The method of determining vitamin B1 by the formation of thiochrome by oxidation and measuring its intensity of fluorescence is the most specific and widely used of various quantitative determination of vitamin B1. Sources of errors in this method were statistically examined at every step and the procedures and errors in this determination method were theoretically made clear.
The procedures and errors in the formalin-azo method of determining vitamin B1 used for the testing of B1 crystals or injections were statistically examined. It was found that this method gave error of only within 1.7% and was advantageous in that it needed no strict control of the reagents used.
The sources of errors in the fluorescent method (potassium ferricyanide and cyanogen bromide methods), formaldehyde-azo method, and ultraviolet absorption measurements for the determination of vitamin B1 were taken up, experimental designs made accordingly, and errors were comparatively examined statistically. The order in which less error occurred were ultraviolet absorption measurements, formaldehyde-azo, cyanogen bromide, and ferricyanide methods.
In continuation of the previous experiments, the oxygen bridge in 2, 3-dimethoxy-5, 4′-dimethyldiphenyl ether (IV) and 2, 3, 2′-trimethoxy-5, 4′-dimethyldiphenyl ether (V) was severed by Grignard reagent (butyl- or tert-butyl-magnesium bromide), in the presence of anhydrous cobaltous chloride. In either case, 3-hydroxy-4, 5-dimethoxy-1-methylbenzene (VI) was obtained as a phenolic substance, the yield being better from (IV) than from (V). The reason for this difference was assumed to be due to the steric hindrance of the methoxyl group substituted in (V).
Relation between the rate of hydration of hydrated methanol solvent and its Rf value and the relation between the basicity and Rf value of acetone-ammonium hydroxide solvent system were examined by paper chromatography. It was thereby found that the Rf values of anions were not greatly influenced by the basicity of the solvent while such was greatly affected by its rate of hydration. It was also found that cations were absorbed in a place different from those of corresponding anions, i.e. anions and cations moved in individual manner, and that the cations hardly influenced the Rf values of anions. Separatory determinations of a various mixtures of ions were also carried out by paper chromatography. Separations effected were: Fe(CN)6--- and Fe(CN)6----, and of CNS-, AsO3---, and Fe(CN)6---- by 60% methanol; Cl-, Br-, I-, and CNS-, and of ClO3- BrO3-, and IO6 by Me2CO: N NH4OH (4:1); Cl- and ClO3- by BuOH: Me2CO: H2O (5:2:3); AsO3--- and AsO4--- by MeOH: N NH4OH (3:1); and AsO3---, AsO4---, and PO4--- by BuOH: MeOH: N NH4OH (5:2:3).
Formation of hydroxycoumarin nucleus from some phenols was examined by the use of the Sonn method and it was found that orcinol underwent condensation with cyanoacetic ester to form 5-methyl-4, 7-dihydroxycoumarin. Acylation of this substance with acid chloride in pyridine resulted in its reaction by the mechanism elaborated in the case of 4, 7-dihydroxycoumarin and it was found that by the selection of a suitable reaction conditions, 3-acyl-5-methyl-4, 7-dihydroxycoumarin could be obtained. In order to examine the influence of this methyl group in 5-position to the antibacterial activity, the effect on Staphylococcus aureus and Escherichia coli was tested with 3-acetyl derivatives. 3-Acetyl-5-methyl-4, 7-dihydroxycoumarin was found to inhibit the growth of Staph. aureus at 40, 000 dilution, being 10times stronger than 3-acetyl-4, 7-dihydroxy- and 3-acetyl-4-hydroxy-coumarins used as the control. 3-Decanoyl derivative could not be examined due to its difficult solubility. None of the compounds showed any apparent effect on E. coli. The structures of the substances prepared in the present series of experiments were determined by comparing their ultraviolet absorption spectra with those of the substances of 4, 7-dihydroxycoumarin series prepared previously. Direct proof of the structure of 5-methyl-4, 7-dihydroxycoumarin was obtained by its alkaline decomposition to oracetophenone.
Reaction of various aldehydes with ethyl ester of α-bromofatty acids, in the presence of metallic zinc, results in the formation of corresponding ethyl esters of β-hydroxylic acids. By heating these esters with equal amount of 80-85% sulfuric acid at 75-80° for 5 hours, dehydrogenation and hydration occur alternately and the most stable γ-butyrolactones are formed. Fifteen kinds of γ-butyrolactones were prepared by this method, three of which were new compounds. All of these compounds were aromatic oil and gave crystalline hydrazinolactones when heated with 50% hydrazine hydrate solution.
