Various substituent groups were introduced into anisole and acetanilide and their reactivity was compared by reacting with chloral in conc. sulfuric acid. The order of reactivity was OCH3>CH3>Cl>NHCOCH3>COOH>NO2 (in H2SO4). It is assumed that the reason why the acetamido group is placed lower than chlorine is because its polarization to Ar-NH+=C(CH3)-O- increases in conc. sulfuric acid.
Polarographic behavior of bismuth subgallate (Dermatol) and its allied substances was examined in detail and its result supports the structure proposed by one (Nagase) of the authors earlier. The observation of these polarographic waves will not only make their determination possible but also offer some help in their identification and purity tests. According to these experimental basis, microdeter-mination of bismuth was carried out by coulometric titration, using gallic acid.
By adsorption chromatography using exsiccated aluminum hydroxide gel, components of mercurochrome were each isolated and their polarographic waves were examined. The conclusions reached agreed with results obtained in past experiments. Each of the fractions adsorbed is dissolved in aqueous sodium hydroxide solution, which contains a large amount of A1O2- that will be useful as a supporting electrolyte, and this can directly be used as a polarographic electrolytic solution. This solution may be acidified and the aluminum hydroxide that precipitates out can be dissolved again to obtain the objective substance. This process offers a suggestion for the separatory purification and polarography of a complicated mixture.
It had been shown in the preceding paper, that hydrogen peroxide is an effective additive in polarization titration, in connection with neutralization titration with platinum electrodes. Examination of various additives in the determination of halogen ions by silver titration also indicates hydrogen peroxide to be effective. On the other hand, an additive is unnecessary when using a minute silver electrode. Silver titration of halogen ion with the use of platinum or silver electrode was carried out by the dead-stop and derivative polarographic titrations. Potentio-metric polarography was carried out to make some theoretical considerations on titration.
When the venom of Formosan cobra (Naja naja atra) is purified by acetone and ammonium sulfate, a strong poisonous component, that gives one spot moving towards the cathode side in paper electrophoresis, is obtained. It has almost no lecithinase action and its minimum lethal dose in mice is 0.08-0.1γ/g., causing respiratory paralysis. As tested by Sanger's DNP method and Akabori's hydrazinolysis method, its N-terminal amino acid was determined as leucine and its C-terminal amino acid as glycine. It was found from the formation of bis-dinitro-phenyl-aspartic and -glutamic acid α-monohydrazides during hydrazinolysis that the peptide chain contains aspartic acid with a free carboxyl group in the β-position and glutamic acid with free carboxyl group in the γ-position. The result of determination of these amino acids is given in Table I. Since these amino acids are present in one molecule each, the peptide composing this strong poisonous component is not branched and one mole each of aspartic and glutamic acids containing free carboxyl group are present in the peptide chain. From the content of these amino acids, the minimum molecular weight of this peptide is assumed to be around 6000. Cystine is not present as a terminal amino acid and it is clear that there is no cystine with a free carboxyl group in the peptide chain since there was no cystine derivatives in the sodium hydrogen carbonate layer during decomposition by Akabori's hydrazinolysis.
Poisonous component of Formosan cobra (Naja naja atra) was purified by acetone and ammonium sulfate to obtain a neurotoxin and its hydrolyzate with hydrochloric acid was submitted to two-dimensional chromatography and paper electrophoresis, from which 13 kinds of amino acid were detected. Since neurotoxin is positive to the Adamkiewitz reaction, it is known to contain tryptophan. Results of these determinations of amino acid are listed in Table I. As will be clear from this Table, neurotoxin contains a large amount of threonine, glycine, aspartic acid, glutamic acid, and arginine. It has been shown in the preceding paper that neurotoxin contains leucine as the N-terminal amino acid and glyine as the C-terminal amino acid in an unbranched polypeptide chain. From the quantitative determination of amino acids, this peptide is thought to be composed of 50 amino acid residues and its minimum molecular weight would be around 6000, while the presence of cystine, either as the terminal amino acid or in the peptide chain with free carboxyl, is denied. From these results, structural formulae presented in Figs. 1 and 2 are proposed for neurotoxin. The fact that neurotoxin can pass through ordinary cellophane membrane suggests that its true molecular weight would not be so large and the lysozyme-type structure with molecular weight of about 6000, as given in Fig. 1, seems to be the most appropriate.
