = 74% of the sample in vitro; < or = 13% in vivo) plus 16 metabolites were profiled, quantified and tentatively identified based on the API-MS and MS/MS data. The formation of RWJ-53050 metabolites are via the 5 pathways: 1. N/O-demethylation, 2. phenylhydroxylation, 3. pyrido-oxidation, 4. dehydration, and 5. conjugation. Pathway 1 formed O-desmethyl-phenyl-RWJ-53050 (M1, < 1-12% in vitro & in vivo), O-desmethyl-benzimidazole-RWJ-53050 (M2), and N-desmethyl-RWJ-53050 (M3) (M2 & M3, < or = 3% in vitro & in vivo). Pathway 2 generated hydroxy-benzimidazole-RWJ-53050 (M4), hydroxy-phenyl-RWJ-53050 (M5), and hydroxy-phenyl-M4 (M9) (< or = 3% in vitro & in vivo). Pathway 3 formed 2 trace oxidized metabolites, hydroxy-pyrido-RWJ-53050 (M6, < or = 1% in vitro) and oxo-pyrido-RWJ-53050 (M8, < 1% in vitro) and in conjunction with pathway 1 produced 2 trace dioxidized metabolites, OH-benzimidazole-M6 (M10) and OH-benzimidazole-M8 (M11) (in vitro). Pathway 4 formed a minor dehydrated metabolite of M6 (M7, 3%, in vitro). Pathway 5 produced 3 in vivo conjugates, M1-glucuronide (M14, 17%), M5-glucuronide (M15, 50%), and M5-sulfate (M16, 10%). RWJ-53050 is substantially metabolized in vitro in the rat and human, and extensively metabolized in vivo in the dog. CYP1A2, CYP3A4 and CYP2D6 are responsible for the formation of oxidized metabolites, M1, M2, M4, M5 and M9.

API-ionspray MS and MS/MS study on the structural characterization of bisbenzylisoquinoline alkaloids.

API-ionspray MS and MS/MS techniques have been utilized to elucidate the structures of 20 bisbenzylisoquinoline alkaloids, consisting of 17 diether and three monoether links of two benzyltetrahydroisoquinoline units, which were isolated and identified previously from a variety of Thalictrum sp. (Ranunculaceae family). Apparent protonated molecular ions ([M+H](+)) and very intense doubly-protonated molecular ion ([M+2H](++), 100% of relative abundance) in Q1 Scan MS spectra and prominent as well as diagnostic product ions for the structural information in MS/MS spectra were observed in nanogram quantities for all investigated alkaloids.

The in vitro metabolism of thalicarpine, an aporphine-benzyltetrahydroisoquinoline alkaloid, in the rat. API-MS/MS identification of thalicarpine and metabolites.

The in vitro metabolism of an antitumor, hypotensive, and antimicrobial aporphine-benzyltetrahydroisoquinoline alkaloid, thalicarpine was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged thalicarpine (46% of the sample) plus eight metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS/MS data. The proposed metabolic pathways for thalicarpine are proposed, and the three metabolic pathways are: (1) N-demethylation; (2) aporphine ring oxidation; and (3) benzylic oxidation/reduction. Pathway 1 formed N-desmethyl thalicarpine (M1, 6%). Pathway 2 produced three minor keto/hydroxy metabolites (M2-M4, each 2-7%). Pathway 3 formed a major (M6, 28%) and three minor (M5, M7 and M8, each 2-3%) benzylic-cleavage metabolites. Thalicarpine is substantially metabolized by this rat hepatic system.

In vitro metabolism of the new anxiolytic agent, RWJ-52763 in human hepatic S9 fraction-API-MS/MS identification of metabolites.

The in vitro metabolism of the anxiolytic agent, RWJ-52763 was studied after incubation with human hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-52763 (64% of the sample) plus six metabolites (M1-M6) were profiled, quantified, and tentatively identified on the basis of API-MS/MS data. The metabolic pathways for RWJ-52763 are proposed, and the two metabolic pathways are: (1) N/O-dealkylation, and (2) phenylhydroxylation. Pathway 1 formed a major N-dealkylated metabolite, N-desethoxy-RWJ-52763 (M1, 22% of the sample) and 2 minor N/O-dealkylated metabolites, O-desmethyl-RWJ-52763 (M2; 2%) and N,N-didesethoxymethyl-RWJ-52763 (M3; 3%). Pathway 2 produced two hydroxyphenyl metabolites, hydroxydifluorophenyl-RWJ-52763 (M4; 4%) and hydroxyphenyl-pyrido-RWJ-52763 (M5; 3%) in small amounts, and in conjunction with step 1 formed a minor N-desethoxymethyl-M4 (M6; 1%). RWJ-52763 is substantially metabolized by this human hepatic S9.

Hepatic biotransformation of the new calcium-mimetic agent, RWJ-68025, in the rat and in man--API-MS/MS identification of metabolites.

The in-vitro biotransformation of a new calcium-mimetic agent and benzenemethanamine analogue, RWJ-68025, was studied after incubation with rat and human hepatic S9 fractions in the presence of an NADPH-generating system. Unchanged RWJ-68025 (44-48% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS and MS/MS data, and ethyl derivatization for phenolic and carboxylic metabolites. Four metabolic pathways for RWJ-68025 were proposed: pathway 1, O-demethylation; pathway 2, phenyl oxidation; pathway 3, methyl oxidation; and pathway 4, N-dealkylation/acetylation. Pathway 1 formed a major metabolite, O-desmethyl-RWJ-68025 (M1; RWJ-68311; 26% in rat; 16% in human fraction). Pathway 2 produced one major (M2; 12-17% in rat and human fraction) and two minor phenolic metabolites (M4 and M5; all <1% in both species), and in conjunction with step 1, formed hydroxy-M1 (M3; 4-5% in both species). Pathways 3 and 4 formed seven minor oxidized metabolites (M6-M12). RWJ-68025 was extensively metabolized in the rat and human hepatic S9 fractions.

