CN1382805A - Biotransfer process for preparing artemisine compounds - Google Patents

Biotransfer process for preparing artemisine compounds Download PDF

Info

Publication number
CN1382805A
CN1382805A CN 02112750 CN02112750A CN1382805A CN 1382805 A CN1382805 A CN 1382805A CN 02112750 CN02112750 CN 02112750 CN 02112750 A CN02112750 A CN 02112750A CN 1382805 A CN1382805 A CN 1382805A
Authority
CN
China
Prior art keywords
alpha
hydroxy
artemisinin
artemethers
och
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN 02112750
Other languages
Chinese (zh)
Other versions
CN1189568C (en
Inventor
余伯阳
陈有根
吴志峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Pharmaceutical University
Original Assignee
China Pharmaceutical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by China Pharmaceutical University filed Critical China Pharmaceutical University
Priority to CNB021127506A priority Critical patent/CN1189568C/en
Publication of CN1382805A publication Critical patent/CN1382805A/en
Application granted granted Critical
Publication of CN1189568C publication Critical patent/CN1189568C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Landscapes

  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

A hydroxylation process for biotransferring the artemisine, hydroartemisine, or artemsic methylether, with griseous streptomyces microbe (ATCC 13273) is disclosed. 12 hydroxylated products are separated from the fermented liquid, which include 3 ones for artemisine, 4 ones for dihydroartemisine and 5 ones for artemic methylether, among them, 10 ones are new compounds contg. mother kernels.

