Antibacterial action, in vitro, of 2-imino-4-thiazolidone (I), 2-thio-4-thiazolidone (II), 2-oxo-4-thiazolidone (III), and 2-hydrazono-4-thiazolidone (IV), possessing fundamental structures of 4-thiazolidone derivatives, was tested and (IV) was found to be better than the other three. 5-Acetaminobenzal-2-imino-(V), 5-acetaminobenzal-2-thio-(VI), and 5-acetamino-2-oxo-4-thiazolidone (VII) were also found not to possess strong inhibitory action against bacteria. For in vitro tests against tubercle bacilli, a basic Kirchner medium in which asparagin was substituted with sodium glutamate and the amount of glycerol was increased to twice the usual volume, was used. Under the assumption that pyridine would be used as a solvent, its effect on human, bovine, and avian type tubercle bacilli was examined and it was found that it had no effect on the bacillary growth at a concentration below 1%.

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By the condensation of p-acetaminoanisole and chloral in conc. sulfuric acid, 5-acetamino-2-methoxyphenyltrichloromethylcarbinol (I), m.p. 236.7° (decomp.), and bis (5′-acetamino-2′-methoxyphenyl) trichloromethylmethane (II), m.p. 240° (decomp.) (Chemical Abstracts, 44, 3952 (1950) gave the decomposition point 227° for a substance containing alcohol of crystallization), were obtained. Recrystallization of (II) from 90% ethanol gives a substance containing 3 1/2 moles of the water of crystallization. Thermal decomposition, action of alcoholic potash, and treating in conc. sulfuric acid at 40°, of (II) result in the liberation of one mole of HCl to give 1, 1-bis (5′-acetamino-2′-methoxyphenyl)-2, 2-dichloroethylene (VI), m.p. 232-233°, whose deacetylated compound (VII) forms pale yellow plates of m.p. 174°. Reduction of (II) with glacial acetic acid and zinc dust gives 1, 1-bis (5′-acetamino-2′-methoxyphenyl) ethane (III), m.p. 226.8°, with a small amount of fluorescent substance of m.p. 229° as a by-product. Deacetylation of (III) gives the amino compound of m.p. 129°, and removal of nitrogen from it gives 1, 1-bis (o-anisyl) ethane (IV), m.p. 68.5°. Chromic acid oxidation of (IV) gives a small amount of 2, 2′-dimethoxybenzophenone and a large amount of quinone (V), m.p. 82.9°, C₁₅H₁₄O₃ (IV) yields a phenol when boiled with hydroiodic acid and red phosphorus.

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Attempts were made to prepare a series of ω-cyclic fatty acids in order to promote the antibiotic action of chaulmoogric acid and its analogs. Some compouds with acid amide bonded to one of the carboxyls of azelaic or brassylic acid were prepared.

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It is often observed that the substitution of a part of a certain compound with allyl group often results in a substance possessing characteristics totally reverse of the original compound. Based on this idea, compounds in which allyl group was introduced into the nitrogen in barbituric acid were prepared. The compounds thus prepared, N-allylbarbital, N, N′-diallylbarbital, N-allylphenobarbital, N, N′-diallylphenobarbital, and N -allylmethylhexabital, were tested as to their antagonistic action against toxicity of barbital, phenobarbital, and methylhexabital, by their lethal dose in mice, and their action on respiration, blood pressure, and heart action in rabbit. However, none of the N-allylbarbitals possessed antagonistic action but rather showed synergistic action.

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The agar substance obtained from Gracilaria confervoides Grev. was decomposed by heat and determination of sulfate ion, periodate oxidation, and measurements of specific viscosity of each fraction were carried out. It has been learned from these results that the agar molecule was a linear polysaccharide composed of galactose bonded at C₁-C₃. Determination of the molecular weight was also carried out by various methods.

