JOURNALOFTHE CHEMICAL SOCIETYPERKIN TRANSACTIONS IOrganic and Bio-organic ChemistryMicrobial Transformations of GlaucineBy Patrick J. Davis, Daniel Wiese, and John P. Rosazza,* College of Pharmacy, The University of Iowa, IowaCity, Iowa 52242, U.S.A.Microbial transformation experiments were conducted with the aporphine alkaloid glaucine. Small-scale screeningexperiments provided a number of rnicro-organisms which produced three metabolites. In preparative scalestudies, Streptomyces griseus (U I 1 1 58) produced norglaucine (4) and 2-O-demethylglaucine (6) (predicentrine)in I 1 and 14% yield, respectively. Fusarium solani (ATCC 12823) produced didehydroglaucine (3) and a norapor-phinone (10) (an artefact) in 60 and 21% yield, respectively. With racemic glaucine, F.solani preferentiallydehydrogenated (+) -(S) -glaucine, and unchanged, optically enriched (-) - (R) -glaucine was recovered fromfermentations. N- and U-dealkylation did not occur in stereoselective fashion.MICROBIAL transformations of alkaloids and othernatural products have been ~tudied.l-~ Groups ofalkaloids investigated to date include morphine deriva-tives’s-’ ergolines,8 and other indole derivative^.^,^In general, systematic studies on the types of reactionpossible with alkaloids have not been conducted,although these compounds offer unusually rich arrays offunctional groups which might be susceptible to chemicaltransformation by micro-organisms. Reactions re-ported include N- and O-dealkylations, hydroxylations,and N-oxidat ions .loExcept for a previous report from these labora-tories,ll no microbial transformation studies with1 W.Charney and H. L. Herzog, ‘ Microbial Transformationsof Steroids,’ Academic Press, Ney York, 1967.2 H. Iizuka and A. Naito, Microbial Transformations ofSteroids and Alkaloids,’ University Park Press, State College,Pennsylvania, 1967.3 G. Fonken and R. S. Johnson, ‘ Chemical Oxidations withMicroorganisms,’ Dekker, New York, 1972.4 L. L. Wallen, F. H. Stodola, and R. W. Jackson, ‘TypeReactions in Fermentation Chemistry,’ Agricultural ResearchService, US. Department of Agriculture, Bulletin, RS-71-13,1959.6 L. A. Mitscher, W. W. Andres, G. 0. Morton, and E. L.Patterson, Experientia, 1968, 24, 133.6 K. Iizuka, M. Yamada, J.Suzuki, I. Seki, K. Aida, S.Okuda, T. Asai, and K. Tsuda, Chem. and Pharm. Bull. (Japan),1962, 10, 67.D. Groger and H. P. Schauder, Exfierientia, 1969, 25, 95. * R. Beukers, A. F. Marx, and M. H. J. Zuidweg, in ‘ DrugDesign,’ ed. E. Ariens, Academic Press, New York, 1972, vol. 3.P. Bellet and T. Van Thuong, Ann. pharm. frang., 1970, 28,245.aporphines have been described. This paper describesthe selective N- and O-dealkylation of glaucine (1) by aStreptomyces species, and its stereoselective dehydrogen-ation by a Fusarium species.RESULTS AND DISCUSSION(+)-(S)-Glaucine was prepared by methylation ofcommercially available (+I-(S)-boldine in good yieldwith sodium hydride and 2 equiv. of methyl tosylate,12or with diazomethane.13 Other literature pro-cedures 14-16 were lower yielding.Didehydroglaucine(3) was prepared from (+)-(S)-glaucine by treatmentwith iodine and sodium acetate in dioxan,17 or in higheryield with palladium-carbon in acetonitrile.lsInitial small-scale screening experiments were con-ducted to discover micro-organisms capable of metabol-izing glaucine (1). Micro-organisms were selected on17, 785.Adrian, J . Medicin. Chcm., 1975, 18, 791.10 R. V. Smith and J. P. Rosazza, Biotechnol. and Bioeng., 1975,11 J. P. Rosazza, A. W. Stocklinski, M. A. Gustafson, and J.12 A. Modiri, Ph.D. Thesis, University of Iowa, 1971, p. 49.13 T. Kakasato and S. Nomura, J . Pharm. SOC. Japan, 1957.77,14 M. Tomita and K. Fukagawa, J . Pharm. SOC. Japan, 1963,1s G. W. Kenner and M.A. Murray, J . Chem. SOC., 1950, 406.16 R. Goutarel, M. M. Janot, V. Prelog, and R. P. A. Sneeden,17 M. P. Cava, A. Venkateswaria, M. Srinivasan, and D. L.18 M. P. Cava, D. L. Edie, and Jose J. Saa, J . Org. Chem., 1975,816.83,293.Helv. Chim. Ada, 1951, 34, 1962.Edie, Tetrahedron, 