The interactions of theophylline with aliphatic diamines and aliphatic monoamines were studied by measurements of solubility in aqueous solution. Diamines such as 1, 3-propanediamine, 1, 4-butanediamine, 1, 6-hexanediamine and ethylenediamine, and monoamines such as triethylamine, diethylamine, n-propylamine, allylamine, 2-methoxylamine, ethanolamine and triethanolamine were used. The solubility of theophylline increased in the presence of amine and increased in proportion to the pK_a of the amine. The amount of the complex formed was found to be proportional to the pK_a of the amine and the apparent stability constant of complex formation tended to increase as the pK_a of the amine increased.

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The hydroalkylation of aniline in a palladium-fused salt system was examined under hydrogen pressure. By using 0.5% Pd-Al₂O₃ and fused salt (NaCl-AlCl₃), N-cyclohexylaniline (4) was formed as the main product at temperatures below 300°C ; at higher temperatures, the cyclohexyl group rearranged to form nuclear alkylated products, o-and p-cyclohexylaniline, (5) and (6). The yield of 4 was 27.7% at 280°C ; those of 5 and 6 were 5.4 and 7.3%, respectively, at 400°C. The reaction mechanism for the hydroalkylation of aniline with this catalytic system is discussed.

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Oxidation of glucose on immobilized enzymes was carried out to study the effect of intraparticle mass transfer of oxygen on the global rate of the reaction. The catalyst employed, which consisted of both glucose oxidase and catalase supported on activated carbon, was prepared by the carbodiimide method. The experiments were performed in 0.1M acetate buffer solution (pH=5.5) and at 25°C. The initial rates of reaction were determined from the absorption rates of oxygen gas, under conditions of no external mass transfer resistances. Nonlinear least-squares analysis gave values of the maximum velocity (V), the Michaelis constant (K_m), the effective diffusivity of oxygen (D₆) and the tortuosity factor of the particles (τ) of V=8.30×10^-6 [mol/ (g-cat·s)], K_m=9.46×10^-5 [mol/cm³], D_e=7.70×10^-6 [cm²/s] and τ=2.0 [-], respectively. The effectiveness factor calculated from these values indicated that the intraparticle mass transfer of oxygen in the liquid significantly influences the global rate of reaction.

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The synthesis of the new heterocycles, 4-amino-[1] benzofuro [2, 3-g] cinnolines, was accomplished by the intramolecular cyclization of the (Z) configuration of arylhydrazono-acetonitriles (5). The latter compounds were obtained via interaction between diazonium salt of 2-aminodibenzofuran (1) and various active methylene compounds following the Japp-KLINGEMANN reaction. The alkaline treatment of azo intermediates (4), which may be isolated in the course of condensation, gave the corresponding arylhydrazonoacetonitriles (5). A study of the configuration and possible isomerisation of 5 is reported. The linear structure of 4-amino-[1] benzofuro [2, 3-g] cinnolines was elucidated by H-nuclear magnetic resonance.

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The total synthesis of a thromboxane A₂ analog (2) and its hydroxy epimer, starting from the exo-adduct (3) of maleic anhydride and furan, has been achieved via a key intermediate (9).

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A series of cross-coupling reactions of aryl Grignard reagents with aromatic iodides catalyzed by bis (acetylacetonato) nickel (II) was studied to establish reaction conditions under which the cross-coupling proceeded selectively and quantitatively. Under the standard conditions thus selected, the coupling reactions proceeded within a few hours by a simple procedure and gave very pure product in high yield. Thus, the Kharash-type cross-coupling reaction was found to be very useful for the synthesis of a variety of polyphenyls. With some reactants of more or less orowded geometry, the reaction resulted in rather low yields of polyphenyls. This cross-coupling was successfully applied to the synthesis of a new compound, 2, 6, 2', 6'-tetraphenylbiphenyl, having a highly non-planar arrangement of π-systems.

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The structures of three triterpenoidal saponins, onjisaponins A (1), B (2) and E (3), were determined on the basis of spectral and chemical evidence as presenegenin-(3)-β-D-glucopyranosido-(28)-2-O-{[β-D-apio-D-furanosyl (1→3)] [β-D-galactopyranosyl (1→4)-β-D-xylopyranosyl (1→4)]-α-L-rhamnopyranosyl}-3-O-(α-L-rhamnopyranosyl)-4-O-(4'-methoxy-cinnamoyl)-β-D-fucopyranoside, presenegenin-(3)-β-D-glucopyranosido-(28)-2-O-[β-D-galactopyranosyl (1→4)-β-D-xylopyranosyl (1→4)-α-L-rhamnopyranosyl]-3-O-(α-L-rhamnopyranosyl)-4-O-(4'-methoxycinnamoyl)-β-D-fucopyranoside (=senegin III) and presenegenin- (3)-O-β-D-glucopyranosido-(28)-2-O-[β-D-galactopyranosyl (1→4)-β-D-xylopyranosyl (1→4)-α-L-rhamnopyranosyl]-4-O-(3', 4', 5'-trimethoxycinnamoyl)-β-D-fucopyranoside, respectively. The ¹³C nuclear magnetic resonance spectra of onjisaponins A, B, E and their derivatives were investigated and each carbon signal was assigned as shown in Table I.

