Testosterone 5alpha-reductase inhibitory active constituents from Anemarrhenae Rhizoma.

The diethyl ether extract of Anemarrhenae Rhizoma (rhizomes of Anemarrhena asphodeloides Bunge) showed testosterone 5alpha-reductase inhibitory activity. Two major constituents, cis-hinokiresinol (1) and 2,6,4'-trihydroxy-4-methoxybenzophenone (2) were identified as the active principles. The inhibitory activity of 1 was superior to that of ethinylestradiol, but that of 2 was weak.

Syntheses and biological evaluation of (18)F-labeled 3-(1-benzyl-piperidin-4-yl)-1-(1-methyl-1H-indol-3-yl)propan-1-ones for in vivo mapping of acetylcholinesterase.

We synthesized novel (18)F-labeled acetylcholinesterase (AChE) inhibitors, 3-[1-(3- and 4-[(18)F]fluoromethylbenzyl)piperidin-4-yl]-1-(1-methyl-1H-i ndol-3-yl )propan-1-ones ([(18)F]1 and [(18)F]2) and 3-[1-(4-[(18)F]fluorobenzyl)piperidin-4-yl]-1-(1-methyl-1H-i ndol-3-yl )propan-1-one ([(18)F]3) in high yields (decay-corrected, 25%-40%) and with high effective specific activities (>37 GBq/micromol). Tissue distribution studies of the [(18)F]1 and the [(18)F]3 in mice showed the nonspecific bindings in brain regions, with metabolic defluorination of the [(18)F]1. The result suggests that these radioligands may not be suitable agents for in vivo mapping of AChE, despite their potent in vitro anti-AChE activities.

Simultaneous determination of a novel calcium entry blocker, monatepil maleate, and its metabolites in rat plasma by means of solid-phase extraction and reversed-phase liquid chromatography with electrochemical detection.

A reversed-phase LC method with electrochemical detection is described for the simultaneous determination of monatepil maleate (AJ-2615, AJ), a novel calcium entry blocker, and its three S-oxidized metabolites in plasma. These compounds were extracted from plasma by solid-phase extraction and injected onto an ODS column. The determination limit in plasma (0.5 ml) was 10 ng/ml for AJ and 5 ng/ml for the three metabolites. The method was applied to the determination of AJ and the metabolites in rat plasma samples.

Identification of rat faecal metabolites of ebastine by B/E linked scanning liquid secondary ion mass spectrometry.

The identification of rat faecal metabolites of a new antihistaminic agent, ebastine, 4'-tert-butyl-4-[4-(diphenylmethoxy)piperidino]butyrophenone, is presented. After oral administration of (14C)ebastine (20 mg kg-1) to rats, 84% of the radioactive dose was excreted in the 24 h faeces. Unchanged drug and five metabolites were isolated from the faeces by thin-layer chromatography and solid-phase extraction, and their structures were identified by liquid secondary ion mass spectrometry using the B/E linked scanning technique. The main metabolic pathways were oxidation of a terminal methyl group to give the hydroxymethyl and carboxyl derivatives, and hydroxylation of a phenyl ring in the diphenylmethoxy moiety. In addition to the oxidative mechanism, metabolism of ebastine involved sulphate conjugation. It is noteworthy that M-4, having both phenolic and alcoholic hydroxyl groups, was sulphated selectively in the latter position.

Determination of gliclazide in serum by high-performance liquid chromatography using solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic method for a routine assay of gliclazide in serum is described. Serum samples spiked with glibenclamide (internal standard) were applied to Bond Elut C18 cartridges. After washing with phosphate buffer (pH 7.5) and water, the cartridge was eluted with 60% methanol. The eluate was evaporated to dryness. The residue was dissolved in methanol and injected onto an octadecyl silica column (5 microns, 150 mm x 4.6 mm I.D.). The mobile phase was 0.04 M potassium dihydrogenphosphate (pH 4.6)-acetonitrile-isopropyl alcohol (5:4:1, v/v). Ultraviolet detection at 227 nm was used. The minimum detectable level of gliclazide was 20 ng/ml.

Determination of AJ-3941, a possible agent for the treatment of cerebrovascular disorders, in plasma and brain by means of high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method with fluorescence detection is described for the determination of AJ-3941 (I), a possible agent for the treatment of cerebrovascular disorders, in plasma and brain tissue. A simple hexane extraction was used for plasma, and for brain homogenate the hexane extract was further purified by solid-phase extraction. The determination limit was ca. 3 ng/ml for both plasma (0.5 ml) and 10% (w/v) brain homogenate (1 ml). The method was applied to the determination of I in plasma and brain samples of experimental animals.

Identification of AJ-2615 and its S-oxidized metabolites in rat plasma by use of tandem-mass spectrometry.

