Pyridine N-oxides bearing electron-donating substituents (III) are efficient catalysts for rearrangement of O-alkyl S-methyl dithiocarbonates (xanthates) (I) to the corresponding S-alkyl S-methyl dithiocarbonates (dithiolcarbonates) (II). Of the catalysts tested, 4-piperidinopyridine N-oxide (IIIh) is the best from the viewpoints of catalytic activity and solubility in I. Heating of I in the presence of catalytic amounts (0.02-0.05 molar eq) of IIIh gave II together with the symmetric S, S-dialkyl and S, S-dimethyl dithiocarbonates in good yields. The catalytic behavior of donor-substituted pyridine N-oxides is discussed on the basis of kinetic and molecular orbital calculation data. The complete calculation of the perturbation equation on the initial stage of the reaction was consistent with the experimentally observed activity of the catalysts.

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Mass spectra of more than one hundled saturated and unsaturated pentacyclic triterpenoids were examined. In general, the presence of nuclear double bond(s), vicinal methyl groups and functional group(s) controls the fragmentation behavior, and therefore the basic carbon skeleton with double bond(s) and functional group(s) of unknown compounds can be identified by consideration of the fragmentation pattern.

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The reaction of aromatic methylenedioxy compounds containing electron-withdrawing groups with dimsyl anion-methyl alcohol, potassium tert-butoxide-alcohols, and metallic sodium-alcohols in dimethyl sulfoxide (DMSO), and with sodium alkoxides-alcohols in hexamethylphosphoramide, gave 3- and 4-hydroxybenzene derivatives in good yield by regioselective attack of the alkoxide ions on the methylenedioxy ring. The formation mechanism of alkoxide ions and the effect of DMSO in the cleavage reaction of the methylenedioxy ring are discussed on the basis of proton nuclear magnetic resonance (¹H-NMR) spectra. The reactions of aromatic methylenedioxy compounds (3 and 22) with sodium cyanide in dipolar aprotic solvents gave 4-cyano-3-hydroxybenzene derivatives (23 and 24) by regioselective attack of the cyanide ion on the methylenedioxy ring. The reactions of aromatic methylenedioxy compounds (28-30) containing no electron-withdrawing group with MeONa-MeOH in dipolar aprotic solvents gave non-regioselective cleavage products (31 and 34).

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A general method for synthesis of a physiologically important skeleton, perhydro-6H-pyrido[2, 1-i]indole, through cyclization of an active methylene group to an N-acyliminium, was developed. Treatment of the ketoester 5 with BF₃·Et₂O in methylene chloride resulted in deacetalization of the ethylene acetal group accomapnied with the expected double cyclization to give the tricyclic product 8 in 83% yield, and 8 was smoothly decarbomethoxylated to give decahydro-6H-pyrido[2, 1-i]indole-2, 6-dione (9). The former compound 8 was converted into derivatives of the isoerythroidine skeleton, 14 and 16.

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For new hydrolyzable tannins, coriariins G, H, I and J. were isolated from the leaf of Coriaria japonica (Coriariaceae). Structures 1, 2 and 3, having a sedoheptulose residue, were assigned for coriariins G, H and I, respectively. Structure 4, having a depsidone-forming dehydrodigalloyl group, was assigned for coriariin J, based on the chemical correlation with coriariin B (5). The orientation of the dehydrodigalloyl group in 5 was confirmed by ¹H-¹³C long-range shift-correlation spectroscopy.

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The structure of davallialactone (1), obtained from the rhizomes of a fern, Davallia mariesii MOORE, was revised on the basis of chemical and spectroscopic evidence. Also, the absolute stereochemistry of 1 was determined to be 5'R, 6'S from a circular dichroism (CD) study of dihydrodavallialactone (2).

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We have developed a simple method for the deprotection of allyl groups used as a protective group for the anomeric oxygen on a sugar moiety by employing tetrakis(triphenylphosphine) palladium in acetic acid. This method is applicable for not only simple but also complex allyl glycosides, such as natural lipid A intermediates and other important lipid A intermediates. Further, this method would be useful for large-scale preparation.

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p-Cymene (1) was metabolized in rabbits and the following four optically active metabolites, 2-(p-tolyl)-1-propanol (3' : R/S=65 : 35), 2-(p-tolyl)propanoic acid (5' : R/S=0 : 100), p-(2-hydroxy-1-methylethyl)benzoic acid (6' : R/S=91 : 9) and p-(1-carboxyethyl)benzoic acid (8' : R/S=30 : 70), were isolated in addition to three optically inactive metabolites, 2-(p-tolyl)-2-propanol (2), p-isopropylbenzoic acid (4'), and p-(1-hydroxy-1-methylethyl)benzoic acid (7').The presumed metabolic pathways of p-cymene in rabbits were confirmed by the administration of the intermediate metabolites (2, 3', 4', and 5'). The enantiomeric ratios of the metabolites, 3' and 6', suggested that ω-hydroxylations of the isopropyl group in 1 and 4' occurred preferentially at the pro-S methyl group. In the metabolism of 1, the S-isomers are predominant in the propanoic and derivatives, but the R-isomers are rich in the propanol derivatives. It is of interest that the metabolism of 4', however, produced predominantly the corresponding propanol derivative (6'; R/S=91 : 9) and propanoic acid derivative (8'; R/S=80 : 20) possessing the same R-configuration. Some optically active p-cymene derivative were also synthesized as standard compounds.