Description

Biotransfer process for preparing artemisine compounds
Technical field
The present invention relates to Biotransfer process for preparing artemisine compounds, is to utilize known microorganisms that Artemisinin and derivative thereof are carried out the method that bio-transformation prepares artemisine compounds specifically.
Background technology
Microbial transformation is to utilize the specific enzymes of microorganisms to finish specific biochemical reaction.Microorganism can produce plurality of enzymes, the multiple reaction of catalysis.For example, the microbial transformation reaction type has: oxidation, reduction, hydrolysis, replace, slough, condensation and scission reaction etc.If can utilize single enzyme, make the specific reaction of its single-minded ground catalysis, just can reduce by product, improve the production efficiency of purpose product.So, if the purpose product that utilizes the microbial transformation preparation to have practicality, at first must screen and produce the bacterial classification of needed reaction at the specific substrate of certain class, simultaneously, in order to improve the production efficiency of object, reduce by product, also need to study the condition of bio-transformation, the composition of pH, concentration of substrate, substratum during as reaction.
At present, external having utilizes bio-transformation to prepare artemisine compounds.For example: persons such as Lee I.-S carry out bio-transformation with bacterial strain Nacardia corallina (ATCC 19070) and Penicilium chrysogenum (ATCC 9480) to Artemisinin, obtain deoxidation Artemisinin (deoxyartmisinin) and 3 Alpha-hydroxy deoxidation Artemisinins (3 α-hydrodeoxyartemisinin), referring to: the microbial transformation research of antimalarial drug sesquiterpenes southernwood element, natural product magazine (Jounal of Natural Products) 1989,52 (2) 337-341; Abourashed, E.A. and Hufford, C.D. select for use bacterial strain Cunninghamella elegans (ATCC 9275) that Artemether is carried out bio-transformation, obtain 3 Alpha-hydroxy Artemethers (3 α-hydroxyartemether), 3 beta-hydroxy Artemethers (5 products such as 3 β-hydroxyartemether), referring to: the bio-transformation of Artemether, natural product magazine (Jounal of Natural Products) 1996,59 (3) 251-253.
Light grey streptomycete ATCC13273 is at three United States Patent (USP)s 5,674,714,5,213,971,5,169, in 755, all is used for inducing cell pigment P-450 enzyme, do not retrieve other purposes.
Summary of the invention
The technical problem to be solved in the present invention is the bioconversion method of research preparation artemisine compounds, especially utilize the bioconversion method preparation to have the bioconversion method of the hydroxylation artemisine compounds of physiologically active, this need select to produce artemisine compounds the bacterial classification of hydroxylation reaction.
In order to improve the production efficiency of object, reduce by product, the present invention also needs to study the bio-transformation condition of artemisine compounds being carried out hydroxylation reaction, as the composition of pH, concentration of substrate, reaction times and substratum when reacting, especially need on the basis of conventional substratum, screen suitable carbon source, nitrogenous source and metal ion.
Technical scheme of the present invention is:
Preparation has the method for the artemisine compounds of following general formula, it is characterized in that: utilize preserving number to be the light grey streptomyces strain microorganism of ATCC-13273 or the varient and the mutant of its functional equivalent, Artemisinin, dihydroarteannuin or Artemether are carried out bio-transformation, obtain following artemisine compounds
Figure A0211275000061
1) 3 Alpha-hydroxy Artemisinins, R 1=OH, R 2=R 3=H, R 4+ R 5==O;
2) 3 beta-hydroxy Artemisinins, R 1=R 3=H, R 2=OH, R 4+ R 5==O;
3) Artemisinin ketone-3, R 1+ R 2==O, R 4+ R 5==O, R 3=H
4) 3 Alpha-hydroxy dihydroarteannuins, R 1=R 4=OH, R 2=R 3=R 5=H;
5) 3 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 5=OH, R 2=R 3=R 4=H;
6) 10 Alpha-hydroxy dihydroarteannuins, R 1=R 2=R 5=H, R 3=R 4=OH;
7) 10 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 2=R 5=H, R 3=R 5=OH;
8) 3 α, 3 beta-dihydroxyl Artemethers, R 1=R 2=OH, R 3=R 5=H; R 4=-OCH 3
9) 3 β, 10 alpha-dihydroxy-Artemethers, R 1=R 5=H, R 2=R 3=OH, R 4=-OCH 3
10) 3 α, 10 alpha-dihydroxy-Artemethers, R 1=R 3=OH, R 2=R 5=H, R 4=-OCH 3
11) 3 Alpha-hydroxy Artemethers, R 1=OH, R 2=R 5=R 3=H, R 4=-OCH 3
12) 3 beta-hydroxy Artemethers, R 1=R 5=R 3=H, R 2=OH, R 4=-OCH 3
The above-mentioned method for preparing artemisine compounds, substratum are mainly formed except that 20% potato decoction liquor, KH 2PO 4, MgSO 47H 2O, trace V B 1Outward, it is characterized in that: carbon source is glucose, starch, sucrose, maltose, semi-lactosi, dextrin, Semen Maydis powder and/or glycerine, nitrogenous source is peptone, yeast extract paste, Semen Maydis powder, bean powder, urea element, volatile salt, SODIUMNITRATE, L-Methionin, DL-α-An Jibingsuan, and metal ion is K +Or Co 2+, Mg 2+, Cu 2+, Mn 2+, Zn 2+
The described method for preparing artemisine compounds, substratum are mainly formed except that 20% potato decoction liquor, KH 2PO 4, MgSO 47H 2O, VB 1Outside the trace, carbon source is selected glucose, starch, dextrin, Semen Maydis powder and/or glycerine, and nitrogenous source is selected yeast extract paste, Semen Maydis powder and/or DL-α-An Jibingsuan, Metal Ion Selective Electrode Mn 2+Better.
The described method for preparing artemisine compounds, substratum are mainly by 20% potato decoction liquor 1000ml, VB 1Trace, metal ion 0.1-0.3mmol/L and following raw material (by weight) are formed:
Carbon source 8-12 part: nitrogenous source 1-5 part: KH 2PO 41-3 part: MgSO 47H 21 part of O.
The described method for preparing artemisine compounds, substratum is mainly by 20% potato decoction liquor 1000ml, VB preferably 1Trace, metal ion 0.1-0.3mmol/L and following raw material (by weight) are formed:
Carbon source 10-11 part: nitrogenous source 3-3.5 part: KH 2PO 42 parts: MgSO 47H 21 part of O.
The described method for preparing artemisine compounds is characterized in that: bio-transformation is to carry out under pH5.0～7.5 conditions; The concentration of Artemisinin, dihydroarteannuin and/or Artemether is 0.2～1mg/mL; The bio-transformation time is 36～108 hours.
The comparatively ideal method for preparing artemisine compounds is characterized in that: bio-transformation is to carry out under the pH6.0 condition, and the concentration of Artemisinin, dihydroarteannuin and/or Artemether is 0.4-0.6mg/mL; The bio-transformation time is 80～90 hours.
The described method for preparing artemisine compounds comprises, utilizes preserving number to be the light grey streptomyces strain microorganism of ATCC-13273 or the varient and the mutant of its functional equivalent, is being 6.0, is containing Mn as carbon source or nitrogenous source, pH with Semen Maydis powder respectively 2+Substratum in, cultivated 84 hours, obtain 3 Alpha-hydroxy Artemisinins.