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Reaction of 3β-acetoxybisnorchol-5-enic acid (1) with lead tetraacetate in benzene gave ⊿²0 and ⊿^{17 (20)} compounds (2a and 2b) and 20α-and 20β-acetoxy compounds (3a and 3b), with a trace of 20-keto compound (4) and a dimer (5). The structure of 5 was determined as 3'β-acetoxypregn-5'-en-20'α-yl 3β-acetoxybisnorchol-5-enate (Chart 1). The use of toluene or xylene in place of benzene resulted in decreased formation of 2b with the formation of the 17-ethyl compound (2c). The reactions were examined under various conditions (Table I) and the reaction mechanisms are discussed.

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4-Chloro-5-(2-hydroxyethylamino)-3 (2H)-pyridazinones were converted upon treatment with base to novel fused ring compounds, 3, 4-dihydro-2H-pyridazino [4, 5-b]-1, 4-oxazin-8 (7H)-ones. When a nitrogen atom in the hydroxyethylamino group or at the 2-position of the 3 (2H)-pyridazinone ring had a remaining hydrogen, the ring closure reaction did not occur. 3, 4-Dihydro-2H-pyridazino [4, 5-b]-1, 4-oxazine was similarly synthesized from corresponding precursor. The presence of a C-6 amino group in the precursor did not affect the ring formation mentioned above, but when the amino group was diazotized, ring formation took place in a different fashion, involving the C-6 diazonium moiety as a leaving group, to give another ring system, 6, 7-dihydro-2H-pyridazino [3, 4-b]-[1, 4] oxazin-3 (5H)-one. Similar phenomena were observed in the cases of precursors having a C-6 nitro group : cyclization occurred involving the elimination of the nitro group to give fused ring products. The method was applied to the formation of a tricyclic heterocycle, 2H-pyridazino [3, 4-b] [1, 4] benzoxazin-3 (5H)-one. Some compounds thus obtained were found to have potent analgesic and significant anti-inflammatory activities in animal models.

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As a part of our study on the reactions of 1-ethoxyisochroman (1) with nucleophilic reagents, the reactions of 1 with amines, amides, thioamides, sulfonamides, urea, thiourea, and heterocyclic compounds were examined.

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The reactions of 2-nitronaphthalenes with active methylene compounds in the presence of a base were investigated. 2-Nitronaphthalene (I) reacted with ethyl cyanoacetate in the presence of potassium hydroxide to produce ethyl N-(1-cyano-2-naphthyl) oxamate (IIa) in 64% yield. Similarly, the reactions of I with methyl cyanoacetate, p-nitrophenyl-acetonitrile, ω-cyanoacetophenone, α-cyanoacetamide and 1-cyanoacetylpyrrolidine in the presence of a base gave the 2-aminonaphthalene-1-carbonitriles (IIb-f) as major products corresponding to IIa. On the other hand, the reaction of 2, 3-dinitronaphthalene (VII) with ethyl cyanoacetate in the presence of potassium hydroxide gave ethyl 2-cyano-2-(3-nitro-1-naphthyl)-acetate (VIIIa). In the reaction of 2-nitronaphthalene-1-carbonitrile (IX) with ethyl cyanoacetate, ethyl 2-cyano-2-(1-cyano-2-naphthyl) acetate (Xa) was obtained.

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Four triterpenoid prosapogenins named CP_7a, CP_8a, CP_9a and CP_10a have been isolated from the alkaline hydrolysate of the crude saponin obtained from the root of Clematis chinensis OSBECK. On the basis of chemical and physicochemical evidence, they were characterized as follows : CP_7a (I), oleanolic acid 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside ; CP_8a (VI), hederagenin 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside ; CP_9a (XI), oleanolic acid 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside ; CP_10a (XIII), hederagenin 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside.

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The nonatriaoontapeptide corresponding to the entire amino acid sequence of ostrich adrenocorticotropic hormone (ACTH) was synthesized by successive condensations of four peptide fragments ; Z (OMe)-(15-19)-OH, Z (OMe)-(11-14)-OH, Boc-(5-10)-OH and Z-(1-4)-NHNH₂, with H-(20-39)-OBzl, a synthetic intermediate of ostrich-type corticotropin-like intermediate lobe peptide, followed by deprotection with 1M trifluoromethanesulfonic acid-thioanisole in TFA. The synthetic peptide exhibited an in vivo steroidogenic potency of 0.8 relative to synthetic human ACTH.

