1) Change of activity in 0.05% aqueous solution of atropine sulfate with passage of time was examined by the anticholinergic action in excised intestine of guinea pigs and by mydriatic action in mice. 2) It was found with tests with excised intestine of guinea pigs that about 30% decrease in activity occurred on heating the solution at 80° for 1hour. The activity also decreased rapidly in the first few days when heated and unheated solutions were kept at 20°, but both solutions showed a definite value after 10 days, and no further change were seen to have occurred after 60 days of storage. The activity of the heated preparation after this lapse of time decreased to 30-40%, and that of unheated preparation to 10-15%. 3) According to tests of mydriatic action in mice, the efficacy of the solution stored for 66 days was no different from that of the fresh solution.
In order to understand the reaction mechanism whereby a solution of dehydroascorbic acid shows fluorescence upon the addition of o-phenylenediamine solution, 2-(l-glycerodihydroxyethyl)-3-ketofuro [2, 3-b] quinoxaline (I), 7-(l-glycerodihydroxyethyl)-13, 14-furo [2, 3-b; 3, 4-b′] diquinoxaline (II) and 3-(l-glycerodihydroxyethyl)-1-ketofuro [3, 4-b]-quinoxaline (III) were prepared and the nature of the fluorescence shown by the aqueous solutions of these compounds, Rf values of the fluorescent substance, ultraviolet absorption spectra, and the properties of respective phenylhydrazones were examined. It was found from these experimental results that the only one that gave completely identical properties as the reaction mixture was that from (I).
Mixing of the solutions of dehydroascorbic acid and o-phenylenediamine at room temperature results in their condensation and appearance of fluorescence. Fundamental experiments were carried out in order to utilize this reaction for the fluorometric determination of total vitamin C, which is the sum of ascorbic and dehydroascorbic acids. A solution of total vitamin C in HPO3 or HPO3-AcOH was adjusted to pH 4.5 with alkali, o-phenylenediamine solution added, and allowed to stand for a definite length of time at a definite temperature. The resultant fluorescence was measured and the amount of total vitamin C determined by comparison with the degree of fluorescence of standard ascorbic acid. In order to eliminate error by blind fluorescence, alkaline treatment and elimination with an adsorbent were devised. Of the substances showing similar reaction, error by reductone could not be completely eliminated but the amount of interference in the presence of reductone in equal quantity as total vitamin C was only 7%.
Fluorometric determination of total vitamin C, based on the condensation reaction of dehydroascorbic acid (DAA) and o-phenylenediamine, was utilized in examining the stability of the aqueous solution of DAA, and it was confirmed that DAA was labile in an acid solution that stabilizes ascorbic acid. Its decomposition velocity constant and temperature coefficient were examined. Fluorometric determination and Roe's dinitro-phenylhydrazine method were compared with 19 samples of fresh plant tissues not containing DGA and showed that both gave identical results.
Protease was extracted with water from hog pancreas, activated with trypsin, and the extract was used for the enzymatic hydrolysis of casein. The hydrolysis rate (α-amino-N/total-N, %) was determined with samples taken out at definite intervals, as well as free threonine and total threonine by the Sinn-Nicolet method, by which the rate of threonine liberation and decomposition during enzymatic hydrolysis were examined. The results are shown in Table I, and the relationship between the rate of threonine liberation and hydrolysis rate is shown in Fig. 1. In the determination of total threonine, hydrolysis with 20% hydrochloric acid results in an easier decomposition of threonine in peptide bond than free threonine. It is assumed that the hydrolysis by heating with 20% hydrochloric acid for 24 hours results in over 11% of threonine in casein being decomposed.
Protease was extracted with water from hog pancreas and activated by standing the solution at 0°C for 72 hours. This extract was used for the enzymatic hydrolysis of casein, and the decomposition rate (α-amino-N/total-N, %) was determined with samples taken at definite intervals. At the same time, free and total methionine were determined by T. F. Lavine's method, free tryptophane by Shimada's method, and total tryptophane with p-dimethylaminobenzaldehyde, to examine the rate of methionine and tryptophane liberation and decomposition during the course of enzymatic hydrolysis. The results are shown in Table I, and the relationship between the rates of methionine and tryptophane liberation is shown in Fig. 1.