The method for preparing artemisine compounds also comprises: utilize preserving number to be the light grey streptomyces strain microorganism of ATCC-13273 or the varient and the mutant of its functional equivalent, be 6.0, containing Mn as carbon source or nitrogenous source, pH with Semen Maydis powder respectively 2+Substratum in, cultivated 84 hours, obtain 3 beta-hydroxy Artemisinins.
The present invention also comprises having immunocompetent artemisine compounds, it is characterized in that having following general formula:
Figure A0211275000081
1) 3 Alpha-hydroxy Artemisinins, R 1=OH, R 2=R 3=H, R 4+ R 5==O;
2) 3 beta-hydroxy Artemisinins, R 1=R 3=H, R 2=OH, R 4+ R 5==O;
3) Artemisinin ketone-3, R 1+ R 2==O, R 4+ R 5==O, R 3=H
4) 3 Alpha-hydroxy dihydroarteannuins, R 1=R 4=OH, R 2=R 3=R 5=H;
5) 3 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 5=OH, R 2=R 3=R 4=H;
6) 10 Alpha-hydroxy dihydroarteannuins, R 1=R 2=R 5=H, R 3=R 4=OH;
7) 10 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 2=R 5=H, R 3=R 5=OH;
8) 3 α, 3 beta-dihydroxyl Artemethers, R 1=R 2=OH, R 3=R 5=H; R 4=-OCH 3
9) 3 β, 10 alpha-dihydroxy-Artemethers, R 1=R 5=H, R 2=R 3=OH, R 4=-OCH 3
10) 3 α, 10 alpha-dihydroxy-Artemethers, R 1=R 3=OH, R 2=R 5=H, R 4=-OCH 3
With microorganism S.griseus Artemisinin, dihydroarteannuin and Artemether have been carried out bio-transformation in the aforesaid method, from fermented liquid, be divided into 12 converted products, wherein 3 is that the converted product, 4 of Artemisinin is the converted product of Artemether for the converted product of dihydroarteannuin, 5.With IR, SCI-MS, ESI-MS, FAB-MS, HREI-MS, 1H-NMR, 13C-NMR, DEPT, HMBC, 1H- 1Multiple wave spectrum means such as H COSY have been carried out the structure evaluation, have identified 12 altogether, and wherein 10 is new compound.
Specific implementation method
Embodiment 1
The screening of tool activity of conversion microbial strains
The present invention has carried out transformation and selection to 14 bacterial classifications (strain), and the result shows: Streptomyces griseus (ATCC-13273) all has stronger conversion capability to Artemisinin, dihydroarteannuin and Artemether.
The used liquid nutrient medium of bacterial screening-bean powder dextrose culture-medium: get glucose 20g, yeast extract 5g, bean powder 5g, NaCl 5g, K2HPO4 5g, distilled water 1000mL is with the HCl accent pH to 7.0 of 6N.Be sub-packed in the 150ml triangular flask, every bottle of 25ml, 121 ℃, 0.15Mpa 20min that sterilizes.
During bacterial screening, preserve the used inclined-plane of bacterial classification solid medium and be add 1.5% agar in the aforesaid liquid substratum, after the heating for dissolving, be sub-packed in the test tube of 15mL tool screw-cap, behind 121 ℃, 0.15Mpa sterilization 20min, tilting cooling forms.
Bacterial classification is preserved and activation
Bacterial classification inoculation is cultivated 3d for 28 ℃ on the inclined-plane solid medium, be stored in then in 4 ℃ the refrigerator.
Actication of culture adopts two step activation methods, referring to: Nair, M.S.R., and Basile, D.V.Bioconversionof artenium B to artmisinn. natural product magazine (Jounal of Natural Prodacts) 1993,56 (9): 1559.Elder generation in liquid nutrient medium, behind 28 ℃ of 200r/min rotating and culturing 48h, transfers bacterial classification inoculation in another liquid nutrient medium, cultivates 24h with condition.
Application of sample and sampling
Every bottle two step activatory bacterium liquid (25ml) adds the Artemisinin solution 100 μ L that make 150mg/mL with the DMF dissolving, makes every mL bacterium liquid contain Artemisinin 0.6mg.Behind condition cultivation 84h, fermented liquid is taken out, add the equivalent ethyl acetate extraction 3 times, reclaim ethyl acetate after merging, the residue small amount of acetone is dissolved for thin-layer chromatography and is differentiated usefulness.
Blank: every bottle two step activatory bacterium liquid adds DMF 100 μ L, behind condition cultivation 84h, fermented liquid is taken out, and adds the equivalent ethyl acetate extraction 3 times, and ethyl acetate is reclaimed in the merging back, and the residue small amount of acetone is dissolved for the thin-layer chromatography discriminating and used.
The thin-layer chromatography of converted product is differentiated
Sample and blank solution after transforming are respectively put on the silica gel g thin-layer plate made from 0.3%CMC-Na, sherwood oil-acetone (3: 1) ascending method is launched, and after solvent is dried in taking-up, sprays the Vanillin-vitriol oil with 5%, conversion results is inspected in hair dryer heating colour developing.
Conversion results sees Table 1
Table .1. is to the screening of Artemisinin tool activity of conversion microbial strains
Organism Metabolie?production
Amycolatopsis?orientalis?subsp.orientalis(ATCC-19795) -
Aspergillus?niger(CGMCC-3.4463) -
Beauveria?sp.(CGMCC-3.3574) -
Caldariomyces?fumago(ATCC-16373) -
Candida?rugosa(ATCC-14830) -
Corynebacterium?mediolarrum(ATCC-14004) -
Cylindrocapon?radicicola(ATCC-11011) +
Gliocbadium?deliquescens(NRRL-1086) -
Nocardia?sp.(NRRL-5646) -
Peeudomonas?putida(ATCC-33015) -
Penicillium?notatum(ATCC-36740) -
Polyporus?versicolor(ATCC-12679) -
Rhizopus?sp(NRRL-1478) +
Streptomyces?griseus(ATCC-13273) ++
ATCC: American type culture collection, NRRL:Northern Regional
Research Laboratories, CGMCC: China Microbial Culture Preservation Commission is common
The microorganism center
From 14 bacterial classifications being screened as can be seen, only have 3 bacterial classifications that sweet wormwood is have the catalyzed conversion effect, wherein the conversion capability with S.griseus is the strongest.After this, with S.griseus dihydroarteannuin and Artemether have been carried out transformation experiment again, the result shows that this bacterial strain also has stronger transformation to dihydroarteannuin and Artemether.Therefore, the present invention selects the bacterial strain that this bacterial strain transforms as preparative-scale.
Embodiment 2
The bio-transformation of artemisine compounds
Experiment material and instrument
This experiment of microorganism microorganism used therefor is that light grey streptomycete Streptomyces griseus preserving number is ATCC-13273, and is at United States Patent (USP) 5,674, open in 714,5,213,971,5,169,755.
Reagent and reagent Artemisinin are pressed the literature method preparation, referring to: Chen Yougen, Yu Baiyang etc., the extraction separation of Artemisinin and precursor compound thereof and evaluation, herbal medicine, 2001; 32 (4): 302-303, and consistent through its structure of spectroscopic identification with bibliographical information; Dihydroarteannuin is available from Beijing the 6th pharmaceutical factory; Artemether is available from Kunming Medicine Stock Co., Ltd.This three compound is made the solution that concentration is 240mg/mL with acetone solution respectively, shakes up, and is standby as conversion of substrate.
Substratum 20% potato decoction liquor 6000ml, Semen Maydis powder 120g, MnSO 41.50g, VB 16g (6N NaOH or HCl transfer pH=6.0).Be sub-packed in 24 1000ml triangular flasks (every bottle of 200ml) and 24 150ml triangular flasks (every bottle of 25ml), 121 ℃, the 0.15Mpa 20min that sterilizes.
Instrument WL-1 type micro melting point apparatus (not proofreading and correct); NICOLET Impact 410 infrared spectrometers; VarianXL-500, XL-400 and BRUKER ACF-300 nuclear magnetic resonance analyser; Finnigan FTMS-2000 mass spectrograph (70eV).
