Reserpine was separated by paper electrophoresis from the total alkaloids of Rauwolfia serpentina BENTH. Electrophoresis was carried out in a special apparatus in which two paper strips were held in a modified Liebig condensers to avoid evaporation and rise of temperature during operation. Pure reserpine, total alkaloids, and three fractions extracted from the total alkaloids with chloroform in different buffer solutions (pH 6.5, 6.7, and 12.0) were used as samples. Migration was carried out in 5N acetic acid as the electrolyte, at 700V./33cm., 0.3-0.4mA./cm., for 2 hours at 25-27°. The spots were detected by fluorescence under ultraviolet light. Nine separate spots were observed on tbe strip of total alkaloids. Migrated distance of pure reserpine and that of fraction C (pH 6.5) were 105 and 102mm., respectively, under the same conditions.

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The present experiment was carried out to attempt determination of reserpine quantitatively on filter paper by fluorometry. Reserpine or its fresh solution does not exhibit fluorescence under ultraviolet rays. Stock solution of reserpine dissolved in chloroform or methanol is comparatively stable, but when 5N acetic acid solution was stored at room temperature, a new absorption maximum at 380mμ was observed which was not detected in the spectrum of reserpine. At the same time, a marked fluorescence was observed under ultraviolet rays and it was assumed that the fluorescence must be due to the degradation product of reserpine.
Same fluorescence was observd under irradiation of ultraviolet ray on the spots of reserpine on filter paper. The fluorescence of spots became more intense when the paper was heated at 105° in an atmosphere of mixed vapor of hydrogen peroxide and acetic acid. Various amounts of reserpine (0.2-1.5γ) were spotted on Whatman No. 1 filter paper, treated as above, cooled, and the fluorescence intensity at 500±50mμ was measured using an instrument designed for the determination of fluorescence intensity on filter paper. As a result, linear relationship was found to exist between the amount of reserpine and intensity of fluorescence.

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Syntheses of 3-phenylbenzo-1, 4-thiazine by the condensation of o-aminothiophenol and ω-bromoacetophenone and by the reduction of ω-(o-nitrophenylthio) acetophenone were carried out and their nature was clarified. Attempt to introduce a basic side chain in the nitrogen was unsuccessful but 3-phenyl-4-(ω-dialkylaminoalkyl) benzo-1, 4-thiazine was obtained by the condensation of ω-bromoacetophenone with o-(ω-dialkylaminoalkyl) aminothiophenol, already introduced with a basic side chain by the reaction of benzothiazolinone with dialkylaminoalkyl chloride, followed by hydrolysis with potassium hydroxide.

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1, 2, 3, 4-Tetrahydrophenothiazine was prepared by the condensation of o-aminothiophenol and 2-chlorocyclohexanone or by the reduction of o-nitrophenyl 2-oxocyclohexyl sulfide. Attempt to introduce a basic side chain into this thiazine compound was unsuccessful and 10-(ω-dialkylaminoalkyl)-1, 2, 3, 4-tetrahydrophenothiazine was finally obtained by the condensation of 2-chlorocyclohexanone and o-(ω-dialkylaminoalkyl) aminothiophenol, already introduced with the required side chain. Dehydrogenation of 1, 2, 3, 4-tetrahydrophenothiazines with sulfur or chloranil afforded the objective phenothiazines, though in a low yield and with liberation of the side chain when a basic side chain was present.

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By the reaction of o-aminothiophenol and ethylene oxide or ethylene chlorohydrin, o-(2-hydroxyethyl) thioaniline was prepared and its dehydrative cyclization by heating its hydrochloride afforded 2, 3-dihydrobenzo-1, 4-thiazine, which was reacted with ω-dialkylaminoalkyl chloride to form 4-(ω-dialkylaminoalkyl)-2, 3-dihydrobenzo-1, 4-thiazine. As another route of synthesis, the same compounds was obtained by the cyclization of N-(ω-dialkylaminoalkyl)-o-(2-hydroxyethyl) thioaniline, formed by the reaction of o-(ω-dialkylaminoalkyl) aminothiophenol with ethylene oxide or ethylene chlorohydrin.

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Synthesis of 1, 4-thiazine, the fundamental skeleton of phenothiazine and benzo-1, 4-thiazine, was examined and 3, 5-diphenylthiazine was prepared from phenacyl sulfide and ammonia and 3, 5-diphenylthiazine dioxide from phenacyl sulfone and ammonia or formamide. These all afforded the corresponding thiamorpholine by reduction.

