N-O glucuronidation: a major human metabolic pathway in the elimination of two novel anticonvulsant drug candidates

1. The urinary metabolites of the anti-convulsant compound 4-amino-1-(2,6-difluorobenzyl)-1H-1,2,3-triazolo[4,5-c]-pyridine hydrochloride (GI265080) obtained following a single oral dose to man have been detected and quantified relative to each other using ¹⁹F-NMR spectroscopy. 2. The human urinary metabolites of GI265080 were isolated using semipreparative HPLC and unequivocally characterized using ¹H-NMR spectroscopy, two-dimensional heteronuclear NMR spectroscopy and mass spectrometry. The assignments of the N-(5)oxide and the N-(5)-O-glucuronide metabolites of GI265080 were further confirmed by independent synthesis. The urinary metabolites obtained following single oral doses to dog and rat have also been isolated and characterized. 3. The human urinary metabolites of GI265080 comprise the N-(5)-oxide, the quaternary N⁺-(5)-glucuronide, the 7-hydroxy glucuronide and a glucuronide conjugate of the N-(5)-oxide. The N-(5)-O-glucuronide conjugate is a novel species in human metabolism and is a significant route of elimination of GI265080 in man. 4. The urinary metabolites of the potential anti-convulsant GW273293 (6-amino-3-(2,3,5-trichlorophenyl)pyrazin-2-ylamine) obtained following a single oral dose to man have also been isolated and characterized. The formation of a novel N-O-glucuronide was also observed and was shown to constitute a significant route of elimination of GW273293 in man.     

Intestinal Azo-reduction and Glucuronide Conjugation of Prontosil

Abstract

1. The lipid-soluble azo-dye Prontosil was excreted in rat bile as an N-glucuronide, and in the urine, after azo-reduction, as free and N⁴-acetylated sulphanilamide.

2. When everted sections of rat intestine were incubated with ³⁵S-Prontosil, the dye was metabolized mostly to the glucuronide and partly to sulphanilamide by the intestinal wall.

3. Both intravenously and intraduodenally administered ³⁵S-Prontosil to biliary cannulated rats was recovered mainly in the bile as the N-glucuronide, whereas intracaecally administered ³⁵S-Prontosil was recovered chiefly in the urine as the products of azo-reduction. Considerably more N-glucuronide was biliary excreted after intraduodenal than intravenous dosage. The results imply that the rat intestinal wall is a major site of glucuronide conjugation of orally administered Prontosil. It is also apparent that the caecal bacteria, and not the liver, is the prime site of azo-reduction of Prontosil, in vivo. Azo-reduction is probably also mediated to some extent by the intestinal wall.

4. Urinary metabolites of ³⁵S-Prontosil from mouse, hamster and guinea-pig were qualitatively the same as the rat, although there were some quantitative differences.     

Biotransformation of a new pyrrolidinone cognition-enhancing agent: isolation and identification of metabolites in human urine

1. The metabolites of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DMPPA; MH-1), in the urine of human volunteers have been investigated.

2. Ten metabolites together with the unchanged drug (MH-1) were isolated by?h.p.l.c. and identified by n.m.r. and mass spectrometry as: three metabolites hydroxylated in the pyrrolidine ring of MH-1 (MH-2, MH-3 and MH-4), three metabolites hydroxylated in the dimethylphenyl ring of MH-1 (MH-6, MH-7 and MH-8), N-[(2,6-dimethyl-phenylcarbamoyl)methyl]-4-hydroxybutyrylamide (MH-5), N-[(2,6-dimethyl-phenyl-carbamoyl)methyl]succinamic acid (MH-9), the 3-O-sulphate of MH-6 (MH-10) and the 3-O-sulphate of N-(2,6-dimethyl-3-hydroxyphenyl)-2-(5-hydroxy-2-oxo-1-pyrrolidinyl)acetamide (MH-11).

3. DMPPA was extensively metabolized. The principal metabolic transformations were hydroxylation of the pyrrolidine ring at the C5 carbon followed by oxidative C-N cleavage, and hydroxylation of the phenyl ring followed by sulphate conjugation.     

