A rapid and simplified method for the determination of a micro-quantity of antimony was devised. A part of the decomposition of organic antimony compounds was modified to lessen its effect on later colorimetric procedure and simplified. Examination of the optimal acidity at which Sb5+ is stable in the decomposition solution indicated it to be 6M hydrochloric acid. The decomposition solution is extracted with isopropyl ether, which has been saturated with 6M hydrochloric acid, by which about 98% of Sb5+ is extracted by one procedure. The coloration reagent is the usual rhodamine B but not the ordinary aqueous solution but a solution in 1M hydrochloric acid since the aqueous solution would be affected by the acid present in the extract and excess of the reagent not bound with Sb5+ would color by itself, interfering in the coloration of the compound of Sb5+ and rhodamine B. Such colorimetric procedures in strong acidity, avoiding drastic change of reaction after decomposition, was found to give a satisfactory result.
It has already been revealed that tannin in crude drugs could be determined by the utilization of the fact that tannic acid undergoes reaction with methylene blue to form water-insoluble product (D-fying of methylene blue) in capillary analysis. The important point in this principle is that the reaction of tannic acid and methylene blue should be quantitative. In order to clarify this point, studies were carried out on the reaction of methylene blue with gallic acid, a componental part of the tannic acid molecule and which is considered to be responsible for its reaction with methylene blue. It was found that the phenolic hydroxyls in the gallic acid were responsible for the reaction of gallic acid and methylene blue, and that the molar ratio of the reaction was 1:1, by preparing capillary image and by determination of sulfur in the reaction product.
Relative reaction of phenol compounds and methylene blue was examined and it was found that phenol and compounds with a hydroxyl in the ortho position, like pyrocatechol, did not undergo D-fication reaction with methylene blue, while those with hydroxyls in mete- and para-positions, such as resorcinol and hydroquinone, those with three hydroxyls, such as pyrogallol and phloroglucinol, and compounds considered to be phenol derivative, such as catechin, rutin, woogonin, baicalin, quercitrin, were found to undergo D-fication. Determination of tannin-bearing crude drugs and tannin materials was carried out by the methylene blue-D-fication and by the use of hide powder, and it was found that the values obtained by the two methods were in parallel and that the value from the former method multiplied by a definite coefficient agreed well with the value obtained by the latter method. It was also clarified that the difference of coefficient in various plants was inevitable from the difference in the composition of tannin in each plant and by the presence of non-tannic substance.
Colorimetric determination of d-catechin with vanillin-hydrochloric acid reagent was carried out. Successful separation of d-catechin and Gambir tannin was effected by paper chromatography, from which d-catechin was eluted out with methanol and submitted to determination. By this means, sum of content of d-catechin and that of its optical isomers was obtained as expressed by d-catechin value.
2-Acetylpropionimidate hydrochloride, an intermediate in the synthesis of sulfisoxazole, does not form the corresponding amidine by ethanolic ammonia but forms a free imido ester. Examination of this amidination reaction showed that the objective 2-acetylpropionamidine (II) can be obtained by warming the free 2-acetylpropionimidate (I) with ammonia water, in the presence of a small amount of ammonium chloride, for 1 hour at 30°. Extension of the period of heating to 4 hours results in the formation of 2-(1-acetylethyl)-4, 5-dimethyl-6-aminopyrimidine (III) and the heating for 4 hours at 60°, in the formation of 2-(1-methyl-2-iminopropyl)-4, 5-dimethyl-6-aminopyrimidine (IV).
Reaction of compounds (A) related to 2-acetylpropionitrile (III), an intermediate in the synthesis of sulfisoxazole, with semicarbazide hydrochloride and hydrazine sulfate was examined. Compounds tested were the ester (I), acid amide (II), nitrile (III), imido ester (IV), and amidine (V) of 2-acetylpropionic acid and following facts were revealed. In reaction of semicarbazide hydrochloride, (I), (II), and (III) formed corresponding semicarbazones (VI), (VII), and (VIII), in the presence of sodium acetate. These semicarbazones underwent cyclization by the action of acid or alkali to form 1-car-bamoylpyrazole derivatives (C). In the case of (IV) and (V), however, (B) is not obtained and (C) is formed directly. (C) derived from (I) or (II) liberates the carbamoyl group by heat to form a pyrazole derivative (E) but (E) is not obtained from (C) derived from (III), (IV), and (V). (I) and (II) react with hydrazine sulfate to form (E) via the intermediate (D), but (III), (IV), and (V) do not afford (D) and (E). Acetylsulfanilyl derivative (XV) of (V), however, undergo these reactions, same as (I) and (II), such as (A)→(B)→(C)→(D)→(E) and (A)→(E).