Some new compounds, four kinds of 7-hydroxycoumarin and two kinds of 4-methyl-umbelliferon derivatives, were prepared. These and 13 kinds of esculetin and umbelliferon derivatives were used in testing anthelmintic action in vitro, using hog ascaris. Umbel-liferon derivatives gave lower melting points with increase of carbon atoms and in that order, increased anthelmintic action. Esculetin derivatives generally showed weak action while the introduction of a methyl radical in the 4-position of umbelliferon butyl ether resulted in the decrease of anthelmintic action.
α-Amino acids possessing a thiazole nucleus, i. e. 4-phenyl-2-thiazole-alanine and 4-p-nitrophenylthiazole-2-alanine, were prepared in order to observe the expected antagonism against histidine and phenylalanine.
With butylidene phthalide, principal aromatic component of Ligusticum acutilobum Sieb. et Zucc., as the chief compound, o-ketocarboxylic acids were obtained by using alkyl cadmium for the syntheses of various alkylidene phthalides given in the chart, which were obtained in good yields by heating the o-ketocarboxylic acids with 50-70% sulfuric acid. Comparison of the aroma of these compounds showed that the aroma tended to become stronger with the increase of carbon atoms in the alkyl group, the aroma of compounds containing propyl and butyl being most similar to that of the plant. These were assumed to be utilizable as perfumes. Substitution of the alkyl group with phenyl or tolyl resulted in the change of the aroma to an unpleasant odor, although the reduction of the pheyl nucleus in the phthalide resulted in an aroma similar to that of Ligusticum acutilobum.
Heating 3-(2-methyl-4-amino-5-pyrimidylmethyl)-4-methyl-5-(2-hydroxyethyl)-2-thiothiazolone (I) with Raney nickel in alcoholic solution results in primary formation of vitamin B1 which undergoes further decomposition and the final decomposition products are varied, including 2-methyl-4-aminomethylpyrimidine. The same reaction carried out with the hydrochloride and acetate of (I) gives, in a short period of time, pure vitamin B1 hydrochloride. O-Acetyl and O, N, N-triacetyl derivatives of (I) also yield acetyl derivatives of vitamin B1 by treatment with Raney nickel. The same treatment of 2-mercapto-4-methyl-5-(2-hydroxyethyl)-thiazole yield 4-methyl-5-(2-hydroxyethyl)-thiazole.
Oxidation of o-tolylcarbinol, prepared in the usual manner, with aqueous potassium persulfate solution gave o-tolualdehyde in 50% yield, although this compound had been considered very difficult to prepare.
Antifungal action of 13 kinds of essential oils were further tested. Antifungal properties were found to be in the order of sandal>vetiver>rose, nerol, citronella, geranium, bay>patchouli, jasmin, petitgrain, anise>Hang-Hang<lemon, sandal being effective in 1:64, 000-1:128, 000 dilutions. The essential oils were somewhat effective against Shigella dysenteriae Shiga, but almost ineffective against Staphylococcus aureus 209P and Escherichia coli, although sandalwood oil was effective against Staph. aureus at 1:64, 000 dilution. The oils were also effective against avian type tubercle bacilli in which the sandalwood oil also showed the highest efficacy, being effective in 1:32, 000 dilution, followed by citronella oil at 1:16, 000 dilution.
Reinvestigation was carried out on the constitution of camellia-saponin, the principal acidic saponin from the fruit of Camellia japonica L. Alkaline hydrolysis of the saponin, m.p. 206.5° (corr.), converted it into prosapogenin I, with liberation of one mole of tiglic acid and probably one mole of glucose. Acid hydrolysis of prosapogenin I gave a basal genin, camellia-sapogenol, m.p. 290-292.5%deg; (corr.), [α]D24: +22.0 (EtOH), and three sugar components, arabinose, galactose, and an uronic acid (most likely, glucuronic acid). Glucose was detected, in addition to the above three sugars, by the first acid hydrolysis, the identification of sugars having been made by means of paper chromatography as well as by their osazone derivatives. The sapogenol hereby obtained is neutral, colors yellow with tetranitromethane, and corresponds to the formula C30H50O4.
