Kinetic and thermodynamic parameters were evaluated for the acylation and the deacylation steps in the hydrolysis of p-nitrophenyl acetate by α-chymotrypsin at pH 7.8 and at temperatures between 15 and 35°C by the use of stopped-flow and ordinary ultraviolet spectrophotometers. In contrast to the temperature dependencies of k₂ and K_s reported in the literature (P.A. Adams and E.R.Swart, Biochem.J., 161, 83(1977)), no kinetic anomaly was observed in either of the steps, but reasonable straight lines were obtained in both Arrhenius and van't Hoff plots. On the other hand, in the chymotryptic hydrolysis of N-benzoyl-L-alanine methyl ester a sharp kinetic anomaly was found. The discrepancy in the case of p-nitrophenyl acetate is discussed in connection with a possible conformational change of the enzyme, an alteration of the rate-limiting step or differences in the experimental procedures. The cause of the anomaly observed in the case of N-benzoyl-L-alanine methyl ester is also discussed in detail.

View full abstract

Cyclization of 13, 14-epoxygeranylgeranyl phenyl sulfide (2) was examined to determine whether 13- or 14-membered ring formation is preferred in the case of a disubstituted epoxide; the cis- and trans-epoxides were both found to cyclize to the 13-membered ring.

View full abstract

3, 5-Diphenylisoxazole and 2, 5-diphenyloxazole were regioselectively carbonylated under an atmosphere of carbon monoxide after the formation of arylpalladium(II) intermediates, to give bioactive o-benzoates substituted by isoxazole and oxazole rings in good yields.

View full abstract

The required left (C1-C9) segment, (R)-2-[(2R, 5S, 6R)-6-[(R)-1-formylethyl]-5-methyltetrahydropyran-2-yl]butanoic acid, for a total synthesis of salinomycin was highly stereoselectively synthesized from D-glucose via a chelation-controlled Grignard reaction and a decarbonylation with Wilkinson's catalyst as important steps. The stereoselective synthesis of the middle (C10-C17) segment, (2R, 4S, 5S, 6R)-6-ethyl-2, 4-dimethyl-7-oxononan-5-olide, starting from D-glucose is also described.

View full abstract

(S)-4[(2R, 5R, 6S)-5-Ethyl-5-hydroxy-6-methyltetrahydropyran-2-yl]pentan-4-olide (5), an alkaline degradation product of salinomycin (1), was synthesized with complete stereoselectivity from D-mannitol and ethyl L-lactate.Compound 5 was then converted to (3R, 4S, 7S)-7-[(2R, 5R, 6S)]-5-ethyl-5-methoxymethoxy-6-methyltetrahydropyran-2-yl]-4, 7-bismethoxymethoxy-3-(tetrahydropyran-2-yloxy)oct-1-yne (3), a C18-C30 segment of 1, via Sharpless asymmetric epoxidation. Another C18-C30 segment, (R)-3-[2RS, 5S]-5-[(2R, 5R, 6S)-5-benzyloxy-5-ethyl-6-methyltetrahydropyran-2-yl]-2-methoxy-5-methyltetrahydrofuran-2-yl]-3-(4-methoxybenzyloxy)prop-1-yne (4), was synthesized more conveniently via coupling between a C19-C22 fragment prepared starting from D-glucose and a C23-C30 fragment prepared starting from D-mannitol and ethyl L-lactate.

View full abstract

The polyether antibiotic salinomycin was synthesized via coupling between the C10-C17 aldehyde, (2R, 4S, 5S, 6S, 7R)-6-ethyl-5, 7-isopropylidenedioxy-2, 4-dimethylnonanal, and C18-C30 acetylenes, for example, (3R, 4R, 7S)-4, 7-bis(tert-butyldimethylsilyloxy)-7-[(2R, 5R, 6S)-5-ethyl-5-(4-methoxybenzyloxy)-6-methyltetra-hydropyran-2-yl]-3-(4-methoxybenzyloxy)oct-1-yne, followed by the aldol condensation with the C1-C9 segment, (R)-2-[(2R, 5S, 6R)-6-[(R)-1-formylethyl]-5-methyltetrahydropyran-2-yl]butanoic acid. In this total synthesis, protection of hydroxy groups with the 4-methoxybenzyl group played an important role.

View full abstract

In order to confirm the structures of some key intermediates employed in the total synthesis of salinomycin (1), natural 1 was cleaved into C1-C9, C10-C30, C10-C17, C21-C30, and C19-C30 segments.

View full abstract

The nuclephilic substitution of 5-(methylsulfonyl)-3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidine (2) with potassium cyanide proceeded smoothly to give 3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidine-5-carbonitrile (6), but the same reaction did not take place in the case of 5-chloro-3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidine (1).Compounds 1 and 2 reacted with ethyl cyanoacetate in the presence of sodium hydride in tetrahydrofuran, giving ethyl α-cyano-3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidine-5-acetate (7). When acetote was used as a ketone, it added across the C⁷, N⁶-double bond, giving 7-acetonyl-5-chloro-6, 7-dihydro- (8a) and 7-acetonyl-6, 7-dihydro-5-(methylsulfonyl)-3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidines (8b), respectively, although the yields were low.The reaction of 1 with Grignard reagents resulted in the formation of addition products such as 7-alkylated 5-chloro-6, 7-dihydro-3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidines (9a-e). The substitution of the methylsulfonyl group of 2 with Grignard reagents proceeded to give the corresponding 5-alkylated 3-phenyl-3H-1, 2, 3-triazolo[4, 5-d]pyrimidines (11a-e).