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An octanucleotide containing a recognition site for Eco RI and its arabinosyladenine (araA) analog, dGGAATTCC and dGGaraAATTCC, were synthesized by the phosphotriester approach with phosphoro-p-anisidate as the protecting group for 3'-phosphodiesters. araA was converted to 5'-dimethoxytrityl-N, 2'-O-benzoyl 3'-p-chlorophenyl phosphate and condensed with N-benzoyldeoxyadenosine 3'-p-chlorophenyl phosphoro-p-anisidate to yield the protected araAdAp. Other deoxynucleotide blocks (dAAp, dGGp) were prepared similarly and condensed with a 5'-deblocked dTTCC block having the 3'-O-benzoyl group after removal of the p-anisidate group with isoamyl nitrite.

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Reaction of 2-or 3-substituted 3-(2-pyridyl)-2-propenals (3a-f) with secondary amines afforded 2-or 1-substituted 3-(dialkylamino) indolizines (2a-1) in the presence of metal halides. It was found that titanium tetrachloride was the best catalyst.

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Various derivatives of 5-fluoro-5, 6-dihydrouracil with an alkoxycarbonyl, substituted carbamoyl, or cyano group at C-5, and one of a variety of substituents, i.e., alkoxy, substituted mercapto, substituted amino, acyl amino, and alkylidene- and arylideneaminooxy at C-6, have been synthesized as a class of potential pro-drugs of autitumor agents, 5-fluorouracil (5-FU) and 1-(2-tetrahydrofuryl)-5-fluorouracil (Ftorafur). Antitumor activity of these compounds against leukemia P388 or L1210 in mice and antifungal activity against Botrytis cinerea are described.

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2-Mesitylenesulfonyldiazomethane (4) was conveniently prepared from 2-mesitylenesulfonylchloride (5) in 4 steps. Reaction of 4 with benzoyl chloride smoothly furnished α-benzoyl α-(2-mesitylenesulfonyl) diazomethane (9). Thermal treatment of 9 in the presence of alcohols generally gave the Wolff rearrangement product (10) as the major product accompanied with the intramolecular C-H insertion product (11). When acetonitrile was used as a reaction solvent, a substantial amount of the desulfonylated product (13) was also formed.

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The structures of three new alkaline degradation products of tetrahydrodesoxyusnic acid (isotype) (Ib) were established to be IIb, III and IV, by chemical and spectral studies. The mechanisms of formations of IIb, III and IV from Ib are discussed.

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The total synthesis of (±)-trypargine (2) was accomplished to confirm the proposed structure, 1-(3'-guanidinopropyl)-1, 2, 3, 4-tetrahydro-β-carboline. Optically active (-)-trypargine (2a) and its enantiomer (2b) were obtained by the optical resolution of the racemate. The stereochemistry at C-1 is discussed on the basis of the optical rotatory dispersion and circular dichroism curves.

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Friedel-Crafts reaction of aromatic compounds with ethyl α-chloro-α-(methylthio)-acetate (1) gave ethyl α-(methylthio) arylacetates (2), which were readily converted into ethyl arylacetates (3) by reductive desulfurization with Raney nickel or zinc dust-acetic acid. The reactions were applied to the syntheses of ibufenac (5) and alclofenac (6), which are anti-inflammatory agents.

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Reaction of N-(benzylideneamino) ephedrine (II) with benzylmagnesium chloride gave N-(1, 2-dimethylethylamino) ephedrine (a mixture of III and IV). On the other hand, reaction of N-[(2-phenylethylidene) amino] ephedrine (VII) with phenyllithium gave IV. Hydrogenolysis of these products gave 1, 2-diphenylethylamine with 40% (S) and 91% (R) optical purity, respectively, and l-ephedrine used as a chiral auxiliary reagent was recovered. Reactions of N-(arylmethylideneamino) ephedrines (VIII and IX) with benzylmagnesium chloride gave chiral hydrazines (X and XI), and reactions with phenyllithium gave XII and XIII.

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For studies on the cyclopentane ring formation from acyclic monoterpenes in the biosynthesis of iridoid glucosides, the following ¹³C-labeled precursors of the acyclic monoterpene series were synthesized : [9-¹³C]-and [4-¹³C]-10-hydroxygeraniol (9), [2-¹³C]-9, 10-dihydroxygeraniol (10), (R)-(+)-and (S)-(-)-[9-¹³C]-10-hydroxycitronellol ((R)-(+)-and (S)-(-)-8), (R)-(+)-and (S)-(-)-[8-¹³C]-9, 10-dihydroxycitronellol ((R)-(+)-and (S)-(-)-11).

