Hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

Rat liver microsomal mixed function oxidase catalyzes the hydroxylation of the cyclohexyl moiety of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to give at least five metabolites. When exposed to alkaline pH at 100° CCNU and its metabolites quantitatively release their cyclohexyl moiety as cyclohexylamine and aminocyclohexanol respectively. The N-(2,4-dinitrophenyl) derivatives of cyclohexylamine and aminocyclohexanols were separated by high pressure liquid chromatography. The metabolites in vitro and in vivo have been identified as trans-2-hydroxy CCNU, cis-3-hydroxy CCNU, trans-3-hydroxy CCNU, cis-4-hydroxy CCNU and trans-4-hydroxy CCNU. Ring hydroxylation axial to the 1-(2-chloroethyl)-1-nitrosourea group (cis-2-, trans-3-, cis-4-) is favored over equatorial attack (trans-2-, cis-3-, trans-4-). Pretreatment of rats with phenobarbital leads to an increased rate of hydroxylation and a change in the relative amounts of the hydroxylated products. The significance of hydroxylation in relation to the antitumor activity of CCNU is discussed.     

N-glucuronidation of the platelet-derived growth factor receptor tyrosine kinase inhibitor 6,7-(dimethoxy-2,4-dihydroindeno[1,2-C]pyrazol-3-yl)-(3-fluoro-phenyl)-amine by human UDP-glucuronosyltransferases.

The potential cancer therapeutic agent, 6,7-(dimethoxy-2, 4-dihydroindeno[1,2-c]pyrazol-3-yl)-(3-fluoro-phenyl)-amine (JNJ-10198409), formed three N-glucuronides that were positively identified by liquid chromatography-tandem mass spectrometry and NMR as N-amine-glucuronide (Glu-A), 1-N-pyrazole-glucuronide (Glu-B), and 2-N-pyrazole-glucuronide (Glu-C). All three N-glucuronides were detected in rat liver microsomes, whereas only Glu-A and -B were found in monkey and human liver microsomes. In contrast to common glucuronides, Glu-B was completely resistant to beta-glucuronidase. Kinetic analyses revealed that glucuronidation of JNJ-10198409 in human liver microsomes exhibited atypical kinetics that may be described by a two-site binding model. For the high affinity binding, K(m) values were 1.2 and 5.0 microM, and V(max) values were 2002 and 2,403 nmol min(-1) mg(-1) for Glu-A and Glu-B, respectively. Kinetic constants of low affinity binding were not determined due to low solubility of the drug. Among the human UDP-glucuronosyltransferases (UGTs) tested, UGT1A9, 1A8, 1A7, and 1A4 were the most active isozymes to produce Glu-A; for the formation of Glu-B, UGT1A9 was the most active enzyme, followed by UGT1A3, 1A7, and 1A4. Glucuronidation of JNJ-10198409 by those UGT1A enzymes followed classic Michaelis-Menten kinetics. In contrast, no glucuronides were formed by all UGT2B isozymes tested, including UGT2B4, 2B7, 2B15, and 2B17. Collectively, these results suggested that glucuronidation of JNJ-10198409 in human liver microsomes is catalyzed by multiple UGT1A enzymes. Since UGT1A enzymes are widely expressed in various tissues, it is anticipated that both hepatic and extrahepatic glucuronidation will likely contribute to the elimination of the drug in humans. Additionally, conjugation at the nitrogens of the pyrazole ring represents a new structural moiety for UGT1A-mediated reactions.     

Metabolism of (R)-(+)-pulegone in F344 rats.

