As an intramolecular model for flavin-indole interaction, the crystal structure of N-2-(3-indolyl)ethyl-7, 8-dimethylisoalloxazine-10-propionamide, a flavinyltryptamine peptide, was determined by the X-ray diffraction method. Although the molecule took an open conformation without indole-flavin interaction, this could be a result of crystal packing effects, because ultraviolet and fluorescence data showed the existence of a prominent intramolecular stacking interaction of the rings in aqueous solution. Proton nuclear magnetic resonance analysis indicated a preferential stacking interaction between the indole ring and the pyrazinoid and pyrimidinoid portions of the flavin ring. These results were supported by the conformation analysis of this molecule based on energy calculations. Polarographic data showed that the indole ring affects the reduction state of the flavin semiquinone form to the hydroquinone form. The results suggest that a tryptophan residue of a flavin enzyme might act not only to fix the flavin coenzyme in place, but also to stimulate the reduction reaction.

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Complex formation of four atropisomers of zinc tetra(ο-pivalamidophenyl)porphyrin with imidazoles (imidazole, 1-methylimidazole, 2-methylimidazole, and benzimidazole) and pyridines (pyridine, 3, 5-dimethylpyridine, and 2, 6-dimethylpyridine) was studied. The ratios of the absorbance of the βband to the αband in the adducts tended to be in the order of αααα>ααββ≥αααβ>αβαβ. This order, which is the same as that of "electronic effect" on the physical properties of the four atropisomers of tetra(ο-pivalamidophenyl)porphyrin, indicates the presence of "electronic effect" in the zinc derivatives of the porphyrin. Among the four isomers, the equilibrium constants of N-base binding tended to be in the order of αβαβ>αααα≥αααβ>ααββ, suggesting the relatively greater importance of "steric effect" of the bulky pivalamido groups rather than "electronic effect" for the coordination of the N-bases. The differences in the equilibrium constants among the atropisomers are attributable to the contributions of both the steric effect and the electronic effect.

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As a part of the conformational studies of aminoacyl adenosine monophosphates, two model compounds, N-2-(3-indolyl)ethyl- and N-2-phenylethyl-5'-deoxy-5'-adenosineacetoamides, were investigated by ultraviolet, circular dichroism and ¹H-nuclear magnetic resonance spectroscopies, and empirical energy calculations. Aromatic ring-ring intramolecular stacking interactions were observed for both compounds. However, the stacking tendency and the ribose puckering accompanying the stacking interaction were different in the two compounds. These conformational differences may be important in the recognition of the tryptophanyl- and phenylalanyladenosine monophosphates by the respective aminoacyladenosine monophosphate synthetases.

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cis-Platinum complexes of aromatic amines were synthesized in water or at the interfacial layer between water and an organic solvent. The position of substitution of aniline derivatives had a marked effect on the synthesis of the complexes. The coordination of 2-substituted anilines needed a long time and the yields were low. That of 3- or 4-substituted anilines was fast, and the yields were high. Disubstituted or N-substituted anilines needed fairly long reaction times and the yields were lower than those of monosubstituted anilines; sometimes unidentified complexes were obtained or no reaction product was formed.Six synthesized cis-dichloro platinum(II) complexes which coordinate bis(4-aminophenol), bis(3-toluidine), bis(2-aminobiphenyl), bis(4-sulfamidoaniline), bis(1-naphthylamine) and bis(4-phenylazo-1-naphthylamine) had high antitumor activities against Sarcoma 180 ascites in female ICR/JCL mice. The dose of platinum of these effective complexes given to the mice was in the range of 7.37-105.76 mg/kg (26.44%-39.96%). This result suggests that the toxicity of the platinum complexes might depend on their carrier ligands.

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A number of N, N-dibenzylaminoacetaldehyde diakyl acetales (1) double-cyclized in 70% perchloric acid or triflic acid (trifluoromethanesulfonic acid) to give good yields of 7, 12-dihydro-5H-6, 12-methanodibenz[c, f]azocines (2) even when a powerful electron-withdrawing substituent, for example a nitro group, was present on the benzene ring. Triflic acid treatment of α-dibenzylaminoketones (3) caused double-cyclization to give 12-substituted derivatives of 2.

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Treatment of spiro[4H-3, 1-benzoxazine-4, 4'-piperidin]-2(1H)-one derivatives with POCl₃ yielded 1, 2, 3, 4-tetrahydro-2, 9-diazaphenanthrene derivatives through a novel rearrangment. The reaction is suggested to processed via the formation of an isocyanate by dehydrative cleavage of the oxazinone ring with POCl₃. It was also found that treatment of 4-(2-methoxycarbonylamino-phenyl)-1, 2, 5, 6-tetrahydropyridine derivatives with trichlorosilane/triethylamine gave the corresponding 10-oxo-1, 2, 3, 4, 9, 10-hexahydro-2, 9-diazaphenanthrene derivatives.