The Role of the Gut Flora in the Metabolism of Prontosil and Neoprontosil in the Rat

1. The urinary excretion of total sulphanilamide (free and acetylated) in rats receiving Prontosil or Neoprontosil orally is considerably reduced when the rats are treated with antibiotics to suppress their intestinal flora. The absorption and metabolism of sulphanilamide are unaffected by such treatment with antibiotics.

2. The lipid-soluble Prontosil after oral or intraperitoneal administration is partly excreted in the bile as a polar conjugate, apparently an N-glucuronide, and this drug appears to be converted into sulphanilamide partly by metabolism by the body tissues and partly by enterofloral metabolism.

3. The polar, water-soluble Neoprontosil is poorly absorbed from the intestine and when given orally is largely reduced to sulphanilamide by the gut flora before absorption. When given intraperitoneally, a large proportion (up to 70%) of the drug is excreted in the bile unchanged, and it would appear that only a small proportion of the drug (ca. 20%) is converted to sulphanilamide in the tissues, the major conversion occurring in the intestine after biliary excretion.

4. The reduction of Neoprontosil by the tissues is stimulated by pretreatment of the animals with phenobarbitone, but its reduction by the gut flora is unaffected by phenobarbitone pretreatment.

5. The conversion of Prontosil and Neoprontosil to sulphanilamide in the rat is an example of drug activation by the intestinal flora.     

Additional Route in the Metabolism of Sulphanilamide

1. Rabbits were found to excrete a substantial portion of a dose of [³⁵S]sul-phanilamide as N-(4-sulphamoylphenyl)glycolamide (20%) and N-(4-sulpha-moylphenyl)oxamic acid (14%), newly discovered metabolites of sulphanilamide. The metabolites were isolated and identified by u.v., i.r., and n.m.r. spectroscopy.

2. Urine of a human dosed with [³⁵S]sulphanilamide contained only a small portion of the dose as glycolamide (2%), and the oxamic acid derivative was not detected.

3. N-(4-Acetylaminobenzenesulphonyl)acetamide and 4-amino-3-hydroxy-benzenesulphonamide, metabolites of sulphanilamide found in the urine of other species, were shown to be produced also by the human.

4. N-Hydroxy-N-(4-sulphamoylphenyl)acetamide was not detected in the urines of rabbits or human dosed with [³⁵S]sulphanilamide. From human urine a very small amount of a metabolite was isolated which was likely to be a conjugate of N⁴-hydroxysulphanilamide.     

DNA cleavage by metabolites of butylated hydroxytoluene

The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di -t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 × 10^–6 M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including Superoxide dismutase (SOD), catalase, polyethylene glycol,t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl₂. The production of Superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals. Such modification of DNA observed in vitro may be relevant to genotoxicity by BHT after metabolic activation in vivo.     

Synthesis of certain N-aryl-N′-(2-pyrimidinyl) guanidine derivatives as potential antimicrobial agents

N-Aryl-N′-(4-hydroxy-6-methyl-2-pyrimidinyl) guanidines (IIa-c) were prepared by cyclization of N-arylbiguanides (Ia-c) with ethyl acetoacetate. Coupling of compounds (IIa-c) with the appropriate diazotized arylamine gave N-aryl-N′-(5-arylhydrazono-6-methyl-4-oxopyrimidin-2-yl) guanidines (IIIa-f). Whereas, their chlorination with phosphorus oxychloride followed by treatment of N-aryl-N′-(4-chloro-6-methyl-2-pyrimidinyl) guanidines (IVa-c) with the appropriate arylamine afforded the corresponding 4-arylamino derivatives (Va-f). Compounds (IIIa-f) were also formed when compounds (Ia-c) were treated with ethyl 2-arylhydrazono-3-oxobutyrates. The antimicrobial testing of some of the prepared compounds against some pathogenic microorganisms revealed that only two have a marked effect against.Escherichia coli.     

The acute toxicity of butylated hydroxytoluene and its metabolites in mice

The acute toxicity of butylated hydroxytoluene (BHT) and four of its metabolites, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien- 1-one(BHT-OOH), 2,6-di-tert-butyl-4-hydroxy-4-methyl-2,5-cyclohexadien- 1-one(BHT-OH), 2,6-di-tert-butyl-p-benzoquinone (DBQ), and 2,6-di-tert- butyl-4-[(methylthio)methyl] phenol (BHT-SCH₃) was studied in young male mice following intraperitoneal administration. The i.p. LD₅₀ values of BHT, BHT-OOH, BHT-OH, DBQ, and BHT-SCH₃ were 3550,190, above 1600, 2270, and 1840 $\text{mg}\text{kg}$, respectively. These results suggest that BHT-OOH probably is the most toxic metabolite of BHT.     

