June 20, 1967
CHEMICAL A N D ENZYMATIC OXIDATIONOF LYSERGIC ACID DIETHYLAMIDE
3191
[CONTRIBUTION FROM THE SATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASESAND FROM THE XATIONAL INSTITUTE O F MENTAL HEALTH, NATIONAL INSTITUTES O F HEALTH]
Studies on the Chemical and Enzymatic Oxidation of Lysergic Acid Diethylamidel BY K. FRETER, J. AXELRODAND B. WITKOP RECEIVED OCTOBER27, 1956 T h e reaction of the trichloroacetate of lysergic acid diethylamide (I) with disulfur dichloride it1 beiizene yielded the symmetric disulfide I1 which on reductive acid hydrolysis and purification by multiple countercurrent distribution gave a non-crystalline compound, which was identical in all respects with a product obtained by enzymatic oxidation of I. on the basis of color reactions, alkaline opening t o a diazotizable amine and ultraviolet absorption spectrum the oxindole structure I11 is suggested for this oxidation product.
Introduction Whereas studies on the metabolic fate of the hallucinogenic (psychotomimetic) lysergic acid diethylamide LSD in intact anirnal~"~have been handicapped by the great potency of I, the formation of a physiologically inactive metabolite (presumably 111) was observed in the oxidation by an enzyme present in liver microsomes.6 This paper describes the formation of I11 from I by reductive hydrolysis of the disulfide 11. CONEt2
CONEti
CONEtz
Conversion of Lysergic Acid Diethylamide (I) to the Disulfide 11.-To a suspension of 300 mg. of LSD ( I ) in 100 nil. of anhydrous benzene was added 500 mg. of anhydrous trichloroacetic acid. -\ clear solution was obtaincd by gentle warming. T o this solution which was cooled to the point of near-solidification was added within 15 minutes a solution of 69 mg. of disulfur dichloride (SpCL) in 10 ml. of benzene. After ten more minutes the strongly yellow rcaction mixture was poured into 700 ml. of petroleum ether (30-40'). The trichloroacetate of the disulfide I1 which separated in yellow flakes was collected and washed thoroughly with petroleum ether and ether. The salt was then transferred into a separatory funnel, decomposed with A' alkali and the free disulfide I1 exhaustively extracted into ethyl acetate. The orange-colored solution was washed, dried and concentrated. The disulfide was recrystallized for analysis from dioxane-water, 1n.p. 184", yield 200 mg. theor.), Rp 0.95 descending technique, 2,4-lutidineamyl alcohol, 1 : 1, saturated with water, Fig. 1. The Ehr-
111
Experimental Purification of the LSD-Metabolite Obtained by Enzymatic Oxidation .-The solution of the crude metabolite from 5 mg. of LSD5 in 0.1 N hydrochloric acid was evaporated t o dryness a t 20" in a vacuum desiccator. The residue was taken up in as little methanol as possible and filtered. The concentrated solution was evenly distributed (micropipet) along the starting line of several sheets of Whatman No. 1filter paper. After completion of the chromatographic process (ascending technique, mixture of 2,4-lutidine-t-amyl alcohol, 1:I, saturated with water) the sheets were dried. Stripes of the area corresponding t o a n &-value of 0.85 were cut out under a n ultraviolet light and eluted by a descending solvent front of methanol. After evaporation of the solvent there remained about 3 mg. of colorless residue which defied crystallization. No crystalline salts could be obtained. The ultraviolet absorption spectrum showed a sharp peak a t 259 mH. (1) Oxidation Mechanisms. XVIII. Previous paper in this series, THISJOURNAL, 78, 2873 (1956). (2) E. S. Boyd, E. Rothlin, J. F. Bonner, I. H. Slater and H. C. Hodge, J . Pharmacol. and Exper. Therap., 113, 6 (1955). (3) A. Stoll, E. Rothlin, J. Rutschmann and W. R . Schalch, E z -
perientio, 11, 396 (1955). (4) V. L a m , A. Cerletti and E. Rothlin, Hclu. Phrsiol. Pharmacol. A ~ l a 13, , 207 (1955). ( 5 ) J. Axelrod, R. 0. Brady, B. Witkop and E . V. Evarts. Nature, 178,143(1956): Symposiumon Psychotomimetic andPsychotherapeutic Agents, Atltlals of the New York Academy of Sciences, in press.