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A series of piperidine derivatives with a 2-oxo-1, 2, 3, 4-tetrahydro-quinazoline or 2, 4-dioxo-1, 2, 3, 4-tetrahydroquinazoline ring at the 4-position were prepared and tested for antihypertensive activity. Among the compounds tested, 1-[2-hydroxy-2-(3, 4-methylenedioxyphenyl)ethyl]-4-(2, 4-dioxo-1, 2, 3, 4-tetrahydro-3-quinazolinyl)piperidine (20) and 1-[2-(4-chlorophenyl)-2-hydroxyethyl]-4-(2-oxo-1, 2, 3, 4-tetrahydro-3-quinazolinylmethyl)piperidine (30) produced relatively strong hypotension in the spontaneously hypertensive rat model.

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1-Methyl-3, 4-benzo-7-thia-2-azabicyclo[3.3.1]nonane 7-oxide has been synthesized by the reaction of quinaldine with methylsulfinylmethyl carbanion, and its structure has been elucidated on the basis of spectral data and some chemical evidence. The conformation of the product has been further investigated by means of proton and carbon-13 nuclear magnetic resonance spectroscopy together with molecular mechanics calculations. The molecular mechanics calculations indicate that the stable conformer has a chair thiane ring with an equatorial sulfoxy group.

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Acid treatment of o-quinol acetates (2a and 2b) derived from 5-hydroxy-1-benzyltetrahydroisoquinolines (3a and 3b) gave the corresponding 3-hydroxyaporphines (4a and 4b) in high yield.Similarly, the 3-hydroxyhomoaporphines (4c, 4d, and 4e) were excusively synthesized from the corresponding 1-phenethyl o-quinol acetates (2c, 2d, and 2e). On the other hand, no C-noraporphine was formed from the 1-aryl o-quinol acetate (2f); instead, the p-quinone (11) was generated.

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New synthetic routes to allochenodeoxycholic (3α, 7α-dihydroxy-5α-cholanic) and alloursodeoxycholic (3α, 7β-dihydroxy-5α-cholanic) acids, and their stereoisomers are described. Treatments of allo 7α-hydroxy-3β-tosyloxy ester with N, N-dimethylformamide and of allo 7α-mesyloxy-3β-tosyloxy and 3β-cathyloxy-7α-mesyloxy esters with potassium superoxide-crown ether afforded the desired 3α, 7α-, 3α, 7β-, and 3β, 7β-dihydroxy stereoisomers, respectively, in high yield. High-performance liquid chromatography was of key importance in characterizing the compounds and determining their purity.

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Three new compounds, 7α, 12β-, 7β, 12α-, and 7β, 12β-dihydroxy-5α-cholanic acids, were synthesized. The principal reactions employed were 1) selective acylation at C-7 of a 7α, 12α-dihydroxy ester with the propionic anhydride-4-dimethylaminopyridine system, 2) potassium superoxide-18-crown-6 ether inversion of the 7α-hydroxyl group, and 3) stereoselective reduction of the 12-ketones with the sodium borohydride-palladium chloride system and tert-butylamineborane complex. High-performance liquid chromatography was of key importance in characterizing the compounds and determining their purity

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Spiro[benzo[b]thiophene-2(3H), 1'-cyclopropan]-3-ones (IIIc-1-IIIc-11) and their aza analogs, such as spiro[cyclopropane-1, 2'(3'H)-thieno[3, 2-c]pyridin]-3'-one (IIIc-12) as well as spiro[cyclopropane-1, 2'(3'H)-thieno[2, 3-b]pyridin]-3'-one (IIIc-13), were synthesized from thiosalicylic acids and from 4- and 2-mercaptonicotinic acids, respectiely, in three steps. Decarboxylation of 4', 5'-dihydrospiro[benzo[b]thiophene-2(3H), 3'(2'H)-furan]-2', 3-diones (IIc) gave 2, 3-dihydrobenzo[b]thieno[3, 2-b]furans (IVc) along with the desired spirocyclopropane compounds (IIIc).The ratios of IIIc to IVc were greatly influenced by the substituents on the benzene ring.

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The skeleton of the title aporphines was synthesized by use of the lead tetraacetate oxidation of 1-(3-hydroxy-4-methoxybenzyl)-1, 2, 3, 4-tetrahydroisoquinolines.

