.icu: rtuum. tniecicu 5, 275-J02 (1971)

Biosynthetic studies on and related cactus alkaloids'

JAN LUNDSTRoM

Ttepurlmeni of Pharmacognosy, Formaceuiiska Fakulieten, Box 680'1, S-11386 Stockholm

In this survey, the following papers will be discussed and will be referred to by the Roman numerals given in the following list. Some unpublished results are also presented.

L J. Lundstrom and S. Agurell. Thin-layer chromatography of the peyote alkaloids. Journal of Chromatography 30, 271 (1967).

II. J. Lundstrom and S. Agurcll. Gas chromatography of peyote alkaloids.A new peyote alkaloid. Journal of Chromatography 36, 105 (1968).

Ill. J. Lundstrom and S, Agurell. Synthesis of specifically labelled substituted .8-, Acta Pluirm. Suecica 7,247 (1970),

IV, S. Agurell, J. Lundstrom and F. Sandberg.Biosynthesis of mescaline in peyote, Tetrahedron Letlers 2433 (1967).

V, J. Lundstrom and S. Agurell, Biosynthesis of mescaline and in peyote. Tetrahedron Letters 4437 (1958).

VI. S, Agurcll and J, Lundstrom, Apparent intermediates in the biosynthesis of mescaline and related tetrahvdroisoquinolines. Chemical Communica- tions 1638 (1968),

1 Inaugural dissertation.

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/ I J. LUNDSTRo?rf

YII. S. Agurell, J. Lundstrom and A. Masoud. Cactaceae alkaloids VII. Alkaloids of Ecliinocereus merkeri. Journal of Pharmaceutical Sciences 58, 1413 (1969).

YIII. J. Lundstrom and S. Agurell. Biosynthesis of mescaline and other peyote alkaloids. Abh. d. Deutsch.Akad, Wissenschaften (Berlin). In press.

IX. J. Lundstrom and S. Agurell. A complete hiosynthctic sequence from tyrosine to mescaline in two cactus species. Tetrahedron Letters 3371 (1969) .

X. J. Lundstrom. Biosynthesis of mescaline and 3,4-dimethoxyphenethyl- amine in Trichocereus pacliatioi Br&R .. 4cia Pharm. Suecica 7, 651 (1970).

XI. J. E. Lindgren, S. Agurell, J. Lundstrom and U. Svensson.Detection of bio- chemical intermediates by mass fragmentography: Mescaline and tetra- hydroisoquinoline precursors. Febs Letters. 13, 21 (1971).

XII. S.Agurell, J. G. Bruhn, J. Lundstrom and U. Svensson. Cactus alkaloids X: Alkaloids of TricJwcereus species and some other cacti. Lloydia. In press.

XIII. J. Lundstrom. Biosynthesis of mescaline and alkaloids in Lopho pliora urilliamsii (Lem.) Coult. Occurrence and bio- synthesis of precursors. Acla Chern, Scand. In press.

XIV. J. Lundstrom and S. Agurell. Biosynthesis of mescaline and tetrahydro- isoquinoline alkaloids in Lophophora williamsii (Lem.) Coult. Acta Pharm. Suecica 8,261 (1971).

XV. J. Lundstrom. Biosynthesis of tetrahydroisoquinoline alkaloids in Lopho- phora uiilliamsii (Lern.)Coult. Acta Pharm. Suecica. In press.

XVI. J. Lundstrom. Identification of new peyote alkaloids.Isomers of the main phenolic thetrahvdroisoquinoliues. Submitted to .4cla Cheiu,Scand.

Mescaline represents one of the earliest known hallucinogenic substances, the structure of which was determined as 3,4,5-trimethoxyphenethyl- amine more than 50 years ago [1 J. The hallucinatory properties of mesca- line combined with the observation of the similarities of its structure to physiologically active e.g. , dopa and have attracted the interest of a large number of scientists [cf. 2, 3J. Adre- naline, noradrenaline and dopamine are all substances that seem to be intimately involved in the function of the brain and it seemed logical to relate the action of mescaline to these substances. The hallucinatory

276

I l'H6sYN'fHEtfC STUDIES ON MESCALINE AND RELATED CACTUS ALI,ALOIDS

OH HO~COOH HO~ HO~ HOV ~H2 HOV ~H2 HOV ~HCH3

MESCALI NE DOPA COPAMI NE ADRENALINE

effects of mescaline resemble in some respect symtorns of various mental disturbances and, in fact, it has been suggested that compounds similar to mescaline or mescaline itself could be formed in the body by an erroneous catecholamine metabolism, thus producing an endogenous psychosis. In the hope of finding the cause and maybe even the cure for various mental illnesses, much research work has been performed on mescaline, including its psychopharmacological effects, its metabolism in the animal body and its use for the production of "model psychoses". Recent reviews covering these fields are given by Hoffer and Osmond [2] and Patel [3]. During the preparation of this manuscript, a review by Kapadia and Fayez [4 J on peyote constituents appeared, covering also biological effects of mescaline and similar substances. The earliest and most well-known source of mescaline is the peyote cactus, Lopho phora williamsii (Lem.) Coulter, used from time imme- morial by Indian tribes in the southern parts of USA and in northern Mexico [5, 6].Besides mescaline, the peyote cactus produces a range of tetrahydroisoquinoline alkaloids exemplified by anhalamine, , lophophorine and anhalinine (see also p. 282) l7].

LOPHOPHORINE ANHALAMINE ANHALININE PELLOTINE

A second source of mescaline was found in the huge column cactus Triclioceretis pachanoi Britton & Rose [8], used by Indians in Peru in their preparation of "cimora", a hallucinogenic drink [6, 9].Mescaline has also recently been found in several other Tricliocereus species [XII, 10,11] . Although considerable information was available at the commencement of this work concerning the metabolism of mescaline and similar sub- stances in animal tissues, little was known about the formation and meta- bolism in the plant [3J. It has long been a matter of general agreement that alkaloids like these arise in nature from amino acid precursors such as and tyrosine [12]. More detailed investigation of the biosynthesis of mescaline and similar compounds in their parent plants was desired, for the aforementioned reasons, in order to obtain a broader understanding of hallucinogenic substances. Furthermore, detailed in-

277

I J. LU~DSTRinI

formation concerning the biochemical reactions leading from primary metabolites, e.g. tyrosine, to mescaline and to its related compounds, would be of importance for an understanding of secondary metabolism in plants. The alkaloids produced by cacti showing a range of structural varieties within limited frameworks appeared suitable for biosynthetic experiments.

