Colorimetric determination of 4-acetamidoantipyrine in plasma was studied and the standard procedure was established. Its principle is based on the oxidative condensation of 4-aminoantipyrine (Emerson's reagent), produced from 4-acetamidoantipyrine by hydrolysis, with phenols in weak alkaline medium to yield an indophenol-type pigment. Of the various kind of phenolic compounds, the simple phenol was found to be the most suitable for this purpose. It has also been found that development of the color is constant and stable over a wide pH range employing borate buffer already reported by Lacoste, et al. A known amount of 4-acetamidoantipyrine added to plasma was measured, using the calibration curve by the standard procedure and the results were quite satisfactory.
The mother liquor left after separation of hydrocortisone (II), 11-epihydrocortisone (III), and 17α, 19, 21-trihydroxypregn-4-ene-3, 20-dione (IV), formed by the microbiological oxidation of Reichstein's substance S (I) by Corticium sasakii further yielded cortisone (V), 6β, 17α, 21-trihydroxypregn-4-ene-3, 20-dione (VI), and prednisolone (VII). Mobility of the monohydroxyl derivatives of substance S at 11β, 11α, 19, 11-oxo-, 6β, and 1-ene-11β, as well as at 14α, 15α, 16α, and 16β, in paper partition chromatography was examined using solvent systems of propylene glycol-toluene·dioxane (7:3) and formamide-benzene·chloroform (1:1), and relationship between the steroidal structure and order of polarity was discussed.
Aromatization of the A-ring in 19-hydroxy-4-ene-3-oxo-steroids was attempted. Dehydrogenation of 17α, 19, 21-trihydroxypregn-4-ene-3, 20-dione 19, 21-diacetate (V), m.p. 165-167°, with selenium dioxide to 17α, 19, 21-trihydroxypregna-1, 4-diene-3, 20-dione 19, 21-diacetate (VI), m.p. 179-181°, and its treatment with potassium hydroxide in ethanol at room temperature afforded formaldehyde and 3, 17, 21-trihydroxy-19-norpregna-1, 3, 5(10)-trien-20-one (VII), m.p. 225.5-229°. The same treatment of 19-hy-droxyandrost-4-ene-3, 17-dione 19-benzoate (IX), m.p. 118-120°, afforded estrone (XI) via androsta-1, 4-dien-19-ol 19-benzoate (X), m.p. 158-160°.
Examinations were made on the conversion of 19-hydroxy-4-ene-3-oxo-steroids to 3-oxo-19-nor-steroids. Oxidation of 19-hydroxyandrost-4-ene-3, 17-dione (II) with chromic acid yielded androst-4-ene-3, 17, 19-trione (III), m. p. 129-133°, and its treatment with sodium hydroxide in hydrous methanol in the cold afforded estr-4-ene-3, 17-dione (IV), m. p. 164-171°. Oxidation of (III) with chromic acid-sulfuric acid in acetone afforded 3, 17-dioxoandrost-4-en-19-oic acid (V), m. p. 155°(decomp.), which formed a methyl ester (VI) of m. p. 135-137° by treatment with diazomethane. (IV) is also obtained by decarboxylation of (V) by heating with hydrochloric acid in methanol. Decarboxylation of (V) was effected by heating in pyridine and estr-5(10)-ene-3, 17-dione (VII), m. p. 142-145°, was obtained. This converted immediately to (IV) on treatment with hydrochloric acid or alkaline methanol.
A new, sensitive colorimetric determination of tetracyclines is described. Tetracyclines react with diphenylborinic acid in ethanol and form yellow chelate compounds of 1:2 combination ratio. This chelate formation was utilized for colorimetry. To the aqueous solution (5cc.) of tetracycline (10-30γ), 5cc. of 1% ethanolic solution of diphenylborinic acid and 99% ethanol are added to make the whole volume 10cc. The blank test is carried out with 5cc. of distilled water, 5cc. of the reagent, and the whole volume brought to 10cc. with 99% ethanol. Optical density at maximum absorption of 415mμ is measured after one hour. Chlor- and oxy-tetracyclines can be determined in a similar manner, using the respective absorption maxima at 417 and 407mμ. The values obtained by the present method and by biological assay on aqueous solution of tetracycline allowed to stand for a long period of time were in good agreement.