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Twenty-eight para-substituted and eleven ortho-substituted 1, 3-diarylureas were prepared.Their proton nuclear magnetic resonance (¹H-NMR) spectra were measured to evaluate the acidity of the ureido protons in the para-substituted ureas, and to estimate the conformations of ortho-substituted molecules. The chemical shifts and acidities of two ureido protons were additively controlled by the two para-substituents in the 1, 3-diarylureas. The sixteen para-substitued diarylureas were nitrosated with gaseous nitrosating reagents (dinitrogen trioxide, dinitrogen tetroxide or nitroxyl chloride) to give unstable isomeric mixtures of 1, 3-diaryl-1-nitrosoureas [R¹-C₆H₄-N(NO)-CO-NH-C₆H₄-R²] and 1, 3-diaryl-3-nitrosoureas [R¹-C₆H₄-NH-CO-N(NO)-C₆H₄-R²]. The isomer ratio of each nitrosated urea was correlated with the difference between the chemical shifts (d[NH]) of the two ureido protons in the ¹H-NMR spectrum of the starting urea. When d[NH] was below 0.25 ppm (with NOCl) or 0.45 ppm (with N₂O₃), good linearity was obtained (r(NOCl)=0.943, n=11; r(N₂O₃)=0.869, n=15). When d[NH] was over 0.25 ppm (NOCl) or 0.45 ppm (N₂O₃), the 1-nitrosated isomer [R¹-C₆H₄-N(NO)-CO-NH-C₆H₄-R²] was the sole product. These principles ould not be extended to dinitrogen tetroxide.Thirteen para-substituted diarylureas gave 1- or 3-nitrosated ureido derivatives as sole products and only four gave nitrosated isomeric mixtures.The ¹H-NMR spectra of 5 types of ortho-substituted 1, 3-diarylureas were measured for comformational analysis. Their conformations seem to be different from those of para-substituted diarylureas. Eleven ortho-substituted 1, 3-diarylureas were nitrosated with nitrosyl chloride and dinitrogen tetroxide. In many cases, the nitroso group was predominantly introduced at the ureido nitrogen, which is less hindered sterically, and isomeric mixtures of 1- and 3-nitrosated ureas were obtained.

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Guanosine fixed in the high-anti conformation by means of an 8, 2'-methylene bridge was prepared. N²-Acetyl-O⁶-ethylguanosine (2) was converted to the 3', 5'-O-(tetraisopropyldisiloxane-1, 3-diyl) derivative (3). Oxidation of 3 to the 2'-keto derivative (4) and successive coupling with methylenetriphenylphosphorane gave the 2'-methylidene derivative (5) and its α-anomer. The 2'-methylidene function of 5 was hydroxylated, and the 2'-hydroxymethyl group was modified to give the phenylthiomethyl derivative (6). Photocyclization of 6 followed by deprotection of the sugar and base protecting groups furnished 8, 2'-methanoguanosine (12). The alpha anomer of 12 was likewise prepared. The circular dichroism spectra of 12, its α-anomer, and related compounds were measured.

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Two new isoprenylated flavans, kazinols E and H, and five new isoprenylated 1, 3-diphenyl-propane derivatives, kazinols C, D, F, G, and K, were isolated from the extract of the root bark of Broussonetia kazinoki SIEB. (Japanese name, Kimekozo, Moraceae). On the basis of chemical and spectral evidence, the structures of kazinols E and H were shown to be 3 and 6, respectively, and the structures of kazinols C, D, F, G, and K to be 1, 2, 4, 5, and 7, respectively.

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A convenient method for synthesis of 2, 4-disubstituted 6H-1, 3-oxazin-6-ones (8) was developed. Acylaminoalkylidene-1, 3-dioxane-4, 6-diones (5a-1), which were prepared from N-acylimidates(3a-1) and Meldrum's acid (4), readily underwent thermolysis upon heating, leading to a variety of 2, 4-disubstituted 6H-1, 3-oxazin-6-ones (8a-1).

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Two new isoprenylated flavonols, named brossoflavonols C (1) and D (2), were isolated from the extract of the root bark of Brossonetia papyrifera (L.) VENT. (Japanese name "Kazinoki", Moracese). The structures of brossoflavonols C and D were shown to be 1 and 2, respectively, on the basis of spectral evidence. The compounds (1 and 2) are the first reported examples of triakenyl flavonols.

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N-Alkylthiocarbonyldopa (1a-e) and dopamine (1f-h) derivatives can be converted into the corresponding 3, 4-dihydroisoquinolines (3) by treatment with 4-nitrobenzyl bromide. The NaBH₄ reduction of 1, 3-disubstituted 3, 4-dihydroisoquinolines (3a-e) gave 1, 3-cis-1, 2, 3, 4-tetrahydroisoquinolines (4a-e). Hydrogenation of 3a-e over PtO₂ also gave 4a-e in good yields. The synthesis of optically active 3a, i and 4a, i is also described.