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Partial thermal decomposition product of the agar from Gracilaria confervoides was permethylated and after methanolysis, it was isolated into various components by liquid chromatography through alumina from which methyl 2, 3, 4, 6-tetramethyl- and 2, 3, 6-trimethylgalactosides, and methyl 2 (or 4)-methyl-3, 6-anhydrogalactoside were obtained. The structure of the thermal decomposition product was assumed from the ratio of these components.

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8-Azaguanine colors bright yellow in alkaline solution with sodium hypobromite solution and this color does not disappear on the addition of sodium sulfite. 8-Azaguanine can be distinguished by this coloration from other 8-azapurines, purines, and pyrimidines.

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Twenty-one kinds of aminovinyl compounds were prepared by the condensation of the quaternary salts of 2, 6-lutidine, 2, 4-lutidine, and 2, 4, 6-collidine respectively with p, p′-dicarbethoxy (p, p′-dibromo-, 2, 2′, 4, 4′-tetrachloro-, or p, p′-dimethyl)-diphenylformamidines, α, α′ (or β, β′)-dinaphthylformamidines, and 3, 3′, 5, 5′-tetrachloro-(or tetrabromo)-dipyridyl-(2, 2′)-formamidines.

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In connection with the imidazolone rearrangement and pyrazolone conversion described in the previous paper, diacetyl disemicarbazone (III), tetramethylpyrazine (IV), 3, 4-dimeth ylpyrazolone-(5) (V), and hydrazine N, N′-dicarboxylic acid amide (VI) were obtained by the reaction of 3, 4-dimethyl-5-aminoisoxazole (I) and semicarbazide (II). Diacetyl-bis (benzoylhydrazone) (VIII) and N, N′-dibenzoylhydrazine (IX) were obtained from (I) and benzoic acid hydrazide, and 5-(p-aminophenylsulfonamido)-3, 4-dimethylisorazole (XVI), m.p. 240-241°, from 5-(p-aminophenylsulfonamido)-3, 4-dimethylisoxazole (XV) and hydrazine hydrate. It was also found that (XVI) can be obtained from p-aminophenylsulfonamido-α-acetylpropionamidine (XVIII) and hydrazine hydrate.

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A proteid substance was found in the urine of healthy young men which effected decrease of calcium level in rabbit serum, increase of circulating leucocytes in rabbit, accelerating calcium deposition in rabbit teeth, and increase of temperature in rabbit osteomyelium. The substance also gave ultraviolet absorption spectrum and polarographic image that were very similar to parotin. This substance was isolated by the method shown in Figs. 1-4, purified by the method shown in Fig. 5, and was obtained in approximately 82.4% purity by the electrophoretic analysis (Figs. 6-7). This substance was named Uroparotin and its physicochemical (Fig. 8 and Table II) and biological (Tables I and III, and Fig. 9) properties were examined. The purified uroparotin gave positive Biuret, xanthoprotein, Adam-Kiewitz, Millon, and Sakaguchi reactions, and negative ninhydrin reaction. It precipitates with general precipitation agents for proteins, such as acids and heavy metals, but does not coagulate by heating. Uroparotin failed to give any effect on the blood pressure of anesthetized cat when injected intravenously in 10mg./kg. dose.

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Ethyl-, propyl-, butyl-, amyl-, isoamyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, and dodecyl-malonic acid dihydrazides were prepared in order to test their antitubercular action. Eight kinds from ethyl- to octyl-malonic acid dihydrazides were found to inhibit the growth of tubercle bacilli of H 37 Rv strain in concentration of 200γ/cc.

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As a preliminary test for the synthesis of terracinoic acid, one of the alkaline decomposition products of Terramycin, 1-keto-3-methyl-2-indanylacetic acid and 1-keto-5-hydroxy-3-methyl-2-indanylacetic acid were successfully prepared. It was also proved that the latter acid and its methyl ester methyl ether were identical with the decarboxyterracinoic acid and its methyl ester methyl ether obtained by Pasternack as one of the alkaline decomposition products of Terramycin.

