AU2011203283B2 - Microbial N- and O-demethylation of a thebaine derivative - Google Patents
Microbial N- and O-demethylation of a thebaine derivative Download PDFInfo
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- 238000010520 demethylation reaction Methods 0.000 title claims abstract description 33
- FQXXSQDCDRQNQE-VMDGZTHMSA-N thebaine Chemical class C([C@@H](N(CC1)C)C2=CC=C3OC)C4=CC=C(OC)C5=C4[C@@]21[C@H]3O5 FQXXSQDCDRQNQE-VMDGZTHMSA-N 0.000 title abstract description 8
- 230000000813 microbial effect Effects 0.000 title description 4
- 238000000034 method Methods 0.000 claims abstract description 37
- 241001290628 Cunninghamella echinulata Species 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 19
- 230000004151 fermentation Effects 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 239000011942 biocatalyst Substances 0.000 claims abstract description 8
- 241000235555 Cunninghamella Species 0.000 claims abstract description 6
- 241000293023 Cunninghamella polymorpha Species 0.000 claims abstract description 6
- 241000235546 Rhizopus stolonifer Species 0.000 claims abstract description 5
- 241000233866 Fungi Species 0.000 claims abstract description 4
- 150000001875 compounds Chemical class 0.000 claims description 27
- RMRJXGBAOAMLHD-IHFGGWKQSA-N buprenorphine Chemical compound C([C@]12[C@H]3OC=4C(O)=CC=C(C2=4)C[C@@H]2[C@]11CC[C@]3([C@H](C1)[C@](C)(O)C(C)(C)C)OC)CN2CC1CC1 RMRJXGBAOAMLHD-IHFGGWKQSA-N 0.000 claims description 11
- 229960001736 buprenorphine Drugs 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 230000017858 demethylation Effects 0.000 claims description 7
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 125000004186 cyclopropylmethyl group Chemical group [H]C([H])(*)C1([H])C([H])([H])C1([H])[H] 0.000 claims description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 4
- 238000007126 N-alkylation reaction Methods 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- -1 cyclopropylmethyl halide Chemical class 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000002425 crystallisation Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 230000029936 alkylation Effects 0.000 claims 3
- 238000005804 alkylation reaction Methods 0.000 claims 3
- 240000008042 Zea mays Species 0.000 claims 1
- 239000012067 demethylated product Substances 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 description 11
- 230000036983 biotransformation Effects 0.000 description 10
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 230000002538 fungal effect Effects 0.000 description 6
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- 239000005720 sucrose Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000037361 pathway Effects 0.000 description 5
- 229930013930 alkaloid Natural products 0.000 description 4
- 150000003797 alkaloid derivatives Chemical class 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- FQXXSQDCDRQNQE-UHFFFAOYSA-N markiertes Thebain Natural products COC1=CC=C2C(N(CC3)C)CC4=CC=C(OC)C5=C4C23C1O5 FQXXSQDCDRQNQE-UHFFFAOYSA-N 0.000 description 3
- 229930003945 thebaine Natural products 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000209149 Zea Species 0.000 description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006900 dealkylation reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- VQJMAIZOEPPELO-KYGIZGOZSA-N (1S,2S,6R,14R,15R,16R)-5-(cyclopropylmethyl)-16-(2-hydroxy-5-methylhexan-2-yl)-15-methoxy-13-oxa-5-azahexacyclo[13.2.2.12,8.01,6.02,14.012,20]icosa-8(20),9,11-trien-11-ol hydrochloride Chemical compound Cl.CO[C@]12CC[C@@]3(C[C@@H]1C(C)(O)CCC(C)C)[C@H]1Cc4ccc(O)c5O[C@@H]2[C@]3(CCN1CC1CC1)c45 VQJMAIZOEPPELO-KYGIZGOZSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 241000523836 Cladophora dalmatica Species 0.000 description 1
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000020335 dealkylation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a process for the N-demethylation or the N- and 0-demethylation of a thebaine derivative which process comprises fermenting the derivative with a biocatalyst selected from the 5 filamentous fungi from Cunninghamella dalmatica NRRL 1394, Cunninghamella echinulata NRRL 1387, Cunninghamella echinulata NRRL 1384, Cunninghamella echinulata 0 ATCC 36190, Cunninghamella echinulata ATCC II 585a, Cunninghamella echinulata ATCC 9244, Cunninghamella polymorpha NRRL 1395, or Rhizopus nigricans Z5/lin a basal fermentation medium at a temperature of at least 250C and not more than 340C, for a period of time of at least 3 days.
