BIOSYSTHESIS O F OPIUM XLKXLOIDS. SUBSTR-ITE SPECIFICITI’ AND ,IBERRIIST BIOSYSTHESIS : ,ATTEhlPTED DETECTIOS O F ORIPXT‘ISE IN P A P A I’E R SO-lILl-IFE R Glli
.iusTRacT.-The iinnatural thebaine analog, oripavine 3-ethyl ether, was efficiently metabolized t o morphine 3-ethyl ether and morphine in the opium poppy. An attempt t o detect oripavine hy an isotope dilution esperinient based on its presumed biosynthesis from reticuline n-as iinsuccessful.
Metabolic reactioiis are in general enzymic and, therefore. $lion- considerable selectkit>-. This selectivity becomes an important factor for biosynthetic studies of natural product because specific incorporation of a labeled substrate usually provides the first dieation that the conipound is a natural precursor. There are, hov,-ever. examples in the literature of uririatural compounds being transformed b!- a plant to a natural product (1-3) and, also, of unnatural modified precwqora being converted to corresponding1)- modified end product^ (3-7). Iiirlp- et ul ( G j have reported the deniethj-lation of several uririatural codeine analogs t o the corresponding morphine analogs by t h e opium poppy. Thebaine (1) is another opium alkaloid n-liich, in contrast to codeine (2) aiid niorphine ( 3 ) . is produced by .5everal members of the genus PapaLer. Some of these species! such as P . orielitale and P . bructratztm, are able t o demeth?-late the phenolic ether t o give oripavine (4),but lack the ability to demethylate the enolic ether in position G of thebaine (scheme I). Although both demethylations appear t o pro-
O
Meow’
O k J
U
1
HO
0
N-CH3
N-CH3
N-CH3
Me0 4
3
2
SCHEME 1 Biotransformations of thebaine.
ceed by the same mechanism is), they require different enzymes. P. somnijerum has enzymes for both 0-3 and 0-6 demethylations and it is, therefore, conceivable that thiq species may contain oripavine as a genuine alkaloid. If present in low concentration compared to morphine and other phenolic alkaloids, it may hitherto T3J
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have e-caped detection. This possibility \\-ab examined by an isotope dilution experiment based on its biosynthesis from reticuline ( 5 ) , which is a precursor of thebaine (9-11). Synthetic oripavine n-as used as a cold carrier. The enolic ether demethj-lation of a n unnatural thebaine analog, oriparine 3-ethyl ether, n as also investigated as ne11 as the 3-0-dealkylation of a higher ether hoinolog of codeine. This n a s part of an effort to study the specificity of enzymes involved in the biosynthesis of opium alkaloidc. Oripavine (4) and labeled oripavine 3-ethyl ether (11) were synthesized as illustrated in scheme 2 .
RESCLTS ASD DISCUSSIOK
Percent Incorporation of 14C Into Compound fed
Amt. fed mCi
I Oripavine 3-ethyl ether". . . . . .~ 0.011 DL-Reticulineb.. , . 0.10 ,
,
Tariety
!
S o . of Plants Oripavines
Soordster Ikkanshu
12 1.5
~
~
3-Ethylmorph.a
-
1 0.003
~
157
~
1
Rel. I4C Amt. in A'-methyl group
Morphine
;3;
~
99.2 98.5
isolated by carrier dilution. b[.T-mefi1yl-1~C].
The incorporation of reticuline into oripavine is so small as to be insignificant. This can hardly be explained on the basis of different reaction rates for enolic and phenolic ether cleavage. Even vastly different reaction rates would give a measurable incorporation of radioactivity into oripavine in view of the large amount of radioactivity administered in the form of reticuline and the high incorporation
SOV-DEC
19S01 BROCHMASS-HASSSES ET
.\L.
OPIUM ALKALOID BIOSTSTHESIS
;:33
into morphine. -Illthough different varieties of P . sowuijeruvz may shov somen-hat different alldoid compositions! it is nevertlielrss reasonable to conclude from this experiment that oripavine is not a genuine alkaloid in the opiunl poppy. If oripavine is derived directly from thebaine hj- phenolic methyl ether cleavage. as i.5 generally believed nnd supported by the work of Hodges et a / . (12), the enzyme n-hich coaverti: codeine t o morphine in P. s o ~ n i i ( f z ~ z iis ? ndifferent from the enzyme demethylating tliebake to cripx-ine, the latter being absent from tlie opium poppy. Howere:., it is also conceivable that thebaine maj- not be a precursor of oripavine, but tlie two alkaloids may be bios!-nthesized along different but parsllel do*
HG
3
+OC*-
__c
4 0@N-CH2 0
O\
N-CH3
Me0
3
N-CH j Me0
12
6
I 0
- 0 N-C-OCH2-CCI3
8
N-C-OCHi-CCI
N- CH3
j
13
7
N-
CH;
11
SCHEXE 2 . Synthesis of oripavine and labeled oripavine 3-ethyl ether.
path-a)-s. Irrespective of d i e t h e r oripariue is derived from thebaine or not, reticuline is a reasonable precursor of both. Work is in progress t o resolve these qwstions regarding the biosynthesis of oriparine.