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Comparative examinations of pharmacological actions were made on derivatives of 1, 2, 3, 4-tetrahydrophenothiazine, 3-phenylbenzo-1, 4-thiazine, and 2, 3-dihydrobenzo-1, 4-thiazine with dialkylaminoalkyl group bonded to nitrogen, thought to have structures similar to Chlorpromazine. In general, toxicity of these derivatives was weaker than that of phenothiazine derivatives and the toxicity became far weaker with the introduction of chlorine into the ring. Antihistaminic action of these derivatives was stronger than their antiacetylcholine action, while some of them extended the duration of Thiopental anesthesia, this action being intensified by the introduction of chlorine into the ring. Temperature depression action was the strongest in phenothiazine derivatives while the compounds in general effected depression of blood pressure and the action was intensified by the presence of chlorine.

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Separation of morphine for its determination in opium preparations by polarography was reëxamined since the earlier process of its extraction was not satisfactory from the point of expenses and procedures. It was thereby found that separation by ion exchange resin was a good process. Using the strongly basic anion exchange resin, Amberlite IRA-411, and with N hydrochloric acid as the eluant, morphine was determined in opium alkaloid hydrochloride injection, opium extract, opium tincture, and opium powder.

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The principle which causes artificial seminal discharge in male bullfrog (Rana nigromaculata HALLOWELL) or the so-called Mainini reaction, was separated by adsorption with benzoic acid from the 0.025% ammonia extract of chorionic villus (human placenta at the age of 2-3 months) and purified by fractional precipitation with ethanol. It was made into a lyophilyzed product having high purity by electrophoretic and ultracentrifugal analyses. The substance having this action was tentatively designated as MG. The product here obtained possessed a potency of 800-1500m.e.d./mg. (frog unit).
From the result of various coloration reactions, MG was assumed to be a glycoprotein and the sugar in MG was determined by paper chromatography as galactose. It was later learned that MG contained 13.19% of galactose and 4.65% of hexosamine, their ratio being about 3:1.

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Various physical constants were determined of the principle (MG) of the Mainini reaction, isolated from human placenta, as a product of approximately single component according to electrophoretic and ultracentrifugal examinations. From the determination of its mobility rate (Table I and Fig. 2), isoelectric point of MG was found to lie between pH 4.1 and 4.2. Its diffusion constant was D_20W=2.53×10^-7cm.²/sec. and sedimentation constant, S_20W=2.55×10^-13(cm./sec.)/(dyne/g.). The molecular weight of MG calculated from these constants is about 100, 000 and about 150, 000 from light scattering analysis. Such a large difference due to the method of determination was assumed to be caused by the high content of sugars. The MG molecule is assumed to take rod form of extremely elongated type, showing friction ratio f/f₀=2.68, axial ratio of prolate 29.4-33.3, and oblate 40-55.

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It had been found that the most general and sensitive metal ion to be used in the precipitation reaction of metal ions and meconic acid derivatives was Hg²⁺ and the precipitate from meconic acid and Hg²⁺ was examined. It was thereby found that this newly formed precipitate was a labile O-hydroxymercurimeconic acid and when it was dissolved in 2% sodium carbonate and reprecipitated with acetic acid, it changed to 6-hydroxymercuricomenic anhydride (?). Further treatment of this compound with 0.2 N hydrochloric acid at room temperature effected transition of mercury, forming 6-chloromercuricomenic acid. The position of the chloromercuri group was determined by deriving the compound to 6-bromocomenic acid with bromine and then to ethyl 6-hydroxy comenate. On boiling O-hydroxymercurimeconic acid with water, it afforded a compound considered to be a different hydroxymercuricomenic anhydride (?), which gave Positive reaction to ferric chloride in 0.2 N hydrochloric acid and liberated mercuric oxide with N sodium hydroxide, forming a different chloromercuricomenic acid.