Sedative and hypnotic actions, and toxicity against mice were tested with 20 kinds of coumarin derivatives synthesized and three kinds of market products. Efficacy and toxicity of each preparation were compared by the values of lethal dose, LD60, hypnotic dose, HD60, and sedative dose, SD60, calculated by the Van der Waerden's Flächenmethode and by the Behrens-Kärber method (cf. Tables and Figs.). Results obtained can be summarized as follows: 1) Efficacy is lost when -CH2- is introduced between the coumarin ring and CO-N(C2H5)2 of coumarin-3-carboxylic acid diethylamide to coumarin-3-acetic acid diethylamide. 2) Exchange of -CO- in coumarin-3-carboxylic acid diethylamide with -O- to isocou-marincarboxylic acid diethylamide did not affect the efficacy to any great extent, but its methyl ester lacked any efficacy. 3) Introduction of -COOH, -COOCH3, or -COOCH2H5, in the 5-position of α-pyrone ring, in place of the benzene ring, in coumarin nucleus, did not add any efficacy. 4) In any case, free carboxylic acid possessed no efficacy. 5) Coumarin itself did not possess any efficacy but 2-thiocoumarin was effective. 6) Hydrogenation of the double bond between 3- and 4-positions decreased the efficacy, with the exception of coumarin-3-acetic acid diethylamide. 7) Efficacy was not shown by the introduction of -NO2 and -NH2 in the 6-position, of isoamyl in the 6-position, and -OH in the 7-position or of -COCH3 in the 3-position of the coumarin ring. 8) The sodium salt of barbital used as the control was more weaker in both efficacy and toxicity than the free base.
Methionine amide acetate was prepared from DL-methionine methyl ester hydrochloride by three processes. Enzymatic hydrolysis was then carried out using Protamylase, a pancreatic preparation, or Takadiastase, from which 61-67% of L-methionine and 56% of D-methionine amide acetate were obtained. Treatment of the latter with hydrobromic acid yielded 77% of D-methionine.
By the combination of equimolar quantities of DL-methionine amide acetate (I) and D-tartaric acid (II) in methanol, the former was resolved into L-methionine amide neutral tartrate and D-methionine amide acidic tartrate, and each tartrate was derived to respective optically active methionine amide acetate and methionine. Combination of (I) and (II) in 2:1 molar ratio in methanol resulted in the separation of L-methionine amide neutral tartrate and D-methionine amide neutral tartrate.
By combining N-p-nitrobenzoyl-threo-β-DL-phenylserine (I) and optically active ephedrine in methanol, (I) was resolved into optically active compound from which optically active phenylserine was derived.
It was clarified in the present experiments that the esterification of N-acylphenylserine resulted in the acyl migration from nitrogen to oxygen. Further acylation was carried out on O-acylphenylserine ester hydrochloride and N-acylphenylserine ester thereby obtained. It was also shown that heating of phenylserine ester hydrochloride with acetic anhydride resulted in the occurrence of catalytic acylation to N, O-diacetylphenylserine methyl ester (XVIII) whose nitration gave a p-nitro compound. Hydrolysis of the nitro compound gave p-nitrophenylserine whose reduction with lithium aluminum hydride yielded 1-p-nitrophenyl-2-acetamino-1, 3-propanediol (XXI), an intermediate in chloramphenicol synthesis.
D-threo-β-Phenylserine was derived to 1-p-nitrophenyl-2-amino-1, 3-propanediol (I) which was found to be an optical antipode of (I) obtained by the hydrolysis of natural chloramphenicol. This confirms the fact that the steric configuration of natural chloramphenicol is the same as that of l-nor-φ-ephedrine.
In view of the clinical results which showed that the crude 6-chloro-4-octylresorcinol possessed a stronger anthelmintic action than the purified product, examinations were made as to a component contained in crude chloroöctylresorcinol. It was found during this examination that alkylresorcinol and chloroalkylresorcinol homologs could separately be identified by the paper chromatography by using a mixture of water and methanol, ethanol, acetone, or other organic solvents miscible with water as a developing agent. Such tests with effective preparation showed the presence of octylresorcinol and assumption was also forwarded that a minute amount of resorcinol was sometimes present. Further tests with column partition chromatography enabled the isolation of pure octylresorcinol. These results have confirmed the clinical results obtained by Yamazaki that a mixture of chloroöctylresorcinol and octylresorcinol possesses stronger anthelmintic action than either of the compounds alone.