The microbial transformation of Artemisinin, dihydroarteannuin and Artemether can directly be carried out, and also can bacterial classification be activated or re-activation by above-mentioned literature method.
Actication of culture: actication of culture adopts two step activation methods, earlier with bacterial classification inoculation in the triangular flask (interior dress 25ml liquid nutrient medium) of 8 150ml, behind 28 ℃ of 200r/min vibration rotating and culturing 48h, transfer respectively in the triangular flask (interior dress 200ml liquid nutrient medium) of other 8 1000ml, cultivate 24h with condition.
Application of sample and sampling: every bottle two step activatory bacterium liquid (200ml) adds the Artemisinin solution 0.5mL that makes 240mg/mL with acetone solution.Behind condition cultivation 84h, fermented liquid is taken out, filter the back and merge, get filtrate 1.6L, with ethyl acetate extraction 3 times (3.2L, 1.6L * 2), combining extraction liquid is used Na 2SO 4After the dehydration, 55 ℃ of reclaim under reduced pressure ethyl acetate of water-bath, residue minimum of chloroform stripping, transformation mixture (I) 0.840g of Artemisinin, standby.Same quadrat method makes transformation mixture (III) 0.935g of dihydroarteannuin (II) 0.967g and Artemether.
The separation of converted product: said mixture I, II and III are respectively with after the minimum of chloroform dissolving, add the 2g silica gel mixed sample, on the chloroform wet method on the sample in the silicagel column that 80g post layer silica gel (200-300 order) is housed, with chloroform-methanol 20: 1-5: 1 gradient elution, obtain 1-3 by I, II obtains 4-7, III obtains 8-12 totally 12 compounds, and all compounds are all used the chloroform recrystallization.
The new compound 1. 3 Alpha-hydroxy Artemisinins that transform to obtain from Artemisinin (3 α-hydroxylartemisinin): R 1=OH, R 2=R 3=H,
R 4+ R 5==O; 2. 3 beta-hydroxy Artemisinins (3 β-hydroxylartemisinin): R 1=R 3=H, R 2=OH,
R 4+ R 5==O; 3. Artemisinin ketone-3 (artemistone-3): R 1+ R 2==O, R 4+ R 5==O, and the new compound 4. 3 Alpha-hydroxy dihydroarteannuins that R3=H obtains from the Dihydroartemisinin conversion (3 α-hydroxy-dihydroartemisinin): R 1=R 4=OH, R 2=R 3=R 5=H; 5. 3 Alpha-hydroxies-α-dihydroarteannuin (3 α-hydroxy-α-dihydroartemisinin): R 1=R 5=OH, R 2=R 3=R 4=H; 6. 10 Alpha-hydroxy dihydroarteannuins (10 α-hydroxy-dihydroartemisinin): R 1=R 2=R 5=H, R 3=R 4=OH; 7. 10 Alpha-hydroxies-α-dihydroarteannuin (10 α-hydroxy-α-dihydroartemisinin): R 1=R 2=R 5=H, R 3=R 5=OH;
Transform new compound 8. 3 α that obtain from Artemether, and 3 beta-dihydroxyl Artemethers (3 α, 3 β-dihydroxyartemether): R 1=R 2=OH,
R 3=R 5=H; R 4=-OCH 39. 3 β, and 10 alpha-dihydroxy-Artemethers (3 β, 10 α-dihydroxyartemether): R 1=R 5=H,
R 2=R 3=OH, R 4=-OCH 310. 3 α, and 10 alpha-dihydroxy-Artemethers (3 α, 10 α-dihydroxyartemether): R 1=R 3=OH,
R 2＝R 5＝H，R 4＝-OCH 3
Transform the known compound 11. 3 Alpha-hydroxy Artemethers that obtain, R from Artemether 1=OH, R 2=R 5=R 3=H, R 4=-OCH 312. 3 beta-hydroxy Artemethers, R 1=R 5=R 3=H, R 2=OH, R 4=-OCH 3
10 All new compounds are identified and spectral data in the above-mentioned converted product:
1,3 Alpha-hydroxy Artemisinins: white needle (CHCl 3); Mp, 161-162 ℃; IR (KBr) V Max3523,1720,1114,1010,989,880,832 (cm -1); 1H-NMR (400MHz, CDCl 3): δ 5.83 (1H, s, H-7), 3.81 (1H, td, J=2.4,3.2Hz, H-3), 3.38 (1H, q, J=7.2Hz, H-11), 2.36 (1H, m, H-5), 2.43 (1H, td, J=4.0,12.8Hz, H-9b), 2.03 (2H, m, H-9a, 4a), 1.89 (2H, m, H-1,2b), 1.54 (1H, br, HO-3), 1.50 (1H, m, H-2), 1.47 (1H, m, H-10a), 1.43 (3H, s, H-15), 1.28 (1H, td, J=2.4,14.4Hz, H-4b), 1.18 (3H, d, J=7.2, H-13), 1.06 (3H, d, J=6.4, H-14); 13C-NMR (400MHz, CDCl 3): δ 172.6 (C-12), 105.5 (C-8), 93.4 (C-7), 79.2 (C-6), 69.1 (C-3), 43.4 (C-1), 41.3 (C-2), 37.6 (C-5), 36.0 (C-9), 32.3 (C-11), 30.7 (C-4), 25.2 (C-15), 24.6 (C-10), 15.8 (C-14), 12.5 (C-13); CIMS m/z 299[M ++ 1] (37), 265 (9), 253 (43), 239 (100), 235 (39), 221 (64), 207 (52); HREIMS[M +] 298.1421 (calc.for C 15H 22O 6, 298.1416).
2,3 beta-hydroxy Artemisinins: white needle (CHCl 3); Mp, 158-159 ℃; IR (KBr) V Max3469,1740,1113,1032,998,882,831 (cm -1); 1H-NMR (400MHz, CDCl 3): δ 5.90 (1H, s, H-7), 3.39 (1H, q, J=6.0Hz, H-11), 3.30 (1H, td, J=3.2,4.0Hz, H-3), 2.47 (1H, td, J=3.6,4.0, H-9a), 2.13 (1H, m, H-4a), 2.09 (2H, m, H-9b, 5), 1.93 (1H, m, H-10a), 1.64 (1H, br, HO-3), 1.53 (2H, m, H-1,9b), 1.47 (3H, s, H-15), 1.41 (1H, m, H-2), 1.23 (1H, d, J=26.8Hz, H-14), 1.19 (1H, m, H-4b), 1.11 (3H, d, J=5.2, H-13); 13C-NMR (400MHz, CDCl 3): δ 171.5 (C-12), 105.4 (C-8), 93.2 (C-7), 78.8 (C-6), 73.5 (C-3), 48.0 (C-1), 44.5 (C-2), 42.2 (C-5), 35.8 (C-9), 32.5 (C-4), 32.2 (C-11), 25.2 (C-15), 24.8 (C-10), 15.5 (C-14), 12.6 (C-13); CIMS m/z 299[M ++ 1] (54), 253 (19), 239 (66), 235 (25), 221 (7), 207 (100); HREIMS[M +] 298.1421 (calc.for C 15H 22O 6, 298.1416).
3, artemisia ketone-3: white needle (CHCl 3); Mp, 164-165 ℃; IR (KBr) V Max, 1739,1714cm -1 1H NMR (400MHz, CDCl 3): δ 6.17 (1H, s, H-7), 3.41 (1H, dt, J=4.8,7.2Hz, H-5), 2.60 (1H, m, H-10b), 2.50 (1H, m, H-9b), 2.20 (1H, m, H-10a), 2.19 (1H, m, H-2), 2.15 (1H, m, H-9a), 2.07 (1H, d, J=4.8Hz, H-4a), 1.84 (1H, m, H-11), 1.82 (1H, m, H-1), 1.66 (1H, d, J=5.2Hz, H-4b), 1.48 (3H, s, H-15), 1.18 (3H, d, J=7.2Hz, H-13), 1.13 (3H, d, J=6.4Hz, H-14); 13C NMR (400MHz, CDCl 3): δ 205.8 (C-3), 170.7 (C-12), 105.8 (C-8), 92.8 (C-7), 77.9 (C-6), 49.5 (C-11), 48.6 (C-1), 43.7 (C-2), 38.3 (C-4), 35.8 (C-9), 33.0 (C-5), 26.1 (C-10), 25.1 (C-15), 12.4 (C-14), 11.9 (C-13); CIMS m/z 297[M ++ 1] (9), 233 (34), 222 (28), 206 (63), 91 (67), 55 (60), 43 (100); HREIMS m/z 296.1359[M +] (calcd for C 15H 20O 6296.1260).
4,3 Alpha-hydroxy dihydroarteannuins: white needle (CHCl 3); M.p.134-136 ℃; IR (KBr) V Max(cm -1): 3500 (OH), 1100,1038,880,827; 1H-NMR (400MHz, CDCl 3): δ 5.64 (1H, s, H-7), 5.26 (1H, d, J=3.2, H-12), 2.60 (3H, s, H-16), 1.41 (3H, s, H-15), 1.05 (3H, d, J=6.2, H-14), 0.95 (3H, d, J=7.2, H-13); 13C-NMR (400MHz, CDCl 3): δ 104.4 (C-8), 94.5 (C-12), 90.9 (C-7), 80.2 (C-6), 74.2 (C-3), 50.0 (C-1), 44.4 (C-2), 43.0 (C-5), 36.2 (C-9), 34.4 (C-4), 31.1 (C-11), 26.2 (C-15), 24.7 (C-10), 16.2 (C-14), 13.1 (C-13); FAB-MS m/z 323[M ++ Na +] (18), 277 (19), 251 (22), 207 (18), 173 (17), 159 (30), 137 (50), 115 (100).
5,3 Alpha-hydroxies-α-dihydroarteannuin: white needle (CHCl 3); M.p.134-136 ℃; IR (KBr) V Max(cm -1): 3500 (OH), 1100,1038,880,827; 1H-NMR (400MHz, CDCl 3): δ 5.45 (1H, s, H-7), 4.74 (1H, d, J=8.8, H-12), 2.32 (3H, s, H-16), 1.42 (3H, s, H-15), 1.06 (3H, d, J=6.2, H-14), 0.94 (3H, d, J=7.2, H-13); 13C-NMR (400MHz, CDCl 3): δ 104.2 (C-8), 96.3 (C-12), 87.4 (C-7), 79.6 (C-6), 73.9 (C-3), 9.0.0 (C-1), 44.1 (C-2), 41.8 (C-5), 36.1 (C-9), 33.7 (C-4), 30.5 (C-11), 25.9 (C-15), 24.6 (C-10), 15.4 (C-14), 12.7 (C-13); FAB-MS m/z 323[M ++ Na +] (18), 277 (19), 251 (22), 207 (18), 173 (17), 159 (30), 137 (50), 115 (100).
6,10 Alpha-hydroxy dihydroarteannuins: white needle (CHCl 3); M.p.130-132 ℃; IR (KBr) V Max(cm -1): 3389 br (OH), 1092,1027,859,826; 1H-NMR (400MHz, CDCl 3): δ 5.53 (1H, s, H-7), 5.25 (1H, d, J=2.8, H-12), 2.61 (3H, s, H-16), 1.41 (3H, s, H-15), 1.02 (3H, d, J=6.4, H-14), 0.93 (3H, d, J=7.2, H-13); 13C-NMR (400MHz, CDCl 3): δ 102.7 (C-8), 94.8 (C-12), 90.8 (C-7), 80.4 (C-6), 70.0 (C-10), 59.8 (C-1), 46.4 (C-9), 44.6 (C-5), 37.1 (C-3), 35.5 (C-2), 34.8 (C-11), 24.5 (C-4), 25.8 (C-15), 21.3 (C-14), 13.1 (C-13); FAB-MS m/z 323[M ++ Na +] (12), 277 (38), 251 (8), 207 (12), 173 (22), 159 (26), 137 (38), 115 (100).
