BIOSYSTHESIS OF USNATURAL PAPATERIXE DERIT',4TITrES IK PAPA VBR SOJ~Al-IFERU1l!f E. B R O C H J I . \ S S - H . ~ S . ~C-T. ~ E S ,CHENand E. E . LINS D e p n r t m e n f of Piinriiiocciiticei Chemistrj, School of Pliarnzacy, C?zisersi::f sf Cd$oriiia, San Frmicisco, California 9414.3 .InsrR.icT.-Dehj-drogenation of various tetrahydropapaverine analogs t o the corresponding, unnatural analogs of papaverine has been studied in P a p a z r soni~ijfeiziiiz. The efficiencies of the unnatural process have been compared Kith the natural conversion of tetrahydropapaverine t o papaverine in an attempt t o gain infurmalion regarding t h e specificity of the enzyme(s) and the mechanism of the aromatization react ion.
Papaverine (6) is produced in the opium poppy by dehydrogenation of tetrahydropapaverine (THP) (1). The reaction is stereobpecific inJ-olving only the L(S)-isomer. but its substrate specificity is less well defined (1). We nou- report the bioy-nthetic conversion of some modified THP analogs to the correspondingly modified papaverine analogs. The modifications consisted of the replacement of one of the niethoxy substituents by an ethoxy group to give four analogs of THP represented by structures 2-5. These compoundc n-ere synthesized TT-ith a 14C label in poqition 3. The correqponding papaverine analogs (7-10) were synthesized for use as carriers in the isotope dilution method.
1; RI= R 2 = R3= R4=;\Ie 2; R1=Et, R2=R3=R4=Me 8 ; R2=Et,R1=R3=R4=lle 1:R 3 = E t , R 1 = R 2 = R S = i \ l e 5 ; R 4 = E t , R1=R2=R3=;\Ie
EXPERI;\IEST,IL 1-Benzyl-isoquinolines and -tetrahydroisoquinolines 17ei-e synthesized a s described previously (2-4). T h e radioactive purit?- of t h e labeled sitbstrates was determined b y radioactivity scanning Kith a Berthold radio scanner (T'arian Aerograph Co.) of thin layer chromatograms developed with two different solvent systems. Physical measurements and spectroscopic d a t a v e r e obtained with nonradioactive samples synthesized b y t h e same methods as the labeled compounds and their identitj- established b y chromatography (glc, tlc). 1H S m r spectra were measured in deuteriochloroform n-ith tetramethylsilane a s internal standard (table l j . Papnrer s o i m i J e r i m , S o o r d s t e r variety, was used throughout. T h e methods for cultivation of the plants, administration of labeled substrates, extraction, separation, purification and controlled degradation of t h e alkaloids have been described in previous communications (4, 5 ) . I n general, t h e weakly basic papaverine analogs were separated from t h e t o t a l alkaloid extract by extraction m-ith chloroform a t pH 1-2 and chromatographed on a column of silica gel n-ith chloroform containing 2 5 methanol and 1% glacial acetic acid. T h e fractions containing papaverine and i t s ethoxy analog n-ere combined and subjected t o preparative tlc on silica gel with t h e follox-ing solvent systems: (1) chloroform-methanol (9:1), (2) benzeneethanol (8:2) and (3) ethyl acetate-methanol (9G:i). Finally, t h e papaverine analogs m-ere crystallized from ether t o constant radioactivity.
736
SOV-DEC
19801
BROCHlLkS11-HASSSES ET -IL.:
BIOSTSTHESIS O F PAP-IVERISE
737
TABLE 1. Spectroscopic d a t a of substrate3 and carrier compounds
I
2
4 121q,2Hj 1
1 46(t,SH)
-
3
1 4l(t,3Hi 4.00 ( q , % H
3.S8
343 (AI- 1 192(11-151)-
,
I
3 SD ~
3 s4
l
3 Si
3 85 s.GH)
1
-
3.84
l -
35i (lI-) 206(11-151)-
I
1 8
3 99
3 7
3 90
1
3 92
3 62
i
3 80
I
I
384
I
9
3 94
4 03
1 3Sit,3H)
1 4 04(q,2H)
3 s4
-
I
I
I
RESULTS -1SD DISCUSSIOS - i s s1ioIv-n i:i table 2 , all four THP analogs n-ere aromatized to the correspond-
ing papayerine analogs with the label intact, but the efficiency of the incorporations 1-aried n-ith :he position of the ethoxy group. K h e n the niodification involved the A-ring of the tetrali!-droisocluirioliiie moiet>-, deli>-drogenation took place n-it11 about the same degree of efficiency a.5 n-it11 THP it.self (1). I t \\-as considerably les.? efficient, lionvever, when the ethos!- group resided in the benzj-lie portion of the molecule. indicating that the incrensed bulkiness prevented optiniuni interaction \\-it11 the enzyme. In ail earlier communication, A1-e h a r e reported ;bat T.LBLE 2.
Precursor Compound
~
Results of feeding experiments with PlipoL'er s o i i i i i i f e r i i i i i .
Percent incorp. of cnmpd.
into
1:
i 1
- I _- ---_ ~ _ _ 8_ _i _9 _ _ lo _ _ -7 11.25 I
i
~
3 4 a
1
i 10.00
I
I ~
I
i
1.91
~
1
1.50 1
Rel. activity in isolated fragment
9i.8 98.6 95.8 98.1
JOURXAL O F 3.4TURAL
738
PRODUCTS
[VOL.
43, xo. G
aromatization can occur to an extent coinparable to that of THP when one of the methoxy groups is demethylated irrespective of whether the phenolic hydroxyl group is in ring A or in ring C. The mechanism of the reaction is still not kn0TT-n in all details, but the size of the substituents of ring C and the orientation of this ring in space as determined by the chiral center at position 1 are determinant factors. Battersby et al. (6) have proposed a stepv-ise mechanism by n-hich the first double bond is introduced hetx-een the nitrogen and C-3 by a stereospecific loss of hydrogen a t C-3 follom-ed by a nonstereospecific isomerization of the imine to the enamine (1,klihydropapaverine). hCKKOWLEDGME?;T This research was supported by a grant from the National Institute on Drug Abuse ( D 4 00014-18).
Receired 20 Ilfalarcii 1980. LITERATURE CITED 1. E. Brochmann-Hanssen, C-H. Chen, C. R . Chen, H-C. Chiang, A. T. Leung and K. Mchlurtrey, J . Chen7. Soc., Perkzit I , 1531 (1976). 2. E. Brochmann-Hanssen and I< Hirai, J . Phnriii. Scz., 57, 940 (1968). 3 A. R . Battersby, R.Binks, R . J. Francis, D. J. hIcCaldin and H. Ramuz, J. Chem. Soc., 3600
(1964).
E. Brochmann-Hanssen, C-C. Fu and G. Zanati, J . Phalarin. Scz., 60, 873 (1971). 6 . E. Brochmann-Hanssen, C-C. Fu, A . Y. Leung and G. Zanati, J . Pharm. Sci., 60, 1 6 i 2
4.
(1971).
6. A. R. Battersby, P. IT. Sheldrake, J. Staunton and 1%.C. Summers, Bzoorg. Chem., 6 , 43 (19ii).
