Reductive cyclization of 2′-nitroacetanilide to 2-methylbenzimidazole was examined and the use of sodium dithionite was found to give a better yield, with added advantage of simplicity of experimental procedures. This method was applied to 4′-substituted 2′-nitroacetanilides and 5 (6)-substituted 2-methylbenzimidazoles were obtained in a satisfactory yield. Examination of the ease or difficulty of reductive cyclization according to electron-repulsion and electron-attraction groups indicated that there is no effect from such substituents present.
Drug receptor in smooth muscles was examined with vascular strips from a guinea pig, chiefly using reversible antagonists, and following facts were revealed. 1) Sensitivity of vascular smooth muscles is similar against adrenaline, phenylephrine, histamine, and barium chloride, and a definite dose-response curve was obtained for each agent. 2) In general, following antagonistic phenomena were observed with reversible antagonists in vascular smooth muscles. (a) Sympatholytics showed reversible, competitive antagonism against adrenaline but showed reversible, non-competitive antagonism against barium chloride and histamine. (b) Analgesics showed reversible, non-competitive antagonism against adrenaline while anticonvulsants of strong bases, such as Benactyzine, showed a reversible, non-competitive antagonism against barium chloride and those of weak bases, such as papaverine, a reversible competitive antagonism. (c) Vasodilators like sodium nitrite and aminophylline showed reversible, non-competitive antagonism against adrenaline. (d) Parasympatholytics showed reversible, non-competitive antagonism against adrenaline, barium chloride, and histamine. 3) The mode of action of various anti-convulsants against vascular smooth muscles, i.e. effectivity ratio of the inhibitory action of various anti-convulsants, and antibarium action suggested that pharmacological properties of vascular smooth muscles is midway between that of tracheal smooth muscles and intestinal smooth muscles.
Drug receptor in smooth muscles was examined with vascular strips from a guinea pig, chiefly using irreversible antagonists, and following facts were revealed. 1) The vascular smooth muscles showed the following phenomena: (a) 2-Haloalkylamines showed irreversible antagonism against adrenaline and tolazoline are protective against this irreversible inhibition but not cysteine. From this result site of action of 2-haloalkylamines is assumed to be the adrenergic receptor. These 2-haloalkylamines showed irreversible antagonism against barium chloride and histamine in about 100 times higher concentration than against adrenaline. Application of a high concentration of barium chloride and 2-haloalkylamines at the same time results in protection against contractive action of barium chloride. This fact suggests that there is a specific receptors for histamine and barium chloride besides the adrenergic receptor in the vascular smooth muscles taking part in the mechanism of muscle contraction. (b) SH-Inhibitors also showed irreversible antagonism against adrenaline, while adrenaline had no protective action against this phenomenon due to its change by heavy metals. Phenylephrine, tolazoline, and cysteine had protective action against this irreversible inhibition. This fact suggests that the site of action of SH inhibitors is the adrenergic receptor, especially its SH group. 2) The foregoing facts suggest that the receptor in the vascular smooth muscles has an SH group, which binds with heavy metals and is protected by cysteine, and a functional group which binds with 2-haloalkylamine and is not protected by cysteine.
Drug receptor in smooth muscles was examined with vascular strip from a guinea pig and following conclusions were drawn with regard to vascular smooth muscles. 1) Adrenaline, noradrenaline, and phenylephrine effect the strongest contraction, followed by histamine and barium chloride. Acetylcholine and posterior pituitary hormone have only a weak activity. 2) Vascular strip which had undergone the maximum contraction by the action of noradrenaline and phenylephrine does not undergo further contraction on addition of adrenaline but the strip contracted by the action of barium chloride, histamine, and acetylcholine undergoes further contraction on addition of adrenaline, to the same degree as that caused by single addition of adrenaline. Higher concentration of ephedrine inhibits the action of adrenaline. 3) Morphine and Aspaminol show reversible, competitive antagonism against barium chloride. Morphine, which does not affect contraction by adrenaline, does not antagonize irreversible, non-competitive anti-adrenaline action of Aspaminol. This point differs from intestinal smooth muscle. 4) Reversible, competitive antagonism against barium chloride appears at pH 6.25 and 5.83, from which the apparent pKa of the barium receptor is calculated as 7.37.