ESPERIMEST-IL XITERIALS A S D XETHODS.-TKO varieties of Papri;er somuiiferuw x e r e used in these esperiments, Ikkanshu and Soordster. The methods for cultivation of the plants, administration of labeled precursors, extraction, separation, purification and controlled degradation of alka-
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loids, and determination of radioactivity have been described in previous communications (9, 10, 13, 14). I n general, the crude alkaloid extracts were separated into weakly basic alkaloids by extraction of an acidic solution n i t h chloroform, moderately basic nonphenolic alkaloids by extraction with chloroform a t pH 13, and phenolic alkaloids by extraction with chloroform-2-propanol (3:l) a t pH 9. Morphine 3-ethyl ether was isolated from the moderately basic nonphenolic fract,ion by column chromatography on alumina with benzene-chloroform (4:l) and purified by crystallization t o constant radioactivity. Morphine and oripavine were separated from the phenolic fraction by column chromatography on silica gel with chloroform containing increasing amounts of methanol ranging from lyOto 207,. Morphine 3-ethyl ether used for carrier dilution was obtained from Merck I&Co. The radioactive purity of labeled precursors was determined by radioactivity scanning with a Berthold radioscanner (T-arian A4erograph Co.) of thin-layer chromatograms prepared with tR-0 different solvent systems. Spectroscopic measurements were made with nonradioactive samples synthesized by the same methods as the labeled compounds, and their identity was established by chromatographic methods (glc, tlcj. Nmr spectra were taken in deuteriochloroform. P R E P A R A T I O S O F L.\BELED
S U B S T R . i T E S A S D NONR.iDIO.\CTIVE
C.iRRIERS.--i~--~~-nleth3'1-14C-(f )-
reticuline was synthesized as described previously (13): specific activity, 0.814 mCi/mmoleMorphine Gmethyl ether (heterocodeine) (6) was prepared from morphine by the method of Barber and Rapoport (15): yield 98%, mp 242-243" (lit. mp 242-243" (14)); ei mass spectrum n i l e 299 (11-j;'H nmr 6 2.5 (s, 3H), 3.5 (s, 3H), 4.9 (d, l H , J=3Hz), 5.5 (m, 2Hj, 6.5 (q, 2H, J=4Hz). The method of demethylation was adapted from Montzka et 01. (16). Heterocodeine (1.72 g) m-as dissolved in 150 ml chloroform, 11.0 g potassium bicarbonate, and 11.5 g of trichloroethylchloroformate were added and the mixture refluxed with magnetic stirring for 3 days. The chloroform solution m-as decanted, the residue dissolved in water, extracted with chloroform and the extracts combined n-ith the original chloroform solution. Removal of the solvent gave a residue which n-as dissolved in 120 ml of methanol, and a solution of 4.2 g of potassium hydroxide and 7.4 g of potassium bicarbonate in 75 ml -water was added. The mixture was stirred a t room temperature for 24 hr, the pH was adjusted to 5 with hydrochloric acid, and the methanol was removed a t reduced pressure. The crystals which separated out were filtered off and purified by column chromatography on silica gel with chloroform containing 2 ' 5 methanol: yield of trichlorocarbethoxy normorphine G-methyl ether (7), 84.4%; homogeneous on tlc. To a solution of compound 7 (1.68 g) in 100 ml acetone --ere added 0.5 g of potassium carbonate and 1 ml of ethyl iodide, and the mixture %-asheated a t reflux x-ith stirring for 24 hr. A%fterevaporation of the solvent, the residue was extracted with chloroform and purified by column chromatography: yield of trichlorocarbethoxynormorphine3-ethyl-6methyl ether ( 8 ) , 95%. Compound 8 (1.7 g) was dissolved in 50 ml of 90y0 acetic acid, 2 g of zinc ponTder was added and the mixture stirred overnight. rlfter addition of n-ater, the solut,ion m s filtered, and the filtrate was adjusted t o p