Description
Regulation 3.2 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: Reckitt Benckiser Healthcare (UK) Limited Actual Inventors: John Alfred Davis Andrew John Carell Address for Service: C/- MADDERNS, GPO Box 2752, Adelaide, South Australia, Australia Invention title: MICROBIAL N- AND O-DEMETHYLATION OF A THEBAINE DERIVATIVE The following statement is a full description of this invention, including the best method of performing it known to us.
la MICROBIAL N- AND O-DEMETHYLATION OF A THEBAINE DERIVATIVE The present invention relates to the synthesis of intermediate compounds which are useful in the synthesis of 5 the alkaloid buprenorphine and, in particular, the synthesis of useful intermediates by the selective microbial N- and 0 demethylation of 7a-[(S)-1-hydroxy-1,2,2-trimethylpropyll 6,14-endo-ethano-6,7,8,14-tetrahydrothebaine. 10 The alkaloid buprenorphine, (chemical name 21-cyclopropyl 7a-[(S)-1-hydroxy-1,2,2,-trimethylpropyl] -6,14-endo-ethano 6,7,8,14-tetrahydrooripavine), is a commercially available mixed agonist/antagonist of pt receptors and can be synthesized relatively efficiently from the alkaloid 15 thebaine. However, two steps in the synthesis involve the N- and 0-dealkylation of thebaine derivatives and require the use of expensive and difficult to handle reagents, or lead to poor yields. 20 Accordingly, a target in improving the synthesis of buprenorphine from thebaine is an improved process for carrying out the dealkylation steps. We have now discovered that certain filamentous fungi 25 exhibit regiospecific N- and 0-demethylation of a thebaine intermediate currently used in the synthesis of buprenorphine. Accordingly, the present invention provides a process for 30 the N-demethylation or the N- and 0-demethylation of the compound of the formula 2
CH
3 0 0
NCH
3
CH
3 0 3 CH HONA
C(CH
3
)
3 (I) which process comprises fermenting the said compound of Formula I with a biocatalyst selected from the filamentous 5 fungi Cunninghamella dalmatica NRRL 1394, Cunninghamella echinulata NRRL 1387, Cunninghamella echinulata NRRL 1384, Cunninghamella echinulata ATCC 36190, 10 Cunninghamella echinulata ATCC 11585a, Cunninghamella echinulata ATCC 9244, Cunninghamella polymorpha NRRL 1395, or Rhizopus nigricans Z5/1 in a basal fermentation medium at a temperature of at least 15 250C, preferably at least 27*C, most preferably at least 280C and not more than 340C, preferably not more than 33 0 C and most preferably not more than 320C for a period of time of at least 3 days, preferably at least 4 days. 20 The preferred filamentous fungi for use in the present invention is Cunninghamel1a echinulata NRRL 1384. The fermentation process is preferably carried out for a period of time of from 7 to 10 days and the fermentation medium is preferably vigorously shaken or stirred in order to assist 3 the biotransformation process. The fermentation preferably takes place at a pH in the range of from 5 to 6. The compounds which are produced by the N-demethylation or 5 N- and 0-demethylation process of the present invention are as follows: N- and 0-demethylation 10 H 0 NH
CH
3 0 H" CH 3
C(CH
3
)
3 (II) 7a-[(S)-1-hydroxy-1,2,2-trimethylpropyll-6,14-endo ethano-6,7,8,14-tetrahydronororipavine 4 N-demethylation
CH
3 0 0 NH
CH
3 0 --CH3 HON\
C(CH
3
)
3 (III) 7a-[(S)-1-hydroxy-1,2,2-trimethylpropyll-6,14 5 endo-ethano-6,7,8,14-tetrahydronorthebaine These products which are produced in the biotransformation process of the present invention may be isolated by procedures known in the art for example by chromatography, 10 crystallisation or extraction procedures. The compound of formula II may be directly converted to buprenorphine by N-alkylation, for example with a cyclopropylmethylhalide, in order to introduce a 15 cyclopropylmethyl group onto the N-atom. The compound of formula III may be converted to buprenorphine in two stages. The first stage comprising an N-alkylation, for example with a cyclopropylmethyl halide, 20 in order to introduce a cyclopropylmethyl group onto the N atom, and the second stage comprising the O-demethylation of the intermediate compound IV produced from the first stage by techniques well known in the art, for example using potassium hydroxide in diethylene glycol at an elevated 5 temperature in the range of 200 to 245 0 C. These reaction schemes are shown in Scheme 1 below: z 0 0 0 ~0 0 CCr I E 00 z 0 oc Ift-I 0 0 7 The present invention also includes within its scope a process for the preparation of buprenorphine from the compound of formula I, which process includes the step of the N-demethylation or the N- and 0-demethylation of a 5 compound of the formula
CH
3 0 0
NCH
3 CH30 H -CH3 HOX
C(CH
3
)
3 (I) which process comprises fermenting the said compound of 10 Formula I with a biocatalyst selected from the filamentous fungi Cunninghamella dalmatica NRRL 1394, Cunninghamella echinulata NRRL 1387, Cunninghamella echinulata NRRL 1384, 15 Cunninghamella echinulata ATCC 36190, Cunninghamel1a echinulata ATCC 11585a, Cunninghamella echinulata ATCC 9244, Cunninghamella polymorpha NRRL 1395, or Rhizopus nigricans ZS/1 20 in a basal fermentation medium at a temperature of at least 25'C, preferably at least 27"C, most preferably at least 28'C and not more than 34*C, preferably not more than 330C and most preferably not more than 32 0 C for a period of time of at least 3 days, preferably at least 4 days.
