Studies on organic fluorine compounds in our laboratory are summarized. New methods for the synthesis of fluorine compound are as follows ; 1) fluorination of alcohols with fluorophosphoranes, 2) trifluoromethylation of aromatic and aliphatic halides with trifluoromethyl halides and copper powder, and 3) cycloaddition reaction with novel fluorine reagents like hexafluorobutyne. Reactions of trifluoromethyl aromatic compounds were investigated thoroughly, and some interesting characters of fluorine compounds were revealed. Trifluoromethylated valence bond isomers of aromatic compounds were synthesized, and some novel reactions of these highly strained compounds were shown. The characteristics that a fluorine atom is a small substituent and that a carbon-fluorine bond is very strong were used to study the metabolism of vitamin D3. Some interesting reactions by use of fluorine compounds as synthetic reagents are also discussed.
The hepatotoxic effects of lithocholylglycine (LCG) and sulfolithocholylglycine (SLCG) were investigated in male rabbits intravenously injected for 7 days with 2 and 20 mg/kg of LCG and SLCG, respectively. GPT, GOT, ALP and leucine aminopeptidase (LAP) activities were measured on days 2, 4 and 7 after the start of the treatments and on day 7 the liver was studied microscopically and the bile acid composition in plasma, urine and gallbladder bile were determined. In the 20 mg/kg LCG group, GPT activities were markedly elevated, in one rabbit (No. 8), GOT, ALP and LAP activities were also elevated. Histopathologically, focal necrosis of liver cells, portal infiltration of round cells, hypertrophy of ductular cells, proliferation of bile ducts and an increase in connective tissue around bile ducts were found. Plasma concentrations of lithocholic acid (LC) and sulfated LC were increased, as was urinary excretion of sulfated LC. In rabbit No. 8, the increase of sulfated LC in plasma and urine was marked and the sulfate-percentage of LC was more than 95%. Moreover, urinary excretion of deoxycholic-, cholic- and chenodeoxycholic acid was markedly higher than in other animals. No clear changes of bile acid concentration and composition in gallbladder bile were detected. In the 2 mg/kg LCG group and in the SLCG groups, no hepatotoxic changes were noted biochemically or histopathologically, and no changes in the bile acid pattern were observed.
In order to obtain more hydrophylic and more potent hemostatics, 2-semicarbazono-1, 2-naphthoquinone analogs with or without sulfonic acid or its salts at 4, 5, 6, 7 or 8 position (III and IV) and 4-semicarbazido-1, 2-naphthoquinone analogs (V) were prepared and their hemostatic activities and acute toxicities were examined. Among these compounds, more hydrophilic 2-semicarbazono-1, 2-naphthoquinone analogs with sulfonic acid group (IIIj and IIIr) as well as less hydrophilic 2-semicarbazono-1, 2-naphthoquinones without sulfonic acid group (IVa and IVh) and 4-semicarbazido-1, 2-naphthoquinone analogs (Ve) showed the activity comparable to that of 2-semicarbazono-1, 2-naphthoquinone (II) or carbazochrome sodium sulfonate (I) in shortening the bleeding time.
Gaschromatography-mass spectrometry (GC-MS) was applied to the analysis of fatty acid composition in milt of chum salmon Oncorhynchus keta. Arachidonic acid and eicosapentaenoic acid (which were precursors of prostaglandin G2 and G3 respectively) were identified. Myristic acid, palmitic acid, arachidic acid, oleic acid, linolenic acid and docosahexaenoic acid were also identified.