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The treatment of succinic acid with lauroyl or stearoyl chloride gave the five-membered β-diketone (II) substituted with a long chain at the α-position, together with a trimer (III) of the acyl chloride. Bicyclo[3.3.1]nonane-2, 4-dione (VI) substituted with a methyl or ethyl group at the C₃-position was prepared from cyclohexane-1, 3-dicarboxylic acid (V) and propionyl or butyryl chloride, respectively. D-Camphoric acid afforded an unexpected product, 4-acyl-2, 2, 3-trimethyl-3-cyclopentenecarboxylic acid (IX), on reaction with acyl chloride. Although acetyl chloride gave no product on treatment with succinic acid, it afforded 3-acetylbicyclo[3.2.1]octane-2, 4-dione (XI), 3-acetylbicyclo[3.3.1]nonane-2, 4-dione (VId), and 2-acetylcyclohexane-1, 3-dione (XII) on reaction with the corresponding dicarboxylic acids.

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The cluster [Fe₄S₄(SR)₄]^2- catalyzed the reductions of nitro compunds, quinones and N-oxide compounds to amines, hydroquinones, and deoxygenated compounds, respectively, in the presence of thiols in CH₃CN solution. The reaction mechanism was also investigated by electron spin resonance spectroscopy, and it was concluded that these reactions proceed via one-electron transfer from the cluster to the substrates.

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The iodination and subsequent dehydroxychlorination of 1, 6-naphthyridin-5(6H)-one gave 5-chloro-8-iodo-1, 6-naphthyridine, which was converted to the 5-methyoxy derivative. Starting from this compound, didehydromatrine was synthesized by using palladium-catalyzed cross-coupling reactions with ethyl acrylate and 3-butyn-1-ol, as key reactions. Similarly, nordehydro-α-matrinidine was synthesized through four steps from 8-bromo-1, 6-naphthyridine, obtained by the bromination o unsubstituted 1, 6-naphthyridine.

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Nipradilol (NIP), 3, 4-dihydro-8-(2-hydroxy-3-isopropylamino)propoxy-3-nitroxy-2H-1-benzopyran, is extensively metabolized in man and dogs. In order to identify the ring-hydroxylated metabolites we have synthesized cis- and trans-4-hydroxy-, and 5-hydroxy-NIP, their denitro derivatives and 7-hydroxy-denitro-NIP. As judged from their spectra, trans-4-hydroxy- and 5-hydroxy-NIP and their denitro derivatives corresponded to the isolated ring-hydroxylated metabolites. Moreover, a pharmacological study in anesthetized spontaneously hypertensive rat showed that the nitroxy derivatives caused a transient hypotensive response, and both the nitroxy derivatives and the denitro derivatives caused long-lasting bradycardia.

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A detailed account is given of the final step of the general 7-alkalation procedure for adenine (1), which consists of the preferential benzylation at the 3-position of 1, regioselective alkylation of the resulting 3-benzyladenine (2) to give 7-alkyl-3-benzyladenine salts (3a-c), and debenzylation of 3a-c leading to 7-alkyladenines (4a-c). Debenzylation of 3a-c (X=Cl or ClO₄) has been achieved by hydrogenolysis using hydrogen and Pd-C catalyst at atmospheric pressure, producing 7-alkyladenines (4a-c) in 38-74% yields. The use of the allyl or γ, γ-dimethylallyl group at the 3-position instead of the benzyl group for the synthesis of 7-methyladenine (4a) by this procedure has no practical value. Alternatively, the salts 3a-c (X=Br, ClO₄, or I) have been debenzylated efficiently by treatment with conc. H₂SO₄ in the presence of toluene at room temperature for 3-6h or at 60°C for 0.5-2h, giving 4a-c in 73-93% yields.

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Protected N⁶-methyl-2'-deoxyadenosine (d-m⁶A), 2-amino-2'-deoxyadenosine (d-a²A), 2'-deoxyinosine (dI), 5-methyl-2'-deoxycytidine (d-m⁵C) and deoxyuridine (dU) were reacted with bis(diisopropylamino)methoxyphosphine in the presence of diisopropylammonium tetrazolide as the activating reagent to gie the corresponding phosphoramidite derivatives in yields of 100, 65, 90, 78 and 65%, respectively. The ³¹P-nuclear magnetic resonance spectra of the products were measured. Using these compounds, dinucleotides and trinucleotides were synthesized on a long-chain alkylamine controlled pore glass (LCA-CPG) in quantitative yields.The stability of 6-methyldeoxyadenosine and N, N-diisobutyryl-2-aminodeoxyadenosine to acid was examined. When protected di- and trinucleotides (m⁶A-T, a²A-T, T-m⁶A-T, T-a²A-T)bound to the support (LCA-CPG) were treated with 3% trichloroacetic acid in dichloromethane, depurination was negligible within 10 min (dinucleotide) or 60 min (trinucleotide).