1972, 28, 4299.40, 3012 J.C.S. Perkin Ithe basis of previous experience,lg*N and of reportsdescribing cultures capable of 0- and N-dealkyl-ations.6,7,21,22 Of sixty cultures screened, eight gavesufficiently large amounts of glaucine metabolites towarrant further study (Table 1).TABLE 1Cultures yielding metabolites of (+)-(S)-glaucine (1)Metabolites7- Micro-organisms (6) (4) (3) Other + + + + +Stysanus stemonites (SC 2831) - -Penicillium claviforme (MR 376) - -Cunninghamella echinulata + -Cunninghamella bainieri + -Streptomyces punipalus + - - +Stveptomyces griseus (UI 1158) + + - Penicillium brevicompactum -Fusasium solani (ATCC 12823) -Two metabolites were isolated from preparative scalefermentations with Styeptomyces griseus (UI 1158).One of these, norglaucine (4) was identified simply onthe basis of comparison of n.m.r.and mass spectral data-(NRRL 3665)(ATCC 3065)(NRRL 3529)- --- + -- + + (ATCC 10418)( 1 ) R' = RZ= R4= R5 = OMe, R3 = Me, R6 =H(2 ) R' = R5=OMe, RZ= R4= OH, R3= Me, R6=H( 3 1 6a, 7- didehydro - ( 1 1( 4 ) R' = RZ= R4 = R5 = OMe, R3 = R6 = H( 5 ) R ' =R2=R4=R5=OMe, R3=Ac, R6=H( 6 ) R' =R4=R5=OMe, R2=OH, R3=Me, R6= H( 7 ) R ' =R2=R5=OMe, R4=OH, R3=Me, R6=H( 8 ) R' = R '=R4= OMe, R5=OH, R3= Me, R6 = H(9) R' =R2=R4=H, R5=R6=OMe, R3 =Mewith published value.^.^^,^ The resolved N-methyln.m.r.signal of glaucine is absent, and the mass spectrumshows retro-Diels-Alder fragmentation *,26 (M - CH2=NH). The m.p. of the N-acetyl derivative (5) wasidentical with the published value.23lS R. V. Smith and J. P. Rosazza, Arch. Biochem. Biophys.,1974,161,551.2o R. V. Smith and J. P. Rosazza, J. Pharm. Sci., 1975, 64,1737.P. Bellet and L. Penasse, Ann. $harm. fralzc., 1960, 18, 337.22 B. Boothroyd, E. J . Napier. and G. A. Somerfield, Biochem.J., 1961, 80, 34.29 S. R. Johns, J. A. Lamberton, C. S. Li, and A. A. Sioumis,Austral. J . Chem., 1970, 23, 423.24 C. Casagrande and G.Ferrari, I1 Farmaco, Ed. Sci., 1970,2!5,442.26 M. Okashi, J. M. Wilson, H. Budzikiewicz, M. Shamma,W. A. Slusarchyk, and C. Djerassi, J . Amer. Chem. Soc., 1963,86, 2807.96 A. H. Jackson and J. A. Martin, J. Chem. Soc. (C), 1966,2181.The second metabolite was 2-0-demethylglaucine(6) (predicentrine; 0-methylboldine 27). The massspectrum indicated that a single methyl ether linkagehad been cleaved (m/e 341). N.m.r. signals for both theN-methyl (6 2.55) and the 1-methoxy-group ( 6 3.58)were Methoxy protons at positions 2, 9,and 10 give a,n unresolved singlet at 8 3.90 in the 60MHz n.m.r. spectrum of glaucine, and are only margin-ally separated in a 100 MHz spectrum. The n.m.r.spectrum of the metabolite was comparable with thepublished spectra of predicentrine (6) ,3O N-methyl-laurotetanine (7),%s3l and thalicmidine (8) 31-33 (2-, 9-,and 10-0-demethylglaucine , respectively).The meta-bolite was probably not N-methyl-laurotetanine (7)since no bathochromic shift occurred in the U.V. spectrumupon the addition of base.34The signals for the aromatic protons at positions 3, 8,and 11 are well separated in the n.m.r. spectrum ofglaucine and that of the metabolite. Thus, it waspossible to locate the site of 0-dealkylation by obtainingn.m.r. spectra of the metabolite while titrating the samplewith Under these conditions signals due toaromatic protons ortho to a phenolate ion experience agreater upfield shift than those of protons adjacent tomet hoxy-groups. For comparison, base- titration n .m.r.spectra were obtained for the metabolite, boldine (2),and (later) synthetic 9-0-methylboldine (6) .27 Theresults for the metabolite are shown in Table 2.ItTABLE 2Relative chemical shifts of protons of the metabolite (6)when titrated with NaOD in (CD,),SO-D,O *Solvent I(CD,),SO 6.52(equiv. NaOD) H-3(CD,)aSO-D,O (0.28) 6.50(CD,),SO-DaO (0.56) 6.55(CD,),SO-DzO (0.84) 6.50(CD3),SO-D,0 (1.10) 6.38A8 (total) -0.148H-8 H-1 i6.90 7.876.87 7.866.90 7.856.85 7.926.90 7.950.00 +o.os* The experiment was conducted according to the procedureof P a ~ h l e r ; ~ ~ the metabolite (30 mg) was dissolved in (CD,),SO(0.5 ml) and 30% NaOD in D,O added in 3 pl portions.was necessary to add base (NaOD) stepwise since it waspossible for signals of H-3 and H-8 to cross one another.The H-3 signal was the only one shifted upfield; that ofH-11 was shifted downfield, and that of H-8 was un-altered.The magnitude of the shift of H-3 was con-,' R. Tschesche, P. Welzek, R. Moll, and G. Legler, Tetra-hedron, 1964, 20, 1435.28 W. H. Baarschers, R. R. Arndt, K. Pachler, J. A. Weisbach,andB. Douglas, J . Chem. Soc. (C), 1964,4778.2s I. R. C. Bick, J. Harley-Mason, N. Sheppard, and M. J.Vernengo, J . Chem. SOC. (C), 1961,1896.3O S . R. Johns, J. A. Lamberton, A. A. Sioumiss, and H. J.Tweeddale, Austral. J . Chsm., 1969, 22, 1277.91 K. G. R. Pachler, R. R. Arndt, and W. H. Baarschers,Tetrahedron, 1966, 21, 2159.92 M. Shamma, R. J. Shine, and D. S. Dudock, Tetrahedron,1967,28,2887.38 M.Shamma, M. J. Hillman, R. Charubala, and B. Pai,Indian J . Chem., 1969, 7 , 1056.a4 M. Shamma. S. Y. Yao, B. R. Psi, and R. Charubala, and B.Pai, J . Org. Chem., 1971, 36, 32531977 3sistent with published values for O-demethyl derivativesof papa~erine.~5The structure of the metabolite was confirmed bycareful methylation of boldine (2) with diazomethane 27to yield the two possible isomeric monophenolic trime-thoxyaporphines (6) and (7). These were easily separ-ated by t.1.c. and gave different colour reactions withdiazotized sulphanilic acid reagent.27 One of them wasidentical with authentic N-methyl-laurotetanine (7),and exhibited the expected bathochromic U.V. shiftupon addition of base.= The other (6) behaved chro-matographically and spectrally (including the NaODn.m.r. titration) like the metabolite.The metaboliteand (6) prepared from boldine formed identical crystal-line hydro bromide^.^^It is interesting that O-dealkylation was restricted t oring A of glaucine, since highly selective O-dealkylationof l0,ll-dimethoxyaporphine (9) is effected by Strep-tomyces species, and by CunnivghameZla species.ll Itappears that O-dealkylation is restricted to an un-hindered methoxy group, as in the O-dealkylation of10,ll -dimethoxyaporphine.llFusarium solani (ATCC 12823) gave two products,both different from those produced by S. griseus. Onewas identical with synthetic didehydroglaucine (3),showing a broad U.V. absorption maximum from 264 to278 nm, characteristic of didehydroaporphine~.~~~ 37The mp.of the metabolite38 and the n.m.r. spec-trum 37938 were in agreement with published values.Lower yields of a noraporphinone (10) were obtained.The product was spectrally identical with a sampledescribed in the literature (i.r.,39940 n.m.r.,4l and U.V. 41)and possessed the same m . ~ . ~ ~Oxidation of glaucine in air is known to yield avariety of products including didehydroglaucine, andthe noraporphinone (10) !3 When didehydroglaucinewas incubated under the same conditions as employedfor the cultivation of micro-organisms, the noraporphin-one was readily detected, in 10% yield as estimated byt .I.c. Glaucine incubated under these conditions pro-duced only traces of didehydroglaucine and the nor-aporphinone.Thus, the noraporphinone (10) was anartefact produced by aerial oxidation of didehydro-glaucine (3).Stereoselectivity has been demonstrated in a varietyof microbial reactions with other s u b ~ t r a t e s . ~ ~ ~ , ~ ~ ~Attempts were made to determine whether S. griseus or36 F. M. Belpaire, M. G. Bogaert, M. T. Rossell, and M.Antennis, Xenobiotic?, 1975, 5, 413.38 M. Shamma, The Isoquinoline Alkaloids,’ vol. 25 of‘ Organic Chemistry; a Series of Monographs,’ eds. A. T. Blom-quist and H. Wasserman, Academic Press, New York, 1972, p.226.38 C. D. Hufford, M. J. Funderbunk, J, M. Morgan, and L. W.3B J. H. Linde and M. S. Ragab, Helv. Ckim. Ada, 1968,51, 683.40 M. A. Buchanan and E. E. Dickey, J.Org. Chem., 1960, 25,P. E. Sonnet and M. Jacobson, J. Pkarm. Sci., 1971, 60,43 J. Cohen, W. Von Langenthal, and W. I. Taylor, J. Org.H. G. Kiryakov, Chem. and Ind., 1968,1807.Robertson, J. Pharm. Sci., 1976, 