Biokinetics of a new prodrug gidazepam and its metabolite]

Pharmacodynamics and pharmacokinetics of a novel tranquilizing agent--gidazepam (I), a prodrug, and its physiologically active metabolite--7-bromo-5-phenyl-1,2-di-hydro-3H-1,4- benzodiazepine-2-one (II) in mice organism were studied. The form of relationship was determined between the dynamics of the anticonvulsant effect of labelled (2-14C-) I and II and the kinetics of the content of 14C-compounds in the experimental animals brain. It was noted that the biophase of the effect and the effector fragment of the scheme of biokinetics for I and II are identical. The effector prognosis of pharmacokinetics of I was realized. The comparison of the main characteristics of biokinetics for the prodrug (I) and drug (II) allowed us to reveal the nature of the quantitative differences of these pharmacological effects.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Enantiomerization of 3-carbethoxy-l,4-benzodiazepin-2-one: combined chiral HPLC and spectroscopic study.

Recently developed chiral HPLC columns CHIRIS AD1 and CHIRIS AD2 have been demonstrated to separate racemic, configurationally unstable ethyl-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-carboxylate (1) and its 3-methyl congener 2; fast on-column enantiomerization of configurationally unstable 1 was observed, however. Addition of 0.1% of AcOH to the eluting mixture inhibits enantiomerization, whereas the same percentage of Et(3)N completely precludes enantioseparation, suggesting base-catalysis by free beta-aminoethyl groups, present in low percentage in chiral stationary phase (CSP). When both CSPs were prepared under conditions of nonexhaustive acylation by N-DNB-alpha-aminoacids, no separation of 1 was observed. The rate of enantiomerization on CHIRIS AD2 was determined at 25 degrees C, the mechanism is discussed, and experimental results correlated with calculated relative stabilities of the tautomers la-c. Absolute (3S) configuration of (+) enantiomers of 1 and 2 was determined by comparison of their eluation profile to that of (+/-)-3 and (3S)-(+)-3, taking into account relative (psia or psie) configuration of the prevailing conformer in solution.     

Synthesis and biological evaluation of 1,4-dihydropyridine calcium channel modulators having a diazen-1-ium-1,2-diolate nitric oxide donor moiety for the potential treatment of congestive heart failure

A group of racemic 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridines possessing nitric oxide donor O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino, or 4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituents were synthesized by coupling the respective 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylic acids with either O(2)-acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate, or O(2)-acetoxymethyl-1-[4-(2-methylsulfonyloxyethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. Compounds having a C-4 2-pyridyl, 4-pyridyl, 2-trifluoromethylphenyl, or benzofurazan-4-yl substituent exhibited more potent smooth muscle calcium channel antagonist activity (IC(50)'s in the 0.37-1.09 microM range) than related analogs having a C-4 3-pyridyl substituent (IC(50)'s=3.03-9.14 microM range) relative to the reference drug nifedipine (IC(50)=9.13 nM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of smooth muscle calcium channel antagonist activity where the relative potency profile was 4-pyridyl>2-pyridyl>3-pyridyl. Replacement of the C-5 methyl ester substituent of methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644) by an O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate, or O(2)-acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituent provided compounds, which exhibited a lower, yet respectable, cardiac positive inotropic effect (IC(50)'s=4.82 and 4.05 microM, respectively) relative to the reference drug Bay K 8644 (IC(50)=0.30 microM). All compounds released nitric oxide upon incubation with either phosphate buffer at pH7, or porcine liver esterase. However, the percentage nitric oxide released was up to 3-fold higher (76%) when these O(2)-acetoxymethyl-1-(alkylamino)diazen-1-ium-1,2-diolates were incubated with guinea pig serum. These results suggest that *NO would be released in vivo, upon cleavage by nonspecific serum esterases, preferentially in the vascular endothelium where it may enhance smooth muscle calcium channel antagonist activity.     

