Microsomal metabolism of N,N-diethyl-m-toluamide (DEET,DET): the extended network of metabolites

1. The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identified by HPLC retention times, UV spectroscopy, mass spectrometry and by comparison with authentic standards. 2. DEET (1a) produces three major metabolites, N-ethyl-m-toluamide (1b), N,N-diethyl-m-(hydroxymethyl)benzamide (2a) and N-ethyl-m-(hydroxymethyl)benzamide (2b), and, at low substrate concentrations or extended reaction times, two minor metabolites, toluamide (1c) and N,N-diethyl-m-formylbenzamide (3a). 1b and 2a are primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low DEET concentrations, ring methyl group oxidation is favoured; at saturation concentrations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of formation of 2b decreases with increasing DEET concentration; 2b is therefore a secondary metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of 1b and 2a. 3. Except for the primary amides, where N-dealkylation is impossible, metabolism of all subsequent compounds, 1b,c, 2a-c, 3a-c and 4a,b, involves an N-deethylation (NEt₂ → NHEt or NHEt → NH₂) competitive with a ring substituent oxidation (CH₃ → CH₂OH, CH₂OH → CHO or CHO → CO₂H). Surprisingly, the aldehydes 3a-c are also reduced to the corresponding alcohols 2a-c (CHO → CH₂OH); CO inhibits the oxidative metabolism of 3a-c, but reduction to 2a-c continues uninhibited. 4. The outcomes of this work are that (1) previously unreported aldehydes 3b and 3c form part of the DEET network of metabolites, (2) the reduction of the aldehydes 3a-c has the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3) amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for N-(1-hydroxyethyl)amide intermediates.     

Oxidative metabolism of the GABAA receptor antagonist t-butylbicycloortho[3H]benzoate

1. The trioxabicyclooctane ring of t-butylbicycloortho[3H]benzoate (TBOB), (CH3)3CC(CH2O)3CC6H5, is cleaved to yield the 3-oxo-benzoate, (CH3)3CC(CHO)(CH2OH)CH2OC(O)C6H5, on O-methylene hydroxylation by microsomes from mouse liver or houseflies in the presence of NADPH. 2. The methyl and phenyl substituents are tentatively identified as additional sites of oxidative metabolism. 3. The 3-oxo-benzoate from oxidative cage opening and the bis-(hydroxymethyl)-benzoate, (CH3)3CC(CH2OH)2CH2OC(O)C6H5, from enzymic reduction of the 3-oxo-benzoate undergo esteratic hydrolysis to benzoic acid. 4. Metabolites of TBOB excreted by rats and houseflies include the bis-(hydroxymethyl)-benzoate and benzoic and hippuric acids. 5. Metabolic hydroxylation of TBOB at O-methylene, alkyl and aryl substituents may serve as a model for detoxication reactions of related potent GABAA receptor antagonists and insecticides.     

Chemical stability and fate of the cytostatic drug ifosfamide and its N-dechloroethylated metabolites in acidic aqueous solutions.

