Biosynthesis of Quinine and Related Alkaloids EDM-ARI)LEETE

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February 1060 I~IOSYNTHESISOF QUINIXE AND l1ELATE:L) XLKALO1I)S 50 Biosynthesis of Quinine and Related Alkaloids EDM-ARI)LEETE

Department oj Chenaisli.y, Ilniversitij of Minnesota, ilfinneapoIis, I1mnesotu 6,i/+

R~eizpdSeptendw ,I, 1968

The antimalarial drug quinine (1) has had a fascinat- years old at the timc of hwvestirig for quinirie extr:ic- ing history.’ In 1820 it was isolated in a crystalline tion. state from the bark of a Cinchona tree by l’elletier T’ortunately from our point of view, cjuiniiic mid and Caventou.2 In 1856 I’erltin, having little infor- other alkaloids are biosynthe*ized in young seedlings. mation on the alkaloid except its molecular formula, This was evident from the work of de Jloerloo~,~ attempted to obtain it by oxidizing allyltoluidine with who cultivated l-year-old Czr~chomsuccii-ubi~ p1:Lrit.: potassium dichromate according to in a11 atmosphere containing radioactive carbon dioxide and obtained radioactive quinine from the p1:mts. 2CioHiBN + 30 + C+NzO, + HzO allyltolkiidiiie yiiinine The biogenetic relationship of the indole illkdoids cin- Seedless to say, this attempt was unsuccessful and choriamine and quiriamine was suggested by Chuttirel, yielded a dirty reddish brown precipitate. However, et UZ.,~ and a plausible biogenetic scheme based on this the results of this experiment led Perkin to investigate view, upon Turner and Wood\vitrd’sg ideaq, arid oil the oxidation of simpler amines, and from the oxidation tracer work to be described is illustrated in Scheme I. of crude aniline (which contained some toluidine) he It is suggested that tryptophan (or possibly trypt- obtained the first synthetic dye, mauve (2),which was amine’O) condenses with the nine-carbon triuldehyde used for dyeing silk and coloring postage stamps in the 3 (the origin of this unit will be discussed later) to 186O’~.~ afford the tetrahydro-@-carbolinederivative 4. Hypo- thetical cleavage of the @-carbolinering and formtition H // of the quinuclidirie ring as illustrated, followcd by unexceptional reductions arid decarboxylation, afford.; cinchonamine, which is a minor alkaloid in Cinchona bark. However it is more abundant in young Cin- chona plants.11 This observation is coilsisterit with its being a precursor of the Cinchona alkaloids which contain a quinoline ring. IZeceritly a dimeric indole alkaloid has been isolated from the leaves of C. letl~jei~i- analZarid named cinchophyllamine (6). It seems probable that this alkaloid is formed by a JIarinich renctioti between the aldehyde 7, which may be formed by the oxidation of cinchonamine, arid tryptamine. The stage at which the methoxy groups in cinchono- 2 phyllamine, and alkaloids such as quinine and quinidine, are introduced is currently unltnown. However, The gross structure of quinine was elucidated (by hydroxylation and methylation are apparently terminal Rabe) in 190g4 and its stereochemistry, as illustrated in formula 1, in 1944.j It was finally synthesized by (7) 1’. de Moerloose and It. Iinyssen, J. Pharm. Bdo.,8, 150 Woodward and Doering in 1944.‘j (1953) ; Pharm. Tijdsch?. Belg., 30, Si (1953) ; 1’. de Moerloose, The major commercial source of quinine is the bark Pharm. WeekbZad, 89, 541 (1954). (8) It. Goutarel, 11%M. Janot, V. I’relog, anti W. I. Taylor, HLLI,, of the tree Cinchona ledgerianu, where it occurs to the Chim. Acta, 33, 150 (1950). extent of 13y0 of the dry weight. Unlike some alkaloids (9) 11. B. Turner and It. B. Woodward, AZizaZoids, 3, 54 (1953). (10) In some cases tryptophan and tryptamine serve equally well quinine is apparently a metabolic end product and is as Iirecursoro of the indole alkaloids; however, with some alkaloids deposited in the outer layer of bark where the cells the carboxyl groul, of tryptol~hanis apparently required for initial conderisations and is lost at a later stage in the biosynthesis of the are dead or dying. Commercial trees are at least 20 alkaloid. For example, trylitophim, but not tryptamine, is a ])re- cursor of the Ergot alkaloids [(a) li. Af. Baxter, 8. I. Kandel, and A. Okany, Chem. Ind. (London), 1453 (1961) 1. Tryptamine tind trypto- (1) Cf. M. G. Kreig, “Green Medicine,” Rand hlcNaliy Co., phan were equally efficient as precursors of the Vinca rosea alkaloids, New York, N. Y., 1964. ajmalicine and tetrahydroalstonine; however, the incorporiition of (2) M. I’elletier and E. Caventou, Ann. Chim. Phys., [2] 15, tryptamine into catharanthine and vindoline, produced by the diinie 291,1337 (1820). plant, was much less than that of trykltophan [(b) E. Leete and 13. AI. (3) W.H. I’erkin, J. Chem. SOC.,14, 230 (1862); English I’ntent Bowman, unpublished work; (c) J. 1’. Kutney, TY. J. Cretney, J. It, 1984 (1856). Hadfield, E. 8. Hall, V. I<. Nelson, and D. C. IYigfield, J. Am. Chem. (4) 1’. Rabe, Ann., 365, 353 (1909). Soc., 90, 3567 (1968)l. (5) V. I’relog and E. Zalan, Heh. Chim. Acta, 27, 535 (1944). (11) E. Leete, unpublished observation. (6) K. B. Woodward and W.von E. Doering, J. Am. Chem. Soc., (12) 1’. I’otier, C. Kan, J. LehlIan, M.M. Janot, H. Budzikiewicz, 66, 849 (1944); 67, 860 (1945). and C. Djerasai, Br(L Sor. Chim. Frmw, 2309 (1966). 60 EDWARDLEETE VOl. 2

