The interaction of arabic acid with counterions was studied by measuring the molal osmotic coefficients and counterion activity coefficients. The molal osmotic coefficients, φ, of various salt solutions were measured with a vapor pressure osmometer. In the monovalent salts, the φ value slightly decreased with decreasing hydration radii of counterions, but in divalent salts, the φ value was similar to each other and was about a half of those of monovalent salts. The counterion activity coefficients, γ₊, for Na and/or Ca arabate with or without added salts were determined by measuring electromotive force with cation-selective electrodes. In the absence of added salts, γ₊ was slightly larger than φ. In the presence of added salts, the additivity rule held very well for Na and Ca arabate solutions. In such a system with two kinds of counterions, Na and Ca, coexisting in arabate solution, Ca ion was preferentially adsorbed onto the polyions and consequently Na ion was less subject to the influence of the polyion. These results suggest that the interaction of arabic acid with counterions is due to the electrostatic free force alone, and not by any other specific force, and further suggest that Manning's theory for cylindrical polyelectrolytes seems to be applicable to the case of a branched polysaccharide, arabate, if the intercharge distance, "b, "can be assumed to be a parameter reflecting a spatial intercharge distance.

View full abstract

Pancreatic lipase activities toward fatty acid vinyl esters solubilized in micelles of five surfactants, sodium taurodeoxycholate (NaTDC), octa-oxyethylene dodecyl ether (ODE), and three polyoxyethylene nonylphenyl ethers (PNE), were kinetically investigated. The kinetic data for all the micellar systems were well interpreted on the basis of fully competitive inhibition mechanisms, where substrate-free micelles act as an inhibitor. For NaTDC micellar systems, the maximum velocity V, the Michaelis constant K_m, and inhibition constant K₄ were determined with respect to four substrates. The K_m/K₄ values for all the substrates were larger than unity but smaller than those previously obtained for sodium deoxycholate micellar systems. For the micellar systems of ODE and two PNEs with average numbers of oxyethylene units of 10 and 15, the Michaelis plots at constant surfactant concentrations were linear, indicating that the K_m/K₄ value is close to unity. On the other hand, for PNE with an average number of oxyethylene units of 30, a similar plot was concave, which suggests that the K_m/K₄ value is larger than unity. The alteration of the K_m/K₄ value with surfactants was closely correlated with the change in stability of the enzyme-micelle solubilizing substrate complex relative to the enzyme-substrate-free micelle complex ; the relative stability was shown to depend on the micellar size. The maximum velocity was also affected by micellar size.

View full abstract

It is well known that the asymmetry of membrane structure is closely related to the membrane function. We could obtain information on the asymmetry of membrane structure if we could analyze accurately. the asymmetric membrane potential, since the asymmetry of membrane structure should affect the membrane potential. It is considered that the origin of the asymmetric membrane potential is the difference in properties between the two membrane surfaces. In this paper, the membrane is treated by dividing it into the two sublayer (surface layer) parts adjacent to each membrane surface and the inner membrane part. The asymmetric membrane potential is investigated theoretically on the base of this model. The results show that the effects of asymmetry with respect to the surface charge density on the surface potential and on the diffusion potential within the membrane compensate for each other. The membrane potential is, therefore, scarcely affected except in the case where the diffusion potential within the membrane is negligible. It is also pointed out that, in an asymmetric membrane system with respect to the surface charge density, diffusion potential within the membrane is generated even if the mobility of a cation in equal to that of an anion. When the diffusion potential within the membrane is not negligible, only the effect of the asymmetry with respect to the standard chemical potential at the membrane surfaces is measurable experimentally. Our theory was applied to the analysis of the membrane potential of a cellulose membrane, and gave good agreement with the experimental results. Various experimental methods to confirm whether the membrane is asymmetric or symmetric are discussed.

View full abstract

Concurrent adsorption of chondroitin sulfate ion (Chs) and calcium ion (Ca²⁺) to hydroxyapatite (HAP) from an aqueous solution of Na₂Chs mixed with CaCl₂ was studied at 25°C. The adsorbed amount of Chs increased with increasing concentration of Ca²⁺ for the following two reasons : (a) Ca²⁺ adsorbed on the surface of HAP offers an adsorption site for Chs owing to its positive charges, and (2) the binding of Ca²⁺ to anionic sites of Chs depresses the inter- and intramolecular electrostatic repulsion between Chs segments, resulting in the dense adsorption of Chs. The apparent amount of Ca²⁺ adsorbed by HAP is also increased in the presence of Chs, because Ca²⁺ is captured by negative charges of Chs adsorbed on the surface of HAP as well as by adsorption sites for Ca²⁺ on the surface of HAP itself. However, it was concluded that the amount of Ca²⁺ adsorbed directly on the surface of HAP in the presence of Chs is almost the same as that in the absence of Chs when the adsorbed amount of Ca²⁺ is shown as a function of the concentration of free Ca²⁺.