Sixteen kinds of β, γ- or α, β, γ-substitution products of γ-butyrolactone, including 13 new compounds, were prepared by heating under stirring with 85% sulfuric acid the ethyl esters of β-hydroxy acids obtained by the Reformatzsky reaction of various ketones and ethyl esters of α-bromo fatty acids. The compounds synthesized were all oily substances possessing individually characteristic aroma, and their hydrazinolactones were generally difficult to crystallize with the exception of the α, γ-substituted products and α, β, γ-substituted products possessing cyclohexyl ring which were comparatively easy to crystallize.
In order to observe the relationship between chemical structure and aroma in γ-butyrolactone derivatives, 27 kinds of γ-methyl-γ-alkylbutyrolactones were prepared by the application of the Grignard reaction between various alkyl halides and ethyl levulinate, twenty of the compounds synthesized being new substances. Majority of them were an aromatic oil but γ-methyl-γ-p-tolyl-, γ-methyl-γ-p-anisyl-, γ-methyl-γ-cyclohexyl-, and γ-methyl-γ-cyclohexylethyl-butyrolactones were obtaind as crystals.
Following conclusions were drawn regarding the relation between chemical structure and aroma in 58 kinds of γ-butyrolactone derivatives synthesized: 1) Derivatives with around 10 carbon atoms possess the most value as essence materials. Above that number of carbons, the aroma becomes weak, the aroma being almost undetectable in compounds with 14-16 carbons. The odor becomes slightly irritating when the number of carbon is below seven. 2) The compounds with alkyl substituents in γ or in γ and γ′ possess the aroma of fruits or flowers, those with substituents in α and γ possessing the aroma similar to mint or spearmint, and those with substituents in α, β, and γ, cnidium- or cumene-like aroma. The aroma of the compounds with substituents in the β- and γ-positions is intermediate between those of γ- and α, γ-substituted compounds. 3) Compounds with flower-like or fruit-like odors change to resinous odor on introduction of substituent of the aromatic radical, and to the intermediate of aliphatic and aromatic substituted compounds when substituted with alicyclic radical. The aroma is found to be inferior when the alkyl chain is branched as compared to the straight chain.
2-[2′-Methyl-4′-aminopyrimidyl-(5′)]-methylformamino-5-hydroxy-Δ2-pentenylaryl disulfides (thiamine aryl disulfides) were prepared by the application of arylsulfenyl chloride, aryl arylthiosulfonate, and aryl thiothiocyanate to the thiol type of vitamin B1, and homothiamine aryl disulfides were prepared in the same manner using homovitamin B1. Thiamine alkyl disulfides were obtained from the thiol type vitamin B1 and alkyl-sulfenyl chloride and alkyl or aralkyl thiosulfonate.
2, 4-Dimethyloxazole forms bromine adduct which changes to bromine substituted derivative at 135°. Nitration of this dimethyloxazole with nitric or nitric and sulfuric acids results in ring fission, showing that the reactivity of C5 to electrophilic reagents falls in the order or selenazole, thiazole, and oxazole. The reactivity at C5 increases in 2-diethylamino-4-methyloxazole and it undergoes coupling with diazonium compounds to form a labile azo compound. Condensation of 2, 4-dimethyloxazole and benzaldehyde gives 2-styryl compound and not 2, 4-distyryl derivative which shows that the reactivity of C2-position to nucleophilic reagent is higher than that of C4, this tendency being common with selenazoles and thiazoles.
Benzoxazole gives 2-aminobenzoxazole when warmed with sodium amide and this compound was found to be identical with the substance obtained from o-aminophenol and cyanogen bromide. 2-Amino-5-ethoxyphenol and cyanogen bromide gave 6-ethoxybenzoxazole whose hydrochloride exhibited a local anesthetic action. 2-Methylbenzoxazole underwent condensation with benzaldehyde in the presence of zinc chloride to form 2-styrylbenzoxazole which was identical with the substance obtained by the condensation of o-aminophenol and cinnamic amide. 2-Methylbenzoxazole underwent condensation with benzyl chloride in the presence of sodium amide to form 2-β-phenylethylbenzoxazole which is also obtained by the catalytic reduction of the afore-mentioned 2-styryl derivative. Benzoxazole ring is opened by the application of benzoyl chloride in the presence of potassium cyanide to form o-benzoylaminobenzoylphenol. 2-Aminobenzoxazole does not undergo diazotation under conditions similar to those used for 2-aminobenzothiazole, and does not form imidazole derivative by the action of bromoacetophenone. Heating of 2-aminobenzoxazole and bromoacetophenone results in resinification, partly giving 2-acetaminophenol formed by ring fission, and not the pyrrol derivative.