Examinations were made on biologically changed substances excreted into urine after oral administration of sulfamethylthiadiazole, using paper chromatography, paper electrophoresis, and combined use of these two procedures. The products detected and identified were sulfamethylthiadiazole, N4-acetylsulfamethylthiadiazole, sulfamethylthiadiazole N4-sulfonate, and sulfamethylthiadiazole N4-glucuronide. Separatory determination of these products was made by the counter-current distribution technique and their quantitative relationship was clarified.
5-(2-Thiacyclobutyl) valeric acid (III) was prepared by the reaction of alkyl 6, 8-dihaloöctanoates (IV and V) with alkali sulfide and by tosylation of mercapto-hydroxy-octanoic acids (I and VII) followed by alkali treatment. Reaction of methyl 6, 8-dichloroöctanoate (IV) with various thiolation agents invariably resulted in the formation of (III) and α-lipoic acid. It was also found that α-lipoic acid (X) or its ester (XIII and XIV) is formed by heating (III) or its ester (XI and XI) with sulfur. Using this new sulfurization reaction, 5-methyl-1, 2-dithiolane-3-valeric acid (XVI) was prepared.
Determination of various saccharides, albumin, and glycine by the hypoiodite method was carried out and examinations were made on the effect of the quantities of sample, alkali, reaction time, and temperature. Determination was possible for D-glucose, D-galactose, D-mannose, L-arabinose, D-xylose, D-ribose, L-rhamnose, maltose, lactose, D-glucuronolactone, and L-ascorbic acid by adding 2cc. of 0.1N iodine and 2cc. of 0.1N sodium hydroxide to 4cc. of the sample solution, allowing the mixture to stand at 15-30° for 20 minutes, acidification of the mixture with hydrochloric acid, and titration of residual iodine with 0.01N sodium thiosulfate. Determinable range of the sample was clarified for each of the sugars. Examinations were also made on the iodine consumption by ketoses, non-reducing sugars, albumin, and glycine. Reaction using 3 and 4cc. of 0.1N sodium hydroxide made it impossible to determine D-mannose, D-ribose, and L-rhamnose, and iodine consumption by ketoses and nonreducing sugars also increased considerably.
Among the acid amides used as solubilization agents nicotinamide shows the best solubilization effect, as reported in the previous work. In the present series of experiments, over 20 kinds of pyridinecarboxamides were prepared and relationship between their structure and solubilization effect was clarified. Substances chosen for solubilization were adrenochrome monosemicarbazone, salicylamide, p-toluidine, and caffeine. It was found that, among the position isomers of the acid amide in pyridine ring, practically no difference was found in 2-, 3-, and 4-substituted compounds, Introduction of alkyl into the nitrogen in acid amide group increased solubilization activity, the effect being better in lower homologs of methyl and ethyl groups. Introduction of aryl groups, such as phenyl or pyridyl, was found to give far stronger effect, while introduction of two alkyls into the nitrogen in the acid amide group resulted in increased solubility in the case of salicylamide and p-toluidine, but a decrease of 30-40% in activity in the case of adrenochrome monosemicarbazone and caffeine. Introduction of a methylene group between the pyridine ring and acid amide group to form pyridine-acetamides resulted in lowering of solubilization activity by 10-40% from that of nicotinamide.
Pyridine-carboxamides were prepared and their solubilization effect was examined in the previous work. In the present series of work, urea acetamide, and pyridine-carboxamide derivatives having two carbonamide groups were synthesized and their solubilization effect was examined. Urea and acetamide derivatives had only a weak solubilization effect while N-2-pyridylacetamide had an effect equal to that of nicotinamide. Nicotinoylureas possessed 1.2-1.5 times stronger solubilization effect than nicotinamide and water-soluble derivatives of theophylline possessed 2.2-6.6 times stronger effect than nicotinamide. Although nicotinuramides possess two carbonamide groups, they have the acetamide linked in series with nicotinamide and possessed the effect only about equal to that of nicotinamide. Pyridinecarboxamides possessed the effect 1.5-2 times greater than that of nicotinamide.