View full abstract

Four novel dimeric and trimeric hydrolyzable tannins, nupharins C (1), D (13), E (15) and F (16), which possess a less favorable α-D-glucopyranose core with an atropisomeric (R)- or (S)-hexahydroxydiphenoyl group at the 3, 6-position, have been isolated from the rhizomes of Nuphar japonicum DC. (Nymphaeaceae). The structures of these compounds were established on the basis of spectroscopic and chemical evidence.

View full abstract

10, 11-Diphenolic 8-hydroxy-5, 6, 7, 8-tetrahydrodibenz[c, e]azocine 3, having a partial structure of adrenaline, was prepared by demethylation of the dimethoxyazocine 12 with boron tribromide. The related monophenolic 8-hydroxyazocines 4 and 5 were prepared by cyclization of the dihalogeno-β-phenylethanolamines 6c and 7c with zerovalent nickel, followed by hydrolysis and debenzylation of the O-protected dibenz[c, e]azocines 13 and 14, respectively.

View full abstract

Negative ion fast atom bombardment mass spectrometry employing a hexamethylphosphoric triamide-glycerol (1 : 1, v/v) mixture as a matrix has been found to facilitate the molecular weight determination of hydrolyzable tannins in their free forms, as well as the detection of diagnostic fragment ions.

View full abstract

The ene reaction product of trifluoroacetone with cyclohexene, 3-(2, 2, 2-trifluoro-1-hydroxy-1-methylethyl)-cyclohexene (1), was converted to trifluorinated analogues of p- and m-menthane derivatives. For introduction of a functional group at suitable positions of the cyclohexene ring, oxidation of 1 was carried out to give 1-(2, 2, 2-trifluoro-1-hydroxy-1-methylethyl)-2-cyclohexen-1-ol (2), 4-(2, 2, 2-trifluoro-1-hydroxy-1-methylethyl)-2-cyclohexen-1-one (3) and 3-(2, 2, 2-trifluoro-1-hydroxy-1-methylethyl)-2-cyclohexen-1-one (4). Selenium dioxide gave only a small amount of 2. The best oxidizing reagent among those examined was tert-butyl hydroperoxide with chromium trioxide or chromium hexacarbonyl as a catalyst. In the presence of basic compounds such as pyridine or dimethylpyrazole, chromium trioxide gave a larger amount of 4 than 3, while in the presence of acetic acid, 3 was formed preferentially though the total yield was very low. Oxidation of 1 with tert-butyl hydroperoxide catalyzed by chromium hexacarbonyl gave 3 in excess over 4 in satisfactory yields. Treatment of the carbonyl compounds (3 or 4) with methylmagnesium iodide gave a p- or m-menthane skeleton with fluorine substituents, and these products were converted to some fluorine analogues of monoterpenes.

View full abstract

(26-¹³C)Desmosterol (1) and its 3-benzoate (8) and 3-tert-butyldimethylsilyl ether (9) were synthesized starting with (1-¹³C)propionic acid and a steroidal C-24 aldehyde (2).

View full abstract

The structures and absolute configurations of the cytokinins 1'-methylzeatin and its 9-riboside, both secreted by Pseudomonas syringae pv savastanoi, have been established as [R-(E)]-2-methyl-4-(9H-purin-6-ylamino)-2-penten-1-ol[(1'R)-2] and [R-(E)]-N-(4-hydroxy-1, 3-dimethyl-2-butenyl)adenosine [(1''R)-4], respectively, as a result of the chiral syntheses of (1'R)- and (1'S)-1'-methylzeatins [(1'R)-2 and (1'S)-2] and of their 9-β-D-ribofuranosides [(1''R)-4 and (1''S)-4] from D- and L-alanines. These zeatin derivatives were tested for cytokinin activity in the tobacco callus and the lettuce seed germination bioassays. The "natural" enantiomer (1'R)-2 was found to be as active as zeatin (1) itself and more active than its 9-riboside [(1''R)-4]. The "unnatural" enantiomer (1'S)-2 and its 9-riboside [(1''S)-4] were less active than the corresponding natural cytokinins, (1'R)-2 and (1''R)-4, respectively.

View full abstract

Various optically pure 3-substituted dihydroorotic acids (9, 12) were synthesized by the N-alkylation and Mitsunobu reaction of optically active benzyl or tert-butyl dihydroorotate (6, 10), followed by deprotection of the ester group.Moreover, optically pure 1-methyl (16, 17) and 1-ethyl (19) dihydroorotic acids were obtained by an optical resolution method.

