When nonionic surface-active agents of polyoxyethylene series are mixed with urea, the mixture solidified and the solid can be used as a powder or tablet. There is no evidence as to whether this solid is an inclusion compound or not. Mixtures of urea with various surfactants were prepared by the method of Barker and others and examination was made to see if hexagonal urea adduct has been formed through X-ray diffraction pattern. It was thereby found that the straight-chain alkyl ethers and esters of polyoxyethylene formed inclusion compound, like fatty acids and polyoxyethylene, while sorbitan alkylate and alkyl aryl ethers of polyoxyethylene did not form the inclusion compound. The latter is due to the fact that the size of sorbitan and benzene ring is larger than the channel of 5Å in diameter for formation of urea adduct. This nature can be utilized for the separation or fractionation of surfactants. For the preparation of urea adduct, it is more convenient to dissolve the two components in a solvent and to evaporate the solution to dryness. When a solvent is not used, formation of an adduct is facile with liquid surfactant but the adduct will not be formed easily without warming when the surfactant is a solid.

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A papaverine-like antispasmodic activity of daidzein (7, 4′-dihydroxyisoflavone) which was isolated from Pueraria roots, a chinese medicament, was estimated using intestinal segments excised from white mice. The potency ratio using papaverine as a standard and acetylcholine as an constructor was given in Table I.
The other isoflavone derivatives isolated from Pueraria roots showed no remarkable anti-acetylcholine action.
The fractions F_A and F_C (see Chart 1) of the extracts of roots gave a contraction of gut.

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In order to devise a method for rapid and facile measurement of lecithinase-A activity, examinations were made on (1) method for preparation of diluted blood suspension for measurement of the enzyme activity, (2) relationship between the amount of lysolecithin and degree of hemolysis, (3) conditions for the formation of lysolecithin, and (4) optimal conditions for enzyme-substrate reaction. Based on these experimental results, a method was devised whereby the activity of lecithinase is calculated from changes of extinction coefficient measured during addition of the reaction mixture of lecithinase-A and egg-yolk lecithin to diluted blood suspension. This method is more sensitive than the existing method which observes the degree of complete hemolysis and the sample required is very small in quantity. This method is also convenient for measuring activity of each fraction during purification through chromatography.

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Addition of 0.8 mole of sulfur powder to 1 mole of the Grignard reagent prepared from 4-trimethylsilyl-1-bromobenzene (I) resulted in the formation of p-trimethylsilylthiophenol (II) in the highest yield, with formation of bis (4-trimethylsilylphenyl) disulfide (III) as a byproduct. (III) is reduced to (II) with zinc dust and acetic acid. Similar reaction of 3-trimethylsilyl-1-bromobenzene (IV) affords m-trimethylsilylthiophenol (V). Application of various active halogen compounds to (II) and (V) afforded the corresponding thioethers.

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Preliminary experiments were carried out in order to prepare p-trimethylsilylaniline (V). Application of sodium azide to p-trimethylsilylbenzoyl chloride (II) affords its azide (III) and heating of its benzene solution results in the formation of its isocyanate (IV). Application of various alcohols, amines, and water to (IV) respectively gives the corresponding urethan and urea derivatives, and bis (4-trimethylsilylphenyl) urea. Hydrolysis of ethyl 4-trimethylsilylcarbanilate with dilute hydrochloric acid affords ethyl carbanilate but hydrolysis with alkali affords (V) in quantitative amount.

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In order to examine chemical properties of p-trimethylsilylphenol (I), reaction of (I) with several kinds of cationoid reagents was carried out. Application of bromine to (I) affords p-bromophenol, while that of benzenediazonium chloride gives 4-hydroxyazobenzene. Nitration of acetate (II) of (I) with acetyl nitrate affords p-nitrophenyl acetate and the Friedel-Crafts reaction of the methyl ether (III) of (I) with anhydrous aluminum chloride and acetic anhydride gives p-methoxyacetophenone. The Fries rearrangement of (II) results in formation of o- and p-hydroxyacetophenone. The Reimer-Tiemann reaction of (I) affords 5-trimethyl-silylsalicylaldehyde. (I) is decomposed by 10% hydrochloric acid to form phenol but remains unchanged with 10% sodium hydroxide. These experimental results indicate that the cationoid reagents that easily undergo substitution at a position para to hydroxyl replaces the trimethylsilyl group in (I) while reagents that undergoes substitution in the ortho position does so in (I) without severance of a bond between silicon and the aromatic ring.

