Indian Journal of Chemistry Vol. 39B, May 2000, pp. 377 - 381

N-Demethyhitlon and N-oxidation of thebaine, an isoquinoline alkaloid by Mucor piriformis

K M Madyastha·" b, G V B. Reddy, H.Nagarajappa & G R Sridhar

aBio-Organic Section. Department of Organic Chemistry. Indian Institute of Science. Bangalore 560 012. India hChemical Biology Unit, Jawarlal Nehru Centre for Advance Scientific Research. Jakkur. Bangalore 560 064. India

Received 27 July 1999; accepted /3 December 1999

Northebaine was identified as the major metabolite formed during the biotransformation of thebaine by Mucor piriformis. Minor metabolites identified are isomeric thebaine N-oxides. Among isomeric thebaine N­oxides, the one with the equatorial oxygen attached to the nitrogen is relatively unstable. Resting cells. grown in the presence of thebaine for 24 hr, efficiently converted thebaine into northebaine (-77% conversion). Similar experiments carried out with northebaine and isomeric thebaine N-oxides as substrates, revealed that while northebaine and the stable thebaine N-oxide are proved to be resistant to further transformation, the unstable thebaine N-oxide nonenzymatically rearranges to 6, 7, 8, 9, 10, 14-hexadehydro-4, 5-epoxy-3. 6-dimethoxy-17-methylthebinan and 6, 7, 8,9, 10, l4-hexadehydro-3. 6-dimethoxythebinan-4-01. These studies support the idea that N-demethylation of thebaine is hot proceeding via the N-oxide intermediate.

The first microbial transformation within the class of morphine alkaloids was reported by Iizuka et al. 1

•2

, on the conversion of thebaine 1 into 14-hydroxycodei­none and 14-hydroxycodeine using spore suspension of Trametes sanguinea. The fungus, Trametes cinnabarina was also shown to have similar activities, except that 14-hydroxycodeinone N-oxide was the additional transformation product formed 3. When the l4-position was blocked by an etheno-bridge as in the case of its Diels-Alder adduct, the microbial system transformed it into its norcompound in very poor yield4

. It was also demonstrated that some of the microorganisms have the ability to carry out N­demethylation of codeines-7 but not morphine and thebaine. However, we have reported in a preliminary communication the ability of Mucor piriformis to carry out N-demethylation of thebaine and its N­variants8

. Earlier it has been documented that morphine, codeine and thebaine undergo microbial­facilitated C-14 hydroxylation 1-3.7.9.10. More recently, a strain of Pseudomonas putida was isolated by enrichment culture technique using morphine as the sole source of carbon and energy' I . This organism was also shown to utilize codeine, but not thebaine as the primary carbon source. These studies have clearly demonstrated that Pseudomonas putida contains a novel NADP+-dependant dehydrogenase, which converts morphine and codeine into morphinone and codeinone, respectively .

The present paper describes an efficient transformation of thebaine into northebaine by the fungus, Mucor piriformis. The paper also provides evidence for the formation of isomeric thebaine N-oxides as minor metabolites. In fact , the conversion of thebaine into its isomeric N-oxides has not been documented before in a microbial system.

Materials and Methods Thebaine 1 was procured from the Government

Opium and Alkaloid Works, Ghazipur, India. Northebaine 2 was prepared from thebaine 1 using diethylazodicarboxylate reagent following the reported procedure '2. Isomeric thebaine N-oxides were synthesised by treatment with metachloroperbenzoic acid as described previously13 and the isomers were purified by column chromatography using 10% methanol in chloroform. 'H NMR and mass spectral analyses of the isomeric thebaine N-oxides were in full agre~ment with literature data13

.14

.

TLC analysis was performed on silica gel G plates (0.-5 mm thick) developed with either chloroform­methanol (85:15, vol/vol , system 1) or benzene­isopropanol (80:20, vol/vol, system 2) as solvent systems. The compounds were visualized by exposure to iodine vapours. HPLC analysis was carried out using Water Associates ALC/GPC 244 instrument. The analysis was performed on a J.l-Porasil silica

378 INDIAN 1. CHEM., SEC. B, MAY 2000

column using chloroform-methanol (80:20 voUvol) containing 0.3% NH40H as the solvent system. Flow rate was maintained at 0.5 mL/min and the e1uents were monitored with an UV detector at 254 nm. The metabolites formed were quantified on the basis of the area under each peak.