31P NMR spectroscopy was used to study the products of the decomposition of the antitumor drug ifosfamide (IF, 1d) and its N-dechloroethylated metabolites, namely, 2,3-didechloroethylIF (1a) and 2- (1b) and 3-dechloroethylIF (1c), in buffered solutions at acidic pH. The first stage of acid hydrolysis of these four oxazaphosphorines is a P-N bond cleavage of the six-membered ring leading to the phosphoramidic acid monoesters (2a-d) of type R'HN(CH(2))(3)OP(O)(OH)NHR, with R and/or R' = H or (CH(2))(2)Cl. The electron-withdrawing chloroethyl group at the endocyclic and/or exocyclic nitrogens counteracts the endocyclic P-N bond hydrolysis. This effect is even more marked when the N-chloroethyl group is in the exocyclic position since the order of stability is 1d > 1c > 1b > 1a. In the second stage of hydrolysis, the remaining P-N bond is cleaved together with an intramolecular attack at the phosphorus atom by the non-P-linked nitrogen of the compounds 2a-d. This leads to the formation of a 2-hydroxyoxazaphosphorine ring with R = H (3a coming from compounds 2a,c) or (CH(2))(2)Cl (3b coming from compounds 2b,d) and to the release of ammonia or chloroethylamine. The third step is the P-N ring opening of the oxazaphosphorines 3a,b leading to the phosphoric acid monoesters, H(2)N(CH(2))(3)OP(O)(OH)(2) (4a) and Cl(CH(2))(2)HN(CH(2))(3)OP(O)(OH)(2) (4b-1), respectively. For the latter compound, the chloroethyl group is partially (at pH 5.5) or totally (at pH 7.0) cyclized into aziridine (4b-2), which is then progressively hydrolyzed into an N-hydroxyethyl group (4b-3). Compounds 3a,b are transient intermediates, which in strongly acidic medium are not observed with (31)P NMR. In this case, cleavage of the P-N bond of the type 2 phosphoramidic acid monoesters leads directly to the type 4 phosphoric acid monoesters. The phosphate anion, derived from P-O bond cleavage of these latter compounds, is only observed at low levels after a long period of hydrolysis. Compounds 1a-c and some of their hydrolytic degradation products (4b-1, 4b-2, diphosphoric diester [Cl(CH(2))(2)NH(CH(2))(3)OP(O)(OH)](2)O (5), and chloroethylamine) did not exhibit, as expected, any antitumor efficacy in vivo against P388 leukemia. (31)P NMR determination of the N-dechloroethylated metabolites of IF or its structural isomer, cyclophosphamide (CP), and their degradation compounds could provide an indirect and accurate estimation of chloroacetaldehyde amounts formed from CP or IF.     

Synthesis and antiviral and cytotoxic activity of iodohydrin and iodomethoxy derivatives of 5-vinyl-2'-deoxyuridines, 2'-fluoro-2'-deoxyuridine, and uridine

A series of new 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine and uridine compounds (11, 16) was synthesized by the regiospecific addition of HOI to the vinyl substituent of 5-vinyl-2'-deoxyuridine (10a), 5-vinyl-2'-fluoro-2'-deoxyuridine (10b), 5-vinyluridine (10c), and (E)-5-(2-iodovinyl)-2'-deoxyuridine (4b). Treatment of the iodohydrins 11a-c with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-iodoethyl) derivatives (12a-c). In contrast, reaction of 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine (11a) with sodium carbonate in methanol afforded a mixture of 5-(1-hydroxy-2-methoxyethyl)-2'-deoxyuridine (13) and 2,3-dihydro-3-hydroxy-5-(2'-deoxy-beta-D-ribofuranosyl)- furano[2,3-d]pyrimidin-6(5H)-one (14). The most active compound, 5-(1-methoxy-2-iodoethyl)-2'-deoxyuridine (12a, ID50 = 0.1 micrograms/mL), which exhibited antiviral activity (HSV-1) 100-fold higher than that of the 5-(1-hydroxy-2-iodoethyl) analogue (11a), was less active than IVDU or acyclovir (ID50 = 0.01-0.1 micrograms/mL range). The C-5 substituent in the 2'-deoxyuridine series was a determinant of cytotoxic activity, as determined in the in vitro L1210 screen, where the relative activity order was CH(OH)CHI2 (16) greater than CH(OMe)CH2I (12a) greater than CH(OH)CH2I (11a) congruent to CH(OH)CH2OMe (13). The 2'-substituent was also a determinant of cytotoxic activity in the 5-(1-hydroxy-2-iodoethyl) (11a-c) and 5-(1-methoxy-2-iodoethyl) series of compounds, where the relative activity profile was 2'-deoxyuridine greater than 2'-fluoro-2'-deoxyuridine greater than uridine (11a greater than 11b greater than or equal to 11c; 12a greater than 12b greater than 12c). The most active cytotoxic agent (16), possessing a 5-(1-hydroxy-2,2-diiodoethyl) substituent (ED50 = 0.77 micrograms/mL), exhibited an activity approaching that of melphalan (ED50 = 0.15 micrograms/mL). All compounds tested, except for 13 and 14, exhibited high affinity (Ki = 0.035-0.22 mM range relative to deoxyuridine, Ki = 0.125) for the murine NBMPR-sensitive erythrocyte nucleoside transport system, suggesting that these iodohydrins are good permeants of cell membranes.     