(R)-(+)-Pulegone, a monoterpene ketone, is a major component of pennyroyal oil. Ingestion of high doses of pennyroyal oil has caused severe toxicity and occasionally death. Studies have shown that metabolites of pulegone were responsible for the toxicity. Previous metabolism studies have used high, near lethal doses and isolation and analysis techniques that may cause degradation of some metabolites. To clarify these issues and further explore the metabolic pathways, a study of (14)C-labeled pulegone in F344 rats at doses from 0.8 to 80 mg/kg has been conducted. High-pressure liquid chromatography (HPLC) analysis of the collected urine showed the metabolism of pulegone to be extensive and complex. Fourteen metabolites were isolated by HPLC and characterized by NMR, UV, and mass spectroscopy. The results demonstrated that pulegone was metabolized by three major pathways: 1) hydroxylation to give monohydroxylated pulegones, followed by glucuronidation or further metabolism; 2) reduction of the carbon-carbon double bond to give diastereomeric menthone/isomenthone, followed by hydroxylation and glucuronidation; and 3) Michael addition of glutathione to pulegone, followed by further metabolism to give diastereomeric 8-(N-acetylcystein-S-yl)menthone/isomenthone. This 1,4-addition not only took place in vivo but also in vitro under catalysis of glutathione S-transferase or mild base. Several hydroxylated products of the two mercapturic acids were also observed. Contrary to the previous study, all but one of the major metabolites characterized in the present study are phase II metabolites, and most of the metabolites in free forms are structurally different from those previously identified phase I metabolites.     

Identification of the main urinary metabolites of omeprazole after an oral dose to rats and dogs

The structures of seven urinary metabolites of omeprazole following high oral doses to rats and dogs were determined unambiguously by combining different analytical and spectroscopic techniques including derivatization and stable isotopes. Omeprazole was metabolized by aromatic hydroxylation at position 6 in the benzimidazole ring followed by glucuronidation. There was also oxidative O-dealkylation of both methoxy groups, and aliphatic hydroxylation of a pyridine methyl group followed by oxidation to the corresponding carboxylic acid. Due to the experimental design, implying no pH control of collected samples, all metabolites were isolated as sulfides. They were formed in both species with quantitative variations in the metabolic pattern. As far as identified metabolites are concerned, aromatic hydroxylation and subsequent glucuronide formation were the major biotransformation routes in the dog. In the rat, aliphatic hydroxylation and the formation of the carboxylic acid represented the major metabolic pathways. The identified metabolites corresponded approximately to 50% (rat) and 70% (dog) of the amount excreted in the 0-24-hr urine (about 12% of the given dose in both species).     

2-benzazepines. 6. Synthesis and pharmacological properties of the metabolites of 9-chloro-7-(2-chlorophenyl)-5H-pyrimido[5,4-d] [2]benzazepine

The 2-benzazepine 9-chloro-7-(2-chlorophenyl)-5H-pyrimido[5,4-d] [2]benzazepine (1) has been selected for development as an anxiolytic agent. In support of this program, we have confirmed by chemical synthesis the structures of three in vitro (rat liver homogenate) metabolites of 1 and confirmed the structure of the major in vivo (dog and man) metabolite of 1, compound 2. Two of the metabolites, arising from hydroxylation of the pyrimidobenzazepine ring at the 5-position (2) and N-oxide formation at the 3-position of the pyrimidobenzazepine ring (3), were found to be as active as 1 in a series of pharmacological tests. The third metabolite, formed by hydroxylation of the 7-phenyl group in the 4-position (4), was found to be inactive in the same pharmacological screens.     

The metabolism of the dual endothelin receptor antagonist macitentan in rat and dog

Abstract

1.?The metabolism of the endothelin receptor antagonist macitentan has been characterized in bile duct-cannulated rats and dogs.

2.?In both species, macitentan was metabolized along five primary pathways, i.e. conjugation with glucose (M9), oxidative depropylation (M6), aliphatic hydroxylation (M7), oxidative cleavage of the ethylene glycol linker (M4) and hydrolysis of the sulfamide moiety (M3). Most of the primary metabolites underwent subsequent biotransformation including conjugation with glucuronic acid or glucose, hydrolysis of the sulfamide group or secondary oxidation of the ethylene glycol moiety.

3.?Though there were species differences in their relative importance, all metabolic pathways were present in rat and dog. The depropylated M6 was the only metabolite present in plasma of both species.

