11. S . Frsrr, N.
PI.JOHNSON
to the oxime, which was found t o melt in part around 95'; on further heating the sample resolidified and remelted a t 194'. The oxime base of I11 is mentioned in the literature* but no details are given. A sample, prepared from authentic I11 and recrystallized from dilute ethanol, melted in part around 95", resolidified and remelted a t 197". I t s mixture with the oxime of the methylation product of IV melted a t 195-196'. Anal. Calcd. for ClaH&204: K, 8.48. For a monohydrate: S , 8.04. Found: (sample dried in t m z ~ oat 100'): N , 8.08.
AND
E. C. HORNINC
Vol. 77
For further proof of identity, the infrared spectra, in KBr discs, of the methylation product of IV, and of the oxime derived from it, were taken* and compared with those of authentic samples of I11 and its oxime. Complete agreement was found. (8) We are indebted to Dr. S. Lieberman, Columbia University f o r permission t o use the College of Physicians and Surgeons, N. Y . , Perkio Elmer 21 spectrophotometer.
R r c i r ~ o s uHILI,, S.Y.
ICONTRIBUTION FROM THE LAUORATORY O F CHEMISTRY O F NATURAL PRODUCTS, NATIONAL H E A R T ISSTITUTE, XATIONAI, INSTITUTES O F HEALTH, LT.s. PUBLIC HEALTHSERVICE, u.s. DEPARTMENT O F HEALTH, EDUCATION ANI1 n'EI.FAR&]
Piptadenia Alkaloids. Indole Bases of P. peregrina (L.) Benth. and Related Species BY h l .
S.F ~ s HS, . 11.JOHNSON
AKD
E. C. HORNING
RECEIVED M A Y 27, 1958 Four indole bases have been found to occur in the seeds and pods of P. peregrina (L.) Benth. and P. macrocarpa Benth. The pods contain S,N-dimethyltryptamine, and the seeds contain bufotenine, bufotenine oxide and S ,N-dimethyltryptamine oxide. In addition, a &hydroxyindole base of unknown structure was found in P. macrocarpa seeds. The identification of these compounds was accomplished by isolation through paper chromatography, and comparison with authentic samples involving Rf data, ultraviolet spectra and fluorescence analysis. The synthesis of these two oxides, hitherto t i w known as components of plant or animal metabolism, is described.
One of the most interesting customs of certain Indian tribes of South America and the Caribbean is the use of a ceremonial snuff inhaled through a bifurcated tube fitting the nose. The use of this snuff during the late fifteenth century was described by Ramon Pane, and a number of later writers have also described its preparation and use. 1,2 All accounts stress the power of the snuff to produce a kind of intoxication during which visions were reputed to occur; excessive doses apparently induced a violent temporary derangement. Seeds of Piptadenia peregrinu (L.) Benth. and possibly P. macrocarpa Benth. were reportedly used for the preparation of the snuff, and the isolation of bufotenine from P. peregrina seeds of Puerto Rican origin was described Further work on the indole bases of Piptadenia species has now been carried out. Because of CUTrent interest in the effects of 5-hydroxyindoles on the central nervous system, it was hoped that some direct means could be found to establish a connection between the suggested source of the s n d , Piptadenia seeds, and authentic snuff. Fortunately, the Smithsonian Institution collection includes under Cat. No. 387781 a Piaroa snuff box of relatively recent origin (1949); the box was found to contain a few milligrams of snuff, and this was used for comparison with "synthetic" laboratory snuff samples prepared in a way that corresponded as closely as possible to the descriptions of Humboldt and Spruce. ? , 4 Using paper chromatography
in a propanol-ammonia system, with Ehrlich's reagent as a spray, one indole area of major intensity and four additional lighter areas were found for the authentic snuff. With laboratory snuff from fieregrina seeds of Puerto Rican origin, the area of major intensity was duplicated (bufotenine), and two additional areas were also identical in Rt value, in color of the sprayed area, and in relative intensity of the color. Two faint areas present for the South American snuff were not observed for the laboratory samples. This evidence indicates that the origin of the authentic snuff was undoubtedly Piptadeniu seeds, although the species could not be identified with certainty. Earlier tests on leaves, bark and seeds of P. peregrina indicated that organic bases were present only in the seeds. These observations were extended through examination of the seeds and seed pods of P. peregrina, P . macrocarpa and P. paniculuta from several areas (Brazil, Florida, Puerto Rico). With the exception of paniculata, all seed samples gave very strong alkaloid tests, while the pods gave tests of varying strength (Table I). Preliminary experiments indicated that the indole bases, including bufotenine, were readily extractable with alcohol or with a tetrahydrofuran-chloroform-ammonia mixture and could be separated by paper chroinntography for purposes of identification. -1 coinbined seed-seed pod sample of P.macrocarpa from Florida was chosen for detailed examination. TABLE I