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Titration curves of meconic acid derivatives with 0.1 N sodium hydroxide were comparatively examined with those of benzene ring compounds of the same type and it was thereby learned that the compound of m.p. 164° reported to be monomethyl meconate was dimethyl meconate. Pyromeconic acid shows far stronger jump in its curve than phenol while there are two clearly distinguishable jumps in the curve of comenic acid. The second jump is the same as that of pyromeconic acid but the 3rd jump due to the hydroxyl group in meconic acid was indistinct. In 6-hydroxycomenic acid, only the 2nd jump was clear and 1st and 3rd jumps were indistinct. Ethyl 6-hydroxycomenate was titrated as a monobasic acid from its two hydroxyl groups. These esters dissolve in N sodium hydroxide and afforded the corresponding acid easily by its acidification. The warming of its ethyl and methyl esters with corresponding alcohol, with hydrochloric acid as a catalyst, caused reversible transesterification but only the esters of comenic acid and O-methyl comenic acid underwent the same reaction when potassium hydrogen carbonate was used as the catalyst.

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The structure, absorption spectrum, and stability of uranyl chelate compounds of β-diketones, benzoylacetone (I), dibenzoylmethane (II), 2-furoylbenzoylmethane (III), and ω-isonicotinylbenzoylmethane (IV) were studied. The composition of such uranyl chelate compounds was found to be 1 mole of uranyl to 2 moles of β-diketone, and (I) and (II) possesses 2.5 moles and (III) 1.5 moles of the water of crystallization. The chelate compounds are soluble in organic solvents such as alcohols, ether, and pyridine, but are sparingly soluble in water. Their ultraviolet spectra in ethanol exhibited absorption maxima at 375 mμ (ε 6, 300) in (I), at 395 mμ (ε 16, 500) in (II), at 404 mμ (ε 23, 300) in (III), and at 400 mμ (ε 17, 700) in (IV). The value of ε tends to become larger as the absorption maximum shifts to a longer wave length range. Stability of the compounds was in the order of (I), (II), (III), (IV), and the stability increases with increased concentration of ethanol.

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Structure, ultraviolet absorption spectrum, and stability of uranyl chelate compounds of o-, m-, and p-nitrodibenzoylmethane (I, II, and III) and m- and p-aminodibenzoylmethane (IV and V) were examined. It was found that the composition of such a chelate compound is made up of 2 moles of the β-diketone to 1 mole of uranyl and that (II) and (III) possess 1.5 moles of the water of crystallization while the others do not. These chelate compounds dissolve in organic solvents such as alcohols, ether, and pyridine. Absorption maximum in their ultraviolet spectrum, measured in ethanol solution, is at 390mμ (ε 12, 300) in (I), at 400mμ (ε 15, 000) in (II), at 406mμ (ε 16, 400) in (III), at 400mμ (ε 16, 400) in (IV), and at 410mμ (ε 30, 000) in (V). The value of ε tends to become larger as the absorption maximum shifts to a longer wave length range. The stability becomes greater in the order of (I), (III), (II), and (IV) is more labile than (II). Stability of (V) could not be determined.

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Structure, ultraviolet absorption spectrum, and stability of uranyl chelate compounds of o-, m-, and p-methoxydibenzoylmethane (I, II, and III) were examined. The structure of these chelate compounds is made up of 2 moles of the β-diketone to 1 mole of uranyl and none of these compounds possess water of crystallization. The compounds are soluble in organic solvents such as alcohols, ether, and pyridine. Absorption maximum in their ultraviolet spectrum is at 400 mμ (ε 14, 500) in (I), at 400 mμ (ε 16, 800) in (II), and at 404 mμ (ε 20, 900). The stability is in the order of (I), (III), (II) and they are more labile than the corresponding nitro compounds or dibenzoylmethanes, In other words, chelate compounds possessing electron-releasing groups as the substituent are more labile than those with electron-attracting groups.

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Colorimetric determination of UO₂²⁺ with β-diketone was examined. The β-diketones used were benzoylacetone (BA), dibenzoylmethane (DBM), isonicotinylbenzoylmathane (INBM), 2-furoylbenzoylmethane (FBM), and o-, m-, and p-methoxydibenzoylmethane (o-, m-, and p-MDBM). Absorption spectra and the effect of the amount of reagents, pH, and ethanol were examined and calibration curve under optimal conditions was prepared. In this analysis, 1cc. of 0.2% β-diketone solution was added to 5cc. of the sample solution of UO₂²⁺, 99% ethanol was added to make the whole volume 10.0cc., and optical density at each absorption maximum was determined. The curves became linear in the range of 0-120γ of FBM, 0-150γ of p-MDBM, 0-160γ of INBM, 0-160γ of DBM and m-MDBM, 0-180γ of o-MDBM, and 0-400γ of BA. indicating that colorimetric determination is possible. Of these β-diketones, the most sensitive was FBM, followed by.p-MDBM and INBM, both being better in sensitivity than DBM. o-MDBM and BA showed lower sensitivity than DBM.