4.?Metabolism was a prerequisite for macitentan excretion as relevant amounts of parent drug were neither detected in bile nor urine. Biliary excretion was the major elimination pathway, while renal elimination was of little importance.     

Metabolism and disposition of a potent and selective GABA-Aalpha2/3 receptor agonist in healthy male volunteers.

[14C]7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine ([14C]-TPA023; 99 microCi/dose) was administered to five young, healthy, fasted male subjects as a single oral dose (3.0 mg) in solution (propylene glycol/water, 10:90 v/v). The parent compound was rapidly absorbed (plasma Tmax approximately 2 h), exhibited an apparent terminal half-life of 6.7 h, and accounted for approximately 53% of the total radioactivity in plasma. After 7 days of collection, the mean total recovery of radioactivity in the excreta was 82.6%, with 53.2% and 29.4% in urine and feces, respectively. Radiochromatographic analysis of the excreta revealed that TPA023 was metabolized extensively, and only trace amounts of unchanged parent were recovered. Radiochromatograms of urine and feces showed that TPA023 underwent metabolism via three pathways (t-butyl hydroxylation, N-deethylation, and direct N-glucuronidation). The products of t-butyl hydroxylation and N-deethylation, together with their corresponding secondary metabolites, accounted for the majority of the radioactivity in the excreta. In addition, approximately 10.3% of the dose was recovered in urine as the triazolo-pyridazine N1-glucuronide of TPA023. The t-butyl hydroxy and N-desethyl metabolites of TPA023, the TPA023 N1-glucuronide, and the triazolo-pyridazine N1-glucuronide of N-desethyl TPA023 were present in plasma. In healthy male subjects, therefore, TPA023 is well absorbed and is metabolized extensively (t-butyl hydroxylation and N-deethylation > glucuronidation), and the metabolites are excreted in urine and feces.     

In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.     

Metabolism, pharmacokinetics and excretion of a potent tachykinin NK1 receptor antagonist (CP-122,721) in rat: characterization of a novel oxidative pathway

The metabolism, pharmacokinetics and excretion of a potent and selective substance P receptor antagonist, (+)-(2S,3S)-3-(2-methoxy-5-trifluoromethoxybenzlamino)-2-phenylpiperidine, CP-122,721, have been studied in rat following oral administration of a single dose of [14C]CP-122,721. Total recovery of the administered dose was 84.1+/-1.1% for male rat and 80.9+/-2.7% for female rat. Approximately 81% of the administered radioactivity recovered in urine and faeces were excreted in the first 72 h. Absorption of CP-122,721 was rapid in both male and female rat, as indicated by the rapid appearance of radioactivity in plasma. The plasma concentrations of total radioactivity were always much greater than unchanged drug, indicating early formation of metabolites. CP-122,721 t1/2 was 3.1 and 2.2 h for male and female rat, respectively. The plasma concentrations of CP-122,721 reached a peak of 941 and 476 ng ml-1 for male and female rat, respectively, at 0.5 h post-dose. Based on AUC0-tlast, only 1.5% of the circulating radioactivity was attributable to unchanged drug (average of male and female rats) and the balance, approximately 98.5% of the plasma radioactivity was due to metabolites. The major metabolic pathways of CP-122,721 were due to O-demethylation, aromatic hydroxylation and indirect glucuronidation. The minor metabolic pathways included aliphatic oxidation at the piperidine moiety and aliphatic oxidation at the benzylic position of the trifluoromethoxy anisole moiety. In addition, a novel oxidative metabolite resulting from ipso substitution by the oxygen atom and trifluoromethoxy elimination followed by glucuronide conjugation was also identified.     

Liquid chromatography-mass spectrometry and liquid chromatography-NMR characterization of in vitro metabolites of a potent and irreversible peptidomimetic inhibitor of rhinovirus 3C protease.