Pharmacokinetics,metabolism and excretion of [14C]-lanicemine (AZD6765), a novel low-trapping N-methyl-d-aspartic acid receptor channel blocker,in healthy subjects

Abstract

1.?(1S)-1-phenyl-2-(pyridin-2-yl)ethanamine (lanicemine; AZD6765) is a low-trapping N-methyl-d-aspartate (NMDA) channel blocker that has been studied as an adjunctive treatment in major depressive disorder. The metabolism and disposition of lanicemine was determined in six healthy male subjects after a single intravenous infusion dose of 150?mg [¹⁴C]-lanicemine.

2.?Blood, urine and feces were collected from all subjects. The ratios of C_max and AUC_(0–∞) of lanicemine to plasma total radioactivity were 84 and 66%, respectively, indicating that lanicemine was the major circulating component with T_1/2 at 16?h. The plasma clearance of lanicemine was 8.3?L/h, revealing that lanicemine is a low-clearance compound. The mean recovery of radioactivity from urine was 93.8% of radioactive dose.

3.?In urine samples, 10 metabolites of lanicemine were identified. Among which, an O-glucuronide conjugate (M1) was the most abundant metabolite (～11% of the dose in excreta). In plasma, the circulatory metabolites were identified as a para-hydroxylated metabolite (M1), an O-glucuronide (M2), an N-carbamoyl glucuronide (M3) and an N-acetylated metabolite (M6). The average amount of each of metabolite was less than 4% of total radioactivity detected in plasma or urine.

4.?In conclusion, lanicemine is a low-clearance compound. The unchanged drug and metabolites are predominantly eliminated via urinary excretion.     

Über die Reaktion von 3,5-Diacetyl-2,6-dimethyl-4H-pyran-4-on mit Natriumalkanolaten

On the Reaction of 3,5-Diacetyl-2,6-dimethyl-4H-pyran-4-one with Sodium Alkoxides The reaction of 3,5-diacetyl-2,6-dimethyl-4H-pyran-4-one (10) with sodium methoxide or ethoxide leads to the formation of the 1-acetyl-2-hydroxy-4-methyl-6-alkoxybenzenes 11a and 11b .     

(+)-SKF-10,047 and dextromethorphan ameliorate conditioned fear stress via dopaminergic systems linked to phenytoin-regulated σ1 sites

Mice exhibited a marked suppression of motility when they were re-placed in the same environment in which they had previously received an electric footshock. (+)-SKF-10,047 ([2S-(2α,6α,11R1)]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride; (+)-N-allylnormetazocine hydrochloride) and dextromethorphan, putative σ receptor agonists, have been reported to reverse this psychological stress-induced motor suppression, defined as conditioned fear stress, through phenytoin-regulated type σ₁ receptors. In the present study, we investigated the involvement of dopaminergic neurons in the ameliorating effects of (+)-SKF-10,047 and dextromethorphan on conditioned fear stress. (+)-SKF-10,047 and dextromethorphan attenuated conditioned fear stress at low doses (4 and 20 mg/kg, respectively) when they were co-administered with phenytoin (10 mg/kg), an anticonvulsant drug. The effects were antagonized by the σ receptor antagonists, NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride) and BMY-14802 (a-(4-fluoro-phenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine-butanol hydrochloride). Furthermore, the effects of (+)-SKF-10,047 or dextromethorphan in combination with phenytoin were blocked by the dopamine D₁ receptor antagonist, SCH 23390 (R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine), and the dopamine D₂ receptor antagonist, (−)-sulpiride, and they were also attenuated by 6-hydroxydopamine-induced lesions of dopaminergic neurons. The ameliorating effects of (+)-SKF-10,047 and dextromethorphan on conditioned fear stress at high doses (5 and 30 mg/kg, respectively) were also blocked by both the dopamine receptor antagonists. These results suggest that the stress-induced motor suppression is restored by the activation of dopaminergic neuronal systems as a result of the stimulation of phenytoin-regulated type σ₁ receptors.     