Scope of the present investigation

"Then this work was initiated, it was known from Leete's early experi- ments [13 J that tyrosine could be converted to mescaline in peyote. A few types of biochemical reactions seemed to be involved in the trans- formation of tyrosine to mescaline: decarboxylation, hydroxylation of the aromatic nucleus and O-methylations. Enzymes from both animal and plant origin which catalyze these types of reactions are known. We decided to investigate rigorously the sequence of ordcr, if any, for these biochemical transformations occurring in the conversion of tyro- sine to mescaline. It was also our intention to investigate thc biosynthesis of the tetrahydroisoquinoline alkaloids of peyote and especially to study the extent to which the biosynthesis of mescaline and these structurally related compounds were linked by common pathways, For the accurate determination of hiosynthetic pathways, compounds, postulated as precursors and intermediates on account of incorporation experiments using radioactive tracers, should also be identified as natu- rally occurring metabolites.In performing this research, sensitive methods such as gas chromatography and gas chromatography-mass spectrometry (GLC-MS) were used and several compounds fitting in the hiosynthetic schemes were identified as occurring naturally in cacti. To broaden our experience in the biosynthesis of mescaline, parallel studies were performcd in the two mescaline producing cacti L urilliamsii and T. pachanoi, Thus, according to our results, and results meanwhile obtained inde- pendently by other groups, Leete [Ll, 15], Paul et al, [ef. 16],Battersby et al, [17, 18J and Kapadia ei al. [cf. 4, 19], the hiosynthetic sequence in the formation of mescaline and the phenethylarnine portion of thc tetrahydroisoquinoline alkaloids of peyote, is one of the most complete sequences in alkaloid biosynthesis known today. In addition to L. uiilliamsi and T. pachanoi, the alkaloid contents of several Trichocereus species were examined and mescaline was found in some of these. Also, some other cacti were investigated and many alka- loidal compounds, previously unknown from cacti or other plants, were identified and valuable information regarding the biosvnthetic work was gained.

Chromatographic methods (TLC, GLC) of great importance for further work are mainly described in papers I and II.

278

r BIOSY::\THETICSTUDIES 0::\ MESCALI::\E AND RELATED CACTUS ALKALOIDS

Methods for the synthesis of labelled compounds are mainly described in paper III. Some additional syntheses of doubly labelled compounds are presented in paper XV. Results on the biosynthesis of mescaline are presented in the papers IY, V, VIII-X and XIII, XIV. The biosynthesis of 3,1~-dimethoxyphenethyl- amine is treated in paper X. Biosynthesis of the portion of the tetralujdroisoquitioline alkaloids is described in papers V, VIII, IX and XIII-XV. Some results on the ring closing units (C-l and C-l + C-9) in the tetrolnjtiroiscquinoline biosuniliesis are presented in papers V and XY. Intermediates of the biosijnihetic pathways, identified by GLC-MS or in "trapping experiments" are discussed in papers ·VI, XIV and XV. The use of the sensitive and very selective analytical method called mass [raqmentoorcphu, in the search for intermediates of biosynthetic routes is discussed in paper XI. Some new teirobudroisoquinoline alkaloids of hiosvnthetic interest are presented in paper XVI. The alkaloid contents of some 'I'richocereus species and other cacti are presented in papers VII and XII.

Cactus alkaloids

Historical remarks on the use of cacti Peyote [5, 6J The earliest reports in the literature on the hallucinogenic effccts of peyote are written by the chroniclers of the Spanish conquest of Mexico. Peyote was probably userl and worshipped by Indian-tribes in the southern parts of North America centuries before that time. Peyote is a popular name, derived from the ancient Aztec word "peyotl", for Loplio phora ioilliamsii (Lem.) Coulter, a small, spineless cactus, sage-green in colour and with white silky hairs on the top. Its natural habitat is the Mexican plateau and southwestern United Statcs. For harvesting, the part of the plant above ground is looped off and cut into two slices, which are dried in the sun. The dehydrated, hard, brownish slices, irregular in form, constitute the "mescal buttons".It is this material, which can be preserved for a very long time, that is usually consumed in ritual ceremonies or for non-religious purposes to pro- duce euphoric effects. Until the end of the 18th century peyote was taken mainly by indiuiduals, as a medicine and to induce visions leading to prophetic utterance, and collec- tively to obtain the desired state of trance for ritual dances. Primitive peyote ceremonies are still found among certain tribes in northern Mex ico (Cora, Huichol,Tarahumare). As a result of considerable movements of Indian-tribes of ::\o1'th America during the 19th century, the use of peyote was spread oyer wide areas to about forty more tribes. The use of peyote was adopted mainly by Xlescalero-Auaches, Kiowas and Chomanches during raids southwards into Xlcxico. On the return

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/ I J. LUNDSTRoM

to the prarrres, an entirely new peyote-cult gradually formed consisting of strange synthesis of old Mexican and Christian elements as well as differe local religious rites. The Native American Church was thus estahlishe-' ;- United States at the beginning of this century.

San Pedro [6, 9]

In the northern coastal region of Peru, the huge column cactus TriCli·' A .,-,,, pacluuioi Britton and Rose, is employed by folk healers in the diagnr cure of illness. This cactus, under the name of San Pedro, is used a,' several ingredients in the preparation of the hallucinogenic drink ''t..imora There is indirect evidence for the longtime use of this narcotic drink \\ concerned with the moon rites of the religion. It is taken for therapeutic. for diagnosis and divination, and to enable an indiv idual to assume ow' of another person's identity.

Structures Although the occurrence of alkaloids in the family Cactaceae is fairly 'wide-spread, the structural types of cactus alkaloids so far known and reported are limited to differently substituted phenethylamine deriva- tives and related [11], General formulas are seen in Fig. 1, and the various types of structures are also well represented by the peyote alkaloids shown in Fig. 2 (p. 282) .

Fig. 1

R,' H-. HO- or CH30-

R2, H- or CHr R), H- or HO- R4:H-. CH3- or (CH)h CHCHr

Side chain hydroxylated phenethylamincs such as (35) were found in Lepidocoru plianilia species [20, 21J. An analogous sub- stituted tetrahydroisoquinoline, , was reported in Carneqia gi- gante-a [20,39].

35.' MACROMERI HE JIS. J7.

280

I BIClSYNTH1?TIC STUDIES ON MESCALINE AND BELATED CACTUS ALKALOIDS

However, the structure 36 for gigantine was found erroneous by Brossi ei al, [22J. Later findings by Kapadia et al, [23J support the structure 37 r . gigantine. The ring closing units (C-1 and its substituents) of known tetrahydro- isoquinoline alkaloids of cacti are: one carbon units (Fig. 1, R, = H), two cirbon units (Fig. 1, R, = CHe) or isoprene units (Fig. 1, R, = (eRe), . CHCH,-). Lopohocerine (38) found in Lophocereus schot ti by ',:erassi et al. [24J is a representative of the last type. Lophocerine under- les oxidative coupling in vivo to the trimeric pilocerein (39) [24, 25J.

o

3'6, LOPHOCERI NE 39. PI LOCEREI NE

Only in one single case has an alkaloid structure diverging from these mentioned been found in cacti. Rosenberg and Paul [26 J reported the imidazol alkaloid dolichotheleine (40) from Dolichothele spliaerica.