64, 789.1389.1254.Chem., 1961, 28, 4143.F. solavti exhibited substrate enantiospecificity in thereactions described with glaucine. For these studies,(RS)-glaucine was prepared by reduction of didehydro-glaucine with sodium cyanotrihydridoborate 46947 atpH 3 4 . All attempts to reduce didehydroglaucinewith dissolving sodium tetrahydridoborate,and hydrogen over palladium-carbon or platinumoxide 49 were disappointing, giving low yields. Glaucinerecovered from fermentations of S. griseus was notoptically active. On the other hand glaucine recoveredfrom F. solani fermentations was 50.8% optically pure(75.4% enantiomerically pure) (-)-(R)-glaucine.Thus,an apparent substrate enantiospecificity for natural(+)-(S)-glaucine operates during dehydrogenation.The time course of the conversion of racemic glaucineinto didehydroglaucine was studied. Cultures of F .solarti were grown as usual, and at various times after6o r - 3-1200-90 Fi P-60 9” A-30-0 1 2 3 4 5 6 7 8 ”DaysTime course of the conversion of racemic glaucine into dide-hydroglaucine by F. solani: (A) glaucine recovery; (B)glaucine optical activity ; (C) didehydroglaucine yieldaddition of (RS)-glaucine substrate the contents ofwhole flasks were extracted; unchanged glaucine anddidehydroglaucine were separated by preparative t .l.c.and their identities were confirmed by chromatographicand U.V.and mass spectral analyses. Their concen-trations were determined spectrophotometrically (u.v.absorptions a t 280 and 302, and 260 and 334 nm,respectively). Optical rotations of the recovered glau-cine were also measured. The results are shown in theFigure.43 M. Tomita, S. Lu, S. Want, C. Lee, and H. Shih, J. Pharm.Soc. Japan, 1968,88, 1143.44 ‘ Steric Course of Microbiological Reactions,’ CIBA Founda-tion Study Group 2, eds. G. E. W. Wolstenholme and C. M.O’Conner, Little Brown and Co., v s t o n , 1969.45 C. J . Sih and J. P. Rosazza, Microbial Transformations inOrganic Synthesis,’ in ‘Applications of Biochemical Systems inPreparative Organic Chemistry,’ Part I, eds.B. Jones, D. Perl-man, and C. J. Sih, Wiley-Interscience, New York, 1976.R. F. Borch, M. D. Bernstein, and H. D. Durst, J. Amer.Chem. Soc., 1971, 93, 2897.47 D. E. Nichols and C. F. Barfknecht, J. Heterocyclic Chem.,1973, 10, 339.48 J. Gadmer, Arch. Phavm., 1911, 249, 698.4@ M. Kupchan, T. H. Yang, M. L. King, and R. T. Borchardt,J . Org. Chem., 1968, 33, 10524 J.C.S. Perkin IOptical enrichment in (-)-(R)-glaucine was confirmed,and the optical rotation approached the theoreticalvalue (-115") after 7 days. At the same time, the over-all concentration of glaucine diminished. After 7 days,only a few percent of the glaucine originally addedremained. Didehydroglaucine yield increased up to6 days, then decreased.The low recoveries of didehy-droglaucine at later times are reproducible. In general,recoveries of both glaucine and dehydroglaucine were low,even at zero time, probably owing to binding of bothcompounds by the mycelium of F. solani. This pheno-menon has been observed with glaucine and otheraporphines studied in our laboratory. Since littleglaucine remains in the fermentation after 7 days, bothisomers are probably utilized by the micro-organism.This is the first report of a microbial dehydrogenationof an aporphine. As with steroids and other compoundsstudied in microbial transformation systems, thisreaction occurred in stereoselective fashion. Withthe intact micro-organism, a multitude of reactions arepossible. The true degree of enantiospecificity of the F.solani dehydrogenase enzyme will not be known untilthe enzyme is purified.It is conceivable that (-)-(R)-glaucine is degraded by a completely differentmetabolic path, or that it is simply dehydrogenatedmore slowly than the (+)-(S)-glaucine isomer. Furtherstudies concerning the metabolic fate of (-)-(R)-glaucineare in progress. When purified, the appropriate enzymemay provide a useful tool for the resolution of racemicmixtures of glaucine and other aporphines.EXPERIMENTALPhysical data were obtained as follows: n.m.r. spectra,Varian T-60 spectrometer, Me,Si internal standard ; lowresolution mass spectra, Finnigan 3200 spectrometer ; highresolution mass spectra determined by Battelle MemorialLaboratories, Columbus, Ohio ; m.p.