Iron chelate compound of dehydroacetic acid, differing from other metal chelates of this acid, is fairly soluble in organic solvents. In the present series of experiments, structure of this chelate was examined and the chelate of iron (II) was found to be composed of 1:2 of iron and dehydroacetic acid, with two moles of water of crystallization. The chelate of iron (III), on the other hand, consists of 1:3 ratio of iron to dehydroacetic acid and is free from crystal water. Examination of iron (III) chelate by the continuous variation method showed that this chelate compound had the same composition even in aqueous solution and its stability constant was found to be 1×1010. Determination of iron ion with dehydroacetic acid by utilization of this chelate formation gave a fairly satisfactory result in the case of iron (III). In this case, the chelate was extracted with chloroform at pH 5.1 and absorbance of the extract solution was measured at 420mμ.
In continuation of previous works, X-ray diffraction pattern, specific gravity, and gram volume of pentobarbital calcium and calcium pyruvate isoniasone were examined. Pentobarbital calcium tablet is similar to calcium p-aminosalicylate in that its tablet disintegration requires longer time with passage of time, while the tablets of calcium pyruvate isoniasone produce cracks and self-destruction in the air. Changes in the state of crystal water were also examined and relationship of these physical data with retarded disintegration and self-destruction of these tablets was also examined. Pentobarbital dihydrate, similar to calcium p-aminosalicylate, becomes amorphous when dehydrated and transition state of hydrate-anhydrate appears over a long period of time. Gram volume of the dihydrate is greater than that of anhydrate. If the tablet is formed from granules partially in amorphous state by drying and other condition, and allowed to absorb moisture, the tablet will not disintegrate for more than 30minutes whereas tablets immediately after tabletting underwent disintegration in a few minutes. Calcium pyruvate isoniasone differs from calcium p-aminosalicylate and forms a hydrate with different crystal lattice, between the stable heptahydrate and trihydrate. Self-disintegration of this tablet is considered to be due to changes in crystal structure between the heptahydrate and trihydrate. Vapor pressure of heptahydrate crystal is high and the gram volume of stable heptahydrate is small, contrary to the case of calcium p-aminosalicylate and pentobarbital calcium, with extremely short period of transition state. Tabletting of granules mixed with the trihydrate will result in sudden appearance of distortion in a tablet by absorption of moisture, accompanying lattice formation. Therefore, such a tablet undergoes complete self-destruction within 20-30minutes in a room with relative humidity of 76% and loses the original form.
Based on the report that formylantipyrine thiosemicarbazone has growth-inhibitory action on tubercle bacilli comparable to that of thioacetazone (p-acetamidobenz-aldehyde 3-thiosemicarbazone), the present series of work was carried out to make further examination on antibacterial activity of antipyrine ring, as one of heterocyclic rings. In the present work, antipyrine derivatives of 4-thiazoline-2-thione, dithiocarbamic acid ester, and rhodanine system were prepared and their antibacterial action in vitro was examined with human-type tubercle bacilli, Staphylococcus aureus, and Esherichia coli. None of these compounds had any marked growth-inhibitory action on the foregoing bacteria.
Fifteen kinds of new compounds of the general formula R-CH=N-NH-CSN-R′R″, where R′ is an antipyrinyl ring, were synthesized, as well as three of their cyclization product, 3-(4-antipyrinyl)-2-arylidenehydrazono-4-methyl-4-thiazolines, and their antibacterial activity was examined. These compounds in general had only a weak activity and only (XVI) showed somewhat weak growth-inhibitory action against tubercle bacilli and Stapylococcus aureus.
Sulpyrine was decomposed with N sodium hydroxide solution and the route of its decomposition was assumed to be through 4, 4′-(N, N′-dimethylmethylenediamino)antipyrine (II) and 4-methylaminoantipyrine (I), rather than in the reverse order of (I) to (II), as assumed by Takahashi, et al. for its decomposition with dilute hydrochloric acid. Sulpyrine is decomposed quantitatively into (N-(4-antipyrinyl)-methylamino)acetonitrile (III) by potassium cyanide and it was found that (III) underwent quantitative decomposition into 1-(N-(4-antipyrinyl)methylamino)acetamidoxime (IV) by hydroxylamine and that (IV) colored reddish violet with iron (III) chloride. This reaction was utilized as a characteristic method for determination of sulpyrine.