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N-Ethoxycarbonyl- and N-benzyloxycarbonyl-5-hydroxy-5-methoxyalkylpyrrolinones (6a-c) and (7a-c) together with minor products 8-10 were obtained from the photooxidation or autoxidation of corresponding 2-furylcarbamates (4a-c) and (5a-c). The catalytic hydrogenation of 6a-c afforded saturated γ-ketoamides (11a-c), while that of 7a-c led to the formation of 5-hydroxypyrrolidones (12a-c).

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Epipodophyllotoxin (EPT) and podophyllotoxin (PT) were prepared from desoxypodophyllotoxin (DPT), obtained from the seeds of Hernandia ovigera L. (Hernandiaceae) EPT was obtaind together with dehydrodesoxypodophyllotoxin (I) by a radical bromination of DPT with N-bromosuccinimide (NBS) followed by hydrolysis of the resultant 1-bromo-DPT, which was confirmed to be a mixture of 1α- and 1β-bromo compounds (7 : 3). EPT was oxidized with pyridinium chlorochromate to give podophyllotoxone (IV). The stereoselective reduction of IV to afford PT was examined with a variety of reagents, and borane-tert-butylamine complex was found to be effective.

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Catalytic hydrogenation of the (22R)-unsaturated loctone 8 and its (22S)-epimer 9 afforded stereoselectively the (22R, 24R)-saturated lactone 10 and the (22S, 24S)-isomer 11, respectively. The C-24 stereochemistry of the hydrogenated products was determined by their conversion into 24-ethylcholesterols.

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An unsymmetrical biphenyl lignan, honokiol (1a), a constituent of Magnoliaceae, and related compounds were synthesized by the reaction of the quinol-acetates 3 with the Grignard reagent 4 followed by Claisen rearrangement.

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Optically pure (R)- and (S)-N-alkyl-1-cyclohexyl-2-phenylethylamine hydrochlorides (9a-r)were synthesized from (R)- and (S)-phenylglycine by means of a simple procedure. The analgesic activity of these compounds was evaluated by the acetic acid writhing method, and 9e, 9f, 9i-k, and 9m showed more potent activity than (-)-pentazocine hydrochloride. Moreover, the analgesic activities of 9a-e, 9h, 9i, 9m, and 9o-r were not antagonized by naloxone.

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A series of (R)-3-amino-4-oxo-2, 3, 4, 5-tetrahydro-1, 5-benzothiazepine-5-acetic acids (4 and 13)and (S)-3-amino-4-oxo-2, 3, 4, 5-tetrahydro-1, 5-benzoxazepine-5-acetic acids (5 and 14) having an(S)-ω-amino-1-carboxyalkylamino group at the 3-position was prepared as part of our search for long-acting angiotensin converting enzyme (ACE) inhibitors. A number of derivatives had potent in vitro and in vivo ACE inhibitory activities. The structure-activity relationship of the series indicated that the duration of in vivo ACE inhibitory activity depends on the length of the carbon chain in the ω-aminoalkylamino substituent at the 3-position. The most prolonged activity was observed with (S)-8-amino-1-carboxyoctylamino derivatives (4d and 5d).

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Two new β-carboline dimer alkaloids, picrasidines-H (1) and (2), have been isolated from the root bark of Picrasma quassioides BENNET (Simaroubaceae). The structures were determined on the basis of spectral analysis.

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Adenosine 3', 5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitiors contained in the roots of Sophora flavescens AITON were identified as flavonoids, kushenol A (9), kurarinone (5), and kuraridin (22). The structure-inhibitory activity relationships were studied in 30 flavonoids. O-Methylation of a hydroxyl group on an aromatic ring decreased the activity. The prenyl group is important for high inhibitory activity. A kinetic study revealed that nor-kurarinone, kurarinol and kurafidin non-competitively inhibit cAMP phosphodiesterase.