Nitration of benzoxazole with sulfuric and nitric acids at -15° chiefly gives 6-nitrobenzoxazole which was confirmed by deriving it to 5-nitroaminophenol by heating it with dil. hydrochloric acid. Nitration of 2-methylbenzoxazole with cold sulfuric and nitric acids gives the 6-nitro derivative and its dimers. Heating of 2-methyl-6-allylbenzoxazole at 230° for 15 minutes results in the occurrence of Claisen rearrangement almost quantitatively to give 2-methyl-6-hydroxy-7 (?)-allylbenzoxazole. The allyl ether of this compound, when heated at 230° for about 20 minutes, forms 2-methyl-6-hydroxy-5, 7-diallylbenzoxazole in a good yield. These results show that benzenoid activity is prominent in the benzene portion of benzoxazole.
The succharin method was applied to the synthesis of amino acids and three kinds of aliphatic N-monoalkylamino acids, sarcosine, N-ethylglycine, and N-methyl-β-alanine, were prepared in a good yield. The interesting fact was that, during the hydrolysis of methyl ester of o-carbomethoxybenzenesulfonyl-N-methyl-β-alanine (IV) with alcoholic alkali hydroxide solution inverse Michael reaction partially occurred forming o-carboxybenzenemethylsulfonamide besides the objective (V).
Alkylbenzylamines and phenethylamines were satisfactorily prepared by the saccharin method. The yield of second alkylation was increased by boiling a mixture of dipotassium salt of (III) and alkylation agent in dehydrated xylene. The yield of alkylation was the maximum in the methyl, decreasing in the order of ethyl, butyl, isobutyl, and sec-butyl. The final saponification was also the most easiest with the methyl, becoming difficult and the yield becoming poorer in the same order as above.
Bromination of quinaldine 1-oxide with N-bromosuccinimide was found to proceed far more rapidly than that of quinaldine itself. By the use of one mole of N-bromo-succinimide, the yield was 22% of ω-dibromoquinaldine 1-oxide, 14% of ω-monobromide, and 35% recovery of quinaldine 1-oxide.
Six kinds of α-(N-tert-aminoethyl)-aminophenylacetic acid esters were prepared by the application of tert-aminoethyl chloride to α-aminophenylacetic acid esters and four kinds of β-(N-tert-aminoethyl)-aminophenylethyl alcohols were obtained by their reduction with lithium aluminum hydride or by the reduction of α-aminophenylacetic acid esters to phenylethyl alcohol with subsequent application of tert-aminoethyl chloride.
Monoalkylaminoethyl alcohol was prepared from monoalkylamine and ethylene oxide, derived to the chloride by the usual method, and reacted with α-aminophenylacetic acid esters or β-aminophenylethyl alcohol to obtain eight kinds of α-(N-sec-aminoethyl)-aminophenylacetic acid esters and eight kinds of β-(N-sec-aminoethyl)-aminophenylethyl alcohols.
Alkyldiethanolamine was prepared from monoalkylamine and ethylene oxide, and by the reaction of the amine chloride with α-aminophenylacetic acid ester or β-aminophenylethyl alcohol, eight kinds of corresponding esters of α-(1-alkylpiperazyl-4)-phenylacetic acid and eight kinds of β-(1-alkylpiperazyl-4)-phenylethyl alcohols were prepared.
Mercuration of dibromofluorescein by heating a mixture of dibromofluorescein and mercury acetate in 1:4 ratio in acetic acid showed that the pH of the medium greatly affected the amount of mercury introduced, besides the length of heating. At pH below 3, the mercury introduced was liable to liberate and the half-masked mercury precipitated out as metallic mercury. At pH over 6, dibromofluorescein was oxidized by mercury acetate and a part of bromine was liberated. At above pH 7, mercuration no longer proceeded and the amount of liberation of black mercury increased. The reaction proceeded most smoothly at around pH 4 and up to 1.5 moles of mercury could be introduced. However, adjustment of molar ratio, heating time, and pH of the medium failed to effect introduction of two moles of mercury by this method to form a dimercury compound.