7,10 Alpha-hydroxies-α-dihydroarteannuin: white needle (CHCl3); M.p.130-132 ℃; IR (KBr) V Max(cm -1): 3389 br (OH), 1092,1027,859,826; 1H-NMR (400MHz, CDCl 3): δ 5.32 (1H, s, H-7), 4.73 (1H, d, J=9.2, H-12), 2.29 (3H, s, H-16), 1.42 (3H, s, H-15), 1.03 (3H, d, J=6.4, H-14), 0.92 (3H, d, J=7.2, H-13); 13C-NMR (400 MHz, CDCl 3): 102.4 (C-8), 94.5 (C-12), 87.5 (C-7), 79.6 (C-6), 69.4 (C-10), 58.8 (C-1), 45.3 (C-9), 44.3 (C-5), 37.0 (C-3), 35.1 (C-2), 30.8 (C-11), 22.1 (C-4), 25.8 (C-15), 21.2 (C-14), 12.7 (C-13); FAB-MS m/z 323[M ++ Na +] (12), 277 (38), 251 (8), 207 (12), 173 (22), 159 (26), 137 (38), 115 (100).
8,3 α, 3 beta-dihydroxyl Artemethers: white needle (CHCl 3); IR (KBr) V Max(cm -1): 3528 br (OH); 1H-NMR (300MHz, CDCl 3): δ 5.77 (1H, s, H-7), 4.77 (1H, d, J=3.2, H-12), 3.48 (3H, s, H-16), 3.06 (1H, t, J=14.6, H-4b), 2.67 (1H, m, H-11), 2.43 (3H, m, H-9b, 4,2), 3.01 (1H, m, H-9a), 1.93 (2H, m, H-5,10b), 1.73 (2H, m, H-1,10a), 1.50 (3H, s, H-15), 1.07 (3H, d, J=6.7, H-14), 0.92 (3H, d, J=7.0, H-13); 13C-NMR (300MHz, CDCl 3): δ 117.6 (C-3), 104.4 (C-8), 103.6 (C-12), 86.91 (C-7), 79.4 (C-6), 56.3 (C-16), 51.6 (C-2), 47.5 (C-1), 44.0 (C-5), 39.9 (C-4), 36.1 (C-9), 30.9 (C-11), 26.0 (C-15), 25.7 (C-10), 12.6 (C-13), 11.8 (C-14); ESI-MSm/z 315[M ++ 1] (5), 284 (12), 283 (100), 224 (21), 227 (9), 195 (8), 179 (14), 161 (29).
9,3 β, 10 alpha-dihydroxy-Artemethers: white needle (CHCl 3); M.p.123-125 ℃; IR (KBr) V Max(cm -1), 3380 br (OH), 1116,858,827; 1H-NMR (300MHz, CDCl 3) δ 5.21 (1H, s, H-7), 4.74 (1H, d, J=2.8, H-12), 4.46 (1H, td, J=11.0,4.8, H-3a), 3.98 (1H, dd, J=7.9,9.4, H-10b), 3.49 (1H, s, HO-3), 3.47 (3H, s, H-16), 2.82 (1H, m, H-11), 2.52 (2H, m, H-9a, 9b), 2.18 (1H, s, HO-10), 1.87 (1H, ddd, J=12.7,12.7,4.8, H-4b), 1.57 (2H, m, H-2,5), 1.45 (3H, s, H-15), 1.32 (1H, dd, J=11.5,7.9, H-1), 1.20 (3H, d, J=7.9, H-13), 1.16 (3H, d, J=5.2, H-14), 1.16 (H, m, H-4a); 13C-NMR (300MHz, CDCl 3): 103.6 (C-12), δ 102.7 (C-8), 88.1 (C-7), 81.0 (C-6), 70.0 (C-3), 69.1 (C-10), 59.1 (d, C-1), 56.0 (s, C-16), 51.3 (C-2), 46.3 (C-9), 45.3 (C-5), 33.1 (C-4), 31.4 (C-11), 25.7 (C-15), 20.9 (C-14), 15.6 (C-13); ESI-MS m/z 353[M ++ Na] (100), 323 (49), 307 (37), 235 (43).
10,3 α, 10 alpha-dihydroxy-Artemethers: white needle (CHCl 3); M.p.121-123 ℃; IR (KBr) V Max(cm -1), 3380 br (OH); 1H-NMR (300MHz, CDCl 3) δ 5.28 (1H, s, H-7), 4.70 (1H, d, J=3.5, H-12), 3.43 (3H, s, H-16), 1.47 (3H, s, H-15), 1.24 (3H, d, J=6.9, H-13), 1.16 (3H, d, J=3.8, H-14); 13C-NMR (300MHz, CDCl 3): 103.6 (C-12), δ 102.4 (C-8), 86.8 (C-7), 81.1 (C-6), 70.1 (C-3), 69.4 (C-9), 56.1 (s, C-16), 51.6 (d, C-1), 46.7 (C-5), 40.3 (C-2), 37.5 (C-4), 32.4 (C-10), 31.4 (C-11), 25.9 (C-15), 17.8 (C-14), 12.8 (C-13); ESI-MS m/z 353[M ++ Na] (88), 323 (62), 307 (100), 235 (64).
Embodiment 3
The selection of carbon source, nitrogenous source, metal ion in the Artemisinin microbial transformation substratum
Microorganism Streptomyces griseus is transformed the principal product that Artemisinin produces, and especially the conversion condition of 3 Alpha-hydroxy Artemisinins screens.
Substratum:
Carbon source is selected substratum: 20% potato decoction liquor 1000ml, carbon source (glucose or soluble starch, sucrose, maltose, citric acid, semi-lactosi, dextrin, Semen Maydis powder, glycerine) 20g, yeast extract paste 5g, bean powder 5g, KH 2PO 43.0g, MgSO 47H 2O 1.5g, VB 1Trace (pH5.0～7.5).
Nitrogenous source is selected substratum: 20% potato decoction liquor 1000ml, glucose 20g, nitrogenous source (peptone or extractum carnis, yeast extract paste, Semen Maydis powder, bean powder 10g, or urea element, volatile salt, SODIUMNITRATE, L-Methionin, DL-α-An Jibingsuan are equivalent to SODIUMNITRATE 2g/L), KH 2PO 43.0g, MgSO 47H 2O 1.5g, VB 1Trace (pH5.0～7.5).
Metal Ion Selective Electrode substratum: 20% potato decoction liquor 1000ml, glucose 20g, yeast extract paste 5g, bean powder 5g, metal ion (K +Or Co 2+, Fe 3+, Mg 2+, Cu 2+, Mn 2+, Zn 2+) 0.2mmol/L, VB 1Trace (pH5.0～7.5).Method
Bacterial classification is preserved and activation: bacterial classification inoculation is cultivated 3d for 28 ℃ on the inclined-plane solid medium, is stored in then in 4 ℃ the refrigerator.Actication of culture adopts two step activation methods, and elder generation in liquid nutrient medium, behind 28 ℃ of 200r/min vibration rotating and culturing 48h, transfers bacterial classification inoculation in another liquid nutrient medium, cultivates 24h with condition.
Application of sample and sampling: every bottle two step activatory bacterium liquid (25ml) adds the Artemisinin solution 100 μ l that make 150mg/ml with the DMF dissolving.Behind condition cultivation 36-120h, fermented liquid is taken out in the timesharing sampling, add the equivalent ethyl acetate extraction 3 times, merge the back and reclaim ethyl acetate, it is 10ml that residue adds chromatographically pure dissolve with methanol constant volume, with the aperture is the millipore filtration filtration of 0.45 μ m, gets subsequent filtrate and measures usefulness for high performance liquid phase.
High performance liquid phase condition determination and method: Waters 600E high performance liquid chromatograph, the online de-aerator of 4 passages, 717 type automatic samplers, 996 diode-array detectors, Waters Symmetry C 18Chromatography column.Moving phase: acetonitrile-water 35: 65; Flow velocity: 1.5ml/min; Detect wavelength: 200nm.
Above-mentioned carbon source is investigated the result and shown: bacterial classification S.griseus conversion capability to Artemisinin in the substratum that with the Semen Maydis powder is carbon source is the strongest; Next is starch, glycerine, dextrin and glucose; Mycelial growth is slow in the substratum that with the citric acid is carbon source, and Artemisinin is not almost had conversion capability.
Nitrogenous source is investigated the result and shown: bacterial classification S.griseus conversion capability to Artemisinin in the substratum that with the Semen Maydis powder is nitrogenous source is the strongest; Next is yeast extract paste and DL-α-An Jibingsuan.
In the 7 metal ion species substratum of investigating containing, bacterial classification S.griseus than higher, comparatively speaking, is containing Mn to the transformation efficiency of Artemisinin 2+Substratum in 3 Alpha-hydroxy Artemisinin yield the highest.
Embodiment 4
The selection of Artemisinin microbial transformation medium pH, incubation time, application of sample amount
Microorganism Streptomyces griseus is transformed the investigation of conditions such as medium pH that Artemisinin produces new product, incubation time, application of sample amount,
Substratum:
Transform and use substratum: 20% potato decoction liquor 1000ml, glucose 20g, yeast extract paste 5g, bean powder 5g, KH 2PO 43.0g, MgSO 47H 2O 1.5g, VB 1Trace.
More than all substratum all be sub-packed in the 150ml triangular flask, every bottle of 25ml, 121 ℃, 0.15Mpa 20min that sterilizes.Method:
