In order to carry out the Huang-Minlon reduction of 2α, 3α-diol, 2α, 3β-diol, and 2β, 3α-diol derivatives of 5β, 25D-spirostan-11-one and their derivatives were prepared from 5β, 25D-spirost-2-en-11-one derivatives.
Following the previous work, synthesis of 2, 3-diols of 5β, 25D-spirostane series, substituted with oxygen at 11-position, was attempted and 2α, 3α-diol, 2β, 3β-diol, 2β, 3α-diol, 2α, 3β-diol, and their derivatives were prepared from the corresponding spirost-2-ene derivatives.
Preparation of 21-phosphate of hydrocortisone was carried out by the following route. Dibenzyl chlorophosphate of hydrocortisone was esterified, debenzylation was repeated twice with sodium iodide or lithium chloride, and hydrocortisone phosphate was obtained as its cyclohexylammonium salt. From its infrared absorption spectrum, assumptions were made on the assignment for phosphorus-oxygen bonding. The compounds (VII), (VIII), (IX), and (V) were deuterated to assign the absorptions originating in OH, and the absorption at 1162cm-1 was assigned to asymmetric stretching vibration and that at 1114cm-1 to symmetric stretching vibration of P-O bond in (V).
Steroids can be made soluble in water by derivation to the salt of a secondary phosphate. Synthesis of a secondary phosphate including a steroid was attempted on hydrocortisone with phosphorylation agent of the ROPOCl2 type but the product obtained was a bis-hydrocortisone ester. With the reagent of (RO)2POCl type, esterification using this reagent with R as phenyl gave a tertiary phosphate which easily liberated one phenyl to form the phosphate of steroid and phenyl by treatment with potassium carbonate at room temperature. With this reagent where R is benzyl, the tertiary phosphate obtained by esterification also lost one benzyl group to form a secondary phosphate by treatment with sodium iodide or lithium chloride.
The structures of enteromycin and related compounds were checked by infrared spectroscopy. The results agree with the structure proposed by Mizuno excepting the stereochemical configuration of the hydroxyiminoacetyl group in demethoxy-enteromycin and the O-methyl-aci-nitroacetyl group in enteromycin. The spectra in dilute carbon tetrachloride solutions reveal an intramolecular hydrogen bonding between the N-O and N-H groups in these compounds but no such interaction is found between the ester carbonyl and amide N-H. This fact can be explained only by the assumption that the configuration of C=C is trans and that of C=N is anti, the latter being opposite to that suggested by Mizuno on chemical grounds.
Effect of chemical structure of surface active agents on the stabilization of acetylsalicylic acid was examined by the use of nonionic surfactants, such as higher alcohol ethers of polyoxyethylene and fatty acid esters of polyoxyethylene sorbitan, in aqueous solution of pH 1 in which acetylsalicylic acid would be present as a nondissociating type. With the series of nonionic surfactants tested, those with stronger degree of lipophilic character seemed to be more effective for stabilization. Examination of activation energy at the time of addition of various surfactants revealed that activation clearly increased in the systems added with surfactant. The quantity of acetylsalicylic acid dissolved by addition of a surfactant was measured and the greater the quantity, the greater was the stabilization. These experiments indicated that stabilization of the non-dissociating form of acetylsalicylic acid by nonionic surface active agents was due to solubilization of this acid into the micelle.
The existing coloration reaction for sugars by the anthrone reagent differs in reactivity according to the kind of sugars but is common to various sugars such as hexose, methylpentose, and pentose. In carrying out the reaction, it is necessary to cause heat generation by mixing of the test solution and the reagent or heating. Coloration of pentose and furfural is somewhat more labile than that of other sugars and the color tends to fade by excessive heating. It was found that there is a furfural-anthrone coloration reaction different from the known sugar-anthrone reaction. In this case, 1cc. of furfural (1-50γ/cc.) and 10cc. of the coloration reagent (0.1% of anthrone in a mixture of 6 volumes of conc. sulfuric acid and 4 volumes of 85% phosphoric acid) are mixed under cooling to avoid generation of heat, allowed to stand in flowing water for 40 minutes, and a deep blue color with absorption maximum at 600mμ is produced. The relationship between furfural concentration and absorbance meets the Beer's law and this coloration can be utilized for determination of furfural. If pentose is heated preliminarily with sulfuric or phosphoric acid, cooled, and the foregoing coloration reaction is carried out, coloration highly characteristic to pentoses is obtained. This coloration also follows the Beer's law.