Three new α-pyrone derivatives from the plant endophytic fungus Penicillium ochrochloronthe and their antibacterial,antifungal, and cytotoxic activities

Three new 3,4,6-trisubstituted α-pyrone derivatives, namely 6-(2′R-hydroxy-3′E,5′E-diene-1′-heptyl)-4-hydroxy-3-methyl-2H-pyran-2-one (1), 6-(2′S-hydroxy-5′E-ene-1′-heptyl)-4-hydroxy-3-methyl-2H-pyran-2-one (2), and 6-(2′S-hydroxy-1′-heptyl)-4 -hydroxy-3-methyl-2H-pyran-2-one (3), together with one known compound trichodermic acid (4), were isolated from the solid-substrate fermentation culture of Penicillium ochrochloronthe associated the roots of Taxus media. Compounds 1–4 displayed the antimicrobial activity selectively against tested fungal and bacterial strains with minimum inhibitory concentration (MIC) values ranging from 12.5 to 100 μg/ml. Furthermore, we found that only compound 4 exhibited moderate cytotoxicity against five human cancer cells (A549, LN229, MGC, LOVO, and MDA231) with IC₅₀ values of 51.45, 23.43, 39.16, 46.97, and 42.85 μg/ml, respectively.     

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy

¹⁹F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of ¹⁹F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the ³H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with ¹⁹F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.     

The metabolism of nitrophenolic and 5-arylazorhodanine anthelmintics by Ascaris suum,Moniezia expansa and by mouse- and sheep-liver enzymes

1. The anthelmintics disophenol (2,6-diiodo-4-nitrophenol), nitroxynil (3-iodo-4-hydroxy-5-nitrobenzonitrile) and nitrodan (3-methyl-5-(4-nitrophenylazo)rhodanine) were reduced in vitro to the corresponding amines by intact Ascaris suum, Moniezia expansa by enzymes prepared from these helminths, and by mouse- and sheep-liver homogenates. Helminth reductases required NADH₂ and glutathione as cofactors and were inhibited about 50% by 2.0 × 10^?7 M allopurinol. Azo bonds of nitrodan and its analogues were not reduced by the helminths but were reduced by mouse- and sheep-liver enzymes.

2. Mouse- and sheep-liver enzymes, in addition to effecting nitro reduction, metabolized nitroxynil by hydrolysis to 3-iodo-4-hydroxy-5-nitrobenzamide and 3-iodo-4-hydroxy-5-nitrobenzoic acid. No hydroxylation products were found. Nitrodan was oxidized by the mammalian microsomal oxidation enzyme system to the thiazolidinedione derivative, but not by helminth enzymes.     

Isolation and identification of metabolites of ofloxacin in rats,dogs and monkeys

1. Metabolites of (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid (ofloxacin) in excreta of rats, dogs and monkeys after oral administration of ¹⁴C-ofloxacin (20?mg/kg) were isolated and identified.

2. Three metabolites of ofloxacin were detected in the excreta of all three species, and identified by t.l.c., u.v., n.m.r. and mass spectrometry as follows: M-1, ester glucuronide of ofloxacin; M-2, unchanged ofloxacin; M-3, (±)-9-fluoro-2,3-dihydro-3-methyl-10-(1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid (desmethyl ofloxacin); M-4, (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]-benzoxazine-6-carboxylic acid piperazine-4-oxide (ofloxacin N-oxide).

3. It is concluded that ofloxacin is metabolized by O-acyl glucuronidation, N-demethylation and N-oxidation.     

Synthesis,isolation and identification of glucuronides and mercapturic acids of a novel antiparasitic agent,licochalcone A

1. Four glucuronic acidconjugates of licochalcone A(Lica),and their metabolites,have been synthesized using rabbit and pig liver microsomes and purified by preparative hplc. 2. The glucuronides were identified as E-Lica 4′-O-β-glucuronide, E and Z-Lica 4-O-β-glucuronide and a mono-glucuronide conjugate of a β-hydroxylated Lica metabolite. The metabolites were identified by hplc-nmr (one and two-dimensional nmr) as well as hplc-ms. 3. At pH 8·5 Lica reacted with N-acetyl-L-cysteine giving the twoepimeric conjugates, which were then isolated by preparative hplc and identified by one and two-dimensional nmr spectroscopic methods. 4. Only twoglucuronic acid conjugates (E- and Z-Lica 4-O-β-glucuronide) were found in the urine of rat after i.p. administration of a single dose of Lica.     