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In order to examine chemical properties of p-trimethylsilylaniline (II), reaction of p-trimethylsilylacetanilide (III) with various kinds of cationoid reagent was carried out. Application of bromine to (III) afforded p-bromoacetanilide, and that of anhydrous aluminum chloride and acetyl chloride gave 4-acetylacetanilide. Nitration of (III) with acetyl nitrate gave 4-trimethylsilyl-2-nitroacetanilide (IV) with formation of 4-nitroacetanilide as a byproduct. These experiments have shown that cationoid reagents which tend to undergo substitution in the position para to the amino group, rather than at ortho, tend to replace trimethylsilyl group in (III), while those that tend to undergo substitution in ortho-position is likely to make ordinary substitution reaction at ortho-position in (III). Therefore, chemical properties of (III) was found to be similar to those of p-trimethylsilylphenol.

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Two kinds of crystals giving different X-ray diffraction patterns have been discovered in thiamine hydrochloride and one of the present authors reported this as polymorphism. At about the same time, relationship between these two modifications was clarified through microscopic studies and the presence of a third modifications was clarified through microscopic studies and the presence of a third modification was pointed out. In order to prove the presence of the third modification, and to clarify the temperature and heat of transition, differential thermal analysis was attempted. As a result, it was revealed that there is no third modification in thiamine hydrochloride, that the β-form crystals transited to the α-form crystals at 193±1°, with absorption of 4-6 Kcal./mole of heat, and that this transition is apparently monotropic.

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Infrared spectra of the two kinds of polymorphic forms of thiamine hydrochloride were examined. The spectra showed difference in the absorption region (at 3μ and around 6μ) related to that of OH, NH₂, and NH₃⁺. Therefore, each was deuterated and the infrared spectra of deuterated derivatives were examined. It was concluded from this result that the β-forms formed a stronger hydrogen bond than the α-form, which suggested that there might be difference in their ability to absorb moisture. Measurement of the velocity and equilibrium of moisture absorption showed that the α-form absorbed moisture at a much faster rate and in a larger quantity than the β-form. In general, commercial thiamine hydrochloride found on the market recently is in α-form but β-form is also found at times. When stipulating the amount of moisture in thiamine hydrochloride, the value must differ with the α- and β-forms.

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Vitamin A-choleic acid was first prepared by Shimizu and Hatakeyama but its presence had been doubtful and its structure had only been presumed. In the present series of work, the palmitate, acetate, and alcohol of vitamin A and desoxycholic acid were dissolved in ethanol or ethanol-methanol mixture and crystals of vitamin A-choleic acid were obtained from each of them. X-Ray powder diffraction pattern showed that vitamin A palmitate-choleic acid had the same structure as methyl palmitate-choleic acid, and that vitamin A acetate- and alcohol-choleic acid had the same structure as decalin-choleic acid. Crystal lattice constants of these substances were calculated from oscillation and Weisenberg photographs. It was learned that the vitamin A palmitate-choleic acid had the same crystal structure as that of fatty acid-choleic acid, and the unit lattice in the other two substances was somewhat larger than that of fatty acid-choleic acid but was approximately the same. Infrared spectrum of vitamin A-choleic acid was the same as that of desoxycholic acid. Vitamin A in these substances is stable and not decomposed by tabletting and, since vitamin A is extracted with ether, it was proved that these substances are the channel-type inclusion compound reported by Giacomello and others.

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Several kinds of 1-methoxy-chlorophenazines were prepared by the Wohl-Aue reaction and it was found that N-oxides formed as a by-product in some cases. 1-Methoxy-6-chlorophenazine was prepared by the condensation of 2-nitro-1-chlorobenzene and o-anisidine, and 1-methoxy-8-chlorophenazine by the condensation of p-chloroaniline and o-nitroanisole, but formation of N-oxide was not observed in this case.
Condensation of 3-nitro-1-chlorobenzene and o-anisidine afforded the 1-methoxy-7-chloro and 1-methoxy-9-chloro compounds, and the N-oxide of the latter. Condensation of m-chloroaniline and o-nitroanisole afforded 1-methoxy-9-chlorophenazine, and that of o-chloroaniline and m-nitroanisole gave 1-methoxy-9-chloro compound and 1-methoxyphenazine 5-oxide. Condensation of p-chloroaniline and m-nitroanisole afforded 1-methoxy-7-chlorophenazine and its 5-oxide, 2-chlorophenazine 10-oxide, and 2-methoxy-8-chlorophenazine 10-oxide.