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Malabathrins A (6), E (11) and F (14), new complex tannins consisting of a C-glucosidic ellagitannin and a flavan 3-ol, have been isolated from the leaves of melastoma malabathricum L., and their structures were determined by chemical and spectroscopic methods including two-dimensional nuclear magnetic resonance spectroscopy.

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3-(1-tert-Butyldimethylsiloxy)ethyl-4-sulfinylazetidin-2-one was reacted with silylated N-, S-, O-, and P-nucleophiles in the presence of a catalytic amount of ZnI₂ to give the corresponding trans-4-heterofunction-substituted azetidin-2-ones in high yelids.

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Diels-Alder reactions of cyclopentadiene with methylenemalonates or O-acetylisonitrosomalonates in the presence of LiClO₄ as the catalyst were examined and the results were compared with those obtained under high pressure without the catalyst. Diels-Alder reaction of cyclopentadiene with dimethyl acetoxymethylenemalonate (1) in the presence of LiClO₄ afforded the [4+2] adduct (2) as a mixture of endo- and exo-isomers, whose ratio was 2.0, irrespective of the solvent or the concentration of LiClO₄. Asymmetric Diels-Alder reaction of cyclopentadiene with di-l-methyl acetoxymethylenemalonate (3) was accelerated remarkably by LiClO₄. The configurations of both endo and exo adducts corresponded to the natural form (D-form). These results suggested strongly that LiClO₄ would behave as a bidentate Lewis acid catalyst just like titanium tetrachloride. the hetero Diels-Alder reaction of cyclopentadiene with O-acetylisonitrosomalonate in LiClO₄-ether produced the adduct (5) in a higher yield than the reaction performed under high pressure.

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The stereoselective total synthesis of (+)-mayolide A (1) was achieved starting from D-mannitol via two crucial steps : stereoselective introduction of a two-carbon unit into the β-position of the butenolide 5 and repeated Claisen rearrangement to produce the side chain. The absolute structure of natural mayolide A from the Okinawa soft coral Sinularia mayi was determined as 2 by the present synthesis.

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Three new hydrolyzable tannins, eucalbanins A (5), B (8) and C (11), have been isolated from the fruit extract of Eucalyptus alba REINW., and their structures were elucidated on the basis of spectral data and chemical correlation with known tannins. Quercetin-3-O-α-L-arabinopyranoside, (+)-catechin, procyanidin b-7, and nine known hydrolyzable tannins [casuarinin, casuariin, pedunculagin, tellimagrandin I (1), gemin D (2), cornusiin B (3), 2, 3-(S)-hexa-hydroxydiphenoyl-D-glucose, penta-O-galloyl-β-D-glucose and oenothein B (4)] have also been isolated.

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Dramatic changes in the activities of squalene-2, 3-epoxide : cycloartenol cyclase and β-amyrin cyclase were observed in germinating pea seeds. By taking advantage of this phenomenon, the two cyclases were purified from pea seedlings. The cyclases were purified to homogeneity by solubilization with Triton X-100, chromatography on hydroxylapatite and diethylaminoethyl (DEAE)-cellulose, isoelectric focusing and gel filtration. Cycloartenol cyclase was purified 471-fold to a specific activity of 167 pkat/mg protein, while β-amyrin cyclase was purified 4290-fold to a specific activity of 28 pkat/mg protein. They each showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 55 and 35 kilodaltons (kDa), respectively. The apparent K_m values for (3S)-squalene-2, 3-epoxide were estimated to be 25 and 50 μM, respectively. The cyclases required Triton X-100 or deoxycholate for their highest activity and each showed a broad pH optimum within the range of pH 6.5-7.5. Inhibition by p-chloromercuribenzene sulfonic acid and N-ethylmaleimide suggested involvement of an SH group at the active site of each enzyme.

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6, 1'-Propanouridine (10), a carbon-bridged cyclouridine fixed in the syn-conformation, was synthesized from D-fructose. Two additional carbon-units were introduced at the 1'-position of 1'-hydroxymethyl-O², 2'-anhydrouridine 13 and inversion of the 2' hydroxyl group was achieved by sequential oxidation-reduction reactions. Finally, the spiro-carbon bridge was constructed by radical cyclization of the 1'-iodopropyl derivative of 5-chlorouridine. Dehydrochlorination followed by deprotection gave the desired 10. The circular dichroism (CD) spectrum of 10 showed a negative Cotton effect ([θ]=-6100) at the main absorption region, whereas 5'-O-tert-butyldimethylsilyl-2', 3'-O-isopropylidene-6, 1'-propanouridine (30) showed almost no Cotton band at the same absorption region. These results suggest that the critical region in which the CD Cotton effect changes from negative to positive is present in the syn region where 10 is located. Correlation of the magnitude and the direction of the sign of the CD Cotton effect and the torsion angle (χ) is also discussed.