A total of 36 kinds of new antipyrine derivatives were synthesized, including 3 kinds of 2-(4-antipyrinylamino)thiazoles (II to IV), 18 kinds of 4-(4-antipyrinyl)thiazoles (VI to XXIII), 5 kinds of 2-(4-antipyrinylamino)-4-(4-antipyrinyl)thiazoles (XXIV to XXVIII), two kinds of 2-(4-antipyrinyl)methylenehydrazinothiazoles (XXIX and XXXI), 3 kinds of 4-(4-antipyrinyl)-2-arylidenehydrazinothiazoles (XXXVII to XXXIX), two kinds of 2-(2-methyl-1-phenyl-5-pyrazolon-4-yl)methylmethylenehydrazinothiazoles (XXXIII and XXXIV), 2-(4-antipyrinyl)methylmethylenehydrazino-4-phenylthiazole (XXXVI), and the starting materials, 4-acetylantipyrine thiosemicarbazone (XXXV), and 1-phenyl-4-acetyl-2-methyl-5-pyrazolone thiosemicarbazone (XXXII), as well as the known 4-(4-antipyrinyl)-2-aminothiazole (V) and 2-(4-antipyrinyl)methylenehydrazino-4-phenylthiazole (XXX). Antibacterial test of these compounds using tubercle bacilli, Staphylococcus aureus, and Escherichia coli showed that compounds having arylidenehydrazino group (XXXIX, XXX, XXXVII to XXXIX, XXXIII, XXXIV, XXXVI) and the starting thiosemicarbazones (XXXV and XXXII) had weak activity against tubercle bacilli. With the gram-positive Staphylococcus aureus, 4-(4-antipyrinyl)-2-salicylidenehydrazinothiazole (XXXVIII) showed growth-inhibitory action at 160, 000 dilution (6.25γ/cc.). Antibacterial action of (XXXVIII) against other gram-positive bacteria such as Bacillus subtilis, Diplococcus pneumoniae, Streptococcus hemolyticus, Cl. tetanii, and Cl. perfrigens, showed that the compound had inhibitory action against D. pneumoniae in 6.25γ/cc. concentration and a weak activity against Strept. hemolyticus but was ineffective to all other bacteria. None of the compounds proved effective against the gram-negative coli bacilli.
Since 4-(4-antipyrinyl)-2-salicylidenehydrazinothiazole showed characteristic action of inhibiting growth of Staphylococcus aureus in 160, 000 dilution (6.25γ/cc.), 18 kinds of new compounds of this series were synthesized in order to examine the changes in antibacterial activity when the salicylidene group was substituted with other arylidene groups and by introduction of an alkyl group in 5-position of the thiazole ring. These compounds and one known compound were tested with human-type tubercle bacilli, Staph. aureus, and E. coli. For tubercle bacilli, compounds with p- or m-hydroxybenzylidene, in place of salicylidene, were more effective, while the activity decreased markedly against Staph. aureus in the case of arylidene groups other than salicylidene and by introduction of an alkyl group in 5-position.
A total of 12 kinds of new antipyrine derivatives with thiazolidinone ring were synthesized, including 5 kinds of 2-(4-antipyrinylimino)-5-arylidene-4-thiazolidinones (II-VI), 3-(4-antipyrinyl)-2-imino-4-thiazolidinones (VII-XI), 3-(4-antipyrinyl)-5, 5-dimethyl-2-oxo-4-thiazolidinone (X: hydrolysis product of XI), 2-[(4-antipyrinyl)methylmethylenehydrazono]-4-thiazolidinone (XII), and 2-[(2-methyl-1-phenyl-5-pyrazolon-4-yl)methyl)methylenehydrazono]4-thiazolidinone (XIII). Antibacterial action in vitro of these compounds were tested and some growth-inhibitory activity against tubercle bacilli was found in compounds (IV), (V), (VI), (XII), and (XIII), but none were effective against Staph. aureus and E. coli.