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Boiling of 2-hydrazino-4-hydroxy-6-methylpyrimidine (I) with glacial acetic acid affords 3, 7-dimethyl-5-hydroxy-1, 2, 4-triazolo [4, 3-a] pyrimidine (VI) and its heating to above 260° converts it into 2, 5-dimethyl-7-hydroxy-1, 2, 4-triazolo [2, 3-a]pyrimidine (IX), identical with the condensation product of 3-methyl-5-amino-1, 2, 4-triazole (VIII) and acetoacetic ester. The structure of 1, 3-bis (5-amino-1, 2, 4-triazol-1-yl)-3-methyl-2-propen-1-one (XI) has been given to the condensation product obtained by reacting 5-amino-1, 2, 4-triazole (V) and acetoacetic ester in the cold, in the presence of sodium hydroxide, but this substance was proved to be the salt (XIII) of 5-methyl-7-hydroxy-1, 2, 4-triazolo [2, 3-a] pyrimidine (IV) and (V).

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Reaction of 2-hydrazino-4, 6-dimethylpyrimidine (IV) and orthoformic acid ester afforded 5, 7-dimethyl-1, 2, 4-triazolo [4, 3-a] pyrimidine (III) while boiling of (IV) with formic acid first gives the formylhydrazino compound (V) which undergoes cyclization to (III). (III) is isomerized during the reaction to 5, 7-dimethyl-1, 2, 4-triazolo [2, 3-a] pyrimidine (II), which agrees with the product obtained from condensation of 5-amino-1, 2, 4-triazole (I) and acetylacetone. Similar reaction of 2-hydrazinopyrimidine (X) afforded 1, 2, 4-triazolo [4, 3-a] pyrimidine (XIII) and 1, 2, 4-triazolo [2, 3-a] pyrimidine (XII). (XIII) was found to undergo isomerization to (XII).

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1) Quinaldic acid hydrazide was reacted with various aldehydes to form the corresponding hydrazones (I to XIII), (XVI) was reacted with 2-nitrofurfural, p-dimethylaminobenzaldehyde, p-hydroxybenzaldehyde, and p-acetamidobenzaldehyde to form the corresponding hydrazones (XVII to XX), and (I) and (VII) were led to their acetylated compounds (XIV and XV) by treatment with acetic anhydride.
2) Oxidation of quinaldic acid with hydroxide in acetic acid solution or hydrolysis of quinaldonitrile 1-oxide with sodium hydroxide in ethanol afforded (XXI). Hydrolysis of (XXIII) with ethanolic sodium hydroxide results in deoxygenation to form cinchoninc acid. Therefore, (XXIII) was converted to (XXV) by the Radziszewsky's method, then to (XXVI) with sulfuric acid and sodium nitrite, and (XXI) and (XXVI) were esterified with diazomethane to be converted to (XXVII) and (XXVIII). Reaction of these with hydrazine hydrate afforded the hydrazides (XXIX and XXX). Condensation of (XXX) with p-dimethylaminobenzaldehyde and p-hydroxybenzaldehyde, and that of (XXX) with p-dimethylaminobenzaldehyde afforded the corresponding hydrazones (XXXI to XXXIII) Examinations were made of the antibacterial power of these compounds against the most potent human-type tubercle bacilli, Frankfurt strain.

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In order to clarify the relationship between chemical structure and the hypoglycemic activity, 22 kinds of new 2-arylsulfonamido-5-alkyl-1, 3, 4-thiadiazoles were synthesized. The sulfonamides of this series which have the power to reduce blood sugar level are generally soluble in dilute alkaline carbonate, similar to N-acylated arenesulfonamides, and this nature can be utilized for their purification.