Bacterial classification is preserved and activation: bacterial classification inoculation is cultivated 3d for 28 ℃ on the inclined-plane solid medium, is stored in then in 4 ℃ the refrigerator.Actication of culture adopts two step activation methods, and elder generation in liquid nutrient medium, behind 28 ℃ of 200r/min vibration rotating and culturing 48h, transfers bacterial classification inoculation in another liquid nutrient medium, cultivates 24h with condition.
Application of sample and sampling: every bottle two step activatory bacterium liquid (25ml) adds Artemisinin solution 30,60,100,130, the 160 μ l that make 150mg/ml with the DMF dissolving.With (except investigating sample time) behind the condition cultivation 84h, fermented liquid is taken out, add the equivalent ethyl acetate extraction 3 times, merge the back and reclaim ethyl acetate, it is 10ml that residue adds chromatographically pure dissolve with methanol constant volume, is the millipore filtration filtration of 0.45 μ m with the aperture, gets subsequent filtrate and measures usefulness for high performance liquid phase.
High performance liquid phase condition determination and method: Waters 600E high performance liquid chromatograph, the online de-aerator of 4 passages, 717 type automatic samplers, 996 diode-array detectors, Waters Symmetry C 18Chromatography column.Moving phase: acetonitrile-water 35: 65; Flow velocity: 1.5ml/min; Detect wavelength: 200nm.
Experimental results show that: the pH value has considerable influence to conversion.Transform substratum and transfer pH with hydrochloric acid or the sodium hydroxide of 1.0mol respectively.Investigated pH4.5,5.0,5.5,6.0,6.5,7.0,7.5, the 8.0 Artemisinins influence to transformation efficiency respectively, pH all helps conversion reaction in 5.0～7.5 scopes as a result.In pH was 5.0～7.5 scope, the growing way of bacterial classification strengthened with the pH value, to the transformation efficiency of Artemisinin also along with increase, but the yield of 3 Alpha-hydroxy Artemisinins with pH be 6.0 o'clock the highest.
The conversion results of different sample times proves: tested 12,36,60,84,108,132,156 7 different sample time Artemisinin transformation efficiency, the result all has higher transformation efficiency during transforming 36-108 hour.Along with the prolongation of incubation time, bacterial classification increases the consumption of Artemisinin, but after cultivating 84h, the bacterial classification growing way weakens.The yield of 3 Alpha-hydroxy Artemisinins is the highest to cultivate the 84h sampling.
Embodiment 5
The biological activity determination of artemisine compounds
Compared the immunocompetence of Artemisinin, 3 α-Artemisinin, 3 β-3 compounds of Artemisinin with the lymphocyte luminescence method, the result shows: converted product 3 Alpha-hydroxy Artemisinins, the 3 β hydroxyl Artemisinins of Artemisinin all have very strong immune-enhancing activity.
1 material and instrument
1.1 medicine and reagent: luminol,3-aminophthalic acid cyclic hydrazide (Luminol, Mark CO, F.R.G, final concentration 10 -5Mmol/L); Dextran F500 (Pharmacia product); Lymphocyte separation medium (Shanghai smart biological High Seience Technology Co., Ltd. of China); Phytohaemagglutinin (PHA, Shanghai Yi Hua clinical medicine scientific ﹠ technical corporation, lot number: 980101); Buddhist ripple myristic acid ethyl ester (PMA), formyl first sulphur bright ammonia acyl phenylalanine (fMLP) is Sigma company product; Lymphocyte (PMN) parting liquid (includes NaCl 140mmol/L, KCl 5mmol/L, MgCl 21mmol/L, CaCl 21mmol/L, NaH 2PO 41mmol/L, HEPES 10mmol/L, Tris 5mmol/L); Auspicious Te Shi dye liquor (bio-engineering research institute is built up in Nanjing); Artemisinin; 3 Alpha-hydroxy Artemisinins, 3 beta-hydroxy Artemisinins are for transforming the new compound that obtains; All the other reagent are homemade analytical pure.
1.2 instrument: SHG-C type biochemiluminescence survey meter (going up marine upright detecting instrument factory); The last ware electronic balance of FA-1104, Shanghai balance equipment factory; HH-2 digital display thermostat water bath, Changzhou Guohua Electric Appliance Co., Ltd.
1.3 animal, the SD rat, body weight 180～250g, male and female are regardless of.
2 methods
2.1 the separation of PMN and preservation: get the fresh anticoagulation of rat, press document (Tao Yi instruction pending trial is compiled the clinical immunology inspection, the first volume, Shanghai, Science and Technology of Shanghai press, 1983, p16-19) carry out PMN and separate.After 40 minutes, get supernatant liquor through the dextran precipitation, isolate PMN through lymphocyte separation medium density gradient centrifugation.Tongue expects that blue procuratorial work PMN vigor was more than 95% in 3 hours, auspicious special Albert'stain Albert method check purity is more than 95%.Adjusting concentration of cell suspension with the Hanks liquid that contains 2% calf serum is 5 * 10 6PMN/mL, 4 ℃ of preservations, the use vigor is constant in 12 hours.
2.2 chemiluminescent mensuration is measured chemoluminescence (CL) reaction with SHG-C type biochemiluminescence survey meter.It is 37 ± 0.5 ℃ that 37 ℃ of recirculated waters make the sample chamber temperature.Get PMN suspension 1mL, add 1uminol 0.2mL, 10min is hatched in 37 ℃ of water-baths, surveys the spontaneous CL 1min of PMN.Add the different concns specimen, contrast adds HBSS 0.1mL, and METHOD FOR CONTINUOUS DETERMINATION CL intensity 5min adds PHA, PMA, fMLP, again METHOD FOR CONTINUOUS DETERMINATION 10-15min then immediately.Measurement result is by the automatic record of biological-chemical photometer 6s integration.(chemoluminescence method such as Zhang Xuejun is measured the active Chinese Medical Journal 1992 of isolating lymphocyte of human peripheral and neutrophil leucocyte, 15:365 to the reference literature method; Zhang Xuejun isoreactivity oxygen scavenqer and calcium channel blocker are to chemiluminescent biological chemistry and the biophysics progress 1990 of influencing of human lymphocyte, 17:444).
2.3 result treatment CL intensity is represented with mV.With the spontaneous CL of PMN is background, and adding the CL maximum that occurs behind stimulant PMA, FMLP and the PHA is peak value.Experimental result is represented with the pure peak value that peak value deducts behind the background.Every part of sample detects all does multiple pipe, and statistic data is taken from the experimental result of sample more than 3 parts.Specimen influences the CL degree of rat PMN and represents with inhibiting rate.
PMN-CL inhibiting rate (%)=[the pure peak value mean of the pure peak value mean/control group of 1-experimental group] * 100%
3, experimental result (referring to table 2):
Table 2, to the inhibiting rate (%) of rat PMN
Concentration (mol/L) sample
10 -510 -6Artemisinin-0.3187 3.74333 Alpha-hydroxy Artemisinin-1.2392-1.20823 beta-hydroxy Artemisinin-9.0381-5.4654
The present invention has carried out external immunocompetence relatively with the Ly-CL method to 3 compounds of artemisine, the result shows: the converted product 3 Alpha-hydroxy Artemisinins and the 3 beta-hydroxy Artemisinins of Artemisinin have significant immunologic enhancement, and are 10 at drug level -5During mol/L, the promoter action of 3 beta-hydroxy Artemisinins is stronger about 30 times than Artemisinin.