By heating dibromofluorescein and mercury acetate, in 1:4 molar ratio, at pH 4-5 for 6 hours, a mercury compound giving analytical values of Hg 37.3% and Br 19.67% is obtained when the reaction medium was around pH 4, and of Hg 34.7% and Br 20.8% when around pH 5, for which formulae (VII) and (VIII) were respectively assumed. When these compounds were made soluble by the application of a calculated amount of sodium hydroxide solution, a mixture of the sodium salts of di-(V) and monohydroxymercuri (VI) compounds was formed. These assumption were drawn from the absorption spectra and confirmed by the following facts: 1) Application of iodine solution and derivation of HgOH to -I followed by ace-tylation resulted in the separation of two kinds of acetates of dibromodiiodo- and dibromomonoiodo-fluorescein. 2) Paper chromatography developed with 3% ammonia water gave two spots of Rfca. 0.07 and 0.38. 3) Separation by column chromatography through activated magnesium oxide gave a dimercury and monomercury compounds. It has been found that the so-callced Mercurochrome is not the sodium salt of mo-nohydroxymercuridibromofluorescein alone but always contains the dimercury compound and a small amount of unreacted dibromofluorescein, by examination with paper chro-matography.
A glass capillary of approximately 0.8mm. inside diameter and approximately 10cm. long, filled with alumina and having a pinhole at one end was used as a means of detecting several kinds of mixed ions by suction of sample solution and coloration reagents into the capillary through the pinhole. Limit of detection of metallic ions by various coloration reagents was examined and the present method was found to be far sharper than the paper spot test and the best suited for the detection of a minute amout of ions. The feature of this method may be said to lie in the fact that only a minute amount of sample and alumina are needed and that the procedure is simple and time short.
By the use of the same method of capillary filled with alumina as given in the previous paper, order of adsorption, limit of detection, separation of several kinds of anions, and the method of microdetection of I-, NO2-, and CN- ions were examined. It was found that the separations of Cl- and PO4---, Cl- and AsO4---, PO4--- and AsO4----, AsO3--- and AsO4---, I-, CrO4--, and PO4---, NO2- and CrO4--, were possible. The coloration reagents used were, besides the known AgNO3 method, CuCl2, Hg(NO3)2, FeCl3, and benzidine. Chromatographic separation of metal complexes were also carried out by the same method and the separation of Cr and Pb, which was previously reported as being comparatively difficult was found to be possible by preparing a tartaric acid complex, while that of cobalt, nickel, and cadmium waa found to be possible by the ammonium complex method.
By the determination of the stepwise change of pH in the absorption tube during cation adsorption and the amount of cerresponding anion adsorbed, precipitation of basic salt of metals was assumed, and following tests were carried out. Hydrogen ion concentration at the time of precipitation was determined with glass electrode pH meter and the relationship between pH at the time of absorption and the amount absorbed were examined by static adsorption, and it was found that the precipitation pH range and the order of adsorption were in good agreement and that the adsorption of cation became maximum in the precipitation pH range. From these facts, it was assumed that the adsorption of metallic ions by alumina was chiefly due to the precipitation of their basic salts. It was also found that the influence of anions was rather great in the adsorption of mercury salt, as the adsorption of mercury became extremely small in the presence of a chloride and this was assumed to be due to the formation of mercuric chloride of small degree of dissociation.
By allowing crystals or alcoholic solution of vitamin D2 to stand in air at a room temperature, 5% of it already undergoes decomposition in one week and the decomposition is accelerated thereafter, especially in crystals. This is assumed to be due mainly to air oxidation as well as by the action of light. It is suggested that the safest way is to store vitamin D2 in a brown bottle filled with inactive gas in an ice box. Several kinds of substances were separated by liquid chromatography as the decomposition product.
