Novel antidiabetic and hypolipidemic agents. 3. Benzofuran-containing thiazolidinediones.

Several thiazolidinedione derivatives having 5-hydroxy-2,3-dihydro-2, 2,4,6,7-pentamethylbenzofuran moieties and their 5-benzyloxy derivatives and 5-hydroxy-2,4,6,7-tetramethylbenzofuran moieties were synthesized and evaluated in db/db mice. Insertion of an N-Me group into the linker between thiazolidinedione and substituted benzofuran pharmacophores showed considerable improvement in their euglycemic activity. Further improvement has been observed when a pyrrolidine moiety is introduced in the structure to give 5-[4-[N-[3(R/S)-5-benzyloxy-2,3-dihydro-2,2,4,6, 7-pentamethylbenzofuran-3-ylmethyl]-(2S)-pyrrolidin-2- ylmethoxy]pheny lene]thiazolidine-2,4-dione (21a). At a 100 mg/kg/day dose of the maleate salt, compound 21a reduced the plasma glucose and triglyceride to the level of lean littermate, i.e. 8 +/- 1 mM, and is the most potent and efficacious compound reported in this series.

Oral bioavailability and pharmacokinetics of the new insulin sensitizer DRF-2189 in Wistar rats.

The pharmacokinetics of the new insulin sensitizing agent, DRF-2189 ([5-[4-[2-(1-indolyl) ethoxy]phenyl]methyl]thiazolidine-2,4-dione, CAS 172647-53-9) were studied in male Wistar rats following oral doses of 1, 3 and 10 mg/kg as suspension in 0.25% carboxymethylcellulose. Drug was extracted from plasma samples using a solvent mixture containing ethylacetate and dichloromethane (3:2) and analyzed by high-performance liquid chromatography with fluorescence detection. DRF-2189 was absorbed slowly, attaining maximum levels at 2-3 h, and was eliminated with a half-life (t1/2) of about 3 h. The Cmax and AUC(0-infinity) increased linearly (r2 = 0.99) with the dose, while the elimination half-life (t1/2) was independent of the dose. An intravenous pharmacokinetic study of DRF-2189 was carried out in Wistar rats at a dose of 3.0 mg/kg. The pharmacokinetic parameters AUC(0-infinity), t1/2, plasma clearance (Cl) and volume of distribution (Vd) were found to be 49.52 micrograms x h/ml, 2.99 h, 16.31 ml/h and 45.11 ml. respectively. Oral bioavailability (f) of DRF-2189 in Wistar rats was 44%. Based on pharmacokinetic studies, DRF-2189 is a good choice for further development.

High-performance liquid chromatographic determination of the insulin sensitizing agent DRF-2189 in rat plasma.

A high-performance liquid chromatographic method for the determination of DRF-2189, using troglitazone as internal standard, is described. A dichloromethane-ethyl acetate solvent mixture (6:4, v/v) was used as the extraction solvent. A Kromasil C18 column with a mobile phase consisting of 0.05 M phosphate buffer-acetonitrile-methanol (22.5:37.5:40) (pH 5.0) was used at a flow-rate of 1.0 ml/min. The eluate was monitored by using fluorescence detection with excitation and emission wavelengths at 292 nm and 325 nm, respectively. Ratio of peak area of analyte to internal standard was used for quantification of plasma samples. Using this method, the absolute recovery of DRF-2189 from rat plasma was >95% and the limit of quantitation was 50 ng/ml. The intra-day relative standard deviation (R.S.D.) ranged from 1.74 to 7.24% at 1 microg/ml and 1.86 to 3.83% at 10 microg/ml. The inter-day R.S.D.s were 8.34 and 4.91% at 1 and 10 microg/ml, respectively. The method was applied to measure plasma concentrations of DRF-2189 in pharmacokinetic studies in Wistar rats.

Novel euglycemic and hypolipidemic agents. 1.