Two anticoagulant factors anticoagulant 1 (A1) and anticoagulant 2 (A2) were isolated from the venom of Agkistrodon acutus by a combination of gel filtration on Sephadex G-75 and chromatographies on carboxymethyl (CM)-Sephadex C-50, diethylaminoethyl (DEAE)-Sephacel. By these procedures, 98.6 mg of Al and 24.8 mg of A2 were obtained from 1 g of crude venom. Anticoagulant activities of these preparations were inhibited by mercaptoethanol but not by soy bean trypsin inhibitor (SBTI), egg white trypsin inhibitor (EWTI), p-chloromercurybenzoate (PCMB), diisopropyl fluorophosphate (DFP), heparin or trasylol. These preparations were homogeneous as judged by disc electrophoresis on polyacrylamide gels at pH 8.3 and pH 4.3, and isoelectric focusing. The molecular weight of A1 and A2 were determined to be 25800 and 24000. By the addition of mercaptoethanol, A1 was dissociated into two subunits (MW 14000 and 12800), and A2 was also dissociated (1300 and 10500). By this treatment, anticoagulant activities of A1 and A2 were both lost. The isoelectric points were found to be pH 5.80 and pH 7.06 by isoelectric focusing with carrier ampholyte, respectively. These factors did not possess caseinolytic, fibrinogenolytic, fibrinolytic, esterolytic, 5'-Nucleotidase, ATPase, phosphomonoesterase, phosphodiesterase, phospholipase A, lethal or hemorrhagic activities. These proteins contained a little carbohydrates.
3-Aminoisocarbostyril derivatives were synthesized in order to investigate their fluorescent properties. 5-Methoxy-, 6-methoxy-, 5, 6-dimethoxy-, 5-chloro-, and 6-chlorophthalides (Ia-Ie) reacted with potassium cyanide to give the corresponding (2-cyanomethyl) benzoic acids (IIa-IIe). Methyl (2-cyanomethyl) benzoates (IIIa-IIIe) were treated with aqueous amines under mild conditions to yield 3-aminoisocarbostyril derivatives having methoxy or chloro groups at the 6- and/or 7-positions. Reactions of IIIa-IIIe with 28% ammonia solution at 70-80°gave 3-aminoisocarbostyrils (IVa-IVe), and with 40% methylamine solution at room temperature gave 3-amino-2-methylisocarbostyrils (Va-Ve). IIIa-IIIe were allowed to stand with hydrazine hydrate at room temperature to give 2, 3-diaminoisocarbostyrils (VIa-VIe) in good yield, whereas IIIa-IIIe as well as VIa-VIe were heated with hydrazine hydrate in a water bath to yield 2-amino-3-hydrazinoisocarbostyril derivatives (VIIa-VIIe).
3-Alkylamino- and 3-arylamino-isocarbostyril derivatives were synthesized in order to investigate their fluorescent properties. Reactions of (2-cyanomethyl) benzoic acid derivatives (Ia-Ie) with primary amines (cyclohexylamine, benzylamine, and aniline) in chlorobenzene gave the corresponding 3-substituted aminoisocarbostyrils (IIa-IIo) having methoxy or chloro groups at the 6- and/or 7-positions. 3-Alkylaminoisocarbostyrils (IIa-IIc, IIf-IIh, and Va-Vf) were also given by reactions of methyl 2-(2'-ethoxy-2'-iminoethyl) benzoates (IVa-IVc) with alkylamines (methylamine, dimethylamine, cyclohexylamine, and benzylamine) in aqueous solution.
Two kinds of hyaluronidases (HD) differing in molecular weight were highly purified from the bovine testicle and their physicochemical and enzymatic properties were studied. By isoelectric focusing both HDs of high (H-HD ; MW 93300) and low (L-HD ; MW 69200) molecular weight were found to be composed of three kinds of subforms with different isoelectric points. It was also recognized that both H-HD and L-HD are glycoproteins consisting of a single polypeptide chain and that the treatment of H-HD with proteinase lowered its molecular weight to achieve its conversion into L-HD. The results obtained by enzyme kinetic study clarified that the affinity of H-HD for the substrate was twice that of L-HD. Therefore, it was considered that the native HD in the bovine testicle is H-HD, which affinity for the substrate was reduced as a result of a lowering of its molecular weight for its conversion into L-HD by the action of hroteinase contained in the bovine testicle extract.
The reduction of α-(N, N-disubstituted amino) propiophenones (3a, b, and c) and their hydrochlorides with sodium borohydride in methanol was examined. The hydrochlorides in contrast to their free bases afforded the corresponding erythro-alcohols (4a, b and c) stereoselectively. It is speculated that the high degree of stereoselectivity was attributed to the initial formation of aminoborine and the formation of the Cram's five-membered cyclic transition state.