Scheme I: Tentative Biosynthetic Scheme for the Cinchona Alkaloids

5 (cinchonamine)

E! H

12, R = OCH3 (quinine) 14 15, R = OCH3 (quinidine) 13,R = H (cinchonidine) 16, R = H (cinchonine) steps in the formation of the indole alkaloids vindoline and cinchonine (16) which are epimeric at this newly and resperpine.13,14 formed asymmetric carbon and also at C-3. Quinine Qujnamine (11) is another minor alkaloid in Cin- and quinidine are the corresponding methoxylated chona plants, and its formation can be rationalized alkaloids. van Tamelen and Haarstad16 have ob- as follows. Electrophilic attack by OH+ at the /? tained 4-acetylquinoline (18) from 2-methyltryptophan position of the indole nucleus of cinchonamine would (17) in 20% yield by treatment with sodium hypo- afford the 3-hydroxyindolenine derivative 8. Cycliza- chlorite. The mechanism of this remarkable trans- tion of the primary alcohol group at the LY position formation is considered to be analogous to the forma- yields quinamine. WitkopIj has actually achieved the tion of quinine and is illustrated in Scheme 11. conversion of cinchonamine to quinamine in vityo The first evidence in favor of this biosynthetic by oxidation with peracetic acid, the hydroxyindolenine scheme for quinine was obtained by feeding r,L-trypto- 8 being a probable intermediate. The indolenine 8 phan-2-I4C to C. succi~ubmplants by means of cotton is also considered to be the source of the Cinchona wicks inserted in the stems of the ~1ants.I~The plants alkaloids, which contain a quinoline nucleus. Hy- were allowed to metabolize the radioactive tryptophan drolysis of the C=N bond and oxidation of the primary for 6 weelis, and were then harvested. Extraction of alcohol group could possibly afford the intermediate 10. the plants yielded radioactive cinchonamine and Cyclization of the primary amino group with the al- quinine. The quinine was degraded according to dehyde group would yield compound 9, the quinoline Scheme 111. The quinine is numbered in an uncon- nucleus in 14 then being formed by dehydration. ventional manner in order to illustrate its biogenetic Reduction of the ketone affords cinchonidine (13) relationship to indole alkaloids of the Corynanthe type,