2′-, 3′-, and 4′-(Piperonyloxy)-2-acetoxyacetophenones (V) have been synthesized by the bromination arid subsequent treatment with sodium acetate of the respective (piperonyloxy) acetophenones (III) which in turn were prepared by the reaction of piperonyl chloride with 2′-, 3′-, and 4′-hydroxyacetophenones, respectively, in the presence of alkali. Alkaline hydrolysis of 2′-, and 4′-(piperonyloxy)-2-acetoxyacetophenones gave the corresponding benzoic acids (VII), while the meta derivative (V) was converted into the hydroxy-ketone (VI) on treatment with boiling water.
Pale yellow microneedles, m.p. 214° (decomp.), were isolated in 0.01-0.02% yield from the fresh leaves of Plantago (Plantago asiatica L.). This substance corresponds to the molecular formula of C21H20O11⋅5H2O, and colors brownish violet to ferric chloride, orange to yellow with magnesium and hydrochloric acid, and yellowish brown to yellow with zinc and hydrochloric acid. Its hydrolysis with 350% hydrochloric acid produced one mole each of scutellarein and glucose. The position of glucose bonding was proved to be 7, since complete methylation of the glycoside with diazomethane and subsequent hydrolysis afforded scutellarein 4′, 5, 6-trimethyl ether, m.p. 196-197°. This glycoside is therefore scutellarein 7-glycoside and was named plantaginin.
Tropane alkaloids widely distributed in some genera of the Solanaceae family and decomposed bases were submitted to paper chromatography with various developing solvents, in comparison with hygrine, xanthine, and betaines, which are known to be fairly widely distributed in the Solanaceae and related families, and synthesized homatropine (Table I). In tropane alkaloids, there were marked differences between the original alkaloids and their degraded bases in their Rf values by various developing solvents and coloration to the Dragendorff reagent. Hygrine, xanthine, and betaines showed different Rf values from those of tropane alkaloids and their degraded bases. This paper chromatographic procedure enabled isolation of nortropine in a good yield from scopolia extract, and isolation and identification of scopine and scopoline from the hydrolyzate of scopolamine. Modification of the Dragendorff reagent for identification of tropane alkaloids by paper chromatography was examined. The use of hydrochloric acid in place of nitric acid and dilution with 10% potassium iodide solution were found to give a formula which is stable over a long period and give comparatively quantitative result in examining the limit of alkaloidal coloration in paper partition chromatography. Limit of detection of alkaloids and betaines, and developed area of atropine in paper partition chromatography were examined by this modified reagent.
The amount of hyoscyamine (I) and scopolamine (II) contained in both Scopolia Extract and Belladonna Extract differ markedly in their ratio in these two extracts (Table I) and it is possible to differentiate these extracts by separatory determination of the two alkaloids. The minute amount of (II) contained in the Belladonna Extract can be easily detected in the Scopolia Extract by paper partition chromatography and it was confirmed that this procedure in capable of distinguishing the extract containing at least within 0.1% of the alkaloid. Norhyoscyamine (III) gives a characteristic and stable lemon yellow coloration to Ninhydrin reagent, a coloring reagent for amino acids, but other tropane alkaloids and a few betaines color reddish violet to brown with this reagent (Table II), so that its presence in the extract can be detected by paper partition chromatography. In an experiment carried out, the presence of (III) in 1g. of Scopolia Extract containing within 0.1% of alkaloids was detected but this was impossible with Belladonna Extract. Consequently, this method was found to be a good measure in distinguishing the two kinds of extract.
Examinations were made on the sterol (needles, m.p. 138-139°, [α]D25-36° (CHCl3)), obtained from the non-saponifiable matter of the crude oil, produced in 1.0-1.2% yield from Pinellia ternata BREITENBACH (Japanese name “Hange”), and this was found to be β-sitosterol. This sterol is present in ca. 3.4-4.0% in the crude oil. A quaternary base was separated from the methanol extract of this plant as the ammonium reineckate and the analysis of its picrate (yellow needles, m.p. 240-241°) corresponded to the molecular formula of C11H16O8N4. From the analytical values and properties of various derivatives, this base was proved to be choline. Re-examination of Suzuki's experiment revealed that this crude drug did not contain any tertiary base which is said to be the antiemetic factor.
Concentration-action curves of acetylcholine, barium chloride, and Oxytocin were plotted and the slopes of acetylcholine and Oxytocin were n=1.0 and that of barium chloride, n=1.5. Atropine showed competitive antagonism with acetylcholine in a very low concentration (10-13 to 10-10 g./cc.), while papaverine and allied spasmolytic agents showed non-competitive antagonism with acetylcholine, barium chloride, and Oxytocin. Aspaminol, a spasmolytic agent with large degree of dissociation, showed competitive antagonism with barium chloride and it was assumed that rat uterus strip is more like intestine than trachea in behavior.