40.00LlCHOTHELEINE

Peyote alkaloids The cactus 'which is by far the richest in alkaloids, at least with respect to the number of different alkaloids, is the peyote cactus or Lophophora williamsii. No less than 28 alkaloids are, up to the present date, known to occur in this cactus and there are still more to be encountered. The structures of the different alkaloids are shown together with their names in Fig. 2. The elucidation of the structure and the synthesis of mescaline and ten other alkaloid constituents of peyote was accomplished by the Aust- rian chemist Spath [ef. 4, 7J during thc period 1919-1939. All major alkaloids were covered in these studies and compounds later found (1965 -present) can mainly be regarded as minor ones or trace constituents, possibly 'with the exception of :1-hydroxy-4,5-dimethoxyphenethylamine

281

I J. LUNDSTRo:\I

"/0

CH,O~I HO~ CH30~i 8 ,.tr.) (tr. ) (tr.) CH,O~NH )mH' CH,O~ NH, CH,O~NH HO CH,O YRAI-II NE [37] 9. 3,,·01 METHOXV- 15. ANHA,-AMI E (7; 23. ISQANI-IALAMINE [XVI] PH?: £TH'(\..AMINE (II]

CH,O~ (t r.) HOV ~H, HO 14 HOfi(1 (t r.l H.tE. THYl TYRAt-ilNE [37) 10, 3,L-OiHYOROXY-5-M!THOXY' CH,O~~H PH!:NE';'HYlAMINE (:C:IlIJ CH,O

17. AN!-iALONfOf NE [7] 2Jf. ISOANHA.LONIDINE [XVI]

CH'0(1(1 1-5 '~ 8 oV N(CH,l, CH,O~ NH, HO CH,O~ HO~ (1,37) 1/, )-HYDROXY-l"S-DiMETHOX)" 2 (tr: ) PKENETHYLAMINE [Vl,29J CHJO~~CH3 CH30~~CH3 HO CH,O

18. ANHALIDI NE [7J 25. ISOANHALIOiNE :XYII

CH'O~I ' (tr.) (t n) CH30V ~HCH3 HO DOPAMI NE. (XlIl) 12. N-M::THYL- 3- HYDROX·,· CHJO~: -L,5- 01 MET HOXY PH!: NETHYL- 17 HOF'l(1 0.5 AM I HE lxvl CH,O~NCH3 CH,O~NCH3 HO CH,O ;9. PELLOTINE [7] 25.ISOPELlOTINE txvn

O~ CH,O(!(J (t to) CH,OV N(CH,l, 0.5 .00 NHCH, HO :EPININE [XII!] IJ. H N-OI METHYL- )-HYOROXY- -',5-01 METHOXYPH!:NEi HV> CH)0f101 ' CH,O~ AMI HE Ixvl O~Nf' 3 CH,O~~H 0.5 Lo ' CH,O

20. ANHAlONINE (I; 27 A HAll INE [71 'o~ CH'0(\(i (t n) 30 00 NH, CH,O~ Nf', CH,O ,4-HYOROXV- 3-METHOX,(- IJ;. MESCAL! HE [1, i J PHENETHYlAMINE [Xlll] CH,Ofl(1 5 <0.5 CH,O~NH CH,O CH,O~i o~ 21, LOPHOPHORINE ('13 28. O-METHYL 3 NHCH) <0.5 CH,OV NHCH, AHHAlONI 01 HE (0] 00 CH,O N- MET HYl-l. - HYDROXY- /5. N-METHYU..\!::SCAlINE [0]

-)- MET H OXY PH E N ET HYL-

AMINE [XIII] < 0.5

ama '" I N (CHl 0,5-2 22. PEYOPHORINE [30} " N,N-DIMETHYL- -4 ·HYD ROXY· ]- MET HDX'f- PHENETHYLAMINE [XlI1} Fig. 2. Alkoloidal constituents of peyote, and their approximative percentages in the alkaloid fraction. Trace omounls are indicaied by (11'.). (The formula of peyophorine (22) should include a 1-melhyl sub stitucnt.)

282

I BIOSY:-\THETIC Sn:DIES 0:-\ }IESCALINE AND RELATED CACTDS ALKALOIDS

(11). ~rcLaughlin and Paul [37J identified (1), N-methyl- tyramine (2) and hordenine (N,N-dimethyltyramine, anhaline, 3) using column and thin layer chromatography. In our search for intermediates in the biosynthesis of peyote alkaloids, we identified 3-hydroxy-4,5-dimethoxyphenethylamine (3-demethylmesca- line, 11) using gas chromatography and gas chromatography-mass spec- trometry (GLC-MS) [Y1J. Fresh cactus material was shown to contain considerable amounts (1-5 %) of this compound [XIV]. Later studies [XV] also revealed the presence of the .Y-methyl (12) and S,N-dimethyl (13) deri vatives of 11. 4-Hydroxy-3-mdhoxyphenethylamine (6, 3- methoxytyramine), an efficient mescaline and tetrahydroisoquinoline precursor, was shown to occur in peyote by employing a trapping experi- ment [XIIIJ. The S-methyl (7) and N,N-dimethyl (8) derivatives of this compound were also encountered by this method [XIII]. In addition, N-methyl--l-hyclroxy-3-metoxyphenethylamine (7) was earlier identified by massf ragmentographv [XIJ. The two diphenolic compounds, dopamine (4) and 3A-dihydroxy-5-methoxy-phenethylamine (10), were shown to occur in peyote by trapping experiments [XIII]. The four tetrahydroisoquinolines23, 24, 25 and 26 were recently identi- fied in the alkaloid fraction of peyote by preparative GLC and GLC-:\IS as described in paper XYI. These compounds can be regarded as isomers of the four major phenolic tetrahydroisoquinolines 16, 17, 18 and 19.1 Biogenetic ally, they are believed to arise from the same pheriethylaminc precursor, 3-hydroxy-J,5-dimethoxyphenethylamine (11). In addition to 3-hydroxy-J,5-dimethoxyphenethylamine (11, 3-de- methylmesc aline ) , Kapadia et al, [29, 30J identified the tetrahydroiso- quinoline peyophoririe (22) containing an -V-ethyl substituent. Kapadia and his coworkers also examined the relatively minor non-basic (quater- nary, amide and acid) constituents of relation to peyote alkaloids and identified 24 additional compounds. These include the quaternary deriva- tives of 18,19 and 21 (Fig. 2), called anhalotirie, peyotirie and lophotirie; iV-formyl and N-acetyl derivatives of several of the compounds of Fig. 2; several cyclic amides of mescaline: and some acidic compounds. Some of these compounds wi ll he discussed in relation to their biosynthetic role (below) , others may be found in the recent review by Kapadia and Fayez [4]. Regarding percentages for the alkaloidal contents of peyote, both for total alkaloids and for separate compounds, the literature is meagre. Rouhier -cf. 5] reports 3.70 % total alkaloids of dried "upper slices of mescal huttons" and 0.41 % for fresh peyote heads. On an average, we found O.J;c (\Y I\Y) total alkaloids in fresh peyote cacti, in good accor- dance with the earlier reported figure. Percentages for the separate alka- loids have not been found in the literature. A special study was thus conducted to determine these average percentages using GLC (peak

1. It would possibly be of biosynthetic interest to inve st ig ate if the compounds 23 and 26 occur optically act lve in pcvote. The non-phenolic tetrahydroisoquinolines 20, 21 and 28 have been isolated optically actin, while the rapid raccmisation of pelJotine (19) and (7) so far h ave made it impossible to inve st igat e wether or not these phenols occur optically act ive in the l ivirig plant [71:.

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I J. LUNDSTRoM

areas). The figures obtained for the percentages of separate compounds of the alkaloid fraction of peyote are given in Fig. 2. The figures are mean values of the alkaloid content of several runs at different times of the year. It should, however, be emphasized that these percentages are obtained from our green-house grown cacti and it is not known whether they are valid for cacti growing in their natural habitat. A variation of the alkaloid contents following the seasons of the year was observed. Plants harvested during the late autumn and the 'winter showed higher percentages of N-demethylated compounds, e.g. anhalamine (16) and anhalonidine (17), compared to the corresponding N-methyl compounds anhalidine (18) and pellotine (19). Similar changes in the alkaloid con- tents were observed in Camegia gigantea viz. carnegine (42) ~ nor- carnegine (solasodine) and very markedly in Lepidocorn pbontha macro- meris between macromerine (35) and its N-demethylated derivatives.