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Pharmacognostical studies were made of Forsythia suspensa VAHL, F. viridissima LINDL., F. koreana NAKAI, and Syringa dilata NAKAI (Oleaceae-family). It was revealed that it is possible to determine the difference in capsules of these plants from morphological and anatomical examinations and that there is a structural difference in the pericarp and peduncle of F. suspensa and F. viridissima.

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A method was devised whereby dielectric constant of a microamount of substances could be measured with simplicity, high sensitivity, and in a high degree of stability by an operation designed to bring the Lissajou's figure of the brawn tube to a straight line. One of the principles of this method lies in the fact that the phase difference in the circuit is compensated by the fine adjustment of the variable condenser inserted in parallel with the sample and to catch this at the moment at which the Lissajou's figure changes from ellipse to a straight line. The sensitivity at the time of measurement is especially extremely high since this moment is selected so that the pair of parallel resonance circuits is in perfect resonance. Another is the fact that the variation in the voltage of electric source changes only the length and brightness of the Lissajou's figure and the moment that the figure itself changes is only when oscillating frequency changes. Moreover, there is no objection, theoretically, of fixing the oscillating frequency and therefore, crystal oscillator is used in this devise. Therefore, stability is good, voltage stabilizer is not required, and there is no variation even if the period of determination extends over 20 to 30 hours, Since this method possesses high sensitivity and high stability, the amount of the sample required is very small, only 0.5-10cc. of a solution or 1-10mg. of a compound for the measurement of dipole moment will suffice. Further, since the measurement is made by visual observation, operation is very simple and sensitivity is good, and the apparatus can be constructed at a comparatively low cost because the circuit is comparatively simple.

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It was shown earlier that antifungal activity of p-hydroxybenzoic esters decreases in spite of increased solubility by the use of Tween 20 and it was concluded that the antifungal effect is participated by the antifungal agent dissolved in an aqueous phase outside the micelle and is dependent on the size of hydrophilic properties of the esters.
In the present series of experiments, solubility of the esters and surface tension in a low-concentration solution (below 0.1%) of Tween 20, and antifungal effect and apparent solubility of butyl p-hydroxybenzoate were also examined (Fig. 3). The solubility of the esters decreases once until the concentration of Tween 20 reaches a definite level and the surface tension becomes the minimum at this concentration. This point is thought to be the critical micelle concentration of a complex of Tween 20 and the ester. The antifungal activity of the butyl ester reaches the maximum at this point. From such facts, the maximum effect of antifungal activity at critical micelle concentration is assumed to be due to increased permeability of the antifungal agent into fungi by the lowering of surface tension.

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Ethyl 2-(ethoxycarbonylmethylaminomethyl) isovalerate (VIII) was prepared from ethyl isovalerate (I), ethyl 2-bromoisovalerate (III), or ethyl 2-cyanoisovalerate (VI). Dieckmann reaction of (VIII) effects cyclization and oxidation to form ethyl 3-hydroxy-4-isopropyl-2-pyrrolecarboxylate (IX). It was found that the Dieckmann reaction of ethyl 2-(ethoxycarbonylmethylaminomethylene) isovalerate (V) under the same conditions afforded (IX) in a better yield.

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Hydroxypyrrole (I) does not react with carbonyl reagents and its reaction with hydrazine hydrate results in the formation of the hydrazide of the ester group and the hydroxyl group remains intact. It does not undergo the Cope reaction. It follows that the formation of a β-keto ester type in (I) is quite little due to the effect of the pyrrole ring. O-Tosylation, O-acetylation, and O-ethoxycarbonylation of (I) respectively afforded (IV), (V), and (VI), all of which were negative to the ferric chloride reaction. Benzylation of (I) afforded a mixture of the O-benzyl compound (VII), m.p. 70-71°, and C-benzyl compound (VIII), m.p. 108°.

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O-Substituted hydroxypyrrole compounds (II, III, and IV) were derived to O-substituted N-ethoxycarbonylhydroxypyrroles (V, VI, and VII) and catalytically hydrogenated over Raney nickel or platinum oxide at high or ordinary pressure to effect ring hydrogenation and liberation of O-substituted hydroxyl to ethyl 1-ethoxycarbonyl-4-isopropyl-2-pyrrolidinecarboxylate (VIII). Hydrolysis of (VIII) gave an amino acid, 4-isopropyl-2-pyrrolidinecarboxylic acid (IX), which colors yellow with ninhydrin. (IX) was also synthesized from methyl 4-isopropyl-2-pyrrolecarboxylate (XI) by another route.