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Acyl derivatives of four kinds of heterocyclic amines of interest in pharmaceutical chemistry, thiazole, pyrimidine, thiazolone, and thiadiazoles, were prepared. The acyl compounds with 2-18 carbone atoms were prepared as 2-acylaminothiazole, 2-acylaminopyrimidine, 2-acylamino-4-thiazolone, and 2-acylamino-1, 3, 4-thiadiazole, and the compounds were screened for antimicrobial activity. Some compounds having growth inhibition against tubercle bacilli in 10^-4 M concentration were found but none showed any activity against other bacteria or virus. In order to introduce an acyl group in N⁴ of N¹-heterocyclic sulfanilamides, N⁴- acyl derivatives of sulfathiazole and sulfisoxazole were prepared and their antimicrobial activity was examined, but none showed any increased antibacterial activity over that of the parent sulfas. Only N⁴-decanoylsulfisoxazole was found to be significantly effective in vivo against Japanese B encephalitis virus.

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With the idea of introducing a hydrophobic group into thiazole-carboxylic acid, possessing a hydrophilic carboxyl group, and thiazolidine-carboxylic acid, forming a part of penicillin structure, 2-acylamino-4-carboxythiazole, 2-acylamino-4-ethoxycarbonylthiazole, 2-acylamino-5-carboxythiazole, 2-acylamino-5-ethoxycarbonylthiazole, and 2-(2-acylaminoethoxycarbonylmethyl)-4-carboxythiazolidine were synthesized and their antimicrobial activity was examined. As a result, there was only one compound which could inhibit the growth of tuberclebacilli in 10^-4M concentration. The compounds of this series were found to show antiviral activity and some compounds showed significant in vivo effect against Japanese B encephalitis virus.

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6-Methoxy-2-benzoxazolinone is an antifungal substance isolated by Hietala and others from corn and wheat. The same substance had been isolated earlier by Koyama from the root of pearl barley and Coix Lachryma-jory L. This compound was newly synthesized by a new and simple process. 2-Benzoxazolinone, without a methoxyl in 6-position, was also prepared and its antifungal effect was compared with that of the former against phytopathogenic fungi.

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Ethyl p-nitrophenyl carbonate has a snailicidal activity. Antifungal activity against phytopathogenic fungi was examined of ethyl 2-nitro-5-methoxyphenyl carbonate and ethyl 2-nitrophenyl carbonate, the intermediates in the synthesis of 6-methoxy-2-benzoxazolinone described in the preceding paper. These compounds were found to have a fairly high antifungal activity, the latter being more effective. About 30 kinds of new and known esters of o-nitrophenyl hydrogen carbonate, thionocarbonate and thiolcarbonates were prepared and variation in antifungal activity due to difference in chemical structure was examined.

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It has been reported that erythro compounds, such as N-acyl-L-ephedrine, 1-phenyl-2-acylamino-1, 3-propanediol, and 1-p-nitrophenyl-2-acylamino-1, 3-propanediol, undergo N-O acyl migration with inversion. However, it has been found that L-erythro-1-p-nitrophenyl-2-benzamido-1, 3-propanediol undergoes N-O acyl migration with retention, and L-erythro-1-p-nitrophenyl-1-benzoyloxy-2-aminopropan-3-ol hydrochloride was obtained.

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In order to substitute the hydroxyl in 3-position of 1-p-nitrophenyl-2-amino-1, 3-propanediol with chlorine, reaction of thionyl chloride with 1-p-nitrophenyl-1-benzoyloxy-2-aminopropan-3-ol hydrochloride was carried out. The L-threo compound afforded L-threo-1-p-nitrophenyl-1-benzoyloxy-2-amino-3-chloropropane hydrochloride but the L-erythro compound afforded L-threo-1-p-nitrophenyl-1-benzoyloxy-2-amino-3-chloropropane hydrochloride when the reaction temperature was high (70-80°) but the corresponding L-erythro compound was obtained at a low reaction temperature.

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Jacob obtained D-threo-1-p-nitrophenyl-1-chloro-2-dichloroacetamido-3-benzoyloxypropane by treatment of L-erythro-1-p-nitrophenyl-2-dichloroacetamido-3-benzoyloxypropan-1-ol with thionyl chloride, and this shows that an inversion had taken place at the C-N center in this reaction. Treatment of L-threo- and L-erythro-1-p-nitrophenyl-2--benzamido-3-benzoyloxypropan-1-ol with thionyl chloride in pyridine afforded L-threo-1-p-nitrophenyl-1-chloro-2-benzamido-3-benzoyloxypropane in both cases and the inversion at C-N center did not take place in this reaction.