Alkaloids of T r i c hoc ere u s species and some other cacti \Vhen this wor k was started, in addition to L. williams ii, mescaline was known from the cacti Tricliocereus pachanoi, T'richocereus terscheckii and Gymnocalycium gibbosum ([8J, cf. [l1J). The N,N-dimethyl deriva- tive of mescaline, trichocereine (41), was known from T. iersclieckii [27 J .

JO. ,-HY OROXY- 3, 5-01 MET HOXY- 41, TRI CHOCEREI NE PHENETHYLAMINE

To obtain a broader ground for our biosynthetic studies, presursor ex- periments were undertaken in T. pacJwnoi parallel to experiments in L. unlliamsii. In the search for intermediates of the biosynthetic pathways to mescaline and other alkaloids, highly valuable information was ob- tained by the identification of the assumed mescaline precursors 4-hydroxy-3-methoxyphenetylamine (6) and 4-hydroxy-3,5-dimethoxy- phenethylamine (SO) in T. pachanoi [VIJ. Later, rigorous investigations on the trace clements of the alkaloid fraction of this cactus [cf. XJ revealed the presence of the tetrahydroisoquinolines anhalonidine (17) and anhali- nine (27), and also the assumed precursor of tetrahydroisoquinolines, 3-hydroxy-4,5-dimethoxy-phenethylamine (11). However, the lastmen- tioned compound occurred in very minute quantities compared to the amount (1-5 %) present in L. williamsii [XIV]. Several Trichocereus species have subsequently been shown to contain mescaline [10, 11, XIIJ. The occurrence of tyramine and its N-methyl- derivatives seems ubiquitous in these cacti [XII]. Paciuicereus pecten-aboriqinum Backeberg, was reported by Spath and Kuffel' [28J to contain carnegine (42), previously known from C. gigan- tea. In a current re-investigation, this cactus was found to contain several alkaloids. The main component of the phenolic alkaloid fraction was

284

I IJIOS1'NTHETIC STUDIES ON }[ AND RELATED CACTUS ALKALOIDS

identified as 3-hydroxy-4-methoxyphenethylamine (31), and not the 4-hydroxy-3-methoxy isomer (6), which is fairly common in cacti [XII]. 3-Hydroxy-4-methoxyphenethylamine might theoretically be a good pre- cursor for tetrahydroisoquinolines, e.g. carnegine (42) and salsoline (46), since it provides an advantageous para-activation for a ring closure [32]. However, such assumptions are not supported by recent results obtained in C. gigantea on the biosynthesis of carnegine [33], nor was this com- pound found to be present or incorporated into alkaloids in T. pachanoi and L. williamsii [X, XIII]. Interesting biosynthetic experiments await to be carried out in Paclujcereus pecten-aboriqinura since also tetrahydro- isoquinolines, trisubstituted in the nucleus (43) like the peyote alkaloids, are present (.'14).

xt. 42. CARHEGI HE 43.

Echitiocereus merkeri Hildm. proved to be rich in alkaloids [YII]. Major alkaloids found were the N-methyl derivatives of 3,4-dimethoxy- phenethylamine (44, 45). The co-presence of 4-hydroxy-3-methoxyphen- ethyl amine (6) and salsoline (46) in this cactus is of biosynthetic in- terest, since salsoline will possibly arise from the isomer 3-hydroxy-4- methoxyphenethylamine (31), found in P. pecten-aborioinnm, rather than from 6. It is not unlikely, that both isomers 6 and 31 are formed in this plant, the former undergoing ring closure to salsoline (46) and the latter bcing methylated to the 3,4-dimethoxyphenethylamines 44 and 45.

45. 6. 46. SAlSOLI HE

Chromatographic and analytical methods In biosynthetic studies, the high number of alkaloids present in peyote necessitates the use of rapid and selective chromatographic systems in the isolation of separate alkaloids.Basic procedures for thin layer (TLC) and gas chromatography (GLC) were worked out when this study was started [I, II]. The TLC systems described in paper I, especially systems A and B, proved to he of great value during this work, both for rapid analysis of alkaloid mixtures and for preparative separation on thick (1-2 mm) plates [XI\'].Additional systems, useful for the separation of phenolic

285

I J.Ll..'XDSTRo)1

phenethylamines and their N-methyl and N,N-dimethyl derivatives, are reported in paper XIII. The separation of primary and secondary amines succeeded satisfactorily by employing Schiff's base formation of the former compounds in acetone containing solvents. TLC systems for mix- tures of peyote alkaloids are also described by MeLaughlin and Paul [35] and by Todd [36]. The rather 10\\' molecular weight of the peyote alkaloids make them specially suitable for separation on GLC. Retention times for peyote alkaloids separated on both analytical and preparative columns are re- ported in paper II. Additional GLC separations (incl. chromatogram figures) are reported in papers VII, XIII and XYI. The combined use of 5 % SE-30 (non-polar) and 5 % XE-60 (polar) columns was found espe- cially useful and satisfactory for almost all separation problems during this study. Preparative GLC separations were in many cases useful, in particular in the purification of alkaloids obtained from biosynthetic experiments [IY, X, XIII]. GLC separations of peyote alkaloids and simi- lar compounds are reported by Kapadia and Rao [38] and by Brown et al. [39J.

Gas chromatography-mass spectrometry (GLC-i\IS) is now a frequently used, sensitive and specific method for the rapid identification of minute (1 /.Lg) amounts of a compound in a complex mixture. This method was of great value and importance in the identification of cactus alkaloids and especially trace constituents of alkaloid fractions, e.g. intermediates of biosynthetic pathways. Results obtained by GLC-MS are reported in papers II, VI, VII, XII, XV and XVI.

.llass fmgmentography is another newly developed method for the identi- fication of trace substances in complex mixtures and the usefulness of this method in biosynthetic studies is discussed in paper XI. This method was introduced by Hammar, Holmstedt and Ryhage [40] and was later modified by Hammar and Hessling [41]. The mass spectrometer is here used as a gas chromatographic detector which continuously monitors 1-3 selected mass numbers of compounds eluted from the gas chromato- graphic column .

.11ass spectra of cactus alkaloids are discussed to some extent in papers III and XVI. Mass spectra of peyote alkaloids have been reviewed recently hy Kapadia and Fayez [4].

Synthesis of labelled compounds In paper III the synthesis of partially methylated di- and triphenolic phenethylarnines labelled with tritium and carbon-Ll in the side chain are described. Compounds labelled with HC w ere obtained by condensing an appropriate benzaldehyde with HC-nitromethane and then reducing the formed nitrostyrcne with lithium aluminium hydride, The use of tritiated lithium aluminium hydride in the reduction step afforded phen-

286

/ I IllUi)l?1Tl1£iTlG BTVD1£iB 0::\ )1ESCALlll'E AND RELATED CACTUS ALKALOIDS

N0 I 2 LiAIH, -T l)::,V... ""' C6H;CHO, 7H,O

T CHJ0(nT HCH,% I ••• NaBHL CHJO "" N(CHJ), C,H,O

T T

)~T CHJO~T lV NHCOCHJ CHJo0 NHCOH HO HO Fig. 3

ethylamines, highly 3H-Iabelled in the side chain. This tritium label was of special value in the later synthesis of doubly labelled N-substituted derivatives of 3-hydroxy-4,5-dimethoxyphenethylamine [XV] . Thus N- [He-formyl] -, N- [l-He-acetyl] -, S- [He-methyl] - and N,N- [He-di- methyl] -phenethylamine-a.Sc-H were prepared according to Fig. 3.