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The antithrombic activity of ginsenoside-Ro isolated from the roots of Panax ginseng C. A.MEYER was evaluated in experimental models of disseminated intravascular coagulation (DIC)induced by infusion of endotoxin or thrombin in rats. Ginsenoside-Ro (50 mg/kg, p.o.) prevented the decrease of fibrinogen in endotoxin-induced DIC. Ginsenoside-Ro also inhibited the formation of fibrin thrombi in the renal glomeruli in thrombin-induced DIC.Ginsenoside-Ro showed a promotive effect on the activation of the fibrinolytic system as determined by the euglobulin lysis time (ELT) assay. The promotive effect of ginsenoside-Ro on the activation of the fibrinolytic system was inhibited by pretreatment with ε-aminocaproic acid (an anti-plasmin agent), but not by pretreatment with promethazine or cyproheptadine (anti-chemical agents).

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The effect of the bridge heterologous combination between antiserum and enzyme-labeled antigen on sensitivity in enzyme immunoassay for 11-deoxycortisol has been investigated. In order to obtain evidence for the bridge length effect, the sensitivity of an assay system using a β-galactosidase-labeled antigen prepared from an 11-deoxycortisol derivative having a carboxy-methylthio moiety as a bridge at C-4 was compared with that in the case of a derivative with an N-(carboxymethyl)carbamoylmethylthio moiety at C-4. The enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. Three anti-11-deoxycortisol antisera elicited in rabbits by immunization with different immunogens were used. It was found that the use of the shorter bridge for enzyme labeling resulted in an increase in sensitivity. This indicates that the bridge length is an important factor influencing the sensitivity of enzyme immunoassay.

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The chemical properties of a fungal B-cell mitogen obtained from the fruit body of Peziza vesiculosa were investigated. The non-dialyzable fraction (vesiculogen) of the hot water extract of P.vesiculosa was separated into mitogenic and non-mitogenic fractions by ammonium sulfate fractionation. The mitogenic fraction (precipitated at 20 to 60% ammonium sulfate) was composed of 75-80% protein and 17-19% carbohydrates. The mitogenic fraction was separated into 4 representative fractions by diethylaminoethyl-Sephadex A-25 and hydroxyapatite chromatographies, sequentially. In Sepharose CL-2B chromatography, all of the fractions showed wide ranges of molecular weight distribution. All of these representative fractions from Sepharose CL-2B were similar in amino acid compositions (rich in Ser, Glu, Gly, and Ala). These findings suggest that the mitogen is a high-molecular-weight, acidic polypeptide, showing charge and molecular weight heterogeneities.

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Dehydroepiandrosterone 3-sulfate (DHAS) binding to a cytoplasmic protein of rabbit uterine cervix was studied in vitro. When [³H]DHAS was incubated with the minced uterine cervix obtained at term pregnancy, it was bound specifically to the cytosol fraction. From Scatchard analysis, the binding capacity and dissociation constant were found to be 33 fmol/mg protein and 5.05×10^-8M, respectively. Incubation of the prelabeled cytosol with isolated nuclei resulted in temperature-dependent transfer of DHAS to the nuclei. The cytoplasmic DHAS-binding protein after concentration with (NH₄)₂SO₄ showed a sedimentation constant of 3.6-4.0S, while the DHAS-binding protein in the rabbit serum gave a value of 4.6S. The cytoplasmic DHAS-binding protein was eluted from Sephacryl S-300 mainly in the fractions corresponding to the molecular weight of 1.7×10⁵, and in the void volume to a small extent in the presence of molybdate and glycerol. Dehydroepiandrosterone (DHA) inhibited approx. 50% of [³H]DHAS-specific binding, whereas 17β-estradoil 3-sulfate, estriol 3-sulfate, estrone 3-sulfate, 17β-estradiol, estriol, and 5α-dihydrotestosterone showed lower or no affinity for the DHAS-specific binding site. The content of the DHAS-binding protein in the cervix was found to increase with the progress of pregnancy.These observations suggest that rabbit uterine cervix contains a receptor-like DHAS-specific binding protein int he cytosol fraction which seems to be closely associated with the progress of pregnancy.

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The cytoplasmic dehydroepiandrosterone 3-sulfate (DHAS)-binding macromolecule in the cytosol fraction of human fetal membrane has been investigated. It was distinguished from serum DHAS-binding component on the basis of binding specificity and molecular weight. Scatchard analysis indicated a single class of binding sites (57 fmol/mg protein) with an apparent dissociation constant of 7.2×10^-8M. The cytoplasmic DHAS-binding macromolecule was separated into two moieties by Sephacyl S-300 chromatography. One was eluted at void volume, and the other hand a molecular weight of about 1.7×10⁵. These two moieties had the same specificity for DHAS, and had no affinity for dehydroepiandrosterone or 17β-estradiol. Progesterone, 5α-dihydrotestosterone and sulfated estrogens such as 17β-estradiol 3-sulfate, estriol 3-sulfate and estrone 3-sulfate showed much lower affinity for the DHAS-binding macromolecule. Themperature-dependent nuclear uptake of [³H]DHAS in a cell-free system required the presence of cytosol, suggesting that binding of DHAS to the cytoplasmic macromolecule is a prerequisite for the transfer of DHAS to the nucleus. These data demonstrate the presence in human fetal membrane of a tissue-specific DHAS-binding macromolecule which has properties similar to those of the DHAS-binding protein previously found in rabbit uterine cervix (A. Ito, K. Sakyo, H. Sano, S. Hirakawa and Y. Mori, Chem. Pharm. Bull., 34, 2118 (1986).