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The coumarin isolated from Ariemisia carvifolia WALL was proved synthetically to be 7, 8-dimethoxycoumarin and not 5, 8-dimethoxycoumarin as previously proposed. 3, 4-Dimethoxy- and 3, 6-dimethoxysalicylaldehydes gave the corresponding coumarins in good yield by the method using phosphorane reagent in N, N-diethylaniline under reflux.

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Two new 3-keto-4-methylene steroids, theonellasterone (3) and conicasterone (4), and a Diels-Alder type dimeric steroid, bistheonellasterone (5), were isolated together with two known 4-methylene sterols, theonellasterol (1) and conicasterol (2), from the Okinawan marine sponge Theonella swinhoei. The structures of these steroids have been elucidated on the basis of chemical and physicochemical evidence. Bistheonellasterone (5) is considered to be biosynthesized through a Diels-Alder cycloaddition of theonellasterone (3) and its Δ⁴-isomer. Very interestingly, theonellasterone (3) and conicasterone (4) were seen under an optical microscope as crystals deposited in the tissue of fresh marine sponge.

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Five oleanane-type triterpenoid saponins, gymnemic acids VIII-XII as antisweet principles were isolated from the leaves of Gymnema sylvestre (Asclepiadaceae). Their structures were established on the basis of spectral and chemical evidence. They were characterized as glucosideuronic acid derivatives of gymnemagenin acylated with acetyl, tigloyl and/or 2-methylbutyroyl moieties.

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The 2"-O-, 3"-O- and 2", 3"-di-O-substituted derivatives (4a-p) of etoposide were prepared by nucleophilic substitution of 4'-O-benzyloxycarbonyletoposide (2) followed by deprotection. Controlled reaction (a limited amount of reagents and low temperature) was required for preparing the mono-O-substituted derivatives.In terms of ED₁₂₅ values, doses which show 125% of T/C against P388 leukemia in mice, both the 2"-O-acetate (4a, ED₁₂₅=0.18 mg/kg) and 3"-O-acetate (4b, 0.23 mg/kg) were nearly as active as etoposide (1, 0.19 mg/kg), while the 2", 3"-di-O-acetate (4c, 1.9 mg/kg) was somewhat less potent. In the replacement with other substituents, antitumor activity of the 2"-O-substituted derivatives was affected much more by the difference of the substituents as compared with that of the corresponding 3"-O-substituted derivatives. In the 2", 3"-di-O-substituted derivatives, the activity was decreased additively on the substituents.

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A new series of iodinated analogues of N-(2-aminoethyl)benzamide was synthesized and evaluated for inhibitory potency and specificity toward monoamine oxidase type-B (MAO-B). Among them, N-(2-aminoethyl)-2-chloro-4-iodobenzamide hydrochloride (2d) showed high inhibitory potency and selectivity against MAO-B. The type of MAO-B inhibition by 2d was non-competitive and the inhibition constant (K_i) was 0.80 μM. Strong and selective in vivo MAO-B inhibition by 2d was also confirmed. The brain MAO-B inhibition by 2d was reversible and the enzyme activity completely returned to the control value 24 h after administration. Compound 2d was, therefore, considered to be a candidate for advanced development as a radioiodinated ligand that may be useful for functional MAO-B studies in the living brain using single photon emission computer tomography.

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N-Fluoroalkyl and 4-fluoropyrrolidinyl eticlopride analogues with high affinity toward central nervous system dopamine D2 receptors in vitro were labelled with positron emitting fluorine-18 (t_1/2=110 min), and their in vivo biodistribution was investigated in rats. N-[¹⁸F]Fluoro-ethyl and -propyl eticlopride derivatives showed poor in vivo selectivity in the rat brain. On the other hand, 4-[¹⁸F]fluoropyrrolidinyl eticlopride exhibited almost constant and relatively high striatal concentration. The striata/cerebellar radioactivity ratio, which corresponds to the ratio of a brain D2 receptor-rich to poor region, gradually increased to 5.2-6.4, 90 min after the injection. The striatal accumulation was selectively inhibited by pre-injection of haloperidol, a dopamine D2 antagonist, without affecting accumulation in other tissues. Thus, the selective striatal accumulation of 4-[¹⁸F]fluoropyrrolidinyl eticlopride in striatal tissue appears to be due to the specific binding to dopamine D2 receoptors.

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Nilvadipine (I), isopropyl 2-cyano-3-methoxycarbonyl-6-methyl-4-(3-nitrophenyl)-1, 4-dihydropyridine-5-carboxylate, has a unique 1, 4-dihydropyridine structure in that the substituents at all five positions of the nucleus differ from one another. It is an excellent calcium antagonist drug in terms of its potency, its duration of action and its selectivity in the blood vascular system.During the development of I, some metabolites were isolated from the urine and bile of both rats and dogs afteroral administration. With data obtained from the metabolism of known 1, 4-dihydropyridines at hand, we proposed the synthesis of a series of compounds (1-11) for comparison with the metabolites isolated from I as a method for structure determination. Indeed, of the compounds synthesized five of them (3-7) were found to coincide with the metabolites from both rat and dog urine and bile isolates.

