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1. Ultraviolet absorption spectra of the aqueous solution of O-benzoylthiamine disulfide (BTDS) was determined at various pH levels. They showed the changes according to pH, the isobestic points being at 239 and 274mμ.2. The structure of BTDS was definitely proved by infrared absorption spectra.3. BTDS is soluble in mineral acids, chloroform, glacial acetic acid and methanol, but sparingly soluble in water and other organic solvents.4. The partition coefficient of BTDS between n-butanol or benzene and 0.1 M phosphate buffer (pH 6.6) is remarkably large as compared with those of related compounds. In O-acyltiamine disulfide homologues, the value increases logarithmically with the rise in the number of carbon atoms of the acyl group.5. RF values is as high as 0.85±0.03, when the developing solvent consisted of butanol, acetic acid and water. It can therefore be isolated from other compounds.6. Aqueous solution of BTDS at a concentration of 2mg/ml is fairly stable even in a weakly alkaline solution.7. The powder of BTDS mixed with alkaline salts (calcium carbonate or sodium bicarbonate) was proved to be fairly stable even when stored under a hot and humid condition.

Studies on the inhibition of the germination and the growth of Bacillus subtilis spores by amino acids and sorbate were carried out and the following results were obtained:(1) Inhibition of the germination.In 18 D-, L- or DL- amino acids studied, D-alanine and D- or DL-serine were found to be strongly inhibitory on the germination using meat extract as nitrogen source. When L-alanine was used as nitrogen source, glycine was also found to have an inhibitory effect as well as the above-mentioned amino acids. Sorbate had no influence on the germination.The rate of germination (by Hachisuka's method) at initial stage of the germination appeared to be a mode of first order reaction, and then the rate constant k calculated from the first order reaction equation was confirmed to be well corresponding to the rate of germination by Hachisuka's method.(2) Inhibition of the growth involving the germination.In 19 D-, L- or DL- amino acids studied, glycine, D-, L- or DL-serine, and D-, L- or DL-threonine were found to have an inhibitory effect. Particularly, the combined use of glycine and serine or that of glycine and threonine showed a strong effect. Combined use of these amino acids would be applied for food preservation with sorbate or other preservatives.

It was found that the reaction of thiamine disulfide monosulfoxide (I) with mercaptan is a competitive reaction between the so-called mercaptan-thiolsulfinate reaction and the apparent oxidation reduction reaction. This reaction offers an excellent method for the preparation of the mixed disulfides of thiamine and t-mercaptan or the unstable mixed disulfides, such as thiamineglutathione disulfides.

The new oxidized produucts of thiol-type thiamines, 2-(2′-methyl-4′-aminopyrimidyl-5′)-methylformamino-5-hydroxy-Δ2-pentenyl-3-sulfonic acid (thiaminic acid) and its O-benzoyl ester, were obtained by the oxidation of thiamine disulfide or its O-benzoyl ester with hydrogen peroxide. Thiamine alkyl disulfide derivatives, S-acyltype thiamine derivatives and cyanothiamine are also oxidized to thiaminic acid derivatives in similar oxidation reactions. However, no formation was found from thiamine alkylsulfide. Therefore, the thiol-type thiamine derivatives which are converted to thiamine by the reaction with sulfhydryl compounds such as cysteine or protein-SH, produce thiaminic acid derivatives under such oxidation reactions. Thiamine cannot take the thiol form in an acid medium and no thiaminic acid can be formed. Furthermore, by similar oxidation of several aryl thiamine disulfide derivatives or by acylation of thiaminic acid, several thiaminic acid derivatives were obtained. Thiaminic acid is an interesting compound which can be considered to occur in the body as an intermediate of thiamine metabolism and is assumed to be an effective form of so-called “active” thiamine derivatives.

The improved methods of quantitative analyses of nicotinic acid and nicotinamide were described, whereby the stable, colored compound obtainable by the reaction of nicotinic acid or nicotinamide and cyanogen bromide in Clark-Lubs buffer, pH 9.6 or 9.3, under a certain condition were photometrically determined. The fractional determination of nicotinic acid and nicotinamide is possible by use of anion exchange resin.

Powdery polymers of vitamin A esters were synthesized by polymerizing acrylic and methacrylic ester of vitamin A under the presence of α, α′-azo-bis-isobutyronitrile as a catalyst at 70° or of n-butyl lithium in hexane at -10 to -20°. The polymer failed to be obtained when phenyl magnesium bromide was used as a catalyst. Crotonic and undecylenic esters of vitamin A were not polymerized by the above methods. Powdery copolymers of vitamin A esters with acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, styrene or 2-vinyl-5-ethyl-pyridine were also synthesized by the same methods.