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The nonatetracontapeptide corresponding to the entire amino acid sequence of thymopoietin III was synthesized by assembling ten peptide fragments in solution followed by deprotection with 1M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1 : 1) in trifluoroacetic acid in the presence of m-cresol. The synthetic nonatetracontapeptide was tested for effect on impaired T-lymphocyte transformation by phytohemagglutinin (PHA) in common variable immunodeficiency.The synthetic nonatetracontapeptide was found to have restorin activity on the T-lymphocyte transformation by PHA. However, the restoring activity of the synthetic nonatetracontapeptide was lower than that of the synthetic thymopoietin I at a dos of 2 μg/ml.

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In order to examine the feasibility of using cyclic peroxides as sensitizer for the hyperthermic treatment of cancer, we studied temperature dependent OH radical generation from two synthetic cyclic peroxides, 4-ethoxy-1, 4-dihydro-2, 3-benzodioxin-1-ol and 4-ethoxy-1, 4-dihydro-2, 3-benzodioxepin-1-ol. The amounts of 5, 5-dimethyl-pyrroline N-oxide (DMPO)-OH adduct formed as a result of decomposition of these cyclic peroxides increased markedly at temperatures of above approximately 30 and 40°C, respectively (degradation temperatures). Peroxide-mediated degradation of cytochrome c was also markedly enhanced above these temperatures. These results suggest that biologicaly effective OH radical can be produced specifically under thermally controlled conditions from certain cyclic peroxides.

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The effects of antitumor-active [β-1, 3-glucan (grifolan)] and inactive [β-1, 6(islandican), α-1, 4-(starch)]glucans in mice were compared. β-1, 6-Glucan enhanced the antitumor activity of grifolan, whereas α-1, 4-glucan showed a suppressive effect on the antitumor activity of grifolan. When these glucans were injected i.p. into normal mice, the glucans disappeared gradually. The rate of disappearance was in the order α-1, 4>β-1, 6>β-1, 3. Grifolan induced peritoneal exudate cells, but β-1, 6 and α-1, 4-glucans did not. Only α-1, 4-glucan was degradated by the lysate of the peritoneal exudate cells. These results indicate that the antitumor activity of β-1, 3-glucan can be positively or negatively modulated by other glucans, and that glucans showing different antitumor effects have a variety of actions and fates in mice.

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Extracellular creatine amidinohydrolase (creatinase, EC 3.5.3.3) produced by Alcaligenes sp.nov. was purified to electrophoretic homogeneity by ion exchange chromatography on diethyl-aminoethyl-cellulose, gel filtration on Sephadex G-75 and hydrophobic chromatography on phenyl-Sepharose CL-4B. The molecular weight of the enzyme was estimated to be 51000 by gel filtration on Sephadex G-200 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed the maximum activity at pH 8 and was stable at pH 5-9. The pI value was 4.7 as determined by isoelectric focusing. The enzyme catalyzed hydrolysis of creatine to sarcosine and urea, and the K_m and V_max values for creatine were 17.2 mM and 105 μmol/min/mg protein, respectively. The enzyme was markedly inactivated by p-chloromercuribenzoate (PCMB). Besides PCMB, the enzyme was inactivated by N-bromosuccinimide, Zn²⁺, Cu²⁺ or Hg²⁺.

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The effects of an active tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on cell membrane components have been examined using two lipophilic fluorophores, pyrene and 12-[6-(N-methylanilino)-2-naphthalenesulfonyloxy]-stearic acid (MANSOS), in Chang liver cells. MANSOS has a high fluorescence quantum yield in water and shows a blue shift (15nm from the value of 445nm in phosphate buffer) of the emission maximum on incorporation into the cell membrane.Pretreatment of cells with TPA for 2 h induced differential changes in the fluorescence parameters of pyrene and MANSOS incorporated into the cell membrane; it decreased the lifetime and harmonic mean of the rotational relaxation time and amplified the quanching by KI of pyrene fluorescence, whereas it increased the harmonic mean of the rotational relaxation time and slightly attenuated the quenching of MANSOS fluorescence.Taken together, the findings suggest different partition characteristics of the two fluorescent probes in the cell membrane components, and differential effects of TPA on the two species of components as judged from the opposite mobility changes of the two probes.