Sodium benzoate and salicylate have been used for a long time as a solubilization agent. It has been found that nicotinamide is a better solubilization agent than sodium nicotinate and, therefore, salicylamide derivatives and benzamides were prepared for examination as a possible solubilization agent. Salicylamides are all sparingly soluble in water and cannot be used as a solubilization agent but they showed better effect than nicotinamide when used in aqueous solution first solubilized with nicotinamide. Benzamides showed solubilization effect about equal to that of nicotinamide, but had 1.6-2.0 times the effect of nicotinamide when used for caffeine. N-2-Pyridylbenzamide hydrochloride had a very strong effect, same as the N-aryl derivatives of nicotinamide and about 4.5 times stronger solubilization effect than nicotinamide when used for adrenochrome monosemicarbazone.
Paper chromatographic examination of thiamine crystal revealed the presence of three spots, (A), (B), and (C), besides that of thiamine. The absorption spectrum, diazo reaction, and thiochrome reaction of (B) were closely similar to those of thiamine and its hydrolysis with hydrochloric acid gave a compound showing activity against Lactobacillus fermenti-36. It is therefore assumed that (B) is acetylthiamine. The absorption spectrum of (C) in aqueous solution of pH 3.0 was similar to that of thiamine but that in 2% sodium hydroxide solution was similar to those of chloroethylthiamine and thiamine anhydride in the same solvent. Consequently, (C) is probably chloroethylthiamine or bromoethylthiamine.
Growth-promoting effect on Lactobacillus fermenti-36 was examined in 12 kinds of compound possessing structure similar to thiamine and none of them showed any activity. Apparent activity is sometimes shown by O-chlorothiamine, O-benzoyl-thiamine, O, S-dibenzoylthiamine, 3-[(2-methyl-4-amino-5-pyrimidinyl) methyl]-4, 5-dimethylthiazolium chloride, and N-[(2-methyl-4-amino-5-pyrimidinyl) methyl]-N-[1-(2-thiacyclobutylidene) ethyl] formamide as well as the compounds which undergo chemical rearrangement to thiamine under the conditions for microbiological assay, i.e. O-acetyl-thiamine, thiamine 2-hydroxyethyl disulfide, thiamine propyl disulfide, thiamine disulfide, and thiamine tetrahydrofurfuryl disulfide, but they have no fundamental activity. Ethylthiamine has the same activity as thiamine.
Further examinations were made through paper bioautography on the apparent growth-promoting action of thiothiamine, thiamine 2-hydroxyethyl disulfide, thiamine propyl disulfide, thiamine disulfide, and thiamine tetrahydrofurfuryl disulfide against Lactobacillus fermenti-36 and these compounds were found to form thiamine secondarily. Inhibition experiments on pyrithiamine showed that inhibition on thiamine corresponding to the foregoing apparent effect was present and it is assumed that there is no fundamental action of growth-promotion in these compounds against this bacteria.
Acetylation of the sodium salts of N1-(5-methyl-3-isoxazolyl) sulfanilamide (VII) and N1-(2-phenyl-3-pyrazolyl) sulfanilamide (VIII) with acetic anhydride gave N1-acetylated derivatives (V and VI) by selective acetylation of N1-position. Acetylation of (VII) and (VIII) with acetic anhydride in the presence of pyridine, however, afforded N4-acetylated derivative (IX) in a good yield from (VII), instead of (V), while (VI) was obtained from (VIII) but in a smaller yield than that from the sodium salt. Boiling of (V) and (VI) in pyridine resulted in facile rearrangement of the acetyl group from N1- to N4-position. This rearrangement reaction was especially easy in (V) which formed N4-acetylated compound in a good yield merely by being allowed to stand in pyridine at ordinary temperature for a few days. Ultraviolet absorption spectra of acetylated derivatives of several kinds of sulfanilamide are also described.