The presence of octylresorcinol in the synthetic product of chloroöctylresorcinol, a powerful anthelmintic, was examined. Distillation of chloroöctylresorcinol, at a not very low pressure, furnished octylresorcinol, resorcinol, and chlororesorcinol from the distillate. Octylresorcinol fraction also yielded resorcinol. These facts indicated that chloroöctylresorcinol underwent thermal decomposition and furnished octylresorcinol as the chief product of dehalogenation. At the same time, it was assumed that chlororesorcinol was furnished as the dealkylation product, and resorcinol as the chief product of dealkylation and dehalogenation.
3-(2′-Methyl-4′-aminopyrimidyl-5′)-methyl-4-methyl-5-β-hydroxyethylthiazol-2-thion was treated with hydrogen peroxide at pH 7.0, 8.0 and in a strongly alkaline range to examine the difference in the products obtained at different pH ranges. At the same time, the formation mechanism was also assumed. Confirmation of the products was made chiefly by the paper partition chromatography, with isolation of some of the products. The products formed were as follows: At pH 7.0-Vitamin B1 disulfide (VI), vitamin B1 (II) (in minute amount), thiochrome (IV) (in minute amount). At pH 8.0-Thiamine-thiazolone (III) (in large amount), vitamin B1 disulfide (VI), thiochrome (IV), vitamin B1 (II) (in minute amount). In strongly alkaline range-Thiamine-thiazolone (III) (in extremely large amount), thiochrome (IV), 2-methyl-4-amino-5-aminomethylpyrimidine (VII) (in minute amount).
It has been found that some kinds of α-aminoisoxazole (I), when heated with hydrazine hydrate, undergo transition to its isomer, pyrazolone-(5) (III). By heating with aqueous solution of hydrazine hydrate, 3, 4-dimethyl-(IV), 3-methyl-4-ethyl-(V), 3-methyl-4-propyl-(IV), 3-methyl-4-benzyl-(VII), and 3, 4-tetramethylene-5-aminoisoxazole (VIII) yielded the corresponding isomeric pyrazolone-(5) compounds (IX), (X), (XI), (XII), and (XIII). By the similar procedure, (VIII) and phenylhydrazine yielded 1-phenyl-3, 4-tetramethylenepyrazolone-(5) (XIX), and (IV) and phenylhydrazine yielded bis-1-phenyl-3, 4-dimethylpyrazolone-(5) (XXII). It is considered that such transition occurred with the ring cleavage of (I) and reaction of hydrazine to form an intermadiate of a β-hydrazones (XXVI) of acid amide. The reaction has been termed, for the sake of convenience, as “pyrazolone transformation”.
During the so-called “pyrazolone transformation” of 3, 4-dimethyl-5-aminoisoxazole (I), some water-soluble, colorless prismatic crystals, m.p. 140-141°, were obtained. Its analytical values indicated the formula of C5H9ON3, and it yielded hydrazine hydrochloride and carbon dioxide by treatment with hydrochloric acid. Alkalization of the mother liquor of these crystals gave tetramethylpyrazine (III). Heating of C5H9ON3 with acetophenone yielded methyl phenyl ketazine, whose condensation products with p-tolyl sulfochloride, p-acetaminophenyl sulfochloride, and phthalic anhydride, suggested that C5H9ON3 might be 3-amino-4, 5-dimethylimidazolone-(2) (VI). Comparative examination of the infrared absorption spectra of C5H9ON3 and 4, 5-dimethylimidazolone-(2) indicated that this assumption was correct. The by-product obtained on the “pyrazolone transformation” of 3-methyl-4-ethyl-5-aminoisoxazole, C6H11ON3, m.p. 119°, also possessed homologous properties as those of (VI) and was assumed to be 3-amino-4-methyl-5-ethylimidazolone-(2).