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The effect of quinidine and its isomer, quinine, on the thermotropic behavior of phosphatidylcholine vesicles was studied by differential scanning calorimetry (DSC) and fluorescence polarization measurement. The effect on the release rates of carboxfluorescein and acrinol was also investigated. Quinidine and quinine lowered the lipid trasition temperatures and increased the release of the drugs contained in the lipid vesicles. This effect seems to be due to a fluidizing action on the membrane.

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The elimination of imipramine (IMP) from the brain regions was investigated in rats after a single 5 mg/kg or 20 mg/kg intraperitoneal administration. At both 5 mg/kg and 20 mg/kg IMP injection, the brain regions could be divided into two classes in terms of IMP elimination. IMP levels in plasma and class A brain regions (cerebellum, mid-brain and medulla, striatum, posterior cortex and frontal cortex) declined according to a monoexponential profile with the same elimination rate constant and the time courses only shifted in parallel depending on the dose. On the other hand, the disappearance of IMP in class B brain regions (hypothalamus, thalamus, hippocampus and nucleus accumbens) followed a biexponential profile. The initial phase was similar to that in the class A brain region at each dose. However, the later phase of the curves was identical regardless of the dose. It is suggested tha the class B brain region may possess the high-affinity and low-capacity binding site for IMP.

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The water vapor sorption isotherms have been established for α-, β-, and γ-cyclodextrin at 40°C. The powder X-ray diffraction patterns of crystal forms of cyclodextrins stored at various levels of relative humidity (RH) were examined and the results are discussed in terms of the hydration numbers. α-Cyclodextrin hydrate was stable at RH as low as 11%. The water vapor desorption isotherm of β-cyclodextrin showed pronounced hysteresis, which extended to lower RH.The hydrate form of β-cyclodextrin was stable over a wide range of RH in the desorption process, like that of α-cyclodextrin. The crystal form of γ-cyclodextrin 7H₂O was obtaiend in addition to the dehydrate form and the 17H₂O hydrate form.

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A solid dispersion technique with enteric coating agents was applied to two drugs which present some pharmaceutical problems. The first drug, digoxin, is poorly water-soluble and chemically unstable in an acidic medium. The solid dispersions suppressed the dissolution rate of the drug in JP X 1st fluid (JP disintegration medium, pH 1.2), and improved the chemical stability.In JP X 2nd fluid (JP disintegration medium, pH 6.8), the solid dispersions showed rapid dissolution and supersaturation. It was concluded that this technique can provide an effective dosage form of digoxin for gastrointestinal absorption. The second drug, dipyridamole, is a poorly water-soluble organic base. The solid dispersion technique was applied to prepare a delayed absorption dosage form with good bioavailability. The solid dispersions showed suppressed a dissolution in an acidic medium, and gave supersaturation in a neutral or alkaline medium. Oral administration of this dosage form resulted in delayed absorption with good bioavailability.

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Using the avidin-biotin binding system, an enzyme immunoassay procedure was developed to measure the placental tissue protein 4 (PP4) in serum. The standard curve covered the range from 0.5 to 100 ng/ml of PP4. The intra- and inter-assay coefficients of variation were less than 5 and 10%, respectively. Recoveries of PP4 added to serum ranged between about 99 and 105%. The result obtained by this assay method correlated well with those obtained by the radioimmunoassay method. The PP4 serum leve was under 10 ng/ml in non-pathological individuals. In pregnancies during 5-40 weeks, the level ranged from 0.9 to 15.0 ng/ml. In ovarian malignancies, the PP4 concentration in serum irrespectively scattered from 0.9 to 21 ng/ml. Significantly high PP4 levels were found in uterine cervical cancer in the early stage and also in endometrial cancer.

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A method for dissolution testing of oily and water-isoluble drug preparations was developed.It was found that dissolution testing of a preparation containing d-α-tocopherol as a model drug could be done by using the paddle method in JP X and the 2nd fluid in JP X containing 20mM sodium glycochenodeoxycholate. The data obtaiend by the methdo were useful for predicting in vivo absorption of the drug. However, in the case of a capsule containing d-α-tocopherol dissolved in cottonseed oil, the drug and vehicle floated on the surface of the disslution test medium, and good dissolution was not observed.

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A gene for human lysozyme with 130 amino acids has been synthesized by ligation of chemically synthesized oligodeoxyribonucleotides with chain lengths of 13-18. The duplex with 401 base pairs was divided into 52 fragments, which were prepared by the phosphotriester solid-phase method involving condensation of dinucleotide units. By-products obtained in the synthesis have been characterized.

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