(corrected) for samplesin open-ended capillaries, obtained with a Thomas-Hooverapparatus ; i.r. spectra, Perkin-Elmer 267 spectrophoto-meter ; optical rotations, Perkin-Elmer 14 1 polarimeter(0.099 8 dm microcell) ; U.V. spectra, Pye-Unicam SP 1800spectrophotometer, Didydium external standard.T.1.c. was performed on 0.25 mm or 1.0 mm thick layersof silica gel GF254 (Merck) on glass plates, activated at 120 "Cfor 30 min prior to use; solvent systems (A) ethyl acetate-ethyl methyl ketone-acetic acid-water (3 : 4 : 1 : 2) ; (B)benzene-methanol (4 : 1) ; (C) benzene-methanol (9 : 1) ;(D) chloroform-diethylamine (9 : 1) ; (E) benzene-meth-anol-ammonia (56%) (80 : 20 : 0.1) ; (F) benzene-methanol(6 : 1). Compounds were located by fluorescence quenchingunder 254 or 366 n.m.U.V. irradiation, or by spraying withthe following reagents : Dragendorff 's,So cerium( IV) ammo-nium sulphate (CAS) (1 % in 50% v/v H,PO,) ; 51 or diazo-tized sulphanilic acid-NaOH (5% in 50% ethanol) .50Column chromatography was performed on silica gel(Baker 3400).(+)-(S)-GZuucine (1) from (+)-(S)-BoZdine (2).12-A sus-pension of boldine (2) (3.27 g, 10 mmol) and sodium hydride(1.0 g, 40 mmol) in dry dimethylformamide (100 ml) was50 J. M. Bobbitt, ' Thin Layer Chromatography,' Reinhold,New York, 1964, p. 84.stirred under nitrogen for 0.5 h. A solution of methyltosylate (4.0 g, 21 mmol) in anhydrous ether (20 ml) wasadded over 15 min. The reaction was monitored by t.1.c.[system (B); CAS reagent].After 12 h the mixture waspoured over ice (70 g) and exhaustively extracted withether to yield a residue (3.7 g) after evaporation. Theproduct was purified by column chromatography [silicagel (90 g; 42 x 2.5 cm); benzene-methanol (20 : 1) a t1.5 ml min-l; 15 ml fractions]. Fractions 17-33 gaveglaucine (2.45 g) as an oil. Further removal of solventunder high vacuum yielded crystalline glaucine (1) (2.0 g,56y0), m.p. 117-119" (lit.,62 120"); Lx. (EtOH) 280( E 1.5 x lo4) and 302 nm (1.4 x lo4); G(CDC1,) 2.55 (3 H,s NMe), 3.67 (3 H, s, l-OMe), 3.88, 3.90, and 3.92 (9 H,3Xs,2-,9-,and10-OMe),6.60(1H,s,H-3),6.80(1H,s,H-8),and 8.12 (1 H, s, H-11); [0(],,2~ (c 5.04 in EtOH) + 111"(lit.,62*63 + 113)"; m/e 355 (goyo), 354(100), 341(15),340(64), 338(15), 330(9), 329(30), 312(18), 308(10), 297(19),281(30), and 266(9) (Found: C, 71.25; H, 7.3; N, 3.75.Calc.for C,,H2,N0,: C, 70.95; H, 7.1; N, 3.9%); hydro-chloride, m.p. 234" (from methanol-water) ( lit.,62 234").DidehydrogZuucine (3) .17-Didehydroglaucine was pre-pared either by treating (+)-(S)-glaucine with iodine indioxan l 7 or by refluxing (+)-(S)-glaucine over palladium-carbon ( 10%) in acetonitrile.18 Reactions were monitoredby t.1.c. [system (B); RP (1) 0.5, RF (3) 0.951; the producthad m.p. 124' (from ethanol) (lit.,39 121-122'); Amx.(EtOH) 260.5 (E 3.36 x lo4) and 334 nm (8.72 x lo3); 389393.03 (CDC1,) (3 H, s, NMe), 3.27 (4 H, s, CH,*CH,), 3.90(3 H, s, 1-OMe), 4.02 (9 H, s, 2-, 9-, and 10-OMe), 6.57(1 H, s, H-7), 6.95 (1 H, s, H-3), 7.03 (1 H, s, H-8), and 9.20(1 H, s, H-11); m/e 354(31y0), 353(100), 388(86), 337(86),335(46), 307(31), 306(31), 294(31), 280(54), 279(57), and176(86) ; all data were consistent with published value.^.^^*^^(RS) -GZaucine [ (RS) -( l)] .469 47-Sodium cyanotrihydr-idoborate (0.2 g, 0.45 mmol) was added to a solution ofdidehydroglaucine (3) (100 mg, 1.3 mmol) in absoluteethanol (30 ml) being purged with nitrogen.Alcoholichydrogen chloride was added to maintain the pH a tcu. 3-4. After 18 h, t.1.c. [system (E)] indicated thatthe reaction was complete. The mixture was evaporatedto dryness in vucuo and the residue suspended in cold water(30 ml) and exhaustively extracted with ethyl acetate.The extracts were concentrated to an oil. Racemicglaucine, (RS)-( l), purified by preparative t.1.c.