The three isomers of bromomandelonitrile, by reaction with methylmagnesium iodide, form corresponding bromophenylacetylcarbinol (I) as well as bromophenyl-methylcarbinol (II). Of these, report has already been made on the para-isomer. In the present series of experiments, the ortho-isomer of (I), b.p5 127-131° (semicarbazone, m. p. 181°), and of (II), b.p5 115-120° (phenylurethan, m. p. 78-79°), were obtained in 36.3% and 15.8% yield, and the meta-isomer of (I), b.p6 143-145° (semicarbazone, m. p. 194.5°), and of (II), b.p6 125-131°, were obtained in 31.5% and 20.8% yield respectively. As a result of comparing the reaction of mandelonitrile and methoxymandelonitrile with methylmagnesium iodide, it is seen that the bromine in the aromatic ring of the cyanohydrin, used as the starting material, affects this kind of reactions favorably for the fomation of (I) and unfavorably for the formation of (II). Approximately similar results were obtained in the reaction of furfural- and 2-thiophenaldehyde cyanohydrin with methylmagnesium iodide.
Mandelonitrile (A) or mandelimidic ester (e.g. ethyl mandelimidate) (B) reacts with methylmagnesium iodide to form phenylacetylcarbinol (I) and phenylmethylcarbinol (II). It has already been shown that the reaction of mandelamide (C) and methylmagnesium idodide afforded (I) but it was later clarified that (II) is also formed as a by-product in this synthesis. However, mandelic acid esters (e.g. ethyl mandelate) (D) reacts with methylmagnesium iodide and affords only 1-phenyl-2-methyl-1, 2-propanediol (III) and not (II).
The reaction of methylmagnesium iodide with cyclopentanone and cyclohexanone cyanohydrins respectively give 1-methylcyclopentanol, b.p100 80-85° (phenylurethan, m. p. 91°), in 70% yield and 1-methylcyclohexanol, b.p21 60-64° (phenylurethan, m. p. 105°), in 71.6% yield, but there is no formation of the corresponding 1-acetyl-cycloalkanol. On the contrary, reaction of the cyanohydrins and phenylmagnesium bromide affords 1-benzoylcyclopentanol, b.p7 140-145° (semicarbazone, m. p. 195°), in 53.6% yield and 1-benzoylcyclohexanol, b.p7 150-154° (oxime, m. p. 121-121.5°), in 32.7% yield. In general, the reaction of the Grignard reagents with aliphatic ketonecyanohydrins progresses towards the formation of tert-carbinols but in the reaction of acetone cyanohydrin with phenylmagnesium bromide, 2-benzoyl-2-propanol, b.p7 110-113° (semicarbazone, m. p. 185°), was obtained in a good yield of 56.7%, similar to the case of alicyclic compounds.
In order to compare with 2-arylthio-3-amino-6-chloropyridine derivatives prepared earlier*, derivatives of 2-arylthio-3-amino-6-methoxy (or ethoxy) pyridine, and 3-amino-4-arylthiopyridine with the chlorine in the earlier compounds substituted with methoxyl or ethoxyl groups, were submitted to the Smiles rearrangement. It was found that free amine resisted rearrangement and that a nucleophilic substitution with CH3O-, formed from methanolic potassium hydroxide used as the rearrangement catalyst, occurred more preferably than the rearrangement. This result indicates the great effect of chlorine in 6-positions in the earlier chlorine derivatives, which underwent facile rearrangement even with free amino group. However, it was found that the acetyl derivatives easily underwent rearrangement even in the cold, as in the case of chlorine derivatives. With reference to 2-aminoarylthiobenzene derivatives, which hardly undergo rearrangement in the cold, the present results indicate that pyridine derivatives possessing amino in 3-position and mercapto group in 2- or 4-position are generally liable to this arrangement. Two kinds of benzopyridothiazine were prepared by the use of this Smiles rearrangement.
Detection of 2, 5-dianilino-N, N′-diphenyl-p-benzoquinone diimine (azophenine) (V) and 2, 5-dianilino-p-benzoquinone (VI) by paper chromatography was examined and good results were obtained with (V) but it was not possible to obtain a suitable Rf value or characteristic coloration reagent for (VI). This result was utilized in detecting the presence of (V) in the periodic acid oxidation product of aniline and the presence of (VI) was assumed from its sublimability. It was also found that a minute amount of azobenzene was present. The products from periodic acid oxidation of aniline were systematically separated and it was proved that the chief components are 2, 5-dianilino-p-benzoquinone imine (IV) in 65% yield and 2-amino-5-anilino-N-phenyl-p-benzoquinone imine (I) in 15% yield. An unknown substances (VII), m. p. 203-205° (decomp.) (in 9% yield), and (VIII), m. p. over 360° (in 6% yield), were also isolated. Content of other pigments was all very small.
The Maadonald's hot-plate method, as the assay for analgesics, was improved by the addition of a special devise to induce jumping and it became possible to assay analgesics quantitatively. In the usual hot-plate method, reaction of the animals (mice) varied too greatly and the reaction threshold value tended to be erroneous due to the difficulty in observing abnormal movement of the hind legs (bending of hind legs, licking of sole). Therefore, this original method was difficult in assaying potency of drugs with weak analgesic action. By the present method, the judgement is easy, measurement error is minimized, and it became possible to assay the potency of weak analgesics quantitatively.