Hantzsch 1,4-dihydropyridines containing a diazen-1-ium-1,2-diolate nitric oxide donor moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of racemic 3-isopropyl 5-[(2-piperazin-1-yl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (12a-c), 3-isopropyl 5-{2-[4-nitrosopiperazinyl]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (14a-c) and 3-isopropyl 5-{2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (22-30) were prepared using modified Hantzsch reactions. This group of compounds (12a-c, 14a-c, and 22-30) exhibited less potent calcium channel antagonist activity (IC(50)=0.11 to 3.35muM range) than the reference drug nifedipine (IC(50)=0.01 microM). The point of attachment of the isomeric C-4 substituent was a determinant of calcium channel antagonist activity providing the potency profile 2-pyridyl3-pyridyl4-pyridyl. The N-nitrosopiperazinyl compounds (14a-c) did not release nitric oxide. The prodrugs 22-30 that have a C-5 2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl ester substituent, upon incubation with guinea pig serum, undergo consecutive cleavage of the O(2)-acetoxymethyl moiety to give a nitric oxide donor diazenium-1-ium-1,2-diolate species that subsequently releases nitric oxide. The extent of nitric oxide released from the diazen-1-ium-1,2-diolate group is dependent upon the nature of the amino functionality attached directly to the diazen-1-ium N-1 position where the nitric oxide release profile is 1,4-piperazinyl>N-Et>N-(n-Bu)>N-Me upon exposure to guinea pig serum esterase(s). The results from this study suggest this class of hybrid calcium channel antagonist/nitric oxide donor prodrugs should release the vasodilator nitric oxide in vivo, preferentially in the vascular endothelium, to enhance the smooth muscle calcium channel antagonist effect to produce a combined synergistic antihypertensive effect.     

Microbiological degradation of diazepam.

In studying the transformation of diazepam (7-chloro-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one) by fungi isolated from soil. N1-demethylation and cleavage of the diazepine ring were observed. Three metabolites: 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, 2-acetamido-2"-benzoyl-4"-chloroacetanilide and 2-acetamido-2"-benzoyl-4"-chloro-N-methylacetanilide were isolated and identified.     

Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

Determination of the major urinary metabolite of flurazepam in man by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of N-1-hydroxyethylflurazepam, the major urinary metabolite of flurazepam, in human urine is described. Urine specimens were incubated enzymatically to deconjugate N-1-hydroxyethylflurazepam glucuronide (metabolite) and were then extracted at pH 9.0 to extract the metabolite. The extracts were chromatographed on a microparticulate silica gel column using automatic sample injection, isocratic elution at ambient temperature and UV monitoring at 254 nm. The internal stanard was 7-chloro-5-(2′-chlorophenyl)-1,3-dihydro-1-2-dimethylaminoethyl-2H-1,4-benzodiazepine-2-one. The recovery from urine, in the 0.5–25.0 μg/ml range, was 96.5 ± 11.5% (S.D.), and the sensitivity limit was 0.5 μg/ml. The method was found to be specific for N-1-hydroxyethylflurazepam in the presence of intact flurazepam and other possible urinary metabolites of flurazepam. The method was successfully applied to urine specimens collected from human subjects following the administration of 30-mg single oral doses of flurazepam dihydrochloride.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

Use of a carrier for quantitation of a new dihydropyridine calcium antagonist (OPC-13340) in human plasma by highly sensitive gas chromatography with negative-ion chemical ionization mass spectrometry

A sensitive gas chromatographic—mass spectrometric method for the quantitation of (±)-methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (OPC-13340, I), a new dihydropyridine calcium antagonist with a potent and long-acting antihypertensive and antianginal effect, was developed in order to elucidate its pharmacokinetics. Dihydropyridine calcium antagonists have been usually quantified by this technique in the negative-ion chemical ionization mode. However, direct application of this method to quantify trace amounts of I in biological fluids completely failed, owing to its adsorption on the column and oxidation of its dihydropyridine ring. Human plasma containing I and (±)-[²H₅]methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (II), the internal standard, was extracted with n-hexane—diethyl ether under weakly basic conditions (pH 8). In order to prevent adsorption of the compounds on the column, (±)-[²H₃]ethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (III), an analogue of I, was added to the extracts as a carrier. In addition, this carrier was also effective in preventing the oxidation of I. The quantitation limit of I in human plasma by this method was found to be less than 30 pg/ml. Thus, the method is sufficiently sensitive to study the pharmacokinetics of I in humans.     

Bioactivation of MPTP: reactive metabolites and possible biochemical sequelae

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.     