View full abstract

Eight bayogenin glycosides, lobatosides A-H, were isolated from the herb of Actinostemma lobatum MAXIM.(Cucurbitaceae). The isolation of lobatosides A-H and the structures of lobatosides A, C, D and H are described.Lobatoside A is 3-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucopyranosyl]bayogenin (2β, 3β, 23-trihydroxyolean-12-en-28-oic acid). Lobatosides C, D and H are dicrotalic acid (3-hydroxy-3-methylglutaric acid) esters of the 28-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] ester, 28-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] ester and 28-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] ester of lobatoside A, respectively. Dicrotalic acid is linked at one end to the C₄-hydroxyl group of the α-L-arabinopyranosyl group in the C₃-linked sugar moiety, and at the other end to the C₄-hydroxyl group of the α-L-rhamnopyranosyl group in the ester-linked sugar moiety to form a macrocyclic structure ("cyclic bisdesmoside"). Lobatoside H was proved to be identical with tubeimoside I isolated from the tuber of Bolbostemma paniculatum (MAXIM.) FRANQUET (Cucurbitaceae).

View full abstract

In Rh(I) (Wilkinson)-catalyzed cyclization of 6-octen-1-als, the formation of cis-cyclohexanols is in contrast to Lewis acid-catalyzed cyclization, which affords predominantly the trans-cyclohexanols. However, 6-octen-1-als with a cyclic acetal (1, 3-dioxane or 1, 3-dioxolane) at the C₃-position were stereoselectively cyclized to only the trans-products.The aldehyde with a chiral protecting group ((4R, 6R)-dimethyl-1, 3-dioxane with the C₂-axis) at the C₃-position was diastereoselectively cyclized to the trans-cyclohexanol, and on a basis of the absolute stereochemistry of the cyclized product, the cyclization mechanism is tentatively proposed. The effect of 4R-methyl-1, 3-dioxane at the C₃-position was also examined.

View full abstract

A series of 2-anilino-1, 6-dihydro-6-oxo-5-pyrimidinecarboxylic acids with various substituents was synthesized and evaluated in the rat passive cutaneous anaphylaxis test for antiallergic activity. High activity by intraperitoneal and oral administrations was observed for the 3-trifluoromethyl and 2-alkoxyanilino derivatives (64, 79, 81, 82 and 85).Structure-activity relationships are discussed.

View full abstract

Cytochrome P450 model and liver microsomal oxidations of drugs were compared using phencyclidine. In general, the chemical reaction systems produced many oxidation products. Besides the formation of the cyclohexane-4-hydroxyl compound (2), hydroxylation of the aromatic ring was favored in the Fenton reaction system (Fe²⁺+H₂O₂). Formation of an m-hydroxylated product (m-5) was the main aromatic oxidation pathway in the Udenfriend reaction (Fe²⁺-ascorbic acid-O₂), and 2, the piperidine-3-hydroxyl compound (3), and the piperidine-4-hydroxyl compound (4) were also formed. In the system using meso-tetraphenylporphinatoiron chloride (Fe(III)TPPC1) with an oxidant, the main product was the piperidine-3-oxo compound (8). In the liver microsomes system, 2, 4, 8, and m-5, which were all generated by the chemical oxidation reactions, were detected as metabolites of phencyclidine. They were formed by cytochrome P450-dependent reactions. Chemical oxidation systems can be used to study drug metabolism; they can reveal some tendencies of the real metabolis reactions, are easy to operate, and yield sufficient amounts of product.

View full abstract

As part of the search for new topical antiinflammatory agents, various 21-substituted corticosteroids having sulfur-containing moieties were prepared and tested for vasoconstrictive activity in humans. A structure-activity relationship study revealed that substitution of the 21-hydroxy group with a lower alkyl-thio group enhanced the activity. The activities of the 21-methylthio (3Ad) and the 21-ethylthio (3Ae) compounds were more potent than that of 9α-fluoro-11β, 21-dihydroxy-16β-methyl-17α-valeroyloxy-1, 4-pregnadiene-3, 20-dione (betamethasone 17-valerate, BV).

View full abstract

A new glucuronide, (22R)-3β, 16β, 22, 26-tetrahydroxycholest-5-ene 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-gluc-uronopyranoside (1), was isolated from the aerial parts of Chinese Solanum lyratum THUNB. The structure of this new steroidal glucuronide was deduced by spectroscopic means and the identity of its aglycone was substantiated by comparison with an authentic sample derived from diosgenin acetate.

View full abstract

A new nor-diterpene lactone, picrodendrin A, was isolated from the bark of Indonesian Picrodendron baccatum(Simaroubaceae), and its structure was established on the basis of spectroscopic data and an X-ray crystal structure analysis.

View full abstract

Six β-carboline alkaloids were isolated from a Chinese medicinal plant, Arenaria kansuensis. The structures of four new alkaloids named arenarines A, B, C, and D were identified based on spectral and chemical evidence.