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The murexide reaction of caffeine was investigated to clarify the pathway of the coloration. From the reaction mixture of caffeine with nitric acid, 1, 3-dimethylalloxan (II) and 1, 3, 7-trimethyl-2, 6-dioxo-8-nitro-1H, 3H, 7H-xanthine (IV) were isolated. Compound II was found to be the key intermediate, since it was converted to a purple-red-colored substance, murexoin (III), by treatment with conc. ammonia. It was found that 1-hydroxy-5, 7-dimethyl-2, 4, 6-trioxo-1H, 5H, 7H-oxazolo [4, 5-d] pyrimidine (I), previously obtained by the oxidation of caffeine with hydrogen peroxide and hydrochloric acid, was also transformed to III with conc. ammonia. Consequently, the murexide reaction of caffeine was shown to have two pathways of coloration depending on the oxidizing agent employed. From the spectral data, a symmetrical structure (III) was assigned to murexoin in solution. Amalic acid, which has been reported as an intermediate of the murexide reaction of caffeine, can be ruled out on the basis of our experimental results.

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Some information about the binding of coenzyme to 20β-hydroxysteroid dehydrogenase [EC 1.1.1.53] from Streptomyces hydrogenans was obtained from the coenzyme specificity and from inhibition studies using various nucleotides. Nicotinamide adenine dinucleotide (NAD) and its analogs served as coenzymes of the enzyme, but NADP did not. When NAD was modified in the adenine ring, the apparent K_m values increased considerably and these values were very high as compared with those of the NAD analogs modified in the nicotinamide moiety. Adenosine 5'-diphosphate (ADP)-ribose, ADP and 5'-AMP were strong competitive inhibitors with respect to coenzyme. ATP and adenosine moderately inhibited the enzyme activity. IMP, IDP, IDP-ribose, 1, N⁶-etheno-adenosine 5'-monophosphate, 2'-AMP and 3'-AMP were very poor inhibitors. The N⁶-amino group of the adenine moiety may be essential for the binding of the coenzyme to the enzyme, and the phosphate group at the 5'-position of ribose of the adenosine moiety may also play an important role in the binding process. The presence of a phosphate group at the 2'-or 3'-position of ribose of the adenosine moiety resulted in a significant decrease in the binding capacity of adenine nucleotides to the enzyme. 2'-Deoxy-5'-AMP and 3'-deoxy-5'-AMP were very poor inhibitors relative to 5'AMP, indicating that there may be an interaction between the 2'-or 3'-hydroxyl group and the coenzyme binding site. Nicotinamide and NMN showed only slight inhibitory action compared with adenine nucleotides. It was suggested that the adenosine moiety of the coenzyme may have an important role in the binding interaction with the enzyme.

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3β-Hydroxysteroid oxidase activity was detected in the broth filtrate of strain H 1109 MY 12. Based on taxonomic studies, this strain was shown to be a new species of genus Streptoverticillium and the name Stv. cholesterolicum is proposed for this strain. The enzyme was purified by ammonium sulfate precipitation and affinity column chromatography on crystalline cholesterol, and the product showed a single band on SDS-polyacrylamide gel electrophoresis. The enzyme showed optimum activity at pH 7.0-7.5 and was stable over a rather wide pH range of 4.0 to 12.5. Cholesterol and dihydrocholesterol were oxidized rapidly. The K_m value for the oxidation of cholesterol by this enzyme was about 0.4mM. The enzyme activity was greately inhibited by HgCl₂, and AgNO₃. FeCl₃ and CuSO₄ were also inhibitory though to lesser extents. Iodine and N-bromosuccinimide completely inhibited the enzyme activity at concentrations of less than 0.01 mM. Neither metal-binding agents nor p-chloromercuribenzoic acid had any inhibitory effect on the oxidation of cholesterol by this enzyme. The molecular weight of the enzyme was estimated to be 56000 by SDS-polyacrylamide gel electrophoresis. The enzyme was proved to be a flavoprotein containing flavin adenine dinucleotide as a prosthetic group.

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Thirteen derivatives of hydroxypyruvaldehyde phenylosazone were tested for effects on bovine erythrocyte membrane. Six drugs, including hydroxypyruvaldehyde phenylosazone itself and the o-CH₃, m-CH₃, p-CH₃, o-Cl and p-Cl derivatives, increased the stability of bovine erythrocyte membrane to hypotonic shock ; the other derivatives with highter molecular weights were essentially without effect. Observations by scanning electron microscopy indicated that non-substituted, m-CH₃ and o-Cl hydroxypyruvaldehyde phenylosazones caused extrusion of the erythrocyte surface, whereas o-CH₃, p-CH₃ and p-Cl derivatives caused invagination of the erythrocyte membrane. The p-Cl derivative can be adsorbed onto, or incorporated into the bovine erythrocyte membrane.