(13) E. Leete, J. Am. Chem. Soc., 82, 6338 (1960). (16) E. E. van Tamelen and V, B. Haarstad, Tetrahedron Letters, (14) A. A. Qureshi and A. I. Scott, Chem. Commun., 948 (1968). 390 (1961). (15) B. \vltkoli, .I. Am. Chem. Soc., 72, 2311 (1950). (17) N. Koivanko and E. Leete, J. Am. Chem. Soc., 84,4919 (1962). Fchruary 1969 BIOSYNTHESISOF QUININEAND RELATEDALKALOIDS 61

Scheme 11: Possible Mechanism of the Formation e.g., corynantheine. Oxidation of quinine yielded of 4-Acetylquinoline from 2-Methyltryptophan quininic acid (19))which was decarboxylated by heating 0 with copper chromite. The resultant 6-methoxy- quinoline (21) was allowed to react with phenyllithium in boiling toluene, yielding 6-methoxy-2-phenyl- quinoline (22)) the position of pheriylation being established by an independent synthesis of authentic ma- 17 terial.18 Oxidation of the methiodide of this compound with potassium permanganate yielded benzoic acid which had essentially the same specific activity as the quinine, indicating that all the radioactivity of the alkaloid was located at C-5. We have now carried out a feeding experiment with tryptophan labeled with 15N on the indole nitrogen and with 14C at the CY carbon of the indole nucleu~.'~The enriched nitrogen and the 14C were introduced into the indole nucleus by the sequence of reactions illustrated in Scheme IV. o-Toluyl chloride was allowed to react

Scheme IV: Synthesis of Ind0le-l-*~",2-1~C

18 + tH,C1 + NaOH 4 COCl Scheme 111: Degradation of the Quinine Derived from Labeled Tryptophan and Geraniol

OH kOOH I .I* "2 "2 H tryptophan 19 J geraniol with ammonium chloride containing 89% excess 15N in the presence of sodium hydroxide to yield o-tolu- HO amide. A Hofmann reaction on this compound afforded o-toluidine which was formylated with formic- 14C acid. The resultant formyl-o-toluidine was con- verted to indole by treatment with potassium t-bu- toxide.20 This doubly labeled indole was then con- verted to tryptophan by established methods.21 The quinine derived from this doubly labeled trypto- J phan was degraded as before. The I5N was determined 6OOH using a modification of the method of Gunther, et aLZ2 The nitrogen gas, obtained by heating the alkaloid or its degradation product (less than 0.5 mg required) in an evacuated sealed tube with calcium oxide and A A cupric oxide, was analyzed in a mass spectrometer. 19 20 Only the nitrogen in the quinoline nucleus was enriched with 15N, and all the 14C was located at C-2. Furthermore, the specific incorporation of the 15N and 14C into these positions was identical (0.97%). These results thus provide convincing evidence in favor of the biosynthetic scheme illustrated in Scheme I.

(18) 0. Dobner, Ann., 249,98 (1888). (19) E. Leek and J. N. Wemple, J. Am. Chem. Soc., in press. (20) F. T. Tyson, "Organic Syntheses," Coll. Vol. 111, John Wiley & Sons, Inc., New Tork, N.Y., 1955, p 480. (21) H. R. Snyder and F. J. Pilgrim, J. Am. Chem. Soc., 70, 3787 (1948). (22) H. Gtinther, H. G. Floss, and H. Simon, Z. Anal. Chem., 218, 401 (1966). I’yrrolnitrin (26), an antifungal compound produced Scheme VI by a Pseucloinorias culture, is apparently produced Schematic Representation of the “Monoterpene Hypothesis” from tryptophan by a similar cleavage of the indole nucleus.23 Under certain conditions of fermentation 3-chloroindole (24) is formed, and it was suggested by Gorman and Lively that the nietabolism of tryptophaii h is initiated by a chloro peroxidase enzyme as illustrated in Scheme V, the first product formed being the 3- x geraniol / nerol Scheme V Hypothetical Scheme for the Biosynthesis of Pyrrolnitrin .I from Tryptophan J