(1) T h e distribution of modern tribes following this custom is shown !>y J . E. Cooper in "Handbook of the South American Indian," Bu-
reau of American Ethnology, Bulletin 143, E. s. Government Printing OITice, Washington. D. C., 1949, Vol. 5 , pp. 536-539. ( 2 ) Summaries of several early accounts are in Safford, J . Washi?tgInn A c t i d . Sca., 6 , 547 (1016). Spruce's description of snuff preparation ( " S o t e s o f a Rotanist on the Amazon and Andes," E d . by A. R. Wallace, t h e h'lacmillan C o . , I.td., London, 1408, Vol. 11, p p , 426-430) differs from t h a t u f Humiioldt in t h e omission o f calcined calcium carbonate. (3) V. L. Stromherg,THIs J O U R N A L , 76, 1707 (1954). (4) We are indebted t o Dr. Herbert W. Krieger, Curator, Division of IZthrlology, Smithsonian In-titiiticm f o r advice on11 help i n s r r u r i n g thr aiitheutic siiiitT wttrple
PRECIPITATION T E s r s FOR A L K A L O I D S Species Source \re,l5
POil\
P peregrzna (I, ) Benth
P peregrina P . peregrana P. macrocarpa Benth P . macrocarpa J' panicdoto Benth a Not rrcrivrd,
Puerto Kico, 1984 Puerto Rico, 1956 Brazil, 1955 Brazil, 1955 Florida, 1953 Brazil, lclT,.i
+-t+
4-
++ 4+++ +++ t - + + +++ +++ + +-ti
L
(I
Nov. 20, 19,5,5
Isnor,~:RASES OF P. peregrinn (L.) BENTI-I.AND RRLATEDSPECIES
5893
The general procedure for identification of the thentic reference compounds. The ultraviolet bases involved a paper chromatographic separation and fluoresence analysis curves are not adequate in (Whatman 3 MM, propanol-ammonia) of the prin- themselves for the establishment of structure (for cipal components. Five distinct zones were eluted example, N,N-dimethyltryptamine and its oxide with ethanol, and these were employed for detailed give almost identical ultraviolet absorption curves identification work. Each component was chro- and fluorescence analysis curves), but these data, matographed separately and in mixture with a ref- in conjunction with the paper chromatographic erence compound. Three or four solvent systems behavior, leave no doubt about the identification of were employed. The Rr values are in Table 11. the amines. The identification of the two N-osA pure sample was eluted in each case from a sep- ides posed a greater problem, since these oxides arate chromatographic run, and these were used for were unknown until the present work. However, ultraviolet absorption spectra determinations and synthetic samples of the oxides were prepared by for fluorescence analysis spectra. These data were peroxide oxidation of the corresponding bases, and compared with corresponding spectra for reference these synthetic substances were found to be idencompounds (Tables I11 and IV). I n this way four tical with two of the Piptadenia components. I n indole components were identified as bufotenine, separate experiments i t was found that reduction of NIX-dimethyltryptamine, bufotenine oxide and both natural and synthetic oxides to the parent N,N-dimethyltryptamine oxide. A fifth indole (E) bases was readily accomplished by zinc-acetic acid, remained unidentified, but its properties (discussed and these chemical data, together with the spectra in the Experimental section) indicated a 5-hydroxy- of the oxidized forms, served to establish the Nindole structure. It was not identical with seroto- oxide structure. While the occurrence o€ N-oxides in plants is not nin, N-methylserotonin, bufotenidine or dehydrounusual, separate experiments were undertaken to bufotenine. determine whether the oxides were indeed present TABLE I1 in both seeds and pods, or whether they were formed PAPER CHROMATOGRAPHIC DATA during the isolation procedure. The rather surprisRr VALUESFOR SOLVENT SYSTEMS ing observation was made that the pods of both P . I II' IIIb IV' peregrina and P. macrocarpa contained only N,N-diN,N-Dimethyltryptamine (A) 0.86" 0 , 9 2 0.67 0.71 methyltryptamine ; bufotenine and the two oxides Bufotenine (B) .82b .89 .62 .51 were not present. It was further found that N,NS,N-Dimethyltryptamine oxide dimethyltryptamine oxidizes rather easily in solu.56" .70 .76 .72 tions exposed to air, and that consequently the (C ) Bufotenine oxide (D) .3!ib .46 .72 .62 isolation of its oxide after extensive experimental . 