Metabolism and disposition of [14C]5-amino-o-cresol in female F344 rats and B6C3F1 mice

The metabolism and disposition of [¹⁴C]5-amino-o-cresol (AOC) in female F344 rats following oral, intravenous, and dermal administration and in female B6C3F1 mice following oral administration were studied. Greater than 80% of a single oral dose (4.0–357 mg kg^?1) or intravenous dose (2.7 mg kg^?1) was excreted in urine within 24 h. When the dosing site was protected from grooming, less than 10% of the dermal dose (2.5 and 26 mg kg^?1, rinsed off after 6 h) was absorbed within 24 h, and most of the absorbed radioactivity was excreted in urine. For the unprotected dermal dose, grooming played a major role in the absorption of AOC. Very little AOC-derived radioactivity was present in the surveyed tissues after 24 or 72 h regardless of route, dose level, or species. Five urinary metabolites were identified: 5-acetamido-1,4-dihydroxy-2-methylbenzene glucuronide, AOC O-glucuronide, AOC O-sulfate, N-acetyl-AOC O-glucuronide, and N-acetyl-AOC O-sulfate.     

Metabolism of 2,6-Dichlorobenzonitrile, 2,6-Dichlorothiobenzamide in Rodents and Goats

1. Twelve ¹⁴C-labelled metabolites were isolated from either urine or bile from either rats (11 metabolites) or goats (7 metabolites) given single oral doses of 2,6-dichlorobenzo[¹⁴C]nitrile (DCBN). Five of these metabolites were also excreted in urine from rats dosed orally with 2,6-dichlorothiobenz[¹⁴C]-amide (DCTBA).

2. All metabolites from either DCBN or DCTBA were benzonitriles with the following ring substitutents: Cl₂, OH (three isomers); Cl₂, (OH)₂; Cl, (OH)₂; Cl, OH, SH; Cl, OH, SCH₃; SCH₃, SOCH₃, OH; Cl₂, S-(N-acetyl)cysteine; Cl, S-(N-acetyl)cysteine: Cl, OH, S-(N-acetyl)cysteine.

3. The thiobenzamide moiety of DCTBA was converted to the nitrile in all the excreted urinary metabolites. No hydrolysis of the nitrile in DCBN to either an amide or an acid was detected.

4. Urine was the major route for excretion; however, enterohepatic circulation occurred.

5. Whole-body autoradiography of ¹⁴C-DCBN and ¹⁴C-DCTBA in mice showed the presence of bound residues in the mucosa of the nasal cavity, trachea, tongue, oesophagus, the kidney, liver and the intestinal contents.     

Antibacterial activity of <Emphasis Type="Italic">Capsicum annuum</Emphasis> extract and synthetic capsaicinoid derivatives against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

The inhibitory effects of the ethyl acetate extract and capsaicin (1) and dihydrocapsaicin (2) isolated from fruits of Capsicum annuum chili pepper type, and synthetic capsaicinoid derivatives (N-(4-hydroxyphenylethyl)decamide (3), (E)-N-(4-hydroxy-3-methoxybenzyl)-3,7-dimethylocta-2,6-dienamide (4), 4-hydroxy-3-methoxy-N-((E)-3,7-dimethylocta-2,6-dienyl)benzamide (5) and N-(4-hydroxy-3-methoxybenzyl)decamide (6) at different concentrations were evaluated against Streptococcus mutans. The minimum inhibitory concentration at which the ethyl acetate extract prevented the growth of S. mutans was 2.5 mg/mL; those of the isolated compounds 1 and 2 were 1.25 μg/mL, while 3 was 5.0 μg/mL, and 4, 5 and 6 were 2.5 μg/mL, respectively.     

Two new anthraquinones from Hedyotis diffusa W.

Two new anthraquinones, 2,6-dihydroxy-3-methyl-4-methoxyanthraquinone (1) and 2-hydroxy-7-hydroxymethyl-3-methoxyanthraquinone (2), were isolated from Hedyotis diffusa W. Their structures were elucidated by means of spectroscopic evidence.