Barium receptor is protected from the action of heavy-metal compounds like mercuric chloride and p-chloromercuribenzoic acid by a high concentration of barium chloride or by morphine, which is a non-competitive, anti-barium agent. It is therefore assumed that barium receptor contains -SH group, as does mouse intestine. Oxytocin is also protected from the action of heavy metals by a high concentration of oxytocin but not by morphine which fact suggests that oxytocin receptor is different from barium receptor.
In experiment with excised uterus strip of a rat, acetylcholine was inhibited noncompetitively by hydrogen ion concentration, while barium choride and oxytocin were inhibited competitively. Oxytocin was far more strongly inhibited than barium chloride by hydrogen ion and this may be considered as inhibition of dissociation of -SH group in the barium receptor. Inhibition of oxytocin is probably due to inhibition of the dissociation of phenol group of tyrosine in the oxytocin molecule.
In experiment with excised uterus strip of a rat, action of oxytocin was not inhibited by majority of amino acids constituting oxytocin and vasopressin but phenoltype compounds, such as nitrophenols, tyramine, and L-tyrosine ethyl ester, all inhibited the action of oxytocin competitively in 10-5 to 5×10-5g./cc. concentration. These had non-competitive inhibition on acetylcholine and barium chloride. Degree of dissociation and inhibitive power of phenol derivatives were approximately in direct proportion so that the active site of oxytocin was assumed to be in tyrosine in the oxytocin molecule. Amine compounds like urethan, glycine ethyl ester, and methionine ethyl ester had no inhibitive action on oxytocin even in a high concentration of 10-4g./cc. and it was therefore assumed that the free amino group of cystine in the oxytocin molecule is not the active site. One of the phenol derivatives, estradiol, indicated competitive inhibition againt oxytocin in 10-6g./cc. concentiation.
1, 1-Bis[(1-phenyl-2-methyl-5-oxo-3-pyrazolin-3-yl)methyl]methylamine was pre pared from ethyl bis[(1-phenyl-2-methyl-4-bromo-5-oxo-3-pyrazolin-3-yl)methyl]-malonate, formed as a by-product during the synthesis of ethyl [(1-phenyl-2-methyl-4-bromo-5-oxo-3-pyrazolin-3-yl)methyl]malonate. The amine was converted to its N, N-dimethyl derivative and 1, 9-dimethyl-2, 8-diphenyl-1, 2, 8, 9, 10a, 11-hexahydro-3H-dipyrazolo [3, 4-b:4′, 3′-g] quinolizine-3, 7(4H, 6H, 10H)-dione was synthesized.
Bischler-Napieralsky reaction of 3-(2-homoveratramidoethyl)- and 1-phenyl-2-methyl-3-[2-(2-phenylacetamido) ethyl]-3-pyrazolin-5-one was carried out and its reaction product was examined. When phosphoryl chloride was used as the reagent, the cyclized product, 1-methyl-2-phenyl-4-veratryl-1, 2, 6, 7-tetrahydro-3H-pyrazolo-[4, 3-c]pyridin-3-one (A) was not obtained and a compound in which the bridged side-chain CH2 had been oxidized to CO, i.e. 1-methyl-2-phenyl-4-veratroyl-1, 2-dihydro-3H-pyrazolo [4, 3-c] pyridin-3-one (B), and its 4-benzoyl compound (C) were obtained. In the case of phosphorus pentachloride, majority of the product was (B) or (C), but (A) was also formed to some extent. By the use of pyridine and phosphorus pentoxide, a cyclization reagent used in the modified Sugasawa method, (A) was obtained in a low yield. A few other cyclized compounds were also prepared.
1-Methyl-2, 4-diphenyl-1, 2, 4, 5, 6, 7-hexahydro-3H-pyrazolo [4, 3-c] pyridin-3-one, prepared from 1-phenyl-2-methyl-3-(2-aminoethyl)-3-pyrazolin-5-one and benzaldehyde by the Pictet-Spengler reaction, was catalytically reduced in the presence of formaldehyde and Raney nickel, and a compound N-methylated in 5-position was obtained. The reduction with formic acid and formaldehyde solution resulted in cleavage of the pyridine ring and the decomposition products, 1-phenyl-2-methyl-3-(2-dimethylaminoethyl)-3-pyrazolin-5-one (m. p. 73°) and benzaldehyde, were obtained. Similar Pictet-Spengler reaction with acetaldehyde also afforded the cyclized product.