Conformation of the two stereoisomers, α and β, of 2, 3-dimethylpiperazine, obtained by the high-pressure, catalytic reaction of ethylenediamine and 2, 3-butanediol, was determined. Since the α-isomer was resolved into d- and l-optical isomers, this must have the trans (aa or ee) conformation, while irresoluble β-isomer must have the cis (ae or ea) conformation. This fact was proved by separate syntheses of cis-and trans-2, 3-dimethylpiperazine.
In order to find the substance present in the cardiac muscle that promotes contraction of cardiac muscles, a method for quantitative bioassay was devised using a special apparatus for perfusion of excised heart of a frog (Rana nigromaculata) and frog unit of effective substance was determined. The effective substance was extracted and purified by the method using acetic acid, ether, ethanol and phenol, and a sample with maximum purity of 600U./mg. was obtained, although this substance seemed to contain still some accompanying impurities. The effective substance would not be a homogeneous. The substance responsible for its action is not a protein, is of comparatively low molecular weight, and is extremely easily soluble in water, soluble in dilute ethanol and dilute acetone, but generally insoluble in organic solvents. It is comparatively stable to heat. The substance which accelerates contraction in excised heart of a frog was not the substance that depresses dog's blood pressure that was present in the crude preparation of the former.
In order to examine the chemical properties of callicrein, examinations were made on the method of purifying callicrein to obtain it in high purity. Callicrein is contained in hog pancreas and submaxillary gland in a large quantity. The acetone powder of hog pancreas was extracted with sodium hydrogen carbonate and the extract was purified through acetone, ethanol, activated charcoal, alumina, salting out with ammonium sulfate, and dialysis, and a sample of 200U/mg. was obtained. However, this substance was not homogeneous yet and seemed to contain two components by paper electrophoretic analysis. The callicrein obtained was inactivated by the serum of a dog. Bioassay of its potency was made by measuring depression of carotid arterial pressure in a dog. Kallikrein (Bayer) was used as the standard. Therefore 1 unit corresponds to 1 Frey unit.
Antibacterial action of 21 kinds of phenazine and phenazine N-oxide derivatives were examined together with the effect of copper on such action. Copper was found to fortify the antibacterial action in 1-hydroxy- and 1, 6-dihydroxyphenazine with hydroxyl radical in α-position but in N-oxide derivatives, such fortifying action of copper was not observed even with those having hydroxyl radical in α-position. As for effect of metals other than copper, a slight fortifying action was seen in zinc, but there was no effect in iron (II) and cobalt.
Antifungal action of 20 kinds of phenazine derivative against Trichophyton rubrum was examined and the effect of copper to it was tested. It was thereby found that the effect of copper is not exerted in this case, differing from the case with bacteria and that antifungal action is stronger in compounds with a methoxyl group.
Examinations were made for conditions in measuring the activity of alkaline phosphatase in snake venom. It was found that phosphodiesterase activity is suppressed by glycine while the activity of phosphomonoesterase increases in the presence of glycine. The activities of 5′-nucleotidase, phosphomonoesterase, and glycerophosphatase are suppressed by Zn2+. Since a fair amount of Zn2+ is contained in crude snake venom, absence of phosphomonoesterase and glycerophosphatase in snake venom has been reported to date. Measurement of activity by the improved method has shown that phosphodiesterase, phosphomonoesterase, 5′-nucleotidase, and glycerophosphatase are present together always in 10 kinds of domestic and Formosan snake venom examined. The amount of these enzymes differed somewhat according to the kind of snake venom and the activity of alkaline phosphatase is generally stronger in snake venom from cobras (Elapidae) than that from asps (Crotalidae). Both phosphomonoesterase and glycerophosphatase are non-specific monoesterases but optimal pH and temperature of the two are different and there is a difference between the two as to their stability to heat and pH.