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A series of 1-amino-5-substituted uracils and their 4-thio or 2, 4-dithio substituted analogues were synthesized and assayed for anti-conflict activity in rats and anesthetic activity in mice. 1-Amino-5-halogenouracils 3b-e, 1-amino-4-thiouracil (9a), and 1-amino-5-halogeno-4-thiouracils 9c, d showed both anti-conflict and anesthetic activities. The most active compound was 1-amino-5-chloro-4-thiouracil (9d) which showed anxilolytic activity at 2 mg/kg of oral administration (p.o.) on a modified Geller-Seifter conflict schedule. Its minimum effective dose (MED) was lower than that of diazepam. The 50 percent effective dose (ED₅₀) for anesthetic activity in mice of the compound (9d) was 32.9 mg/kg, p.o.

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Based on studies of structure-activity relationship, trans-4-aminomethylcyclohexanecarbonyl-L-phenylalanine-4-carboxymethylanilide (Tra-Phe-APAA) was designed as a selective plasma kallikrein inhibitor and synthesized. Tra-Phe-APAA inhibited plasma kallikrein with a K_i value of 0.81 μM, while it inhibited glandular kallikrein, plasmin, urokinase, factor Xa and thrombin with K_i values of >500, 390, 200, >500, and >500 μM, respectively. However, its stereoisomer, Tra-D-Phe-APPA did not exhibit any detectable inhibitory activity against the above enzymes.

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A series of 6-[2-(imidazo[1, 2-α]pyridin-2-yl)ethyl]benzothiazoles (II) and benzimidazole analogues (III) was synthesized and tested for histamine H₂-receptor antagonist, gastric antisecretory and anti-stress ulcer activity. A benzimidazole derivative (IIIa) exhibited strong antisecretory activity, whereas the corresponding benzothiazole derivative (IIb) lacked this potency in in vivo test. In contrast to compound IIIa, however, compound IIb demonstrated good inhibition against stress induced ulcer. The structure-activity relationships of these compounds are discussed.

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A novel series of 2, 2-dialkyl-1'-(N-substituted aminoalkyl)-spiro-[chroman-4, 4'-imidazolidine]-2', 5'-diones was synthesized and evaluated for antiarrhythmic activity in chloroform- or/and aconitine-induced ventricular arrhythmia in mice. Among these compounds, (-)-6-chloro-2, 2-dimethyl-1'-[3-(4-hydroxypiperidino)propyl]-spiro-[chroman-4, 4'-imidazolidine]-2', 5'-dione was found to be more effective than reference agents and was selected for further development.

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We investigated effects of Shosaikoto treatment on cholesterol metabolism in macrophages. Although macrophages, harvested from mice treated with Shosaikoto, took up a small amount of control low density lipoprotein (LDL) (thiobarbituric acid-reactive substance (TBA-RS) value was 0.27 pmol/mg of protein) as control macrophages, they took up more LDL modified with CuSO₄ (TBA-RS value was 6.12 pmol/mg of protein) than control macrophages. Degradation of both control LDL and oxidized LDL was enhanced in Shosaikoto treated macophages. In the presence of control LDL or in the absence of LDL, incorporation of [³H]oleic acid into chlesteryl oleate was significantly reduced in Shosaikoto treated macrophages. This suggests that acyl-coenzyme A : cholesterol acyltransferase (ACAT) activity in macrophages was partly inhibited by Shosaikoto treatment. On the other hand, in the present of oxidized LDL, cholesteryl ester accumulated in Shosaikoto treated macrophages as much as in controls. However, cholesteryl oleate efflux from macrophages in the presence of high density lipoprotein (HDL) was enhanced in Shosaikoto treated macrophages. These result indicate that Shosaikoto facilitates oxidized LDL catabolism in macrophages, resulting in the augmentation of oxidized LDL uptake and the elimination of cholesterol from macrophages by HDL. These Shosaikoto effects may prevent foam cell formation and the progression of atherosclerotic lesions.

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In a previous paper, we reported the isolation and structure determination of three new oleanene glycosides, subprosides I, II and III, together with four known glycosides, kudzusaponin A₃, abrisaponin I, soyasaponin II and dehydrosoyasaponin I from Sophora subprostrata CHUX et T. CHEN (Leguminosae). In a continuing study on this crude drug, we report the characterization of four more new minor glycosides, subprosides IV (1), V (2), VI (3) and VII (4). Their chemical structures could be represented as 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl abrisapogenol D 30-O-β-D-glucopyranoside (1), 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl wistariasapo-genol B 30-O-β-D-glucopyranoside (2), 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl kudzusapogenol A 21-O-α-L-rhamno-pyranoside (3) and 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl soyasapogenol B 22-O-β-D-glucopyranosyl-(1→2)-β-D-xylopyranoside (4) on the basis of chemical and physiochemical evidence.