[system(E)], exhibited no optical rotation; m.p. 136-137" (frommethanol) (lit.,63 137-139"); G(CDC1.J 2.53 (3 H, s, NMe),3.65 (3 H, s, 1-OMe), 3.90 (9 H, s, 2-, 9-, and 10-OMe),6.57 (1 H, s, H-3), 6.77 (1 H, s, H-8), and 8.05 (1 H, s, H-11);m/e 355(62y0), 354(100), 341(10), and 340(45) (Found: C,71.1; H, 7.15; N, 3.85%).Synthesis of 2-O-MethyZboldine [N-MethyZ-luurotetanine(7)] and 9-O-MethyZboZdine [Predicentrine (S)] . 27-Etherealdiazomethane (3 mmol) was added to a solution of boldine(2) (1 .O g, 3 mmol) in methanol (40 ml) cooled in an ice-bath,and the mixture was allowed to warm to room temperature.After 4.5 h more diazomethane (1.5 mmol) was added, andthe solution was set aside a t room temperature overnight.The reaction was monitored by t.1.c.[system (D) ; diazotizedsulphanilic acid spray showed boldine (2) , R p 0.35, brown;9-O-methylboldine (6), RF 0.80, orange ; glaucine ( 1) , RF51 N. R. Farnsworth, R. N. Blomster, D. Damratoski, W. A.Meer, and L. V. Cammarato. Lloydia, 1964,27, 302.6* R. Fisher, Arch. Pharm., 1901, 239, 426.68 J. Gadamer, Arch. Pharm., 1911, 249, 6801977 50.98, grey ; and 2-0-methylboldine (7), RF 0.60, red-brown].The mixture was evaporated to dryness and the productschromatographed on a silica gel column (150 g, 7 0 x 2.6cm) eluted with chloroform-diethylamine (100 : 1) a t2 ml min-l (15 ml fractions). Fractions 68-120 contained9-0-methylboldine (6) (350 mg, 35y0), and fractions 133-165 2-0-methylboldine (7) (100 mg, loyo), identical chrom-atographically with N-methyl-laurotetanine, kindly sup-plied by Dr.M. P. Cava. 9-0-Methylboldine hydro-bromide had m.p. 209-211" (lit.,30 209"); 30 the m.p. of amixture with predicentrine hydrobromide prepared fromthe metabolite obtained from S. griseus showed no depres-sion (Found: C, 56.65; H, 5.75; N, 3.2. Calc. for C2oH24-BrNO,: C, 56.9; H, 5.75; N, 3.3%).Fermentation Procedures.-Cultures used in this study aremaintained in the University of Iowa College of Pharmacyculture collection, and are stored at 4 "C in a refrigerator.Organisms were grown according to a two-stage ferment-ation procedure in a medium of the following composition:soybean meal (5 g), glucose (20 g), yeast extract (5 g),NaCl (5 g), K,HPO, (5 g), distilled water to 1 000 ml; pHadjusted to 7.0 with ~ N - H C ~ .Media were sterilized in anautoclave a t 121 'C for 15 min.Fermentations were conducted on rotary shakers,operating a t 250 rev. min-1 (1 in stroke) a t 27 "C in Erlen-meyer flasks holding one-fifth of their volume of medium.The surface growth from slants was suspended in sterilemedium (5 ml) and used to inoculate stage 1 cultures, whichwere incubated as described for 72 h. The thick 72 h stageI growth was used as inoculum for stage I1 cultures, theinoculum volume being 10% of the volume of stage I1fermentation medium in all cases. Substrates were addedto 24 h old stage I1 cultures, either in dimethylformamide(250 mg in 1 ml), or by solubilizing in a convenient amountof deionized water with hydrochloric acid, followed byadjustment of the solution to pH 7 prior to addition.Unless otherwise specified, the final substrate concentrationin stage I1 cultures was 0.5 mg ml-l.Substrate-containingstage I1 cultures were sampled a t intervals for t.1.c. analyses.Fermentation Sampling.-Samples (5 ml) were withdrawna t intervals, adjusted to pH 8.5 with saturated aqueoussodium hydrogen carbonate, extracted with ethyl acetateor ether (0.5 ml) and centrifuged if necessary, and spottedon t.1.c. plates.Screening of Micro-organisms.-Small-scale fermentationswere conducted in Erlenmeyer flasks (125 ml) to determinethe abilities of sixty selected micro-organisms to metabolize(+)-(S)-glaucine ( 1 ) .Cultures were grown as describedabove, and a total of 12.5 mg of glaucine (1) was added toeach 24 h stage I1 culture. Substrate-containing flaskswere incubated with shaking, and samples (5 ml) werewithdrawn 24 and 72 h after substrate addition. Of thesixty organisms screened, fourteen metabolized glaucine.Eight of these cultures gave sufficiently high levels ofmetabolites to warrant further study.The experiment was repeated with controls and with onlythe active cultures. Controls consisted of substrate addedto sterile medium and of fermentation blanks containingno substrate. The most active metabolizing cultures wereStreptomyces griseus (UI 1158) and Fusarium solani (ATCC12823).Prefiaratiue-scale Production of Norglaucine (4) and 2-0-Demethylglaucine (6) by Streptomyces griseus.