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L-threo-1-p-Nitrophenyl-1-benzoyloxy-2-amino-3-chloropropane produced L-threo-and L-erythro-2-phenyl-4-(a-hydroxyp-nitrobenzyl)-2-oxazoline whose treatment with thionyl chloride afforded L-threo-1-p-nitrophenyl-1-benzoyloxy-2-amino-3-chloropro-pane hydrochloride from L-threo-oxazoline, and its D-threo compound from L-erythro-oxazoline. This shows that inversion of the C-N center took place only in L-erythro-oxazoline and indicates that it would be possible to convert the L-threo base to D-threo base by combianation of the above reaction with the reaction in which inversion of C-O center is effected, which was described in a previous paper.

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Due to the necessity of following N-glucuronide formed as a metabolic product of sulfanilamides, a process of determining each component from a mixture of free glucuronic acid, ester-type glucuronide, and ether-type glucuronide, in co-existence with N-glucuronide was examined. For this purpose, reduction with sodium borohydride, decomposition with sodium hydroxide, and oxidation with sodium hypobromite were carried out and it was found that free glucuronic acid and each of conjugated glucuronic acids could be determined separately by the suitable combination of these processes and by selecting reaction conditions. Recovery tests were carried out with simple mixture of these four substances in solution and by addition of these four to normal urine, and fairly satisfactory result was obtained.

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The increased amount of glucuronic acid found in the urine of a rabbit after administration of sulfapyridine was considered to be due to N-glucuronide formed as a metabolic product and the present series of experiments was carried out. The amount of various types of glucuronic acid in the urine of normal rabbit and that administered sulfapyridine was examined by the method of separatory determination of free acid, ester- and ether-type glucuronides, and amine N-glucuronide as described in the preceding paper. It was thereby found that majority of the increased excretion of total glucuronic acid was due to increased excretion of N-glucuronides, which comprised 35-41% of sulfapyridine administered. The lyophilized powder of the urine of a rabbit given sulfapyridine and synthesized sodium sulfapyridine N⁴-glucosiduronate was submitted to chromatography on filter paper buffered with the Universal Buffer Mixture. The Rf values on the chromatogram were found to agree at each pH and the N-glucuronide in the urine was thereby established.

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By application of measuring dissolved gas by carbon dioxide evolution, the content of air and oxygen in a mixture of propylene glycol and related substances (polyethylene glycol 400, glycerol, and ethanol) with water was measured and satisfactory result was obtained. In a mixture of propylene glycol and water, the amount of oxygen decreased with increasing concentration up to 0-60% of the glycol but increased gradually in a concentration range of 70-100% of the glycol (Fig. 1). In a mixture of polyethylene glycol 400 and water, oxygen content decreased with increasing concentration up to 0-60% of the glycol while it increased in the concentration range of 70-100% of the glycol (Fig. 2). In a mixture of glycerol and water, the amount of oxygen decreased with increasing concentration of glycerol (Fig. 3). In a mixture of ethanol and water, the oxygen content remained approximately the same in a concentration range of 0-40% of ethanol while the amount of oxygen increased rapidly with increasing concentration above 50% of ethanol (Fig. 4). The content of air was approximately parallel to that of oxygen in all mixtures.

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Condensation of 3, 4-tetramethylene-5-aminoisoxazole and p-acetamidobenzenesulfonyl chloride afforded 3, 4-tetramethylene-5-sulfanilamidoisoxazole which easily formed a stable sodium salt. Derivatives of this compound with acetyl, propionyl, benzoyl, methyl, benzyl, and dimethylcarbamoylmethyl group bonded to the nitrogen in 1-position were also prepared.

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Application of cyanogen bromide to the aqueous solution (pH 6.0-7.0) of thiamine monohydrochloride (I′) or mononitrate (I″) at 10-20° results in the formation of thiochrome, cyanothiamine, and hydrogen bromide, as well as hydrochloric acid from (I′) and nitric acid from (I″). Four days after the reaction, the amount of thiochrome and cyanothiamine formed became about 10% of the thiamine present before the reaction. The pH of the solution falls rapidly to around 5.0 immediately after the reaction but the fall becomes gradual thereafter, and finally reaches pH 3.42 in (I′) and pH 3.62 in (I′). The free acid formed in the reaction mixture becomes about one mole for thiamine present before the reaction.