In vitro metabolism of AG7088 [trans-(4S,2'R,5'S,3"'S)-4-[2'-4-(4-fluorobenzyl)-6'-methyl-5'-[(5"-methylisoxazole-3"-carbonylamino]-4-oxoheptanoylamino]-5-(2"'-oxopyrrolidin-3-"'-yl)pent-2-enoic acid ethyl ester] was studied in liver microsomes isolated from mice, rats, rabbits, dogs, monkeys, and humans. The structures of the metabolites were characterized by liquid chromatography (LC)-tandem mass spectrometry and LC-NMR methods. Hydrolysis of the ethyl ester to produce metabolite M4 (AG7185) is the predominant pathway in all species, with the greatest activity observed in rodents and rabbits, followed by monkeys, dogs, and humans. Several hydroxylation products were identified as minor metabolites, including diastereomers M1 and M2, with a hydroxy group at the P1-lactam moiety, and M3, with a hydroxy group at the methyl position of the methylisoxazole ring. Rodent and rabbit liver microsomes formed almost exclusively the acid metabolite M4 (AG7185), with very little hydroxylated metabolites, whereas monkey liver microsomes formed more secondary metabolites (i.e., acid analogs of the hydroxylated metabolites). The overall metabolic profile of AG7088 formed in dog liver microsomes closely resembled that of human liver microsomes; therefore, this species may be the most appropriate animal model relative to humans for exposure to AG7088 and its metabolites.     

S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen

AIMS: To characterize the kinetics of S-naproxen ('naproxen') acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. METHODS: Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. RESULTS: Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent K(m) values (+/-SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 +/- 13 microm (16, 43) and 473 +/- 108 microm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent K(m) (72 microm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective 'probe' fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis-Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. CONCLUSION: UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug.     

Biotransformation of nimodipine in rat, dog, and monkey

14C-labelled (+/-) 3-isopropyl5-(2-methoxyethyl)1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl)-pyridine-3,5-dicarboxylate (nimodipine, Bay e 9736, Nimotop; CAS 66085-59-4) was administered orally to rat, dog, and monkey (each 5, 10, or 20 mg/kg) and intraduodenally to rat (5 mg/kg). Urine was collected over a period of 24 h (bile 6 h). Dog bile was obtained from the gall bladder 4 h after oral dosing. Rat plasma was taken 1 h p. appl. of the unlabelled compound and additionally at different times following administration of [14C]nimodipine. The metabolite profiles in the excreta were established by TLC (radioscan/autoradiography). The unchanged drug was neither detectable in urine nor in bile, but was present in rat plasma. Nimodipine was extensively metabolized. 18 metabolites were isolated by LC, HPLC, and preparative TLC and identified by comparison with the reference substances using two-dimensional TLC, HPLC, GC/radio-GC, 1H-NMR-spectroscopy, MS, and GC/MS. About 75% of the renally excreted biotransformation products, more than 50% of the metabolites present in the bile (rat, dog) and approx. 80% of the plasma metabolites (rat only) have been identified. The large number of metabolites was produced by some common biotransformation reactions: dehydrogenation of the 1,4-dihydropyridine system, oxidative ester cleavage, oxidative O-demethylation and subsequent oxidation of the resulting primary alcohol to the carboxylic acid, hydroxylation of the methyl groups at 2- or 6-position, hydroxylation of one methyl group of the isopropyl ester moiety, reduction of the aromatic nitro group, and glucuronidation as phase II-reaction.     

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an αhydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.     