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Anti-tauro 1β-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1β, 3α, 7α, 12α-tetrahydroxy-5β-cholan-24-oyl)-2-aminopropionic acid-bovine serum albumin (BSA) conjugate. The antisera raised had high affinity (1.25-1.46×10⁹ M^-1) and specificity for conjugated 1β-hydroxycholic acid; cross-reactivity for glyco 1β-hydroxycholic acid was 100% and that for the glycine and taurine conjugates of other 1β-hydroxylated bile acids ranged from 11.30 to 0.23%. Urinary concentrations of conjugated 1β-hydroxycholic acid were determined by radioimmunoassay in newborns, 0-20 d after birth, in amounts ranging from 0.29 to 18.51 μg/ml and in women in late pregnancy (<1.13 μg/ml), as well as in normal women (<0.12 μg/ml).

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[1β-³H]16α-Hydroxyandrostenedione (16α-OHA) (715 mCi/mmol) was prepared from commercially available [1β-³H]androstenedione (A) by the microbiological method with Streptomyces roseochromogenes and its structure and purity were determined by chromatographic and reverse isotope dilution methods. When [1β-³H]16α-OHA was incubated with human placental microsomes and reduced nicotinamide adenine dinucleotide phosphate (NADPH), ³H₂O-release into the medium was dependent upon protein concentration and incubation time. An apparent K_m and and V_max of the microsomal aromatase for the [1β-³H]substrate were 650 nM and 34 pmol/min/mg protein, respectively. In this assay, aromatase activity could be determined as low as 0.1 nmol estrogen formation/min/mg protein. 3-Deoxyandrostenedione, a potent competitive inhibitor of the A aromatization, also blocked the 16α-OHA aromatization in a competitive manner with K_i of 15 nM.

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A method for the preparation of insulin derivatives which have protected sulfhydryl group(s) at definite site(s) on the molecule is described. Porcine insulin reacts with S-acetylmercaptosuccinic anhydride to afford four species of insulin derivatives that have 2 (or 3)-acetylmercapto-3-carboxypropanoyl group(s) at i) Gly(A1), ii) Gly(A1) and Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Lys(B29) positions. The derivatives are efficiently separated in a preparative scale by anion-exchange high-performance liquid chromatography on a TSKgel DEAE-2SW column. The four derivatives are all readily deacetylated with hydroxylamine to give the corresponding sulfhydryl insulin derivatives.

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After internalization of hemoglobin-haptoglobin complex (Hb-Hp) via receptor-mediated endocytosis (RME) into liver parenchymal cells, organelles containing the complex distribute in the microsome fraction (Ms). Prior to the catabolism, Hb-Hp dissociates symmetrically into two 82000-dalton (82 kDa) subunits. In the present investigation, the first event of Hb-Hp metabolism in Ms were further examined after [³H-heme, ¹⁴C-globin]Hb-Ho or [¹²⁵I-Hb]Hp injection to rats. Shortly after the internalization of Hb-Hp, this complex in Ms was intact. At 60 min after injection, radioactive materials of Ms extracted by freezing and thawing (F&T) with yield of 15% were composed of Hb-Hp, 82 kilodaltons (kDa) subunits and Hb metabolites with a ratio of 1 : 6 : 13. The heme metabolites were identified as [³H]bilirubin by high performance liquid chromatography (HPLC). The ratio of Hb-Hp/82 kDa subunits/Hb metabolites in microsome residue of the F&T was 40 : 8 : 1. The radioactivity in Ms at 60 min localized microsome subfraction except Golgi light fraction. In electron microscope radioautography of microsome subfraction using [¹²⁵I]Hb-Hp, silver grains were observed over or within morphologically heterogenous vesicles, e.g. vesicles containing very low density lipoprotein (VLDL) particles with appendage like multi-vesicular body (MVB) or compartment of uncoupling of receptor and ligand (CURL) in Goligi light and intermediate fractions.These studies suggest that Hb-Hp internalized by RME is dissociated symmetrically into two 82 kDa subunits in organelles of Ms, and that organelles with MVB or CURL-like structures are associated with Hb-Hp metabolism.

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β-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-β-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds.In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that β-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B.From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents.Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-β-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-β-D-glucuronic acid-CH Sepharose 4B. Furthermore, the adsorbed enzyme was eluted by the addition of urea as well as ethylene glycol which are known as reagents which weaken hydrogen bonding. The results suggested that the interaction between the enzyme and the adsorbents with an amide bonding may be affected by the electrostatic force in the adsorbents under a high concentration of salt, although the electrostatic force decreases under the high concentration of salt.We also investigated whether or not the adsorbed enzyme was eluted by sodium cholate, cholic acid and triton X-100 known as hydrophobic reagents.it was assumed from the results of these chromatographiers that the presence of amide bonding in adsorbents with glycosides as the ligand may be essential for the adsorption of the enzyme and that the glycosidic parts of the ligands have an effect on adsorption, however, it may not be essential for adsorption.

