Salts of Luteolin.

It was found (Trans., 1899, 75, 433) that as a rule phenolic colouring matters containing two hydroxyls in the ortho-position relatively to one another decompose the alkali acetates, forming mono-substituted salts. The products then obtained from luteolin in this manner, being gelatinous, were not fully examined. Experiments now show that if a saturated alcoholic solution of luteolin is treated with potassium acetate, and then, while boiling, with a few drops of water, a crystalline salt separates on cooling. This was collected, washed with water, and dried at 160°.

K=12·03.

K = 11.97.

6

=

0-4990 gave 0.1340 K2SO. 0.5053 0.1350 K2SO 0.6559 decomposed with acid gave 0.5800 C15H100. C15H100688.42. CH2OK requires K = 12.03; C15H1006-88-27 per cent. Monopotassium luteolin forms fine yellow needles, and is decomposed by boiling water with separation of luteolin. It closely resembles the corresponding quercetin salt.

The sodium compound is obtained by employing a dilute alcoholic solution of sodium acetate in place of the potassium salt. If crystals do not separate on cooling, water is added drop by drop, and the mixture boiled after each addition. The product is washed first with alcohol, then with water, and finally with alcohol.

Analyses were made with distinct preparations.

0.3696 gave 0.0424 Na2SO4. Na 3.71.

0.4968 0.0584 Na,SO. Na=3.80.

C30H19012Na requires Na= 3.87 per cent.

The sodium salt of luteolin is thus analogous to the potassium salts of rhamnetin and rhamnazin (loc. cit.) as it is a mono-sodium derivative of a double molecule of the colouring matter.

Summary of Results.

On methylation, luteolin from weld yields, not only the normal trimethyl ether, but a second substance, which is regarded as methylluteolin trimethyl ether. This appears to be produced by the entrance of a methyl into the ring of the phloroglucinol nucleus during the methylation process, rather than to result from the presence of methylluteolin (as glucoside) in weld itself, and it is pointed out that luteolin from the Genista tinctoria (loc. cit.) and luteolin monomethyl ether from parsley (Vongerichten, loc. cit.), also yield this compound. By the ethylation of luteolin, Herzig (loc. cit.) has found that, in

addition to the expected triethyl ether, a tetraethyl ether is formed to some extent, so that, in these respects, this colouring matter behaves analogously to resacetophenone (Gregor, Monatsh., 1894, 15, 437) and B-resorcylic acid (Trans., 1895, 67, 995). Curiously enough also genistein (this vol., p. 1310) forms two similarly constituted dimethyl ethers. Other members of the flavone group do not appear to possess this property, for, as a general rule, the hydroxyl adjacent to the carbonyl group is not etherified in the ordinary manner of working, although Friedländer (Ber., 1897, 30, 2154) speaks of a chrysin dimethyl ether received from Piccard for purposes of comparison. Although there is little doubt that methylluteolin trimethyl ether has the constitution assigned to it, it is proposed to further investigate this substance in the hope of isolating methylphloroglucinol from among its decomposition products. Much luteolin will be necessary for this purpose, and as this is difficult to prepare, the publication of the results may be delayed for some time. The decomposition of luteolin into acetylcatechol and phloroglucinol practically establishes the tetrahydroxyflavone constitution previously assigned to it (loc. cit.). In addition to luteolin, weld contains a trace of apigenin, and to this is no doubt due the slightly lower melting point previously assigned to acetylluteolin.

CLOTHWORKERS' RESEARCH LABORATORY,

DYEING DEPARTMENT,

YORKSHIRE College.

CXXVII. Contributions to the Knowledge of Fluorescent Compounds. Part I The Nitro-derivatives of

Fluorescein.

By JOHN THEODORE HEWITT and BRYAN W. Perkins.

IT is a remarkable fact that whereas the tetrabromo- and tetraiododerivatives of fluorescein show marked fluorescence in alkaline solutions, the corresponding nitro-compound exhibits no trace of this property. This fact has already been commented on by Richard Meyer (Zeit. physikal. Chem., 1897, 24, 468) and by one of the authors of the present communication (Proc., 1900, 16, 3; Zeit. physikal. Chem., 1900, 34, 1). Meyer, noticing that tetraiodofluorescein fluoresces less than tetrabromofluorescein, and this, in turn, less than fluorescein itself, would have been inclined to attribute the decrease in the fluorescence to an

increase in the mass of the substituent groups, were it not that an alkaline solution of tetranitrofluorescein shows no trace of fluorescence whatever, although NO2 = 46, whilst Br=80 and I-127. The view put forward by one of the present authors, that in the greater number of cases the fluorescence of organic compounds is caused by a doubly symmetrical tautomerism, furnishes a satisfactory explanation of the fluorescence of fluorescein, eosin, and tetraiodofluorescein where the only tautomerism possible is indicated in the following scheme :

whereas, for a nitro-derivative in which a nitro-group stands in the ortho-position relatively to a hydroxyl-group, it is not only possible, but indeed probable, that the nitrophenol group itself reacts tautometrically, producing a salt in which the metal is attached to the nitroxyl, and not to the hydroxyl grouping. The sodium salt of tetranitrofluorescein should then possess one of following constitutions :

In this case, the configuration would be stable enough to preclude tautomerism, due to the opening of the lactone ring, the oxygen of the hydroxyl groups having been previously transformed into the quinonoid form and sodium ions, thus losing their chance of migrating to the carboxyl group of the lactone ring.