-S.griseus(UI 1158) was grown according to the usual fermentationprocedure. Stage I1 fermentations were conducted in 4.0 1of medium in 500 ml Erlenmeyer flasks. ( + )-(S)-Glaucine(1.5 g) in dimethylformamide (2 ml) was distributed evenlyamong forty stage I1 culture flasks, and the substrate-con-taining cultures were monitored by t.1.c. [system (B)].After 90 h the contents of all flasks were combined, adjustedto pH 7.5 with ammonia solution (58y0), and exhaustivelyextracted with ether (liquid-liquid extractor). The ex-tracts were dried (Na,SO,) and evaporated to dryness inuacuo to give a crude extract (5.0 g). The extract wasadsorbed on a silica column (300 g; 42 x 5.5 cm) elutedwith benzene-methanol (20 : 1) a t 2.5 ml min-l (18 mlfractions).Fractions 100-185 contained pure (6) (209mg, 14%), and fractions 241-331 pure (4) (165 mg, 11%).Characterization of Norglaucine (4) .-The metabolite infractions 241-331 could not be induced to crystallize. How-ever, a crystalline acetyl derivative was obtained by treatingthe metabolite with pyridine-acetic anhydride; m.p. 102"(lit.,23 for N-acetylnorglaucine, 102-104'). The n.m.r.spectrum of the metabolite was similar to the publishedspectrum of norglaucine (D,O-pyridine) ,23 and identicalwith that of glaucine (CDCl,) except for the absence of anNMe signal a t 6 2.55. The metabolite showed m/e 341.161 2(calc. for C,oH,3NO,: 341.162 7 ) ; m/e 341(84y0), 340(100),327(17), 326(74), 325(22), 324(22), 312(8), 310(53), 298(38),and 294(36).Characterization of 2-0-Demethylglaucine (6) .-The meta-bolite could not be induced to crystallize, but showedG(CDC1,) 2.58 (3 H, s, NMe), 3.58 (3 H, s, 1-OMe), 3.90(6 H, s, 9- and 10-OMe), 5.50br (1 H, s, 2-OH, exchangeswith D20), 6.65 (1 H, s, H-3), 6.82 (1 H, s, H-8), and 7.95(1 H, s, H-11); Am..(EtOH) 2 8 G 3 1 2 nm (no batho-chromic shift on addition of 5% NaOH); m/e 341.161 5(calc. for C,,H,,NO,: 341.162 7 ) ; m/e 341(84%), 340(100),327(17), 326(74), 325(22), 324(22), 310(53), 298(38), 294(36),and 283(40); the hydrobromide had m.p. 208-211'; them.p. of a mixture with synthetic material was not depressed(lit.,30 209-212'). I.r., u.v., n.m.r., and mass spectralcomparisons of the metabolite with synthetic (6) provedthe compounds to be identical.N.m.r.-monitored Titrations of the Metabolite (6) withSodium Deutero~ide.~l--Base-titration n.m.r.spectra wereobtained with [2H,]dimethyl sulphoxide as solvent andadding sodium deuteroxide (30% in D,O) in 5 equal por-tions. Titrations were conducted with boldine (2), syn-thetic (6), and the metabolite (6). The results are shown inTable 2.Prefiarative-scale Production of Didehydroglaucine (3)and the Noraporphinone (10) by Fusarium solani (ATCC12823).-F. solani (ATCC 12823) was grown according tothe usual fermentation procedure. Stage I1 fermentationswere conducted in 2.3 1 of medium held in 500 ml Erlen-meyer flasks. (+)-(S)-Glaucine (1) (1.15 g) in dimethyl-formamide (2.3 ml) was distributed evenly among 23 stageI1 culture flasks.The reaction was monitored by t.1.c.