Metabolism of pantoprazole involving conjugation with glutathione in rats

We have investigated the metabolism of pantoprazole and have provided an explanation for the formation mechanism of its metabolites. Metabolites found in the urine of rats after oral administration of pantoprazole sodium (25 mg kg(-1)) were analysed by liquid chromatography/ion trap mass spectrometry (LC/MS(n)). The N -acetylcysteine derivatives of benzimidazole (M1) and pyridine (M2), four pyridine-related metabolites (M3-M6), and three benzimidazole-related metabolites (M7-M9) were found, none of which had been reported previously. Five of the metabolites (M1, M2, M3, M7, and M8) were isolated from the urine of rats after oral administration of pantoprazole sodium by semipreparative HPLC. Structures of these metabolites were identified by a combination analysis of LC/MS(n) and (1)H NMR spectra. Structures of the remaining four metabolites (M4, M5, M6, and M9) were tentatively assigned through LC/MS(n). The metabolites M2, M3, M4, M5 and M6 and the other metabolites (M1, M7, M8, and M9) reflected the fate of the pyridine moiety and the benzimidazole moiety, respectively. The proposed formation route of M3-M6 was via initial reduction to mercaptopyridine followed by S-methylation, O-demethylation, and S-oxidation to the corresponding sulfoxide or sulfone. Meanwhile, M8 and M9 were formed via initial reduction to the 5-difluoromethoxy-1H benzoimidazole-2-thiol (M7) followed by hydroxylation and S-methylation. The metabolism of pantoprazole included an attack by glutathione on the benzimidazole-2-carbon and pyridine-7'-carbon. It is an important metabolic pathway of pantoprazole in rats.     

Metabolic patterns of JWH‐210, RCS‐4, and THC in pig urine elucidated using LC‐HR‐MS/MS: Do they reflect patterns in humans?

The knowledge of pharmacokinetic (PK) properties of synthetic cannabinoids (SCs) is important for interpretation of analytical results found for example in intoxicated individuals. In the absence of human data from controlled studies, animal models elucidating SC PK have to be established. Pigs providing large biofluid sample volumes were tested for prediction of human PK data. In this context, the metabolic fate of two model SCs, namely 4‐ethylnaphthalen‐1‐yl‐(1‐pentylindol‐3‐yl)methanone (JWH‐210) and 2‐(4‐methoxyphenyl)‐1‐(1‐pentyl‐indol‐3‐yl)methanone (RCS‐4), was elucidated in addition to Δ⁹‐tetrahydrocannabinol (THC). After intravenous administration of the compounds, hourly collected pig urine was analyzed by liquid chromatography‐high resolution mass spectrometry. The following pathways were observed: for JWH‐210, hydroxylation at the ethyl side chain or pentyl chain and combinations of them followed by glucuronidation; for RCS‐4, hydroxylation at the methoxyphenyl moiety or pentyl chain followed by glucuronidation as well as O ‐demethylation followed by glucuronidation or sulfation; for THC, THC glucuronidation, 11‐hydroxylation, followed by carboxylation and glucuronidation. For both SCs, parent compounds could not be detected in urine in contrast to THC. These results were consistent with those obtained from human hepatocyte and/or human case studies. Urinary markers for the consumption of JWH‐210 were the glucuronide of the N ‐hydroxypentyl metabolite (detectable for 3–4 h) and of RCS‐4 the glucuronides of the N ‐hydroxypentyl, hydroxy‐methoxyphenyl (detectable for at least 6 h), and the O ‐demethyl‐hydroxy metabolites (detectable for 4 h). Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro metabolism of (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol, a synthetic bicyclic cannabinoid analog

The oxidative metabolism of CP-55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3- hydroxypropyl)cyclohexanol] was studied in mouse liver S-9 microsomal preparations. [3H]CP-55,940 was incubated in a microsomal supernatant enriched with the appropriate cofactors for cytochrome P-450 oxidative metabolism. HPLC separation of petroleum ether/diethyl ether (1:1) extracts facilitated the identification of metabolites by GC/MS after derivatization with BSTFA or [2H18]BSTFA. The mass spectral data indicated that five monohydroxylated metabolites had been formed that differed with respect to the position of hydroxylation on the 1',1'-dimethylheptyl side chain. Two additional compounds were detected whose mass spectral data suggested that these metabolites were hydroxylated at two positions on the side chain. Side chain hydroxylation is consistent with the metabolic profile of delta 9-tetrhydrocannabinol (delta 9-THC) and other cannabinoid compounds. It is possible that these side chain-hydroxylated metabolites retain activity, as is the case with similar metabolites formed from delta 9- and delta 8-THC, and thereby contribute to the pharmacological profile seen with this potent synthetic cannabimimetic agent.     

