There are numerous organic compounds which possess both hydroxyl and amino groups in their molecule. Since O and NH₂ both have a mass number of 16, it becomes a problem which had been eliminated in fragmentation. The analysis of their fragmentation is often carried out by their labeling with ²H, ¹⁸O or ¹⁵N but labeling with isotopes is rather limited and the procedure is often difficult. Following previous reports on the mass spectrometry of aromatic compounds having both hydroxyl and amino groups in their molecule, particularly in those of antipyretics such as acetophenetidine (4-ethoxyacetanilide), pyrinazine (4-hydroxyacetanilide), and 4-aminosalicylic acid, their spectra were measured with a doublefocusing high-resolution mass spectrometer and their fragmentation was examined.

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Effect of {2-[4-(p-chlorophenyl)hexahydro-1H-azepin-1-yl] ethyl} guanidine sulfate (G-72) on several skeletal muscle praparations was examined, and following results were obtained. G-72 blocked neuromuscular transmission in sciatic-sartorius of frog, and this block was antagonized by guanidine and tetraethylammonium (TEA), but not by neostigmine. The mode of neuromuscular blocking action of G-72 was more similar to that of procaine than to those of decamethonium or d-tubocurarine. In rat diaphragm, it seemed that G-72 mainly acted on the neuromuscular junction and muscle, and its block was not antagonized by neostigmine, tetraethylammonium, decamethonium, d-tubocurarine and potassium ion. G-72 produced flaccid paralysis in hen sciatic-gastrocnemius and chicken. In some cases, G-72 and neostigmine were antagonistic, but not in other cases in hen sciatic-gastrocnemius. On frog rectus, G-72 showed dual action, i.e., competitive and non-competitive antagonism to ACh. G-72 had local anesthetic action as potent as procaine, and its action was more lasting than that of procaine as measured by intradermal weal test in guinea pigs. The concentration of G-72 required to block nerve-muscle preparation was higher in rat diaphragm than in frog sciatic-sartorius, but such difference in effective concentration was not observed with d-tubocurarine, decamethonium and procaine. From the results of experiments in hen sciatic-gastrocnemius and frog rectus, it may be concluded that G-72 would be a muscle relaxant of mixed type of type II and III (type II∼III).

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Inhibitory effects of several hopolone analogs and 2-methoxyphenol (IX) on catechol-O-methyltransferase (COMT) were examined, comparing with those of catechol (I) and pyrogallol (II) as controls, and the following results were obtained. COMT was partially purified by the method of Mavrides et al., and the enzyme activity was measured by the modified method of Pisano, using solvent extraction with isoamyl alcohol instead of chromatographic procedure. The inhibitions of I, tropolone (III), sodium salt of 4-isopropyltropolone (IV), 5-nitrosotropolone (V), 5-aminotropolone (VI), and 2-hydrazino-5-(p-methoxyphenoxy) tropone (VII) were competitive, and those of II and IX were mixed type, and their K_i values were determined. On the other hand, 2-methoxytropone (VIII) had no effect on COMT activity, suggesting that the hydroxyl in III was indispensable for the inhibitory effect. From the fact that VIII as metabolite of III could not be detected in the incubation medium, it was suggested that the mode of inhibition of III was different from those of polyphenols.

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Labeling of biotin with sulfur-35 was carried out in a medium containing Aspergillus niger with 4.5mCi of H₂³⁵SO₄ and dethiobiotin. A large amount of ³⁵S-amino acid besides ³⁵S-biotin and its related compounds (biotin fraction) were obtained. Following amino acids were identified by a two-dimensional paper chromatography : ³⁵S-Cysteinic acid, ³⁵S-cystine, ³⁵S-homocystine, ³⁵S-methionine, ³⁵S-methionine sulfoxide, and ³⁵S-methionine sulfone. About one-half of the added ³⁵S (46%) remained in the supernatant as non-reacted H₂³⁵SO₄ and or non-hydrolysed proteins, and a greater amount (89%) of radioactvities taken up by the cell were incorporated into ³⁵S-amino acids. In the biotin fraction, 0.7% of the total radioactivity was found. Biotin fraction was isolated as avidin complex, dialysed with ammonium carbonate, and identified chromatographically from radioactivity and by bioassay. From this experiment, biotin was found mainly in an oxidized form such as l-sulfoxide and l-sulfone.