View full abstract

Aqueous and nonaqueous polarographic properties of 4-substituted 2, 6-dimethylbenzonitrile N-oxides (stable nitrile N-oxides) have been studied and compared with those of the substituted pyridine N-oxides and benzylidenemethylamine N-oxides (nitrones) investigated previously by us. The first reduction wave in both the aqueous and N, N-dimethylformamide (DMF) solvent systems is due to the deoxygenation reaction of the nitrile N-oxide group except when certain substituents are present (see text). This conclusion has also been verified by controlled potential electrolysis in aqueous solution and by cyclic voltammetry in DMF solvent. A plot of the Hammett σ constants of the substituents against E_1/2 values was linear with a positive slope for both the aqueous and DMF solvent systems. The slope is smaller than in the case of pyridine N-oxides and nitrones, this being reasonably attributable to the triple bond nature of the C〓N〓O group. Half-wave reduction potentials of the nitrile N-oxides are positively shifted compared with those of pyridine N-oxides, particularly in an aqueous solvent. Molecular orbital calculations were applied to interpret the substituent effect on the reduction potentials of the N-oxides.

View full abstract

A pyrrolo-1, 4-benzodiazepine skeleton which constitutes the common part of a series of antibiotics exhibiting antitumor activity was synthesized by insertion of carbon monoxide into o-bromoprolylaniline derivatives in the presence of a catalytic amount of Pd (OAc)₂ and PPh₃. In this reaction, hexamethylphosphoramide (HMPA) gave a good result as the solvent and the use of a high pressure of carbon monoxide (5 atom) increased the yield of the desired pyrrolo-1, 4-benzodiazepine.

View full abstract

Thermal reaction of 2, 2-dimethyl-2H, 4H-1, 3-dioxin-4-ones (1) with amines was studied. Acylketenes 2, generated by heating of 1, reacted with anilines and benzylamine to give the corresponding β-ketocarboxamides (3, 4, and 5) in good yields. The reaction of 1 with ammonia gave 3-amino-2-alkenamides (7), which were hydrolyzed to β-ketocarboxamides (6). The former products 7 were readily transformed to the 6-substituted 2-methyl-3H-pyrimidin-4-ones (9) via the 3-acetamido-2-alkenamides (8). Acylation of O-benzylhydroxylamine with 1 gave the β-ketohydroxamic acids 10. Debenzylation of 10 followed by cyclization gave rise to 5-alkyl-3-hydroxyisoxazoles (12). The reaction of 1 with amides gave the corresponding N-acylated amides (13).

View full abstract

The oxidation of various 5H-N-alkyl-10, 11-substituted-dibenz [b, f] azepines with m-chloroperbenzoic acid (m-CPBA) was examined. The oxidation of 5H-N-benzyl-10-substituted-dibenz [b, f] azepine (V) gave N-benzyl-9-formyl-9-substituted-9H, 10H-acridine (VI), while the oxidation of 5H-N-benzyl-10, 11-disubstituted-dibenz [b, f] azepine (VII) gave the corresponding 10, 11-oxide (VIII). The chemical reactivities with several nucleophiles and the acid-catalyzed reactions of 5H-dibenz [b, f] azepine 10, 11-oxides are also discussed.

View full abstract

Chemical synthesis of (22E, 24R)- and (22E, 24S)-1, 24-dihydroxy-Δ²²-vitamin D₃ has been achieved starting with the commercially available dinorcholenic acid acetate. Synthesis involved introduction of the 1-hydroxy group by a reduction of the 1, 2-epoxide generated by epoxidation of the 1, 4, 6-trien-3-one. The side chain on the steroid was then constructed by means of a Wittig reaction followed by introduction of the Δ⁷ bond by standard methods and its protection with 1-phenyl-1, 2, 4-triazoline-3, 5-dione. Subsequent reduction of the hydroxy groups in the steroid side chain followed by reduction of the Diels-Alder addition products yielded the both 24-isomers. The 5, 7-dienes were irradiated and the corresponding vitamin D compounds isolated. Nuclear magnetic resonance was used to identify individual isomers. The (22E, 24S)-1, 24-hydroxyvitamin D₃ compound bound equally well to the chick intestinal cytosol receptor as 1, 25-dihydroxyvitamin D₃, while the 24R-isomer was approximately ten times less active. In vivo, both isomers were less active than 1, 25-dihydroxyvitamin D₃ ; however, the 24S-isomer was considerably more active than the 24R-isomer approaching the activity of 1, 25-dihydroxyvitamin D₃.

View full abstract

Six biologically active minor bases (P-II-P-VII) related to paragracine (P-I) were isolated from Parazoanthus gracilis LWOWSKY and their structures were determined by spectral analyses as well as on the basis of chemical interconversions.

View full abstract

Two new phenylpropanoid glycosides, named cistanosides C and D, were isolated from the whole plant of Cistanche salsa (C. A. MEY.) G. BECK (Orobanchaceae), together with 2'-acetyl acteoside and osmanthuside B. The structures of cistanosides C and D were established as 2-(4-hydroxy-3-methoxyphenyl) ethyl O-α-L-rhamnopyranosyl-(1→3)-O-(4-O-caffeoyl)-β-D-glucopyranoside (II) and 2-(4-hydroxy-3-methoxyphenyl) ethyl O-α-L-rhamnopyranosyl-(1→3)-O-(4-O-feruloyl)-β-D-glucopyranoside (III), respectively, on the basis of chemical and spectral data.

View full abstract

Treatment of quinoline 1-oxide (1) with phenylacetic acid and acetic anhydride in boiling benzene for 10 h gave benzaldehyde (2), quinoline (3), 2-benzylquinoline (4), 2-benzoylquinoline (5), phenyl-di (2-quinolyl) carbinol (6), di (2-quinolyl) ketone (7), and N-(2-quinolyl)-2-benzylidene-1, 2-dihydroquinoline (8) in small yields. The reaction of 1 with phenylacetic anhydride in boiling benzene for 10 h afforded 1, 3-diphenyl-1-(2-quinolyl) acetone (9) and dibenzyl ketone (10) together with small amounts of 2, 3, and 5. Further, it was found that phenylacetic anhydride undergoes decarboxylative coupling upon heating with tertiary amines to give 10. The mechanisms of these reactions are discussed.