Studies of the mode of action of antitumor triazenes and triazines. 6. 1-Aryl-3-(hydroxymethyl)-3-methyltriazenes: synthesis, chemistry, and antitumor properties

1-Aryl-3-(hydroxymethyl)-3-alkyltriazenes [ArN = NN(CH3)CH2OH] have been synthesized by diazonium coupling to the carbinolamine (RNHCH2OH), generated in situ from the alkylamine and formaldehyde mixtures. The (hydroxymethyl)triazene structure has been confirmed by IR, NMR, and mass spectral analysis and also by the preparation of a crystalline benzoate derivative. The mass spectra of the (hydroxymethyl)triazenes suggest that they fragment by loss of formaldehyde to give the methyltriazene, which is also the product of hydrolysis in solution. The degradation of the (hydroxymethyl)triazenes in solution has been followed by UV spectroscopy and by HPLC analysis, and the half-lives were determined under a variety of conditions. The half-lives of the corresponding methyl- and (hydroxymethyl)triazenes are very similar. Both methyl- and (hydroxymethyl)triazenes decompose on silica plates during TLC analysis to give products consistent with known diazo-migration reactions. The (hydroxymethyl)triazenes have pronounced antitumor activity against the TLX5 tumor in vivo; in vivo-in vitro bioassay experiments suggest that the (hydroxymethyl)triazenes exert their in vivo antitumor activity via the degradation product, the alkyltriazene.     

Analgesic narcotic antagonists. 5. 7,7-Dimethyldihydrocodeinones and 7,7-dimethyldihydromorphinones

Treatment of dihydrocodeinone (1a) or the 8 beta-methyl (1b) or 8 beta-ethyl (1c) analogues with formaldehyde-Ca(OH)2 in aqueous dioxane gave the corresponding 7,7-bis(hydroxymethyl)-6 beta-ols 2a-c. Ditosylation of 2, followed by LiEt3BH reduction, gave either the 7,7-dimethyl-6 beta-ol (6a) or 7 alpha-methyl-6 beta, 7 beta-oxetane compounds (5b,c). Compounds 5b and 5c were cleaved to 6b or 6c using LiAlH4-AlCl3. The configuration of the C6-alcohol group of 6a was confirmed by an oxidation-reduction sequence which gave the 7,7-dimethyl-5 alpha-ol 8a. Oxidation of 6 gave the C6-ketones 7a-c, which were converted to N-(cycloalkylmethyl) derivatives 11 and 12 and their corresponding 3-hydroxy compounds 14 and 15. The 3-methoxy-7,7-dimethyl-6-ones 7 were as active as dihydrocodeinone in agonist assays. One compound of this series, N-(cyclopropylmethyl)-7,7-dimethyldihydronorcodeinone (11a), was a potent mixed agonist-narcotic antagonist.     

Design and synthesis of novel 5-substituted acyclic pyrimidine nucleosides as potent and selective inhibitors of hepatitis B virus