8 The conversion of the N-demethylated or the N- and 0 demethylated compounds of Formula II and Formula III as described above may be carried out by the processes as 5 described above. The present invention will be further described with reference to the following Examples. 10 Examples 1 to 8 A total of 38 fungal type strains, 6 unknown fungal isolates from environmental samples and 2 strains of S. cerevisciae were tested for their ability to demethylate the compounds 15 of formula I described above. Each organism was scored according to the number and intensity of the spots observed on thin layer chromatograms. Eight candidate strains were selected for further testing. 20 The compound of Formula I was subjected to biotransformation using each of the eight fungal strains under standard conditions as follows: Standard biotransformation procedure: All organisms were 25 grown in liquid fermentation media (50 ml) at 25'C for 7-10 days with vigorous shaking (250 rpm) in a New Brunswick Scientific orbital incubator. The fungal fermentation medium consisted of 2%(w/v) glucose, 0.5%(w/v) Corn Steep Solids, pH6.2. After all biotransformations were complete 30 the media were sampled (1 ml) and extracted with an equal volume of ethyl acetate. The extent of alkaloid demethylation was assessed by thin layer chromatography 9 (TLC) in a solvent system comprising ethyl acetate::triethylamine (19::1) and developed with a CAN dip. Quantitative analysis of putative positive results was carried out by HPLC (Waters 2690 separations module, 996 5 photo-diode array). Samples were prepared as above, dried under a stream of N 2 gas and re-dissolved n a methanol based mobile phase (methanol, 600 ml; ammonium acetate, lg; distilled water, 160 ml; 0.1M acetic acid, 1ml). Reverse phase HPLC was performed on an ODS-A column (250mm x 4.6mm, 10 YMC Co. Ltd., Japan). Sample size injected was 20 pl. Flow rate and operating pressure was 0.6 ml and c.1900 psi, respectively. Detection was 288 nm. The results are given in Table 1 below. 15 Table 1 Example Organism Biotransformation No. products II(%) III(%) 1 C. dalmatica NRRL 1394 1.7 16.4 2 C. echinulata NRRL 1387 1.9 23.6 3 C. echinulata NRRL 1384 6.3 39.4 4 C. echinculata ATCC 36190 15.8 19.2 5 C. echinculata ATCC 11585a 8.8 19.2 6 C. echinculata ATCC 9244 10.2 21.7 7 C. polymorpha NRRL 1395 10.4 31.5 8 R. nigracans Z5/1 - 10.6 10 Example 9 Each Cunninghamella type strain (see Table 2) was grown in a basal fermentation medium consisting of a single defined 5 carbon source (glucose, sucrose, galactose or maltose) and a relatively undefined mixture of complex carbohydrates, amino acids and vitamins as contained in corn steep liquor. Biotransformations of compound I were carried out at 28 0 C or 32 0 C for 7 days and the results are summarised in Table 2. 