View full abstract

A general synthetic route to Lythraceae alkaloids based on a regio- and stereoselective [3+2] cycloaddition reaction has been developed. The [3+2] cycloaddition of the nitrone 5 with the olefins 14 and 26 gave the adducts 15 and 27 which, upon treatment with methanesulfonyl chloride followed by reductive cleavage of the N-O bonds and acetylation, gave the trans-quinolizidines 18 and 30 and the cis-isomers 20 and 32 in reasonable yields. The cis-isomers were converted into the simple phenylquinolizidine alkaloid, (±)-2, and the ester alkaloid, (±)-10-epi-desmethoxyabresoline (4). On the other hand, (±)-1, (±)-lasubine II (23), and (±)-abresoline (3) were synthesized efficiently from the trans-isomers, after inversion of the configuration at the C-2 position by means of the Mitsunobu reaction.

View full abstract

The benzoylation of 3, 4-dihydro-4-quinazolinecarbonitrile (3) with benzoyl chloride in pyridine gave quinazoline Reissert compound (1, 3-benzoyl-3, 4-dihydro-4-quinazolinecarbonitrile) in 82% yield. The alkaline hydrolysis of 1 in methanol resulted in the formation of quinazoline (2) and benzoic acid (4). Acid hydrolysis gave 2-benzamido-2-(2-aminophenyl) acetonitrile (6), 4, and 2. The HCl salt of 1 existed predominantly in the cyclic amidinium structure of the type 8. Compound 1 reacted with sodium hydride in dimethylformamide to yield 4-quinazolinecarbonitrile (9), α-phenyl-4-quinazolinylmethyl benzoate (10), and O-benzoylbenzoin (11). In the present paper we compare the chemical properties of 1 with those of isoquinoline Reissert compound (13, 2-benzoyl-1, 2-dihydro-1-isoquinolinecarbonitrile).

View full abstract

Three new triterpene acids were isolated from the twigs and leaves of Hedyotis lawsoniae, together with sixteen known ones. The new compounds were determined to be 3β, 23-dihydroxyurs-12-en-28-oic acid, 3β, 24-dihydroxyurs-12-en-28-oic acid, and 2α, 3β, 24-trihydroxyurs-12-en-28-oic acid on the basis of spectroscopic evidence.

View full abstract

Five new sesquiterpene glycosides, macroclinisides A (II), B (III), C (IV), D (V), E (VI), have been isolated from Macroclinidium trilobum MAKINO together with glucozaluzanin C (I). The structures were determined on the basis of chemical and spectral data. Macrocliniside C has antitumor activity in mice.

View full abstract

To develop antagonists which show selectivity in blocking neurotransmitters, several aminophosphonoic acids, 2-amino-5-phosphonopentanoic acid (IVb), 2-amino-4-(2-phosphonomethylphenyl) butyric acid (VIII), 2-(2-amino-2-carboxy) ethylphenylphosphonic acid (XIc), and N-benzylproline-4-phosphonic acid (XIX), were synthesized. Compounds IVb, VIII, and XIc were prepared from the corresponding halides (V, Xa, and XIa, respectively) by treatment with sodium diethyl acetamidomalonate (VI). Compound XIX was synthesized via 1, 3-dipolar cycloaddition of ethyl N-benzyl-N-phenylthiomethylglycinate (XV) to diethyl vinylphosphonate (XVI).

View full abstract

Bifunctional photo-activable fluorescent thiol reagents of a new type were synthesized. A maleimide group was bonded to an azidocoumarin group via a methylene chain as a spacer. Reagents of this type react first with a cysteine residue of a protein through the maleimide group, and then form another bond with an amino acid side chain of the protein upon irradiation with light, through a nitrene group formed from the azide. Although the reagent is non-fluorescent, the products are highly fluorescent. The fluorescence characteristics of model compounds of these reagents are also described.

View full abstract

The synthesis of N-carboxy α-dehydroamino acid anhydrides (ΔNCA) from benzyloxycarbonyl-α-dehydroamino acids and the subsequent conversion of these products into new α-dehydroamino acid and dehydrooligopeptide derivatives are described. It was found that the new ΔNCA derivatives were very useful synthons for dehydropeptides. The racemization behavior and configurational determination of all the new dehydrooligopeptides thus obtained are discussed.

View full abstract

The oxidation of 1-acylindoles, 1, 15, 20, and 22, with (hexamethylphosphoramide) oxodiperoxomolybdenum (VI), MoO₅·HMPA, in dry methanol gives a variety of products depending on the indole substituents ; trans- and cis-1-acyl-3-hydroxy-2-methoxyindolines 2 and 3 are obtained from 2, 3-unsubstituted 1-acylindoles 1, 1-acetyl-2-hydroxy-3-methoxy-3-methylindoline (16) from 1-acetyl-3-methylindole (15), 1-acetyl-2, 3-dihydroxy-2, 3-dimethylindoline (21) from 1-acetyl-2, 3-dimethylindole (20), and 1-acetyl-2-hydroxy-2-methylindoxyl (23) from 1-acetyl-2-methylindole (22). Reaction of 2 and 3 with acids gives 1-acylindoxyl 10, respectively, while reaction of 16 with the acid gives 3-methyloxindole (19). Treatment of 3-acetoxy-1-acetyl-2-methoxyindoline (5) with stannic chloride gives 1-acetylindoxyl (10a) and 1-acetyl-3-chloroindole (14).

