Lysine glucose ester, a kind of sugar ester of basic amino acid, was synthesized and its properties were examined. The synthesis started with 1, 2:3, 5-di-O-benzylidene-D-glucofuranose, N2, N6-bis(benzyloxycarbonyl)-L-lysine, and dicyclohexylcarbodiimide, which were reacted in molar ratio of 1:2:1 in pyridine, and the product, 6-O-[N2, N6-bis(benzyloxycarbonyl)-L-lysyl]-1, 2:3, 5-di-O-benzylidene-α-D-glucofuranose, was to be isolated and derived to the objective substance by hydrogenolysis. However, attempted liberation of the protective group from this product resulted in decomposition, and glucose and lysine alone were found in the reaction mixture. This seems to indicate that the objective lysine glucose ester is extremely labile in the aqueous solution and is rapidly hydrolysed into respective components after formation. A small amount of Ninhydrin-positive substance, other than lysine, was formed in this case and this was considered to be a peptide composed of lysine. This behavior is similar to glycine glucose ester, the sugar esters of neutral amino acid described in the preceding paper, and it is therefore considered that the sugar esters of neutral and basic amino acids are such a labile substance that their isolation would be difficult.
The two isomers of monoglucose aspartate, i.e. 6-O-(L-α-aspartyl)-D-glucose and 6-O-(L-β-aspartyl)-D-glucose, were synthesized and their properties were examined. In both cases, 1, 2:3, 5-O-dibenzylidene-D-glucofuranose was used as the glucose moiety. For the α-ester, this was condensed with α benzyl N-benzyloxycarbonyl-L-aspartate in dicyclohexylcarbodiimide and its product was hydrogenolyzed. For the β-ester, the glucofuranose was reacted with 1-benzyl N-benzyloxycarbonyl-L-aspartate 4-chloride and the product was hydrogenolyzed. Both of these esters were far more stable than the lysine glucose or glycine ester and did not undergo hydrolysis in aqueous solution. However, the esters were easily hydrolyzed in alkaline solution and 50% decomposition in 0.001M solution at pH 10, at 40°, was less than 5 minutes.
Roemerine, nuciferine, and nornuciferine, a nornuciferine, a new aporphine-type tertiary phenolic base, were isolated from the leaves and petioles of domestic lotus, Nelumbo nucifera GAERTN. (Japanese name, “Hasu”). The composition of this base corresponded to C18H19O2N=C16H12(O-CH3)(OH)(N-CH3) and O-methylnornuciferine was found to be entirely identical with nuciferine (1, 2-dimethoxyaporphine) (I). Consequently, it was found that the formula (II) or (III) would be forwarded for nornuciferine. In the present series of work, the product obtained by degradation of O-ethylnornuciferine, methoxy-ethoxyphenanthrene, was proved to be 3-ethoxy-4-methoxyphenanthrene (XVb) and, consequently, the structure of nornuciferine was established as 1-methoxy-2-hydroxyaporphine (III).
The structure of 1-phenethyl-3-cyano-2(1H)-pyridone, reported in Part II of this series, was confirmed as (I) by the route shown in Chart 1. Further, (XII) was synthesized from (I) by the Curtius reaction and, since its diazonium salt was stable, 1-phenethyl-3-substituted-2(1H)-pyridones were prepared by its Sandmeyer reaction. Measurement of dipole moments of these compounds further confirmed the ortho effect of the dipole moment of these 3-substituted 2-pyridones, which had been assumed with small number of examples in the preceding work.
Reaction of DL-methionine ethyl ester to the active derivatives of α-lipoic acid (I), i.e. α-lipoic butylcarbonic anhydride (III), O-α-lipoyl phosphorodichloridate (IV), benzyl α-lipoate (V), and S-benzyl thio-α-lipoate (VI), and hydrolysis of the product with sodium hydroxide affords N-α-lipoyl-DL-methionine (VIII), which is also obtained by the reaction of 6, 8-dichloroöctanoyl chloride (IX) and DL-methionine ethyl ester with subsequent application of sodium sulfide. As the mixed acid anhydride of (I), butyl- (III), isobutyl- (XI), octyl- (XII), and isoöctyl-carbonic anhydride (XIII) were prepared and their reaction with copper complex compound of L-lysine afforded N6-α-lipoyl-L-lysine (XIV). Reaction of 6, 8-dichloroöctanoic alkylcarbonic anhydride and copper complex compound of L-lysine gives N6-(6, 8-dichloroöctanoyl)-L-lysine (XVIII) which reacts with sodium sulfide to form (XIV). α-Lipoic alkylcarbonic anhydride reacts with N2-acetyl-L-lysinamide (XXIII), obtained by catalytic reduction of N2-acetyl-N6-benzyloxycarbonyl-L-lysinamide (XXII), to form N2-acetyl-N6-α-lipoyl-L-lysinamide (XXIV).