A novel class of 5-substituted acyclic pyrimidine nucleosides, 1-[(2-hydroxyethoxy)methyl]-5-(1-azidovinyl)uracil (9a), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-(1-azidovinyl)uracil (9b), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azidovinyl)uracil (9c), were synthesized by regiospecific addition of bromine azide to the 5-vinyl substituent of the respective 5-vinyluracils (2a-c) followed by treatment of the obtained 5-(1-azido-2-bromoethyl) compounds (3a-c) with t-BuOK, to affect the base-catalyzed elimination of HBr. Thermal decomposition of 9b and 9c at 110 degrees C in dioxane yielded corresponding 5-[2-(1-azirinyl)]uracil analogues (10b,c). The 5-(1-azidovinyl)uracil derivatives 9a-c were found to exhibit potent and selective in vitro anti-HBV activity against duck hepatitis B virus (DHBV) infected primary duck hepatocytes at low concentrations (EC(50) = 0.01-0.1 microg/mL range). The most active anti-DHBV agent (9c), possessing a [4-hydroxy-3-(hydroxymethyl)-1-butyl] substituent at N-1, exhibited an activity (EC(50) of 0.01-0.05 microg/mL) comparable to that of reference compound (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3-TC) (EC(50) = 0.01-0.05 microg/mL). In contrast, related 5-[2-(1-azirinyl)]uracil analogues (10b,c) were devoid of anti-DHBV activity, indicating that an acyclic side chain at C-5 position of the pyrimidine ring is essential for anti-HBV activity. The pyrimidine nucleosides (9a-c, 10b,c) exhibited no cytotoxic activity against a panel of 60 human cancer cell lines. All of the compounds investigated did not show any detectable toxicity to several stationary and proliferating host cell lines or to mitogen stimulated proliferating human T lymphocytes, up to the highest concentration tested.     

Reactions with pyrrolidine-2,4-diones, Part 4: Synthesis of some 3-substituted 1,5-diphenylpyrrolidine-2,4-diones as potential antimicrobial, anti-HIV-1 and antineoplastic agents.

The condensation of 1,5-diphenylpyrrolidine-2,4-dione (1) with ethyl orthoformate yielded 3-ethoxymethylene-1,5-diphenylpyrrolidine-2,4-dione (2). Reaction of the latter with hydrazine hydrate, secondary amines 7a-c or urea afforded the corresponding 3-substituted aminomethylene-1,5-diphenylpyrrolidene-2,4-diones 3, 8a-c or 9. On the other hand, condensation of 3 with veratraldehyde (5a) yielded 3-[(3,4-dimethoxybenzylidene)hydrazinomethylene]-1,5-diphenylpyrrolidine- 2,4-dione (6). Whereas, cyclization of 9 with the reactive malonate ester 11 produced 3-[(5-butyl-4-hydroxy-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-1-yl) methylene]-1,5-diphenylpyrrolidine-2,4-dione (12). The condensation of some selected aromatic aldehydes 5a-c and addition of morpholine (7c) or piperidine (7d) to some of the resulting 3-arylidene-1,5-diphenylpyrrolidine-2,4-diones 13b, c gave the respective 3-substituted methyl-4-hydroxy-1,5-diphenyl-delta 3-pyrrolin-2-ones 14a-c. Selected members of the new series were screened for their in vitro antimicrobial, anti-HIV-1 and antineoplastic activities. Two compounds 14a, b showed pronounced inhibitory activities against Gram-positive bacteria; whereas, in the in vitro anti-HIV-1 screening, only one compound 13c displayed a moderate activity. However, in the antineoplastic screening protocol, the tested compounds were devoid of activity.     

In vitro human metabolism and interactions of repellent N,N-diethyl-m-toluamide.

Oxidative metabolism of the insect repellent N,N-diethyl-m-toluamide (DEET) by pooled human liver microsomes (HLM), rat liver microsomes (RLM), and mouse liver microsomes (MLM) was investigated. DEET is metabolized by cytochromes P450 (P450s) leading to the production of a ring methyl oxidation product, N,N-diethyl-m-hydroxymethylbenzamide (BALC), and an N-deethylated product, N-ethyl-m-toluamide (ET). Both the affinities and intrinsic clearance of HLM for ring hydroxylation are greater than those for N-deethylation. Pooled HLM show significantly lower affinities (K(m)) than RLM for metabolism of DEET to either of the primary metabolites (BALC and ET). Among 15 cDNA-expressed P450 enzymes examined, CYP1A2, 2B6, 2D6*1 (Val(374)), and 2E1 metabolized DEET to the BALC metabolite, whereas CYP3A4, 3A5, 2A6, and 2C19 produced the ET metabolite. CYP2B6 is the principal cytochrome P450 involved in the metabolism of DEET to its major BALC metabolite, whereas CYP2C19 had the greatest activity for the formation of the ET metabolite. Use of phenotyped HLMs demonstrated that individuals with high levels of CYP2B6, 3A4, 2C19, and 2A6 have the greatest potential to metabolize DEET. Mice treated with DEET demonstrated induced levels of the CYP2B family, increased hydroxylation, and a 2.4-fold increase in the metabolism of chlorpyrifos to chlorpyrifos-oxon, a potent anticholinesterase. Preincubation of human CYP2B6 with chlorpyrifos completely inhibited the metabolism of DEET. Preincubation of human or rodent microsomes with chlorpyrifos, permethrin, and pyridostigmine bromide alone or in combination can lead to either stimulation or inhibition of DEET metabolism.     