View full abstract

Regio-and stereocontrolled terminal functionalization of protected 2-prenyl-and 2-geranylhydroquinones (2) was achieved by way of terminal methallylic sulfides (4), which were easily obtained via benzenesulfenyl chloride addition followed by dehydrochlorination or via allylic chlorination with SO₂Cl₂ followed by sulfenylation, leading to terminal trans-allylic alcohols (5) which are promising intermediates for the synthesis of polyisoprenoidquinones. In this transformation, interesting steric effects of the hydroquinone nucleus with the adjacent arylic methyl group influencing the site-selectivity in the reactions of the isoprenoid side chain were observed.

View full abstract

Physiologically active polyisoprenoidquinones, ubiquinone-10 (coenzyme Q₁₀), phylloquinone (vitamin K₁), and menaquinone-4 (vitamin K₂₍₂₀₎) were synthesized by a chain-extending method utilizing protected hydroquinones with the omega-hydroxyprenyl or omega-hydroxygeranyl side chain. Conditions for reductive desulfurization subsequent to allylic homologation were investigated.

View full abstract

Various amino-and guanidino-substituted naphthoates and tetrahydronaphthoates were synthesized and evaluated for inhibitory activities against trypsin, plasmin, kallikrein, thrombin and Cl esterase. Among these compounds, phenyl 4-guanidino-1-naphthoate (IIIj) and phenyl 6-guanidino-1-naphthoate (IIIl) exhibited potent and selective trypsin inhibition (IC₅₀ : 4×10^-7 and 5×10^-8 M, respectively) and phenyl 7-guanidino-1, 2, 3, 4-tetrahydro-1-naphthoate (Vb) and p-ethoxycarbonylphenyl 7-guanidino-1, 2, 3, 4-tetrahydro-1-naphthoate (Vg) had selective inhibitory activities against thrombin (IC₅₀ : 4×10^-5 and 1×10^-5 M, respectively).

View full abstract

A series of novel 6-[ω-(4-aryl-1-piperazinyl) alkyl]-5, 6, 7, 8-tetrahydro-1, 6-naphthyridines was synthesized and evaluated for antivertigo activity by testing their ability to inhibit spontaneous nystagmus in cats. Structure-activity relationships are discussed. Many of the compounds having the 4-(2-alkoxyphenyl) piperazine group as the 4-arylpiperazine moiety showed more potent antivertigo activity than diphenidol. Among them, 2-{2-[4-(2-ethoxyphenyl)-1-piperazinyl] ethyl}-1, 2, 3, 4, 6, 7, 8, 9-octahydrobenzo [b] [1, 6] naphthyridine (NK 422, 41) was selected as a promising antivertigo agent. NK 422 also exhibited a more potent inhibitory effect on apomorphine-induced vomiting in dogs than diphenidol.

View full abstract

Six steroidal glycosides, tentatively named glycosides O-1, O-2 (1), O-3 (2), O-4 (3), O-5 (4) and O-6 (5), were isolated from the subterranean part of Ophiopogon ohwii OKUYAMA (Liliaceae) and another six steroidal glycosides, tentatively named glycosides J-1, J-2 (6), J-3 (7), J-4 (8), J-5 (9) and J-6 (10), were isolated from the subterranean part of O. jaburan (KUNTH) LODD. Glycosides O-1 and J-1 were identified as so-called β-sitosterol β-D-glucopyranoside. The structures of 1-10 were established as ophiopogonin B (1), ophiopogonin D (2), ruscogenin 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-β-D-fucopyranoside (3), ruscogenin 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-α-L-arabinopyranoside (=glycoside E of O. planiscapus NAKAI) (4), 26-O-β-D-glucopyranosyl 22-hydroxy-25(R)-furost-5-en-1β, 3β, 26-triol 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-β-D-fucopyranoside (5), ophiopogonin D (=glycoside O-3 (2)) (6), a mixture of 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-β-D-fucopyranosido-3-O-β-D-glucopyranosides of ruscogenin and neoruscogenin (7), neoruscogenin 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-α-L-arabinopyranosido-3-O-β-D-glucopyranoside (8), a mixture of 26-O-β-D-glucopyranosyl 22-hydroxy-25(R)-furost-5-en-1β, 3β, 26-triol 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-β-D-fucopyranosido-3-O-β-D-glucopyranoside and 26-O-β-D-glucopyranosyl 22-hydroxyfurost-5, 25(27)-dien-1β, 3β, 26-triol 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-β-D-fucopyranosido-3-O-β-D-glucopyranoside (9), and 26-O-β-D-glucopyranosyl 22-hydroxyfurost-5, 25(27)-dien-1β, 3β, 26-triol 1-O-α-L-rhamnopyranosyl(1→2)-4-O-sulfo-α-L-arabinopyranosido-3-O-β-D-glucopyranoside (10). It is interesting that the main saponins, ophiopogonins B and D, found in Ophiopogonis Tuber (obtained from O. japonicus KER-GAWLER) were also found in O. ohwii OKUYAMA and the latter saponin was also found in O. jaburan (KUNTH) LODD. Further, several steroidal glycosides carrying sulfate on the sugar moiety were found in Liliaceous plants belonging to the genus Ophiopogon.