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It has been known that bromo compounds are debrominated in vivo by four types of mechanisms, i.e., reduction, hydrolysis, dehydrobromination, and mercapturic acid synthesis. Of these, reductive debromination was found recently by Curry, Tamminen, and Butler to occur in two bromoacylurea hypnotics, Bromural and Carbromal. The present investigation was undertaken in order to reconfirm whether or not this reaction is common in substances with the structure of =CBrCONHCONH-, using Bromural, Carbromal, and Acetylcarbromal. Rabbits were used as experimental animals. The detection and determination of debrominated metabolites in urine, blood, and various organs were carried out by gas and thin-layer chromatographies. The metabolites obtained from urine extracts of rabbits receiving above three drugs were purified by column chromatography, isolated as crystals, and identified as 3-methylbutyrylurea (from Bromural), and 2-ethyl-butyrylurea (from Carbromal and Acetylcarbromal), respectively. It seemed very likely that acetylcarbromal was first hydrolyzed to Carbromal and then reduced to 2-ethylbutyrylurea. It may reasonably be concluded that the bromine atom in α-position of ureid compound was displaced with hydrogen, the ureid chain remaining unchanged. It seems very interesting that this reductive debromination occurs also in a rabbit, like in man, dog, and mouse.

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It has long been recognized that β-glucuronidase can hydrolyze the glycosidic linkage of a wide variety of O-glucuronides in vitro but no direct evidence has been available concerning their in vivo hydrolysis. The present investigation was undertaken to confirm whether or not the glucuronide, when administered intravenously, could be hydrolyzed by β-glucuronidase in vivo. For this purpose, p-nitrophenyl glucuronide was injected intravenously into a rat, and 24-hour urine was analyzed. This result showed that most of the administered dose was excreted unchanged, but a small portion was excreted as p-nitrophenol by the action of 4-glucuronidase in the bladder. By the effect of CCl₄ on the β-glucuronidase activity in vivo and in vitro, an evidence was obtained that such a difficult hydrolysis of the glucuronide in vivo was perhaps attributable to its difficult penetration into the cell.

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Antispasmodic action was examined in 67 kinds of basic amides and alkamine esters of alkoxybenzoic acid, using the excised intestine of male mice and by the Magnus method. In general, these compounds had a strong papaverine-like action and some of the basic amides showed atropine-like activity. The compound with a strong antispasmodic activity, 3-bromo4-butoxybenzoic acid dibutylaminoethylamide (PA-1), was examined by the Magnus method, using mouse intestine and it was found that the anti-barium chloride action of PA-1 was 3.94 times that of papaverine and that its anti-acetylcholine action was 0.0071 times that of atropine. PA-1 has a marked inhibitory action on rabbit intestine in vivo and also has a surface anesthetic activity. In the circulating system, PA-1 acted inhibitorily against isolated heart of a toad but had no effect on the vascular system of a toad hind leg. PA-1 showed transitory fall of blood pressure in the dog. Acute toxicity of PA-1 was 218.5 mg./kg. by subcutaneous injection in mice.

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Examinations were made on the sodium salts of sucrose, lactose, and maltose sulfates on their in vitro action on pepsin inhibition and their inhibitory action of pepticulceration in pylorus-ligated rats. It was there by found that even these low-molecular compounds like disaccharide sulfates had a strong anti-pepsin and anti-ulcer activities, comparable to those of sulfate esters of polysaccharides when highly sulfated, and these activities were found to increase with increasing number of the sulfate radical. Comparison among the disaccharides showed that the nonreducing sugar-type sucrose sulfate, having three primary hydroxyls in its molecule, had the strongest activity, and the reducing-type disaccharides, lactose and maltose sulfates, showed approximately the same effect. These facts seemed to suggest that the sulfate radical bonded to the primary hydroxyl took part in the appearance of the activity rather than that bonded to the secondary hydroxyl. Since the disaccharide sulfates showed a strong inhibitory action comparable to that of polysaccharide sulfates, the chief group responsible for the appearance of anti-ulcer effect of the sulfate esters of saccharides is the sulfate radical and that the degree of molecular polymerization was a supplementary factor in this effect.

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A new, non-phenolic alkaloid was isolated from a Formosan Stephania japonica MIERS and was named prometaphanine. From its various chemical degradation and spectral data, the structure of this alkaloid was determined as shown by formula (I), including its absolute configuration.