Enantioselective and diastereoselective hydroxylation of bufuralol. Absolute configuration of the 7-(1-hydroxyethyl)-2-[1-hydroxy-2-(tert-butylamino)ethyl]benzofurans, the benzylic hydroxylation metabolites.

Asymmetric synthesis of the diastereomeric 7-(1-hydroxyethyl)-2-[1-hydroxy-2-(tert-butylamino)ethyl]benzofurans (2), the benzylic hydroxylation metabolites of bufuralol (1), is described, and the absolute configurations of these diastereomers are assigned. 1"-Oxobufuralol (3) was reduced with a complex of (2S)-(-)-2-amino-3-methyl-1,1-diphenylbutan-1-ol and borane, yielding 2, which had a 95:5 ratio of the possible 1"R and 1"S isomers as determined by HPLC. Separation of the resulting diastereomers was facilitated by derivatization with the enantiomers of 1-phenethyl isocyanate (PEIC). The absolute configurations 1'S,1"R and 1'R,1"R were assigned to the diastereomers formed in excess, 2c and 2b, on the basis of the known stereochemistry of reduction of closely related alkyl phenyl ketones to R alcohols by using this chiral borane reagent. The circular dichroism spectra of the four isomeric benzylic alcohols were in agreement with these assignments. In the presence of the rat liver microsomal fraction, benzylic hydroxylation of bufuralol was significantly product stereoselective favoring formation of diastereomers with the 1"R absolute stereochemistry at the new chiral center in products from (1'R)-1 by a ratio of 4.5:1 [(1'R,1"R)-2:(1'R,1"S)-2] and by nearly 8:1 [(1'S,1"R)-2:(1'S,1"S)-2] from (1'S)-1. (1'R)-Bufuralol was more rapidly hydroxylated than was (1'S)-1, by about 3-fold. In the presence of human liver microsomes, (1'R)-bufuralol was also more rapidly hydroxylated than was (1'S)-1, by ca. 2.5-fold. However, product stereoselectivity from the 1'R enantiomer was reversed from that observed in the rat liver microsomal oxidation, with more (1"S)-carbinol being formed than 1"R isomer by nearly 4-fold. From (1'S)-1, about equal amounts of the two possible hydroxybufuralol diastereomers were formed. The results from the human liver microsomal studies are consistent with observed enantioselectivity of hydroxylation of bufuralol in vivo in humans.     

R,S-1-(2-甲氧基苯基)-4-［3-(萘-1-氧基)-2-羟基丙基］哌嗪在大鼠血浆中代谢产物的研究

目的研究R,S-1-(2-甲氧基苯基)-4-［3-(萘-1-氧基)-2-羟基丙基］哌嗪(naftopidil，NAF)在大鼠血浆中的代谢产物。方法用LC/MS法对大鼠口服NAF后的血浆样品进行分析，根据检测到的代谢产物与原形药的质谱行为及类似结构化合物的代谢规律，推测可能的代谢产物。合成对照品，通过比较代谢产物和合成对照品的色谱和质谱行为，对I相代谢物予以确认。通过质谱信息及酶水解的方法间接鉴定II相代谢物。结果大鼠血浆中发现I相代谢物：R,S-1-(2-羟基苯基)-4-［3-(萘-1-氧基)-2-羟基丙基］哌嗪(DMN)、R,S-1-(2-甲氧基-4-羟基苯基)-4-［3-(萘-1-氧基)-2-羟基丙基］哌嗪(PHN)，R,S-1-(2-甲氧基苯基)-4-［3-(4-羟基萘-1-氧基)-2-羟基丙基］哌嗪(NHN)及II相代谢物：NAF和NHN与<>-D-葡糖醛酸形成的结合物。结论NAF在大鼠血浆中的主要代谢途径是苯环、萘环羟基化和苯环邻位甲氧基的脱甲基化。此外，萘羟化代谢物和原形药与内源性分子<>-D-葡糖醛酸形成结合物也是原形药的代谢方式之一。     