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Two different glycophorins were isolated from rabbit erythrocyte membranes. Crude glycophorin fraction prepared by extraction with lithium diiodosalicylate and partition in aqueous phenol contained water-soluble glycolipid in addition to glycophorins. After removal of the glycolipid from glycophorins by ion-exchange chromatography, glycophorins were effectively fractionated by two different techniques : gel chromatography on Bio-Gel A 1.5m and ion-exchange chromatography in the presence of a nonionic detergent, Ammonyx LO. Two glycophorins were isolated in a ratio of about 1 : 2 (10 mg and 18 mg/3 g lyophilized erythrocyte membranes). The minor component, designated glycophorin RA, was a sialoglycoprotein (42% protein and 58% carbohydrate) and the major one, designated glycophorin RB, was of smaller size and lower carbohydrate content (57% protein and 43% carbohydrate). Glycophorins RA and RB were also different in chemical composition and amino-terminal sequence. Further evidence that the structures of the two glycophorins are unrelated to each other was obtained by Ouchterlony immunodiffusion with lectins ; glycophorin RB precipitated with concanavalin A and Ricinus communis agglutinin, whereas glycophorin RA did not.

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A mass fragmentographic method for the quantitative determination of ferulic acid, a main metabolite of γ-oryzanol in rabbit and dog plasma, was developed. After its isolation by Amberlite XAD-2 chromatography and by solvent extraction, ferulic acid was converted to the heptafluorobutyryl ethyl ester derivative and analyzed by using 4-hydroxy-3-trideuteromethoxycinnamic acid (ferulic acid-d₃) as an internal standard. The recovery of ferulic acid from plasma samples was 100.5±4.5% (mean±standard deviation, n=20) in the range of 5.0-150.0 ng/ml plasma. γ-Oryzanol was administered orally to rabbits and dogs at doses of 25, 50 and 100 mg/kg body weight as a solution of sesame oil, and then the plasma concentrations of ferulic acid were determined by mass fragmentography. A good correlation between the administered dose of γ-oryzanol and the area under the concentration curve (AUC) of ferulic acid was obtained in both animals, so the AUC of ferulic acid may be used as an index for estimating the extent of gastrointestinal absorption of γ-oryzanol.

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In the present study, as a continuation of the previous work on a new mucosal dosage form for insulin using hydroxypropyl cellulose-H (HPC) and Carbopol-934 (CP), we attempted to develop a dosage form containing a local anesthetic for toothache, using lidocaine as a model drug in HPC and CP as the peripheral base. The dissolution profile of lidocaine was investigated with various mixing ratios of freeze-dried HPC/CP (FD-HPC/CP) to lidocaine in the core base, and the absorption when this dosage form was applied to the human gingiva was also studied. It was found that the rate in the dissolution test was delayed with increase in the amount of FD-HPC/CP in the core base and that the absorption of lidocaine was about 30% after 1 hour and then increased by about 10% of the initial amount per hour for 4 hours in preparations containing 5 and 10 mg of FD-HPC/CP.

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The effects of methylcellulose (MC) and hydroxypropylcellulose (HPC) on oil-in-water (o/w) type ointment bases and the crystalline conversion from the anhydrous form of prednisolone (A-PD) to its hydrated form (C-PD) in such o/w type ointment bases were studied. The weight loss of the base, that is the volume of water released from the base, was measured in dry conditions and at a low temperature (5°C). The weight loss of the base without MC or HPC (No.1 base) was about 7% in 10d. On the other hand, the weight losses of the base containing MC (No.3 base), and that containing HPC (No.6 base) were about 1% and 4% under the same conditions, respectively. The yield value of each of the bases Nos.1, 3, and 6 was measured with a spreading meter. The yield value of No.1 base was smaller than that of the other bases for 24h after preparation at room temperature (25°C). The yield value of No.3 base became lower and that of No.6 base became slightly greater than that of 24 h for 7 d after preparation upon storage at room temperature and humidity (25°C). However, the yield value of No.1 base increased after 7 d. The crystalline conversion of A-PD in o/w type ointment base was measured by the use of an X-ray diffractometer. A-PD was entirely converted into C-PD in No.1 base in only 1 d at 5°C. On the other hand, when MC or HPC was incorporated in the base, the conversion of A-PD to C-PD was retarded. The periods required for complete crystalline conversion from A-PD to C-PD in the bases (Nos. 2-4) containing MC and the bases (Nos.5-7) containing HPC were about 20-30 d and 3-18 d, respectively. Furthermore, in vitro release of PD from No.1 base without MC and from No.2 base containing MC were studied for 24 h after preparation at 5°C. Since A-PD was entirely converted into C-PD after 24 h at 5°C, the amount of PD released from No.1 base was less than that of immediately after preparation, whereas the release pattern of PD from No.2 base was the same as that from the base immediately after preparation since crystalline conversion did not occur in No.2 base. In addition, the release of PD from No.4 base was studied for 10 d after preparation at 5°C. The amount of PD released from No.4 base after 10 d was the same as that of immediately after preparation, since crystalline conversion of PD did not occur in this base. Water-soluble polymers such as MC and HPC act as protective colloids, and so the structure of the emulsion in the ointment base is stabilized. The structure of the emulsion in the base including water-soluble polymer is stabilized even at low temperature where the o/w type emulsion structure normally deteriorates. It is suggested that water-soluble polymer is an effective additive to o/w type ointment bases.