Biosynthetic pathways It was early a matter of general agreement that secondary products such as phenethylamine and tetrahydroisoquinoline derivatives found in plants must be biogenetically related to primary metabolites such as the aro- matic amino acids phenylalanine and tyrosine. In his early review on cactus alkaloids and related compounds, Reti [12] visualized the trans- formations of the primary metabolites to alkaloids as simple processes of decarboxylation, oxidation, 0- and N-methylation, and ring closure with formaldehyde or acetyldehyde equivalents. Largely based on Gug- genheim's fundamental treatise on the formation of natural amines [44J, Reti further proposed hypothetical pathways for the formation of peyote alkaloids i.e. tyrosine -+ dopa ---+ dopamine -+ 3,4,5-trihydroxyphenethyl- amine ---+ mescaline. Although these pathways later proved fairly rea- sonable they were unsubstantiated by experimental evidence and were offered mainly as a working hypothesis. There is now substantial experimental evidence for biosynthetic path- ways in the formation of peyote alkaloids according to Scheme 1.

287

I J. LUNDSTROl\{

HO~COOH HO~ HOV NH2 HOV NH2 HO 3<;. 33.

~COOH

HOV ~H2

32.

HOmH2

t. 5. Scheme 1

Biosynthetic experiments supporting these pathways are reported in the papers IV, V, VIII-X, XIII-XV. Parallel work by other groups on these biosynthetic sequences and mainly by the Paul group [cf. 16) will be considered and referred to at appropriate points. The elucidation of the biosynthetic routes of Scheme 1 was mainly accomplished by the feeding of labelled substrates. However, the identi- fication of possible intermediates of these pathways in cacti [VI, XI, XIII] were of great importance, and in fact the pathways were to a large extent correctly predicted from these results [VI]. The biochemical reactions of the pathways in Scheme 1 have been discussed at length in papers X, XIII-XV. In the following text some additional remarks and results are presented.

Amino acids as precursors for cactus alkaloids The incorporation of tyrosine into mescaline in Leete's experiment [13] was the first evidence for the earlier anticipations that mescaline and other peyote alkaloids are derived from aromatic amino acids. In a second report Leete [14] found a negligible incorporation of phenylalanine into mescaline and consequently suggested that peyote lacks the enzymes for converting phenylalanine into tyrosine. Inconsistent with this finding was the study by Rosenberg et al. [45 J which reported an incorporation of 0.025 % of phenylalanine and indicated that phenylalanine hydroxylation may be possible in peyote. In addition to these results, we found the minute incorporations 0.008 %, 0.003 % of phenylalanine into mescaline in T. pachanoi [46]. In plants as well as in microorganisms, phenylalanine and tyrosine derive independently from prephenate [cf. 47]. The hydroxylation of

288

I DWiH?iTtlI'irIC STrDlES 0:\)IESO.LI:\E A:\,D RELATED CACTUS ALKALOIDS

phenylalanine into tyrosine, a reaction of utmost importance in mammals, seems to be of minor importance in plants r cf. 47]. There is some evidence that phenylalanine will not be hydroxylated to tyrosine in plants [cf. 14]. On the other hand, it is reported that phenylalanine hydroxylations will take place in plants [cf. 47], e.g. in spinach, especially under aerobic conditions. Although the importance of this reaction in plants is not yet fully understood, it certainly seems to be a process of a minor order. The results obtained in cacti also would support this view. The alternative pathways from tyrosine to dopamine (Scheme 1) either via dopa or via tyramine were pointed out early during these investiga- tions [IY, 4,5]. The results of incorporation experiments obtained by Rosenberg et al, [45] and also those reported by us [IV, IX] indicate that the tyramine and the dopa routes would be of about equal importance in peyote.Results obtained in T. pachanoi [X] and recent investigations of these pathways in peyote [XIII] question the role of dopa in the dop- amine formation in these cacti. The route tyrosine -7 dopa -~ dopamine would seem to be of minor importance only. Likewise a further participa- tion of substituted amino acids like 47-50 seems unlikely. In fact, a special search carried out by Kapadia et al. [48 J for compounds like 48-50 in peyote gave no evidence for their presence.

~COOH CH30~COOH CH30~COOH

HOV ~HCH3 HOV ~H2 CH30V ~H2 CH30 CH30

47. 4S. 49. so.

Biosynthesis of mescaline The feeding of all possible hydroxy- and methoxy-substituted labelled phenethvlamines to the two mescaline producing cacti L. williamsii and T. paclianoi revealed the specific pathways for the alkaloid biosynthesis as shown in Scheme 1. The incorporation rates indicated alternative routes for the introduction of the 5-hydroxy group. However, for the accurate determination of biosynthetic pathways like these, it is necessary to establish the natural occurrence of incorporated precursors, since a compound not occurring naturally but theoretically plausible might accidently be transformed into the proper alkaloid. Meta- bolic intermediates seldom accumulate but arc quickly metabolized and consequently they may be difficult to detect.The sensitive methods of gas chromatography and gas chromatography-mass spectrometry proved to be of great value in the detection of some intermediates of the bio- synthetic routes leading to mescaline and the tetrahydroisoquinolines IVI]. In T. paclianoi, [VI] two substances were identified as 4-hydroxy-3- methoxyphenethylamine (6) and 4-hydroxy-3,5-dimethoxyphenethyl- amine (30) respectively, and both these substances fit well into Scheme 1, thus supporting the results of incorporated compounds.

289

/ I J. Lt:NDSTRil},f

~COOH

HOV ~H2

32.

HO~COOH HOV NH2 I. -...... > 33. HO~ HOV NH2

4.

HO~ HOV NH2 HO 34.

Scheme 2

Another approach to overcome this problem is the inverse isotope dilu- tion method often referred to as the "trapping experiment". This method was used in paper XIII to establish the natural occurrence of some incor- porated compounds of Scheme 1! particularly non-extractable catechol- amine derivatives. Dopamine (4) and 3,4-dihydroxy-5-methoxyphenethyl- amine (10), both key substances in the biosynthesis of peyote alkaloids, were shown to occur in L. williamsii. Convincing evidence for the pre- sence in peyote of 4-hydroxy-3-methoxyphenethylamine (6) was also obtained [XIII]. No radio-activity could be traced in dopa (33) added as carrier to a tyrosine-t=C feeding experiment. Nor was it possible to estab-

290

I Iish the presence of 3,-!,6-trihydroxyphenethylamine (34) by a trapping experiment using dopamine-He as precursor [XIII]. Thus, as a convincing additive proof of the validity of Scheme 1, all intermediates incorporated into mescaline, apart from dopa and 3,4,5- trihydroxyphenethylamine, are shown to be naturally occurring in either of the cacti L. williamsii or T. pachanoi. Of the alternative pathways in the formation of dopamine, the route via dopa was found to be of minor importance [XIII]. In addition, the route dopamine -+ 3,4,6-trihydroxy- phenethylamine (34) ---+ 3,4-dihydroxy-5-methoxJ'phenethylamine (10) appears to be a minor pathway, as discussed in papers X and XIII. Bio- synthetic routes considering the major and these possible minor path- ways in the biosynthesis of mescaline are shown in Scheme 2. Papers X and XIII give a discussion of the preference for meta-O-methylation along the route to mescaline, including some discussions of O-methylating enzymes involved [X].