Seventeen kinds of new antipyrine derivatives were synthesized, including those possessing an amidoxime group (IV-X), imidazo[1, 2-a]pyridine ring (XI-XIV), and 3-(4-antipyrinyl)pyrazole (XV), 5-(4-antipyrinyl)-2-methylthio-1, 3, 4-thiadiazole (XVIII), 4-3-(chloropropionyl)antipyrine (XIX), 4-acrylantipyrine (XX), 4-(3-chloropropionamido)antipyrine (XXI), and 4-(5-nitrofurfurylideneamino)antipyrine (XXII). These compounds, and the known antipyric acid hydrazide and formylantipyrine oxime for comparison, were tested in vitro against human-type tubercle bacilli, Staph. aureus, and E. coli. Growth inhibition against tubercle bacilli, though weak, was found in the compounds with amidoxime (IV-VIII) and (XXII), but not in others, while only (XXII) was effective in inhibiting the growth of Staph. aureus, also to a weak degree. None of the compounds were effective against E. coli.
1) Tannin content in Geranium nepalense SWEET is determined by the difference in extinction of the aqueous solution of the tannin extract at pH 2.5 and pH 10 by the Maranvile-Geldschmid method and its value is converted into tannin content by the hide powder method: Tannin content (%)=L(0.493E-0.031)/S where L is the volume of extract solution (cc.), S is the weight (g.) of the sample, and E is the difference in extinction at 320mμ. 2) Seasonal variation of tannin content was examined and the maximum content was found to appear from the end of June to end of August. Individual variation in tannin content was great due to selection of the plant as wild species.
Separation and purification of Racemomycin complex, produced by a mutant strain of Streptomyces recemochromogenus SUGAI, SHINOBU et OTANI, was reported earlier but reëxaminations were made on some insufficient points. It was found that the sulfates of Racemomycin A, B, and C can be separated and purified by column chromatography using cellulose powder. Racemomycin A, B, and C have β-lysine and reseonine as the constituent component of their molecule and are assumed to be chemically similar antibiotics but they show difference in their biological activity. It was assumed that Racemomycin-A is an antibiotic with strong antibacterial activity and without acute toxicity or delayed toxicity. All racemomycins A, B, and C are labile to acid and alkalis, and require great care in their preservation.
Reductive action of hydrazine hydrate on quinones was examined and systematic reduction was carried out on 16 kinds of various substituted quinones by which the corresponding hydroxyl compounds were obtained in a good yield. This reduction did not cause any side reaction even in quinones with functional groups and hydrazine hydrate was found to be a mild agent, better suited than the existing reducing agents.
The Krafft point of thiamine alkylsulfates was calculated by determination of their conductivity. An increase of ca. 15° over that of sodium alkylsulfates with the same number of carbon atoms was recognized and the Krafft point was found to be affected markedly even by gegenions. The Krafft point lowered by addition of alcohols with 1-4 carbon atoms and the relationship between the degree of lowering (b) and concentration of alcohol (M) was found to be represented by equation (1). The logarithm of the degree of lowering (b) was found to increase in proportion to the increasing number of the alcohol. In thiamine hydrochloride solution of a definite concentration, there was a marked elevation and a solution above 0.03M in concentration no longer showed the Krafft phenomenon. Surface tension and critical micelle concentration (CMC) were measured at temperatures above the Krafft point and the CMC and surface tension were both found to be lower than those of sodium alkylsulfates. Since micelle formation becomes stronger in thiamine hydrochloride solution, fixation of gegenion on micelle surface was found to be the important condition for micelle formation.
It had been found that combination of higher alkylsulfates to several kinds of pharmaceutics with ionic nitrogen resulted in appearance of surface activity and formation of salts with wide range of application to pharmaceutical preparations. It was also observed with thiamine alkylsulfates that micelle formation and the Krafft point change markedly by the change of gegenions in the alkylsulfate. In the present series of work, systematic formation of alkylsulfate of various amines was carried out to examine the changes in physical properties by salt formation. Examinations made on alkylamines and amino acids were first examined and salts listed in Tables I and II were obtained. Measurement of the Krafft point showed the marked changes caused by the kind of gegenions. Salt formation was also revealed through measurement of infrared absorption spectra of alkylsulfates of amino acids. As shown in Fig. 3, combination of alkylsulfate with the amino group resulted in disappearance of absorption due to COO- in the amino acid taking an amphoteric ionic structure and appearance of a strong absorption band due to COOH at around 1590cm-1.