View full abstract

Further studies have been done on the constituents of the stems, underground parts and leaves of two Palmae plants, Rhapis exelsa HENRY and R. humilis BL. We isolated and identified dioscin, Pb, deltonin, methyl proto-dioscin, methyl proto-Pb and methyl proto-deltonin from the stems, dioscin, methyl proto-dioscin and methyl proto-Pb from the underground parts, and saponaretin (=isovitexin), methyl proto-dioscin, methyl proto-Pb and methyl proto-rhapissaponin from the leaves of R. exelsa. On the other hand, we isolated and identified prosapogenin A of dioscin, dioscin, deltonin, methyl proto-prosapogenin A of dioscin, methyl proto-dioscin and methyl protodeltonin from the stems, dioscin, methyl proto-prosapogenin A of dioscin, methyl proto-dioscin and methyl proto-Pb from the underground parts, and saponaretin, vitexin, isoorientin, methyl proto-prosapogenin A of dioscin, methyl proto-dioscin and methyl proto-Pb from the leaves of R. humilis. Methyl proto-rhapissaponin is a new furostanol saponin and its structure has been established to be 26-O-β-D-glucopyranosyl 22-O-methyl-25(R)-furost-5-en-3β, 22, 26-triol 3-O-[β-D-glucopyranosyl(1→4)-α-L-rhamnopyranosyl(1→4)]-[α-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside. This is the second report of the isolation of steroidal saponins from Palmae plants, and the results are interesting from the standpoint of chemotaxonomy.

View full abstract

In order to separate cortisone acetate, dexamethasone acetate, fluorometholone, and betamethasone valerate in ointments and a cream from the base and excipients, a silica gel column pretreatment was carried out (pre-screening procedure) and then glucocorticoids were successfully determined by high-performance liquid chromatography on a LiChrosorb RP-18 column. By the use of the pre-screening procedure, the base, excipients, and other active ingredients (except glycyrrhetinic acid) in commercial preparations were almost completely removed, and hence a lowering of the efficiency of the LiChrosorb RP-18 column and blocking of the flow of the mobile phase in column caused by the base remaining on the top of the column could be avoided. The contents of glucocorticoids in various commercial preparations were found to be 95.7 to 102.5% of the labeled amounts.

View full abstract

A sensitive fluorimetric method using p-cresol as a hydrogen-donating substrate is described for th assay of thyroid peroxidase in a crude preparation of human thyroid tissue. The fluorescence of the product formed enzymatically from p-cresol is measured at 410 nm with excitation at 325 nm in an alkaline solution (pH 12.3). The specific activities of thyroid peroxidase are 2 to 5 times higher in tissues from patients with Graves' disease than in normal tissues from patients with adenoma of the thyroid. This method is simple and sensitive enough to allow the enzyme assay to be carried out with as little as 10 mg of thyroid tissue.

View full abstract

The synthesis of the 3-glucuronides of 20α-cortolone and their 20β-epimers is described. The cortol 20, 21-diacetates (3, 10) and cortolone 20, 21-diacetates (6, 12) were the key intermediates. Sodium borohydride reduction of the carbonyl group at C-20 in 21-acetoxy-3α, 11β, 17α-trihydroxy-5β-pregnan-20-one 3-tert-butyldimethylsilyl ether (1) or its 11-oxo derivative (4) followed by acetylation of the product with acetic anhydride gave the silyl ether-acetates (2, 5), which, on removal of the protecting group at C-3 with sulfuric acid, were converted into the desired 20β-intermediates (3, 6). On the other hand, the 20α-acetates, 10 and 12, were synthesized from methyl 20α-acetoxy-3α-tert-butyldimethylsilyloxy-17α-hydroxy-11-oxo-5β-pregnan-21-oate (7). Introduction of the glucuronyl residue at the C-3 position was carried out by means of the Koenigs-Knorr reaction.

View full abstract

A simple, sensitive and precise method for serum ferroxidase assay was developed by determining Fe (III) produced enzymatically from Fe (II) added as a substrate in an acetate buffer (pH 6.0). The Fe (III) formed was determined by flow injection analysis (FIA) using thiocyanate as a detection reagent. The reproducibility of this method throughout all steps was satisfactory (coefficient of variation, C.V.=3.4%), and the normal value of human serum ferroxidase activity was 0.30±0.02μmol Fe (III)/min/ml (mean±standard deviation). This method was applied to study the behavior of human and rabbit serum ferroxidases on Sephadex G-150 column chromatography.

View full abstract

Alkaline proteinase produced by one of the monospore isolates of Streptomyces violaceorectus MC675-A8, a producer of the alkaline metalloendopeptidases named alkinonases A and AF, was purified by affinity chromatography on N-benzyloxycarbonylglycylleucylaminohexyl-Sepharose to electrophoretic homogeneity. The enzyme was inactivated by phenylmethanesulfonyl fluoride and diisopropylfluorophosphate, but not by chelating agents or sulfhydryl reagents, and was distinct from alkinonases A and AF. The optimum pH for casein hydrolysis was 9.5-10.5, and the enzyme was stable within a pH range of 5.0-12.0. The molecular weight was estimated to be 30000. The peptidase activity of the enzyme was greatest on N-benzyloxycarbonylglycylprolylleucylglycine ethyl ester among the synthetic substrates tested in this work. Amidase activity was observed towards peptide amides such as N-benzyloxycarbonylglycylleucine amide and N-benzyloxycarbonylglycylphenylalanine amide. The enzyme showed anti-inflammatory activity against carrageenan-induced edema in rats, as did alkinonases A and AF. Bradykinin was hydrolyzed by the enzyme to arginylprolylprolylglycylphenylalanylserylprolylphenylalanine and arginine. Since the enzyme showed a serine proteinase nature, the enzyme was named alkaline proteinase S, and the producing microbe was designated as Streptomyces violaceorectus MC675-A8-S.