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Effects of human placental calphobindin II (CPB-II) on the protein C activation and prothrombin activation on the cell surface of cultured calf pulmonary arterial endothelial cells have been investigated. CPB-II inhibited thrombin generation by factor Xa bound to the surface of the cultured endothelial cells in a dose-dependent manner. The amount (IC₅₀) of CPB-II causing the inhibition at 50% was estimated to be approximately 10 nM. CPB-II was found to be ineffective, however, in the protein C activation by thrombin-thrombomodulin (TM) complex on the cell surface. Assay using purified TM revealed that CPB-II was able to exhibit the inhibitory potency for the protein C activation exclusively in the reconstituted system with negatively charged phospholipids. These results suggest that the neutral phospholipids participate in the protein C activation through the thrombin-TM system on the endothelial cell surface. The ability of CPB-II to inhibit procoagulant activity without affecting anticoagulant activity on the cultured endothelial cells is probably related to its potential physiological function, while it is able to exert various degrees of influence upon these activities in blood coagulation by interacting with negatively charged phospholipids in vitro.

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A novel type of arylsulfotransferase which was obtained from a human intestinal bacterium catalyzes the sulfation of polyphenols related to tannins. Isoamyl gallate and (-)-epigallocatechin gallate were found to be sulfated rapidly using p-nitrophenylsulfate as a donor substrate. In the case of isoamyl gallate, two sulfated products, 3-monosulfate and 4-monosulfate, were isolated when an equimolar amount of p-nitrophenylsulfate was incubated with isoamyl gallate. In the case of (-)-epigallocatechin gallate. 4'-monosulfate was isolated at an equimolar incubation of donor and acceptor. Thus, arylsulfotransferase was useful for the convenient preparation of sulfate esters of these polyphenols.

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Photodynamic deoxyribonucleic acid (DNA) strand breaking activities of 9 widely used drugs suspected of inducing photodermatological toxicity in humans, were examined. Enoxacin and afloqualone induced single strand breaks in supercoiled plasmid DNA following irradiation with common fluorescent lamps. The ED₅₀ (effective dose which change 50% of supercoiled closed circular DNA to the open circular form) of enoxacin was 2 mM and that of afloqualone was approximately 860 μM. DNA strand breaking activity of photoirradiated enoxacin was markedly inhibited by NaN₃ and partly by superoxide dismutase and D-mannitol. That of afloqualone was inhibited by NaN₃ primarily and by catalase partly.These results suggest that enoxacin and afloqualone are active photosensitizers and that these drugs induce phototoxicity in biological systems through type II photodynamic mechanisms. The experimental system used in assessing the photodynamic activity of drugs (i.e., induction of single strand breaks in plasmid DNA) may also be useful for screening phototoxic compounds in our environment and for determining the active oxygen species involved.

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α- And β-glucoside derivatives of validamine and valienamine were prepared by enzymatic transglucosidation using α- and β-glucosidase of rhodotorula lactosa. The structures of these derivatives have been elucidated by ¹³C- and ¹H-nuclear magnetic resonance spectral analysis. Thus, 7-α-glucoside, 7-α-isomaltoside, and 4-α-glucoside of validamine and 7-α-glucoside, 7-α-isomaltoside, 4-α-glucoside, and 4-α-isomaltoside of valienamine were obtained from maltose and validamine or valienamine using α-glucosidase. 7-β-Glucoside, 2-β-glucoside, and 4-β-glucoside of validamine or valienamine were obtained from cellobiose and validamine or valienamine using β-glucosidase. These derivatives were tested for α-glucosidase inhibitory activity on rat small intestinal glycosidases.

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In order to study the mechanism of propranolol-quinidine interaction, the effects of quinidine on proprranolol pharmacokinetics were examined in male Wistar rats. The concurrent oral administration of quinidine (10 mg/kg) markedly increased the plasma concentration of propranolol (2.5 mg/kg), and the area under the propranolol concentration-time curve increased about 3.6-fold. These results are consistent with previous observations in man and indicate the possible usefulness of the male Wistar rat as an animal model for investigating the mechanisms of the drug interaction. When propranolol was given intravenously, a concurrent administration of quinidine increased the apparent distribution volume of propranolol, mainly by decreasing its plasma protein binding. However, the systemic clearance of propranolol was not significantly altered by quinidine. Thus, quinidine increased the availability of oral propranolol from 13.8±2.2 to 44.2±4.6% (p<0.01). Furthermore, quinidine delayed the elimination of propranolol from the isolated perfused rat liver.These results indicate that quinidine reduces the presystemic elimination of propranolol in the liver, thereby increasing its systemic availability after oral administration.

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Effect of the local anesthetics dibucaine, tetracaine, lidocaine and procaine on the water permeability of phospholipid membrane was examined using liposomes composed of bovine heart cardiolipin and egg yolk phosphatidylcholine in a molar ratio of 2/98 by monitoring the osmotic shrinkage of liposomes in hypertonic glucose solution at pH 7.3 and 30°C. These local anesthetics greatly accelerated the water permeability by destabilizing the membrane structure. The effect was found to be governed by the hydrophobicity of the anesthetics. There was also a significant correlation between the membrane destabilizing actions and the anesthetic activities.