The aqueous suspension of precipitated calcium carbonate obtained by the reaction of calcium hydroxide and carbon dioxide differs somewhat in alkalinity from that of the solution obtained by the reaction of soluble calcium salt and aqueous solution of carbonate. It is not easy to measure this difference by acid titration apart from the main constituent, calcium carbonate. Studies were made on the method of measuring such a minute amount of basic component by measuring loss by combustion using thermobalance and with consideration of analytical results from chemical determination. A fairly satisfactory result was thereby obtained.
Pale yellowish white, fluffy needles, m.p. 245°, were obtained in 0.31% yield from the fresh leaves of Tilia japonica SIMK. This substance colors purplish brown to ferric chloride and pale yellow to magnesium and hydrochloric acid, and to zinc and hydrochloric acid. Its molecular formula corresponded to C22H22O10⋅2 1/2H2O, and it had optical rotation of [α]D9 -64.57°. It is not decomposed by dilute mineral acids but undergoes decomposition on being boiled in 35% hydrochloric acid. It dissolved in hot water and the solution forms a gel when cooled. Formation of one mole each of acacetin and glucose by hydrolysis showed that the substance is acacetin 7-glucoside and it was named tilianin. The methanolic extract of the leaves of Tilia japonica was treated with 5% lead acetate and its filtrate afforded pale yellow needles, m.p. 171-172°, in 0.05% yield. This substance was identified with rutin.
Sulfonphthalein dyes combine with Neutral Red in 1:1 ratio to form a salt. These salts, especially those with Bromophenol Blue and Bromocresol Green, were found to be a good indicator in two-phase titration of ionic surfactants. These salts dissolve in chloroform, in the presence of a buffer solution of pH 6-7, to form a pale yellow solution and the color changes to red in the presence of a trace of anionic surfactant or to green in the presence of cationic surfactant. These indicators are prepared into 0.01% solution in chloroform, its 0.5cc. is added to 20cc. of the test solution of ca. 0.005M concentration, and the solution is titrated with the corresponding 0.005M solution of ionic surfactant of known potency, in the presence of 10cc. each of chloroform and buffer solution. This method is not affected by the presence of fatty acid soap and nonionic surfactants, so that its use may be anticipated in various fields.
Twelve kinds of fluoresceine dyes were examined for their adaptability as an indicator for two-phase titration with cationic surfactant as the titrant. It was thereby found that Rose Bengale and Eosine B could be used like Eosine and Erythrosine for an indicator. Using the method with Rose Bengale as an indicator, stability of four kinds of representative anionic detergents at 40° and 100°, under various pH's, was examined. Alkyl sulfate-type detergents, like sodium dodecylsulfate and Tergitol 7, are stable in neutral and alkaline state but labile in acid media. Tergitol is less stable than the straight-chain alkyl sulfates. Aerosol OT undergoes decomposition both in acid and alkaline state, and this is due to hydrolysis of the carboxylic acid ester moiety. Sodium dodecylbenzenesulfonate was stable under any conditions.
In order to elucidate the role of coumarin derivatives in effecting fall of body temperature, 2-thiocoumarin was taken up and relationship between its hypothermic action and distribution in brain tissues, chemical form in the brain, and its effect on brain tissue respiration were examined. It was found that a part of 2-thiocoumarin administered is distributed in the brain tissue as such and a part in the form of hydroxycoumarin. The sulfur atom eliminated from 2-thiocoumarin converted to organic or inorganic sulfate or entered the sulfur-containing amino acid in proteins. The amount of biological products of 2-thiocoumarin distributed in the brain tissue and the effect of lowering body temperature were in complete parallelism, the greatest hypothermic reaction being observed at the peak of distribution in the brain. 2-Thiocoumarin inhibited tissue respiration in the telencephalon, and diencephalon and midbrain.