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Fries rearrangement of α-naphthyl palmitate and stearate was examined and 2-hexadecanoyl-1-naphthol (III), m.p. 82-83°, and 2-octadecanoyl-1-naphthol (IV), m.p. 85-86°, were respectively obtained. Aluminum chloride and stannic chloride were used as the reagent and it was found that a good result could be obtained with the latter reagent without any solvent. 2, 4-Dinitrophenylhydrazones (V and VI) and corresponding sulfonic acids (VII and VIII) were obtained as derivatives of (III) and (IV).

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Free type and bonded type biotin in several kinds of crude drugs, including cinnamon and liquorice, were examined by microbiological assay method. It was found that although bonded biotin was not contained in the cinnamon, other crude drugs contained both types. In order to find out whether the biotin-like factors contained in the crude drugs, as shown by the microbiological assay, were really biotin or not, paper partition chromatography was carried out. To determine the Rf value of standard biotin, it was developed with water-saturated butanol and examined by bioassay by which it was found that biotin was developed into two groups giving Rf values of 0.2-0.3 and 0.6-0.7, and both helping growth of Lactobacillus arabinosus. Biotin-like factors of liquorice, royal jelly and Apis melifica were examined under the same conditions by which growth factors were found in those giving Rf 0.2-0.3 from liquorice and royal jelly, and Rf 0.2-0.3 and 0.6-0.7 from Apis melifica.

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Paper partition chromatography of biotin was carried out with a mixture of AcOH: H₂O:BuOH (1:5:4) as the developer. Later examination by microbiological assay showed that the compound had been developed into two groups (Rf 0.6-0.7 and Rf 0.8-0.9) of substances which helped the growth of Lactobacillus arabinosus. Two-dimensional paper chromatography of the two components showed that the growth factor of larger Rf value changed to that of a smaller Rf value. It was observed that such phenomenon was affected by the heating and irradiation of ultraviolet light after spotting of biotin on the filter paper.

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Syntheses of the Brooker-type dyes were reported in Part XV and XVI of this series, but some counter-argument has been made against the conclusion of the Brooker-type structure because of the difficulty of the mutual reaction of negative anilino groups and the possibility of another substitution in this synthetic method. The conclusion of the Brooker-type dyes was made from the analytical values and absorption spectra but further proof of the formation of a Brooker-type dye by the substitution of an anilino group in the meso-position was affored by the application of ω-phenylaminovinylbenzothiazolium ethiodide or ω-phenylamino-7-methylbenzothiazolium ethiodide on binuclear trimethinethiocyanine dye possessing inactive methyl in the terminal group and anilino group in the meso-position, to a Brooker-type thiocyanine with a terminal methyl, whose absorption spectrum was similar to or the same as that of binuclear trimethine dyes. It was also experimentally proved that reaction of negative anilino groups does occur by the fact that 1, 1′-diethyl-2, 2′-trimethinethiocyanine iodide was obtained from ω-phenylaminovinyl-benzothiazolium ethiodide and benzothiazole-2-aldehyde p-dimethylaminoanil ethiodide.

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Isolation of snake venoms by paper microelectrophoresis was carried out on 11 kinds of venomous snakes, Naja naja atra Cantor (I), Naja naja naja (II), Naja hanah (III), Bungarus multicinctus Blyth (IV), Vipera russellii formosensis Maki (V), Trimeresurus mucrosquamatus Cantor (VI), Trimeresurus gramineus stjnegeri Schmidt (VII), Trimeresurus flavoviridis flavoviridis Hallowell (VIII), Trimeresurus okinavensis Boulenger (IX), Agkistrodon acutus Guenther (X), and Agkistrodon halys blomochoffi Boie (XI). It was found that the venom from same species gave similar pattern. The venom from Naja species was composed of two or three bases and that of Agkistrodon species contained a large amount of acid proteins. Each protein on the filter paper from the venom of Formosan cobra (I) was cut out and their enzymatic action was examined. It was found that lecithinase was present in electrically neutral portion while cholinesterase was at pH 6.0, slightly to the cathodic side. Cobra venom purified by fractional precipitation with ammonium sulfate and acetone precipitation was found to be proteose, being about eight times more poisonous than the original venom, but showed no activity of lecithinase, cholinesterase, and l-ophio amino acid oxidase, being electrically almost neutral. The kinds of amino acids constituting the venoms of (I) and (X), which showed completely different electric characteristics, were examined by two-dimentional paper chromatography and paper ionophoresis by which 15 kinds of amino acids, including leucine and alanine, were detected. No difference in the kinds of amino acids constituting these two venoms was found.