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Two kinds of sustained release morphine suppositories have been prepared; one is an oleaginous base suppository (MSC) containing a controlled release morphine tablet (MST : MS Contin), and the other is a hollow-type suppository (MSCH) containing MST and morphine powder packed in its hollow space. In vitro release tests and in vivo rectal absorption experiments in rabbits were performed. The profiles of morphine release from MST and MSC in vitro were similar, and revealed that suppository bases had no effect on the release profile of morphine from the preparation. Morphine release from MSCH was rapid in the early phase, and then enclosed morphine was slowly and continuously released from MST. Pharmacokinetics of morphine from the suppository were compared with the orally administered MST, and it was found that there was no difference in the maximum plasma concentration (C_max) and the peak time (T_max) between MSC and MST, but the mean residence time (MRT) of MSC was approximately three times longer than that of MST, and the extent of bioavailability (BA) of MSC was significantly larger than that of MST (71.6±14.2% and 11.9±4.0%, respectively). C_max can be altered arbitrarily by changing the morphine content in the hollow space of MSCH. As in the case of MSC, the plasma concentration of morphine from MSCH was maintained.It is concluded from the above results that MSC is a satisfactory sustained release morphine suppository for the treatment of cancer pain, administering it twice a day, and that MSCH is effective due to its fast analgesic effect and sustained release nature not only for cancer pain but also for surgical operations.

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Fourier transform infrared/attenuated total reflection (FT-IR/ATR) spectroscopy was used to examine the effect of fatty acids, fatty amines and propylene glycol (PG) on the molecular mobility of rat stratum corneum lipids and keratinized proteins, using a hydrophobic solute, indomethacin, and a polar solute, 5- and 6-carboxyfluorescein (CF). Treatment of the skin with either oleic acid or oleylamine resulted in significant CH₂ C-H asymmetric stretching band shifts and broadening. The extent of spectral alteration varied with the chemical structure of the penetrant. The penetrants increased the lipophilic indomethacin flux and shortened the lag times through the skin in vitro. The plot of frequency changes vs. indomethacin flux or lag time demonstrated a linear relationship, thus indicating that spectral alteration in CH₂ C-H stretching regions of stratum corneum lipids may provide a reliable index for characterizing penetrants. The data also showed that the hydrophilic group which attached to the CH₂ group in the penetrant molecules did not play a part in the membrane permeability enhancing action. Oleic acid and oleylamine appeared to induce a conformational alteration of the keratinized proteins from α-helix to beta sheet. Such alteration was also observed with PG treatment. Accumulation of CF was significantly increased by the PG pretreatment of the skin, thus suggesting that PG-induced protein conformational changes could be related to the enhancement of CF accumulation.

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A monoclonal antibody (mAb) reactive with a crosslinking reagent, N-(m-maleimidobenzoyl)dipalmitoylphos-phatidylethanolamine (mMBPE), in liposomes was produced from a hybridoma clone established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells from a BALB/c mouse hyperimmunized with the antibody-coated liposomes containing mMBPE. Using this mAb (termed AL-6), the quantity of immunoliposomes bound on target tumor cells was assessed by flow cytofluorometry. The results obtained using fluorescein isothiocyanate-coupled AL-6 allowed the enumeration not only of the immunoliposomes bound on all tumor cells but also those on individual target tumor cells. The relevance of this assay method was confirmed by a comparison with another assay method of cell-bound liposomes using immunoliposomes containing carboxyfluorescein in the vesicles.

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The influence of the addition of powder on the viscoelasticity of carboxycinylpolymer (CVP) hydrogel was studied by the oscillation method. The powder-filled hydrogels (PFHs) were prepared using Hiviswako 103 and 105 (CVP of rich side chains and poor side chains, respectively), and six powders [zinc oxide (ZnO), titanium dioxide (TiO₂), magnesium stearate (StMg), talc, synthetic aluminum silicate (SiAl), and hydrated silicone dioxide (Cp)].The profiles of storage modulus (G') and loss modulus (G") of each PFH differed depending on powder and CVP. log G' and log G" changed little with TiO₂, monotonously increased with talc, StMg and Cp, and showed fairly complex behaviors differing with polymer species with znO and SiAl. Plotting according to the kerner equation suggested that powder bridge structures were formed in the PFHs.The possible structures formed in the PFHs were presumed as follows on the basis of their viscoelasticity change and microscopic observation. With TiO₂ : the original hydrogel network was not ruptured and powders were dispersed uniformly in the gel network. With talc, StMg and Cp : the original hydrogel networks and the powder bridge structures coexisted. With ZnO and SiAl : the original hydrogel networks were ruptured and powder bridge structures were constructed.

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We designed a new enteric coated preparation which is pH independent and functions by pancreatic lipase activity in the duodenum. Triolein (TO) and trilaurin (TL) were selected as lipase sensitive components and ethylcellulose (EC) was used as the support film for TO and TL.Tablets (330 mg, d=10 mm) containing a model drug, sulfamethizole (SMZ), were coated with 1% each of TO, TL and EC solution by the fluidized bed coating technique. Disintegration tests were carried out in the media including JPXI 1st fluid (pH 1.2, JP-1), 2nd fluid (pH 6.8, JP-2) and JP-2 with gall powder and pancreatic lipase (JP-2-GL). The lag time of disintegration of the tablet (TOTL-Tab) coated 5-7 mg/tab with TO, TL and EC was about 10 min and all of the tablets disintegrated completely within 30 min in JP-2-GL. However, in the other media, which did not contain lipase, TOTL-Tab did not disintegrate for at least 2 h. It was confirmed that TO and TL in the coating film were digested by lipase. In addition, the tensil strength of the film decreased quickly after incubation in JP-2-GL.These results suggest that the application of TO, TL and EC to tablet coating is useful for an enteric release preparation sensitive to pancreatic lipase, even if patients have low gastric acidity or are taking antacids.