An allied compound of chloramphenicol, in which the nitro group in the benzene nucleus was substituted with two nitro groups in the meta positions, was prepared. No effective action was found to be exerted by the compound against Salmonella typhi.
dl-1-[4′-Methylthiazolyl-(5′)]-2-acetamido-1, 3-propanediol, a compound in which the benzene nucleus in chloramphenicol had been substituted with a thiazole nucleus, was prepared. No antibacterial effect was exerted by this compound against Salmonella typhi.
Glutamylcholine was prepared from N-carbobenzoxyglutamic anhydride and choline chloride. Examination of the product by the paper partition chromatography showed the formation of two kinds of esters. Considering this reaction formulae, they were assumed to be the esters of α-and γ-choline. In order to examine the general properties of α-and γ-derivatives of glutamic acid, their methyl, ethyl, and propyl esters, amides, and hydrazides were prepared. The paper partition chromatogram always gave smaller Rf values for γ-derivatives than those of α-derivatives. Coloration with ninhydrin were reddish purple for γ-, and yellowish violet to violet for α-derivatives. From these results, it was assumed that the two esters mentioned above were glutamyl-(γ)-choline with spot at Rf 0.07-0.09, and glutamyl-(α)-choline with a spot at Rf 0.11-0.12.
Reaction of N-carbobenzoxyglutamic anhydride and choline chloride yielded carbo-benzoxyglutamylcholine whose reduction to glutamylcholine and its ammoniac decomposition gave l-isoglutamine (I) and l-2-ketopyrrolidine-5-carboxylic acid (II). The crystals which precipitate out on reduction (assumed as a mixture of glutamyl-γ-choline (V) and glutamic acid, by the paper partition chromatography), and hydrazine yielded l-glutamic-γ-hydrazide (III). The ammoniac decomposition of the intermediate, N-carbobenzoxyglutamylcholine, furnished N-carbobenzoxy-l-isoglutamine (VI) whose mother liquor gave l-glutamine (VII) on reduction. Hydrolysis of glutamylcholine with hydrochloric acid yielded l-glutamic acid from which it was seen that no racemization took place during this reaction. From foregoing experimental results, the glutamylcholine obtained by the afore-mentioned method was proved to a mixture of l-glutamyl-α-choline (IV) and l-glutamyl-γ-choline.
Pharmacological tests were carried out on 15 kinds of synthetic glucosides, including 11 new compounds, in the Pharmacological Laboratory of University of Kyoto. As a result, they were all found to have the action of slackening and paralyzing the muscles, although their actions were weaker than that of Myanesin. It was also found that Van't Hoff's Law of additive action of optical rotations could be applied to these glucosides and their acetylated products.
The flow rates of α- and β-isomers of m-cresol, phenol, and guaiacol glucosides were determined by the paper chromatography, using various developing solvents by which the difference of the flow rate was made to the maximum distance of 0.24. By the utilization of this difference in flow rate, a small amount of crude crystalline mixture of α- and β-isomers were separated into respective isomers and purified. According to the present experiments, the flow rate of α-compound was smaller than that of the β-compound, which is contrary to the conclusion of Jeanes that generally the flow rate of α-isomer is larger than that of β-isomer in sugars, and the same could be said of glycosides. Instead of the usual p-toluenesulfonic acid, various kinds of ion exchange resins were used as catalysts in the preparation of β-D-glycoside was obtained by the fusion of β-pentaacetylglucose and phenol. The catalysts used were recovered and their regeneration was found to permit repeated use.
Fresh bark of Ehretia thyrsiflora Nakai contains 0.25-0.50% of allantoine, which is in larger quantities in the lower part of the tree and lesser in the upper part. Root bark contains a large amount of allantoine, the content being 0.53-0.86%. The fresh tree barks also contain 5.3% of succharose.
The present method of determining potency of anthelmintics utillizes the worm excretion rate and egg extermination rate, but neither of the values alone is enough to express the potency of a drug. For this reason, various indeces were formulated and the following gave a fairly satisfactory results: Index=Total points before prescription-Total points after prescription/Total points before prescription where 0 point is the state in which no egg is found in one field of vision under the microscope, 2 points for 1-5 eggs, 3 points for 6-10 eggs, 4-points for 11-15 eggs, and increasing by 1 point thereafter for every 5 eggs found.