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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.

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.

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.

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. Protein-bound compound, protein-SS-C3H7 (III) formed by the reaction of egg albumin and C3H7SSC3H7 (I) or C3H7SSOC3H7 (II) as well as S-propylmercapto-L-cysteine (IV), reacts with thiamine to form thiamine propyl disulfide (TPD), whereby thiamine was masked.2. The thiamine-masking reaction of III and IV did not proceed stoichiometrically as II; the masking activity of III was 15% of that of II and that of IV about 40%. On the contrary, I had scarcely any activity.3. The thiamine-masking rate of III was about the same, whether it was produced from. I or II. The masking activity was dependent on the amount of C3H7SH bound with the protein.4. It was confirmed that the thiamine-masking activity was increased with the rise in the concentration of either III (or IV) or thiamine, suggesting the existence of chemical equilibrium.5. From the above findings the thiamine-masking activity of III or IV is considered to be due to the thiol disulfide exchange reaction between the thiol form of thiamine and these disulfides.

Publisher Summary The chemical oxidation of thiol-form thiamine derivatives makes it clear that various derivatives, which are easily converted to thiamine in vivo, provide thiaminic acid as a final product. It is confirmed that thiamine alkyl sulfides, which are hard to revert to thiamine in vivo, afford the corresponding sulfoxides and sulfones. Hypothiaminic acid as an intermediate in the oxidation is synthesized from thiamine disulfide monosulfoxide, the formation of which is the first step in the oxidation of thiamine disulfide. The monosulfoxide and hypothiaminic ester are biologically active. However, these hypothiaminic and thiaminic acids, the toxicity of which is extremely low, have neither thiamine nor antithiamine activity but have a degree of analgesic and antiinflammatory effect similar to that of easily absorbable thiamine derivatives. Although no evidence has been obtained in support of the formation of these oxidation products as metabolites, it has been assumed that their formation may occur as with the metabolic reactions of cysteine and cystine.

1. The formation of protein-thiamine complex (mixed disulfide) in the reaction of iodine-oxidized egg albumin and thiol-type thiamine or O-benzoylthiamine (reaction II) proceeds according to pseudo-first order kinetics in its initial stage where SS groups of the protein is in excess to the thiamine thiol.2. Rate constant is larger in the O-benzoylthiamine reaction than in that of thiamine at each temperature, but the constants are both smaller than that of the reaction between disulfide-type thiamine and SH groups of heat-denatured albumin (reaction I).3. Activation energies for the reactions of thiamine and O-benzoylthiamine with oxidized protein are both about 30 kcal per mole. This shows that the reaction of the type II needs the larger energy than the reaction of type I.4. Applying the absolute reaction rate theory, thermodynamic quantities of the reactions in their activated state were calculated.Entrorpy of activation is negative for reaction II, and positive in reaction I. However, heat of activation and free energy of activation are positive and fairly large in the two reactions, showing the endothermic nature of the reactions. It is further confirmed from these kinetic data that the thiamine-protein complex is formed by the covalent bound through chemical interactions.

Egg albumin oxidized with iodine reacts with the thiol form of thiamine or O-benzoylthiamine (OBT), forming a protein-thiamine mixed disulfide. The amount of the disulfide formation was markedly decreased by heat or urea denaturation before oxidation. The reactivity was greatest, when the SH-groups of the native protein was oxidized directly. Denaturation after oxidation did not cause any significant change in reactivity. When the SH-groups of the native protein was oxidized after denaturation, the reactivity was lowered with the degree of denaturation. The finding seems to be due to the decreased formation of the intramolecular -S-S-linkages in the protein molecule which are involved in the reaction with the thiol forms of thiamine derivatives owing to the denaturation prior to oxidation.

1. When 10μmoles of thiamine disulfide (TDS) or O-benzoylthiamine disulfide was added to 0.2μmoles of SH-blocked urea-denatured bovine serum albumin and the mixture was incubated at pH 9.0 and 37°, the formation of protein-thiamine mixed disulfide, at most 4.5 moles per mole protein, was observed.2. This reaction was almost completely inhibited by SH-reagents, such as p-chloromercuribenzoate (PCMB) and N-ethylmaleimide, about 1/10 mole of TDS, whereas it was markedly accelerated by the addition of thiamine, corresponding to 1/50 to 1/10 mole of TDS.3. In the reaction with iodine-oxidized egg albumin, the formation of similar compound and its inhibition by PCMB were observed.4. From these results, it was assumed that protein-thiamine mixed disulfides were formed from protein SS-groups and symmetric disulfide-type thiamines by the similar disulfide-disulfide exchange reaction of low molecules catalyzed by a trace amount of thiols.5. Asymmetric disulfide-type thiamines such as thiamine benzyl disulfide and thiamine propyl disulfide were found to produce both protein-bound thiamine and protein-bound alkylmercaptan under the same conditions.6. Considering the results of this paper and those of the previous findings, it was concluded that there are three types of exchange reaction between thiamine derivatives and proteins.

Polyampholites of vinylpyridine or its alkyl derivatives with acrylic or methacrylic acid and the copolymers of these compounds with other vinyl derivatives were synthesized and studied as protective coating agents for tablets. Results showed that the synthesized copolymers had adequate viscoelasticity, protective qualities, and disintegration characteristics to serve as protective coating agents.