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1 THE BENZOYLATION OF ALKAPTON URINE. BY K. J. P. ORTON AND ARCHIBALD E. GARROD. IN a paper on the excretion of diamines in cystinuria, by P. J. Cammidge and one of us', it was mentioned that when the process of benzoylation, recommended by Udranszky and Baumann2 for the detection of cadaverine and putrescine in urine, was applied to that of an alkaptonuric patient a product was forrmed which, like the benzoyl compounds of the diamines, was precipitated when a hot alcoholic extract of the precipitate was allowed to fall into an excess of water. No such product was obtainred when an aqueous solution of homogentisic acid was similarly treated, but it was formed by the benzoylation of ordinary urine to which that acid had been added. As then obtained the substance was crystalline, melted at C. and was found to contain nitrogen. We have recently undertaken a further investigation of the product id question, and the results of this investigation are embodied in the present communication. The materials employed were the urine of the same alkaptonuric child, and the lead salt of homogentisic acid prepared therefrom. Method of preparation. A litre of the alkapton urine was shaken with 15 c.c. of benzoyl chloride and 150 c.c. of a 10 0/0 solution of sodium hydroxide. The sodium hydroxide was added in instalments, in such a way that an alkaline reaction was maintained, whereas excessive alkalinity was avoided. The shaking was continued until the odour of benzoyl chloride was completely lost, in the course of which process the liquid became quite black, owing to the oxidation of some of the contained homogentisic acid. The precipitate formed, which consisted, in part, of earthy phosphates, was collected upon a filter, washed with water until the washings were colourless, and 1 P. J. Cammidge and A. E. Garrod. Journal of Pathology and Bacteriology, VL p Zeitschr. f. physiol. Chemie, xiii. p

2 90 K. J. P. OR TON AND A. E. GARROD. when dry was repeatedly extracted with boiling alcohol. The hot alcoholic extract was allowed to run, through a warmed filter, into excess of water, when a dense, white, flocculent precipitate was immediately formed in the more or less milky fluid. This was collected upon a filter, redissolved in alcohol and the solution was again thrown into water. After this second separation in water the precipitate was again dissolved in boiling alcohol, and, as the alcohol cooled, the product separated in crystalline form, and after several recrystallisations from bot alcohol, was obtained in the form of fine colourless needles. The preliminary precipitation from water facilitated the obtaining of a pure product, by removing impurities which interfere with the crystallisation from alcohol In the above manner we were able to obtain nearly a gramme of the pure product from 1700 c.c. of the alkapton urine. The nature and constitution of the product. Further investigation showed that the substance so obtained is the amide of dibenzoylhomogentisic acid, the relation of which to homogentisic acid is shown by the following formulse: Homogentisic acid Dibenzoyl-homogentisic Dibensoyl-homogentisamide C8H8o4 acid, CuH1,0o C0H1705N CH2.CO.OH CH2. CO. OH CH2.CO.NH2 OH. CO.C6H5 O.CO.06H5 HO CH5.CO.O C6H5.CO.O Analyses of the product gave the following results: I gram yielded grm. 002 and 0'0895 grm. H20. II ,,,, 4 c.c. of moist N at 19 and 754 mm. III ,,,, 41 c.c.,,,, and 765 mm. Calculated for C2AH705N I II III C = H = '68 - N = ' From it we were able to obtain dibenzoyl-homogentisic acid, and to reconvert this acid into the original amide.

BFNTZOYLATION OF ALKAPTON URINE. Properties of dibenzoyl-homogentisamide. This substance crystallises from alcohol in colourless needles, which are often flattened, and which C6H5.CO.O CH2. CO. NH2 O.

3 BFNTZOYLATION OF ALKAPTON URINE. Properties of dibenzoyl-homogentisamide. This substance crystallises from alcohol in colourless needles, which are often flattened, and which C6H5.CO.O CH2. CO. NH2 O.CO.C6H5 melt at 204 C. without showing any signs of decomposition. In cold alcohol the amide is almost insoluble, but it dissolves fairly readily in hot alcohol. It is but little soluble in benzene, chloroform, or ether; readily in cold glacial acetic acid. Boiling aqueous solutions of alkalis decompose it very slowly, with evolution of ammonia, but with alcoholic solutions of alkalis the action is rapid. In either case the dibenzoylhomogentisic acid formed is rapidly hydrolysed by the alkali. After being heated for many hours with concentrated aqueous hydrochloric acid the amide is practically unchanged, but on boiling with alcoholic solutions of hydrochloric or sulphuric acid the odour of ethyl benzoate becomes obvious, and on adding water a solid substance separates which is neutral in reaction, and after being crystallised from petroleum (B. P ) melts at 1200 C., which is the melting-point of ethyl homogentisate. In concentrated sulphuric acid the amide promptly dissolves and is readily decomposed. On pouling the acid solution on to ice benzoic acid crystallises out, and the liquid has all the properties of a solution of homogentisic acid, including the reduction of Fehling's solution. On the other hand we have obtained dibenzoyl-homogentisic acid by the action of nitric acid, saturated with nitrous acid, upon the amide. Preparation of dibenzoyt-homogentisamide by benzoylation of ammoniacal solutions of honmogentisic acid. It was found that not only was the amide formed by the benzoylation of ordinary urine to which homogentisic acid had been added, but also by the like treatment of solutions of homogentisic acid with ammonia or ammonium carbonate. The product so obtained melted at 2030 C. and was identical in all its properties with that got directly from the alkapton urine. The identity was further shown by the results of analysis gram yielded grm. C02 and grm. H1O. C = 70.37O/0 and H = 4.79G/b. The formula C.H1705N requires C = 70 40/o and H = /0. 91