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A substance giving postitive Sakaguchi reaction was examined from the extract of Cristaria plicata Leach by paper microelectrophoresis, microbiological assay, Sakaguchi reaction, and ion exchange resins. Several kinds of guanidyl derivatives were found which had not been found in the extract of shell-fish, such as arginine, agmatin, alkain, and octopine. One of such derivatives was not adsorbed by the ion exchanger, Amberlite IRC-50, and this was a peptide-like, almost neutral substance which precipitated by phosphotungstic acid. When boiled in acid, this formed a guanidyl derivative which showed no arginine-activity with Streptococcus faecalis. The portion which did not precipitate by phosphotungstic acid also showed the presence of a substance with positive Sakaguchi reaction. This substance is almost neutral. Other substances which were detected included the peptide of arginine and a substance with positive Sakaguchi reaction which moved towards the acid side. Ninhydrin-coloring substances were examined with two-dimensional paper chromatography and 13 kinds of amino acids, including glutamic acid and aspartic acid, were found besides a fair amount of glutamine and asparagine, and a large amount of the peptide of glutamic acid.

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Effect of the oil of Thuja dolabrata and its components on pathogenic and nonpathogenic hyphomycetes was tested. Of the several components of this oil, hinokitiol was the most effective, being effective up to 64, 000 to 128, 000 dilutions against Trichophyton, Epidermophyton, and Anchorion, followed by l-rhodinic acid and thujopsol. Non-pathogenic fungi were found to be very resistant and hinokitiol was effective only in 8, 000-16, 000 dilutions. Based on the assumption that the antifungal action of hinokitiol was due to its formation of complex salts, effect of its iron, zinc, and copper salts were tested and it was found that, as in the case of oxine, the addition of iron complex resulted in complete invalidation, while that of zinc complex reduced the effect to one-quarter, and the addition of copper complex failed to bring out any difference. This specificity of the copper complex was assumed to be due to its substitution with the iron complex, as in the case of oxine.

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Growth inhibitory action against 24 kinds of bacteria, including Streptococcus hemolyticus, Diplococcus pneumoniae Type I, and Brucella melitensis, were tested with 13 kinds of compounds reported in Parts I to V of this series which showed strong growth inhibition against Staphylococcus aureus 209 P, Bacillus coli communior, and Bacillus subtilis. None of them showed a very strong growth inhibition but 6-chloro-4-heptylresorcinol, 2-chloro-3-methyl-4-thiocyano-6-isopropylphenol, p-nitrothymol, and 2, 4-dihydroxy-3-chloro-5-hexylbenzaldehyde thiosemicarbazone were able to completely inhibit the growth of Streptococcus hemolyticus at 160, 000 dilution, 6-chloro-4-cyclohexylresorcinol, 2-chloro-4-acetylthymol, p-nitrosophenol, 2, 4-dihydroxy-3-chloro-5-amylbenzaldehyde thiosemicarbazone, propyl olivetolcarboxylate, and sphaerophorin in 80, 000 dilution. 6-Chloro-4-heptylresorcinol, 2-chloro-3-methyl-4-thiocyano-6-isopropylphenol, and 2, 4-dihydroxy-3-chloro-5-hexylbenzaldehyde thiosemicarbazone completely inhibited the growth of Diplococcus pneumoniae Type I in 160, 000 dilution, and 2-chloro-4-acetylthymol, p-nitrothymol, propyl olivetolcarboxylate, p-quinonedichloroimide, and sphaerophorin in 80, 000 dilution. 6-Chloro-4-heptylresorcinol, p-nitrosophenol, 2, 4-dihydroxy-3-chloro-5-hexylbenzaldehyde thiosemicarbazone, sphaerophorin, and divaricatic acid complety inhibited the growth of Bacillus anthracis in 80, 000 dilution. It should be noted that, as shown in the accompanying Table, sphaerophorin was the only one among the 13 compounds tested which was able to uniformly inhibit the growth of the bacteria tested, except six, although in 80, 000 dilution.