27b .68 .49 manipulation may be an artifact. Comparable exCnknourn (E) Whatman 3 MM, washed. a S & S 507, unwashed. periments demonstrated the presence of bufotenine oxide and dimethyltryptamine oxide, along with TABLE I11 bufotenine, in both peregrina and macrocarpa seeds. SPECTRA DATA ULTRAVIOLET Since the oxidation of bufotenine was never observed Maxima, mu Compound 1 2 3 in the absence of a specific oxidizing agent, it may be presumed that this oxide is indeed present in the 274 283 291 N,N-Dimethyltryptamine seeds. The unidentified indole base (E) (Tables 11, 275 283 291 (A) IV) was also found in Brazilian macrocarpa seeds, N,N-Dimethyltryptamine oxide 274 282 290 but it was not observed as a component of the pods. 274 282 291 ( C) These determinations raise several interesting Bufotenine 279 301 314" questions of comparative biochemistry. A recent 279 301 315" (B) investigation by Page5 of the indole bases in normal 278 301 314" Bufotenine oxide human urine indicated the presence of serotonin, N275 802 314" ( D) methylserotonin, bufotenine and two additional Shoulder. unidentified indole bases. Titus and Udenfriend,G TABLE IV working with toads, established the presence of serotonin along with bufotenine in the secretion of FLUORESCENCE ANALYSIS DATA the parotid gland, and in separate studies UdenPosition of maxima, ma Compound Exitation Emission friend developed the hypothesis that tryptophan is N,N-Dimethyltryptamine (A) 283 350 converted to serotonin via 5-hydroxytryptophan, N,N-Dimethyltryptamine oxide (C) 283 349 and that the end product of this metabolic seBufotenine (B) 300 340 quence is 5-hydroxyindoleacetic acid. The presence Bufotenine oxide (D) 308 330 of bufotenine in such varied sources as Piptadenin 305 335 (E) seeds, toads, certain mushrooms and human urine The validity of these identifications may be es- indicates a ubiquitous nature. This compound is timated by examination of the data for bufotenine evidently associated with serotonin as a product of and N,N-dimethyltryptamine. The first com- tryptophan metabolism, but its metabolic route of pound previously was isolated in crystalline form synthesis and its function are unknown. It may from P. peregrina. The solvent systems and papers possibly be derived from serotonin by methylation, employed gave excellent resolution and good repro- yet the distribution of N,N-dimethyltryptamine, ducibility for indole bases; in this case the l i t values (3) F. M. Bumpus and I H Page, J . Biol. Chem., 212, 111 (195.5). and colors were identical for the natural and au(0) E Titiis and S. IJdenfrientl, F e d r m l t o n Proc., 13, 411 (1'154).
5 894
M. S. FISH,N. 11.1. JOHNSON
bufotenine and the two amine oxides in Piptadenia seeds and pods indicates that a common substance was supplied to the growing seed pod, and that a peroxidic (OH)@type of oxidation leading to bufotenine and to bufotenine oxide occurred in the seed. Serotonin seems less likely to be this common substance than N,N-dimethyltryptamine or tryptamine, These data point to the existence of a new pathway of tryptophan metabolism. The part sequence N,N-dimethyltryptamine + bufotenine -.t bufotenine oxide is suggested by the distribution pattern. The immediate precursor of N,N-dimethyltryptamine and the relationship of (E) to this sequence is unknown. Current speculations on the role of tryptophan metabolites in the central nervous system stress the possible role of serotonin; in view of Page's isolation of bufotenine from normal human urine it may be of equal importance to study the related problems involved in bufotenine synthesis and metabolism.' I n connection with the present isoIation of two amine oxides as components in tryptophan metabolism, two recent notes should be cited. Recent studies by Tsuyuki, Stahmann and Casida8 have shown that octamethylpyrophosphoramide (an insecticide) may be oxidized enzymatically and by chemical agents to a potent anticholinesterase (I). This metabolic transformation product is unstable in both acid and alkali by reason of its rearrangement to a less active but more stable substance (IIA) which in turn may be hydrolyzed easily to formaldehyde and heptamethylpyrophosphoramide (111). This reaction was interpreted as follows (-4)
AND
E. C. HORNING
Vol. 77
planation for the general reaction of N-dealkylation often observed in studies of drug metabolism' and it also may occur for normal metabolites. For example, the possible products from a similar reaction with bufotenine oxide and N,N-dimethyltryptamine oxide would have the part structures -CH,CH,N(
CHiOH CHB
or
CH? --cH,cH~N