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The percutaneous absorption of ketoprofen (KPF) from gel patches containing d-limonene and ethanol was investigated in rats. Plasma levels of KPF varied with the kind of polymers which constitute the gel patch, and the highest level was observed when the copolymer of ethylacrylate (EA) and diethyleneglycolmethacrylate (DEGMA) was used as a vehicle. The amount of KPF permeating through the rat skin from the gel patch was well correlated with that of ethanol. Permeations were enhanced with increase in the amount of d-limonene distributed from the vehicle to the skin tissue. The amount of d-limonene accumulated in the skin varied greatly with the kind of polymers; the highest accumulation was observed with the EA-DEGMA copolymer, and decreased with increasing affinity of d-limonene to the polymers. The reason EA-DEGMA copolymer showed the highest percutaneous absorption of KPF from gel patches containing d-limonene may be the hydrophilic nature of this polymer which showed the lowest affinity to d-limonene.

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Phospholipid nanosphere dispersion was prepared from phosphatidylcholine with or without vitamin E by the heating method. The dispersed particles had diameters of 8.6-150 nm. These nanosphere dispersions were powdered with sugars such as sucrose, lactose and mannitol by spray drying in an aqueous system. The resultant powders, except coformulation with mannitol, consisted of spherical, homogeneous and freely flowing particles, in which the sugar was amorphous. The powder yielded a nanosphere dispersion having almost the same particle size and optical density as the original dispersion, when rehydrated with water. The particle size of rehydrated nanospheres with vitamin E increased with the increasing amount of vitamin E coformulated. A water-soluble drug, 5-fluorouracil, could be entrapped in the nanospheres by rehydrating the powdered nanospheres with an aqueous solution of the drug. All the steps from the preparation of the original nanosphere dispersion to spray drying were performed in an aqueous system without using any organic solvent. The procedures described here should be suitable for the production of stable powdered nanosphere which can be rehydrated to form phospholipid nanosphere dispersions as required.

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The dose-dependent pharmacokinetics of glycyrrhizin (GLZ) was investigated by measuring drug disappearance from plasma and biliary excretion in rats. The decline in plasma concentration was biexponential after an i.v. dose of 5, 10, 20, or 50 mg/kg. Dosage, however, had a marked effect on the pharmacokinetics, with a greater-than-proportional increase in area under the plasma concentration curve (AUC) at doses of 20 and 50 mg/kg, even though the increase was proportional at doses of 5 and 10 mg/kg. There was also a significant increase of the steady-state distribution volume (Vd_ss), as well as significant decreases in total body (CL_tot) and biliary (CL_B) clearances, at 20 and 50 mg/kg from those at 5-10 and 5-20 mg/kg, respectively. The AUC, Vd_ss, and renal clearance (CL_R) at a given dose showed no significant difference between rats with and without bile fistulas. The plasma unbound fraction (f_p) (0.006-0.026) increased with increasing plasma GLZ concentration over the observed range (2-900 μg/ml). No significant change in Vd_ss for unbound GLZ was observed between the doses, indicating that the distribution of GLZ into tissues is not changed by an increase in dose. On the other hand, a dose dependency in CL_tot for unbound GLZ was observed and confirmed to be attributed to dose dependency in CL_B for unbound GLZ since there was no significant difference in CL_R or metabolic clearance for unbound GLZ between the doses. The biliary excretion rates (V_B) at steady-state plasma unbound levels fitted reasonably well a Michaelis-Menten type equation with a maximum V_B of 98.77 μg/min and a Michaelis constant of 1.83 μg/ml. It was suggested that the saturable V_B may result in the dose dependence of CL_B. Thus, the pharmacokinetics of GLZ in rats is dose-dependent.

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4-p-Bromophenyl-5-hydroxymethyl-3-methoxycarbonyl-2-isoxazoline-2-oxide methanesulfonate (1c) reacted with excess titanium tetrabromide in dichloromethane to yield a novel ring transformation product, 4-p-bromophenyl-5-hydroxy-3-methoxycarbonyl-5, 6-dihydro-4H-1, 2-oxazine (2). The structural determination of 2 by single-crystal X-ray analysis is reported.

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Thermal reactions of clavulones, I, II, and III gave a novel prostanoid with a 10-acetoxy-11-ene system through a [3, 3]-sigmatropic rearrangement of allylic acetate.

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(3R, 4R, 5R)-1-(tert-Butoxycarbonyl)-3, 4-isopropylidenedioxy-5-methoxymethyl-2-pyrrolidinone (6), a useful chiral intermediate for the preparation of calyculins, was synthesized starting from (S)-pyroglutaminol via the O-methylation of 1c with diazomethane in the presence of fluoboric acid and cis-dihydroxylation of the α, β-unsaturated lactam (4) as the key reactions.

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