[system (E) ; Dragendorff reagent spray (all compoundspositive) or CAS spray (didehydroglaucine, RF 0.95, yellow;glaucine, RF 0.75, blue; aporphinone (lo), Rp 0.4, colour-less). After 6 days the cultures were adjusted to pH 8.3with saturated sodium hydrogen carbonate solution, andexhaustively extracted with ether (liquid-liquid extractor).The extract was dried (Na,SO,) and dried in uacuo. Theresidue (1.5 g) was subjected to column chromatography onsilica gel (350 g; 47 x 5.5 cm) eluted with benzenemethanol-ammonium hydroxide (57%) (200 : 1.0 : 0.1) a t0.5 ml min-1 (15 ml fractions). Fractions 225-263 gavJ.C.S. Perkin Ipure didehydroglaucine (700 mg, 61%) ; fractions 1 600-1 860 gave the noraporphinone (10) (250 mg, 21%).The metabolitewas similar to synthetic didehydroglaucine (3), givingcrystals from 95% ethanol; m.p.121.5-122" (lit.,,* 121-122"); A,, 264 and 334 nm; 37 6 (CDCl,) 38 3.08 (3 H, s ,NMe), 3.28 (4 H, s, CH,*CH,), 3.93 (3 H, s, 1-OMe), 4.05(9 H, s, 2-, 9-, and 10-OMe), 6.67 (1 H, s, D,O-exchanged,H-7), 6.93 (1 H, s, H-3), 7.05 (1 H, s, H-8), and 9.10 (1 H, s,H-11); m/e 353(100%), 352(100), 338(88), 337(76), 335(35),307(29), 306(29), 294(25), 280(51), 279(51), and 176(63).Characterization of 4,5,6,6a-tetradehydro- 1,2 9,lO-tetra-methoxynorafiorfihin-7-one (lo). 1.r. [vmZ 1 645 (GO),1 735, and 1 590 cm-11 4O and U.V.data [Lx. 215, 246, 276,299sh, 320-395, and 4 2 0 4 4 5 nm; Amin 233 and 261 nm] 41were identical with those published. The n.m.r. spectrumshowed G(CDC1,) 4.05 (s, OCH,), 4.07 (s, OCH,), 4.10 (s,2 OCH,), 7.20 (1 H, s), 7.8 (1 H, d), 8.1 (1 H, s), and 8.9(2 H, m); 6 (CF,*CO,D) 3.4, 3.47, 3.57, and 3.60 (4 s,4 OCH,), 7.21 (1 H, s), 7.75 (1 H, s ) , 8.33 (2 H, q), and 8.71(1 H, s) . The compound was insoluble in water, turning redon addition of concentrated hydrochloric acid. In chloro-form it shows a green fluorescence. It formed reddishcrystals (from 95% EtOH), m.p. 225-229" (lit.,42 227-229").Transformations of (RS)-Glaucine [(RS)-( I)].-Stage I1cultures of F. solani (ATCC 12823) and S. griseus weregrown in the usual way in 500 ml Erlenmeyer flasks.Thesubstrate, (RS)-(1) (50 mg per flask), was then added indimethylformamide (250 mg ml-1). Sampling was per-formed in the usual way and the reaction was monitored byt.1.c. as described under ' Preparative-scale Productions.'After 6 days approximately half the glaucine remained.Fermentations were adjusted to pH 8.0 with saturatedsodium hydrogen carbonate solution, and exhaustivelyextracted with ethyl acetate; the extract was dried (Na,-SO,) and taken to dryness in vacz4o. Residual glaucineCharacterization of didehydroglaucine (3).was purified by preparative t.1.c. [system (B)] and identifiedby mass and U.V. spectra, and ko-chromatography withstandards. Quantities recovered were : from S. griseus,31 mg, [a]D26 0.81" (c 3.71 in abs. EtOH); from F. solani,15 mg, [ol]D26 -56.4" (c 2.24 in abs. EtOH) (enantiomericpurity 75.4%).Transformation of (RS)-GZaucine[(RS)-( l)] by Fusariumso1ani.-Further experiments were conducted to evaluatethe time course of enrichment in (-)-(I?) glaucine byF. solani, grown according to the usual fermentationprocedure. Stage I1 fermentations were conducted in 1 1Erlenmeyer flasks. (RS)-Glaucine (100 mg in 0.4 ml ofdimethylformamide) was added to each of six flasks and asterile media control after 24 h. Whole flasks wereharvested at day intervals up to 8 days. The control wasalso harvested after 8 days. The contents of each flaskwere adjusted to pH 8.5 with saturated aqueous sodiumhydrogen carbonate, and extracted three times with ethylacetate (50 ml). The extracts were dried (Na,SO,) andevaporated to dryness in vacuo. Preparative t.1.c. was thenperformed [system (B)]. Bands corresponding to glaucineand didehydroglaucine were isolated and eluted withmethanol; the products were dried in vacuo and identifiedby chromatography and U.V. spectra. For optical rotationmeasurements the glaucine was taken to a convenientvolume in absolute ethanol (usually 0.5-1.0 ml, corres-ponding to a ca. 5% solution). For recovery data, U.V.extinctions were measured and compared with the valuesE~~~ 1.512 x lo4 and t302 1.445 x lo4 for glaucine, and E~~~3.36 x lo4 and E,,~ 8.72 x lo3 for didehydroglaucine.U.V. spectra of all samples were run in 95% ethanol. Theresults of these experiments are shown in the Figure.Cancer Institute.through a Lilly Foundation Fellowship.This work was supported by a grant from the NationalP. J. D. gratefully acknowledges support[6/1074 Received, 6th June, 1976