27 Corynanthe unit /

/ Hf 23 1 / C’ .4spidosperma unit 22 Iboga unit 24 2s alkaloid corynantheine) is produced by cleavage of the cycloperitnrie ring at the position iridiciited nith a dotted line. The numbering of this unit corresponds to the numbering of corynantheine to indicate the ultimate origin of each carbon atom. We~ikert~~a190 put for- ward ingenious mechanisms for the rearr:ingement of 26 the Corynarithe unit to the Aspidosperma arid Iboga units which arise by migration of the isopropyl side chloroindolenine derivative 23. A fragmentation re- chain at C-13. Examples of alkaloids which cont:tin action affords 3-chloroindole, while opening the in- these various units are illustrated in Scheme 1-11. dolenine ring yields 25. Formation of the pyrrole ring, C-22 has been lost in the formation of some illd do id^, oxidation of the aromat’icamino group to a nitro group, for example, in cephaeline arid ibogaine. Initid ex- arid ehlorinat’ion afford pyrrolnitrin. periments which favored the morioterprrie hypotheii.; The origin of t’he nine-carbon aldehyde 3 became were carried out with labeled mevalonic acid,L6--11 fairly obvious from related work on the biosynthesis of the precursor of terpenes. The expected pattcrn of the indole alkaloids of I’inca i’osea, which had been labeling TY~Sfound in the indole allinloids of 1*?)jca carried out by Scott, drigoni, Battersby, and our- )’osea and other species. selves. Most of the indole allialoids contain a nine- The next obviou9 step was to test an actual moiio- or ten-carbon unit, in addition to the tryptophan-de- terpene as the precursor of the ubiquitous ninc- or ten- rived portion. There has been much speculation on t’he carbon unit, and four research gro~ps~j-~~independently origin of this nit.^^ 3,4-Dihydroxyphenylalanirie, prepared labeled geraniol arid administered it to 1’inc.a prephenic acid, acetic acid, arid mevalonic acid have all been considered as precursors of this unit. In 1961, (27) E. IT’enkert and R. Wickberg, J. Am. Chcm. Soc., 87, 1580, WenkertZ5 and Thomasz6 indeperident’ly suggested 5810 11965). (28j T. iloiiey. I. G. Wright, E’. .\IcCapra, arid A, I. Scott, Proc. that this nontrypt’ophan-derived unit is formed from :i Sail. Acad. Sei. c’. S.. 53, 901 (1965). cyclopentanomonoterperie (27) (Scheme VI) which (29) F. McCapra, T. Money, -4.I. Scott, aiid I. G. IT’right, Chcm. Cornmiin., 537 (1967). could be formed from geraniol or its isomer nerol. (30) T. lloney, I. G. TYriglit, 1;. McCapra, A. I. Scott, and IC. 9. The Corynant’heunit (so called because it occurs in tjhe Hall, J. Am. Cham. Soc., 90, 4144 (1968). (31) H. Goeggel arid D. Arigoni, Chem. Comnauic., 538 (1905). (23) M. Gorman and D. H. Lively in “Antibiotics,” 1-01, 11, (32) A. 11. Battersby, lt. T. Brown, 11. S. Kapil, A. 0.I’luiiltett, D. Gottlieb and I-’. D. Shaw, Ed., Springer-T’erlag, Berlin, 1967, aid J. B. Taylor, ibid.,46 (1966). 11 433. (33) A. l<,Battersby, 1%.T. Browrl, It. 5.Kapil, J. A. Knight, J. A, (24) For an historical account of this speculation see E. Leete in Martin, and A. 0.l’lunkett, ibid., 888 (1966). “Biogenesis of Natural Compounds,” 1’. Berrifeld, Ed., 2nd ed, (34) 1’. Loew, H. Goeggel. and D. Arigoni, ibid.,347 (1966). Pergamon Press, Oxford, England, 1967, Chapter 17. (35) A. I<. Battersby, 11. T. Brown, J. 4.Knight, J. A. AI:!rtin, (25) E. Wenltert, J. Am. Chem. Soc., 84, 98 (1962) [manuscript :rrid A. 0.I’lunkett, ibid.,346 (1966). received Jan 28, 19611. (36) E. 9. Hall, F,AIcCapra, T. Money, K. I’ulaimoto, T. 11. (26) A. F. Thomas, Tetrahedron Letters, 544 (1961) [rnanuscril>t Hansori, B. S. Nootoo, G. T. l’hilips, and A. I. Scott, ihid., 348 (19(jW. received Aug 18, 19611. (37) 1;. Leete and S. Iyeda, Tetrahedwnl Lcti(,rs, 4915 (1960). Fcbruary 1069 BIOSYXTHESISOF QUININICAND 1IE:Lbmm ALKALOIDS (22