Calcium pyruvate isoniazone comes in three kinds of crystal form, spindle-shaped, prismatic, and granular, and these three kinds of crystals were found to differ in their solubility in water and in viscosity-temperature curves of their saturated aqueous solution. These curves cross at 27° and 71°. Spindle-shaped crystals are stable below 27°, prismatic crystals in the temperature range of 27-71°, and granular crystals are stable at temperatures above 71°. Crystals in the temperature range of metastable state change into the crystal form stable at that temperature. X-Ray diffraction patterns of these three kinds of crystal differ and the difference in crystal form of calcium pyruvate isoniazone is thought to be due to the difference in crystal structure.
Measurement of hydration of calcium pyruvate isonicotinoylhydrazone hydrate was carried out and examinations were made on the amount of water contained and mode of dehydration in three kinds of spindle, prismatic, and granular crystals (tentatively designated respectively as crystals A, B, and C). Dissociating vapor pressure of each hydrate was measured in the temperature range of 20-50°, and it was clarified that 2, 3, 6, and 7 moles of water were present in A crystal, 2, 4, and 5 moles in B crystal, and 2, 4, and 4.5 moles of water in C crystal. Empirical equation, log p=A-B/T was applied to the relationship between dissociating vapor pressure and temperature, constants A and B for each system were calculated, and kinetic constants (heat of dissociation ΔH, change of free energy ΔG, and entropy change ΔS) were calculated. Comparison of dissociating vapor pressure of each hydrate and average humidity in various points in Japan showed that heptahydrate of A crystal, pentahydrate of B crystal, and tetrahemihydrate of C crystal are the most stable against moisture.
Examinations were made on the nitration of 3, 4-dihydro-5, 7-dimethyl-1(2H)-naphthalenone (I) and 1, 2, 3, 4-tetrahydro-5, 7-dimethyl-naphthalene (II). Using the nitro compounds (V and IX) so obtained as the starting materials, compounds (XIX to XXXIII) possessing α-dialkylamino acylamino group in the 6- or 8-position were synthesized. Among these compounds, tetralin derivatives had stronger local anaesthetic action than tetralone derivatives.
Microdetermination of organic mercurials by combustion method is described. The sample is weighed into a quartz boat and burned in a quartz combustion tube having two inlets, while passing oxygen. Mercury in organic compound will change into mercuric oxide or further undergo decomposition by the temperature inside the heating furnace to form mercury vapor which will be adsorbed on gold wire filled in the absorption funnel. The amount of mercury is calculated from changes in the funnel weight. Test carried out with purified mercury showed that the amount of mercury is completely adsorbed on the gold wire. If the sample does not contain halogen or sulfur, empty combustion tube (Fig. 1) is used, while a tube filled with calcium oxide is used when one or two of these elements is present (Fig. 2). This method is more simple in procedure than the existing method and takes shorter time, requiring only about 40 minutes.
Pharmaceutics sparingly soluble in water but possessing a hydroxyl group were led into quaternary ammonium-type derivatives to give water solubility. The compounds were first led to bromoacetate by application of bromoacetyl bromide in pyridine and addition of tertiary amine to its product gave the quaternary ammonium salts. Pharmaceutics used were hydrocortisone (Ia), prednisolone (Ib), and dexamethasone (Ic). The amines used were trimethylamine, triethylamine, N4-methyl-morpholine, pyridine, and triethanolamine for (Ia) and triethylamine for (Ib) and (Ic), using acetone as the solvent for addition reaction, becuase the use of alcohol as a solvent resulted in alcoholysis and the objective could not be obtained. The quaternary ammonium-type derivatives (III) synthesized formed crystals with the exception of pyridine adduct and their pharmacological activity was about equal to that of sodium hydrogensuccinate derivatives.
Thermostatic dehydration rate of A, B, and C crystals of calcium pyruvate isoniazone hydrate was measured in a temperature range of 30-130°. From the dehydration rate, αi, obtained in this experiment, linear fraction of dehydration is defined as ξi=1-(1-αi)1/3. ξi increases in proportion to time. Activation energy of each of the hydrated crystals was calculated and it was found that 4 moles of water in A crystal, 1 mole of water in B crystal, and 2.5 moles of water in C crystal are more easily liberated than other water of crystallization. Of these three kinds of crystals, B crystal was found to be the easiest to be dehydrated and C crystal, the hardest to become an anhydrate.