View full abstract

Forty preparations of the extracts from 28 kinds of Asian herbs were tested for ability to inhibit the activities of murine retroviral reverse transcriptase and human deoxyribonucleic acid (DNA) polymerases. Among the 40 extracts, 35 inhibited reverse transcriptase activity and 29 inhibited DNA polymerase α activity. The inhibitory potencies of these extracts were expressed as the 50% inhibition concentrations (IC₅₀), at which the enzyme activities were inhibited by 50%. Very strong inhibitions were observed with the extracts from Millettia pachycarpa (Leguminosae) and Mallotus apelta (Euphorbiaceae) as shown by their low IC₅₀ values for reverse transcriptase (0.4-0.5 μg/ml) and DNA polymerase α(0.9-1.4 μg/ml). Enzyme kinetic analysis revealed that the mode of inhibition of reverse transcriptase by these two extracts was competitive with respect to the template·primer [poly(rA)·oligo(dT)] and noncompetitive with respect to deoxythymidine triphosphate (dTTP) substrate. Besides reverse transcriptase and DNA polymerase α, DNA polymerase I and ribonucleic acid (RNA) polymerase from E. coli were inhibited by these two extracts. These results indicate that the herb extracts contain as yet unidentified substance(s) which inhibit the activities of reverse transcriptase and cellular DNA polymerases.

View full abstract

To clarify the structure-activity correlation of flavonoids for inhibition of aldose reductase, about fifty flavonoid compounds were screened. The presence of hydrophobic substituents on the A ring and hydrophilic substituents on the B ring of the flavonoid skeleton was suggested to improve the potency of inhibitory activity. The activities of extracts of Scutellaria baicalensis, Andrographis paniculata and Gutierrezia microcephala are also described.

View full abstract

Two new biphenyls, 3, 4, 3', 4'-tetrahydroxy-5, 5'-diisopropyl-2, 2'-dimethylbiphenyl (4a) and 3, 4, 4'-trihydroxy-5, 5'-diisopropyl-2, 2'-dimethylbiphenyl (5), were isolated from the leaves of thyme (Thymus vulgaris L.). Their structures were elucidated by spectroscopic methods and chemical transformations. Both biphenyls 4a and 5 showed more effective deodorant activity against methyl mercaptan than did rosmanol, carnosol or sodium copper chlorophylline.

View full abstract

In order to develop new drugs from natural products, constituents of natural medicines were examined for their effectiveness in the KCN-induced anoxia model in mice. Methanol extract from a Chinese medicine, evodia (fruits of Evodia rutaecarpa BENTH. or E. officinalis DODE), had a significant effect in the KCN-induced anoxia model in mice and therefore the active constituents were further examined. The results indicated that the antianoxic action of evodia was found in the fraction containing evodiamine. Further analysis of the active constituent indicated that evodiamine and rutaecarpine, indole-alkaloids found in large amounts in the Chinese medicine evodia, were mainly responsible for the antianoxic action.

View full abstract

A fluorometric method for the determination of 1-naphthol (NP) with 3-amino-2(1H)-quinolinethione (AQT) was developed. The method is based on the oxidation of NP by Fremy's salt and the reaction with AQT in an acid medium, followed by extraction of a red fluorescent product (excitation maxima, 535 and 575 nm; emission maximum, 600 nm) with carbon tetrachloride after making the reaction mixture strongly basic. The calibration curve was linear in the range from 0.01 to 1.2 μg/ml of NP. This method was applied for the fluorometric determination of Carbaryl (NAC, 1-naphthyl N-methylcarbamate)(0.01-1.8 μg/ml), which was hydrolyzed with potassium hydroxide and determined by the same method as NP. The coefficient of variation was 13% (n=10) or 1.5% (n=10) for 0.3 μg/ml of NP or NAC, respectively.

View full abstract

The fluorescence quenching reaction between fluorescein mercury or halogeno-fluorescein mercury compounds (fl.Hg, 2, 7- or 2, 4-dichloro-fl.Hg, 3', 4', 5', 6'-tetrachloro-fl.Hg, mercurochrome) and β-lactam antibiotics (ampicillin (AB-PC) and cephalexin (CEX)) was investigated, and mercurochrome was selected for the detection of β-lactam antibiotics;the detection limit was about 0.8 μg/ml.A fluorimetric assay of β-lactam antibiotics was established by measuring the fluorescence of mercurochrome and mercurochrome-β-lactam antibiotics solutions in weakly basic media to determine the degree of fluorescence quenching.The maximum emission wavelength of mercurochrome solution was at 544 nm with excitation at 470 nm. The calibration graphs were linear over the ranges of about 0-6 μg/ml β-lactam antibiotics penicillins (AB-PC, penicillin G, sulbenicillin, amoxicillin, cyclacillin, oxacillin, hetacillin and piperacillin) and cepham antibiotics (CEX, cefazolin, cephaloglycin, cephaloridine and cefpyramide), and the relative standard deviation was 2.7% for 1.4 μg/ml of AB-PC(n=5).This fluorescence quenching reaction between mercurochrome and β-lactam antibiotics was applied in a survey of decomposition and remaining potency of β-lactam antibiotics.

View full abstract

A simple chemical method for introducing biotin into nucleic acids has been developed for the synthesis of non-isotopic hybridization probes. The method is based on the reaction of biotin hydrazide with amino residues of nucleic acids by using glutaraldehyde as a bifunctional coupling reagent.Biotin-labelled deoxyribonucleic acid (DNA) was detected by the use of alkaline phosphatase-labelled avidin, and alkaline phosphatase activity was measured by colorimetric and chemiluminescence methods. The chemiluminescence method using the nicotinamide adenine dinucleotide phosphate (NADP)/alcohol/alcohol dehydrogenase/microperoxidase/isoluminol system gave the highest sensitivity. A few picograms of λ-phage DNA coated on a microtiter plate well could be detected by this method.