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Treatment of 3-nitro-2, 6-dimethyl-4-pyridinol (I) with phosphoryl chloride gives 3-nitro-4-chloro-2, 6-dimethyl-pyridine (II) in 61% yield, and the use of phosphoryl chloride and excess of phosphorus pentachloride results in chlorination of the side chain to give 2-tri-chloromethyl-3-nitro-4-chloro-6-methylpyridine (III) in 45% yield. In a similar manner, use of excess phosphorus pentachloride on 2, 6-dimethylpyridine (V) gave a minute amount of 4-chloro-2, 6-dimethylpyridine (VI) and 2-chloromethyl-6-methylpyridine (VII), as well as 2, 6-bis (trichloromethyl) pyridine (VIII) in 3% yield, and 2-trichloromethyl-3-nitro-6-methylpyridine (X) in 38% yield from 3-nitro-2, 6-dimethylpyridine (IX). The same treatment of 2, 6-dime-thyl-4-pyridinol (XI) gave 4-chloro-2, 6-dimethylpyridine (VI) in 68% yield and a minute amount of 2-trichloromethyl-4-chloro-6-methylpyridine (XII).

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An automatic melting point apparatus (Fig.1 and 2) was designed with the air bath furnace and sample holder which were previously reported.^{5, 6)} About 2 mg. of a sample is powdered and spread on one side of a piece of glass plate (10×75mm.), covered with another plate, and inserted into the furnace. The sample is irradiated by a lamp and the reflected scattering light is introduced into a phototube through a small lens. The temperature of the furnace is automatically elevated by turning the dial of a bimetal regulator, at the rate of about 6°/minute (Fig. 3). The heaters of the furnace are automatically intensified with increasing temperature by removing resistance by turns. The rise in temperature is stopped when the sample is melted and hence the scattering light decreased extremely, and the temperature is read on a mercury thermometer which is set close under the sample. The standard deviations of melting points measured in a wide range of temperature (Table I) proved that the apparatus was precise enough in the identification of organic compounds.

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A mechanism of color reaction of sulpyrine and secondary amines by potassium 1, 2-naphthoquinone-4-sulfonate has been studied. Reaction products of potassium 1, 2-naphthoquinone-4-sulfonate with sulpyrine, 4-methylaminoantipyrine, 4-aminoantipyrine, N-methylaniline, and N-methylbenzylamine were isolated by extraction with chloroform, followed by alumina chromatography, and recrystallization. Structures of these color reaction products were investigated by elementary and N-methyl analysis, nuclear magnetic resonance spectra, and visible absorption spectra. On the basis of these data, reaction mechanism of coloration of sulpyrine was considered as the initial hydrolysis of sulpyrine to 4-methylaminoantipyrine, attack of potassium 1, 2-naphthoquinone-4-sulfonate on nitrogen atom in the 4-position of 4-methylaminoantipyrine as an electrophilic reagent to form addition product of 1, 2-naphthoquinone-4-sulfonate tentatively, and desulfonation to give N-methyl-N-(1, 2-naphthoquino-4-yl)-4-aminoantipyrine as a pigment quantitatively. In the same way as sulpyrine, mechanism of color reaction of N-methylaniline by potassium 1.2-naphthoquinone-4-sulfonate was found to be the initial reaction of 1, 2-naphthoquinone-4-sulfonate with N-methylaniline to form 4-(N-methylanilino)-1, 2-naphthoquinone as the consequence of desulfonation. Coloration of other aromatic and aliphatic secondary amines was investigated, and secondary amines were found to react generally with potassium 1, 2-naphthoquinone-4-sulfonate to form colored substances. Mechanisms of these colorations have been interpreted in terms of electronic theory.

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5-Hydroxysparteine (I), the stereoisomer of retamine (II), was prepared by the reduction of 5-oxosparteine (V) with sodium borohydride. The hydroxyl group in 5-position with equatorial configuration was obtained almost selectively. By the utilization of the cyclic enamine activity of Δ⁵-dehydrosparteine (VI), 5-hydroxymethylsparteine (IX) was prepared via 5-ethoxycarbonyl-Δ¹⁽⁶⁾-dehydrosparteinium ion (VII) and -sparteine (VIII). In this case, both equatorial and axial hydroxymethyl compounds (IXa and b) were obtained.

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2-Benzyloxy-3, 4-dimethoxyphenethylamine (VIII) was synthesized from 2-benzyloxy-3, 4-dimethoxybenzaldehyde (VI) via ω-nitrostyrene derivative (VII) by the conventional method and condensation of VIII with p-methoxyphenacetyl chloride gave the amide (IX). The Bischler-Napieralski reaction of IX afforded the 3, 4-dihydroisoquinoline base but this was unexpectedly found to be the oxidation product (XI). Treatment of XI with methyl iodide, followed by reduction with sodium borohydride to the O-benzyl derivative (XII), and its debenzylation afforded the title compound (XIII).