The total alkaloids obtained from opium by solvent extraction was packed in a column, connected to the top of an acid column (pH 2.2) which in turn was connected to the top of an alkali column (pH 12.5), all standing vertically (Fig. 1). These were developed with ether to separate the alkaloids into amphoteric and non-amphoteric bases, the acid column was separated, and this column was eluted by the procedure described in Part I of this series to collect fractions of crude morphine, narcotine, papaverine, crude thebaine, and crude codeine. Crude morphine, thebaine, and codeine were purified by rechromatography through columns of respective pH of 3.5, 5.0, and 3.8. The pure alkaloids thereby obtained were determined by non-aqueous titration. The manner of separation of these alkaloids is presented in Chart 1.
Examinations were made on the nature of adsorption by filter paper, which is considered to be one of the reasons for variation of Rf values in partition paper chromatography, and following facts were found: 1) Examinations were made on the adsorption mechanism of a filter paper and, with considerations on adsorption, the formula representing the Rf value was established (equation (7) in the text). In a solvent system with large λs (adsorption equilibrium constant between fiber and stationary phase), result of paper chromatography would not be parallel to the counter-current distribution method unless this equation (7) is adopted. In order to prove this fact, the value of Rfu1 (Rf value in which the partition ratio will be 1/2⋅k when adsorption is taken into consideration of equation (10) in the text) was calculated and compared with the value of Rfu0 (equation (9) in the text), showing that the former is almost constant. 2) It was shown that the phenomena of tailing, double spots, and strained shape of the spot could be explained on the basis of adsorption mechanism. 3) The degree to which adsorption plays a part in partition mechanism was considered to be correlated to Vs (volume of stationary phase contained in unit weight of the filter paper) and λs, from equation (7), and equation (11) was derived from it to obtain the relationship between these two values, showing the limit at which adsorption can be neglected. From the relationship between these two within this limit, upper limit of λs of each Rf value was calculated (Table IV). It was theoretically clarified that λs≤0.05 must be satisfied in order that adsorption could be neglected throughout all Rf values and that adsorption cannot be neglected in Toyo Roshi No. 51 if λs>0.5. It was experimentally proved that Vs≅0.5 is the lower limit of Vs. 4) Measurement of λ (adsorption equilibrium constant between fiber and developing solution in adsorption-type paper chromatography) by the use of various solvents showed that adsorption on the filter paper cannot be neglected when using a solvent system with water as the stationary phase in substances sparingly soluble in water or those having strong affinity to fiber even if soluble in water. On the other hand, use of formamide or ethylene glycol as the stationary phase in such cases was found to decrease adsorptivity markedly. These facts seem to prove that the equation for calculation of Rfu values established without regard to adsorption, as shown in Part I of this series, was not a mistake.
The Rf values in partition-type paper chromatography contains numerous variables affected by temperature (equation (1) in the text) and its fluctuation is complicated. In order to simplify this, the value was converted to Rf value of (Vs+λs)/Vm=1/2 (Rfu value) and its relation to temperature was examined. It was found that the Rfu value is affected by the mutual solubility curve of the solvent in a two-componental solvent system and, when the solubility increases with temperature (especially when there is an upper critical solution temperature), it converges near a specific point around 0.7 with increasing temperature. When the solubility decreases with increasing temperature (especially when there is a lower critical solution temperature), the curve diverges from the specific point. It is considered that utilization of this phenomenon would make it possible to predict a suitable temperature for separation of substances.
Pectin soluble in cold water (pectin-I) and that soluble in hot water (pectin-II) were isolated from the fruit rind of Citrus Unshiu MARCOVITCH. It was clarified that pectin-I has weakly bonded galactan alone and pectin-II, weakly bonded galactan and araban.
Acid hydrolysis of pectin-II, isolated from the fruit rind of Citrus Unshiu MARCOVITCH, gave an acid substance which was identified as an oligogalacturonic acid consisting of eight D-galacturonic acid residues, from the formation of methylglycoside methyl ester and acetate, and from the result of periodate oxidation.
Pectin-II, isolated from the fruit rind of Citrus unshiu MARCOVITCH, was hydrolyzed with an acid, oligogalacturonic acid thereby formed was oxidized with sodium periodate, and a hydrazone of its oxidation product was obtained by reaction with phenylhydrazine or isonicotinoylhydrazine. Reduction of this oxidation product with sodium borohydride and hydrolysis of the reduction product with hydrochloric acid afforded glycolic aldehyde, L-glyceric acid, and D-threonic acid. A structure was proposed for the oligogalacturonic acid from these derivatives and decomposition product.