View full abstract

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), β-galactosidase (β-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl β-D-galactopyranoside and 4-methylumbelliferyl β-D-galactopyranoside were used for β-GAL, and 3, 3', 5, 5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (K_a=2×10¹⁰M^-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods. It was found that the assay using the HRP label was the most sensitive, and the AP, GOD and β-GAL labels gave nearly equal sensitivities. An interesting finding was obtained on the so-called bridging phenomenon, which is a factor influencing the sensitivity of hapten enzyme immunoassays.

View full abstract

Antitumor glucan, grifolan LE (GRN LE), from Grifola frondosa was chemically modified to examine the structure-function relationship of the products. Modification by periodate, borohydride and acid hydrolysis of side chains of GRN LE did not alter properties such as helical conformation and antitumor activity of GRN LE. Introduction of carboxylic acid groups into the side chains by oxidation with periodate and with sodium chlorite (GRN LE-PC), and substitution with carboxymethyl (CM) or hydroxyethyl (HE) groups abolished the gel-forming ability of GRN LE.Significant antitumor activity was observed in all of the derivtives having gel-forming ability as well as some derivatives having no such ability. These results suggested that essential factors required for antitumor activity were (1→3)-β-D-glucosy linkages and high molecular weight, and that accessory groups could be linked to the main chain without loss of antitumor activity in a higher ratio than that of gel-forming ability.

View full abstract

A high cholesterol/cholate diet induced hypercholesterolemia and fatty liver in both spontaneously hypertensive rats(SHR) and normotensive control rats (WKY). However, in contrast to previous concepts, the levels of cholesterol ester, triacylglycerol and phosphatidylcholine in plasma as well as triacylglycerol in liver were higher in WKY than in SHR fed a normal diet. The high cholesterol/cholate diet elevated the levels of plasma cholesterol, plasma cholesterol ester and hepatic triacylglycerol, and the extent of elevation was significantly higher in WKY than in SHR. Increases both in monoene/saturated ratios, an indication of elevated Δ⁹-desaturase activity, and in linoleate/arachidonate ratios, a possible indication of impaired desaturation-elongation activity, were observed in hepatic and plasma lipids of both strains fed the high cholesterol/cholate diet. The increases in monoene/saturated ratios were similar in both strains, but the increases in the linoleate/arachidonate ratios were higher for the plasma cholesterol esters of WKY than of SHR.The n-6/n-3 ratios of plasma and hepatic lipids were higher in WKY than in SHR throughout the experiments. These diet-induced changes observed in hepatic and plasma lipids were not reflected in the aortic lipids.Thus, hypertension per se does not promote the development of hyperlipemia and fatty liver induced by a high cholestrol/cholate diet. Our results also suggest that the metabolism of polyenoic fatty acids is different between SHR and WKY.

View full abstract

New 5'-nucleotidase-inhibitory polyphenols named NPF-86IA, NPF-86IB, NPF-86IIA and NPF-86IIB were isolated from the seeds of Areca catechu L. The ability of the inhibitors to precipitate gelatin was investigated by microturbidimetry. These inhibitors produced weak turbidity. As 5'-nucleotidase is a kind of phosphatase, we examined the effects of these inhibitors on alkaline and acidic phosphatases. While they showed moderate inhibitory effects on the activity of acidic phosphatases, they did not have any significant effect on the activity of alkaline phosphatase. Therefore, they showed a higher inhibitory effect on the 5'-nucleotidase than the other phosphatases. Murine macrophages were directly stimulated by the 5'-nucleotidase inhibitors.

View full abstract

In order to confirm the structure of α-trichechol, the major bile alcohol of the West Indian manatee, chemical synthesis of 5β-cholestane-3α, 6β, 7α, 25, 26-pentol was carried out. The chain of 3α-hydroxy-5β-chol-6-en-24-oic acid was elongated by an Arndt-Eistert reaction to form 3α-hydroxy-26, 27-dinor-5β-cholest-6-en-25-oic acid. The unsaturated C₂₅ bile acid was converted into 3α, 6β, 7α-trihydroxy-25-homo-5β-cholan-25-oic acid by 1, 2-glycol formation of the Δ⁶-double bond. The acetylated derivative of the trihydroxy C₂₅ bile acid was then converted into 3α, 6β, 7α, 26-tetraacetoxy-27-nor-5β-cholestan-25-one by successive treatment with thionyl chloride, diazomethane, and acetic acid. A Grignard reaction of the 25-oxo compound with methylmagnesium iodide afforded the desired bile alcohol, 5β-cholestane-3α, 6β, 7α, 25, 26-pentol. By direct comparison with the synthetic pentahydroxy bile alcohol, the structure of the naturally occurring α-trichechol was determined to be 5β-cholestane-3α, 6β, 7α, 25, 26-pentol.