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To examine the influence of drugs on the physicochemical stability of semisynthetic fatty suppositories, model suppositories containing various kinds of drugs were prepared with Witepsol H-15 and storage experiments were performed. It was shown that the stability of pharmaceutical properties was largely dependent on the solubility of drugs in the fatty vehicle (S_o). That is, the rate of polymorphic transition of the vehicle measured by X-ray diffraction was accelerated by lipid-soluble drugs but was not much affected by lipid-insoluble drugs. The melting points measured by DTA generally decreased with inrreasing S_o, but they all increased by about 2 or 3°C during storage. The softening time decreased with increasing S_o. The release properties measured at 37°C were dependent on the solubility of the drugs in the dissolution fluid (S_w) initially but became dependent on S_o during storage. This phenomenon is discussed on the basis of the transportion mechanism of drugs in the lipid phase.

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The effects of pH, albumin and urate on the inactivation behavior of rabbit muscle creatine phosphokinase (CPK) have been investigated at 39°C. The conditions under which the inactivation of CPK shows apparent first order kinetics and biphasic behavior have been explored and regression equations are presented along with half-lives. A circular dichroism (CD) study showed no evidence of spectral change of CPK in the range of pH 6.00-8.00, and a kinetic study showed that CPK is most stable at near neutral pH. Both rabbit serum albumin (RSA) and urate significantly enhance the stability of the CPK activity and retard CPK coagulation during incubation. No evidence for any intermolecular interactions between RSA and CPK in pH 7.40, 50 mM phosphate buffer solution was obtained in the CD study. CPK is presumably just dispersed in the matrices of RSA and stabilized by protein colloid. On the other hand, urate is likely to form a polymeric structure in the buffer at around the physiological urate level in circulatory blood (2.5-3.5×10^-4M). CD spectra of CPK can be observed with a maximum around 287 nm, induced by urate. The molar ellipticity coefficient depends on the concentration of urate present in the solution.

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The inactivation profiles of rabbit muscle creatine phosphokinase (CPK) in heparinized whole blood and plasma under various pH conditions at 39°C have been investigated. Under pH 7.40, at 39°C, the inactivation profile of CPK in heparinized whole blood follows apparent first-order kinetics. The inactivation rate constant is 0.054 h^-1 and is very close to that observed in patients with acute myocardial infarction (reported by Steele and Cohn) but much less than that resulting from exercise (reported by King et al.). The nature of the inactivation of CPK in vitro is presumably thermal denaturation, and there is no evidence to show that blood cells or dissolved oxygen are involved. The inactivation profiles of CPK in heparinized whole blood at pH other than 7.40 appear to be biphasic. The regression equations and the half-lives are presented.

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A radioreceptorassay with ³H-diazepam and benzodiazepine receptors was used to determine the levels of receptor reactive substances (diazepam activity) in various brain regions after i.p. injection of six benzodiazepines (2 mg/kg) into rats. All the compounds tested gave nearly the same regional distribution. The correlations between diazepam activities and pharmacological or clinical potencies were better than those between in vitro binding data and central nervous system activities. This result indicates that the determination of diazepam activity in the brain by the radioreceptorassay technique is useful for evaluating the pharmacological and clinical potencies of benzodiazepines and related compounds.

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The mode of antiproliferative action of two 5-fluorocytosine nucleosides, 5-fluoro-cytidine (FCR) and 5-fluoro-2'-deoxycytidine (FCdR), was examined using mouse leukemia L5178Y cells in vitro. FCR and FCdR were markedly active against L5178Y cells, though the cells were deficient in cytidine deaminase activity. Both compounds increased the incorporation of ¹⁴C-labeled thymidine into the acid-insoluble fraction of L5178Y cells and decreased labeled deoxycytidine incorporation. In reversal studies, the antiproliferative effects of both compounds were almost abolished by simultaneous addition of thymidine or deoxyuridine. Deoxycytidine completely reversed the growth inhibition caused by FCdR, but not that caused by FCR. These results demonstrate that the cytotoxicity of both compounds is due to inhibition of thymidylate synthetase, presumably through formation of 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) after deamination by deoxycytidylate deaminase in the pyrimidine de novo pathway.