1. Symmetrical disulfides, such as O-benzoylthiamine disulfide (BTDS) or thiamine disulfide (TDS) are reduced to free thiamine when it is allowed to react with heat- or urea-denatured egg or serum albumin under certain conditions, whereby a protein-thiamine complex is partially formed. The complex can be separated by deproteinization with metaphosphoric acid, which can also stop the reducing reaction of protein-SH. The complex thus separated can easily be converted into free thiamine by reduction with thiosulfate, followed by determination by thiochrome method. Applying this method, reactions of BTDS or its related compounds with protein-SH were examined and the following results were obtained.2. Egg albumin in native state scarcely reacts with the thiamine derivatves. Further, denatured protein, whose SH group had been oxidized by iodine or blocked by P-chloromercuribenzoate, loses its reactivity, scarcely producing the complex.3. Serum albumin reacts both in native and denatured states, producing a complex.4. The complex formation with denaturedprotein takes place only with symmetrical disulfides. Asymmetric thiamine derivatives such as thiamine propyl disulfide, produce only free thiamine. It was also suggested that the same complex was formed by a reaction of thiamine or O-benzoylthiamine (OBT) with oxidized protein.5. In the reaction of BTDS with heat-denatured protein, the optimun pH of the reaction, the effects of the protein amount, the concentration of added BTDS, temperature and time were investigated and the relationship between complex formation and production of free thiamine was clarified. In any case, complex formation was proportional to the amount of free thiamine formation, increasing with the progress of the reaction.6. The protein-thiamine complex is, therefore, considered to be due to chemical binding of the thiamine derivatives with protein-SH as protein-S-S-thiamine.

Oxidation of S-alkylthiamines and S-alkoxycarbonylthiamines, which are difficult to convert to thiamine by sulfhydryl compounds, was investigated. S-alkylthiamines were oxidized with hydrogen peroxide and perbenzoic acid in glacial acetic acid to afford the corresponding sulfoxides and sulfones in accordance with the amount of the peroxide. α-Acyloxysulfide-type thiamine, which was presumed to be produced via the Pummere's reaction, obtained by the reaction of sulfoxide with acid anhydride.O-Benzoyl-S-ethoxycarbonylthiamine, one of S-alkoxycarbonylthiamines, was oxidized with hydrogen peroxide to give O-benzoylthiaminic acid.

In order to study more in detail the formation of thiaminic acids by the oxidation of thiol type thiamine derivatives, the oxidation condition of O-benzoylthiamine disulfide (BIDS) was investigated and the following results were obtained. In the oxidation with hydrogen peroxide, nearly the same yield was obtained in lower fatty acids such as acetic, formic, propionic or butyric acid and in its solution in water or various organic solvents. Further optimal condition was that more than 5 moles of H2O2 per mole of BTDS was allowed to react at about 40° for 2 to 4 days, whereby the yield was about 50%, showing that at least one mole of O-benzoylthiaminic acid (BTnA) was produced from one mole of BTDS. Furthermore, it was observed that BTnA was also obtained by H2O2 oxidation in an aqueous solution (pH 4-9) or by oxidation of bromine, permanganate or bichromate though in a fairly reduced yield.

A new sustained-release micropellet was prepared which takes advantage of the relationship of gelatin solubility to the hardness of the gelatin caused by formalin treatment. This new gelatin micropellet could be produced more easily than the coated micropellet employed in the usual sustained-release dosage forms.

Various O-acyl thiamine disulfides were parenterally given in rabbits, the retentivity in the blood was compared, and the following findings were obtained.1. The proximate elimination rate constant and the biological half-life were calculated by treating reaction-kinetically the decreasing process of the blood total thiamine level to estimate the retentivity of each compound in the body by exact numerical values.2. It was clarified that the retentivity obtained by this procedure had a correlation with the uptake by erythrocytes, determined in vitro.3. Among the derivatives examined, O-butyrylthiamine disulfide was proved to be of value as a new thiamine derivative, in respect to the maintenance of the high blood level, stability, solubility and safty.

Polymers of vinylpyridine and of its derivatives and copolymers of vinylpyridine with other series of vinyl compounds were experimentally synthesized. The purpose of the synthesis was to obtain such protective coating agents exhibiting protective activity on drugs and solubility in gastric juice. Solubility, water vapor permeability through the film, and viscoelasticity were tested for each compound. The practical coatings on tablets were also carried out and disintegration time of coated tablets and shelf life of active ingredients were subsequently investigated. All the compounds showed excellent properties as protective coating agents.