The structural elucidation of the metabolites of a new calcium entry blocker, AJ-2615 (AJ), in rat plasma is described. Metabolites in a crude plasma extract from spontaneously hypertensive rats were identified without chromatographic separation by fast atom bombardment tandem mass spectrometry. When the plasma extract was examined by using parent ion scans, the presence of the oxidized metabolites of AJ was suggested. These metabolites were identified as the S-oxide and the S,S-dioxide by comparing their daughter ion spectra with those of authentic samples. The presence of the two diastereomeric S-oxides of AJ in the plasma extract was ascertained by high-performance liquid chromatography. Their relative configurations were determined by infrared and proton nuclear magnetic resonance spectra.

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection.

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm x 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)-acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.

Synthesis and biological activity of 11-[4-(cinnamyl)-1-piperazinyl]- 6,11-dihydrodibenz[b,e]oxepin derivatives, potential agents for the treatment of cerebrovascular disorders.

A series of 11-[4-(cinnamyl)-1-piperazinyl]-6,11-dihydrodibenz[b,e] oxepins and related compounds were synthesized and evaluated for their protective activities against complete ischemia, normobaric hypoxia, lipidperoxidation and convulsion. Structure-activity relationship studies of this series led to the finding of (E)-1-(3-fluoro-6,11-dihydrodibenz[b,e]oxepin-11-yl)-4-(3- phenyl-2-propenyl)piperazine dimaleate (50), AJ-3941 with the most appropriate property for combined pharmacological activities. Compound 50 also shows an inhibitory effect against cerebral edema as well when orally given to rats.

A new class of calcium antagonists. 2. Synthesis and biological activity of 11-[[4-[4-(4-fluorophenyl)-1-piperazinyl]butyryl]amino]-6,11- dihydrodibenzo[b,e]-thiepin maleate and related compounds.

A series of [(epsilon-aminoalkanoyl)amino]-6,11- dihydrodibenzo[b,e]thiepins and -5H-dibenzo[a,d]cycloheptenes and related compounds were synthesized and evaluated for calcium antagonistic activity by calcium-induced constriction of potassium-depolarized rat aorta. Semiempirical molecular orbital calculations of the dibenzotricyclic systems indicated that calcium antagonistic activity increased with a decrease of the angle between the planes of the two phenyl rings. AM1 net charge calculations showed that a neutral or positive charge distribution in the bridge portion was necessary for activity. 11-[[4-[4-(4-Fluorophenyl)-1- piperazinyl]butyryl]amino]-6,11-dihydrodibenzo[b,e]thiepin maleate (16, AJ-2615) showed a more gradual and longer lasting antihypertensive effect than diltiazem and nifedipine in spontaneously hypertensive rats (SHR) administered orally. Compound 16 also possessed antianginal effects in methacholine-induced ST elevation and vasopressin-induced ST depression tests in rats. The alteration of the dibenzotricyclic system of 16 to 5H-dibenzo[a,d]cycloheptene (19, 5-[[4-[4-(4-fluorophenyl)-1-piperazinyl]-butyryl]amino]-5H- dibenzo[a,d]cycloheptene) resulted in selectivity for cardiac tissue over vascular tissue, thereby conferring antianginal activity without an effect on blood pressure. Antianginal potencies of 16 and 19 were equal to or somewhat more potent than those of diltiazem.

Novel benzamides as selective and potent gastric prokinetic agents. 1. Synthesis and structure-activity relationships of N-[(2-morpholinyl)alkyl]benzamides.

With the purpose of obtaining more potent and selective gastric prokinetic than metoclopramide (1), a new series of N-[(2-morpholinyl)alkyl]benzamides (17-52) were synthesized and their gastric prokinetic activity was evaluated by determining effects on the gastric emptying of phenol red semisolid meal and of resin pellets solid meal in rats and mice. The morpholinyl moiety was newly designed after consideration of the side-chain structure of cisapride (2) and produced the desired activity when coupled with the 4-amino-5-chloro-2-methoxybenzoyl group of both metoclopramide and cisapride. Modification of the substituents of the benzoyl group markedly influenced the activity. In particular, 4-amino-N-[(4-benzyl-2-morpholinyl)methyl]-5-chloro-2-methoxybenzamide (17) and the 4-(dimethylamino) and 2-ethoxy analogues (25 and 29) of 17 showed potent and selective gastric prokinetic activity along with a weak dopamine D2 receptor antagonistic activity.

Synthesis and antiinflammatory activity of cis- and trans-6,6a,7,8,9,10,10a,11-octahydro-11-oxodibenzo[b,e]thiepinacetic and -oxepinacetic acids.