10 Of the four strains tested, C. echinulata NRRL 1384 is a significantly better biocatalyst than the others with respect to both N-demethylation of compound I and N- and 0 didemethylation of this same substrate. There is no 15 significant difference between the levels of N-demethylation at either temperature, the mean conversions to product III being 55% ± 6.3% at 28 0 C and 47% ± 6.5% at 32 0 C, respectively. However, didemethylation, to produce the product II, shows marked temperature dependence. At 28 0 C the 20 maximal formation of product II is approximately 20%. In contrast, at the elevated temperature of 32 0 C formation of this compound can be inhibited by up to 53%, although changing the carbon source from glucose to galactose, maltose or sucrose can partially suppress this temperature 25 dependence. The other biocatalysts, C. echinulata ATCC 36190, C. echinulata NRRL 1387 and C.echinulata NRRL 1395, show no temperature dependence with respect to their ability to 30 demethylate compound I. Although the N-demethylation pathways leading to the formation of product III are more active than the didemethylation pathways in these organisms, 11 they are still at least 50% less active than the equivalent pathways in C. echinulata NRRL 1384. Table 2 5 280C 280C 32"C 320C STRAIN SUGAR %II %III %II %III ATCC36190 Glucose 11.2 ± 31.7 ± 11.6 ± 37.1 ± 2.9 6.2 2.0 7.7 Sucrose 14.5 ± 33.6 i 11.7 ± 36.2 ± 0.8 7.4 0.5 3.0 Galactose 10.91 35.74 12.4 ± 37.5 ± 0.9 3.7 Maltose 13.2 ± 35.3 i 15.1 ± 46.2 ± 0.3 0.5 1.9 1.4 NRRL1384 Glucose 20.5 ± 58.6 ± 9.5 ± 3.0 41.3 ± 1.8 1.6 8.3 Sucrose 20.2 ± 53.9 t 12.1 ± 42.4 ± 1.2 7.3 1.3 1.4 Galactose 21.6 ± 61.7 ± 16.4 ± 55.0 ± 1.1 7.2 1.5 3.6 Maltose 15.6 ± 47.2 + 13.2 ± 50.0 ± 1.1 0.3 1.7 5.6 NRRL1387 Glucose 6.5 0.9 24.6 + 5.4 +/- 23.1 +/ 5.4 0.3 0.6 Sucrose 3.8 ± 2.4 13.5 ± 4.2 +/- 21.1 +/ 12.1 1.6 3.5 Galactose 3.7 ± 0.4 11.6 + 6.8 +/- 34.3 +/ 1_ _ 3.3 0.4 3.7 Maltose 3.4 ± 0.4 12.1 + 3.7 +/- 14.4 +/ 7.8 2.7 11.1 NRRL1395 Glucose 6.9 ± 1.0 27.5 ± 5.6 ± 0.3 28.3 ± 3.6 0.6 Sucrose 6.6 ± 0.2 25.5 ± 5.5 ± 0.1 24.5 ± 0.3 0.6 Galactose 5.6 ± 0.6 26.8 ± 6.2 ± 0.6 31.2 ± 3.4 0.8 Maltose 8.0 ± 1.2 29.3 ± 6.5 ± 0.3 28.3 ± 2.5 1 2.2 # n = 1 (n = 3 unless otherwise stated) 12 Example 10 The effect of pH on C. echinulata NRRL 1384 mediated 5 demethylation was assessed in GCM medium at 28 0 C. This medium was found to promote both N-demethylation and N- and 0-didemethylation (data not shown) and this temperature was chosen in order for us to evaluate the effect of pH on both demethylation pathways without biasing the outcome of the 10 biotransformation due to the temperature dependence previously identified. The data are summarised in Table 3. The optimum pH for promoting didemethylation is between pH5.0 and pH6.0 with the maximal formation of product II being 24.6% ± 6.3% at pH5.0. Above pH6.0 the efficiency of 15 the process falls rapidly, whilst at more acidic pH's fungal growth, and hence biotransformation efficiency of the organism, is affected in an unpredictable way. This latter feature of the biotransformation process correlates with the observation that at pH3.0 fungal growth is completely 20 inhibited. N-demethylation of compound I operates over a broader pH range. The maximum formation of product III of 56.9% ± 14.2% is observed at pH5.0. However, mean product III 25 levels of 44.4% and 50.3% at pH's 4.0 and 6.0 indicate that this pathway is robust and that N-demethylation of compound I is the biologically most favourable process in this organism.
