JP4580980B2 - コルヒチノイド化合物のバイオトランスフォーメーション - Google Patents
コルヒチノイド化合物のバイオトランスフォーメーション Download PDFInfo
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- 230000036983 biotransformation Effects 0.000 title claims abstract description 24
- 150000001875 compounds Chemical class 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 43
- 230000008569 process Effects 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract 2
- 238000000855 fermentation Methods 0.000 claims description 19
- 230000004151 fermentation Effects 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 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 13
- 229910052799 carbon Inorganic materials 0.000 claims description 13
- 239000008103 glucose Substances 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 11
- 108010080698 Peptones Proteins 0.000 claims description 11
- 235000019319 peptone Nutrition 0.000 claims description 11
- 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 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 8
- 230000013595 glycosylation Effects 0.000 claims description 7
- 238000006206 glycosylation reaction Methods 0.000 claims description 7
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- JRRUSQGIRBEMRN-HNNXBMFYSA-N n-[(7s)-3-hydroxy-1,2,10-trimethoxy-9-oxo-6,7-dihydro-5h-benzo[a]heptalen-7-yl]acetamide Chemical compound O=C1C(OC)=CC=C2C3=C(OC)C(OC)=C(O)C=C3CC[C@H](NC(C)=O)C2=C1 JRRUSQGIRBEMRN-HNNXBMFYSA-N 0.000 claims description 6
- 241000194107 Bacillus megaterium Species 0.000 claims description 5
- 229930091371 Fructose Natural products 0.000 claims description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 5
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- 125000002252 acyl group Chemical group 0.000 claims description 2
- 238000010924 continuous production Methods 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
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- PKYOHQGXPPVIGD-HNNXBMFYSA-N n-[(7s)-3-hydroxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-6,7-dihydro-5h-benzo[a]heptalen-7-yl]acetamide Chemical compound O=C1C(SC)=CC=C2C3=C(OC)C(OC)=C(O)C=C3CC[C@H](NC(C)=O)C2=C1 PKYOHQGXPPVIGD-HNNXBMFYSA-N 0.000 claims 2
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000002609 medium Substances 0.000 description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 16
- 239000000758 substrate Substances 0.000 description 15
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- CMEGANPVAXDBPL-INIZCTEOSA-N n-[(7s)-1,2,3-trimethoxy-10-methylsulfanyl-9-oxo-6,7-dihydro-5h-benzo[a]heptalen-7-yl]acetamide Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(SC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC CMEGANPVAXDBPL-INIZCTEOSA-N 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 239000012137 tryptone Substances 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 229960001338 colchicine Drugs 0.000 description 6
- 235000011187 glycerol Nutrition 0.000 description 6
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- 108010076119 Caseins Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
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- 229920002472 Starch Polymers 0.000 description 3
- 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 3