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Ten kinds of pyrimidine series sulfanilamides, possessing methyl and methoxy groups in the pyrimidine ring, were prepared and their in vitro antibacterial activity was tested.

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Twenty-one kinds of fluorine-containing pyrimidinylsulfanilamide derivatives, possessing CF3_, CH₂F, or CF₃CH₂O group in 2- or 4-position of the pyrimidine ring, were prepared, their pK values were measured, and their in vitro antibacterial activity was tested.

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From the non-alkaloidal fraction of Rhazya stricta DECAISNE, ursolic acid, magnesium quinate, and two new flavonoid glycosides (rhazianoside A and B) were isolated. Hydrolysis of rhazianoside A (III), C₃₄H₄₂O₂₁, m.p. 250∼251.5°, with diluted acid yielded isorhamnetin, rhamnose, and galactose. Methylation of III with diazomethane followed by hydrolysis with diluted acid afforded 3, 7-dihydroxy-3', 4', 5-trimethoxyflavone. It is thereby suggested that the sugar residue of III was attached to C-3 and 7-position of isorhamnetin. Rhazianoside B (IV), C₃₄H₄₂O₂₁·3H₂O, m.p. 186∼187°, was considered to be an isomer closely related to III.

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The terpenic constituents of the leaf-oil of Chamaecyparis taiwanensis MASAM. were examined and 11 monoterpenes, 19 sesquiterpenes, and a diterpene have been identified. On the basis of the present results, the identity of "chamene" and "sesquichamene", both of which have been isolated earlier, with ocimene and thujopsene, respectively, is suggested. The biogenetic significance of the sesquiterpenic constituents of this also discussed.

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Examination was made on the seeds of Alpinia Kumatake MAKINO (A.formosana K.SCHUMANN) and quercetin, kaempferol, rhamnocitrin, and a new flavonoid derivative (kumatakenin), yellow needles of m.p. 246∼247°, were isolated by thinlayer chromatography and column chromatography. Analyses of kumatakenin agreed with C₁₇H₁₄O₆, involving two methoxyl and two hydroxyl groups. It gives 3, 5, 7, 4'-tetrahydroxyflavone (kaempferol) by demethylation and 3, 5, 4'-trihydroxy-7-methoxyflavone (rhamnocitrin) by partial demethylation. UV and NMR spectra of kumatakenin indicated that it is a kaempferol derivative with hydroxyl groups in 5- and 4'-positions, and methoxyl groups in 3- and 7-positions. Thus, the structure of kumatakenin has been established as 5, 4'-dihydroxy-3, 7-dimethoxyflavone.

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Some considerations were made on the action mechanism of 2-naphthalenealkylamines, with O-7 carbons in the alkyl chain, and their N-alkyl- or N-aralkyl-N-methyl derivatives by measuring their kinetic activity, applying Fergusson's rule to their pharmacological activity. It was thereby found that the atropine-like and antihistamine activities of these compounds are specific action and contribution of the physicochemical properties of the base is very small. On the other hand, their papaverine-like action in the intestine is not so specific like atropine-like activity although in a strong base like the compounds of this series, a certain contribution of the cation is expected. Local anesthetic action was found to be non-specific, being dependent on the physicochemical properties of the non-ionic base or neutral molecule. This fact was further endorsed by the relationship between surface anesthetic action and pH.

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Biological change of sulfisomezole (N¹-(5-methyl-3-isoxazole) sulfanilamide) in the human body was examined. Human urine after administration of sulfisomezole was mixed with activated charcoal, the adsorbed charcoal was eluted with ammonia-alkaline solvent, and the solvent was concentrated in a reduced pressure. The concentrated solution was passed through a column of Dowex 50-X8, the column was eluted with water, and the aqueous eluate was passed through a column of Amberlite IRA-68. The column was eluted with diluted ammonia water, the precipitate formed by the addition of lead acetate was removed, and the substance precipitated by basic lead acetate was collected by filtration. The precipitate was dissolved in ammonium sulfate solution and filtered, and the filtrate was passed through SE-Sephadex C-25 medium. The solution was neutralized with 0.1N sodium hydrogencarbonate and submitted to paper partition chromatography. The exlracted solution was passed through columns of Amberlite IRP-64 and Dowex 4, and evaporation of the effluent afforded a crystalline residue. Hydrolysis of this crystalline residue afforded sulfisomezole and glucuronic acid in 1 : 1 molar ratio. Their binding locus was examined by the comparison of Rf values of the conjugate in paper partition chromatography developed with neutral and alkaline media, study of its solubility, and the absorption bands of SO₂ at 1138 and 971cm^-1 in the infrared spectrum of These evidences indicated that this compound is an imide-type conjugate the glucuronic acid attached to the nitrogen in the isoxazole ring. Therefore, the biological product of sulfisomezole is a sodium sulfisomezole 2'-glucosiduronate containing 1 mole of water of crystallization.