NMR ('H and '3C) spectra, mass spectra and infrared IR spectra were obtained as described previously's.

Culture conditions. The organism used in the present study was maintained either on potato dextrose agar (PDA) slants or modified Czapek-Dox'6 agar slants, stored at 4°C and subcultured periodically. Fermentations were carried out in modified Czapek-Dox medium. The pH of the medium was maintained between 6.0-6.3.

Biotransformation of thebaine 1. Erlenmeyer flasks (500 mL) containing 100 mL of sterile modified Czapek-Dox medium'6 and 1 mM thebaine (in 0.5 mL of acetone-methanol; I: I vol/vol) were inoculated with I mL of spore suspension of Mucor piriformis (2.7xlOs spores countlmL) from a 5··days old culture. The flasks were incubated at 29-30°C on a rotary shaker (220 rpm) for 4 days. A control experiment was also run with the substrate but · without organism. At the end of the incubation period, the contents from all the flasks were pooled, basified to pH 8.0-9.0 with IN NH40H and filtered through cheese cloth. The mycelia and the filtrate were separately extracted with chloroform-methanol (2: I , vol/vol) and the two extracts were mixed. The total extract was dried over sodium sulphate, evaporated to dryness in vacuo and the residue was subjected to column chromatography to isolate metabolites.

Time course studies . In the time course experiment the transformation of thebaine was carried out as described above. At the end of every 24 hr an aliquot (5 mL) was taken aseptically, extracted and the residue was subjected to HPLC analysis to estimate the amount of thebaine metabolised.

Transformation by resting cells. The organism was grown for 24 hr in the presence of 1 mM thebaine as already described . The mycelia were filtered and washed well with 0.5% saline, distilled water and phosphate buffer (50 mM, pH 7.2) successively. The cell suspension in the same buffer (2 g, wet weight in 50 mL) was incubated wi th thebaine ( 10 mg) and glucose (J 00 mg) at 30°C 01'1 a rotary shaker for 24 hr. Control experiments were carried out without the substrate and the organism. After the incubation period, the contents of the flasks were extracted as

described earlier. The organic phase was subjected to HPLC analysis to monitor the transformation. Transformation of northebaine as well as isomeric thebaine N-oxides by resting cells was also carried out in a similar way.

Results A fermentation time course revealed that most of

the thebaine added was transformed by 96 hr and hence, incubation was terminated.

A batch of 30 flasks was inoculated with Mucor piriformis and at the end of incubation, the contents were pooled and processed as described under Materials and Methods . The broth extract (1.26 g), upon examination by TLC (system I) , revealed the presence of one major (Rr 0.5) and five minor metabolites (Rr 0.21, 0.27, 0.77, 0.87 and 0 .9). The crude broth extract (1.26 g) was subjected to column chromatography on silica gel (30 g) and the metabolites with Rr 0.9 and 0 .87 eluted with chloroform. This fraction was further subjected to repeated preparative TLC (5% methanol in chloroform) to separate metabolites with Rr 0 .9 (12 mg) and Rr 0.87 (84 mg). Eluting the column with 3% methanol in chloroform yielded the major metabolite (Rr 0.5, system I) along with a minor metabolite (Rr 0 .77, system I) . The major metabolite (624 mg) was separated from the minor metabolite (36 mg) by preparative TLC (system 1). The minor metabolite (Rr 0.77) had IR, NMR and MS data which corresponded well with an authentic sample of thebaine 1. Subsequently, the column was eluted with 10-15% methanol in chloroform to obtain the metabolites with Rr 0.27 (30 mg) and Rr 0 .21 (12 mg). All the purified metabolites were subjected to various spectral analyses for characterisation. Compound with Rr 0.5: Recrystallization of this compound (from chloroform-methanol) gave pale yellow crystals, m.p . J58-59°C, [a]o23 -200 (c 0. 1, 5% methanol in chloroform); IR (Nujol/cm-'): 3400 (-NH); 'H NMR (90 MHz, COCl3): 06.68 (lH, d, 7.2 Hz, H-I), 6.58 (IH, d, 7.2 Hz, H-2), 5.48 (lH, d, 7.2 Hz, H-8), 5.26 (IH, s, H-5) , 5.02 (lH, d, 7.2 Hz, H-7), 3.86 (3H, s, H3CO-3), 3.6 (3H, s, H3CO-6), '3C NMR (100 MHz, CDCI3+ DMSO-d6):o 153.49 (C6), 144.41 (C4),