Claims (10)

1, preparation has the method for the artemisine compounds of following general formula, it is characterized in that: utilize preserving number to be the light grey streptomyces strain microorganism of ATCC-13273 or the varient and the mutant of its functional equivalent, Artemisinin, dihydroarteannuin or Artemether are carried out bio-transformation, obtain following artemisine compounds
1) 3 Alpha-hydroxy Artemisinins, R 1=OH, R 2=R 3=H, R 4+ R 5==O;
2) 3 beta-hydroxy Artemisinins, R 1=R 3=H, R 2=OH, R 4+ R 5==O;
3) Artemisinin ketone-3, R 1+ R 2==O, R 4+ R 5==O, R 3=H
4) 3 Alpha-hydroxy dihydroarteannuins, R 1=R 4=OH, R 2=R 3=R 5=H;
5) 3 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 5=OH, R 2=R 3=R 4=H;
6) 10 Alpha-hydroxy dihydroarteannuins, R 1=R 2=R 5=H, R 3=R 4=OH;
7) 10 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 2=R 5=H, R 3=R 5=OH;
8) 3 α, 3 beta-dihydroxyl Artemethers, R 1=R 2=OH, R 3=R 5=H; R 4=-OCH 3
9) 3 β, 10 alpha-dihydroxy-Artemethers, R 1=R 5=H, R 2=R 3=OH, R 4=-OCH 3
10) 3 α, 10 alpha-dihydroxy-Artemethers, R 1=R 3=OH, R 2=R 5=H, R 4=-OCH 3
11) 3 Alpha-hydroxy Artemethers, R 1=OH, R 2=R 5=R 3=H, R 4=-OCH 3
12) 3 beta-hydroxy Artemethers, R 1=R 5=R 3=H, R 2=OH, R 4=-OCH 3
2, according to the described method for preparing artemisine compounds of claim 1, substratum is mainly formed except that 20% potato decoction liquor, KH 2PO 4, MgSO 47H 2O, trace V B 1Outward, it is characterized in that: carbon source is glucose, starch, sucrose, maltose, semi-lactosi, dextrin, Semen Maydis powder and/or glycerine, nitrogenous source is peptone, yeast extract paste, Semen Maydis powder, bean powder, urea element, volatile salt, SODIUMNITRATE, L-Methionin, DL-α-An Jibingsuan, and metal ion is K +Or Co 2+, Mg 2+, Cu 2+, Mn 2+, Zn 2+
3, according to claim 1 or the 2 described methods that prepare artemisine compounds, substratum is mainly formed except that 20% potato decoction liquor, KH 2PO 4, MgSO 47H 2O, trace V B 1Outward, it is characterized in that: carbon source is glucose, starch, dextrin, Semen Maydis powder and/or glycerine, and nitrogenous source is yeast extract paste, Semen Maydis powder and/or DL-α-An Jibingsuan, and metal ion is Mn 2+
4, according to claim 1 or the 2 described methods that prepare artemisine compounds, substratum mainly is made up of 20% potato decoction liquor 1000ml, VB1 trace, metal ion 0.1-0.3mmol/L and following raw material (by weight):
Carbon source 8-12 part: nitrogenous source 1-5 part: KH 2PO 41-3 part: MgSO 47H 21 part of O.
5, according to the described method for preparing artemisine compounds of claim 4, substratum is mainly by 20% potato decoction liquor 1000ml, VB 1Trace, metal ion 0.1-0.3mmol/L and following raw material (by weight) are formed:
Carbon source 10-11 part: nitrogenous source 3-3.5 part: KH 2PO 42 parts: MgSO 47H 21 part of O.
6, according to claim 1 or the 2 described methods that prepare artemisine compounds, it is characterized in that: bio-transformation is to carry out under pH5.0～7.5 conditions; The concentration of Artemisinin, dihydroarteannuin and/or Artemether is 0.2～1mg/mL; The bio-transformation time is 36～108 hours.
7, according to the described method for preparing artemisine compounds of claim 6, it is characterized in that: bio-transformation is to carry out under the pH6.0 condition, and the concentration of Artemisinin, dihydroarteannuin and/or Artemether is 0.4-0.6mg/mL; The bio-transformation time is 80～90 hours.
8, according to the described method for preparing artemisine compounds of claim 1-2, it is characterized in that: utilize preserving number to be the light grey streptomyces strain microorganism of ATCC-13273 or the varient and the mutant of its functional equivalent, be 6.0, containing Mn as carbon source or nitrogenous source, pH with Semen Maydis powder respectively 2+Substratum in, cultivated 84 hours, obtain 3 Alpha-hydroxy Artemisinins.
9, according to the described method for preparing artemisine compounds of claim 1-2, it is characterized in that: utilize preserving number to be the light grey streptomyces strain microorganism of ATCC-13273 or the varient and the mutant of its functional equivalent, be 6.0, containing Mn as carbon source or nitrogenous source, pH with Semen Maydis powder respectively 2+Substratum in, cultivated 84 hours, obtain 3 beta-hydroxy Artemisinins.
10, have immunocompetent artemisine compounds, it is characterized in that having following general formula:
Figure A0211275000041
1) 3 Alpha-hydroxy Artemisinins, R 1=OH, R 2=R 3=H, R 4+ R 5==O;
2) 3 beta-hydroxy Artemisinins, R 1=R 3=H, R 2=OH, R 4+ R 5==O;
3) Artemisinin ketone-3, R 1+ R 2==O, R 4+ R 5==O, R 3=H
4) 3 Alpha-hydroxy dihydroarteannuins, R 1=R 4=OH, R 2=R 3=R 5=H;
5) 3 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 5=OH, R 2=R 3=R 4=H;
6) 10 Alpha-hydroxy dihydroarteannuins, R 1=R 2=R 5=H, R 3=R 4=OH;
7) 10 Alpha-hydroxies-α-dihydroarteannuin, R 1=R 2=R 5=H, R 3=R 5=OH;
8) 3 α, 3 beta-dihydroxyl Artemethers, R 1=R 2=OH, R 3=R 5=H; R 4=-OCH 3
9) 3 β, 10 alpha-dihydroxy-Artemethers, R 1=R 5=H, R 2=R 3=OH, R 4=-OCH 3
10) 3 α, 10 alpha-dihydroxy-Artemethers, R 1=R 3=OH, R 2=R 5=H, R 4=-OCH 3
CNB021127506A 2002-03-12 2002-03-12 Biotransfer process for preparing artemisine compounds Expired - Fee Related CN1189568C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB021127506A CN1189568C (en) 2002-03-12 2002-03-12 Biotransfer process for preparing artemisine compounds