In order to clarify the relationship between chemical structure and pharmacological action, 14 compounds of monohalogenated benzhydryl aminoethyl ether series of the general formula X- CH-O-CH2CH2Y (X=F, Cl, Br; Y=NMe2, NEt2, piperidino, morpholino, 1-pyrrolidinyl) were synthesized and their pharmacological activities were examined chiefly on antihistamine, antiacetylcholine, and antibarium actions. In general, monofluorinated derivatives showed marked antihistamine activity either by the Magnus or anti-histamine shock method. Chloromorpholine and bromomorpholine derivatives showed marked lowering of toxicity and an abnormally strong activity against histamine shock compared to that by Magnus method. These compounds may prove to be a new antihistamine agents and examinations are being made to see if they also possess sedative, antitussive, and hypotensive actions.
Conditions for purification of crude hyaluronidase was examined by the use of zone electrophoresis with starch as the carrier and by column chromatography with ion exchange resin, Amberlite IRC-50, and carboxymethylcellulose. These methods were found to be better than the existing methods in the degree of purification, yield, and reproducibility. Some doubts were found for the theory of Hahn that the isoelectric point of hyaluronidase was at pH 5.7, by the behavior of hyaluronidase to zone electrophoresis and adsorption to various adsorbents.
Specific activity of crude hyaluronidase was successfully raised about 100 fold in a good yield by the combination of purification processes in the order of carboxymethylcellulose chromatography, salting out with ammonium sulfate, zone electrophoresis, ion exchanger chromatography with Amberlite IRC-50, and salting out with ammonium sulfate. The purified hyaluronidase proved to be a homogeneous by ultracentrifugation and electrophoresis.
The effect of various stabilizers for aqueous solution of hyaluronidase was examined with the samples purified 10 and 100 folds, under conditions used for crude preparations in previous works. It was found that hyaluronidase became markedly labile as purification progressed, that sodium chloride had stabilizing effect against the purified enzyme but the effect was insufficient when used alone, and that the gelatin showed a very good stabilizing effect in the presence of sodium chloride.
Relationship between enzymic action and spreading activity was examined using hyaluronidase sample thought to be homogeneous by electrophoresis and ultracentrifugation. Dose-response curve of this sample showed lineality when logarithm of the dose was taken. Heat treatment at 65° resulted in rapid disappearance of enzymic activity but spreading activity remained. Enzymic activity showed a marked decrease by tryptic digestion but the spreading activity was not greatly affected. Enzymic activity lowered only slightly by digestion with chymotrypsin but there was no change in spreading activity.
Examinations were made on the difference in the active site of enzymic and spreading activities of hyaluronidase, using a sample considered to be homogeneous by electrophoresis and ultracentrifugation. Both activities showed marked decrease by iodination, while alone decreased markedly by the coupling of diazobenzenesulfonic acid resulted in disappearance of enzymic activity but the spreading activity remained. Enzymic activity lowered to 30% of the control by acetylation but there was no change in spreading activity. Both activities were almost unaffected by guanidination with O-methylisourea. This hyaluronidase sample does not contain -SH or -S-S- linkage.
Human urine during oral administration of sulfathiazole was examined by paper chromatography, using neutral, acid, and alkaline developing solvents, and by paper electrophoresis, and sulfathiazole, acetylsulfathiazole, sulfathiazole-N4-sulfonate, sulfathiazole-N4-glucuronide, and an unknown substance were detected. These products, except the last one, was proved by comparison with authentic samples prepared by a synthetic route. In another experiment, sulfathiazole, acetylsulfathiazole, and an unknown glucuronic acid conjugate were isolated but not other substances, possibly because of the minute quantity and poor stability. In this connection, a new and simple apparatus for two-dimensional, ascending paper chromatography was devised, in which the filter paper is wrapped in a plastic sheet.