Pyrazolopyridine derivatives with nitrogen in 6- or 4-position were synthesized. For that with nitrogen in 6-position, 1-phenyl-2-methyl-3-amino-3-pyrazolin-5-one (I) and ethyl acetoacetate (II) were reacted according to the method of Fierz-David, and the cyclized product, 1, 4-dimethyl-2-phenyl-6-hydroxy-1, 2-dihydro-3H-pyrazolo [3, 4-b] pyridin-3-one, was obtained in one step. The anil compound was prepared from (I) and (II) by the Conrad-Limpach reaction and its compound was further cyclized at 250° to afford 1, 6-dimethyl-2-phenyl-4-hydroxy-1, 2-dihydro-3H-pyrazolo [3, 4-b] pyridin-3-one, which was further derived to the true pyrazolo-pyridine compound. The derivative with nitrogen in 4-position was started from 3-pyrazoline-3-carboxaldehyde derivative and reacted according to Sugasawa's method and ethyl 1, 5-dimethyl-2-phenyl-3-oxo-1, 2-dihydro-3H-pyrazolo [4, 3-b] pyridine-6-carboxylate was identified as its platinum salt.
3-Aminoquinoline and 3-aminoquinoline 1-oxide were derived to 3-azidoquinoline and 3-azidoquinoline 1-oxide (VIII) via the respective hydrazino compounds. 5-Azidoquinoline 1-oxide (XX) was similarly prepared. 2-Azidoquinoline 1-oxide (XV) was obtained by oxidation of 2-chloroquinoline with 30% hydrogen peroxide in monochloroacetic acid or trifluoroacetic acid to its 1-oxide (XI) and its reaction with sodium azide, though in a minute amount. Recrystallization of (XV) from benzene gave 2-aminoquinoline 1-oxide. The azide group in (VIII) and (XX) has little activity and its properties are similar to that of aromatic azide compounds.
In order to examine the biological activity of nicotinic acid N-oxide, biological change of this compound in a rat was examined. Method for determination of nicotinic acid N-oxide has not been established as yet. In the present series of work, a method was devised for determination of nicotinic acid in a supernatant after reduction of the N-oxide group with iron and acetic acid, and removal of iron as a hydroxide by centrifugal precipitation. This method was found to be applicable for determination of nicotinic acid N-oxide in urine. This method was applied to the determination of urinary excretion after oral administration of 1.0g./kg. of nicotinic acid in a rat and it was found that about 50% of the dose administered was excreted in the urine, of which ca. 20% was in the form of nicotinic acid and 10-20% in the form of nicotinuric acid. Besides these, 1-methyl-6-oxo-1, 6-dihydronicotinamide and a small amount of 1-methylnicotinamide were detected from the urine. In the case of intravenous injection of 100mg./kg. of nicotinic acid N-oxide, almost 50% of this N-oxide, 20-25% of nicotinic acid, and 20-25% of nicotinuric acid were detected from the urine, as well as a trace of 1-methyl-6-oxo-1, 6-dihydronicotinamide and 1-methylnicotinamide.
Following the previous work on biological changes of nicotinic acid N-oxide in a rat, in which about one-half of the compound was found to be reduced to nicotinic acid, perfusion test with excised liver was carried out and only a few percentages of nicotinic acid N-oxide was found to have been reduced in rabbit and entirely unchanged in a rat liver. Enzymatic examination of in vitro reduction of nicotinic acid N-oxide was made and following points were clarified as the mechanism of the reduction system. When using liver as the enzyme source, the main enzyme protein was present in the supernatant of cell fraction by the Schneider method and specific activity increased about two-fold by ammonium sulfate fractionation (0.28-0.50 saturation). Besides this protein, there is activation action in the proteinic factor originating from mitochondria. A low-molecular activation substance is necessary as a co-factor and, as far as the present examinations are concerned, ATP and ADP are most effective, followed by DPN and DPNH. Both the oxidation and reduction forms of TPN were devoid of this activity. It is interesting to note that ATP and ADP take part in the reduction reaction and that AMP and adenine have strong inhibitory action in a system using ATP as the activator. However, it was not possible to clarify the mechanism of this reaction.
Nicotinic acid N-oxide is utilized, although to a very small extent, by Lactobacillus arabinosus and Leuconostoc mesenteroides which require nicotinic acid. It was also found that nicotinic acid N-oxide may be substituted to a certain extent for nicotinic acid in three strains of coli bacillus mutants requiring nicotinic acid. Examination of baterial enzymes in these microörganisms suggested that these organisms adaptively form enzyme systems that reduce the N-oxide on addition of nicotinic acid N-oxide and utilize the nicotinic acid thereby formed.