The NIH shift in relation to mescaline biosynthesis An alter native approach to the studies on the course of aryl hydrcxyla- tions and O-methylations in alkaloid biosynthesis might be provided by the "NIH shift" [68]. The KIH shift - now a wel l-es tahlixhed pheno- menon - implies the migration of ring substituents during the course of enzymatic aryl hydroxylaticns [69, d. 68 J. Substrates, tritiated or deu- terated at the site of the hydroxylation, will, dependent the nature and position of other substituents, to a certain extent retain the label by a migration of the heavy isotope to an adjacent position in the aromatic ring [701. The complete loss of para- or ortho-deuterium or tritium atoms of phenolic compounds has been found during hydroxylation at the posi- tion of label. During para- or ortho-hydroxylation of ani soles, approxima- tely 50 % of deuterium at the position of hydroxylation will be retained [68 J. Thus, the feeding of various tritiated phenvlalaniries and tyro sines to peyote as have been suggested by Daly and Jerina 168] might provide information as to the course of hydroxylations and O-methylations in the biosynthesis of mescaline. In this respect, the following experiments were made [46]. L-3,5-3H- Tyrosine (500 p.e) injected together with u.t-tyrosine-o-t+C (50 /J.e) into a peyote cactus afforded mescaline containing only He-label (0.10 % incorp.), This is in agreement with the results reported earlier lYIII-X] that hydroxylation will not occur in para-O-methylated compounds, i.e. 3-hydroxy -1-111 eth oxyphen eth y Iami n e and 3,-l-d imcthoxyphenethyl amine on the route to mescaline. The poor utilization of phenylalanine in the biosynthesis of peyote alkaloids made a demonstration of the NIH shift in the hydroxylation of phenylalanine in these cacti difficult. Although, by the feeding of L-p-3H-phenylalanine (500 Ice) and o.t-phenylalanine- a-He (25 p.e) to a T. pachanoi cactus, radioactive -l-hydroxy-Bvmethoxy- phenethylamine was obtained (0.004 % He, 0.001 % "H incorporations) . .Approximately 50 % of the 3H label of this compound was located to the 5-position of the aromatic ring, the rest was scattered oyer the molecule.

291

------

/ I .7.LUNDSTRoM

Biosynthesis of N -methylated phenethylamines in peyote

The biosynthetic sequence tyrosine -7 tyramine -7 N-methyltyramine ---7 hordenine was established by Leete et al. [49] in barley almost 20 years ago. The results by Me Laughlin and Paul [50] suggested that this metabolic route is also operable in peyote. Experiments reported in pa- pers IV and XIII verify this finding. In addition to dopamine, the N-methylderiyatiye, epinine un, was also shown by trapping to occur in peyote [XIII]. Further, 4-hydroxy-3- methoxyphenethylamine (6), its N-methyl (7) and N,N-dimethyl (8) derivatives were all found to originate from dopamine-He [XIII]. Thus, hiosynthetic routes according to Scheme 3 may he proposed [XIII].

HO~ CH30Plrl __ HOV NH2-- HOV ~H2 HO~H2 -- 4. 6.

HO~ HO~HCH3 HOV NHCH3

2, 5.

8. J. 51, Scheme 3

It is not known at present if X-methylated derivatives as such or after demethylation ean be hydroxylated in peyote, e.g. :2---75, or O-methylated- e.g. 5 --7/.1 The in vitro oxidation of hordenine (S) to N-methylepinine (51) has, however, been reported by Daly et al. [51J. The co-presence of ,v-methyl mescaline (15) and also the X-methyl (12) and N,N-dimethyl (13) derivatives of 3-hydroxy-4,5-dimethoxy- phenethylamine (11) in peyote tend to extend the validity of Scheme 3 to other phenethylarnines of the hicsynthetic route shown in Scheme 1. The major part of the biosynthetic pathways seem, however, to involve primary amines. In T. pachanoi no N-methylated alkaloids apart from hordenine have been detected [11].

1 Results on the biosynthesis of pellotine (19) indicate that hyd roxyl at ions and O-methylalions occur with N-methylated compounds in peyote in routes analogous to those of Scheme 1 [XV].

292

I BIOSYXTHETIC STt:DIES 0)1 )!ESCALIXE AXD RELATED CACTt:S ALKALOIDS

Biosynthesis of 3,4-dimethoxyphenethylamine 3,4-Dimethoxyphenethylamine has been identified in both L. williamsii [II] and T. paclianoi [VI].Although this compound has earlier been reported to be incorporated efficiently into mescaline l45], it is now fully understood that 3,4-dimethoxyphenethylamine is not a direct pre- cursor of either mescaline or tetrahydroisoquinoline alkaloids [cf. X] . \Vhereas only 4-hydroxy-3-methoxyphenethylamine (6) was found to be incorporated into mescaline, both the 4-hydroxy-3-methoxy (6) and 3-hydroxy-4-methoxy (31) isomers (Scheme 4.) "were efficiently methylat- ed to 3,4-dimethoxyphenethylamine (9). However, convincing experi- mental evidence was obtained, showing no initial pal'a-O-methylations of dopamine to occur in these cacti [X,XIII]. The results might indicate that 3-hydroxy-4-methoxyphenethylamine (31) is a compound foreign to these mescaline producing cacti but it can still fit into the O-methylat- ing enzyme system.

___ to mescaline / 6. CH30~ CH30V ~H2 .> 9.

HO~ CH30V ~H2 JI.

Scheme II

Biosynthetic routes in relation to catecholamine metabolism in animals The structural similarities of mescaline and its precursors to catechol- amines of animal origin prompt a comparison of the routes of mescaline biosynthesis in plants and the biosynthesis and metabolism of catechol- amines in animals. Main differences in the biosynthesis of tyrosine in plants and animals have already been discussed. Dopamine appears to be a key substance hoth in catecholamine metabolism [52] and in the biosynthesis of cactus alkaloids. The main route of dopamine formation in mammals [52] is tyrosine ---0>- dopa ---0>- dopamine, whereas this route is at most of minor importance in mescaline producing cacti. On the other hand, the con-

293

/ I J. LV:s'DSTRu:\f

version of tyramine to dopamine in mammals by a rather unspecific "tyramine hydroxylase" is a minor pathway [52], but in these cacti the hydroxylation of tyramine --;> dopamine is a main route. There is a high preference for a meta-O-methylation of dopamine both in T. pachanoi and L. zvilliamsii [X, XIII]. This preference for meta-O- methylations of catccholamines is also pronounced in the animal orga- nism in vivo [52, 53 J. In vitro, O-methylation of substrates of catechol-O- methyl transferase such as catccolarnines and their metabolites affords mixtures of meta- and para-O-methyl derivatives [d. 53].However, as recently reported by Cr-eveling et al. [53], the extent of para-methylation relative to meta-methylation is low with substrates containing an ionized ring substituent, i.c. the side chain of dopamine. The methylation product of dopamine, 4-hydroxy-3-methoxyphenethylaminc (3-methoxytyramine, 6) is a regularly present metabolite in humans [54J and is an important mescaline precursor in cacti as well. Hydroxylation of a catecholamine derivative in the 5-position, as in the biosynthesis of cactus alkaloids, is a reaction not yet known to occur in vivo in anim.al metabolism. There are, however, some indications for such reactions in vitro. Daly, Inscoe and Axelrod [51] reported the con- version of 3-methoxytyrosol, mctanephrine and N-methylmetanephrine to 3,4,5-trioxyphenethyl derivatives by a rabbit liver microsomal prcpara- tion. Benington and Morin [55] demonstrated the hydroxylation of 4- hydroxy-3-methoxyphcncthylamine in the 5-position using rat or rabbit liver homogenate. These finrlings lend some support to the hypothesis that mescaline-like substances could be formed by an aberrant catechol- amine metabolism in humans. The efficient detoxication of mammalian catecholamines hy monoamine oxidase (~IAO) deamination has no counterpart in plants.Most probably, mescaline and many other alkaloids arc not end products of metabolic chains, hut wi ll be transformed and transported further, maybe at a slow rate. However, little is known about such transformations and they were not investigated in this study.