Gyrase inhibitors. 3. Synthesis and reactions of 1,4-dihydro-4-oxo-(1)benzothieno(3,2-b)pyridin-3-carboxylic acid esters

The title compound 4a is synthesized from potassium 3-aminobenzo[b]thiophene-2-carboxylate (1) by Gould-Jacobs reaction. Compound 4a reacts with alkyl halides and sodium hydride in DMF to yield the N-alkylpyridones 5a-c as well as the 4-alkoxypyridines 6a-c; with phosphoryl chloride the 4-chloropyridine 7a is obtained. The carboxylic acids 4b, 5d, 5e, 6d and 7b are received by alkaline saponification of the esters 4a, 5a-c, 6a-c and 7a. The carbinoles 8 and 9, formed by boranate reduction of the esters 6a and 7a, are transformed to the aldehydes 10 and 11 by activated manganese dioxide oxidation. The aldoxime 12 from the carbaldehyde 11 is dehydrated to yield the nitrile 13. The carbaldehyde 11 reacts with methyl beta-aminocrotonate to yield the 1,4-dihydropyridine (DHP) 14, which is dehydrogenated to give the 3,4'-bipyridine 15. The pyridone 4a reacts with tosylisocyanate to yield the 4-tosylaminopyridine 16a. The antibacterial activity of the carboxylic acids 4b, 5d, 5e and 6d is proved. The growth of Escherichia coli and Bacillus megaterium is inhibited by 5d in the same range as nalidixic acid does.     

Opioid agonists and antagonists. 6,6-Hydrazi and 6-oximino derivatives of 14-hydroxydihydromorphinones

Naloxone (1a), naltrexone (1b), and oxymorphone (1c) were converted to the corresponding 6,6-diaziridines (4a-c), oximes (5a-c), and oxime O-methyl ethers (6a-c). The antagonist derivatives (R = CH2CH = CH2 and R = CH2-c-C3H7) were less active than the parent ketones in the tail-flick assay vs. morphine, by 2-10-fold, except for 6a, which was much less active. The agonist analogues (R = Me) were more active than morphine but less active than dihydromorphine in standard agonist assays. None were significantly longer in duration of action. Thus structural changes at the C-6 position to produce diaziridines, oximes, and oxime O-methyl ethers provide compounds retaining expected opioid activity.     

Synthesis and antifungal activity of novel pyrano[2',3':4,5]thiazolo[2,3-b]quinazolines, pyrido[2',3':4,5]thiazolo[2,3-b]quinazolines and pyrazolo[2',3':4,5]thiazolo[2,3-b]quinazolines

The starting materials thiazolo[2,3-b]quinazolines (5a,b) were obtained in one pot synthesis by treating octahydroquinazoline (2) with chloroacetic acid and aromatic aldehydes. Thiazoloquinazoline (5) was reacted with CH2(CN)2/piperidine and CH2(CN)2/NaOH (CH3OH), to furnish pyrano[2',3':4,5]thiazolo[2,3-b]quinazolines (6a,b) and pyrido[2',3':4,5]thiazolo[2,3-b]quinazoline (7), respectively. Refluxing of 5a with NH2CSNH2/KOH and hydrazines in ethanol furnished the corresponding, [1,3]thiazino[4'5':4,5]thiazolo[2,3-b]quinazoline (10) and pyrazolo[3',4':4,5]thiazolo[2,3-b]quinazolines (11a,b), respectively. Antifungal activity was shown for some of the synthesized compounds.     