It has been found that the resting cells obtained by stationary cultivation of Escherichia coli K-12 cultured in a liquid medium containing 1% each of peptone, bouillon, and glucose, reduce nicotinic acid N-oxide in the presence of a hydrogen donor like formate and glucose. Examinations of culture conditions revealed that cells obtained on shaking culture and cultured in a medium containing nitrate ion lacked the activity to reduce nicotinic acid N-oxide. The resting cells lacking this reducing activity, incubated with nicotinic acid hydrazide, in the presence of casamino acid and formate at 37°C, was found to regain the activity of reducing nicotinic acid N-oxide after about 1 hour. Cells having reducing activity were treated with sonic waves in order to take out the reductase for nicotinic acid N-oxide in cell-free state. The supernatant obtained by centrifugation at 18000g for 30 minutes did not show any reduction of nicotinic acid N-oxide by itself but coupling of nitrate-adapted cells, lacking in the ability to reduce nicotinic acid N-oxide, with hydrogen transfer system resulted in marked reduction in the presence of methyl viologen. This fact suggests that, although the reductase is present in this supernatant, it lacks the hydrogen (or electron) transfer system and this system is regenerated by addition of nitrateadapted system, lacking in nicotinic acid N-oxide reductase, thereby resulting in the reduction of nicotinic acid N-oxide.
1. The metabolic end product of nitrogen compounds by the homogenate of lungworm, Metastrongylus elongatus, using various amino acids as substrate, was ammonia. Urea and uric acid were not detected. 2. The activities of arginase and crease were not found in this lung-worm homogenate. 3. The formation of ammonia was attributed mainly to hydrolysis of α-NH2 group in amino acids, when arginine was used as a substrate. The ammonia formation from nucleotides was observed, as is the case in amino acids, but was not perceptible from amine. 4. Transamidinase and transaminase activities were recognized.
A number of 2-[(1, 2, 3, 4-tetrahydro-2-naphthyl)aminomethyl]propionic acid derivatives were prepared and tested of their oxytocic activity. Methyl 2-[(1, 2, 3, 4-tetrahydro-2-naphthyl)aminomethyl]propionate showed the maximum activity so far observed in this series.
Oxidation of cycloheptatriene (IV) with selenium dioxide in hydrous dioxane afforded tropone (I) which was derived to its acylhydrazones. The benzoylhydrazone of (I) was isolated in two forms of keto (V) and enol (VI) types. Of the substituted benzoylhydrazone, p-methyl- (VIII) and p-methoxy-benzoylhydrazone (IX) were isolated in keto form, and p-nitro- (X), m-nitro- (XI), and 3, 5-dinitro-benzoylhydrazones (XII) were isolated in enol form crystals, isonicotinoylhydrazone (XIII) also came in enol type.
Condensation of 2-hydrazinotropone (I) and its derivatives with aliphatinc aldehydes or ketones afforded hydrazones which were heated with polyphosphoric acid or dilute sulfuric acid to form cyclohepta [b] pyrrol-8(1H)-one (XVI). Halogenation of 3-methyl-(XVII) and 2, 3-dimethyl-cyclohepta [b] pyrrol-8(1H)-one (XIX) was examined and it was found that their bromination with N-bromosuccinimide resulted in introduction of bromine into the ring, instead of bromination of the side chain, first in the 2-position when it is vacant and further in other positions, while the 7- and then 5-position of the seven-membered ring are brominated when there is a methyl group in 2-position.
From the leaves of Isodon trichocarpus KUDO, enmein, m. p. 297-298°(decomp.) was isolated together with a new bitter principle, dihydroenmein, which had heretofore been obtained by catalytic reduction of enmein. Dihydroenmein was not isolated by recrystallization or chromatography, but was isolated in an amount corresponding to 30% of crude enmein from the neutral portion left after removal of enmein as an acid substance after oxidation of crude enmein with potassium permanganate.
In order to measure granular strength, examinations were made on the granulation of granules, amount charged, number of balls, period of pulverization, and manner of indication of granulation, using a ball mill. It was thereby found that pulverization by ball mill is practical as a method for measurement of granular strength.
Growth-inhibitory activity was examined in the newly synthesized 8 kinds of diphenyl sulfone derivatives, 17 kinds of diaminodiphenyl sulfone derivatives, and 20 kinds of heterocyclic diaryl sulfones (methylthiazole, benzothiazole, 1-methylimidazole, benzimidazole, lepidine, quinoline, and indole systems), using Mycobacterium 607, Escherichia coli 026, and Staphylococcus 209P. 4, 3′, 5′-Trinitrodiphenyl sulfone was found to have comparatively strong growth-inhibitory action against Mycobacterium and Staphylococcus.