A series of [[(heterocyclyl)ethoxy]benzyl]-2,4-thiazolidinediones have been synthesized by the condensation of corresponding aldehyde 1 and 2,4-thiazolidinedione followed by hydrogenation. Both unsaturated thiazolidinedione 2 and its saturated counterpart 3 have shown antihyperglycemic activity. Many of these compounds have shown superior euglycemic and hypolipidemic activity compared to troglitazone (CS 045). The indole analogue DRF-2189 (3g) was found to be a very potent insulin sensitizer, comparable to BRL-49653 in genetically obese C57BL/6J-ob/ob and 57BL/KsJ-db/db mice. Pharmacokinetic and tissue distribution studies conducted on BRL-49653 and DRF-2189 (3g) indicate that these drugs are well-distributed in target tissues. On the basis of euglycemic activity as well as enhanced selectivity against reduction of triglycerides in plasma, DRF-2189 (3g) has been selected for further evaluation.

An efficient route to N6 deoxyadenosine adducts of diol epoxides of carcinogenic polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are metabolized to a wide variety of oxidized derivatives, including highly reactive diol epoxides which alkylate DNA. The reaction lacks regio- or stereospecificity but occurs primarily at the exocyclic amino groups of deoxyguanosine and deoxyadenosine. An efficient route to N6 adducts of deoxyadenosine is described using as examples those arising from trans opening of the anti-tetrahydrodiol epoxides of naphthalene, benzo[a]pyrene, and benzo[c]phenanthrene. The adducts were synthesized in 50-92% yields by reaction of 6-fluoropurine 2'-deoxyriboside with aminotriols formed by trans opening of racemic dihydrodiol epoxides using liquid NH3. The diastereomeric adducts were separated by HPLC and their absolute configurations were assigned by circular dichroism. 1H NMR studies revealed significant differences in conformation of the tetrahydroaromatic ring between the sterically unrestricted naphthalene derivative and the sterically congested derivatives of benzo[a]pyrene and benzo[c]phenanthrene. These differences may have a bearing on the higher carcinogenicity shown by the latter hydrocarbons. Undecadeoxyoligonucleotides bearing regio- and stereochemically defined adenine N6-anti-trans-benzo[a]pyrene adducts have been prepared.

A thermospray liquid chromatography/mass spectrometry method for analysis of human urine for the major malondialdehyde-guanine adduct.

A method is described for detection and quantitation of the major malondialdehyde-guanine adduct (M1G) based on thermospray liquid chromatography/mass spectrometry. A stable isotope analog of M1G ([2H2]M1G) was used as an internal standard. Thermospray mass spectra of M1G and [2H2]M1G showed intense protonated molecular (MH+) ions that were suitable for use in quantitation of M1G. M1G was purified from human urine and reduced with NaBH4 to a dihydro derivative that was cleanly separated from the contaminants in the urine. The detection limit of reduced M1G by thermospray liquid chromatography/mass spectrometry in the selected ion monitoring mode was 250 fmol on column. Six human urine samples were analyzed, and the concentrations of M1G were below the limit of detection of the assay (500 fmol/mL).

Thermospray liquid chromatographic-mass spectrometric analysis of anti-AIDS nucleosides: quantification of 2',3'-dideoxycytidine in plasma samples.

Thermospray liquid chromatography-mass spectrometry was investigated as a method for quantification of 2',3'-dideoxycytidine (DDC) from human plasma. A stable isotope analog of DDC ([15N2,2H2]DDC) was used as an internal standard. Selected ion monitoring of the protonated molecular ions for DDC and the internal standard was used to record mass chromatograms. The areas of the peaks in the mass chromatograms were used for quantification. The detection limit of DDC in this assay was 50 pg on-column. The calibration curve was linear over the desired range, 0.25-20 ng/ml. The major advantages of this assay over others are: no derivatization, high sensitivity, high specificity and short assay time.

Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes.

The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihydroxyeicosatetraenoic acids generated by omega/omega-1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B4 in double-bond geometry alone. The other two differed from leukotriene B4 in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids.

Metabolism of the antipsychotic drug tiospirone in humans.