View full abstract

A simple assay method for testosterone hydroxylase activity by thin-layer chromatographyultraviolet spectrophotometry was developed. By using this method, the effects of various H₂-receptor antagonists (cimetidine, CIM ; metiamide, MET ; ranitidine, RAN ; famotidine, FAM) or imidazole (IMZ) on the hydroxylations of testosterone by mouse liver microsomal enzymes were studied in vitro. CIM and MET inhibited the 6β-, 7α-and 16α-hydroxylations in a dose-dependent manner. RAN and FAM had little inhibitory effect on any hydroxylation. IMZ inhibited the 6β-hydroxylation to the same degree as CIM and MET, while the effects on the 7α-and 16α-hydroxylations were very weak. The kinetic data indicated that these drugs inhibited the three hydroxylation reactions competitively and the affinities for each hydroxylase varied from drug to drug. The results suggest that the inhibitory actions of CIM or MET on the hydroxylations of testosterone are due to the imidazole ring structure, and the side chain structures may play a role in the enhancement of affinity to the enzymes, particularly the 7α-and 16α-hydroxylases. On the other hand, RAN and FAM containing a ring structure different from imidazole had little effect on any testosterone hydroxylase.

View full abstract

Complexes of ethylenediamine-N, N-diacetic acid (EDDA) with radioactive metal ions and ³H-labeled EDDA were prepared and the biodistribution of the radioactivity in mice bearing Ehrlich tumor was studied. Radioactive complexes studied were those of ⁵¹Cr, ⁵⁷Co, ⁵⁹Fe, ⁶⁴Cu, and ⁶⁷Ga. μ-Oxo ⁵⁷Co EDDA was prepared by treatment of ⁵⁷Co EDDA with hydrogen peroxide. The tumor/blood ratios of radioactivity indicated that ⁵⁷Co EDDA and μ-oxo ⁵⁷Co EDDA were concentrated in the tumor tissues, whereas other complexes and ³H-EDDA were not. The tumor tissues were clearly visualized in scintigrams after the administration of the ⁵⁷Co complexes to mice.

View full abstract

A procedure for the preparation of chicken liver ribonucleases (RNases) is described, involving affinity chromatography on Sepharose coupled with 5'-amino-5'-deoxyuridine 2'(3')-phosphate. Two kinds of RNases having acidic pH optima were obtained. One RNase exhibited preferential nucleolytic activity toward poly C rather than poly U and the other was specific for poly U. The former RNase may be identical to that reported by Levy et al. and the latter resembles acid RNase detected in several mammalian tissues.

View full abstract

Two molecular species of proline endopeptidase (PEP I and II) were concurrently isolated from normal human plasma. They were clearly separated on ammonium sulfate fractionation of plasma followed by chromatographies on DEAE-Sephadex and Con A-Sepharose and thereafter were partly purified by separate but almost identical procedures which included chromatographies on Sephacryl S-300, hydroxyapatite and carbobenzoxy-Gly-Pro-AH-Sepharose. The apparent molecular weights of PEP I and PEP II were 130 and 62 kilodaltons, respectively, as judged by gel filtration on TSK-Gel G 4000 SW. PEP I had a high affinity for Con A-Sepharose, but PEP II did not. Both enzymes were apparently highly specific for peptides such as angiotensin I, bradykinin, substance P and succinyltrialanine p-nitroanilide, cleaving them after proline or alanine residues. However, both enzymes were inert toward elastin and albumin. The K_m and optimal pH values toward succinylglycyl-L-proline 4-methylcoumaryl-7-amide were 2.3 mM and 5.5-6.5 for PEP I and 0.71 mM and 6.0-7.0 for PEP II. Both enzymes were extremely sensitive to diisopropyl fluorophosphate, but no effect was observed with phenylmethanesulfonyl fluoride, aprotinin, soybean trypsin inhibitor, iodoacetamide or ethylenediaminetetraacetate. Elastatinal significantly inhibited PEP II while PEP I activity was only slightly affected. In addition, PEP II was strongly inactivated by p-chloromercuribenzoate and divalent metal ions such as Hg²⁺ and Zn²⁺, whereas PEP I was only slightly inactivated by these reagents. Thus, the enzymes seem to be quite distinct. PEP II appears to be very similar to PEPs purified previously from brain, pituitary and kidney tissues in terms of enzymatic properties, physicochemical properties and substrate specificity. On the other hand, PEP I is different from other PEPs in molecular nature and some enzymatic properties, but is indistinguishable in substrate specificity and enzyme classification.