OF
1 2 3 4 5 6 7 8 9 1 0 Fig. l.--&VdufS and analytical deterniination of the various compounds; solvent system 2,,4-lutitIiiie-t-3~ii~l alcohol (1:1, saturatcd witli water): 1 = LSD ( I ) ; 2 = lysergic acid; 3 = disulfide (11) of L S D ; 4 = disulfide of lysergic acid; 5 = hydrolysate of disulfide of lysergic acid; 6 = oxidation of lysergic acid; 7 = hydrolysate of 11; 8 = enzymatic oxidation product of I ; 9 = oxidation of LSD (I) by peracetic acid to a labile Ehrlich-positive compound A and a stable Ehrlich-negative compound B ; 10 = stable Ehrlich-negative oxidation product B of I from Ehrlichpositive oxidation product A by the action of acid. -hlcthods for characterization: F = fluorescence under ultraviolet light; C = color developing after spraying with cinnamaldehyde solution; Y = yellow origiiial color.
K. FRETER, J. AXELROD AND B. WITKOP
3192
Vol. 79
lich reaction was negative, the Keller reaction (concentrated aqueous solutions of varying pH before and after extraction sulfuric acid containing a trace of ferric ion) gave a green with various organic solvents were determined by measurecolor, as a result of the combination of the blue color, char- ment of the per cent. transmission of the maximum of fluoacteristic of &substituted indoles, and of the yellow color of rescence at 430 mp in a Bowman-Aminco spectrofluorophothe disulfide 11. Reduction with zinc dust in pyridinetometer? adjusting the transmission to 100 at the beginning glacial acetic acid according to Kuhn and Wintersteine gave of extraction. a colorless solution which formed a n insoluble red-brown RF-Values: identical, 0.85, cf. Fig. 1. mercaptide with lead acetate and on standing reoxidized to Ehrlich Reaction: both negative. the yellow solution of the disulfide. The ultraviolet specAzotest.-Both compounds, after treatment with hot trum of 11 showed the expected displacement to longer wave aqueous alcoholic alkali, but not before, showed a diazotizlength: 324 (log t 3.54); lysergic acid tartrate, able amino group which coupled after diazotization with an Ao$:” 312 (log t 3.93). alkaline solution of p-naphthol. Reaction of Folin and Ciocalteus: both positive. A i d . Calcd. for C40H4sN~0&.2H20: C, 04.53; H , Oxidation of LSD ( I ) with Peracetic Acid.-Five milli0.98; N , 11.31; S, 8.60. Found: C, 64.72; H , 6.79; N, gram samples of LSD (I) were oxidized with 1 ml. of a mix11.46; S, 8.83. No weight loss was observed when the material was dried ture of glacial acetic acid and 30% hydrogen peroxide (“Supr:roxol,” C.P.)in a ratio of 5:l at room temperature at 100° in vucuo. for ten minutes up t o one hour. T h e reaction mixtures were Dr. A . Hofmann, Sandoz A. G., Basle, Switzerland, had the kindness to inform us t h a t the same disulfide, m.p. freeze-dried and chromatographed using the usual solvent mixture. Two discrete spots were observed (Fig. 1). The 182’, [a]2 0 ~ 1020” (pyridine), analyzing for C ~ O H ~ ~ N ~ O ~ S ~ (recrystallized from dioxane-water, dried at 90” zlt vucuo) compound A with RF 0.5 was obtained on brief oxidation. 249 and has been obtained .in his laboratory. A direct comparison I t gave a positive Ehrlich reaction and had 310 mp. On standing in solution or, more rapidly on treatof our, with his, sample established the identity. Hydrolysis of the Disulfide 11.-Refluxing of I1 with 2070 ment with acid, rearrangement to an Ehrlich-negative cotnpound B, RF 0.85 occurred. acetic acid after a short time led to the evolution of hydrogen Similarly, the action of peracetic acid on lysergic acid sulfide which was trapped as lead sulfide. Although the rate of liberation of H,S decreased markedly i t was still produced two different compounds with RF-values of 0.3 (Ehrlich-positive) and 0.4 (Ehrlich-negative, Fig. 1 ) . noticeable after 6 hr. On a preparative scale, 200 mg. of Lysergic Acid Disulfide.