Metabolism of the antipsychotic drug tiospirone was studied in humans after a single 60-mg oral dose of [14C]tiospirone. Metabolites were isolated from a 0-24 hr pooled urine from eight subjects, which represented 39% of the dose, and purified to homogeneity by HPLC. Purified metabolites were identified by desorption chemical ionization mass spectrometry in the positive ion mode with methane as a reagent gas. Structures of the metabolites were confirmed by coelution on HPLC in several systems with synthetic standards. In addition to unchanged tiospirone, five metabolites of tiospirone were identified and one additional metabolite was partially identified. Based on the structures of these metabolites, five routes of metabolism of tiospirone were identified: N-dealkylation of the butyl side chain attached to the piperazinyl nitrogen, hydroxylation alpha to the glutarimidyl carbonyl at the 6'-position on the spiro ring, hydroxylation at the 3'-position on the spiro ring, oxidation at sulfur resulting in the formation of sulfones, and oxidation at carbon alpha to the piperazinyl nitrogen resulting in the formation of a lactam-sulfone. The major urinary metabolites were benzisothiazole piperazine sulfone and its lactam derivative, accounting for 5.0 and 4.3% of the dose, respectively. The identified metabolites accounted for 50% of the total radioactivity in the urine (approximately 20% of the dose). The remaining radioactive components were extremely heterogeneous and could not be isolated in sufficient quantities to characterize. A scheme for the metabolism of tiospirone in humans is proposed.

In vitro metabolism of the antianxiety drug buspirone as a predictor of its metabolism in vivo.

1. Metabolism of the antianxiety drug buspirone was studied by in vitro incubations with rat liver microsomes and hepatocytes. Metabolites were isolated and purified by h.p.l.c. The purified metabolites were identified by co-elution on h.p.l.c. with authentic standards and by g.l.c.-electron impact mass spectrometry of their trimethylsilyl (TMS) derivatives. 2. Five metabolites of buspirone were identified in the microsomal incubates and seven in the hepatocyte incubates. The major metabolites arose from aromatic hydroxylation at C-5, N-dealkylation of the butyl chain, and hydroxylation at C-6' and C-3' on the azaspirodecanedione moiety. 3. Metabolism of buspirone by rat liver microsomes was NADPH-dependent and was completely inhibited by cytochrome P-450 inhibitors SKF-525A and metyrapone. 4. Metabolites of buspirone formed in vitro were good predictors of the primary metabolites formed in vivo. 5. Hepatocytes and phenobarbital-induced rat liver microsomes were better predictors of in vivo metabolism of buspirone than non-induced rat liver microsomes. These in vitro systems should provide excellent models for studying the metabolism of other azaspirodecanedione-containing drugs.

Characterization of in vitro metabolites of the antipsychotic drug tiospirone by mass spectrometry.

Metabolism of the antipsychotic drug tiospirone was studied in vitro with phenobarbital-induced rat liver microsomes. Metabolites were isolated and purified to homogeneity by high-performance liquid chromatography. It was possible to characterize the metabolites as trimethylsilyl (TMS) derivatives by gas chromatography/electron impact mass specrometry (GC/EIMS) so long as the sulfur was present in the reduced form. However, sulfoxide and sulfone analogs of tiospirone underwent reductive decomposition on the GC column. In addition, no molecular ions were observed in the EI spectra of these analogs. Desorption chemical ionization/mass spectrometry (DCI/MS) in the positive ion mode with methane as reagent gas successfully distinguished sulfoxides and sulfones from their parent sulfides. Protonated molecular ions were observed together with structurally significant fragment ions. Five microsomal metabolites of tiospirone were characterized by a combination of GC/EIMS and DCI/MS. From the structures of the metabolites three major pathways of metabolism were identified: N-dealkylation of the butyl side chain at the piperazinyl nitrogen, hydroxylation alpha to the glutarimidyl carbonyl at C-6 on the azaspirodecanedione ring, and sulfoxide formation on the benzisothiazole moiety.

Structural characterization of urinary metabolites of the antiarrhythmic drug encainide in human subjects.