Some improvements were effected in the method of Sinsheimer for separating phosphodiesterase from snake venom by acetone fractionation. Application of this improved procedure to snake venom from domestic asp (Agkistrodon halys blomhoffi BOIE) effected separation of phosphodiesterase by a comparatively easy procedure. However, this method cannot be termed suitable considering the increased specific activity of phosphodiesterase, recovery of the enzyme, and utilization of enzymes other than phosphodiesterase. A method was devised for obtaining phosphodiesterase of high purity in a good yield from asp venom by the use of column chromatography with cellulose-calcium phosphate gel as the adsorption agent. The sample of phosphodiesterase obtained by this method contains non-specific phosphomonoesterase but not 5′-nucleotidase that it can be used for structural studies of nucleic acid. Use of ammonium sulfate fractionation to the mixture of L-amino acid oxidase and lecithinase-A, separated from phosphodiesterase by this column chromatography, afforded L-amino acid oxidase free from lecithinase-A. The purity of L-amino acid oxidase so obtained was about 11 times that of the crude snake venom. The crude snake venom contains a substance that suppresses the action of L-amino acid oxidase and this substance is adsorbed on calcium phosphate gel.
The venom from Formosan cobra (Naja naja atra CANTOR), differing from that of domestic asp (Agkistrodon halys blomhoffi BOIE), is adsorbed easily on alumina-C γ-gel, calcium phosphate gel (Ca-P-gel), as well as carboxymethylcellulose (CM-cellulose), Amberlite IRC-50 (XE-64), and phosphate-cellulose, and any of the ion exchangers can be utilized for obtaining phosphodiesterase free from phosphomonoesterase from cobra venom. The sample of purified phosphodiesterase from the cobra venom does not contain 5′-nucleotidase or phosphomonoesterase, being approximately 25 times purer than the crude venom, and was obtained in approximately 10% yield. Column chromatography with CM-cellulose was applied to the separatino of alkaline phosphatase from cobra venom and stepwise elution analysis and gradient elution analysis, using acetate buffer solution as the eluate, were attempted. By these means, glycerophosphatase, phosphodiesterase, and 5′-nucleotidase were separated in good yields. It was found that the active portion of phosphodiesterase was separated into two by this column chromatography and the presence of two kinds of phosphodiesterase was evidenced by rechromatography.
Ethyl 2-methyl-6-benzothiazolecarboxylate was principally prepared by thiocyanation, reduction, and ring formation from thiazole with acetic anhydride in the presence of zinc dust from ethyl p-aminobenzoate. 2-Methyl-6-benzothiazolecarbo-hydrazide was readily synthesized by condensation of the above ethyl ester with hydrazine hydrate. The acid hydrazide obtained was condensed with ten organic acid chlorides. The ten new condensed products so obtained were submitted to bacteriological screening in order to find new antifungal and antitubercal agents. Ethyl 2-amino-6-benzothiazolecarboxylate from ethyl 3-thiocyano-4-aminobenzoate was prepared only by fusion under intramolecular condensation.
Vitamins A and D were solubilized in water by the use of various amounts of several non-ionic surface active agents, each solution was heated to 100°, and change in optical density of the solution at different temperatures during cooling was measured by the Coleman spectrophotometer to examine the state of clouding formation. Results obtained were as follows: 1) Turbidity of the solution differed in aqueous solution of the surfactant alone and that in which vitamin A or D had been solubilized, even if the surfactant and its concentration were the same. Turbidity was found to depend on the substance solubilized. 2) The polarity of the solubilized substance seemed to affect turbidity since it was the worst in vitamin A alcohol and vitamin D, followed by A acetate, and practically no change was observed with vitamin A palmitate. This agrees with the fact that there are few changes in non-polar cetane and severe change is seen in polar octyl alcohol. 3) As for the properties of the surfactant, the polymerization degree of ethylene oxide affects turbidity when lipophilic group is the same, the greater the polymerization degree, the less the turbidity. 4) Some of the surfactant was found to give double cloud point, irrespective of the substance being solubilized. 5) When clouding becomes too severe, there occurs separation into two layers or precipitation, which returns to the original state on cooling and mixing.