View full abstract

Technical grade dinitrotoluene (DNT), an important industrial nitroaromatic compound, not only induces methemoglobinemia but is a potent hepatocarcinogen in man and rats. The purpose of the present study was to identify the metabolites produced from five DNT isomers (2, 3-, 2, 4-, 2, 5-, 2, 6-and 3, 4-DNT) by Escherichia coli (E. coli) strain W3110, isolated from human intestine. Data obtained from thin-layer and gas chromatographies or liquid scintillation counting indicated that the metabolites produced by the E. coli were two monoaminonitrotoluenes and hydroxylaminonitrotoluenes in all cases. This finding indicates that DNT is reduced via hydroxylaminonitrotoluenes to monoaminonitrotoluenes in E. coli. In addition, it was found that the reduction reactivities of DNT isomers were 3->2-for 2, 3-DNT, 4->2-for 2, 4-DNT, 5->2-for 2, 5-DNT and 3->4-for 3, 4-DNT. The role of bacterial reduction in the toxic actions of DNT is discussed.

View full abstract

It was observed that the crystal lattice was disordered when crystalline lactose (α-monohydrate) was dehydrated to the α-anhydrate by heating in air. The disorder parameter of the α-anhydrate obtained by desiccating α-monohydrate in methanol was smaller than that of the product obtained by heating it in air. This disorder was also induced by grinding the α-monohydrate, and the free energy level of the water of crystallization in the ground sample seemed to be higher than that in the intact sample. No structural change of amorphous lactose was observed at 30°C in a P₂O₅ desiccator for 30 d. However, during storage at 30°C, 60% R.H. for 24h, crystal growth of α-monohydrate in the solid state occurred and the degree of the crystallinity reached ca. 75%. Further crystal growth hardly proceeded. Crystals of β-anhydrate were also formed. The disorder parameter of this transformed lactose was larger than that of the intact sample. The degree of stress relaxation of lactose was reported to be small, but that of amorphous lactose was nearly equal to that of crystalline cellulose. The tablet hardness of amorphous lactose was ca. ten times that of crystalline lactose.

View full abstract

Adsorption of amino-acetamide derivatives by silica gel from 1-octanol solution, the 1-octanol/water partition coefficient and the capacity factor were investigated. The physico-chemical parameters obtained were well correlated with each other. On the other hand, there was no correlation between the Hammett constant and adsorption parameters. The results indicate that adsorbability measurement may provide useful information for quantitative structure-activity relationship (QSAR) studies.

View full abstract

Isonicotinoyl-γ-aminobutyric acid (GABA) (IG) and nicotinoyl-GABA (NG), candidate prodrugs of GABA, were assessed by measuring various pharmacological responses such as anticonvulsant effect, prolongation of pentobarbital sleeping time and depressive effect on rearing or ambulation in general behavior, in relation to the GABA level in the mouse brain. The GABA level after the intraperitoneal administration of IG at a dose of 1000 mg/kg increased significantly from 2.30±0.02μmol/g wet wt. in the control to 2.93±0.05μmol/g wet wt., while NG caused only a slight increase in GABA level. IG showed a stronger anticonvulsant effect, greater prolongation of pentobarbital sleeping time and greater depressive effect on rearing in general behavior than NG did. The pharmacological effect of IG or NG corresponded well to the GABA level in the brain.

View full abstract

Adsorption characteristics of bile salt anions by activated carbon beads consisting of about 67% activated carbon powder in agar were studies in vitro and compared with those of a cholestyramine preparation from the standpoint of usefulness for sequestering bile salts in the intestine. Although the rate of adsorption of bile salts by the activated carbon powder in the beads was somewhat reduced as compared with that of the naked powder, the adsorption capacity was essentially retained in the beads, and was enhanced by the presence of physiologic anions. The interfering effect of physiologic anions on the adsorption by cholestyramine resin preparation was greater for trihydroxy bile salts than for dihydroxy analogs. Thus, the beads showed a greater capacity for trihydroxy bile salts than the resin preparation. For dihydroxy analogs the opposite relationship was observed, i.e., the capacity of the resin preparation was greater than that of the beads. In the presence of triolein, the adsorption of bile salts by the powder was greatly impaired, whereas that by the beads was only slightly reduced. Encapsulation of the powder by agar apparently permitted selective adsorption of bile salts in the presence of the lipid.

View full abstract

In the hyperlipidemia induced by fructose in rats, 5-tridecylpyrazole-3-carboxylic acid (TDPC) reduced serum triglyceride (TG) and cholesterol (CH) levels dose-dependently. The hypolipidemic profile of this compound was quite similar to that of clofibrate. In a time-course experiment, TDPC lowered serum lipid levels shortly after the treatment, and the maximum effect was observed at 4-8h, which is quite similar to the action of clofibrate. However, there were some differences between TDPC and clofibrate in the effects on lipoprotein patterns ; TDPC increased the ratio of high density lipoprotein (HDL) to very low density lipoprotein+lower density lipoprotein (VLDL+LDL)-CH in a dose-dependent manner but clofibrate did not. The hypolipidemic effect of TDPC was due to a decrease in serum lower density lipoprotein rather than high density lipoprotein. In addition, TDPC remarkably increased the rate of disappearance of exogenous triglyceride. No adverse effects of TDPC were found on parameters such as relative liver weight and hepatic lipid content.

View full abstract

We investigated which laboratory animal would be the most suitable for studies on the mechanism of adriamycin (ADR)-induced lipid peroxidation. Lipid peroxide (LPO) levels of the main organs on the 3rd day after ADR administration (when a remarkable increase of LPO level in mouse heart was seen) were determined. Both the normal LPO levels and the increase ratios of LPO levels after ADR administration in all the studied tissues of mice, except for the serum, were higher than those in rats and guinea pigs. Next, strain differences in 9 strains of male mice (DDY, ICR, C3H, BALB/c, C57BL, DBA/2, BDF₁, CDF₁ and B6C3F₁) were examined, and clear strain differences were recognized. Further, sex and weekly age differences of LPO tissue levels in CDF₁ strain mice were shown. It was concluded that DBA/2 and CDF₁ strains of male mice were the most suitable laboratory animals for studies of the mechanism of the lipid peroxidation induced by ADR.