A series of cis- and trans-6,6a,7,8,9,10,10a,11-octahydro-11- oxodibenzo[b,e]thiepinacetic acids (6-9) and -oxepinacetic acids (10-13) were prepared and their antiinflammatory activity was examined in the rat carrageenan hind paw edema test. The antiinflammatory activity of these compounds depended on their stereochemical features (C6a, C10a, and C2'). The 6a,10a-trans compounds exhibited considerable antiinflammatory activity, whereas the 6a,10a-cis compounds were inactive. Among the trans compounds, 6,6a,7,8,9,10,10a,11-octahydro-11-oxodibenzo[b,e]thiepin-3-p ropionic acid (9a) and its oxepin analogue (13a) showed an antiinflammatory activity superior to that of indomethacin. The phenethyl ester (25) of 9a showed potent antiinflammatory activity, and its safety index (UD50/ED50) was over 14 times higher than that of indomethacin. The phenethyl ester (25) is the most favorable compound with high antiinflammatory activity and little ulcerogenicity.

Competitive binding enzyme immunoassay for zonisamide, a new antiepileptic drug, with selected paired-enzyme labeled antigen and antibody.

We assessed the competitive binding between zonisamide (ZNS) in serum samples and beta-galactosidase-labeled ZNS derivatives, using competing antibodies to ZNS derivatives, and selected the best enzyme-labeled antigen and antibody for accurate enzyme immunoassay (EIA) of ZNS in serum without interference from its metabolites or from other antiepileptic drugs. This EIA, based on use of antibody linked to bacterial cell walls, has advantages over HPLC in simplicity, speed (50 samples per hour), and lack of requirement for special equipment. The concentrations of ZNS in serum as measured by the EIA correlated well with those by HPLC (n = 33, r = 0.977).

Propioxatins A and B, new enkephalinase B inhibitors. IV. Characterization of the active site of the enzyme using synthetic propioxatin analogues.

Propioxatins A and B are inhibitors of enkephalinase B, which hydrolyzes enkephalin at the Gly-Gly bond. In order to clarify the structure-activity relationships of propioxatin, several compounds were synthesized and their inhibitory activity for not only enkephalinase B but also enkephalinase A was examined. The hydroxamic acid group in propioxatin was primarily essential for coordinating the metal ion in the active site of the enzyme. Among devalyl propioxatin A derivatives, the proline-containing compounds inhibited enkephalinase B and others inhibited both enzymes. An alteration of the character of the P3' amino acid valine in propioxatin A, e.g. amidation of carboxylic acid or replacement of the side chain, caused a 2 to 400-fold decrease of the inhibitory activity for enkephalinase B or an appearance of enkephalinase A inhibition with Ki values in the micromolar range. Substitution of the proline by alanine also resulted in a 1,000-fold loss of inhibitory activity for enkephalinase B. Propioxatin A was the most potent and specific inhibitor of enkephalinase B among the synthesized compounds. These potent and specific inhibitory effects were caused by the P2' proline residue, the P3' valine side chain and its free carboxylic acid. Each of the S1', S2', and S3' subsites in an enkephalinase B active site has a large and hydrophobic pocket, but the arrangement might be unique. The results could explain why enkephalinase B does not hydrolyze longer peptides.

Determination of proscillaridin in plasma by radioimmunoassay.

The development of a sensitive and selective radioimmunoassay for determination of proscillaridin in plasma is described. Antiserum against proscillaridin was obtained from guinea pig immunized with an immunogen prepared by conjugating methylproscillaridin, 14 beta-hydroxy-3 beta-[(4-O-methyl-alpha-L-rhamnosyl)oxy]bufa-4,20,20, 22-trienolid to bovine serum albumin. Methyl N-[3-[(14 beta-hydroxybufa-4,20,22-trienolid-3 beta-yl)oxy-carbonyl] propanoyl]-L-[3',5'-125I2] diiodotyrosinate ([125I] SST) was used as a radioactive ligand. [125I] SST and antiserum were added to the recovered sample from plasma using a Bond Elut column. After overnight incubation, the antibody-bound and free [125I] SST were separated using polyethylene glycol. The mean coefficients of variation of intra and interassay at four different plasma concentrations were 4.0 and 5.0%, respectively. The assay was able to determine as little as 125 pg/ml of proscillaridin in plasma by using 1.5 ml of sample. Beagle dogs were orally administered one tablet (Talusin) containing 0.25 mg of proscillaridin. Proscillaridin was rapidly absorbed, exhibiting plasma maximum concentration of 2.06 ng/ml at 20 min. Thereafter, the plasma level declined biphasically with half-lives of 0.6 and 25.4 h.

Synthesis and antiinflammatory activity of [(cycloalkylmethyl)phenyl]acetic acids and related compounds.

[(Cycloalkylmethyl)phenyl]acetic acid derivatives and related compounds were synthesized to test their antiinflammatory and analgesic activities. Some of the compounds in this series were found to have good activity in the carrageenan edema test. Among them, sodium 2-[4-[(2-oxocyclopentyl)methyl] phenyl]propionate dihydrate (15) and 2-[4-[(2-oxocyclohexylidene)methyl]phenyl]propionic acid (13b) showed potent analgesic and antiadjuvant arthritis activities with excellent antipyretic properties.