Some improvement was attempted in the Schütze method for determination of oxygen in organic compounds by oxidizing carbon monoxide with iodine pentoxide and measuring the weight of carbon dioxide thereby formed. A new type of filling tube, which can be heated from inside, was used for the oxidation, and a method of preparing iodine pentoxide to be placed in the lower layer of the tube and silver granules to be filled in the upper layer was examined. Conditions for determination was examined for burning the sample by heating the oxidation tube to 150° from inside, and the life of silver granules was also examined. The advantage or disadvantage of this method was compared with the generally practiced iodometry and heated silver method.
Dissociation vapor pressure of diphenylhydantoin sodium hydrates was measured at a temperature between 20° and 50° using a differential tensimeter and it was found that mono-, tetra-, hepta-, octa-, and hendecahydrates existed in diphenylhydantoin sodium. An empirical formula log P=A-B/T can be given to relationship between the dissociation vapor pressure and temperature and the values of constants A and B for each hydrate system were calculated. It was proved that the transition temperature of 37.6°, 45.2°, and 51.3°, presumed from the solubility curve and viscosity-temperature curve in the preceding paper, agreed respectively with the transition temperature of hendecahydrate-octahydrate, octahydrate-heptahydrate, and heptahydrate-tetrahydrate. Comparison of monthly average humidity in seven areas in Japan and dissociation vapor pressure of the hydrates indicated that the heptahydrate was the most stable in air in a country like Japan with high average humidity.
A method was established whereby coumarin compounds in crude drugs or other material could be determined colorimetrically by cleavage of the lactone ring in coumarins with alkali and coloration with the Emerson reagent. The present method can be applied with simple procedure to the determination of coumarin derivatives having no substituent in the position para to the phenolic hydroxyl formed by the cleave of lactone ring. Free umbelliferone contained in the gum-resins, asafetida, galbanum, and ammoniac, was separated by paper partition chromatography (Figs. 5 and 6), area with spots appearing on the papergram under ultraviolet ray was cut out, and extracted with ethanol. This ethanolic extract was submitted to colorimetric determination by the above method, using the Beckman Model DU spectrophotometer (Tables IV and VI). Free umbelliferone was detected in asafetida, in approximately the same amount as that in galbanum, although the presence of the free form had been denied. The presence of ferulic acid was detected only in asafetida.
Epinephrine was boiled in mineral acid and a new substance was formed by its decomposition. In the case of hydrochloric acid, crystals of m.p. 264-265° (decomp.), comparatively sparingly soluble in water, were obtained in approx. 30-40% yield. The yield was lower with other acids. This substance had a molecular weight of 344.5 and corresponded to formula C17H18O4NCI⋅1/2H2O. Treatment with acetic anhydride afforded a pentaacetyl compound (I) of m.p. 182-183°, C27H27O9N; I.R. γmaxNujol 6.06μ (-CO-N-). Treatment with dimethyl sulfate gave a pentamethyl compound (II), m.p. 263-264°, C24H33O8NS, which formed a methiodide (III), m.p. 284-285°, C23H30O4I on the application of aqueous solution of potassium iodide. Since the basification of the decomposition mother liquor afforded methylamine, this decomposition reaction may be represented by the following equation: 2 C9H13ON-CH3NH2-2 H2O=C17H17O4N A tentative designation of adnamine was given to this substance. Adnamine colors scarlet with alkali hydroxide while the crystals (IV) of m.p. 230-233° (decomp.), obtained by catalytic reduction of its hydrochloride by absorption of 1 mole of hydrogen, does not show any such coloration.
Methyladnamine methosulfate (II) was submitted to the Hofmann degradation and crystals (VI) of m.p. 230°, C20H20O4, were obtained. Its catalytic reduction afforded a dihydro compound (VII), m.p. 177°, C20H22O4, possessing four methoxyl groups. The gas evolved during the Hofmann degradation was proved to be trimethylamine by its derivation to chloroaurate. Ozonolysis of (VI) formed formaldehyde which was established as formaldimedone. The infrared absorption spectrum of (VI) exhibited a maximum (in Nujol) at 11.45μ (=C=CH2), which indicates that the amino is present as the terminal group in the side chain. Oxidation of (II) or (VI) with chromium trioxide in glacial acetic acid afforded 2, 3, 6, 7-tetramethoxyanthraquinone (VIII) which was also obtained on oxidation with alkaline potassium permanganate. Since the oxidation of (II) gave 4, 5, 4′, 5′-tetramethoxybenzophenone-2, 2′-dicarboxylic acid (IX), m.p. 245-260°, (II) must be 5-dimethylaminomethyl-2, 3, 6, 7-tetramethoxydibenzo [a, e]-cycloheptatriene methosulfate.