The utility of 1-[18F]fluoro-3-iodopropane for the synthesis of certain dopamine D-1 and benzodiazepine receptor radioligands

No-carrier-added (NCA) R(+)-7-chloro-8-hydroxy-3-(3′-[¹⁸F]fluoropropyl)-1-phenyl-2,3,4,5-tetrahydro-3-benzazepine (2b) (an analog of dopamine D-1 receptor ligand SCH 23390), ethyl 8-fluoro-5,6-dihydro-5-(3′-fluoropropyl)-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (4b) and 3′-[¹⁸F]fluoropropyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (6b) (analogs of the benzodiazepine RO 15-1788) were synthesized by alkylation of the corresponding nor-compound with NCA 1-[¹⁸F]fluoro-3-iodopropane in 10–15% yield (EOB) in ~110min and with a mass of 2–3nmol. Compound 2 is less potent (~ 12–14 times) than SCH 23390 in binding to rat striatal membranes in vitro. Compounds 2b, 4b and 6b exhibit no specific anatomical distribution to mouse brain. These results suggest that the substituent at position 3 of SCH 23390, and position 5 and carboxylate group of RO 15-1788 are critical determinants both of affinity and selectivity for receptor binding, and underscores the evaluation necessary when even minor changes (C1 to C3) are made in bioactive compounds.     

Three further naphthoquinones produced by Fusarium solani

Three naphthoquinone pigments are described which were produced by Fusarium solani. They are 2,3-dihydro-5,8-dihydroxy-6-methoxy-2-hydroxymethyl-3-(2-hydroxypropyl)-1,4-naphthalenedione, 2,3-dihydro-5-hydroxy-4-hydroxymethyl-8-methoxy-naphtho[1,2-b]furan-6,9-dione and 5,8-dihydroxy-2-methoxy-6-hydroxymethyl-7-(2-hydroxypropyl)-1,4-naphthalenedione. One of these pigments was shown to be the precursor of the other two.     

Stereoselective reactions of (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene derivatives form 27-nor-3,20,23,26-tetrahydroxy-cholesten-22-ones and related bromo ketones

The previously reported analog of pregnenolone having a 3,4-dihydro-2H-pyran attached via a Cz.sbnd;C bond to the C-20 position (1), stereoselectively reacts with m-chloroperoxybenzoic acid in methanol at -5 degrees C. Acid-catalyzed hydrolysis of the isolated intermediates gives good yields of mostly a new 27-norcholesterol analog: (20R,23R)-3,20,23,26-tetrahydroxy-27-norcholest-5-en-22-one-3-acetate (2a, and a smaller amount of its 23S enantiomer 2b). Three different conditions of epoxidation and methanolysis followed by acid-catalyzed hydrolysis typically produce approximately 2:1 ratios of the 23R:23S diastereoisomers with a C-23 hydroxy group at the new asymmetric center. Bromine also reacts stereoselectively with (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene (4) giving mostly (20R,23R)-23-bromo-3,20,26-trihydroxy-27-norcholest-5-en-22-one (7a). Thus both major steroidal products 2a and 7a have the same C-23R configuration. Assignment of molecular structures and the absolute configurations to 1 and 2a were based on elemental analysis, mass spectra, nuclear magnetic resonance, FTIR infrared spectroscopic analysis and X-ray crystallography. Mechanisms are discussed for stereochemical selectivity during epoxidation and bromination of the 3,4-dihydro-2H-pyranyl ring in 1 and 4.     

3H]Ro 15-1788 binding sites to brain membrane of the saltwater Mugil cephalus

The equilibrium binding parameters of the benzodiazepine antagonist [3H]Ro 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a]-1,4 benzodiazepine) were evaluated in brain membranes of the saltwater teleost fish, Mugil cephalus. To test receptor subtype specificity, displacement studies were carried out by competitive binding of [3H]Ro 15-1788 against six benzodiazepine receptor ligands, flunitrazepam [5-(2-fluoro-phenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one], alpidem [N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-acetamide], zolpidem [N,N-6 trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate], and beta-CCM (methyl beta-carboline-3-carboxylate). Saturation studies showed that [3H]Ro 15-1788 bound saturatably, reversibly and with a high affinity to a single class of binding sites (Kd value of 1.18-1.5 nM and Bmax values of 124-1671 fmol/mg of protein, depending on brain regions). The highest concentration of benzodiazepine recognition sites labeled with [3H]Ro 15-1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. The rank order of displacement efficacy of unlabelled ligands observed suggested that central-type benzodiazepine receptors are present in one class of binding sites (Type I-like) in brain membranes of Mugil cephalus. Moreover, the uptake of 36Cl- into M. cephalus brain membrane vesicles was only marginally stimulated by concentrations of GABA that significantly enhanced the 36Cl- uptake into mammalian brain membrane vesicles. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Studies on dehydro-l-ascorbic acid 3-oxime 2-phenylhydrazone: Conversion into substituted triazoles