Synthesis and pharmacological activity of some 9-substituted delta 8-tetrahydrocannabinol analogues

Several 9-substituted delta 8-tetrahydrocannabinol (delta 8-THC) analogues were synthesized and evaluated for biological activity in mice. Compounds with phenyl (2b) and butyl (2c) substituents were prepared by the addition of phenyllithium and n-butyllithium, respectively, to (-)-9-nor-9-oxohexahydrocannabinol (1), followed by dehydration, whereas, isopropyl (2d), PhCH2 (2e), and Ph(CH2)2 (2f) derivatives were synthesized via the Grignard reaction with subsequent dehydration. Compounds with C2H5CH(OH) (2g) and CH3CH(OH) (2h) substituents at C-9 were prepared from (-)-9-nor-9-formyl-delta 8-tetrahydrocannabinol acetate (3) by the reaction of ethyl and methyl Grignard reagents, respectively. Biological activity indicated that a methyl group at the C-9 position is, thus far, optimum for producing hypoactivity and hypothermia in mice. In addition, hydroxyethyl substitution at position 9 reduced and antinociceptive activity of delta 8-THC, in contrast to the increased activity reported for hydroxymethyl substitution.     

Metabolites of [3-13C]1,2-dibromo-3-chloropropane in male rats studied by 13C and 1H-13C correlated two-dimensional NMR spectroscopy

Metabolism of 1,2-dibromo-3-chloropropane (DBCP) was examined by direct 13C and 1H-13C correlated two-dimensional NMR spectroscopy of bile and urine of male albino rats treated intraperitoneally with [3-13C]DBCP at 81 mg/kg. The 3-13C label was introduced at 99% enrichment by coupling [13C]paraformaldehyde with vinyllithium to give [1-13C]allyl alcohol which was converted to allyl chloride with carbon tetrachloride/triphenylphosphine and then brominated. Fifteen 13C NMR signals were observed for biliary metabolites and twelve for urinary metabolites. Nine of the biliary metabolite 13C NMR signals were very similar or identical to those for nine urinary metabolites. The DBCP-derived moieties of five metabolites were identified by comparison of their 13C NMR chemical shifts, 13C multiplicities [obtained via the distortionless enhancement by polarization transfer (DEPT) pulse sequence], and chemical shifts of the directly-attached protons (obtained via two-dimensional NMR) with those of authentic standards. They were E- and Z-RSCH2CH = 13CHCl, RSCH2CHOH13CH2Cl, RSCH2CHOH13CH2OH and RS13CH2CHOHCH2OH, where R is probably glutathionyl in bile and N-acetylcysteinyl in urine. The mechanism proposed for formation of both the E- and Z-isomers of RSCH2CH = 13CHCl involves radical-initiated dehydrobromination followed by reaction of the intermediate allylic bromides with glutathione (GSH). The RSCH2CHOHCH2Cl conjugate may arise from direct GSH conjugation and hydrolysis of the secondary bromine via a thiiranium ion intermediate. The proposed origin of the RSCH2CHOHCH2OH conjugate labeled at either carbon-1 or carbon-3 is oxidation of DBCP at the bromomethyl or chloromethyl substituent, respectively, followed by two spontaneous dehydrohalogenations to give the highly reactive 2-bromopropenal, and addition of GSH followed by reduction of the aldehyde functionality. An alternative mechanism for the formation of the RSCH2CHOHCH2Cl and RSCH2CHOHCH2OH derivatives involves carbon-2 oxidation to give 1-bromo-3-chloroacetone followed by reaction with GSH and reduction of the ketone functionality with or without hydrolysis of the chloro substituent. 2-Bromopropenal, 1-bromo-3-chloroacetone, or GSH conjugates derived from these intermediates may be involved in the male reproductive toxicity, nephrotoxicity and genotoxicity of DBCP.     