In-vitro metabolism of isotetrandrine, a bisbenzylisoquinoline alkaloid, in rat hepatic S9 fraction by high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry.

The objective of this study was to investigate the in-vitro metabolism of isotetrandrine, a bisbenzylisoquinoline alkaloid, using rat hepatic S9 fraction and to profile and identify its metabolites using high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry (HPLC-MS) and tandem mass spectrometry (MS/MS). Isotetrandrine was incubated at a concentration of 100 microg mL(-1) with male rat hepatic S9 fraction in the presence of an NADPH generating system (Tris buffer, pH 7.4, 37 degrees C). Samples were removed at 60 min after reaction initiation. Unchanged isotetrandrine (approximately 63% of the sample) and four metabolites were profiled, characterized and tentatively identified using solvent extraction, methyl derivatization, and HPLC-MS and MS/MS techniques. Isotetrandrine metabolites were mainly formed via two main pathways, N-demethylation and isoquinoline ring oxidation. The first pathway produced a major metabolite, N-desmethyl isotetrandrine (approximately 16% of the sample). The second pathway produced three minor oxidized metabolites, hydroxy-isotetrandrine (approximately 6% of the sample), oxo-isotetrandrine (approximately 7% of the sample), and oxohydroxy-isotetrandrine (approximately 7% of the sample). Diazomethane treatment of these metabolites did not produce any methyl derivatives and therefore the hydroxylated sites of the metabolites were tentatively assigned at the heterocyclic moieties of the isoquinoline rings. In conclusion, isotetrandrine is substantially metabolized in this in-vitro rat hepatic system.

In-vitro metabolism of the new anxiolytic agent, RWJ-50172, in rat hepatic S9 fraction and microbial transformation in fungi, Cunninghamella sp.

The in-vitro biotransformation of the anxiolytic agent, RWJ-50172 was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system, and incubating with Cunninghamella echinulata in soy-bean medium. Unchanged RWJ-50172 (80% of the sample in rat; 86% in fungi) plus 6 metabolites (M1-M6) were profiled, quantified and tentatively identified on the basis of API-MS/MS data. The metabolic pathways for RWJ-50172 are proposed, and the four metabolic pathways are: pyrido-oxidation (pathway A), phenylhydroxylation (B), dehydration (C) and reduction (D). Pathway A formed hydroxy-pyrido-RWJ-50172 (M1, 10% of the sample in both rat and fungi) as the only major metabolite, which further dehydrated to form dehydro-RWJ-50172 in trace quantities in rat. Pathway B produced hydroxyphenyl-RWJ-50172 (M2) in small amounts (4%) in rat, and in conjunction with step A formed dihydroxy-RWJ-50172 as a trace metabolite in rat. Step D produced a minor benzimidazole-reduced metabolite in fungi. RWJ-50172 is substantially metabolized by this rat hepatic S9 fraction and fungi.

Electrospray tandem mass spectrometry for structural characterization of aporphine- benzylisoquinoline alkaloids.

Electrospray mass spectrometry and tandem mass spectrometry techniques were utilized to elucidate the structures of ten aporphine-benzylisoquinoline alkaloids, consisting of monoether link between aporphine and benzyltetrahydroisoquinoline units, which were isolated and identified previously from a variety of Thalictrum sp. (Ranunculaceae family) based mainly on the UV, IR, CD, NMR, EI-MS, CI-MS, derivatization, and chemical degradation techniques. In this investigation, protonated molecules, [M+H]+ ions, for nine tertiary alkaloids, a molecular ion, [M+'] ion, for a quaternary alkaloid, and very intense doubly- protonated molecules, [M+2H]2+ ions (100% of relative abundance) in Q1 Scan MS spectra, and prominent as well as diagnostic product ions for structural information in the tandem MS/MS spectra were observed for all investigated alkaloids each in nanogram quantities. More than 10 microg quantities of each investigated alkaloid or other isoquinoline and aporphine analogs needed for the CI-MS, EI-MS and FAB-MS analysis from the previous studies.

Potential anxiolytic agents. Part 4: novel orally-active N(5)-substituted pyrido[1,2-a]benzimidazoles with high GABA-A receptor affinity.

A series of pyrido[1,2-a]benzimidazoles (PBIs) with substitution on the N(5)-nitrogen has been synthesized and found to possess high affinity for the benzodiazepine (BZD) site on the GABA-A receptor. The compounds evaluated include those bearing a heteroalkyl group and heterocyclic rings. The most promising of these compounds is ethoxymethyl analogue 24, which has an IC(50) of 0.1 nM for the BZD site on the GABA-A receptor and has been advanced to human clinical trials.

N-alkyl-4-[(8-azabicyclo[3.2.1]-oct-3-ylidene)phenylmethyl]benzamides, micro and delta opioid agonists: a micro address.

The tertiary amide delta opioid agonist 2 is a potent antinociceptive agent. Compound 2 was metabolized in vitro and in vivo to secondary amide 3, a potent and selective micro opioid agonist. The SAR of a series of N-alkyl-4-[(8-azabicyclo[3.2.1]-oct-3-ylidene)phenylmethyl]benzamides was examined.