13 Table III Initial pH %Conversion III II pH3.0 (n = 3) No growth No growth pH4.0 16.0 ± 7.2 44.4 i 3.8 pH5.0 24.6 ± 6.3 56.9 ± 14.2 pH6.0 21.0 ± 5.3 50.3 8.9 pH7.0 (n = 3) 11.6 ± 5.7 40.8 ± 11.0 N = 6 unless otherwise stated 5
Claims (13)
1. A process for the N-demethylation or the N- and 0 demethylation of the compound of the formula 5 CH 3 0 0 NCH 3 CH 3 0 -~CH 3 HON C(CH 3 ) 3 (I) which process comprises fermenting the said compound of 10 Formula I with a biocatalyst selected from the filamentous fungi from Cunninghamella dalmatica NRRL 1394, Cunninghamella echinulata NRRL 1387, Cunninghamella echinulata NRRL 1384, 15 Cunninghamella echinulata ATCC 36190, Cunninghamella echinulata ATCC 11585a, Cunninghamella echinulata ATCC 9244, Cunninghamella polymorpha NRRL 1395, or Rhizopus nigricans Z5/1 20 in a basal fermentation medium at a temperature of at least 250C and not more than 34'C, for a period of time of at least 3 days. 15
2. A process as claimed in claim 1 wherein the biocatalyst is Cunninghamella echinulata NRRL 1384.
3. A process as claimed in claim 1 or claim 2 wherein the 5 fermentation is carried out for a period of from 7 to 10 days.
4. A process as claimed in any one of the preceding claims wherein the fermentation is carried out with vigorous 10 shaking or stirring of the fermentation medium.
5. A process as claimed in any one of the preceding claims wherein the fermentation medium comprises 2% w/v glucose, 0.5% w/v Corn Steep solids and has a pH of 6.2. 15
6. A process as claimed in any one of the preceding claims wherein the N- and/or N- and O-demethylated products are separated from the fermentation medium by chromatography, crystallisation or extraction procedures. 20
7. A process as claimed in any one of claims 1 to 4 or 6 wherein the fermentation is carried out at a pH in the range of from 5 to 6. 25
8. A process for the preparation of buprenorphine, which process includes the step of the N-demethylation or the N and 0-demethylation of a compound of the formula 16 CH 3 0 0 NCH 3 CH 3 0 % ,CH 3 C(CH 3 ) 3 (I) 5 which process comprises fermenting the said compound of Formula I with a biocatalyst selected from the filamentous fungi Cunninghamella dalmatica NRRL 1394, Cunninghamella echinulata NRRL 1387, 10 Cunninghamella echinulata NRRL 1384, Cunninghamella echinulata ATCC 36190, Cunninghamella echinulata ATCC 11585a, Cunninghamella echinulata ATCC 9244, Cunninghamella polymorpha NRRL 1395, or 15 Rhizopus nigricans Z5/1 in a basal fermentation medium at a temperature of at least 25 0 C and not more than 340C for a period of time of at least 3 days. 20
9. A process as claimed in claim 8 wherein the compound produced by the N- and O-demethylation step is a compound having the following formula 17 H 0 NH CH 3 0 -CH3 HOA C(CH 3 ) 3 (II) which is isolated from the fermentation medium and converted 5 to buprenorphine by N-alkylation in order to introduce a cyclopropylmethyl group onto the N-atom.
10. A process as claimed in claim 9 wherein the N alkylation is carried out using a cyclopropylmethyl halide. 10
11. A process as claimed in claim 8 wherein the compound produced by the N-demethylation step is a compound having the following formula CH 3 0 0 NH CH 3 O - -CH 3 HO-A C(CH 3 )3 (III) 15 18 which is isolated from the fermentation medium and converted to buprenorphine by the following reaction stages: (a) subjecting the compound of formula (III) to N alkylation in order to introduce a cyclopropylmethyl 5 group into the N-atom; and (b) subjecting the compound produced in step (a) to 0 demethylation in order to produce buprenorphine.
12. A process as claimed in claim 11 wherein the N 10 alkylation is carried out using a cyclopropylmethyl halide.
13. A process as claimed in claim 11 wherein the 0 demethylation is carried out with potassium hydroxide in diethylene glycol at an elevated temperature of from 15 200 to 2450C.
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| GB0210638.3 | 2002-05-10 | ||
| AU2009251172A AU2009251172A1 (en) | 2002-05-10 | 2009-12-23 | Microbial N- and O-demethylation of a thebaine derivative |
| AU2011203283A AU2011203283B2 (en) | 2002-05-10 | 2011-07-04 | Microbial N- and O-demethylation of a thebaine derivative |
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| WO1997044317A2 (en) * | 1996-05-21 | 1997-11-27 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Novel methods of o-demethylation and n-deprotection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1997044317A2 (en) * | 1996-05-21 | 1997-11-27 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Novel methods of o-demethylation and n-deprotection |
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