Metabolism of the antiarrhythmic drug encainide was studied in human subjects after a single 50-mg oral dose. Encainide labeled on the carbonyl carbon with 14C and at the benzylic (2'-1-ethyl) carbon with 13C was administered to four normal healthy male subjects. A large proportion of the radioactive dose (42%) was excreted in the urine in the first 24 hr. The total urinary excretion was 47.0 +/- 4.6% and total fecal excretion was 38.7 +/- 5.7% over 5 days. The conjugated metabolites excreted in the urine were hydrolyzed with beta-glucuronidase/arylsulfatase, and were isolated and purified by HPLC. Structural characterization was carried out by a combination of fast atom bombardment-mass spectrometry, gas chromatography/electron impact mass spectrometry, and 1H-NMR spectroscopy. Structures of the metabolites were confirmed by co-elution on HPLC with authentic standards when available. Six metabolites of encainide were identified from the hydrolyzed urine together with unchanged drug. In addition to already known metabolites O-demethyl-encainide, 3-methoxy-O-demethyl-encainide, and N,O-di-demethyl-encainide, three new metabolites were identified: N-demethyl-3-methoxy-O-demethyl-encainide, 3-hydroxy-encainide, and O-demethyl-encainide-lactam. These metabolites accounted for greater than 90% of the radioactivity excreted in the urine. Four major routes of metabolism were identified: first, O-demethylation of the aromatic methyl ether; second, formation of methylated catechol derivatives; third, N-demethylation of the piperidyl nitrogen; and fourth, oxidation at carbon alpha to the piperidyl nitrogen. A plausible scheme for the metabolism of encainide in human subjects is proposed.

Metabolism of the antianxiety drug buspirone in the rat.

The metabolism of an orally administered, 10-mg single dose of the antianxiety drug buspirone was studied in the rat. Samples of bile and urine were collected for 6 hr and were treated with beta-glucuronidase/arylsulfatase. The deconjugated metabolites were isolated and purified by HPLC. Structural analysis was carried out by combined gas chromatography/electron impact mass spectrometry as their trimethylsilyl derivatives and by 1H-NMR spectroscopy. Structures of the metabolites were further confirmed by co-elution on HPLC with authentic standards when possible. In addition to the already known metabolites 5-hydroxy-buspirone and 1-pyrimidinylpiperazine, seven major metabolites were unambiguously identified together with unchanged drug. Ten minor metabolites were partially characterized. Hydroxylation alpha to the glutaramidyl carbon at the 6'-position on the bicyclo ring system, hydroxylation on the pyrimidine aromatic ring, and N-dealkylation of the butyl side chain were observed as major routes of metabolism. Minor routes of metabolism observed were: 3'-hydroxylation on the bicyclo ring system and formation of the methylated catechol derivatives. The identified metabolites accounted for greater than 90% of the total metabolites excreted in the rat bile and urine samples.

Metabolism of the antianxiety drug buspirone in human subjects.

The metabolism of an oral dose (20 mg) of the antianxiety drug buspirone labeled with 14C/15N was studied in human subjects. 15N was incorporated in the molecule to facilitate structural characterization of the metabolites by mass spectrometry. Urine samples were collected at intervals up to 24 hr and analyzed for radioactivity. Cumulative urinary excretion accounted for 50% of the dose in 24 hr. The urine was hydrolyzed with beta-glucuronidase/arylsulfatase and the deconjugated metabolites were isolated and purified by HPLC. The purified metabolites were identified by GC/MS, 1H-NMR, and comparison with authentic standards when available. Seven metabolites of buspirone were identified unambiguously, together with unchanged drug. Hydroxylation alpha to the glutarimidyl carbonyl at the 6'-position on the spiro ring system, hydroxylation at the 5-position on the pyrimidine ring, and N-dealkylation of the butyl-substituted side chain were major routes of metabolism. The identified metabolites accounted for 88% of the total radioactivity in the urine. A scheme for metabolism of buspirone in human subjects has been proposed.

Resolution of enantiomers of the antiarrhythmic drug encainide and its major metabolites by chiral derivatization and high-performance liquid chromatography.

Commercially available chiral columns were unable to provide adequate resolution of enantiomers of the antiarrhythmic drug encainide or its major metabolites. The homochiral derivatizing agent, (-)-menthyl chloroformate, was found to react at the tertiary piperidine nitrogen of racemic encainide providing two menthyl carbamate diastereomers. The individual diastereomers could be separated with baseline resolution on normal-phase high-performance liquid chromatography on a silica column. Structures of the derivatives were confirmed by electron impact mass spectrometry and 1H NMR spectroscopy. The method was adapted for the chiral analysis of the major metabolites of encainide. The limit of sensitivity for racemic encainide was 10 ng on column and it was possible to detect a mixture containing (+)- and (-)-encainide in a ratio of 1:99. Preliminary studies indicated that (-)-encainide was O-demethylated to a greater extent than the (+)-enantiomer by rat liver microsomes.