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Conditions for the paper electrophoresis were examined with food coloring matter and the reproducibility of the migration distance was obtained. Effect of the different kinds of filter papers (Toyo filter paper) on the distance of migration was found to be rather marked as shown in Table II.

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Influence of the individual change of electric pressure and current on the mobility in electrophoresis and reproducibility of mobility values were examined with observations on the merits of various electrophoretic apparatus used to date. The writer proposes the designation “dark current” for the electric current that flows in areas other than the filter paper, such as the current that flows through the zone of contact between filter paper and the glass plate. This “dark current” was found to be not negligible in electrophoresis.

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Benzylpenillic acid, d-α-benzylpenicilloic acid α-anilide, benzylpenaldic acid anilide, phenaceturic acid anilide, and benzylpenilloic acid were isolated as the decomposition products of benzylpenicillin aniline salt in an aqueous solution. Of these, the yield of benzylpenillic acid was 35-40% and its formation was assumed to be due to the presence of aniline. At the same time some examinations were made on the formation mechanism of benzylpenillic acid.

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As the decomposition product of benzylpenicillin benzylamine salt in an aqueous solution, phenaceturic acid benzylamide was isolated, and benzylpenaldic acid p-methyl-sulfone benzylamide as that of benzylpenicillin p-aminomethylphenylmethylsulfonate. The products common to both were the corresponding amine salts of d-penicillamine and benzylpenicilloic acid.

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As the application of the paper chromatography to natural coumarin derivatives described in the previous paper⁺, presence of daphnin was examined by this means in Daphne Kiusiana Miq., Wikstoroemia Ganpi (Sieb. et Zucc.) Maxm., and Edgeworthia papyrifera Sieb. et Zucc. The presence of daphnin in Daphne Kiusiana was confirmed by the concurrent use of the existing macromethod and paper partition chromatography. The presence of daphnin in the other two plants were clarified by the detection of its glycoside by paper chromatography of a sample obtained by removing as much impurities as possible by lead method or by direct digestion with a solvent, and by the detection of the aglycone by paper chromatography from the sulfuric acid hydrolyzate of the glycoside.

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Antibacterial action of 45 kinds of compounds against Staphylococcus aureus 209P, Bacillus coli communior, and Bacillus subtilis was tested and it was found that the 14 kinds of compounds of isonicotinic acid hydrazone series possessed only a weak antibacterial action. Of the eight kinds of 4-thiosemicarbazones, 2, 4-dihydroxy-5-amylbenz-aldehyde allylthiosemicarbazone was effective in preventing the growth of Staph. aureus 209P in 80, 000 dilution, and of Bac. subtilis in 320, 000 dilution. Seven kinds of 4-hydroxybenzoic acids and its esters were rather weak in growth inhibition. Of the 16 kinds of 4-hydroxy-3-halobenzoic acids and their esters, isoamyl 4-hydroxy-3-iodoben-zoate completely inhibited the growth of Staph. aureus 209P in 160, 000 dilution, and the hexyl ester in 80, 000 dilution. Isoamyl 4-hydroxy-3-chlorobenzoate and hexyl 4-hydroxy-3-iodobenzoate both completely inhibited the growth of Bac. subtilis in 80, 000 dilutions.