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- 230000004913 activation Effects 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
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- 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 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 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 2
- 240000008042 Zea mays Species 0.000 description 2
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- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
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- 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
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- SWWQQSDRUYSMAR-UHFFFAOYSA-N 1-[(4-hydroxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol;hydrochloride Chemical group Cl.C1=CC(O)=CC=C1CC1C2=CC(O)=C(O)C=C2CCN1 SWWQQSDRUYSMAR-UHFFFAOYSA-N 0.000 description 1
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- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- -1 K + Chemical class 0.000 description 1
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- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- LEQAKWQJCITZNK-AXHKHJLKSA-N N-[(7S)-1,2-dimethoxy-10-(methylthio)-9-oxo-3-[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6,7-dihydro-5H-benzo[a]heptalen-7-yl]acetamide Chemical compound C1([C@@H](NC(C)=O)CCC2=C3)=CC(=O)C(SC)=CC=C1C2=C(OC)C(OC)=C3O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O LEQAKWQJCITZNK-AXHKHJLKSA-N 0.000 description 1
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- UXTMROKLAAOEQO-UHFFFAOYSA-N chloroform;ethanol Chemical compound CCO.ClC(Cl)Cl UXTMROKLAAOEQO-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Pain & Pain Management (AREA)
- Physical Education & Sports Medicine (AREA)
- Transplantation (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Neurology (AREA)
- Dermatology (AREA)
- Rheumatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
の3−O−グリコシルコルヒチノイド化合物の製造方法であって、バチルス・メガテリウム(Bacillus megaterium)を用いて、R1がOH又はメトキシである化合物をバイオトランスフォーメーションすることを含み、脱メチル化コルヒチノイドを使用してグリコシル化酵素系を誘導することを特徴とする方法を提供する。
バイオトランスフォーメーションの最初の14〜18時間、好ましくは15〜16時間において、変換されるべきコルヒチノイド基質のアリコート(300〜800mg/l、好ましくは500〜600mg/l)を、開始時及び1〜3時間毎、好ましくは1.5〜2.5時間毎に添加し、
各基質供給時に、以下の原料:
a)最終濃度が2〜4g/l、好ましくは2.5〜3.5g/lのペプトン、トリプトン、又はカゼイン加水分解物(硫酸アンモニウム1〜3m/l、好ましくは1.5〜2.5g/lも組み合わせて)、
b)最終濃度が5〜15g/l、好ましくは8〜12g/lのブドウ糖又は果糖
を含有する溶液を添加する。
バチルス・メガテリウムの冷凍培地のアリコートを使用して、3−O−デメチルチオコルヒチンを添加して最終濃度0.4g/lとした培養液SF2(表)250m/lを収容する1000m/lの三角フラスコ内に、種培養(即ち、前培養)を播種した。上記培地をロータリーシェーカー上で、30℃にて250rpmで一夜インキュベーションした。インキュベーション後、前培養500m/lを、チオコルヒチンを添加して最終濃度0.5g/lとした新鮮な培養液SF2(表1参照)9.5lを収容する14lの発酵槽内に滅菌下で移した。撹拌−エアレーションの適切なレベルを保持して(撹拌は900rpm以下、培地の増殖に応じてエアレーション1〜1.8vvm)、発酵を30℃で行った。発酵の最初の14時間の間、2時間毎にチオコルヒチン(最終濃度0.5g/l)、ペプトン(2g/l)、硫酸アンモニウム(2g/l)及びブドウ糖(10g/l)を培養に添加した。各添加前(即ち、2時間毎)に、培養ブロスからのサンプルを採取して、以下の分析を行った。
600nmにおける光学密度(OD)としての増殖レベル、
LB寒天培地上の株の無菌性及び純度、
顕微鏡形態(Gram株)、
TLC及びHPLCによるチオコルチコシド含有量の分析。
HPLC分析による測定でチオコルチコシド約52gを含有していた発酵の最終培養ブロス(総体積:約10l)を、0.22μmのセラミックカートリッジ上でクロスフロー精密濾過して、ブロスから細胞を分離した。XAD1180(Rohm and Haas)吸収樹脂で充填したカラム上に、浸透液を吸収させた。水で洗浄した後、生成物をメタノールで溶出した。メタノール溶出物を真空下で濃縮させ乾燥した後、メタノールに再溶解した。塩化メチレンで抽出した後、アルコール画分を濃縮させ乾燥し、エタノール−塩化メチレンの1:1混合物に再溶解した。シリカゲルによる清澄化後、溶液を真空下で濃縮し、次いで塩化メチレンをエタノールと交換した。得られた懸濁液を濃縮し、放置して結晶化させた。固体をエタノール−クロロホルム混合物中に更に再溶解して、シリカゲル上で清澄化する工程の後、エタノールによる第二の結晶化を行った。精製後、総量49.9gの生成物が得られ、精製収率は96%、純度は99.5%であった。