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Three kinds of biological products were found in the human urine after administration of sulfamonomethoxine (I). The unchanged I and N⁴-acetylsulfamonomethoxine were identified by comparison with authentic samples by paper chromatography. The other was extracted by the following procedure and identified as sulfamonomethoxine-N¹-glucosiduronic acid, N¹-[N¹-(4-methoxy-6-pyrimidinyl) sulfanilamido] glucopyranosiduronic acid, which was synthesized by a separate method. Extraction of the biological product of I was made by adsorption on activated charcoal, its elution with ammonia alkaline solvent, and concentration of the extract solution. The extract solution was treated consecutively with Dowex 50W-X8 and Amberlite IRA-68, by precipitation With lead acetate and basic lead acetate, paper partition thromatography, and further treatments with Duolite C-10 and Dowex 4, finally affording a crystalline residue. For the synthesis, potassium salt of I was condensed with methyl (2, 3, 4-tri-O-acetyl-α-D-bromo) glucopyranosiduronate, the condensate was hydrolyzed with ammonia, and purified by treatments with Dowex 50W-X8, Amberlite IRA-68, Duolite C-10, and Dowex 4. The extracted crystals and the synthesized products agreed well in elemental analytical values, paper chromatographic results, and ultraviolet and infrared spectral data. Absorption band of SO₂ at 1160cm^-1 in their infrared spectra indicated that the glucuronic acid is bonded at N¹-position.

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N¹-(4, 6-Dimethoxy-, 6-chloro-4-methoxy-, 4-methoxy-6-methylamino-, and 4-methoxy-5-pyrimidinyl)sulfanilamides were synthesized, and their antibacterial activity was examined. Only N¹-(4-methoxy-5-pyrimidinyl) sulfanylamide showed a slight activity against Shigella flexneri 2a.

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Effect of hexylguanidine sulfate on rat diaphragm and intradermal weal test in guinea pig was examined, and following results were obtained. In rat diaphragm, it seemed that hexylguanidine acted on the neuromuscular junction and muscle. Antagonism between hexylguanidine and potassium ion, neostigmine, tetraethylammonium, d-tubocurarine and decamethonium was tested and it was found that hexylguanidine was not antagonized by them. Effect of hexylguanidine on rat diaphragm was different from that on sciatic-sartorius of frog in site and mode of action previously reported. The concentration of hexylguanidine required to block nerve-muscle preparation was higher in rat diaphragm than in frog sciatic-sartorius, but such difference in effective concentration was not observed with d-tubocurarine, decamethonium and procaine. Hexylguanidine had local anesthetic action as potent as procaine, and its action was more lasting than that of procaine in intradermal weal test of guinea pig.

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Ethereal extract of the roots of Angelica polymorpha MAXIM. afforded eight crystalline compounds upon column chromatography over silica gel. Seven of these were known coumarins ; oxypeucedanin (I), osthol (II), imperatorin (III). psoralen (IV), bergapten (V), oxypeucedanin hydrate (VI). and byak-angelicin (VII). The other (VIII) was identified as 3'-acetate of hamaudol⁴⁾, a 2-methylchromone derivative isolated from the roots of Angelica japonica A.GRAY³⁾.

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The chemical shift of methyl-proton of bisflavone derivatives in NMR spectra (in pyridine) was compared with that of apigenin series flavones (Table II) and the shifts were assigned as shown in Tables I and III. From the consideration of the chemical shift of the methoxyls at 5"- and 7"-positions in bisflavones of the biflavonyl ether type, possibility of C₄'-O-C₈" bonding in the structure of hinokiflavone (I) is denied and it is clear that the hitherto proposed bonding of C₄'-O-C₈" would be the correct one. If the bonding C₄'-O-C₈" is correct, the steric structure in Chart 1 indicates that 5"-position would receive stronger paramagnetic effect than the methoxyl in 5-position and 7"-position would receive weaker paramagnetic effect than the 7-position, which fact agrees with the values found by NMR spectral measurements.

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