Scheme VI1 phosphate; however, geraniol emulsified in water \.r.ith dilkaloiils nith the Corynanthe Unit Tween 80 (polyoxyethyleiiesorbitan monooleate3y)w:i* efficiently incorporated into the indole alkaloids. hit- tersby also showed that geraniol is an efficient prccurwr of the nine-carbon Corynarithe unit in the isoquirioline alkaloid cephaeli~ie.~~Independe~itly,~’ 13attcriby13 arid we4*fed radioactive geraniol to Cinchona phits. Hattersby fed gerani01-2-’~Cto C. Zetlqeriaua platiti nnd obtained radioactive quinine (0.001% incorporiLtiori). OCH, vincoside A Kuhn-Roth oxidation on the derived dihydroquiriine corynantheine 20 (Scheme 111) afforded radioactive propionic acid (98% of the total activity of the alkaloid) arid innctivc acetic acid, indicating that all the activity was located at C-20. We fed geraniol-3-14C to C. succirubta plants and obtained radioactive quinine (0.001~incorporn- tion), and a similar dcgrad:itioti indicated that :dl thc cHlooc~o CH,OOC ,&o activity was located at C-19. a jmalicine ipecoside So far, we have no information on the steps between geraniol arid the ultimate Corynarithe unit found in the Cinchona dkaloids. However, in other species it is riow clear that the riaturally occurring cyc1opent:Lrio- monoterpene loganin (Scheme VIII) is an important intermediate. In 1966 Batter~by~~reported that this

I compound, labeled with tritium on its 0-methyl group, OCHJ was significantly iricorporated into the following I’ima wsea alkaloids: catharanthine, vindoline, serpentine, OH ajmalicine, arid perivine. Degradations indicated thxt cephaeline corynoxeine all the activity mas located on the C-22 carbomethoxy group of these alkaloids. Loganiri labeled internally Alkaloids with the Asuidosl)eriiia Unit with I4C WRS obtained biosynthetically by feeding a 8 : 1 mixture of gerani01-2-~~Carid ner01-2-’~Cto Jreny- antiles tufoliata plants. The ndministration of this labeled loganin to Vinca mea plant3 also resulted in specific labeling of the indole alkaloids at the expected positio~is.~~Arigotii4j obtained coniplementary results using loganin labeled specifically at C-8. The presence /z vincamine of loganin in Vinca ?mea plants was established by vi ndoli ne radiochemical dilution,43arid it will he of great interest

.\lkaloids with the Ilioaa Unit to learn whether it is of widespread occurrence in the many species which produce indole alldoids. A possible route from geraniol (or nerol) to loganin is illustrated in Scheme VIII. Batter~by~~suggested a route via citronellal arid iridodial, a known natural product, arid a potential precursor of piperidine alka- catharanthine loids such as skytanthi~ie.~~However, when we fed