Following previous experiments on the cholinesterase inhibition by trimethylamine, action of various bacteria to the addition of trimethylamine N-oxide in the medium was examined. Trimethylamine N-oxide was added to bouillon medium in a ratio of 500mg% and various bacteria were cultured in such medium for 48 hours at 37°. The most powerful reducibility was exhibited by Escherichia coli, followed by Arizona, Ballerupp, and Alkalescens. The inhibition of cholinesterase activity, when trimethylamine N-oxide was added to the medium, was greater with bacteria having strong power to reduce trimethylamine N-oxide, smaller in bacteria with weaker reducibility. When trimethylamine N-oxide was not added to the medium, inhibition of cholinesterase activity was independent of the reducibility of the bacteria and determined by the bacterial species. Relationship between the amount of trimethylamine N-oxide added, the amount of trimethylamine produced, and degree of inhibition of cholinesterase activity was examined with Escherichia coli.
Escherichia coli and Achoromobacter iophagum were incubated in a medium of a homogenate of 12 kinds of fish, crustaceae, and gastropods native to the Hokkaido. The pH of the culture filtrate, the amount of volatile basic nitrogen, trimethylaminenitrogen, and degree of cholinesterase inhibition were examined, as in the case of using squid as the medium. Periodical variation of measured values was approximately the same as in the case of squid, while the trimethylamine-nitrogen and degree of cholinesterase inhibition. in the case of E. coli, were the greatest with squid, followed in the order of mackerel-pike, crab, octopus, shrimp, “Kinki”, and cod. With Ach. iophagum, the order was squid, shrimp, “Kinki”, horse-mackerel, Atka-mackerel, and crab. In general, gastropods and crustacea showed stronger inhibition of cholinesterase with microorganisms.
Substances that inhibit enzymes, which have SH as the active group, were examined systematically in order to aid the studies on chemotherapeutics, and inhibitory action of various metal ions, metalloid ions, and organic compounds containing such metal elements against Papain was compared. The substances tested were mercury and tin as the metal, arsenic as the metalloid as their salts, phenylmercuric acetate, ethylmercuric phosphate, diethyltin sulfate, and ethylarsinic acid, as the organometallic compounds. It was thereby found that there was a threshold of inhibition by mercury compounds, at 2×10-5M of mercuric chloride and ethylmercuric phosphate, and at 4×10-5M of phenylmercuric acetate. There was almost no effect on enzyme activity by the addition of arsenic oxide or ethylarsinic acid, while diethyltin sulfate causes a weak inhibition. In the case of stannous chloride and stannous sulfate, on the contrary, some activation of enzyme activity was observed.
Process for the manufacture of p-nitro-ω-bromoacetophenone, especially p-nitroacetophenone, was studied as the material for the synthesis of chioramphenicol and an extremely easy and economical industrial process was established. Some observations are described on the relationship between conditions and yield of nitration of ethylbenzene, synthesis of p-nitroacetophenone by the liquid-phase, catalytic oxidation of 1-ethyl-4-nitrobenzene with oxygen at normal pressure, with various catalysts, and its catalyst poisons.
Some data are given for the process of industrial manufacture of o-nitrobenzoic acid, 2, 4-dinitro-6-ethylphenol, 4-ethyl-1, 3-phenylene diisocyanate, and indole, comparatively easily and in a good yield, with the purpose of utilizing the by-product, 1-ethyl-2-nitrobenzene.
Preparation of 2, 3-pyrazinedicarboxylic acid by electrolytic oxidation of quinoxaline, in the presence of potassium permanganate was studied, using a diaphragm. The most suitable electrode material, optimal concentration of the alkali, amount of permanganate added, electric density, electrolytic temperature, manner of quinoxaline addition, and amount of electricity were determined.
Absorption spectra in the visible region of 39 kinds of Phenylpyrazolone-azomethine derivatives were measured in methanol and quantitative explanation of the effect of changes in the structure of pyrazolone-azomethine molecule on the visible absorptions was discussed, based on the theory of Porter and Weissberger. Further considerations were made on the effect steric configuration of these molecules on the absorption, from van der Waals radii, bond angle, and bond distance. The position of the x-band due to change of substituent in the 3-position in the pyrazolone ring was in the order of NH2>RO-C6H4-NH>C6H5⋅NH>RSO2NH or RCONH>C6H5, and the absorption maximum of the x-band appeared in the shortest wave length region in the case of the amino group. There was no systematic change in the position of y-bands. When sulfonamide radical is further introduced into the phenyl group in the 1-position of the pyrazolone ring, the x-band shifts very slightly to a longer wave length side. It was assumed that steric configurations like those indicated in (VIa), (VIIa), (VIIIa), and (IXa) seemed to be the closest to the actual state of the molecule.