Biosynthesis of the phenethylamine portion of tetrahydroisoquinolines The first experimental evidences for thc eariier assumption that the tetra- hydroisoquinoline alkaloids of peyote might originate from aromatic amino acids were reported by Battersby and Garratt [56], who incor- poratcd tyrosine into pellotine, and by Leete [14J, who found a signifi- cant incorporation of tyrosine into anhalonidine. Incorporations of dop- amine into pellotine [17l and of tyramine, dopamine and 3,4,5-trihydroxy- phcncthylamine into anhalamine [\'1 were subsequently reported, and it seemed possible that the tetrahydroisoquinoline alkaloids of peyote would be biosynthcsized by routes similar to those leading to mescaline. The identification of 3-hydroxy-4,6-dimethoxyphenethylamine (11) in peyote suggested to us that this compound would be the direct precursor of the phenolic tetrahydrosioquinoline alkaloids [VI] and in fact, the compound 11 was anticipated as the direct tetrahydroisoquinoline pro-

29i:

I BIOHXTHETIC STUDIES OX MESCALrNEAND RELATED CACTUS ALKALOIDS

genitor already by Spath during his structural work on peyote alkaloids [57]. Our simultaneous identification of -l-hydroxy-Sdi-dimethoxyphen- ethyl amine in T. pachanoi [VI], an intriguing mescaline precursor, fur- ther suggested that the O-methylation of 3,4-dihydroxy-5-methoxyphen- ethylamirie (10) would be the determining step for either a mescaline or a tetrahydroisoquinoline formation [\'1]. Compound 10 was anticipated to arise from -!-hydroxy-3-methoxyphenethylamine (6) identified in T. pachanoi [\'1]. There is now substantial experimental evidence for common bio- synthetic routes from tyrosine to mescaline and to the main phenolic tetrahydroisoquinoline alkaloids 16-19 up to the point of the O-methyla- tion of 3,-!-dihydroxy-5-methoxyphenethylamine (10, Scheme 1). Methyla- tion on the meta hydroxy group will give 30 and, on further methylation, mescaline (14).On the other hand, a para-O-methylation will give 11 and subsequent ring closure reactions with suitable one and two carbon units 'will yield the tetrahydroisoquinoline alkaloids anh alamine (16) and an- halonidine (17) : Scheme 5.

10.

30. / / II.~ CH30~ CH30V NH2 CH30 17. 14. 16. Scheme 5

In our first investigation of the alkaloid contents of T. paclianoi, we found neither 3-hydroxy--!,5-dimethoxyphenethylamine (11) nor tetra- hydroisoquinolines [\,1J and this was understood as para-O-methylation of 3,4-dihydroxy-5-methoxyphenethylamine (10) to 11 did not occur in this cactus, this seemingly being a main difference in the methylation activities compared to L. williamsii. Later, trace amounts of 11 and also the tetrahydroisoquinoline alkaloids anhalonidine (17) and anhalinine (27) were found in T. pachanoi [11,X]. The much higher incorporation rates of 3,-!,5-trihydroxyphenethylamine (34.) and 3,4-dihydroxy-5-

295

I J. L(:~DSTRo~I

methoxyphenethylamine (10) into mescaline in T. paclianoi in relation to those obtained in L. toilliomsii [VIIIJ, taken together with the results on the O-methylation of dopamine in T. pachanoi [X}, indicate that meta-O- methylations occur along the biosynthetic route more rapidly in T. pacha- noi, This fact might be a reason why compound 11 and tetrahydroiso- quinolines are not formed in T. pachanoi to a significant degree. Con- siderable amounts of 3-hydroxy-4,5-dimethoxyphenethylamine (11) are present in peyote [XIVJ and a certain poolsize of this compound might be a prerequisite for tetrahydroisoquinoline formation. However, the feeding of a comparatively large amount (55 mg) of the tetrahydroiso- quinoline precursor 11 to T. pachonoi, did not result in a significant alteration of the tetrahydroisoquinoline contents of the cactus (GLC). Possibly, a great number of factors are connected with the formation of tetrahydroisoquinoline alkaloids, such as available ring closing units, different kinds of enzymes etc. In another attempt to alter the metabolic routes of phenethylamines in T. pachanoi, inhibitors of catechol-O-methyltransferase of the kind recently reported by Creveling and Daly [58J were injected into cacti. The experiments were so far not successful. The use of tissue cultures instead of intact plants might be a better approach to such studies and also for time-course studies on the alkaloid production. Alternative routes for the tetrahydroisoquinoline formation in peyote, e.g. a cyclization of a disubstituted phenethylamine and with the intro- duction of the third oxygen taking place into a isoquinolinic compound, might be taken into consideration, although incorporation figures of dif- ferent precursors give no evidence for such a process. An analogous route has however been suggested in the biosynthesis of narcotine [59 J. With ring closures taking place on the disubstituted level, one might expect at least trace amounts of dioxygenated tetrahydroisoquinoline alkaloids. Rigorous investigation of the tetrahydroisoquinoline content of peyote gave no evidence for the presence of such compounds. Instead, trace amounts of the trisubstituted isomeric forms of the major phenolic tetra- hydroisoquinoline alkaloids were found [XVIJ. The presence of these four isomers 23-26 (Fig. 2, p. 282) might be taken as an additional proof of the role of 3-hydroxy-4,5-dimethoxyphenethylamine in the cyc- lization process leading to tetrahydroisoquinolines in peyote. It has been observed both by Khanna et al. [16J and by us [XIVJ that while the incorporation of 3-hydroxy-4,5-dimethoxyphenethylamine (11) into anhalonidine (17) and anhalamine (16) was very efficient, the in- corporation of 11 into pellotine (19) was considerably less and only about 1/10 of that into anhalonidine. Battersby et al. [181 reported very low incorporations of 11 into pellotine and from these figures it was ques- tioned whether 3-hydroxy-4,5-dimethoxyphenethylamine really was a precursor of pellotine. In fact, from the results of the Battersby group [18J and of the Khanna group [16J it is evident that precursors appearing more remote in the biosynthetic sequence of Scheme 1, e.g. dopamine (4-), 4-hydroxy-3-methoxyphenethylamine (6) and 3,4-dihydroxy-5-methoxy- phenethylamine (10), are incorporated to a higher extent than 11.