The reduction of various salts of 1-(4-benzyloxy and hydroxy)-2-(4-benzylpiperidinopropan-1-one (4a and b) with sodium borohydride gave the corresponding erythro-alcohols (5a and b) stereoselectively, and the relative configuration of the threo- and erythro-alcohols (5a and b) was determined by the stereospecific transformation to the cis- and trans-1-(4-methoxyphenyl)-2-methylethylene oxides (7). In addition, the reactions of the trans-epoxide (7), and 4-acetoxy- and 4-nitroderivatives (8a and b) with 4-benzylpiperidine, in order to prepare 2-piperidino-1-propanol derivatives which would be readily transformed to the 5a and b were examined, but these reactions gave mainly the regioisomer, 1-piperidino-2-propanol derivatives (9a, b and c).
Ac4-proteinase, one of proteinases of the venom of Agkistrodon acutus, was purified by a combination of gel filtration on Sephadex G-75 and chromatographies on carboxymethyl (CM) Sephadex C-50, diethylaminoethyl (DEAE) Sephacel, and DE52 Cellulose. By these procedures, 9.2 mg of purified preparation was obtained from 1 g of crude venom. Ac1-, Ac2- and Ac3-Proteinases obtained from the venom, possessed both lethal and hemorrhagic activities, but Ac4-proteinase had the only hemorrhagic activity. Hemorrhagic and proteolytic activities were inhibited by ethylenediaminetetraacetic acid (EDTA), Cysteine or anti-serum but not by diisopropyl fluorophosphate (DFP), soy bean trypsin inhibitor (SBTI) or chicken egg white trypsin inhibitor (EWTI). The preparation was homogeneous as judged by disc electrophoresis over polyacrylamide gel at pH 8.3, SDS polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight of this protein was determined to be approximately 33000, and the isoelectric point was found to be pH 4.4 by isoelectric focusing with carrier ampholyte (pH 3-10). The minimum hemorrhagic dose and proteolytic activities of this protein were 0.307 μg and 0.362 unit/mg, respectively. This protein did not contain any carbohydrates.
In order to elucidate the mechanism of action of new antitumor agents like bis-(haloalkyl) piperidine (CAP) derivatives, the influence of three typical derivatives, 1-(β-chloroethyl)-2-chloromethylpiperidine hydrobromide (CAP-1), 1-(γ-chloropropyl)-2-chloromethylpiperidine hydrobromide (CAP-2), and 2, 6-bis (iodomethyl) piperidine hydrochloride (CAP-12), on the synthesis of nucleic acid and protein was examined by use of rat ascites hepatoma AH-13 cells, and the binding mode of these compounds to deoxyribonucleic acid (DNA) was also studied by the ultracentrifugation of purified phage P 22 DNA in alkaline sucrose gradient. The incorporation of 3H-thymidine was decreased but those of 3H-uridine and 3H-leucine increased by all compounds, and CAP-1 acted most significantly. On the other hand, the contents of nucleic acid and protein per cell all increased to approximately 160% of the control value in 24 hr. Furthermore, the DNA treated with CAP-1 was shifted to higher density position of alkaline sucrose than the untreated DNA, while in the case of CAP-2 and CAP-12, the DNA was scarcely shifted. From these findings, it was deduced that the antitumor activity of these compounds results from inhibition of DNA synthesis and production of unbalanced cell growth after formation of the interstrand crosslinkage of DNA by CAP-1 and some other alkylation to DNA by CAP-2 or CAP-12.
To investigate the 17β-substituent effect upon enzymatic O-methylation of catechol estrogen, substrates such as 2-hydroxyestradiol 17β-sulfate (IV) and the corresponding glucuronide (VII), were prepared. The former was obtained by catalytic reduction of potassium 2, 3-dibenzyloxyestra-1, 3, 5 (10)-trien-17β-yl sulfate (III). The latter conjugate was prepared by alkaline hydrolysis of methyl [2, 3-dibenzyloxyestra-1, 3, 5 (10)-trien-17β-yl-2, 3, 4-tri-O-acetyl-β-D-glucopyranosid] uronate (V), followed by catalytic reduction.