View full abstract

The inhibitory effect of an anabolic steroid, stanozolol, on testicular microsomal cytochrome P-450 (17α-hydroxylase/C_{17, 20}-lyase)(P-450_17α/lyase) and the nature of the interaction were compared with those of other anabolic steroids, furazabol and mestanolone. Stanozolol markedly inhibited Δ¹⁶-C₁₉-steroid synthesizing activity, 17α-hydroxylase and C_{17, 20}-lyase activities, which were mediated by oxygenase activities of testicular microsomal cytochrome P-450_17α/lyase. In addition, stanozolol was a competitive inhibitor of 17α-hydroxylase (K_i=6.31 μM) and C_{17, 20}-lyase (K_i=1.30 μM) activities in the reconstituted enzyme system.The interaction of cytochrome P-450_17α/lyase with stanozolol induced a type I difference spectrum (peak at 387 nm and trough at 418 nm) with a dissociation constant (K_s) of 1.47 μM.

View full abstract

Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (M_r) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The M_r 66000 enzyme had the properties of tissue-type PA (t-PA), while the M_r 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1, 4-piperazinediethanesulphonic acid buffer and purified by (NH₄)₂SO₄ precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed M_r 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, M_r 44000, more effectively than it did the M_r 66000 epidermal PA. It was stable at 60°C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.

View full abstract

The kinetics of the degradation and tautomerization of cefotetan in aqueous solution was studied at 25°C and ionic strength 0.6 over the pH range of 2.0-12.0. The degradation rates of cefotetan and its tautomer were found to be identical and the rate law reflected the spontaneous hydrolysis of each molecular species and the hydroxide ion-catalyzed hydrolysis of the dianionic species. The tautomerization was remarkable especially in the alkaline region and the equilibrium of the tautomerization shifted toward the tautomer as the hydroxide ion concentration was increased.Divalent metal ions were found to catalyze the tautomerization by chelation. Electron transfer triggered by the attack of hydroxide ion at the amide of the 1, 3-dithiethane moiety and electron withdrawal by hydronium ion from the nitrogen atom on the isothiazole ring of the anion intermediate formed from the tautomer by hydroxide ion were proposed as the mechanisms of the forward and reverse tautomerizations, respectively. The latter reaction is considered to be accompanied with epimerization. The apparent activation energies of the degradation at pH 3.0, 7.0 and 9.0 and those of the forward and reverse tautomerizations at pH 9.0 were 23.3, 22.9, 31.4, 35.5 and 24.4 kcal mol^-1, respectively.

View full abstract

Permeation of sulfisoxazole (SIX) across the excised rat skin were studied using two chamber cell with four electrodes, under three successive experimental conditions : without current for 3 h (treatment I), with current for 4 h (treatment II) and without current for 3 h (treatment III). Transport of SIX was significantly increased by iontophoresis. The enhancement ratio of SIX flux were reasonably predicted by Coldman's equation. There was no significant difference (p<0.05) between the flux in treatent I and treatment III. On the basis of the flux reversibility, it was concluded that skin alteration did not occur when the applied electric potential was below 5.025 V. Although a prominent current-induced volume flow (from the anodal side to the cathodal side) was observed during current exposure, SIX flux was not influenced by the volume flow. The flux enhancement of SIX was mainly dependent on transdermal potential difference.

View full abstract

The ^99mTc and ⁵⁷Co complexes of ethylenediamine-N, N-diacetic acid (EDDA) are accumulated in tumor tissue. The complexes and related radioactive compounds were administered to experimental animals bearing Ehrlich tumor, and the blood, tumor, abscess, and other tissues were separated, fractionated and analyzed. In blood, the EDDA complexes of ^99mTc and ⁵⁷Co were in dialyzable forms, whereas other tumor-nonlocalizing compounds were in undialyzable or protein-bound forms. The tumor/blood and tumor/muscle ratios of the radioactivity showed that the complexes had the high affinity for tumor tissues. Density gradient centrifugation analysis of the ascites tumor tissues showed that a significant amount of the radioactivity of the complexes was present in tumor cells. Subcellular fractionation of solid tumor tissue showed that the radioactivity was present in nuclear fraction.

View full abstract

The effects of temperature, hematocrit (Hct), lipid level in plasma and cyclosporin A (CyA) level in whole blood on the concentration of CyA in plasma measured by high-performance liquid chromatography were studied in vitro. With rise in blood storage temperature before cells were removed, the concentration of CyA in plasma was increased in the temperature range between 10°C and 37°C, but was decreased between 4°C and 10°C. With rise in Hct, the concentration of CyA in plasma was decreased, and it was more markedly decreased at the blood storage temperature of 4°C than at 37°C. A lipid supplementation study showed that the concentration of CyA in plasma was increased with rise in plasma triglyceride level and in plasma cholesterol level at the storage temperature of 4°C but not at 37°C. Studies of the effect of CyA concentration in blood on the CyA distribution in blood demonstrated that the cellular/plasma concentration (C/P) ratio at low levels (<200 μg/ml) of plasma CyA ranged from 4 to 10 and was about 2 times higher than that at higher concentrations at 4°C, but the ratio was relatively constant at 37°C. The saturation capacity of the cellular fraction for CyA showed considerable individual variation, but there was no difference between the capacities at 4°C and 37°C. The separation of plasma after equilibration at 37°C made it possible to avoid the variations in the distribution of CyA in whole blood associated with changes in Hct, lipid level in plasma and CyA level in whole blood, and to obtain a measurement reflecting the physiologically significant concentration of CyA in plasma.