296

I BIOSY::\THETIC STT:DIES 0::\ :'IIESCALI:SE A:SD RELATED CACTT:S ALKALOIDS

An apparent explanation of these combined results was obtained in the feeding of N- [HC-methyl] -3-hydroxy-4,5-dimethoxyphenethylamine-a,,B- 3H (Fig. 3, p. 287) to peyote [XVJ. This compound was incorporated very efficiently with retention of the 3H/HC ratio into pellotine. As N-methyl- 3-hydroxy-4,5-dimethoxyphenetbylamine (12) also was identified as naturally occurring in peyote, it might be obvious that the main bio- synthetic route to pellotine (19) includes the cyclization of this com- pound with a suitable two carbon unit (Scheme 6). The interconversion anhalonidine ~ pellotine was reported by Battersby et al, [18J and this was also in part verif'ied by results reported in paper XV. It is possible that 12 also is the direct precursor of anhalidine (17) and that a similar intercoriversion anhalamine (16) ~ anhalidine (17) is present in peyote. The efficient incorporation of intact N-methyl-3-hydroxy-4,5- dimethoxyphenethylamine (12) into pellotine, taking into consideration the low incorporation of 11, and earlier mentioned observations on iV-methylation processes in peyote, might indicate that parallel hydroxyl a- tions and O-meth;ylations occur with N-methylated analogues along the biosynthetic route of Scheme 1 [XVJ.

II. 12.

CH30WI ~ CH30Wl CH30~ NH ~ CH30 ~ NCH3 HO HO

I~ IS.

Scheme 6

Origin of the one carbon and two carbon units of tetrahydroisoquinoline alkaloids Tetrahydroisoquinoline alkaloids have long been assumed to originate in nature from suitable substituted phenethylamine derivatives by Mannich- type condensations with keto compounds. In his structural work on the peyote alkaloids, Spath [7J suggests that the tetrahydroisoquinoline alkaloids present are formed in vivo by condensations of appropriate phenethylamines with aldehydes, e.g. acetaldehyde or formaldehyde. This view of the formation of tetrahydroisoquinoline alkaloids was earlier expressed by Winterstein and Trier [60J and by Robinson [61J. In their classical experiment, Schopf and Bayerle [62J achieved the condensations of acetaldehyde with dopamine (4) and with epinirie (:5) under physio-

297 J. LC\DSTRoM logical conditions and produced the tctrahydroisoquinolines salsolinol (60) and N-methylsalsolinol (61; Fig. -1) in excellent yields.

Fi«. 4. -

~ Oc.QH ~~ ..AyN H 10 H S7. 58. 53. Donovan and Kenneally [6-11 reported the incorporation of acetate-Iv-+C into eleagnine (67) in Eleaqnus angusiifolia: the radioactivity was almost exolusively situated at C-l, indicating an intact incorporation. Stolle and Greger [65] demonstrated that the two carbon unit of (68) originates from C-2 and C-3 of pyruvate rather than: from acetate in Peganum liarmalo, In addition, Slay tor and MeFar lain -[66] showed that N-acetyltryptamine is a direct precursor of harrnane (59) in Passiilora edulis.

208

/ BIOSY~THETIC STL'DIES O~ :lIESCALI~E AXD RELATED CACT1:S ALKALOIDS

Leete and Braunstein [15] found a relatively specific incorporation of pyruvate-d-r+C into the C-9 methyl of arihalon idine (17), and taking into account the aforementioned results on the harman alkaloids these authors proposed the following biosynthetic sequence: pyruvate -7 acetyl CoA -7 53. X-acetyl-3-hydroxy-4,6-dimethoxyphenethylamine (53) would thus be the immediate precursor of anhalonidine (17).

CH30~ eH30~NH HO eOOH

52. 53. 54.

This compound (53), and also S-formyl-3-hydroxy-4,5-dimethoxyphen- ethyl amine (52), were in fact previously identified by Kapadia and Fales 167J in a non-basic fraction of peyote. Bischler-Napier alski type reactions of these compounds yielding the tetrahydroisoquinolines anhalonidine (17) and anhalamine (16) appeared to be attractive biosynthetic routes. On the basis of these facts, the doubly labelled compounds 52 and 53 were prepared, labelled with tritium in the side chain (ct, (3) and with HC in the acetate or formate group (see Fig. 3) and fed to peyote cacti [XV]. However, only small 3H-incorporations into anhalamine and anhalonidine from these compounds were observed. Evidently, the amides i'i2 and 5.'] are not precursors of the tetrahydroisoquinoline alkaloids of peyote and incorporation of these compounds will only occur after a previous hydro- lysis to 3-h~'droxy-4,5-dill1ethoxyphenethylaminc (11).In a very recent report, Kapadia, Rao, Leete and others [19 J obtained a similar result by the feeding of HC-Iabelled 53 to peyote. The S-mcthyl group of methionine has been reported to be incorporated into C-1 of anhalamine (16) [V1. An oxidative cyclization of the N-methyl derivative of an appropriate phenethylamine has an analogy in the ber- berine-bridge formation [cf. L? 1. However. the doubly labelled N-methyl and N,S-dimcthyl derivatives of 3-hydroxy-4,5-dimethoxyphenethylamine (see Fig. 3) were not incorporated into the peyote alkaloids according to this theory [XYj. The incorporation of the methyl of methionine into C-l of anhalamine may be explained by the intermediate labelling of a suitable ring closing unit via the one-carbon pool. Kapadia and Fales [67J have suggested pyruvate as the source of the two carbon unit of anhalonidine, thus considering a direct involvement of pyruvate in the cyclization process suggested by Hahn [631. Glyoxalate was analogously suggested as the source of C-1 of anhalamine. In a recent paper, Kapadia et al. [19J presented evidence in support of these theories. ;)-Hydroxy-4,5-dimethoxyphenethylamine (11) was found to readily react with pyruvic acid and glyoxalic acid to yield almost quan ti tatively the cyc lization products 54 and 55. The feeding of these labelled compounds (;)4. and 55) to peyote cacti afforded incorporations of 54 into anhalamine (16; 6.8 ~~) and of 55 into anhalonidine (17, 6.0 '7c ). Both compounds

299

/ J. LC~DSTR(nr

54 and 55 were also identified as naturally occurring in the amino acid fraction of peyote. Kapadia et al. [19] further reports that incubation of 55 with fresh peyote slices readily yielded the decarboxylation product 8-hydroxy-6,7 -dimethoxy-1- methyl-3,4-dihydroisoquinoline which might he the direct precursor of anhalonidine via a stereospecific reduction by XADPH.The detection of this dihydroisoquinoline in the alkaloid fraction of peyote would support these proposals. The results on the incorporations of glycine, alanine and pyluvate re- ported in paper XV might partly be seen in support of the results obtained by Kapadia and his co-workers.

Acknowledgements These studies were supported by the Swedish :\atural Science Research Council and by the Faculty of Pharmacy, University of Uppsala, Stockholm. The work was mainly carried out at the Department of Pharmacognosy and the valuable encouragement supplied by the Head of the Department, Professor Finn Sand- berg, is gratefully ack nowlcrlgcd. Most of all I owe my thanks to Dr. Stig Agurcll, who introduced me to the field of alkaloid biosynthesis by a proposal of cooperation in the work involved. I am greatly indebted to my co-authors Jan E. Lindgren, Asaad Masoud, Jan G. Bruhn and Ulla Svensson. Further, I wish to thank Drs. J. Lars G. Nilsson and Kurt Leander for valuable help and profitable discussions. I am indebted to Dr. John W. Daly, ~IH, Bethesda, for a sample of tritiated phenylalanine. Finally, I wish to thank my wife, Mariette Lundstrom, for her tolerance in indulging my preoccupation with alkaloids.

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