The dihydro compound (IV), abtained by catalytic reduction of adnamine hydrochloride, was methylated with dimethyl sulfate and sodium hydroxide, and the methosulfate (XIII), m.p. 258-260°, C24H35NS, so obtained was submitted to the Hofmann degradation, affording the des-N compound (XIV), m.p. 184-186°, C20H22O4. This was catalytically reduced to the dihydro compound (XV), m.p. 128-129°, C20H24O4. It was confirmed through admixture, and ultraviolet and infrared absorption spectra that (XIII), (XIV), and (XV) were respectively identical with 5-dimethylaminomethyl-2, 3, 7, 8-tetramethoxydibenzo [a, d] cyclohepta-1, 4-diene methosulfate, 5-methylene-2, 3, 7, 8-tetramethoxydibenzo [a, d] cyclohepta-1, 4-diene, and 5-methyl-2, 3, 7, 8-tetramethoxydibenzo [a, d] cyclohepta-1, 4-diene synthesized by Battersby and others. It was found by consideration of experimental results reported to date that adnamine is 5-methylaminomethyl-2, 3, 7, 8-tetrahydroxydibenzo [a, e] cycloheptatriene. The seven-membered ring in methyladnamine is hardly hydrogenated but the dihydro compound (VII) of its des-N compound forms (XV) in 20% yield on catalytic reduction over palladium charcoal, at 190° and 65 atm., together with recovery of 10% of (VIII).
The oxidation reaction reported in Parts IX and X of this series is a reaction in weak acidity of pH 4.2-4.5. In the present series of experiments, the effect of pH on the oxidation of aniline with periodic acid was examined in detail. 1) Reaction in strong acidity (pH 1.0): In the initial stage of the reaction, precipitate of emeraldine is formed but if the mixture is allowed to stand until consumption of IO4- becomes constant, then a black powder is obtained. This powder, as well as the substance obtained by its treatment with ammonia, forms a precipitate insoluble in 80% acetic acid and other common solvents. In this case, IO3- formed from IO4- also takes part in this reaction. 2) Reaction in strong alkalinity (pH 12.0): Reaction does not occur at all. 3) Reaction in weak alkalinity (pH 9.0-9.4): The reaction is very slow and it requires about 120 hours until consumption of IO4- becomes constant. The chief component of the oxidation products is dianilino-p-benzoquinone. In addition, bibliographically unknown two substances, reddish brown needles of m.p. 151° (decomp.) and yellow plates of m.p. 174°, were obtained as the oxidation products of aniline.
The metabolite of isonicotinoylhydrazide (INAH), first discovered by the present authors in 1953, is a Schiff base, isonicotinoylhydrazonopyruvic acid, and this was later found to have almost the same minimal inhibitive concentration against tubercle bacilli as that of INAH, while its toxicity was much weaker. This point was further endorsed by clinical tests and the compound has been commercialized. The present paper discusses the question of the lowering of toxicity, which formed the basis for the use of isonicotinoylhydrazonopyruvic acid. As shown in Tables III and IV, the acute toxicity LD50 in mice is 3533mg./kg. of the sodium salt by subcutaneous injection and 1780mg./kg. by oral administration. This is 1/29 of that of INAH by parenteral route and 1/6.4 by the oral route. In connection with this fact, it has experimentally been proved that INAH and pyruvic acid are antagonistic in the point of toxicity. It is interesting that the toxicity of the Schiff base is far weaker than that of the concurrent use of INAH and pyruvic acid.
In an earlier work of this series, numerous arylthio derivatives of 3-amino-6-chloro (methoxy, ethoxy) pyridine-2-thiol and 3-aminopyridine-4-thiol were synthesized and conditions for Smiles rearrangement to take place were examined. As a result, it was observed that these compounds were more liable to undergo rearrangement than corresponding benzene derivatives and that the rearrangement took place most easily in 3-amino-6-chloropyridine-2-thiol derivaties. This was explained as the promotion of the rearrangement by the additive effect of +I effect of the chlorine atom and +E effect of the ring nitrogen. In the present series of work experimental examinations were made on the promotion of the rearrangement by a halogen atom by preparing 3-amino-5-chloropyridine-2-thiol and 3-amino-5-bromopyridine-4-thiol and submitting them to Smiles rearrangement. As anticipated, halogen atom was found to effect promotion of the rearrangement irrespective of the position of halogen relative to the amino group or sulfur atom.