(39) Cf.J. F. Parr and A. G. Normau, Hotan. Gnz., 126, 86 (1965), for a discussion of the use of surfactants in plant systems. (40) A. It. Battersby and B. Gregory, Chem. C‘ommi~n.,134 (1968). 16 (41) The author and Professor Battershy had a brief conversation ibogaine at Stockholm airport in June 1966, during which time we each thld the other that we had fed radioactive geraniol to Cincshona phiits. Needless to say this informhtion caused my grnduate student, Jini yosea plants. Specific labeling was found in vindoline, Wemple, to work even harder to comlilete this piece of reseawh. ajmalicine, and catharanthine, in agreement with the (42) E. Leete and J. N. Wemple, J. Am. Chcm. Soc., 88, 4743 general hypothesis depicted in Scheme VI. There was (1966). (43) A. It. Battersby, It. T.Brown, 1%.S. Kapil, J. A. SI:irtin, and no significant difference in the incorporation of geraniol A. 0. I’lunkett, Chem. Commun., 890 (1966). (44) A. It. Battersby, It. 8.Kapil, J. A. Martin, :tnd L. 110,ibid.. and its geometric isomer nero1.33s38 Battersby ad- 133 (1968). ministered geraniol, in preliminary work, as its pyro- (45) 1’. Loew and D. Arigoni, ihid., 137 (1968). (46) A. It. Battersby, Pitre A& Chem., 14, 117 (1967). (38) A. It. Battersby, J. C. Byrne, K. S. Kapil, J. A. Martin, (47) H. Auda, H. It. Juneja, E. J. Eisenbraun, G. It. Wdler, I\-. I?. T. G. Pnyne, D. Arigoni, and 1’. Loew, Chem. Comm., 951 (1968). Kays, arid H. H. Appel, J. Am. Chem. Soc., 89, 2476 (196i), EDWARDIZETE Vol. 2

Scheme VI11

Hypothetical Biosynthetic Route from Geraniol to the Corynarithe Unit

c' - (yo - (yH0 - q:Ho CHO iridodial geraniol citronellal

1 tI

____t CHO qH, \ CHO \ CHO H+ CHO COOCHJ COOCH3 skytanthine 28 I OH 11 I OH i CHCH,l II

COOCH, COOCH3 I 29 loganin COOCH,l plumieride /cox J CH, 0.glucose 0-glucose

CHO

COOCH, COOCHB 3 22 30 secologanin

iridodial, labeled at the position indicated with an could be phosphorylated (X = POSH,) arid a frag- asterisk, to Vinca mea plants, the incorporation of mentation occur, as illustrated, to yield secologanin. activity into the alkaloids was insignifica~it.~~We A secologariin residue is present in ipecoside40 and thus favor a route to loganin via the dialdehyde vincoside50 (Scheme VII), llinor modifications of the 28. Enolization of this dialdehyde to 29 would make functional groups in secologanin could afford the ten- the two aldehyde carbons radiochemically equivalent. carbon unit present in other alkaloids containing a This suggestion was also made by Schmid49 to rational- Corynanthe unit. Loss of the glucose residue and the ize his results on the biosynthesis of the cyclopentano- carboxymethyl group affords the nine-carbon aldehyde monoterpene plumieride (Scheme VIII). The pattern 3 which was utilized in our original scheme for the bio- of labeling found in plumieride and the ubiquitous ten- synthesis of quinine (Scheme I). It now seems clear carbon unit present in the indole alkaloids after feeding that rearrangement of the Corynanthe unit to the mevalonic acjd-2-14C requires that these two positions Aspidosperma and Iboga types occurs after reaction of (C-17 and C-22) do become equivalent at some stage the former unit with trypt~phan.~~,~~5l in the biosynthetic sequen~e.~O-~~Beyond the dialdehyde 28 the metabolic steps are quite plausible and The support of the '\'atzonal Institutes of Health through Research unexceptional. It was suggested4'? that the cyclo- Grant G.ZI-l~l246 as gratefully acknowledged. pentane ring of loganin could be cleaved at the required (50) G. N. Smith (Chem. Commztn., 912 (1968)) isolated from Vinca position by initial hydroxylation of the methyl group rosea plants an alkaloid which he called strictosidine, having the structure of vincoside but with undetermined stereochemistry. at C-8. The resultant hydroxyloganin (30, X = H) More recently, Battersby and coworkers (A. It. Battersby, A. 11. Burnett, and P. G. Parsons, ibid., 1282 (1968)) have isolated from the same species vincoside and isovincoside (epimeric at (2-3). (48) E. Leete and 11. hf. Bowman, Phytochemisiry, in pres. (51) J. 1'. Kutney, C. Ehret, V. 1%.Nelson, and D. C. Wigfield, (49) D. A. Yeowell and H. Schmtd, Ezperienfia, 20, 250 (1964). J. Am. Chem. Soc., 90, 5929 (1968).