Continuing the previous work, salt formation between various amino compounds and higher alkylsulfates was observed systematically and physical changes accompanying substitution of gegenions were assumed from measurement of the Krafft point. Salts indicated in Tables I, II, and III were obtained in pure form from sulfanilamides, basic vitamins, antibiotics, and numerous other pharmaceutics. The Krafft point of these substances rose in proportion to the increasing number of carbon atoms in the alkyl chain of alkylsulfates, similarly as was observed in the case of thiamine alkylsulfates. The Krafft point also changed markedly according to the kind of gegenions and there were also numerous salts in which the Krafft point was not measurable due either to the occurrence of the Krafft phenomenon at an extremely low temperature or non-appearance of the Krafft phenomenon even in boiling water. It is consequently concluded that hydrophilic-lipophilic balance is undergoing changes in a rather wider range than the general conception of the solubility of surfactant in water and hydrophilic-lipophilic balance.
Surface tension of alkylsulfates of various amino compounds obtained in a pure state up till now was measured at 60° and critical micelle concentration was calculated from relationship curve of the logarithm of concentration (M) and surface tension. It was thereby found that tetracaine alkylsulfate, quinine monoalkylsulfate, and the dialkylsulfates of bivalent bases, such as ethylenediamine and thiamine disulfide had a very strong ability to form a micelle. Alkylsulfates which possessed a hydrophillic group such as COOH, alcoholic hydroxyl, SO2NH2, etc. in gegenions tended to have weaker ability for micelle formation irrespective of the Krafft point. It follows, therefore, that ability of micelle formation varies in accordance with changes in hydrophilic-lipophilic balance accompanying changes in gegenions.
In order to obtain diffusion coefficient of oxygen at various concentrations of propylene glycol-water and glycerol-water mixture, polarographic examination of these mixtures was made. The diffusion coefficient of propylene glycol-water mixture calculated by interpolation of measured values in the Ilkovic formula was greater than the values obtained from the Stokes-Einstein or Wilke formula and the difference became greater, the higher the concentration, with the minimum point at around 70-80%. In the glycerol-water mixture, the values by any of these formulae agreed well and the diffusion coefficient decreased with increasing concentration. These facts suggest that a specific association occurred in the propylene glycol-water mixture from around 50-60% and some sudden change had taken place in the internal structure.
For the determination of pentose by colorimetry, specificity and reproducibility of various methods were examined. It was concluded from these experiments that the methods using ferric chloride-phloroglucinol reagent and aniline-acetic acid reagent were the most suitable. When using pentosan as the sample, determination can be made by the method using ferric chloride-phloroglucinol reagent. Coloration by this method, in comparison with D-xylose, was 74% in D-ribose, 56% in L-arabinose, and 23% in D-glucurone, hexose and methylpentose being below 2%. Pentose can be determined up to 20-160μg./cc. The known composition of the aniline-acetic acid reagent was modified and the degree of coloration by this method, in comparison with D-xylose, was 80% in D-ribose, 67% in L-arabinose, and 5% in D-galactose and D-glucurone, other hexoses and methylpentose being below 2%. Pentose can be determined up to 10-80μg./2cc. In both cases, the presence of glucosamine, albumin, and sodium chloride do not interfere in the coloration.
Hydrogenation of ethyl 2-cyanolevulinate with Raney nickel gave, together with ethyl 5-methyl-3-pyrrolecarboxylate (III), ethyl 2-aminomethyl-4-hydroxyvalerate (II). cis- and trans-5-Methyl-3-pyrrolidinecarboxylic acids (IXa and IXb) prepared either by conversion of (II) with thionyl chloride into ethyl 2-aminomethyl-4-chlorovalerate (VI) and subsequent treatment with barium hydroxide or by treatment of (II) with acetic anhydride and anhydrous sodium acetate, followed by hydrolysis. The ethyl ester of (IXa) gave with palladium-carbon the pyrrole (III) and inversely cis-5-methyl-3-pyrrolidinecarboxylic acid (IXa) was prepared by simultaneous hydro-lysis and decarboxylation of diethyl cis-5-methyl-1, 3-pyrrolidinedicarboxylate (XV), which was obtained by hydrogenation of diethyl 5-methyl-1, 3-pyrroledicarboxylate (XIV) prepared from (III) in the usual manner.