The effect of thiamine or Sorbic acid on of bake's yeast was studied and the following findings were obtained.1. A large amount of thiamine stimulated the respiration. Butyryl thiamine disulfide or thiamine diphosphate had rather stronger effect than thiamine hydrochloride. The maximum stimulating effect appeared at the final concentration of 5.5 mM.2. Combined use of thiamine and sorbic acid exhibited the remarkable inhibition of the respiration, i.e., thiamine stimulates the sorbic acid inhibition, and the inhibition increased proportionally to the concentration of the vitamin. Butyryl thiamine disulfide and thiamine diphosphate showed the same interaction, but benzoate, salicylate, dehydroacetate or propionate in place of sorbic acid had no such effect. The remarkable inhibition also appeared with glucose, acetate or pyruvate.3. Sorbic acid incorporation in the cells was not influenced by thiamine. With the cells containing the high level of coenzyme A the stimulating effect of inhibition by thiamine was hardly seen. The results were various according to the lots of yeast. It was confirmed that the different levels of coenzyme A in the cells were the main reason of this variety. It became thus clear that the fact which sorbic acid inhibition was reinforced with the addition of thiamine was related to the coenzyme A level in the cells.4. The yeast growth was also markedly inhibited by the combined addition of thiamine and sorbic acid in the shaking culture, but in the stationary culture the characteristic inhibition was not recognized.

Refluxing of monosulfoxide (Ia) of O-benzoylthiamine disulfide in aqueous ethanol or an alkaline decomposition of the compound Ia provided a new compound, O-benzoylhypothiaminic acid (II) which is presumed to be an intermediate in the course of formation of O-benzoylthiaminic acid (VI). An alkaline hydrolysis of II afforded hypothiaminic acid (V). These hypothiaminic acids were oxidized to the corresponding thiaminic acids by hydrogen peroxide. From the fact that, in refluxing the compound Ia in aqueous ethanol, O-benzoylthiamine disulfide (III) was formed besides the compound II, it seemed reasonable to assume that the decomposition pathway is disproportionation and the alkaline decomposition is similar hydrolysis to that of carboxylic esters to give the sulfinic acid (II) and thiol (IV).

1. The amounts of protein-thiamine mixed disulfide produced by the reaction of thiamine or O-benzoyl thiamine (OBT) with oxidized egg albumin at various pH levels were determined. The formation of the bound form of thiamine was detected at pH about 7.0, increasing suddenly at pH above 8.2, and reaching a nearly constant value at pH above 10.0. The pH-curve was in good agreement with the curve of pH and formation of the thiol form, showing the parallelism between the production of the bound form and the concentration of the thiol type thiamine.2. OBT showed already the reactivity at pH around 6.0, and more amounts were bound with protein than thiamine at any pH levels.3. The equilibrium concentrations of the thiol form of thiamine and of OBT were compared in weakly alkaline range by measuring the anodic wave height of polarography. The anodic wave height of OBT was found to be far higher than that of thiamine, showing that OBT was more liable to take the thiol form. These findings suggest that the difference in the reactivities of thiamine and OBT in weakly alkaline range is mainly due to the difference in the formation of the thiol form.

Reducting conditions of BTDS by cysteine and thiosulfate were investigated and the basal assay procedure was established.1. The optimum condition for reduction with cysteine is the use of 5mg of cysteine hydrochloride for 1 to 100μg BTDS at pH 8.4 to 8.8 and 37° for 60 minutes. The lower the pH of the solution, the more cysteine is required. At pH 7.0 more than 10mg of cysteine is necessary.2. O-Benzoylthiamine disulfide (BTDS) is converted into O-benzoylthiamine and thiamine by cysteine reduction, while O-benzoylthiamine alone is produced by thiosulfate reduction. O-Benzoylthiamine thus formed is converted into thlochrome by alkaline oxidation with cyanogen bromide. The fluorescence intensity produced from thiamine was practically the same as that from O-benzoylthiamine.3. The optimum condition for reduction with thiosulfate is the use of 40mg of sodium thiosulfate for 1 to 100μg of BTDS at pH 4.0 to 5.0, heating at 60° for 20 minutes.4. Adsorbability of O-benzoylthiamine to permutit is poor. However, it become satisfactory after Takadiastase treatment.5. The thiamine formed from BTDS by reduction and Takadiastase treatment is well adsorbed on permutit but has a tendency to be eluted less. 25ml of the eluting solution is not enough but 40ml is sufficient for the recovery more than 95 per cent.

A disintegration test of protective coated preparations in human bodies was carried out by determining the riboflavin amount excreted in urine after administration of the riboflavin tablets coated with the previously reported protective-coating agents. Though the rate of the riboflavin excretion was somewhat slow, there was no indication of unusual disintegration of the coated tablets in human bodies.