l-threo-2,3-Hexodiulosono-1,4-lactone 3-oxime 2-(phenylhydrazone) (1) gave 2-(p-bromophenyl)-4-(l-threo-1,2,3-trihydroxypropyl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone (2), and this gave a diacetyl and a dibenzoyl derivative. On treatment of 2 with liquid ammonia, methylamine, or dimethylamine, the corresponding triazole-5-carboxamides (5–7) were obtained. Periodate oxidation of 5 gave 2-(p-bromophenyl)-4-formyl-1,2,3-triazole-5-carboxamide (10), and, on reduction, 10 gave 2-(p-bromophenyl)-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamide, characterized as its monoacetate. Condensation of 10 with phenylhydrazine gave the triazole hydrazone. Acetonation of 2 gave the isopropylidene derivative. Reaction of 2 with HBr-HOAc gave 4-(l-threo-2-O-acetyl-3-bromo-1,2-dihydroxypropyl)-2-(p-bromophenyl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone. Similar treatment of 1 with HBr-HOAc gave 5-O-acetyl-5-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 3-oxime 2-(phenylhydrazone). This was converted into 4-(l-threo-2-O-acetyl-3-bromo-1,2-dihydroxypropyl)-2-phenyl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone on treatment with boiling acetic anhydride. On reaction of 1 with benzoyl chloride in pyridine, dehydrative cyclization occurred, with the formation of 4-(l-threo-2,3-dibenzoyloxy-1-hydroxypropyl)-2-phenyl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone, which was converted into the amide on treatment with ammonia.     

Regioselective reactions of 1,2-dehydroprogesterone: syntheses of pregnane derivatives as possible contragestational agents

A few pregnane derivatives were synthesized from 1,2-dehydroprogesterone (1). Ring A of 1,2-dehydroprogesterone was aromatized without affecting C-20, and the resulting acetoxy compound (2) after hydrolysis yielded 1-hydroxy-4-methyl-19-norpregna-1,3,5(10)-trien-20-one (3). Reactions of the phenol (3) with alkyl halides yielded the ethers 6a-6b and 7. Opening of the oxirane ring in 7 with secondary amines furnished the aminoalcohols 8a-8b. Friedelcraft's reaction of 3 with maleic anhydride and chloracetyl chloride led to the formation of 9 and 10, respectively. Base-catalyzed ring closure of 10 yielded 1-acetyl-12a-methyl-8-oxo-5[H]-1,2,3,3a,3b,4,8,9,10b,11,12, 12a-dodecahydrocyclopenta (7,8)-phenanthro (3,4-b) furan (11), which reacted with aromatic aldehydes regioselectively to furnish 12a-12b. Reaction of 1 with triethylorthoformate in the presence of boron trifluoride etherate involved the participation of C-21, and the carbonyl at C-3 remained unaffected. The product 13 was identified as 21-[2-hydroxyvinyl]-21-norpregna-1,4-diene-3,20-dione. Reductive amination with sodium cyanoborohydride in the presence of ammonium acetate did not attack ring A and smoothly furnished the amine 14 which, on reaction with succinic anhydride, gave 20-succinamylpregna-1,4-dien-3-one (15).     

First synthesis of double headed 1,3,4-oxadiazino[6,5-b]indole acyclo C-nucleosides

Condensation of 1,3-dihydro-2,3-dioxo-2H-indoles (la-c) with galactaric acid bis hydrazide (2) gave the corresponding galactaric acid bis[2-(1,2-dihydro-2-oxo-3H-indol-3-ylidene)hydrazides] (3a-c). Acetylation of the latter compounds with acetic anhydride in the presence of pyridine at ambient temperature gave the 2,3,4,5-tetra-O-acetylgalactaric acid bis[2-(1,2-dihydro-2-oxo-1-substituted-3H-indol-3-ylidene)hydrazides] (4b-d). Heterocyclization of the tetra-O-acetates 4b-d by heating with thionyl chloride afforded the double headed acyclo C-nucleosides: 1,2,3,4-tetra-O-acetyl- 1,4-bis[9-substituted-1,3,4-oxadiazino[6,5-b]indol-2-yl-1-ium]-galacto-tetritol dichlorides (5b-d). Structures of the prepared compounds were elucidated from their spectral properties.