View full abstract

Aqueous methyl p-hydroxybenzoate (MPHB) solutions containing various amounts of α-cyclodextrin (α-CD) were freeze-dried. The crystalline states of MPHB and the freeze-dried products have been investigated. From a comparison of the powder X-ray diffraction patterns, it was found that rapid freezing provided amorphous products, while slow freezing provided crystalline inclusion complexes. To estimate the amorphous MPHB fraction, crystalline MPHB was removed by means of sublimation treatment, and residual MPHB was determined by ultraviolet spectrophotometry. It was found that the amount of amorphous MPHB in a rapidly frozen sample was greater than that in a slowly frozen sample.Amorphous MPHB molecules were considered to exist in two different states, namely the included state in the α-CD cavity and the dispersed state in the intermolecular hydrogen-bonding network of α-CDs. From studies with linear oligosaccharides, it was suggested that the freezing condition influenced the amount of MPHB molecules dispersed in the intermolecular hydrogen-bonding network.

View full abstract

The molecular interaction in a ground mixture of α-cyclodextrin (α-CD) and methyl p-hydroxybenzoate (MPHB) has been studied as a function of the mixing molar ratio. The states of MPHB molecules were investigated by means by powder X-ray diffraction, differential scanning calorimetry measurement and the determination of remaining MPHB after sublimation. The results indicated that in the ground mixture the MPHB molecules could exist in a mono-molecular dispersed state in the hydrogen bonding network of α-CD as well as in the α-CD cavity. It was suggested that two types of crystalline MPHB, fine crystals embedded in the α-CD matrix and intact crystals, were also present in the ground mixture of molar ratio 5 (MPHB/α-CD) or above.

View full abstract

The dissolution of n-propyl p-hydroxybenzoate was conducted under the sink condition, and was simulated from the changes of surface area during the dissolution process and by means of the cube root law. The surface area of the samples was estimated by using a LUZEX image analyzer. The two simulation methods gave almost the same dissolution rate constants.Dissolution of systems prepared by mixing sieved samples at various weight ratios was also carried out, and these dissolution processes were simulated by means of the cube root law. The apparent mean diameter of the mixed systems was well estimated in terms of particle size and weight ratio of the components. Hence, the mixed systems were converted into several imaginary equivalent systems, and the applicability of the cube root law and the validity of the estimation of apparent mean diameter of the mixed system were examined.

View full abstract

This study was concerned with whether drug-entrapping liposomes can be prepared by using lyophilized, empty dipalmitoylphosphatidylcholine (DPPC)-liposomes. It was found that drug-entrapping liposomes could be prepared by warming a mixture of lyophilized empty DPPC-liposomes and aqueous drug solution over the solid-liquid crystal phase-transition temperature (T_c), indicating that warming (>T_c) resealed the defect in the bilayer membranes after incorporation of the drug into the empty liposomes. The pharmaceutical potential of this new method was examined, and the results proved that drug-containing liposomes could be prepared readily or immediately before use, and with reproducibly high efficiency (40-70%) of entrapment.

View full abstract

Rectal absorption of bacampicillin hydrochloride (BAPC) was found to show the best bioavailability with Witepsol H-15 as suppository base among various Witepsol bases. However, an effective plasma concentration of drug (above 0.5 μg/ml) was only maintained for 2 h, so sustained-release suppositories of BAPC were studied. Bacampicillin reacts with acidic polymer electrolytes such as pectic acid (Pc), chondroitin sulfate (Cd) and precipitates as its adduct with the polymer in an aqueous solution. The dissolution rate of BAPC from the adducts in a solution was slower than that of BAPC itself. The absorptions of BAPC from the suppositories containing the adducts were prolonged, but the bioavailabilities were decreased compared to that from the suppository containing BAPC alone.Similar prolonged absorption could be obtained simply by mixing Pc or Cd with BAPC in a base. Further, the absorption rate was found to be controlled by the amount of the polymer addition, and both a high plasma level and excellent bioavailability were obtained. This desirable outcome may be due to the simultaneous occurrence of rapid absorption of BAPC itself and formation of the adducts.