Isolation and identification of 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3: metabolites of 1alpha,24(R)-dihydroxyvitamin D3 produced in rat kidney

1alpha,24(R)-Dihydroxyvitamin D3 [1alpha,24(R)(OH)2D3], a synthetic vitamin D3 analog, has been developed as a drug for topical use in the treatment of psoriasis. At present, the target tissue metabolism of 1alpha,24(R)(OH)2D3 is not understood completely. In our present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in the isolated perfused rat kidney. The results indicated that 1alpha,24(R)(OH)2D3 is metabolized in rat kidney into several metabolites, of which 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxovitamin D3, 1alpha,23(S),25-trihydroxy-24-oxovitamin D3, and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D3 are similar to the previously known metabolites of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. In addition to these aforementioned metabolites, we also identified two new metabolites, namely 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3. The two new metabolites do not possess the C-25 hydroxyl group. Thus, the metabolism of 1alpha,24(R)(OH)2D3 into both 25-hydroxylated and non-25-hydroxylated metabolites suggests that 1alpha,24(R)(OH)2D3 is metabolized in the rat kidney through two pathways. The first pathway is initiated by C-25 hydroxylation and proceeds further via the C-24 oxidation pathway. The second pathway directly proceeds via the C-24 oxidation pathway without prior hydroxylation at the C-25 position. Furthermore, we demonstrated that rat kidney did not convert 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] into 1alpha,25(OH)2D3. This finding indicates that the rat kidney does not possess the classical vitamin D3-25-hydroxylase (CYP27) activity. However, from our present study it is apparent that prior hydroxylation of 1alpha(OH)D3 at the C-24 position in the 'R' orientation allows 25-hydroxylation to occur. At present, the enzyme responsible for the C-25 hydroxylation of 1alpha,24(R)(OH)2D3 is unknown. Our observation that the side chain of 1alpha,24(R)(OH)2D3 underwent 24-ketonization and 23-hydroxylation even in the absence of the C-25 hydroxyl group suggests that 1alpha,25(OH)2D3-24-hydroxylase (CYP24) can perform some steps of the C-24 oxidation pathway without prior C-25 hydroxylation. Thus, we speculate that CYP24 may be playing a dual role in the metabolism of 1alpha,24(R)(OH)2D3.     

Studies on zwitter-ionization of drugs. I. Synthesis and pharmacological activities of N-alkylcarboxylic acid derivatives of 4-(2-chlorodibenz-[b,f][1,4]oxazepin-11-yl)piperazine, 4-(2-chlorodibenzo[b, f]-[1,4]thiazepin-11-yl)piperazine, and 4-(11H-dibenz-[b,e]azepin-6-yl)piperazine]

The piperazine N-alkylcarboxylic acids of 2-chlorodibenz[b,f][1,4] oxazepine (3a), 2-chlorodibenzo[b,f] [1,4] thiazepine (3b), and dipenz[b,e] azepine (3c) from the corresponding piperazines (1a-c, R1 = H) were synthesized via the piperazine N-alkylcarboxylates (2a-c). The pharmacological activities of the piperazine N-alkylcarboxylic acids (3a-c) were evaluated. Compared with the parent compounds (1a-c), 3a-c (n = 1-5) showed weak inhibitory activities on the uptake of noradrenaline and 5-hydroxytryptamine (5-HT) into hypothalamus vesicles and moderate antagonistic actions to 5-HT2 and H1 in several tissues.     

In vitro metabolism of mifepristone (RU-486) in rat,monkey and human hepatic S9 fractions: identification of three new mifepristone metabolites

1. In vitro metabolism of the antiprogestin drug mifepristone (RU-486) was studied after incubation with rat, monkey and human hepatic S9 fractions in the presence of an NADPH-generating system. 2. Unchanged mifepristone (~45% of the sample(s) in rat; ~70% in monkey; ~65% in human) plus six metabolites, three known and three new, were profiled, quantified and tentatively identified on the basis of MS and MS/MS data. 3. The proposed metabolic pathways for mifepristone are proposed, and the two metabolic steps are (A) N-demethylation and (B) methyl oxidation. 4. Step A formed N-desmethyl mifepristone (M1) in major amounts (~35% s in rat, 16% in monkey and human) and N,N-didesmethyl mifepristone (M2) in minor amounts (< 5% s in all species). Step B, or in conjunction with step A, produced four minor trace metabolites, namely hydroxymethyl mifepristone (M3), hydroxymethyl M1 (M4), hydroxymethyl M2 (M5) and formyl mifepristone (M6).     