H 11-12 with ammonia and a little potassium hydroxide and extracted with chloroform. The combined extracts n-ere washed n-ith ammonia containing a little potassium hydroxide, then twice with water, dried and evaporated to dryness. The residue was purified by chromatography on silica gel with chloroform containing 2% methanol: yield of normorphine 3-ethyl-6-methyl ether (9), 62%, e.i. mass spectrum ntle 313 (>I+);1H nmr 6 1.36 (t, 3H, J = 8 Hz), 3.52 (s, 3H), 4.08 (q, 2H, J = 8 Hz), 6.45 (d, lH, J = 9 Hz), 6.63 (d, l H , J = 9 Hz). ;\:J~etl!yl-I4C-morphine 3-ethyl-G-methyl ether (10) was obtained by methylation m-ith l4C-paraforma1dehyde and formic acid according t o -4ndersen and Woods (17j. The reaction product was purified by column chromatography on silica gel with ethyl acetate-methanolammonia (98:2:0.5) t o give an oily liquid which crystallized on standing: mp 125"; homogeneous on tlc; yield 76%; ei mass spectrum m / e 327 (h1lj; 'H nmr 6 1.29 (t, 3H, J = 8 Hz), 3.38 (s, 3H), 3.47 (s, 3H), 4.04 (q, 2H, J = 8 Hz), 6.49 (d, 2H, J = 8 Hz), 6.58 (d, 2H, J = 8 H z ) . ~~~--~-nlethyl-14C-morphine 3-ethyl-6-methyl ether (10) was oxidized with activated manganese dioxide (18) as described by Barber and Rapoport for synthesis of thebaine (15). The reaction product (11) was crystallized from ethanol: yield 8.57,; mp 139-141"; homogeneous on tlc, ei mass spectrum nzje 325 (M+j: 1H nmr 6 1.3G (t, 3H, J = 8 Hz), 2.51 (s, 3H), 3.62 (s, 3H), 4.15 (9, 2H, J = 8 Hz), 5.04 (d, l H , J = 8 , H z ) , 5,29 (s, l H ) , 5.60 (d, l H , J = 8 Hzj, 6.65 (d, l H , J = 8 Hz), 6.67 (d, I H , J = 8 Hzj. Specific activity 0.173 mCi/mmole. Oripavine (14)was prepared via morphine-3-acetate Gmethyl ether (12) and oripavine 3acetate (13) (1.3): total yield from morphine, 367,; mp 199-201"; ei mass spectrum nile 297 (hI-); identical m-ith authentic oripavine by tlc; lH nmr identical with that of thebaine minus the 3-methoxy group. .4CKXOWLEDGRIENT This research was supported by a grant from the National Institute on Drug Abuse (DA 00014-18). Received 10 March 1980.
LITER-ITURE C I T E D 1. T. J. Gilbertson and E . Leete, J . .4ni. Clieiir. Soc., 89, 7085 (1967). 3 -. E. Brochmann-Hanssen, B. Sielsen and G. -iadahl, J . Pliariii. Sci., 56, 1207 (l9FT). :3. E. Brochniann-Hanssen, C-H. Chen, C . R. Chen, C-H. Chiang, *I.T. Leung and K. McNurtre.v, J . Clieiii. Soc., Perkin I , 1531 (1975). E. Leete, G. B . Bodem and 11.F. Manuel, Pliyiocheiiiisir~,10, 2687 11971). 11.L. Rueppel and H . Rapoport, J . . A I H . C l i e i i i . Soc., 92, 5526 11970): 93, 7021 (1971). G . IT. Kirby, 8.R . Nossey and P. Steinreich, J . Chem. Soc., Perkin I, 1642 (1972). E. Leete, J. Org. Chern., 44, 165 (1979). 8. J . F. Horn, A. G. Paul and H. Rapoport, J . dm. C h e m . S o c . , 100, lS95 (197s). 9. -1.R.Battersby, R . Binks, I?. J. Francis, D. J . McCaldin and H . Ramuz, J . Cheiir. Soc., 3600 (1964). 10. D. H . R . Barton, G . K.Kirb!-, K.Steglich, G. 11.Thomas, h.R.Battersby, T. A . Dobson and H . Raniuz, J . Clievi. Soc., 1965, 2423. 11. R.0. Martin, 11. E . Il-arren and H. Rapoport. BiocheiiiisfrT, 6, 2355 (1967). 12. C. C. Hodgea, J . S.Horn and H . Rapoport, Piijiociieiiiistr~,6, 1939 (197T). 13. E. Brochmann-Hanssen, C.-C. Fu and G . Zanati, J . Pharni. Sci., 60, 873 (19711. 14. E. Brochmann-Hanssen, C-C. Fu, -I.T.Leung and G . Zanati, J . Pharni. Sci., 60, 1672 il9Tlj. 15. R.B . Barber and H. Rapoport, J . .lied. Ciienr., 18, 1074 (19i5). 16. T. A. Nontzka, J. D. Natiskella and R . A. Partyka, Teirahedroiz Leif., 1325 (1974). 17. K. 8. hndersen and L. -1.K o o d s , J . Org. C l i e i i i . , 24: 2 i 4 (1959). 18. J. dttenhurron; -4. F. B. Cameron, J. H. Chapman, R . 31.Evans, B . A. Hens, .I.B . -I. Jensen and T. Walker, J . Cheiii. S O L . 1094 , (1952).