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By the application of the sodium salts of formylacetophenone, formylacetone, formylbenzyl cyanide, 3-formylcamphor, and ethyl formylacetate on barbituric acid, thiobarbituric acid, rhodanine, quinaldine methiodide, quinaldine ethiodide, and 2-methylbenzothiazole ethiodide, corresponding methineoxonol and merocarbocyanine dyes were prepared.

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Methineoxonol dyes were obtained by the application of formamide on barbituric and thiobarbituric acids, and anilidomethylene compounds by the application of form-anilide to these acids. Asymmetric methineoxonol dyes were obtained by the application of rhodanine or 2, 4-dioxothiazolidine to acetanilidomethylene compounds obtained by the acetylation of several kinds of anilidomethylene compounds. Application of p-dimethylaminobenzaldehyde, p-acetaminobenzaldehyde, or m-nitrobenzaldehyde to barbituric acid, thiobarbituric acid, rhodanine, pseudothiohydantoin, or 2, 4-dioxothiazolidine gave the corresponding benzal compounds.

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Nitration of methyl dehydroabietate with conc. nitric acid in acetic anhydride results in the formation of a 8-nitro compound, m.p. 193-194°, [α]_D²⁵: +94° (acetone), and a 6-nitro compound, m.p. 135-137°, [α]_D²⁶: +80° (alcohol). The 6-nitro compound can easily be reduced to the corresponding 6-amino compound by catalytic reduction with palladium-carbon but the 8-nitro compound is resistant to this reduction. The condensation product obtained by heating at 100° an equimolar amount of methyl 6-aminodehydroabietate and ethyl ethoxymethylenemalonate was boiled in Dowtherm oil from which a compound, C₂₇H₃₅O₅N, of prisms, m.p. 156-158°, was obtained. Since the ultraviolet absorption spectrum of this substance is similar to that of 3-carbethoxy-4-hydroxyquinoline, the structure of this crystalline substance was assumed to be the corresponding derivative of 3-carbethoxy-4-hydroxyquinoline. The yield of this substance was extremely poor, being about 5% of the theoretical amount.

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Diquinoline 6, 7′-oxide, diquinoline 7, 7′-oxide, and diquinoline 7, 8′-oxide were respectively synthesized by the Ullmann condensation of 7-bromo- and 6-hydroxyquinoline, 7-bromo and 7-hydroxyquinoline, and of 7-bromo and 8-hydroxyquinoline. Tetrahydro derivatives of these compounds were also prepared.

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Colorimetric determination of alkaloids possessing methylenedioxy group by chromotropic acid was examined and this was found to effect simplification of procedures and microdetermination compared to the existing phloroglucinol method. This method makes it possible to determine the substances in an amount 5-10γ/cc. as formaldehyde and the accuracy is within an error of ±1%. This method is also advantageous in that colored compounds and substances containing impurities could be determined in an almost colorless state by high dilution.

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The intermediate, obtained by the application of potassium thiocyanate to α, β-dichloro (dibromo) ethyl acetate (I), undergoes cyclization when allowed to stand with the addition of ammonia water and forms 2-aminothiazole which was confirmed as its picrate. Condensation of (I) and hydrazodicarbonylthiamide yielded 2, 2′-hydrazothiazole hydrochloride whose reductive decomposition gave 2-aminothiazole.

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During the electrolytic reduction of 3, 4-dimethoxy-β-nitrostyrene to β-(3, 4-dimethoxyphenyl)-ethylamine, an amine giving positive Beilstein reaction was obtained. By making a double diaphragm, an amine not containing halogen was obtained in good yield.

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Efficiency of the quantitative bridge for coloring matter has already been reported. For the determination of colorless substances, it is recommended that a developer which makes the Rf value of the substance to be near 1 be used and to use coloring matter whose Rf value is the same as that of the sample under the same conditions. The position of the sample is calculated from the relationship between the solvent front and the quantitative bridge.

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