発酵の開始時に、最終濃度4g/lのチオコルヒチン全部を一回で添加して、実施例1に記載した手順を繰り返した。その結果、増殖は非常に乏しく、インキュベーションの数時間後に事実上停止した。顕微鏡分析により、明らかな細胞溶解を検出した。TLC及びHPLC分析では、有意なバイオトランスフォーメーションは全く示されなかった。
予備的な種培養中に3−O−デメチルチオコルヒチンを全く添加せずに、実施例1に記載した手順を繰り返した(酵素誘導なし)。バイオトランスフォーメーションは、実施例1と比較してより遅く、28時間後に停止し、チオコルチコシドの最終変換収率は61%、総生産性は3.3g/l、比生産性は0.118g/l時間であった。
発酵の開始時に、最終濃度1g/lのチオコルヒチン全部を一回で添加して、WO98/15642に記載されている発酵培地STを使用して、実施例4に記載した手順を繰り返した。バイオトランスフォーメーションは、実施例1と比較してより遅く、28時間後に停止し、チオコルチコシドの最終変換収率は90%、総生産性は1.22g/l、比生産性は0.044g/l時間であった。
培地の処方
1)LB寒天培地(滅菌:121℃×20’)−pH7
トリプトン 10g/l
酵母エキス 5g/l
NaCl 10g/l
寒天(Agar Agar) 15g/l
2)ブロスSF2(滅菌:121℃×20’)−pH7
ブドウ糖 40g/l
ペプトン 20g/l
酵母エキス 5g/l
NaCl 3g/l
(NH4)2SO4 3g/l
K2HPO4 8g/l
KH2PO4 3g/l
MgSO4.7H2O 0.5g/l
Claims (9)
- 式(I)
の3−O−グリコシルコルヒチノイド化合物の製造方法であって、バチルス・メガテリウム(Bacillus megaterium)を用いて、R1がメトキシである式(I)の化合物をバイオトランスフォーメーションすることを含み、R 1 がメトキシである式(I)の化合物を窒素源及び炭素源と組み合わせて、多数回に分割して、グリコシル化酵素系を誘導するために使用される3−O−デメチルコルヒチン(DMC)又は3−O−デメチルチオコルヒチン(DMTC)を含有する予備的な種培養中に供給することを特徴とする方法。 - 3−O−デメチルコルヒチン又は3−O−デメチルチオコルヒチンが、200〜600mg/lの範囲の量で添加される、請求項1記載の方法。
- 窒素源及び炭素源が、各々、ペプトン及びブドウ糖から選択される、請求項1又は2記載の方法。
- 発酵バッチに添加されるR 1 がメトキシである式(I)の化合物全体が、2〜4g/lである、請求項1〜3のいずれか一項記載の方法。
- 生成物の回収が、吸収樹脂を用いて行われる、請求項1〜4のいずれか一項記載の方法。
- 生成物を含有する最終発酵ブロスの主部分(75%〜90%)を回収し、新しい発酵培養液を生物反応器内の該ブロスの残留部分に添加し、新しいバッチを開始することによって、半連続工程にて実施される、請求項1〜5のいずれか一項記載の方法。
- 3−O−デメチルコルヒチン又は3−O−デメチルチオコルヒチンを種培養中に予備的に添加する工程と、
バイオトランスフォーメーションの最初の14〜18時間において、変換されるべきR 1 がメトキシである式(I)の化合物のアリコート300〜800mg/lを、開始時及び1〜3時間毎に添加する工程と、
R 1 がメトキシである式(I)の化合物の各供給時に、以下の原料
a)最終濃度が2〜4g/lのペプトン、トリプトン、又はカゼイン加水分解物と、硫酸アンモニウム1〜3g/lとの組み合わせ、
b)最終濃度が5〜15g/lのブドウ糖又は果糖、
を含有する溶液を添加する工程と、
を含む、請求項1〜6のいずれか一項記載の方法。 - バイオトランスフォーメーションが、25〜35℃にてpH4〜8で実施される、請求項1〜7のいずれか一項に記載の方法。
- 培地1lにつき、1分間で空気1〜2リットル(vvm)のエアレーションレベルが使用される、請求項1〜8のいずれか一項記載の方法。
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US20110178180A1 (en) * | 2010-01-18 | 2011-07-21 | Kurt Nielsen | Deuterium-enriched colchicine, thiocolchicine, and derivatives thereof; methods of preparation; and use thereof |
EP2619315A2 (en) * | 2010-09-22 | 2013-07-31 | Elysian Life Sciences Private Limited | A microbial method for the biotransformation of colchicinoid compounds |
WO2015097567A1 (en) | 2013-12-23 | 2015-07-02 | Alkaloids Corporation | Process for the conversion of colchicinoids to their 3-glycosylated derivatives via their respective 3-demethyl analogues |
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DK1745140T3 (da) | 2009-12-07 |
IL179141A (en) | 2010-12-30 |
EP1745140A1 (en) | 2007-01-24 |
CA2566437A1 (en) | 2005-11-17 |
PL1745140T3 (pl) | 2010-01-29 |
AU2004319347B2 (en) | 2011-02-10 |
US7910334B2 (en) | 2011-03-22 |
CA2566437C (en) | 2013-01-08 |
AU2004319347A1 (en) | 2005-11-17 |
NO20065157L (no) | 2006-11-09 |
HK1103304A1 (en) | 2007-12-14 |
WO2005108595A1 (en) | 2005-11-17 |
KR101116424B1 (ko) | 2012-03-07 |
US20090011479A1 (en) | 2009-01-08 |
KR20070015196A (ko) | 2007-02-01 |
PT1745140E (pt) | 2009-09-30 |
JP2007536916A (ja) | 2007-12-20 |
SI1745140T1 (sl) | 2009-12-31 |
DE602004022874D1 (de) | 2009-10-08 |
ES2329132T3 (es) | 2009-11-23 |
IL179141A0 (en) | 2007-03-08 |
ATE440958T1 (de) | 2009-09-15 |
NO336051B1 (no) | 2015-04-27 |
CN1954081B (zh) | 2012-07-18 |
EP1745140B1 (en) | 2009-08-26 |
CN1954081A (zh) | 2007-04-25 |
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