1) In order to examine the action (ability for amino acid formation) of various kinds of protease at the pH of gastric juice (2-5, sometimes over 5, at the time of digestion), comparative studies were made on the acid-fast digestive enzyme, Vernase, obtained from Aspergillus oryzae var. microsporus TPR-18, saccharated pepsin, papain, pancreatin, protease of Streptomyces griseus, and human gastric juice. 2) Specificity of various enzymes to peptide bond was examined by application of milk casein as a substrate to various enzymes at pH 3 and 6 until formation of amino acid became constant, secondary application of other enzymes to it, and measuring the amount of amino acid increased. 3) The protease activity at various pH's was measured in Vernase, saccharated pepsin, gastric juice, gastric juice and Vernase, and gastric juice and saccharated pepsin. From these results, examinations were made on the activity of various enzymes in gastric juice and on synergetic action of gastric juice and other various enzymes. It was concluded from these experimental results that (i) the ability for formation of amino acid is higher in Vernase at pH 3 and in Vernase and protease from Streptomyces by reaction with numerous kinds of peptide bond; (ii) two kinds of protease with optimal pH of 3 and 5-6 are present in gastric juice, other than protease; (iii) saccharated pepsin reacts at pH 1-3, the optimal pH being 2.3; (iv) Vernase and gastric juice work synergetically against milk casein coagulated at around its isoelectric point; and that (v) saccharated pepsin and gastric juice do not work synergetically.
Diazotization of 2-amino-4-methylthiazole (I) followed by bromine substitution afforded 2-bromo-4-methylthiazole (II) and 2, 5-dibromo-4-methylthiazole (III). In order to obtain (II) alone, the method of Ganapathi was followed but the product was a mixture of (II) and (III). Further examinations showed that the objective could be attained by removal of excess of nitrogen oxide gas, formed at the time of diazotization, with dry air, completely outside the reaction system.
The fruits of Lindera citriodora (Lauraceae) give an essential oil (3.4% yield, Table I), which contains d-limonene and citral (ca. 65%, Table II). Citral distillate is composed of citral A (geranial) and B (neral), and the former seems to be in majority (Table III).
Reduction of 4-iminovioluric acid, and its 3-methyl and 1, 3-dimethyl derivatives, with zinc dust was carried out in the presence of aqueous ammonia, ammonium carbonate, or ammonium chloride. The reduction with zinc dust and ammonium carbonate seemed to be especially adapted for 1, 3-dimethyl derivative. Electrolytic reduction of 5-phenylazo-6-aminouracil in formic acid afforded 5-formamido-6-aminouracil.
Isoimperatorin (I) was isolated as white needles, m. p. 110-111°, and 5-methoxy-8-hydroxypsoralen (II) as yellow crystalline powder, m. p. 210-212°, from the fruits of Angelica japonica A. GRAY (Umbelliferae), and umbelliferone was detected by microchemical method. (I) had been isolated first from the root of Peucedanum ostruthium KOCH and it is identical with auraptin, obtained from sweet orange oil, but this is the first example of its isolation from the plants of Angelica genus. (II) is formed on decomposition of byak-angelicin with glacial acetic acid added with conc. sulfuric acid and was first isolated from the tree bark of Phebalium nudum HOOK. (Rutaceae), but this is the first time that it was found in Umbelliferae plant.
Schöpf and Robinson have earlier proposed the hypothesis that protoberberine-type alkaloid (I) is biosynthesized from two moles of β-(3, 4-dihydroxyphenyl)-L-alanlne (DOPA) (II) and 1 mole of formaldehyde. The present series of experiments were therefore carried out to see if tyrosine took part in biosynthesis of berberine (III), using Coptis japonica MAKING as the test plant. The plant was cultivated hydroponically in Houghland solution containing DL-tyrosine [2-14C], each component was separately extracted, and their specific radioactivity was measured. It was thereby found that berberine was comparatively well-labeled but there was no radioactivity in the side-chain carbons and this fact showed that berberine was not labeled uniformly. The radioactivity was also found in glucose, a metabolite of tyrosine, and its formation was greater than that of berberine, the secondary metabolite.
The Curtius reaction of 3- and 4-carboxycarbostyril respectively gives 3- and 4-aminocarbostyril, while that of 3- and 4-carboxyisocarbostyril gives 4-aminoisocar-bostyril but not the corresponding 3-isomer. In the case of 3-carboxyisocarbostyril decomposition of the urethan derived from it with hydrochloric acid produced 1, 3(2H, 4H)isoquinolinedione. Animal experiments with mice showed that 3-aminocarbostyril and 4-aminoisocarbostyril are amyostatic poison but 4-aminocarbostyril lacks this activity.