142.67 (C3), 131.72 (C'2), 125.28 (C'4) , 124.68 (C II ),

119.64 (C,), 115.98 (C2), 113 .72 (Cs), 95.19 (C7),

87.25 (Cs), 56.03 (3-0CH3), 54.90 (C9), 52.38 (6-OCH3), 44.5 (C 13), 36.8 (C,o), 34.22 (C ,s), 33.30 (C'6); MS (70 eV): m/z 297 (M+, 100%), 282 (45%), 266 (22%), 253 (19%); 239 (19%), 223 (11%), 211

MADYASTHA etal.: N-DIMETHYLATION & N-OXIDATION OF THEBAINE BY MUCOR PIRIFORMIS 379

(13%); HRMS: Found: M+ 297.1406. C ISHI9NO) requires M+ 297.1365. On the basis of spectral analyses, the compound was identified as northebaine 2. The IH NMR spectral data of compound 2 corresponded well to that reported for northebaine l2.

Metabolite with Rr 0.21: IH NMR (200 MHz, CDCh): () 6.66 (1 H, d, 8Hz, H-l), 6.56 (I H, d, 8Hz, H-2), 5.76 (IH, d, 6Hz, H-8), 5.35 (lH, s, H-5), 5.04 (1H, d, 6Hz, H-7), 3.81 (3H, s, H)CO-3), 3.55 (3H, s, H)CO-6), 3.39 (3H, s, N-CH); MS (70 ev): rnJz 327 (M+, 10%), 311 (M+ -0, 100%), 294 (36%), 279 (18%),254 (91 %),239 (80%), 225 (14%), 211 (26%), 196 (13%); HRMS: Found: 327.1495. CI9H21N04 requires M+ 327.1471. On the basis of spectral analyses, the compound was identified as thebaine N­oxide 3 with an axial oxygen atom attached to the nitrogen atom. The IH NMR and MS data for this compound were in agreement with the literature J3

values .

Metabolite with Rr 0.27: Since this compound was unstable, its spectral analyses were carried out immediately following its isolation. IH NMR (200 MHz, CDCl): () 6.66 (2H, qAB, H-l and H-2), 5.86 (1H, d, 6Hz, H-8), 5.34 (lH, s, H-5), 5.08 (IH, d, 6Hz, H-7), 3.83 (3H, s, N-CH). The NMR data were in agreement with that reported in the literature l3 for thebaine N-oxide 4 with an equatorial oxygen attached to nitrogen. The data also corresponded well to thebaine N-oxide 4 prepared chemically.

Metabolite with Rr 0.87: mp 172-73°C (from CHCl)-MeOH); IH NMR (90 MHz, CDCl): () 6.56 (2H, qAB, H-I and H-2), 6.34 (IH, d, 1O.8Hz, H-IO), 6.18 (1H, d, 10.8 Hz, H-9), 5.64 (lH, d, 7.2 Hz, H-8), 5.12 (IH, d, 7.2 Hz, H-7), 3.84 (3H, s, H)CO-3), 3.7 (3H, s, H)CO-6), 2.98 (3H, s, N-CH) ,; J3C NMR (100 MHz, CDCh): () 156.5 (C6), 146.2 (C4), 141.5 (C), 135.1 (C I4), 129.9 (CJ2), 125.0 (C IO), 124.7 (C II ), 124.5 (C9), 117.6 (Cs), 117.1 (C I), 112.1 (C2), 109.5 (C5), 95.7 (C7), 58.9 (C J3), 56.1 (3-0CH), 55.2 (6-OCH3), 49.0 (C I6), 40.7 (C I5), 36.3 (N-CH3) MS: rnJz 309 (M+, 100%), 294 (63%), 279 (33%), 266 (27%), 251 (20%), 235 (l6%), 222 (II %); HRMS: Found: 309.1357. C I9H I9NO) requires M+ 309.1365. Based on spectral analyses, the compound was identified as 6, 7, 8, 9, 10, 14-hexadehydro-4, 5-epoxy-3, 6-dimethoxy-17-methylthebinan 5. The spectral data matched well with that reported earlier for this compound l4.