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L-Ascorbic acid 2-O-phosphate, synthesized from 5, 6-O-isopropylidene L-ascorbic acid by reaction with phosphoryl chloride in alkaline water-pyridine solution, crystallized as colorless plates of the dipiperazinium salt trihydrate, C₆H₉O₉P·2C₄H₁0N₂·3H₂O. Its molecular structure was determined by X-ray analysis.

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Two novel fused ring systems made up of 1, 4-thiazine and 3 (2H)-pyridazinone were synthesized. 3, 4-Dihydro-7-methyl-2H-pyridazino [4, 5-b]-1, 4-thiazin-8 (7H)-one was obobtained from the intramolecular cyclization of 4-chloro-5-(2-chloroethylamino)-2-methyl-3 (2H)-pyridazinone with sodium sulfide. An isomer, 3, 4-dihydro-6-methyl-2H-pyridazino-[4, 5-b]-1, 4-thiazin-5 (6H)-one, was directly afforded by the condensation of 4, 5-dichloro-2-methyl-3 (2H)-pyridazinone with sodium 2-aminoethanthiolate.

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Pyrimidine derivatives containing a 1, 3-dicarbonyl side chain at the 2-or 4-position were synthesized by the Claisen condensation of pyrimidine 2- or 4-carboxylic esters with ethyl acetate, acetophenone, and phenylacetonitrile. The keto-enol tautomerism of the products is also reported.

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The reaction of 2-(methylamino) benzamide with levulinic acid gave 3a, 4-dimethyl-2, 3, 3a, 4-tetrahydropyrrolo [2, 1-b] quinazoline-1, 9-dione (2). 3a-Methyl-2, 3, 3a, 4-tetrahydropyrrolo [2, 1-b] quinazoline-1, 9-dione (6) was prepared by the method shown in Chart 3. The compounds 2 and 6 were different from authentic samples A and B prepared by the method reported by previous workers. The real structures of A and B were found to be 3a-methyl-2, 3, 3a, 4-tetrahydropyrrolo [1, 2-a] quinazoline-1, 5-dione (9) and 3-(2, 3-dimethyl-4-oxo-1, 2, 3, 4-tetrahydro-2-quinazolinyl) propionic acid (10), respectively.

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Facile oxidations of dihydrazones of α-diketones to acetylenes were performed with a catalytic amount of bis (salicylidene) ethylenediaminatocobalt (II) and bis (3-methoxysalicylidene) ethylenediaminatocobalt (II) under mild conditions in 88-98% yields.

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Various carboxyl-terminal peptides of hCG-β were prepared from the corresponding blocked peptides by TFA treatment followed by hydrogenation. The prepared peptides were tested for inhibitory action on the binding between ¹²⁵I-hCG and the antiserum against the carboxyl-terminal portion of hCG-β. The results suggest that an antigenic site of this antiserum may exist around the amino-terminal portion of the carboxylterminal hexadecapeptide.

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A new caffeoyl glycoside of 3, 4-dihydroxy-β-phenethyl alcohol, designated as forsythiaside (1), was isolated from the fruits of Forsythia suspensa VAHL (Oleaceae). The structure of 1 is proposed to be 3, 4-dihydroxy-β-phenethyl-O-α-L-rhamnopyranosyl-(1→6)-4-O-caffeoyl-β-D-glucopyranside on the basis of analysis of the carbon-13 nuclear magnetic resonance spectrum.

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The stability of the diazoalkane N, N-dimethyl-4-diazomethyl benzenesulfonamide (DDBS), used as a UV-labelling agent, was investigated. The decomposition of DDBS in solvent depended mainly on the temperature and slightly on light and moisture. Thus, the decomposition in a benzene solution kept in a refrigerator and shielded from light and moisture was very slight. DDBS in solid form stored in container shielded from light was hardly decomposed in 30 weeks. DDBS has excellent stability compared with other UV-labelling agents for acidic substances.

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The stimulatory effect of an N-succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilidehydrolyzing protease (STA-protease), which was partially purified from Pronase, on cyclic adenosine 3', 5'-monophosphate (cyclic AMP) phosphodiesterase of bovine heart was investigated by measuring the changes in kinetic constants. The phosphodiesterase used showed hydrolytic activity towards cyclic AMP at a concentration of 1 or 100 μM. STA-protease stimulated low K_m phosphodiesterase but not high K_m phosphodiesterase activity, in the presence of ethylene glycol bis (β-aminoethylether)-N, N'-tetraacetic acid (EGTA). No stimulation of the low K_m enzyme was observed when theophylline was used instead of EGTA. Proteases such as Pronase, trypsin, chymotrypsin, elastase, and substilisin BPN' stimulated the low K_m enzyme to various extents. Although STA-protease showed the lowest caseinolytic activity among proteases tested, its stimulatory activity towards the low K_m enzyme was rather high. STA-protease increased the K_m and V_max values of the low K_m enzyme, whereas trypsin decreased the K_m value without causing significant change in V_max. The stimulatory mechanism of STA-protease probably differs from that of trypsin.