1) Bovine submaxillary gland was extracted with water at pH 8.0 and precipitated by adjusting the aquous solution to pH 4.5. This pH 4.5-precipitate fraction (nitrogen content, 11.49%) was found to have strong activity of decreasing the phosphorus level in rabbit serum but, differing from parotin, it did not seem to affect the level of serum calcium. 2) The potency of decreasing serum phosphorus of pH 4.5-precipitate fraction was found to decrease on standing at a room temperature for one hour at pH 2.0, and to decrease further by being heated for one hour at 60° at pH 2.0. This potency was not decreased when left at a room temperature at pH 8.5 but clearly decreased when heated for one hour at 60° at pH 8.0. 3) Fractional precipitation of pH 4.5-precipitate fraction with ammonium sulfate resulted in the collection of a portion giving strong effect of decreasing serum phosphorus level in a precipitate obtained at 12.5% ammonium sulfate concentration. 4) Purification of the pH 4.5-precipitate fraction by use of the isoelectric point and fractional precipitation by ammonium sulfate gave at 12.5% ammonium sulfate concentration a precipitate which showed the strongest effect in lowering serum phosphorus. (Rabbit, 2.5mg./kg. dose intravenous administration gave lowering rate of 24.5%). 5) The precipitate of 12.5% ammonium sulfate concentration was different from mucin or lecithin, or from the pH 5.8-precipitate extracted from the lymphatic gland near the bovine submaxillary gland.
1) The pH 4.5-precipitate fraction obtained from the bovine submaxillary gland greatly affects serum phosphorus- but not serum calcium level. However, fractional precipitation with sodium sulfate gives a precipitate fraction at 15.4% concentration of the salt which possesses a strong effect of lowering serum calcium level. 2) Dialysis of various fractions, possessing a strong effect of lowering serum inorganic phosphorus level but weak in lowering serum calcium, against tap water for 24 hours at a room temperature results in the increase of serum calcium level lowering effect in the non-dialyzable fraction, while the effect of serum phosphorus level lowering effect showed no change. 3) Dialysis of the fraction which precipitates at 12.5% ammonium sulfate concentration gives a non-dialyzable fraction with strong effect of lowering serum calcium level and this was found to have about 80% purity by electrophoretic analysis. This shows that the purification had actually been effected as compared to the original pH 4.5-precipitate which was found to be approximately 25% purity. 4) The precipitate at 12.5% concentration of ammonium sulfate did not possess amylase effect.
1) It was confirmed through counter-current distribution method that the pH 4.5-precipitate fraction from the submaxillary gland contained two components, one fraction effective for serum calcium level and the other for serum phosphorus. 2) The fraction effective for serum phosphorus level underwent denaturation by alcohol, even at a low temperature, but no change in the effect was observed in a fraction effective for serum calcium level by the same alcohol at a low temperature. 3) The fraction effective for serum calcium level does not precipitate out when alcohol is added to its aqueous solution at a concentration of 60% but precipitates out at 80% concentration. 4) The fraction effective for serum calcium level showed the same ultraviolet absorption spectrum as that of parotin but at the present stage, the product showed only a purity of 63% by electrophoresis. 5) The fraction effective for serum calcium level showed about the same effect as parotin against circulating leucocyte counts. On the contrary, the same effect was far weaker in the fraction effective for serum phosphorus level. 9) The foregoing results have shown that the extract from submaxillary gland contains a substance which lowers serum phosphorus as well as serum calcium, and since the latter is similar to parotin in its physicochemical properties as well as its effect in promoting calcification of incisor tooth and increase of leucocyte in rabbit, these might be one proof of the internal secretion theory of the salivary gland that salivary gland hormone is formed in the parotid gland and that submaxillary gland cooperates in its formation.
Dulcin and acetophenetidine are polarographically inactive compounds but o-nitrodulcin and o-nitro acetophenetidine, obtained by nitration with nitric acid, become polarographically active. It was found possible to determine dulcin and acetophenetidine by polarography if thereaction conditions of nitration were made definite. Therefore, conditions for nitration, extraction of nitro compounds from the reaction mixture, and determination by polarography were examined and satisfactory results were obtained for the quantitaive determination of dulcin and acetophenetidine.
Thiaxanthone was nitrated with nitric acid or mixed acids at various temperatures in order to find the best conditions for the formation of nitro compounds with less formation of 5-dioxide. Application of sulfuric acid to thiaxanthone gave a monosulfonic acid the position of which was proved to be at 2 by comparison with the sulfonamide derived from 2-aminothiaxanthone.
1, 1-Di-(2′-thienyl)-2-methyl-3-tert-aminopropylene-1 (D), differing from the compound (C), prepared by Adamson, et al. in its position of the methyl group, was prepared and its pharmacological effect was examined. Its activity was found to be only one-tenth that of morphine.