Metabolite with Rr 0.9: IH NMR (90 MHz, CDCh): () 6.72 (IH, d, 7.2 Hz, H-l), 6.56 (lH, d,

7.2Hz, H-2), 6.32 (IH, d, 7.2 Hz, H-IO), 6.16 (1H, d, 7.2Hz, H-9), 5.2 (IH, d, 7.2Hz, H-8), 5.28 (lH, d, 7.2 Hz H-7), 4.72 (lH, s, H-5), 3.88 (3H, s, H)CO-3), 3.72 (3H, s, H)CO-6). The spectral data corresponded well with that of 6, 7, 8, 9, 10, 14-hexadehydro-3, 6-dimethoxythebinan-4-o 1 6 isolated and characterised earlierl4 .

Transformation with resting cells. Incubation of thebaine 1 with resting cells indicated that 77% of thebaine was transformed into northebaine 2. Amounts of thebaine N-oxides formed were insignificant «I %). A similar experiment carried out using northebaine 2 as substrate indicated that northebaine was not further transformed. When thebaine N-oxides 3 and 4 were independently incubated with resting cells, it was observed that the stable N-oxide 3 was resistant to further transformation; the unstable N-oxide 4 was transformed into compounds 5 and 6. Control experiments without cells showed the formation of compounds 5 and 6 from unstable N-oxide 4, suggesting that the transformation is non-enzymatic (Chart 1). Both N-oxides, upon incubation with Mucor piriformis, did not yield northebaine 2, suggesting that N-oxides are not intermediates in the conversion of thebaine 1 to northebaine 2.

Discussion Present studies have demonstrated that Mucor

piriformis very efficiently carries out N-demethylation of thebaine 1. It is significant that the northebaine 2 formed is not further metabolised. This particular feature of Mucor piriformis can be of great utility in organic synthesis. One of the crucial chemical steps in the multi-step synthesis of morphine agonists and antagonists is the N-demethylation reaction. It is interesting to note that thebaine 1 is the starting material used for synthesis of these ~harmaceutically and therapeutically important drugs l . A commercial seven step synthesis of norox.ymorphone commencing from the alkaloid thebaine 1 also involves N­demethlyation step IS. Since chemical N-demethylation reactions require harsh reaction conditions as well as

19 . b' I expensive, toxic and hazardous reagents , micro la method could offer a promising approach. Although microorganisms have been successfully utilised in critical synthetic steps of some pharmaceutically important steroids2o, they have not been applied to a comparable degree in preparation of non-steroidal substances like alkaloids.

The present investigation has also demonstrated

380 INDIAN J CHEM, SEC B, MAY 2000

1

Major pathway )

~co

2 Minor pathway

3, 4

Non enzymatic

1

5 6

Chart I-Transformations of thelaine by Mucor piriformis

that Mucor piriformis converts thebaine (1) into its

isomeric N-oxides 3 and 4 as minor metabolites (Chart I). The N-oxide with an equatorial oxygen attached to nitrogen (Str. 4) is unstable and rearranges to compounds 5 and 6 (Chart I) . It was suggested earlier that the striking difference in stability of two thebaine N-oxides is most likely due to the steric orientation of the oxygen atom attached to nitrogen 14.

The characterisation of isomeric thebaine N-oxides 3 and 4 and compounds 5 and 6 is consistent with earlier reports on the i ~olation of these compounds from the higher plant Papaver bracteatum l4

. Since both northebaine 2 and thebaine N-oxides 3 and 4 were isolated from the fermentation broth, intermediacy of N-oxide was suspected in N­demethlyation reaction . However, resting cells

experiments carried out using 3 and 4 clearly support the idea that N-demethylation is unlikely to occur via N-oxide intermediate. Although some of the earlier reports indicate the possible involvement of N-oxide as an intermediate in the N-demethylation reaction, it is now widely accepted that this reaction proceeds via a transient N-hydroxymethyl intermed iate5

.

Acknowledgement One of us (GYBR) is thankful to lISc, Bangalore

for the award of a research fellowship. Financial assistance from CSIR, New Delhi and JNCASR, Bangalore is gratefully acknowledged.

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MADYASTHA et 01.: TRANSFORMATION OF THEBAINE INTO NORTHEBAI E BY MUCOR PIRIFORMIS 381

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