If this view were correct, an exactly similar argument should apply to dinitrofluorescein, but although the so-called hydrate of dinitrofluorescein, which gives blue, non-fluorescent, alkaline solutions, has been prepared and analysed by von Baeyer (Annalen, 1876, 183, 32), the anhydrous substance of the formula C20H10(NO2)2O5 does not appear to have been analysed. In purifying dinitrofluorescein, von Baeyer acetylated the crude product, recrystallised the diacetyl derivative from alcohol or ethyl acetate, and hydrolysed this by boiling with a solution of potassium or sodium hydroxide. On precipitation of the 4 X

VOL. LXXVII.

resulting blue solution with an acid, a yellow substance was obtained, which, even after recrystallisation, again dissolved in alkalis with a blue colour, von Baeyer's numbers for the compound agreeing with those required for the formula C20H12(NO2)2O6. These results we can confirm, but by carrying out the hydrolysis of the diacetyl derivative with sulphuric acid instead of with an alkali, we have succeeded in obtaining the anhydrous dinitrofluorescein; we have also introduced various modifications in methods of purification which considerably simplify the process.

Although the anhydrous dinitro-derivative of fluorescein has been obtained and dissolves in cold alkali with the production of an orangebrown solution which evidently contains a salt of a substance of the formula C20H10 (NO2)2O5, the nitro-groups act in the way which might be expected from the generally accepted views as to the constitution of salts of nitrophenols, and preclude the double tautomerism required for fluorescence which would otherwise result from the opening of the lactone ring. The fact that a solution, even if unstable, of a sodium salt of dinitrofluorescein has been obtained, is of considerable importance, since we have thus been able to examine a derivative of fluorescein which, although still possessing the pyrone ring intact, nevertheless shows no fluorescence in alkaline solutions. The real significance of this result will be observed when it is stated that not only are the usual salts of dinitrofluorescein derived from a hydrate, but that in the case of tetranitrofluorescein it has not been possible to isolate any substance other than a hydrate having the formula CH10(NO2)406Any arguments which might be drawn from the non-fluorescence of the salts of the so-called tetranitrofluorescein are hence invalid.

Preparation of Dinitrofluorescein Hydrate.-In the nitration of fluorescein, von Baeyer's directions with regard to temperature need careful attention. By following his instructions in adding 10 grams of fuming nitric acid to 5 grams of fluorescein dissolved in 100 grams of concentrated sulphuric acid cooled to 0°, and pouring into water immediately after the last addition of nitric acid has been effected, a bright yellow precipitate separates containing very little unaltered fluorescein, much of the dinitro-compound, and always some of the tetranitroderivative. On the other hand, if the solution is allowed to stand overnight, tetranitrofluorescein is practically the only product, as it also is if the temperature is allowed to rise unduly. A very convenient method of separating the tetranitro- and dinitro-derivatives from one another is either to digest the mixture with a solution of sodium acetate, or to dissolve the whole precipitate obtained by pouring into water in hot, dilute sodium hydroxide, and then to precipitate the dinitro-compound with acetic acid, the solution being allowed to cool

before filtering, as the dinitrofluorescein hydrate is fairly soluble in hot water. The colour exhibited by an alkaline solution of the residue shows that it is an almost pure dinitro-compound; for most purposes, it is merely necessary to recrystallise the substance from hot alcohol, whereby small, orange, rhomboidal plates are obtained, which dissolve in cold alkali with a beautiful blue colour. The solution, however, occasionally shows a slight green fluorescence, due to the presence of unattacked fluorescein. If this is the case, it is best to acetylate the product with acetic anhydride, and to recrystallise the acetyl derivative from glacial acetic acid until a sample gives an alkaline solution from which all fluorescence is absent. We confirmed the composition of the dinitrofluorescein hydrate so obtained by analysis:

0.1393 gave 0.2799 CO2 and 0·0335 H2O. C=54·80; H=2·67. 0.2155 12.1 c.c. moist nitrogen at 15° and 768 mm. N = 6·65. C20H12O10N2 requires C=54·52; H2·75; N=6.38 per cent. Diacetyldinitrofluorescein.-On acetylating dinitrofluorescein hydrate, water is split off, and the diacetyl derivative of dinitrofluorescein produced. This acetyl derivative also results on the acetylation of the crude product formed by adding nitric acid to the sulphuric acid solution of dinitrofluorescein, as von Baeyer has previously shown, whilst naturally it may also be obtained, in better yield and of greater purity, by the action of acetic anhydride on the residue left after the crude nitration product is extracted with a dilute sodium acetate solution. It crystallises in colourless rosettes of needles from glacial acetic acid. On analysis:

0.1729 gave 0·3632 CO2 and 0·0450 H2O. C=57·28; H=2·87.

C24H14011N2 requires C=56·91; H=2·76 per cent.

When diacetyldinitrofluorescein is boiled continuously for 10 hours with a mixture of ethyl alcohol and water to which ethyl acetate is added to increase its solubility, a yellow solution is obtained from which, on evaporation of the ethyl acetate and cooling of the mother liquors, small, yellow crystals separate out which partially dissolve in hot sodium carbonate or cold sodium hydroxide (a residue of unattacked diacetyl derivative being left) with an orange colour. On boiling with caustic soda, a blue colour is produced, as might be expected. The substance might be dinitrofluorescein from the colour of its solutions, although this is impossible, since the continued boiling with ethyl alcohol and water would convert any such compound into the hydrate, which dissolves with a blue shade in cold sodium hydroxide. Since the diacetyldinitrofluorescein is insoluble in soda, there seems only one possibility left, namely, that water has been added on to the substance, and a hydrate of the constitution