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Various kinds of Grignard reagent were reacted with acridine (I) in ether solution, heated at 100° for 1.5-3 hours in a sealed tube, and 9-substituted acridans (II) were obtained in good yield (Table I). Oxidation of (II) with alkaline potassium ferricyanide afforded 9-substituted acridines (III) (Table II) and this process seemed suitable as a laboratory process for preparation of (III). (III) is easily reduced to (II) with aluminum foil in methanolic solution of sodium methoxide. (II) is well crystallizable and forms crystals of a definite melting point that it is a suitable derivative of (III) for identification purposes.

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Acridine (I) forms a structurally unknown addition compound (II) with ammonium thiocyanate. (II) forms 9-acridyl methyl ketone by reaction with methyl ketone. The reaction of (II) with acetone, methyl ethyl ketone, methyl isobutyl ketone, and acetophenone respectively produces 9-acridylacetone, 9-acridylmethyl ethyl ketone, 9-acridylmethyl isobutyl ketone, and 9-phenacylacridine. Diethyl ketone and diisobutyl ketone failed to undergo this reaction.

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Nitration of biphenyl (I) with acetyl nitrate gave a mixture of o-nitro- (II) and p-nitro-biphenyl (III) in 58:42 formation ratio, with a total yield of 87%. The formation of (II) was larger than nitration by other methods (Table I) and the present method seemed to be suitable as a process for preparing (II).

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Following the discovery of the presence of a water-soluble quaternary base, magnoflorine, in the basic component of Chinese Fang-chi, the acid and neutral components in it were examined. The acid component contained aristolochic acid (I), aristolochic acid-B, m.p. 275-276° (decomp.), and aristolochic acid-C, m.p. 280° (decomp.). Crystalline neutral substances isolated were aristolo-lactam (III), allantoin, and β-sitosterol. These results have shown that Chinese Fang-chi is a crude drug originating in the plants of Aristolochia genus of Aristolochiaceae family.

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Examinations were made on an Indian crude drug found in the Bombay market, which was said to originate in Stephania hernandifolia WALP. Isotrilobine (I) was obtained as the non-phenolic base and a new base of m.p. 205-206°; [α]²⁹_D:+268° (c=0.312, CHCl₃), was isolated as a phenolic base. This new base corresponded to the formula of C₃₅H₃₄O₆N₂, containing one methoxyl and two N-methyl groups. Its ultraviolet spectrum agreed with that of trilobine- or hydromenisarine-type bases (Fig. 1). It colored blue to sulfuric and nitric acid mixture and is positive to the dibenzo-p-dioxin reaction. It was therefore assumed that the new base belonged to the menisarine-type, and formulated as (II), one of R₁, R₂, and R₃ being a methyl, and the other two, hydrogen. β-Sitosterol was isolated and identified from the neutral portion. This crude drug did not contain magnoflorine, the aporphine-type quaternary base found in a large number of plants of the Menispermaceae family and related species. Presence of two kinds of different water-soluble, quaternary bases was assumed from the result of paper chromatography.

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Friedelanol, m.p. 309-310°, was isolated and proved from the leaves of Euonymus radicans SIEB. (Celastraceae). The epimer of this substance, epifriedelanol, has been isolated and detected in a fairly wide range of natural substances but this is the first time that a presence of friedelanol has been detected in plants. The mixture of triterpenes obtained from the leaves of E. japonica THUNB. was submitted to chromatography over activated alumina and the mixture was separated into friedelin m.p. 259-262°, epifriedelanol m.p. 282-284°, and friedelanol m.p. 309-311°.

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Relationship between coloration reaction and chemical structure was examined in various steroids by heating in 85% formic acid. The condition for coloration seems to be the presence of a hydroxyl in 3-position and a double bond in 5-position or a radical in 5-position which tends to undergo dehydration to form a double bond, with a hydroxyl or carbonyl in 17- or 20-position. When the substituent in 3-position is a carbonyl and there is a double bond in 4-position, or the A-ring is an aromatic ring with hydroxyl in 3-position, presence of a tertiary hydroxyl in 17-position (excluding the case where carbonyl is present in 20-position) and a secondary hydroxyl in 17α-position required.
It was found that by heating a steroid with 85% formic acid and heating glacial acetic acid solution of bromine, only the steroid with tertiary hydroxyl in 17-position shows coloration while other steroids are colored by heating with 85% formic acid but the color fades on addition of glacial acetic acid solution of bromine. The color developed in compounds of 5-en-3β-ol type is bluish violet and that of compounds of 4-en-3-one type is yellowish red, with absorption maximum at 540-560mμ in the former and at 420-440mμ in the latter.

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