-The reaction of lysergic acid disulfide I1 was dissolved in 30% acetic acid and equidistributed into 5 small glass tubes each of which contained with disulfur dichloride in benzene in the presence of tri100 mg. of C.P. zinc dust. The tubes were sealed in vucuo chloroacetic acid was carried out as described for the prepaand heated t o 110” for 16 hr. The contents of the tubes ration of 11. The crude product separated during the reacwas cooled, lyophilized and the residue taken up in a small tion as a yellow microcrystalline powder, showing increasing volume of methanol, filtered, mixed with a fivefold volume decomposition above 150”,soluble in alcohol, slightly soluble in water, insoluble in ethyl acetate, acetone and benzene; of ether, and filtered again. The filtrate was extracted with the Ehrlich reaction was negative. The crude product dilute HCI, the acid solution made alkaline and extracted with ethyl acetate. After washing, drying and concentra- contained 9.66% of sulfur, calculated 10.7070. A satistion, petroleum ether precipitated from the organic solution factory process for recrystallization was not found. The 30 mg. of a greyish-brown powder, whose color reactions and hydrolysis of this disulfide under the same conditions as RF-value were the same as those of the metabolite. How- used for I1 gave several products (chromatogram, see Fig. 1) ever, the analysis by ultraviolet fluorescence spectropho- none of which was identical with either product from the tometry in a Bowman-Aminco spectrofluorophotometer7 in- oxidation of lysergic acid with peracetic acid. dicated the presence of impurities, among them residual diResults and Discussion sulfide. The ultraviolet spectrum of this crude hydrolysis In principle there can be visualized three simple 320 m p with a shoulder at 260 m p . product showed A, .411 attempts at further purification by crystallization, salt pathways to effect the conversion of LSD (I) to formation or fractional precipitation failed. The close RF- the oxindole derivative I11 : (1) oxidation of I with values of the impurities in various solvent combinations peracetic acidg,a process which is known to oxidize (Fig. 1) prevented successful resolution by paper chroniatography. T h e crude hydrolysate was then purified b y skatole (IV) to atroxindole (VII). The first intermediate is believed to be the /3-hydroxyindolenine countercurrent distribution in a 25 unit Craig machine utilizing as stationary lower phase 10 rnl. of 0.2 31 phosphate V L cwhich in aqueous peracetic acid may add the buffer, pH 7.4 atid as moving upper phase heptaue contain- elements of water to yield the glycol VI (R = H) ing 7Y0 of f-amyl alcohol (both solvent systems being mutually saturated). The crude hydrolysis product (30 mg.) which undergoes acid-catalyzed loss of water (k showcd thc following distribution: tubes 1-6 contaiiied a a beiizylcarbonium ion) to the oxindole VII. 1)rorvn impurity wliicli was not investigated; tulles 8-15 With perbeiizoic acid in chloroform the coniiiion contaiiied tlie colorless solution of 111, A,,,, 259 in@; tubes iiiterinediate V may add peracid to form V I (R = 20-2;i contained yellow solution., of disulfide. The solution in tubes 8-15 was the same as the product of cnzyrnatic OCOC61-16) undergoing ring-fission with loss of benzoate anion to give VIII.” Two products oxidation of I in the following respects: Ultraviolet absorption spectrum: ”:?A: 259 rnp, identical are formed from I and peracetic acid; an Ehrlichwith the enzymatic product. positive compound A rearranging on standing or Ultraviolet fluorescence spectrum: identical; A,, of fluorescence 430 mp with A,, of the exciting beam a t 335 by the action of acid to an Ehrlich-negative compound B. If the glycol analog of VI in the lysergic mp. Distribution Coefficients .-The relative concentrations in acid series were stable one would have to assume
Biphasic systems Inorganic
Orgrnic
Relative concn. of metabolite I11 By enBY hy- zymatic droly- oxidasis of tion I1 of I
Phosphate buffer pH 7.8 Heptane-5% isoamyl alcohol 70 Phosphate buffer pH 7.8 Ethylene dichloride 14 0.1 N hydrochloric acid Ethyl acetate 42
69 15 39
(6) R . K u h n a n d A R’interstein, B e y , , 66, 1737 (19.32) (7) I