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A single stimulation with isoproterenol (IPR) resulted in a 1.5-fold increase in the total protein in saliva from rat submandibular glands at 1 day after the injection. Large mobile (LM) protein began to be secreted from the glands early after IPR injection, reaching a maximum at 2 days after injection, when the total protein in saliva had returned to the normal level. The secretion of this protein continued for 22 days after injection, indicating that the life of LM protein template is long or that the effect of IPR remains during this period. The increase of LM protein after IPR stimulation was markedly suppressed by β-adrenergic receptor blocking drugs. When both an α-adrenergic receptor blocking drug and adrenaline were injected together into rats, a small amount of the LM protein appeared and an increase in the wet weight of submandibular glands was seen. Repeated injection of these drugs resulted in a remarkable increase of LM protein. These results suggest that the appearance of LM protein was not a consequence of direct action of IPR but was due to a stimulatory effect of IPR on β-adrenergic receptors and that chronic blocking of α-adrenergic receptors has a significant stimulative effect on the production of LM protein through β-adrenergic receptors.

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Some physicochemical and biochemical properties of the purified large mobile (LM) protein appearing in submandibular glands of isoproterenol-treated rats were studied. The molecular weight of this protein was found by sedimentation equilibrium and gel chromatography on Sepharose 6B to be 12700-13000. The partial specific volume was estimated to be 0.72 and the Stokes radius to be 28.8 Å. The frictional ratio of the LM protein was found to be 1.69 by viscometry by assuming that the hydration of the protein is zero and adopting a prolate ellipsoidal model. The circular dichroism spectra of the LM protein showed that most of the peptide chain took random coil (64%) and β-structure (30%) conformations. These results suggest that the LM protein has a slightly elongated shape. The LM protein did not show measurable activities of deoxyribonuclease, ribonuclease, lysozyme, nerve growth factor or phospholipase.

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The antitumor activity of an immunomodulating material, vesiculogen, from a fungus, Peziza vesiculosa, on the transplantable sarcoma 180 was examined in mice. Vesiculogen showed antitumor activity against both solid and ascites forms of sarcoma 180.

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Radioactive sulfur was introduced into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules by chemical means. The thio group in 4-thiouracil residues, which were introduced into RNA and DNA by sulfhydrolysis, was exchanged with radioactive Na₂S via a 4-thiocyanatouracil intermediate. Specific activity of the ³⁵S-labeled RNA and DNA was around 10⁵ cpm/ODU₂60. The labeled RNA and DNA were successfully applied as substrates for the assay of nucleolytic enzymes or ribonuclease inhibitor. The sensitivity of the assay was 100 times better than that of the current method using non-labeled nucleic acids as the substrate.

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The effects of basic amino acid and sodium salts of diclofenac on the rectal absorption process of ampicillin sodium were studied. All diclofenac salts enhanced the permeation of ampicillin sodium through the membrane in rabbits, rats and dogs. The absorption of ampicillin sodium sigmoidally increased with increase of the concentration of diclofenac salts and the maximum plasma level was attained at 1.5% incorporation for all salts. The permeation of ampicillin sodium increased in the following order ; arginine salt<histidine salt<lysine salt«sodium salt. This order did not change with the animal species studied. From the results of repeated administration experiments, it was found that the leakiness of the barrier induced by diclofenac sodium recovered to the normal condition within about 20 h after the administration, but in the case of diclofenac lysine, the membrane returned to the normal state within only about 6.5 h.

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The effects of ethyl alcohol on the interaction of gliclazide and other sulfonylureas with bovine serum albumin (BSA) were studied by use of an equilibrium dialysis technique. The strongest inhibition of gliclazide-binding with BSA was induced at the drug level which corresponded to the effective dose in the presence of ethyl alcohol (1 mg/ml), resulting in a 2.5-fold higher level of free gliclazide than that of the control. The ratio (D_f/D_t) of free gliclazide concentration to total gliclazide concentration in the medium was constant (about 30%) at concentrations of ethyl alcohol over 0.5 mg/ml. The ratio of D_f/D_t of tolubutamide, glyclazide, acetohexamide and glycopyramide with BSA was raised (+Δ2.1-19.8%) in each case, whereas that of chlorpropamide or tolazamide was lowered (-Δ2.9-6.2%) by the addition of ethyl alcohol. The addition of ethyl alcohol to the medium reduced the gliclazide-binding ability in the primary binding site on the BSA molecule and reduced the association constant to 2.5×10³ M<-1> (56.8%) from 4.4×10³M^-1 of the control. In conclusion, it is suggested that one of the reasons for the enhancing effect of ethyl alcohol on the hypoglycemic action of sulfonylurea is the inhibition of binding of the drug with blood protein.

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