In vitro metabolism of mifepristone (RU-486) in rat, monkey and human hepatic S9 fractions: identification of three new mifepristone metabolites

1. In vitro metabolism of the antiprogestin drug mifepristone (RU-486) was studied after incubation with rat, monkey and human hepatic S9 fractions in the presence of an NADPH-generating system. 2. Unchanged mifepristone (approximately 45% of the sample(s) in rat; approximately 70% in monkey; approximately 65% in human) plus six metabolites, three known and three new, were profiled, quantified and tentatively identified on the basis of MS and MS/MS data. 3. The proposed metabolic pathways for mifepristone are proposed, and the two metabolic steps are (A) N-demethylation and (B) methyl oxidation. 4. Step A formed N-desmethyl mifepristone (M1) in major amounts (approximately 35% s in rat, 16% in monkey and human) and N,N-didesmethyl mifepristone (M2) in minor amounts (< 5% s in all species). Step B, or in conjunction with step A, produced four minor/trace metabolites, namely hydroxymethyl mifepristone (M3), hydroxymethyl M1 (M4), hydroxymethyl M2 (M5) and formyl mifepristone (M6).     

Excretion, metabolism, and pharmacokinetics of CP-945,598, a selective cannabinoid receptor antagonist, in rats, mice, and dogs

1-(8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl)-4-(ethylamino)piperidine-4-carboxamide (CP-945,598) is an orally active antagonist of the cannabinoid CB-1 receptor that progressed into phase 3 human clinical trials for the treatment of obesity. In this study, we investigated the metabolic fate and disposition of CP-945,598 in rats, Tg-RasH2 mice, and dogs after oral administration of a single dose of [(14)C]CP-945,598. Total mean recoveries of the radioactive dose were 97.7, 97.8, and 99.3% from mice, rats, and dogs, respectively. The major route of excretion in all three species was via the feces, but on the basis of separate studies in bile duct-cannulated rats and dogs, this probably reflects excretion in bile rather than incomplete absorption. CP-945,598 underwent extensive metabolism in all three species, because no unchanged parent compound was detected in the urine across species. The primary metabolic pathway of CP-945,598 involved N-deethylation to form an N-desethyl metabolite (M1). M1 was subsequently metabolized by amide hydrolysis, oxidation, and ribose conjugation to numerous novel and unusual metabolites. The major circulating and excretory metabolites were species-dependent; however, several common metabolites were observed in more than one species. In addition to parent compound, M1, M3, M4, and M5 in rats, M1, M3, and M4 in mice, and M1 and M2 in dogs were identified as the major circulating metabolites. Gender-related differences were also apparent in the quantitative and qualitative nature of the metabolites in rats. An unprecedented metabolite, M4, formed by deamidation of M1 or M3 (N-hydroxy-M1), but not by decarboxylation of M2, was identified in all species. M4 was nonenzymatically converted to M5.     

Synthesis, reactions, and CNS-actions of 6,7-annealed 8-oxatropane derivatives]

The butadiene derivatives 10a-c react with the oxa-bicyclooctanone 9 to give the cyclohexene annulated oxatropanes 11a-c. The acetoxyderivatives 11b and 11c can be transformed into the cyclohexadiene or benzene derivatives 13 and 12, respectively. 11a reacts with HN3 to afford the tricyclic oxazepanone 14 which can be reduced to give 15a; the oxime-tosylate 16b reacts with Al(CH3)3/DIBALH to yield the oxazepane 15c. Treatment of the oxabicyclooctanes 9 and 19 with benzonitrile oxide or diazomethane affords the isoxazoline or pyrazoline annulated oxatropanes 21, 22, and 20, respectively. 21 was reduced to the amino alcohol 23. 15c and 23 show CNS-activity in the mouse.