Two new acetyl derivatives of digitoxin, diacetyldigitoxin (III) and triacetyldigi-toxin (IV), were prepared by selective acetylation of digitoxin followed by chromatography on alumina. Tetraacetyldigitoxin (V) was also obtained in a crystalline form for the first time. Physical constants and color reaction of these three acetates were examined. Probable structures of the new acetates (III and IV) are given on the basis of the results of paper chromatography and mild acid hydrolysis.
There are hardly any methods for the determination of eugenol other than the measurement of ultraviolet absorption and nonaqueous titration. Eugenol possesses a substituent in the position para to phenolic hydroxyl and structurally, it appears to be negative to indophenol reaction. Following the previous works on Colorimetric determination of pharmaceutics by the use of indophenol reaction, eugenol was found to be positive to the indophenol reaction. It has already been found that, when the substituent in the para-position of phenolic hydroxyl is easily liberated, the compounds show positive indophenol reaction. It may, therefore, be assumed that, in the case of eugenol, the allyl group in the para-position is easily liberated and the compound shows positive indophenol reaction. Satisfactory results were obtained by colorimetric determination of eugenol and also that in clove oil by application of 0.05% dimethyl-p-phenylenediamine hydrochloride and 0.02% of sodium hypochlorite to eugenol dissolved in Sörensen phosphate buffer of pH 8.0, extraction of the indophenol pigment thereby formed with isobutanol, and measurement of absor-bancy of the extract solution at 610mμ.
Colorimetric determination of testosterone was devised by the use of blue coloration which testosterone and its esters show with ferrous ammonium sulfate. The reagent is prepared by dissolving 1g. of Mohr's salt in 20cc. of water, adding 1cc. of sulfuric acid, followed by 1cc. of 30% hydrogen peroxide, and boiling this mixture. The solution is cooled, diluted to 50cc. with water, and 3cc. of this solution is made up to 100cc. with sulfuric acid. A mixture of this reagent and testosterone or its esters are heated at 100° for 4 minutes in a water bath, cooled in an ice-water bath for 2 minutes, and 2cc, of water is added gently. This is shaken vigorously for 15 seconds by which testosterone shows characteristic blue color, The limit of detection by this coloration is 0.5γ of testosterone. When 20-2.5γ of testosterone was measured in a 10-mm. cell, linearity, parallelism, and fiducial limit were all found to be satisfactory.
Coloration of the oximes and/or semicarbazones of 2-acyl-4-alkylpyridines (acyl=acetyl, propionyl, butyryl, and valeryl; alkyl=methyl and ethyl) with metal ions (iron (II), iron (III), copper (II), nickel (III), and cobalt (II)) was examined. These metals form a complex salt in the solution and gives colorations as listed in Table I. Composition of these complex salts in solution was determined by the continuous variation method (Figs. 1 and 2) and absorption spectra of these complex salts were also measured (Figs. 3-18).
Two kinds of mixed samples containing morphine, codeine, thebaine, narcotine, and papaverine were prepared according to the formula given for assay of alkaloidal content in opium and that described in Pharmacopoeia Kelvetica (1941) for analytical purpose. It was learned that determination can be made with an accuracy of within 3%. Composition of the synthetic samples were as follows: Morphine:codeine:thebaine:narcotine:papaverine=(i) 33:3.3:1:16.5:2.35 and (ii) 30:1:1:13:2. In both cases, determination is possible with the minimum amount of 1mg. of thebaine. Separatory determination of these alkaloids is carried out as follows: A sample is basified with sodium hydroxide and shaken with chloroform to transit morphine to the sodium hydroxide layer and other alkaloids to the chloroform layer (narcotine would be present in both layers and will have to be assayed with a separate sample). Sodium hydroxide layer is neutralized and morphine in it is determined. A part of the chloroform layer is shaken with 0.2% tartaric acid to transit codeine and thebaine into the aqueous layer, and thebaine is determined with a part of this aqueous solution. Another part of the aqueous solution is treated as described in Part XI of this series to separate thebaine and codeine is determined with residual solution. A separate sample is basified with ammonium hydroxide, extracted with chloroform, and papaverine and narcotine are separatory determined by the methods described in Parts VIII and X of this serles.
Heating of phenanthridine 5-oxide (I) with phenyl isocyanate (II) gives 6-anilino-phenanthridine (III). The reaction route is assumed to be as shown in Chart 1. (III) was identified by its synthesis by another route shown in Chart 3.