View full abstract

The thermolysis of 1-methyl-2-vinylpiperidine N-oxide (13) resulted in Meisenheimer [1, 2] rearrangement to give the hexahydro-1, 2-oxazepine (23) and no [2, 3]-sigmatropic rearrangement product (24). Similarly, the 1-methyl-1, 2, 5, 6-tetrahydropyridine N-oxides (15), on heating, gave only the tetrahydro-1, 2-oxazepines (26, 27), together with the Hofmann-type elimination products (28). The thermolysis of 1-benzyl-2-phenyl-(20a) and 1-benzyl-2-vinyl-piperidine N-oxide (20b) afforded the corresponding 1-benzyloxypiperidines (32) as well as the 1, 2-oxazepines (31). These results are different from those observed for open-chain allylamine N-oxides and 1-alkyl-2-vinylpiperidine N-imides and N-ylides, which are known to undergo only [2, 3]-sigmatropic rearrangement.

View full abstract

A radioimmunoassay for N^α-(N-acetylmuramyl-alanyl-D-isoglutaminyl)-N^ε-stearyl-lysine (MDP-Lys-L18) was established. Antibody to MDP-Lys-L18 was obtained from rabbits by immunization with MDP-Lys-L18 conjugated with bovine serum albumin (BSA). ³H-MDP-Lys-L18 labelled at C₉ and C₁₀ of the stearyl residue was used. The ammonium sulfate method was adopted for the separation of bound and free fractions. Though the protein binding of MDP-Lys-L18, which was due to the stearyl residue in the molecule, influenced the sensitivity of the radioimmunoassay, the lower limit for the quantitative determination of MDP-Lys-L18 in serum was estimated to be 500pg/ml and the coefficient of variation of this assay was between 3 and 5%.

View full abstract

Commercially available phenytoin crystals were fractionated by passing them through 44-350 μm sieves, then blended with diluents to prepare phenytoin-diluent physical mixture or made into solid dispersions with polyethylene glycol 4000 (PEG 4000). Dissolution studies of phenytoin in artificial gastric juice suggested that less than 1 to 10 ratio of phenytoin to PEG 4000 was required to disperse amorphous phenytoin completely in the carrier. A comparative dissolution study of various sizes of phenytoin crystals revealed the existence of a critical particle size between 74-149 μm, but phenytoin in solid dispersions showed far faster dissolution and higher solubility than any of the particle fractions. A three-way cross-over study was carried out for phenytoin crystals of particle size 44-53 μm, phenytoin-diluent physical mixture and the phenytoin solid dispersion in five healthy human volunteers. The solid dispersion gave the highest bioavailability of phenytoin, followed by the physical mixture. Thus, phenytoin solid dispersion in PEG 4000 appears to have the clinical advantages of quick release and excellent bioavailability. It also appears that there is a critical particle size for dissolution of phenytoin from powders in vitro. The phenytoin solid dispersion gave a larger bioavailability than crystals below the critical size in vivo.

View full abstract

Barleriaquinone (BQ) has been isolated from the roots of Barleria buxifolia LINN., and its structure has been established as 1-hydroxy-7-methylanthraquinone by degradative and spectroscopic methods.

View full abstract

The structure of the compound 2, 2, 4-dioxo-3-nitroso-1, 2, 4, 5-tetrahydropyrazolo [1, 5-a]-quinoxaline, which was previously obtained from the reaction of 3-hydrazinocarbonyl-methylene-2-oxo-1, 2, 3, 4-tetrahydroquinoxaline (1a) with an excess of nitrous acid, was revised to 3-(1, 2, 4-oxadiazolin-5-on-3-yl)-2-oxo-1, 2-dihydroquinoxaline (7) on the basis of comparison with an authentic sample prepared from 3-cyano-2-oxo-1, 2-dihydroquinoxaline (8) via an unambiguous route.

View full abstract

Substance P analogs, [Arg¹¹]-SP and SP (1-10)-Arg-OH, were synthesized by the liquid phase method. These peptides were purified by ion-exchange chromatography and partition chromatography. The biological activity of the peptides was evaluated in vitro on guinea-pig ileum. Replacement of Met¹¹ in SP with Arg diminished the potency of SP activity. The analogs were found to be inactive as antagonists.

View full abstract

The reaction of haloketenes with 1, 3-diaza-1, 3-diene compounds, prepared by condensation of 2-amino-heterocycles with aromatic aldehydes, gave the [2+4] cycloadducts, fused pyrimidinones (6-17). However, haloketenes underwent the cycloaddition with 2-(p-anisylideneamino)-benzimidazole (5) to give both pyrimido [1, 2-a] benzimidazoles (18 and 19) and 2-azetidinones (20-22).

View full abstract

Lead tetraacetate oxidation of (±)-predicentrine (4) in acetic acid and in CH₂Cl₂ gave (±)-4-O-acetylsrilankine (11) and the o-quinol acetate (6), respectively. The latter (6) was transformed into (±)-O, O-diacetylsrilankine (13) under Thiele's conditions. Hydrolysis of 11 or 13 afforded (±)-srilankine (1), methylation of which yielded (±)-cataline (2).

View full abstract