Further studies on the oxidative cleavage of the pentyl side-chain of cannabinoids: identification of new biotransformation pathways in the metabolism of 3'-hydroxy-delta-9-tetrahydrocannabinol by the mouse

1. Oxidative degradation of the pentyl side-chain of cannabinoids leading to compounds containing even numbers of carbon atoms was studied by investigating the in vivo metabolism of 2'- and 3'-hydroxy-delta-9-tetrahydrocannabinol (THC). 2. The hydroxy cannabinoids were administered i.p. to mice, the livers were removed after 1 h and extracted metabolites were identified by g.l.c.-mass spectrometry. 3. The major metabolic route for both compounds was hydroxylation at the allylic 11-position followed by oxidation to a carboxylic acid. Additional hydroxylation occurred at C-8. 4. Little oxidative degradation of the side-chain was found for 2'-hydroxy-delta-9-THC but abundant metabolites were formed by this route from the 3'-hydroxy compound. 5. The major metabolites of this type were acids containing three carbon atoms in the chain. These are the normal products of beta-oxidation but their formation from an (omega-2)-hydroxy intermediate appears novel. 6. Other metabolites contained two carbon atoms in the side-chain but were alcohols rather than acids and were again apparently formed by novel mechanisms. 7. The results indicate that the major route leading to cannabinoid metabolites with two-carbon side-chains (loss of three carbon atoms) is initiated by omega-2 hydroxylation.     

Regioselectivity of phase II metabolism of luteolin and quercetin by UDP-glucuronosyl transferases

The regioselectivity of phase II conjugation of flavonoids is expected to be of importance for their biological activity. In the present study, the regioselectivity of phase II biotransformation of the model flavonoids luteolin and quercetin by UDP-glucuronosyltransferases was investigated. Identification of the metabolites formed in microsomal incubations with luteolin or quercetin was done using HPLC, LC-MS, and (1)H NMR. The results obtained demonstrate the major sites for glucuronidation to be the 7-, 3-, 3'-, or 4'-hydroxyl moiety. Using these unequivocal identifications, the regioselectivity of the glucuronidation of luteolin and quercetin by microsomal samples from different origin, i.e., rat and human intestine and liver, as well as by various individual human UDP-glucuronosyltransferase isoenzymes was characterized. The results obtained reveal that regioselectivity is dependent on the model flavonoid of interest, glucuronidation of luteolin and quercetin not following the same pattern, depending on the isoenzyme of UDP-glucuronosyltransferases (UGT) involved. Human UGT1A1, UGT1A8, and UGT1A9 were shown to be especially active in conjugation of both flavonoids, whereas UGT1A4 and UGT1A10 and the isoenzymes from the UGTB family, UGT2B7 and UGT2B15, were less efficient. Due to the different regioselectivity and activity displayed by the various UDP-glucuronosyltransferases, regioselectivity and rate of flavonoid conjugation varies with species and organ. Qualitative comparison of the regioselectivities of glucuronidation obtained with human intestine and liver microsomes to those obtained with human UGT isoenzymes indicates that, in human liver, especially UGT1A9 and, in intestine, UGT1A1 and UGT1A8 are involved in glucuronidation of quercetin and luteolin. Taking into account the fact that the anti-oxidant action as well as the pro-oxidant toxicity of these catechol-type flavonoids is especially related to their 3',4'-dihydroxyl moiety, it is of interest to note that the human intestine UGT's appear to be especially effective in conjugating this 3',4' catechol unit. This would imply that upon glucuronidation along the transport across the intestinal border, the flavonoids loose a significant part of these biological activities.