6-Cyanophenanthridine (I) is derived to 6-cyanophenanthridine 5-oxide (II) by treatment with hydrogen peroxide (100°) in glacial acetic acid but to phenanthridine-6-carboxamide (III) by treatment with hydrogen peroxide (room temperature) in alkaline medium. 6-Phenanthridinecarboxamide 5-oxide (IV) is formed from (III) by the former conditions and from (II) by the latter conditions. (III) and (IV) are also obtained by respective hydrolysis of (I) and (II) with 90% sulfuric acid (cf. Chart 1). The carboxamide group in (III) and (IV) so produced differs somewhat in the two compounds. (III) changes into 6-phenanthridinecarboxylic acid (V) by treatment with nitrous acid while (IV) remains inert to this treatment. The Hofmann reaction of (III) affords 6-aminophenanthridine (VI) while (IV) is resistant to this reaction and forms 6-aminophenanthridine 5-oxide (VII) in a small yield, with almost 50% recovery of the starting compound (Chart 2). There is a possibility that a cyclic structure involving hydrogen bonding would be formed between -CONH2 and N-O groups in (IV), as shown in Chart 3, and this might be one of the reasons for resistance of this compound to the foregoing reaction. Treatment of (V) with hydrogen peroxide in glacial acetic acid failed to produce the anticipated 6-phenanthridinecarboxylic acid 5-oxide (VIII) and phenanthridone (IX) and 6-hydroxyphenanthridine 5-oxide (X) were formed (Chart 4). It should be added that (VI) can easily be prepared by heating 6-phenoxyphenan-thridine with urea.
17α-Ethynylestradiol, when heated with Iron-Kober test solution for 3 minutes, shows maximum absorption at 560mμ, differing from other estrogens such as estrone and 17β-estradiol. In addition, estrone and 17β-estradiol do not absorb at 560mμ so that they can be detected easily even if present. This coloration reaction can be utilized for colorimetric determination since the absorbancy and amount of 17α-ethynylestradiol are in linear relationship in the range of 10-40γ of 17α-ethynyl-estradiol. There is no significant difference in the determined value even in the presence of 50γ of estrone. The procedure is carried out exactly the same as with Iron-Kober T. S. reagent A, used for coloration reaction of 17β-estradiol. Optimal conditions for colorimetric determination of 17α-ethynylestradiol were examined, since there has been no appropriate method of determination for this compound, by varying heating time and period of standing. It was thereby found that this is a practicable method of analysis.
Phosphorylcholine forms a blue precipitate with Molybdenum Blue, obtained by reduction of phosphomolybdic acid with tin (II) chloride and colorimetric determination by this coloration reaction was devised. In this method, 0.4cc. of Molybdenum Blue solution (an equivolume mixture of 12% phosphomolybdic acid solution and 2% solution of SnCl2⋅2H2O in 2.5N hydrochloric acid) is added to 1cc. of the sample solution (0.1-0.4μmole), the mixture is cooled in ice for 2 hours, and the precipitate formed is collected by centrifugation. After washing the precipitate with 0.5% solution of SnCl2⋅2H2O in 0.1N hydrochloric acid, it is dissolved in 10cc. of equal mixture of acetone and 2.5N hydrochloric acid and coloni metric determination of this solution is carried out after 10-30 min. at 725mμ. Combination of this method with the cellulose column chromatography enabled rapid determination of phosphorylcholine in rat liver.
The aqueous digest solution of the corm of Lycoris radiata or its extract was treated with weakly acidic ion exchange resin (Amberlite IRC-50) to adsorb the alkaloids and separate them from non-alkaloidal substances. The alkaloids were desorbed with ammoniacal ethanol and the ethanol was evaporated from this solution of total alkaloids. The residual solution was passed through double columns of Celite, with phosphate buffer (pH 7.0) as the stationary phase. The column was eluted with chloroform to separate lycorine and determination by optical method gave a satisfactory result.
In order to examine hypoglycemic action, arylsulfonylurea derivatives were prepared by the method of Marshall and others. Compounds of general formula R-C6H4-SO2-NHCONH-R′ were synthesized with R=CH3CONHCH2- and R′=CH3, C2H5, C3H7, C4H9, iso-C4H9, C5H11, C6H13, CH2CH2C6H5, CH2C6H5, C6H5, C6H4CH3, and C6H4OC2H5; R=H2NCH2 and R′=C4H9, C6H13, CH2CH2C6H5, and CH2C6H5; R=CH3CONHCH2CH2 and R′=C4H9; R=NH2CH2CH2 and R′=C4H9, R=CH3(NHCOCH3)CH and R′=C4H9; and R=CHO and R′=C4H9.