View full abstract

The metabolism of 2, 4-dinitrotoluene (2, 4-DNT), 2, 4-dinitrobenzyl alcohol (2, 4-DNB), 2, 4-dinitrobenzaldehyde(2, 4-DNBAl), 2, 6-DNT, 2, 6-DNB and 2, 6-DNBAl in the microsomal and cytosol fractions prepared from unfortified male Wistar and male Sprague-Dawley (S.D.) rat livers was investigated. Data obtained by high-performance liquid chromatography (HPLC) indicated that the products of dinitrotoluenes (2, 4-DNT and 2, 6-DNT), dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB), and dinitrobenzaldehydes (2, 4-DNBAl and 2, 6-DNBAl) in the microsomal and cytosol preparations containing nicotinamide adenine dinucleotide phosphate (NAD(P)) and reduced NAD(P)(NAD(P)H) were dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB), dinitrobenzaldehydes (2, 4-DNBAl and 2, 6-DNBAl), and dinitrobenzoic acids (2, 4-DNBA and 2, 6-DNBA), and dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB), respectively. From these results, it was concluded that the dinitrobenzaldehydes (2, 4-DNBAl and 2, 6-DNBAl) were intermediates in the oxidations of dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB) to dinitrobenzoic acids (2, 4-DNBA and 2, 6-DNBA), and that the oxidations of dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB) to dinitrobenzaldehydes (2, 4-DNBAl and 2, 6-DNBAl) and the reductions of dinitrobenzaldehydes to dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB) were reversible The result of the consecutive oxidations of 2, 6-DNT in male Wistar rat livers, in the presence of various inhibitors suggests that oxidation of 2, 6-DNT to 2, 6-DNB is done mainly by microsomal cytochrome P-450, oxidation of 2, 6-DNB to 2, 6-DNBAl is mediated by microsomal cytochrome P-450 and nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase, oxidation of 2, 6-DNBAl to 2, 6-DNBA may be mediated by NAD-dependent aldehyde oxidase, and reduction of 2, 6-DNBAl to 2, 6-DNB may be mediated by reduced NAD(NADH)-dependent aldehyde reductase. From the comparative investigation of these reaction activities, it was found that : (a) the activity in the 2, 6-DNT oxidation to 2, 6-DNB was higher than that in the 2, 4-DNT oxidation to 2, 4-DNB in both strains, and the activity in Wistar rat was higher than that in S.D. rat; (b) the activities for the reductions of the dinitrobenzaldehydes (2, 4-DNBAl and 2, 6-DNBAl) to dinitrobenzyl alcohols (2, 4-DNB and 2, 6-DNB) were the highest, among the reactions examined, in both strains, and the reduction activity of 2, 4-DNBAl to 2, 4-DNB in Wistar rat was particularly high; (c) the activity for 2, 6-DNB oxidation to 2, 6-DNBAl was higher than that for 2, 4-DNB oxidation to 2, 4-DNBAl in both strains, and the activity in Wistar rat was higher than that in S.D. rat; (d) the activity for 2, 6-DNBAl oxidation to 2, 6-DNBA was much less than that for 2, 4-DNBAl oxidation to 2, 4-DNBA in both strains, and in particular, the activity for oxidation of 2, 6-DNBAl to 2, 6-DNBA in Wistar rat was low. The present results indicate that the metabolism of DNT isomers differs in different strains of rat.

View full abstract

The stereochemistry in determining the migratory aptitude in the ring-expansion of bicyclo[3.3.1]nonane-2, 6-dione 6-ethylene acetal (7) is discussed. The Tiffeneau-Demjanov ring-expansion of 6β-aminomethyl-6α-hydroxy-bicyclo[3.3.1]nonan-2-one 2-ethylene acetal (5, endo-alcohol) gave the homologous ketones (13 and 14) in the ratio of ca. 8 : 1, together with the endo-oxide (8). The reaction of the epimeric isomer, 6α-aminomethyl-6β-alcohol (6, exo-alcohol) gave the ketones 13 and 14 in the ratio of 2 : 1. The difference in the selectivity between two epimers was well interpreted in terms of least motion theory and the conformational stability of the intermediates. Hydrolysis of 13 and 14 led to two novel tricyclic systems, an isotwistane (15) and a protoadamantane (17), respectively.

View full abstract

Binding of zonisamide, a new antiepileptic sulfonamide derivative, was examined to human erythrocytes, their lysate and their carbonic anhydrase by centrifugation for cells or by ultrafiltration for the others. Scatchard plots revealed that the binding to intact and lysed cells was composed of high- and low-affinity components and that to carbonic anhydrase, of the high-affinity component alone. Parameters for high-affinity binding were similar in all three preparations and those for low-affinity binding were similar in the former two preparations. Dissociation constants for these bindings to erythrocytes were smaller than the dissociation constant for serum albumin. These results may explain the concentration of sulfonamides in red cells, and suggest the participation of cellular protein component(s) in addition to previously known carbonic anhydrase in the binding.Acetazolamide, sulthiame, zonisamide, hydrochlorothiazide and sulfanilamide inhibited carbonic anhydrase in a non-competitive manner to different extents. The K_i values of these sulfonamides were of the order of 0.1-0.2 of their respective K_d values determined by ultrafiltration, suggesting that under the present conditions, physicochemical interactions between sulfonamides and carbonic anhydrase primarily occur at common sites that affect the activity of the enzyme.

View full abstract

The reaction of 3-benzyloxy-4-hydroxy-2-methoxyxanthone with ethyl-2-bromo-3-(4-benzyloxy-3-methoxyphenyl)-3-oxopropionate in the presence of potassium tert-butoxide afforded the condensation product (8) which was converted to the debenzylation product (9) by hydrogenolysis. Reduction of 9 with lithium borohydride followed by treatment of resulting alcohols (10a, b) with concentrated hydrochloric acid in acetic acid provided kielcorin.

View full abstract