Attempts were made to indicate the color of 39 kinds of phenylpyrazolone-azomethine derivatives by the color chart of International Committee on Illumination, a standard method for the indication of colors, by measuring spectral transmittance. Calculation of trichromatic coefficient was made by the weight ordinated method, using the standard light source C, of the International Committee on Illumination, and considerations were made on the relationship between the dominant wave length obtained and chemical structure of the methine molecule. The effect of various substituents on the dominant wave length agreed with results reported previously, and the relationship between purity and brightness was found to be as shown in Fig. 4.
Emulsion tests were carried out on 34 kinds of compounds and their properties as a stabilizer was examined from their effect on photographic properties at the time of addition to the emulsion and from their inhibition of chemical ripening. Results so obtained are presented in Table III, and effect of these compounds on the inhibition of chemical ripening is compared in Table IV. From these data, relationship between chemical structure and photographic action was examined. Of the three compounds obtained by the application of ethyl iodide to silver salt of 5-hydroxy-7-methyl-1, 2, 4-triazolo[4, 3-a]pyrimidine, the two that were not ethoxyl compound was found to be the N-ethyl compound from their infrared absorption spectra.
Silver potential titration was carried out on compounds used for emulsion tests and relationship between potential and photographic properties was examined. In general, the loss noble the potential, the stronger became stabilization action, but too base a potential was found to be accompanied with side effects, such as desensitization on addition, and good stabilizers were found to have medium potential. If the reaction product of the compound and silver ion was to be taken as a sparingly soluble silver salt, it was concluded that good stabilizers would not form a silver salt in photographic emulsion in the amount added.
Decomposition of pyrazinamide in the digestive tract and the liver was examined and it was found that although this compound is stable in artificial gastric and enteric solutions, a part of it was decomposed to pyrazinoic acid by the enzyme solution of rabbit enteric membrane, acetone-dried powder of bovine enteric membrane, and homogenate of a rabbit liver. Perfusion experiments were conducted with excised liver of a rabbit and the concentration of pyrazinamide and pyrazinoic acid in the perfusion solution was determined at definite intervals by colorimetry. It was thereby found that a fair amount of pyrazinamide (ca. 100γ/cc.) was decomposed 5 minutes after the start of perfusion and that it changed almost completely into pyrazinoic acid by two hours of perfusion.
Decomposition of pyrazinamide by enteric bacteria was examined and it was found that while this compound is easily decomposed to pyrazinoic acid by Escherichia coli communior and Streptococcus faecalis R, it is not decomposed by Lactobacillus acidophilus. Further examinations were made on the relationship between the rate of decomposition and pH, and on velocity of decomposition and it was found that decomposition by E. coli is accelerated the most at around pH 5.0 but to a fair extent in neutral to slightly alkaline range and that the decomposition is almost complete in 5 hours at pH 5-9 range when 10mg./cc. of dried bacteria is used for 300γ/cc. of pyrazinamide. The decomposition by S. faecalis is accelerated the most at around pH 7.0 and 300γ/cc. of pyrazinamide is decomposed almost completely with 10mg./cc. of dried bacteria in 7 hours.
Nitration of methyl deisopropylallodehydroabietate with acetic anhydride and nitric acid afforded a mixture of mononitro compounds and from its catalytic reduction product, 6-, 7-, and 8-aminodeisopropylallodehydroabietic acids were isolated as their methyl esters.
2-Ethoxycarbonyl-3, 3-diphenylallylamines were synthesized. These compounds fall under the common law for analgesics in general, planned with characteristics of methadone and pethidine hydrochloride. Pharmacological tests revealed the absence of the anticipated strong analgesic and antitussive actions.
1-Ethoxycarbonyl-3, 3-diphenylallylamines, possessing the characteristics of methadone, dithienylaminobutene, and the compound recently adopted by U.S. Pat., with a tertiary amine in the α-position of fatty acid, were prepared and submitted to pharmacological tests but none of them showed any noticeable analgesic or antitussive action.
Narcotics of dialkylaminodithienylbutene series were oxidatively decomposed with 30% hydrogen peroxide and 2-thienyl ketone was obtained as its neutral portion. This ketone was derived to 2, 4-dinitrophenylhydrazone and its determination was carried out by paper chromatography or by visible light spectrum. The dialkylamine obtained from the volatile basic portion was identified by paper chromatography. This method will be possible for determination of narcotics of dialkylaminodithienylbutene and discrimination of dimethylamino, methylethylamino, and diethylamino homologs.