Ethyl acetoacetate (I) and ethyl benzoylacetate (II) were each isonitrosated, reductively acylated with zinc and phenylacetyl chloride or benzoyl chloride, and submitted to dehydrative cyclization with phosphoryl chloride to form ethyl 2-R′-5-R-4-oxazole carboxylate (V:R=CH3, R′=C6H5CH2; VI:R=CH3, R′=C6H5; VII:R=C6H5, R′=C6H5CH2; VIII:R=R′=C6H5), and the same compound (IX) with R=C6H5 and R′=CH3 was prepared by the route described in Part I of this series. These esters were derived to the corresponding acids and hydrazides. N, O-Dibenzoyl-3-phenylserine ethyl ester (XII) was obtained by heating erythro-3-phenylserine ethyl ester hydrochloride (XI) and its threo isomer (XI) with benzoyl chloride in benzene. In the case of the threo compound (XI), an oxazolone (XIII) formed as a by-product. Catalytic reduction of ethyl α-benzoylhippurate (IV) gave erythro-N-benzoyl-3-phenylserine ethyl ester (XIV) whose treatment with phosphoryl chloride gave ethyl threo-2, 5-diphenyl-2-oxazoline-4-carboxylate (XV) when the reaction was carried out at room temperature and (XIII) when the reaction mixture was heated on a water bath. Application of phosphoryl chloride to (XII) at 50° afforded (XIII).
Reaction of ethyl benzoxyacetoacetate (I) and ethyl acetoxybenzoylacetate (II) with carboxamide afforded 2-phenyl-4-methyl-5-oxazolecarboxylic acid (III) from (I) and 2-methyl-4-phenyl-5-oxazolecarboxylic acid (IV) from (II), both in a low yield (Table I). (III), (IV), and ethyl 2, 4-diphenyl-5-oxazolecarboxylate (IV) were obtained in approximately 50% yield respectively from (I), (II), and ethyl benzoyloxybenzoylacetate (V) by the method of Cornforth, et al. Ethyl 4-methyl-5-phenyl-2-oxazolecarboxylate (IX) and 4, 5-diphenyl-2-oxazolecarboxylate (X) were prepared by treatment of the corresponding keto-amine hydrochlorides by the procedure described in Part I and their hydrazides were also prepared. In order to examine synthetic process for aminoöxazoles, benzylurethans (XI to XIV) (Table II) were prepared and their Curtius decomposition was examined. By heating (XII) with conc. hydrochloric acid, 2-amino-5-phenyloxazole (XV) was obtained in a good yield, while catalytic reduction of (XII) gave 2-amino-5-phenyloxazolidine (XVI), m.p. 110-112°, which was also obtained by reduction of (XV). The objective amines were not obtained from (XII), (XIII), or (XIV) by the Curtius reaction carried out in the present series of experiments.
The crude heavy oil of NTU shale oil, on shaking with diluted sulfuric acid, was separated in three layers; the upper layer consisted of refined heavy oil (60%), the acid layer, and the brownish viscous middle layer (40%). Basic components (“middle layer base”) extracted from the latter and it was concluded that the bases consisted of long-chained 2, 4, 6-trialkylpyridines on the basis of experimental data; oxidation with permanganate gave 2, 4, 6-pyridinetricarboxylic acid and aliphatic carboxylic acids, and reduction with sodium and ethylenediamine afforded a secondary amine.
The “Middle-layer Bases” in NTU Shale Oil have been characterized as long-chained 2, 4, 6-trialkylpyridines. Three methyl groups in the structure of collidine were extended through metallation with phenylsodium and then alkylated with alkyl halides; thus long-chained 2, 4, 6-alkylpyridines were synthesized. The identity between the synthetic products and natural “Middle-layer Bases” was demonstrated by means of gas chromatography. It was found that “Middle-layer Base” in general, has the the constitution which has appropriate long alkyl groups (e.g. C5H11-C10H21) in three directions from the central nitrogen atom or nitrogen-containing ring.