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB021127506A CN1189568C (en) 2002-03-12 2002-03-12 Biotransfer process for preparing artemisine compounds

Publications (2)

Publication Number Publication Date
CN1382805A true CN1382805A (en) 2002-12-04
CN1189568C CN1189568C (en) 2005-02-16

Family

ID=4742224

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB021127506A Expired - Fee Related CN1189568C (en) 2002-03-12 2002-03-12 Biotransfer process for preparing artemisine compounds

Country Status (1)

Country Link
CN (1) CN1189568C (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102887908A (en) * 2011-07-22 2013-01-23 江苏斯威森生物医药工程研究中心有限公司 Simple process for preparing artesunate by one-pot method by taking artemisinin as raw material
CN105037384A (en) * 2015-05-13 2015-11-11 中国药科大学 Novel hydroxyl dihydroartemisinin derivative and application thereof
CN105859742A (en) * 2016-04-29 2016-08-17 中国药科大学 Preparation and application of novel hydroxy dihydroartemisinin sulfur-containing ester derivative
CN106518889A (en) * 2016-10-28 2017-03-22 广东省中医院 Dihydroxy artemisinin as well as preparation method and application thereof
CN111423451A (en) * 2020-05-11 2020-07-17 中国药科大学 14-hydroxy dihydroartemisinin and derivative, preparation method and application thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102887908A (en) * 2011-07-22 2013-01-23 江苏斯威森生物医药工程研究中心有限公司 Simple process for preparing artesunate by one-pot method by taking artemisinin as raw material
CN105037384A (en) * 2015-05-13 2015-11-11 中国药科大学 Novel hydroxyl dihydroartemisinin derivative and application thereof
CN105859742A (en) * 2016-04-29 2016-08-17 中国药科大学 Preparation and application of novel hydroxy dihydroartemisinin sulfur-containing ester derivative
CN106518889A (en) * 2016-10-28 2017-03-22 广东省中医院 Dihydroxy artemisinin as well as preparation method and application thereof
CN106518889B (en) * 2016-10-28 2019-01-29 广东省中医院 Dihydroxy qinghaosu, preparation method and its application
CN111423451A (en) * 2020-05-11 2020-07-17 中国药科大学 14-hydroxy dihydroartemisinin and derivative, preparation method and application thereof

Also Published As

Publication number Publication date
CN1189568C (en) 2005-02-16

Similar Documents

Publication Publication Date Title
KR102102241B1 (en) Euglena spp. microalgae, polysaccharide manufacturing method, and organic compound manufacturing method
Sandjo et al. Tanzawaic acids I–L: Four new polyketides from Penicillium sp. IBWF104-06
CN1590555A (en) Hydroxylation of compactin to pravastatin by micromonospora
CN1189568C (en) Biotransfer process for preparing artemisine compounds
Kossuga et al. Two polyketides from a co-culture of two marine-derived fungal strains
CN1614003A (en) Culture of Chansi mould strains and extraction and use of anagentic compound therefrom
CN1028542C (en) Anticancer antibiotic MZ43-37F11 substance, the production and uses thereof
CN1082052C (en) Process for producing cyclodepsipeptide compounds and novel cyclodepsipeptide
JPH03505817A (en) Method for preparing gamma and delta lactones
CN1724643A (en) New strain and method of producing pacilitaxel using said strain
WO2013153554A1 (en) Fermentation process for the production of rapamycin
CN1827633A (en) Process for extracting N6-(2-ethoxyl) adenosine from Paecilomyces Cicadae Samson culture
CN1908179A (en) Reduction reaction for carbon carbon double bond of lactone compound utilizing microorganism
CN1139660C (en) Substances WK-5344A and WK-5344B and process for producing the same
US5378611A (en) Process for producing 6β,14α-dihydroxy-4-androstene-3, 17-dione amid 14α-hydroxy-4-androstene-3,6,17-trione from 4-androstene-3, 17-dione using myrothecium sp. ferm bp-4432
CN87102651A (en) Antibioltic F-0769 and production method thereof and as producing promotor, feedstuff synergistic agent and carcinostatic agent
US6682914B2 (en) Bioactive tetracyclic diterpene compounds and methods of preparation and use thereof
EP0504711B1 (en) Compound UCA1064-B
US20040266835A1 (en) Inhibitors against complex II of electron transport system
CN1033591C (en) Production of pradimicin antibiotics
EP0657412B1 (en) Saintopin derivative
US4968610A (en) Process for the preparation of chanoclavine
CN1401780A (en) Method for preparing corydamine by biotransformation and use thereof in preparing analgesic
CN108277170B (en) New mycobacterium aureofaciens and application thereof in preparation of 9-hydroxyprogesterone
JPH09107982A (en) Biologically pure cultured substance of microorganism, method for forming lactone, method for forming diol, method for forming compound and method for forming cyclic ether

Legal Events

Date Code Title Description
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20050216

Termination date: 20190312