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A new method of synthesizing 2-carboxy-4-isopropyl-3-pyrroleacrylic acid (II) was established by the mutual dehydration of aminoketones, such as 1-amino-3-methyl-2-butanone and ethyl oxalylcrotonate. Catalytic reduction of (II) with platinum oxide catalyst at room temperature effected reduction of the double bond in acrylic acid to form 2-carboxy-4-isopropyl-3-pyrrolepropionic acid (III). Another method of ethoxycarbonylation of methyl 3-acehyl-4-isopropyl-2-pyrrolecarboxylate (V) and methyl 1-ethoxycarbonyl-3-acetyl-4-isopropyl-2-pyrrolecarboxylate (VI) was attempted but did not materialize.

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Methyl 2-methoxycarbonyl-4-isopropyl-3-pyrrolepropionate (II) was derived to its potassium salt, reacted with ethyl chloroformate to form methyl 1-ethoxycarbonyl-2-methoxy carbonyl-4-isopropyl-3-pyrrolepropionate (III), which was reduced with platinum oxide catalyst to effect reduction to the pyre olidine ring, and finally hydrolyzed to obtain 2-carboxy-4-isopropyl-3-pyrrolidinepropionic acid (V), corresponding to homodihydrokainic acid.

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Methyl 1-ethoxycarbonyl-2-methoxycarbonyl-4-isopropyl-3-pyrrolidinepropionate (I) was derived to 2-ethoxycarbonyl-4-isopropyl-2-azabicyclo[3.3.0]octan-8-one (III) by the Dieckmann cyclization reaction and submitted to isonitrosation, Beckmann rearrangement, and hydrolysis to obtain dihydrokainic acid (VI), which was purified and confirmed through its methyl ester.

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By the reaction of N-acetyl-p-toluidine and chloral in conc. sulfuric acid, with the addition of phosphoryl chloride, at 25-35°, an acid ArCHClCOOH and a small amount of ArCHClCCl₃ were obtained. The latter does not undergo decomposition under these conditions so that the formation of the acid must be due to the decomposition of chloral to an acid and its bonding with the aromatic ring. When the same reaction is carried out at 70°, there is formation of ArCHO and Ar₂CHCOOH as by-products which are formed secondarily from the corresponding hydroxy acid formed from ArCHClCOOH. Since these aldehyde and acid are not formed at a low temperature, the acid formed by the decomposition of chloral must be ⁺CHClCOOH and not ⁺CH(OH)COOH.
It may be assumed that chloral undergoes the same decomposition in conc. sulfuric acid as in the presence of alkali, even in a slight degree, to form ^-CCl₃ and ⁺CHO, which makes it possible to understand the formation of chloralide by the foregoing mechanism. In this reaction, ^-CCl₃ combines with ⁺CHClCOOH to form CCl₃CHClCOOH, which changes to the corresponding α-hydroxy acid, and finally combines with chloral to form the chloralide. At lower temperatures, chloralide is hardly formed and the fact that it is formed in a large amount under conditions that allows the formation of ArCHO and Ar₂CHCOOH secondarily from ArCHClCOOH supports this assumption.

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Oleanolic acid was isolated from the pericarp of Forsythia suspensa VAHL by methanol extraction.

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Determination of aminopyrine in a mixed preparation of aminopyrine, phenacetine, and caffeine through infrared absorption spectrum was examined, It was found that aminopyrine alone could be determined easily as a one-componental system taking the absorption at 14.46 μ as the key band, and that the accuracy of the determination was 100±2% against the weight taken. This analytic procedure is simple, takes only a short time, and only a small amount of the sample is required.

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8-Azaguanine is fairly stable to both acids and alkalis but undergoes decomposition if heated with either of them in a sealed tube. Alkaline decomposition afforded 4(5)-amino-1, 2, 3-triazole as the product.

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From the yellowish crystalline powder, obtained by the hydrolysis of 1-ethoxy-carbonyl-2-carboxy-3-cyanomethyl-4-isopropylpyrrolidine, dihydrokainic acid, C₁₀H₁₇O₄N, was successfully isolated and purified as colorless prisms, m.p. 253° (decomp.). Infrared absorption spectrum and Rf value of this substance were measured.

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