16-Dehydrogitoxigenin 3-acetates (VIII and IX) were newly synthesized, their optical rotation, absorption spectra, coloration reaction, and isomerization phenomena were compared with those of gitoxigenone (III and IV), and these acetates were found to have characteristics in common originating in the carbonyl at 16-position and unsaturated lactone ring at 17-position. These two isomeric acetates (VIII and IX) were reduced with sodium borohydride to form 16- and 17-epimers (VII, X, XI, and XII) of gitoxigen 3-acetate by which the mode of isomerization of the 16-dehydro compound from (VIII) to (IX) was clarified. This has given experimental basis for Cardwell's theory that Jacobs' α-oxoisodigitoxigenone is 17β-3, 16-dehydrogitoxigenin (III) and that β-oxoisodigitoxigenone is 17α-3, 16-dehydrogitoxigenin (IV).
Gravimetric analysis of santonin in crude drugs was modified to use the chlorine or bromine derivative of methane for extraction solvent and alumina column chromatography for purification, by which analytical accuracy was improved. Santonin content was measured by this modified method using 630 stocks of Artemisia kurramensis, now being cultivated to improve the quality, by individuals and according to month of the year. The content of santonin was found to be as follows: Average content throughout the year (June to November; leaves, flowers, buds), 0.555%; first-year growth, 0.498%; second-year growth, 0.628%; third-year growth, 0.538%; period at which the highest content is observed, maximum and minimum content, and average content in first-year growth, September, 1.053-0.414%, 0.762%; second year growth, August, 1.234-0.432%, 0.846%; third-year growth, August, 1.242-0.262%, 0.788%. Statistically, individual difference in the content is great and there is plenty of room for quality improvement. Weight ratio of the leaves, flowers, and buds to the whole herb is in an average of 60-65% (September to November) and there was no difference in the content by method of drying (in sunlight or in the shade). This strain was found to have santonin content 2-2.5 times that of domestic Artemisia maritima LINN. (Mibuyomogi).
Among 413 stocks of the domestic Artemisia kurramensis QAZ. cultivated experimentally (1956-1957), 19 strains were found to contain l-β-santonin in 0.013-1.117%, which was identified by the melting point, ultraviolet and infrared spectral data, and elemental analysis. Another 23 strains were found to contain 0.011-0.448% of l-α- and l-β-santonins, which were also identified by spectral data, elemental analysis, and paper chromatography. Content ratio of l-α- and l-β-santonins was measured by infrared spectra. These individuals have 2n=18 chromosome number in their pollen mother cell and there was no great difference in outward appearance from the strains containing l-α-santonin.
Content of volatile oil in the domestic Artemisia kurramensis cultivated experimentally was found to be 0.618% in the flowers, buds, and leaves (September, 1958), and 0.584% in whole herb (September, 1960); the constants of this volatile oil being: d425 0.9186, nD20 1, 4620, [α]D28.5 32.7°; acid value 0.58, ester value 7.47. The volatile oil components were separated by column chromatography and identified by paper chromatography, chromatostrip, and formation of derivatives. The same components were proved by gas chromatography and determined by area measurement as containing 2.76% of d-α-pinene, 20.09% of cineol, 62.74% of β-thujone, 10.85% of l-camphor and 0.89% of α-terpineol, with assumption of the presence of β-pinene and an unknown, low-boiling fraction. The strain of domestic Artemisia maritima LINN. (Mibuyomogi) used for comparison yielded 0.247% of volatile oil from the whole herb (Hokkaido strain, July, 1958), the data of the oil being: d4250.9356, nD201.4702, [α]D29155.0; acid value 1.31, ester value 3.84. Gas chromatography suggested the volatile oil components to be β-pinene, cineol, β-thujone, camphor, and five other unknown substances (especially high-boiling fraction).
External morphology of 84 individuals of Artemisia kurramensis, cultivated experimentally, was examined (May to November, 1957). It was thereby observed that the domestic species differed from the original description of this plant by Qazilbash in the point of color of young, and grown leaves, tubulous flowers, and number of involucral scales. Further detailed examinations were made on the outward appearance, height, color of stems, leaves, and petals, number of stipules, size of tubulous flower, number of involucral scales and tubulous flowers in one caput, number of chromosome number in pollen mother cell and santonin content in 14 individuals (April to November, 1960). The domestic strains seem to be divisible into three strains by the number of tubulous flowers in one caput, color of leaves, stems, and petals and number of involucral scales.