4 92 K. J. P. ORTON AND A. E. GARROD. This method of preparation shows that ammonia is necessary for the formation of the substance, and the benzoylation of other substituted phenyl-acetic acids (such as para-intro-phenyl-acetic acid) in the presence of ammonia always leads to the formation of considerable quantities of the corresponding amides. C6H5.CO.0 CH2. CO. OH 0.CO.C8H5 Dibenzoyl-homogentisic acid. This acid is readily obtained from dibenzoyl-homogentisamide in the following manner-0-5 gram of the amide is added to 5 c.c. of nitric acid (sp. gr. 1P4) saturated with nitrous acid1; the mixture is cautiously warmed on the water-bath for one minute. Nitrogen is rapidly evolved, and the reaction is ended when the whole of the amide has gone into solution. The hot acid solution is then immediately poured on to about 30 grammes of ice. Practically pure dibenzoyl-homogentisic acid separates, melting at It is best recrystallised from 50 0/0 alcohol, from which it separates in short needles which melt at C gram yielded grm. CO2 and grm. H20. Calculated for C22H16O6 Found C = /0 H = ,, The acid is readily soluble in alcohol, ether, benzene and chloroform, but is almost insoluble in water and in petroleum. Its alkaline solutions darken on exposure to air and reduce Fehling's soluition on warming. On boiling with alcoholic solutions of acids ethyl benzoate is formed, and a neutral substance, melting at 1200, which is probably ethyl-homogentisate. When* the acid is treated with benzoyl chloride and a 100/0 solution of sodium hydroxide, in the presence of ammonia, dibenzoyl-homogentisamide is again formed, melting at 2030 and possessing all the characteristic properties of that compound. Several attempts were 1 This is best effected by adding a few crystals of sodium nitrite to the nitric acid, and then pouring off the liquid from the sodium nitrate formed.

5 BENZOYLATION OF ALKAPTON URINE. made to prepare dibenzoyl-homogentisic acid directly from homogentisic acid, but without success. This acid could not be obtained either by benzoylating by the Schotten-Baumann method, or by prolonged heating, at 1400, of a mixture of homogentisic acid and benzoyl chloride. Preparation of homogentisic acid from its lead salt. In the preparation of the free homogentisic acid required for the above investigations a method was employed which has considerable advantages over those previously made use of by Wolkow and Baumann, and by Huppert2, respectively. Both these processes involve the evaporation of dilute solutions of the acid, set free by the action of hydrogen sulphide upon the lead salt suspended in cold or dissolved in hot water. Not only are the processes very lengthy, but some loss of homogentisic acid is unavoidable; whereas the following plan will be found to be convenient and to give a quantitative yield of the pure acid. Five grams of the very finely powdered, hydrated lead salt are covered with 50 c.c. of ether, through which a current of hydrogen sulphide is passed. The salt is rapidly decomposed, and sufficient heat is developed to cause the ether to boil. After filtering from the lead sulphide formed, the ether is evaporated, leaving colourless stellar aggregates of crystals of the acid, which when so prepared melts at 1520 C. An additional means of detecting the presence of homogentisic acid in urine. The formation in so simple a manner of a substance having such distinctive properties as are possessed by dibenzoyl-homogentisamide, provides an additional and valuable means of detecting the presence of homogentisic acid in urine. The substance-can be readily prepared in crystalline form and may be recognised by its melting-point and other characters. CONCLUSIONS. 1. In the benzoylation of alkapton urine with benzoyl chloride and sodium hydroxide a product is formed which is at once thrown down when a hot alcoholic extract of the precipitate is poured into water. 1 Zeitschr.f. physiol. Chemie, xv. p Deutsches Archiv f. klin. Med., ixv. (FestsChrift), p

6 94 K. J. P. ORTON AND A. E. GARROD. 2. In a pure state this product crystallises in colourless needles which melt at 2040 C. 3. It may also be prepared by the benzoylation in a similar manner of a solution of homogentisic acid, in the presence of ammonia. 4. The product in question is the amide of dibenzoyl-homogentisic acid. 5. From it dibenzoyl-homogentisic acid may be obtained by the action of nitric acid saturated with nitrous acid. 6. When this acid is treated with benzoyl-chloride and a solution of sodiuim hydroxide, in the presence of ammonia, dibenzoyl-homogentisamide is again obtained. 7. The formation of this substance, the properties of which are characteristic, supplies a useful addition to the means at our disposal, for the detection of the presence of homogentisic acid in urine.

JUNIOR COLLEGE CHEMISTRY DEPARTMENT EXPERIMENT 14 SECOND YEAR PRACTICAL. Name: Group: Date:

JUNIOR COLLEGE CHEMISTRY DEPARTMENT EXPERIMENT 14 SECOND YEAR PRACTICAL Name: Group: Date: This practical will serve as (i) an introduction to aromatic chemistry and (ii) a revision of some of the reactions