Passage of ketene through the solution of 2-picoline 1-oxide in acetone or chloroform, in the presence of sulfuric acid as a catalyst, afforded ω-acetoxy-2-picoline. This is thought to be similar to the reaction of 2-picoline 1-oxid with acetic anhydride but the kind of the solvent used and the presence or absence of a catalyst greatly affected the yield. The maximum yield obtained never exceeded 50%.
As a simple method for measuring the insulin content in solution, paper chromatography was adopted. In the case of a solution containing a large amount of impurities, such as the pancreatic extract, it is submitted to pretreatment of adjusting the pH so as to precipitate and remove the large amount of occluded proteins, desalted by dialysis with fish skin or cellophane membrane, concentrated under a reduced pressure by rotary evaporator, and a definite amount of the concentrated solution is submitted to paper partition chromatography. It is streaked at one end of a filter paper (40×40cm.), developed with a solvent system of acetic acid: butanol: water (1:6:7), and the insulin band separated on the papergram is colored by 0.02% solution of bromocresol green. The intensity of the color thereby developed is estimated by the measurement of the colored eluate of the insulin band by photoelectric colorimeter and insulin unit is calculated from the straight linear relationship between the concentration of the pigment and units of insulin. This method was applied to the extract solution obtained in a plant using whale pancreas as the starting material and the content of insulin was found to be 1000-1500 I.U./kg. in fin whale and 2300 I.U./kg. in sperm whale.
Local anesthetics of alkoxynaphthylamine derivatives, having a dialkylaminoacyl group, had been synthesized from 2-naphthol. In the present series of work, 1-alkoxy-4-dialkylaminoacylamidonaphthalene hydrochlorides were synthesized starting with 2-naphthol. 2-Naphthol was nitrated through the alkoxynaphthalenes (alkyl=methyl, ethyl, butyl, isopentyl), reduced to 1-alkoxy-4-aminonaphthalene, and reacted with chloroacetyl chloride, 2-bromopropionyl bromide, or 2-bromobutyryl bromide, in the presence of potassium acetate, in glacial acetic acid, to form 1-alkoxy-4-haloacylamidonaphthalenes. These were reacted with dimethyl- or diethylamine in benzene, by heating in a sealed tube to 100°, and 1-alkoxy-4-dialkylaminoacylamidonaphthalenes were obtained. Passage of dry hydrogen chloride gas through their ether solution afforded the objective compounds, which numbered 24 in all.
Following previous work on alkoxynaphthylamine derivatives possessing dialkylaminoacyl radical, some derivatives possessing dialkylaminoethyl radical were prepared. Alkoxynaphthylamine (methyl, ethyl, butyl, isopentyl) was condensed with dialkylaminoethyl chloride, in toluene with sodium amide as the condensation agent, and 1-dialkylaminoethylamino-2-alkoxy- and 4-dialkylaminoethylamino-1-alkoxynaphthalenes were prepared.
Application of diazo titration to potentiometry was attempted and it was found that it is possible as long as optimal conditions were chosen for titration, such as the use of sodium nitrite sodium nitrite solution of comparatively high concentration and titration carried out at a low temperature. Satisfactory results were obtained by the application of this method to diazo titration of sulfanilamide and its derivatives. Theoretical evidence of titration was supplied by potentiometric polarographic procedures.
In order to study the chloropromazine-like action, we synthesized 2-imino-3-(2-dimethylaminoethyl) benzothiazolines and 2-imino-3-(3-dimethylaminopropyl) benzothiazolines by the condensation of 2-aminobenzothiazoles and 2-(dimethylamino) ethyl-, 3-(dimethylamino) propyl chloride hydrochloride. In comparison to them, we synthesized also 2-(2-dimethylaminoethyl)-, 2-(3-dimethylaminopropyl)-aminobenzothiazole by the condensation of 2-(dimethylamino) ethyl-, 3-(dimethylaminopropyl)-amine and 2-bromobenzothiazoles which were obtained from 2-aminobenzothiazoles by Craig's reaction.