CN1521258A - Novel epothilones compound and its preparation method and application - Google Patents
Novel epothilones compound and its preparation method and application Download PDFInfo
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- CN1521258A CN1521258A CNA031030319A CN03103031A CN1521258A CN 1521258 A CN1521258 A CN 1521258A CN A031030319 A CNA031030319 A CN A031030319A CN 03103031 A CN03103031 A CN 03103031A CN 1521258 A CN1521258 A CN 1521258A
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Abstract
The present invention relates to one kind of polyketone compounds and its preparation process. Further, the present invention also relates to the application of these compounds in preparing medicine composition for resisting tumor, inhibiting overgrowth of cell and stopping cell growth.
Description
Technical field
The present invention relates to the novel polyketide of a class, be particularly related to a series of novel epothilone compounds, and the preparation method of this compounds and this compound are antitumor in preparation, suppress cell transition and increase and end application in the pharmaceutical composition of cell growth.
Background technology
Epothilones A (EpoA) and epothilone B (EpoB) are polyketide deutero-16 membered macrolide novel cpds, separate from soil bacteria Sorangium cellulosum bacterial strain Soce90 at first, and its structural formula is as follows.They are for separated at first, the esperamicin of synthetic and conclusive evidence, (Hofle et al., 1996, Angew.Chem.Int.Ed.Engl.35 (13/14): 1567-1569; Gerth et al., 1996.J.Antibiotics 49 (6): 560-563).Polyketide is present in the multiple organism at nature, and is synthetic by polyketide synthase.Polyketide synthase is called PKS (polyketide synthase) enzyme usually for short.
EpoA：R＝H；????EpoB：R＝CH
3
Epothilones A and B have huge potential to the treatment cancer.Though structurally dissimilar, and the mechanism of action of esperamicin family and famous cancer drug taxol (paclitaxel, Taxol) very similar, comprise and induce tubulin polymerization, and stabilize microtubules forms (microtubule assembly).These compounds show the powerful cell killing power to different carcinoma clone.Particularly, they are to having the tumor cell line of multiple cancer drug resistance (MDR), especially to the tumor cell line of anti-taxol and other cancer therapy drug, demonstrate noticeable effect (Altmann et al., 2000.Biochem.Biophys.Acta.1470 (3): M79-91; Bollag et al., Cancer Res.55 (11): 2325-2333).
The deoxidation counterpart of Epothilones A and B, be also referred to as Epothilone C and D, successfully complete synthesis by chemistry, but also can produce the fermented product extract of bacterial strain S.cellulosum and many similar structures of other esperamicin as minor component detect together from natural esperamicin.Compare with epothilone B, epothilone d shows similar antitumour activity, but cytotoxicity is less.
Describe reorganization modular PKS gene in recombinant host cell among U.S. Pat 5843718 and the US6033883 and produced novel polyketide, and in the host cell body, as streptomycete (Streptomyces), introduce allos PKS gene in intestinal bacteria (E.coli) and the slime bacteria (Myxobacterial) and produce the recombination method of polyketide, wherein the biosynthetic enzyme of esperamicin (polyketide synthase PKS) is by 5 gene product (epoA, B, C, D and E) formed multi-functional, multiprotein complex.In the host cell that can not produce esperamicin, in streptomycete Streptomyces coelicolor and slime bacteria Myxococcus xanthus, by introducing allos esperamicin gene and expressing and the existing report of production Epothilones A and B (Tang, et al.Science287:640-642; Julien ﹠amp; Shah, 2002, Amtimicrobial.Agent Chemother.46 (9): 2772-2778 lists in this with for referencial use).
At present, people just are being devoted to develop as the esperamicin of effective more chemotherapy agents and the analogue of its dependency structure, as modifying naturally occurring epothilone compounds by chemistry is semi-synthetic.The conversion reaction that Epothilones A and B transform into the corresponding lactam analogue has been described among the PCT publication WO99/27890.
Summary of the invention
The object of the present invention is to provide a series of novel epothilone compounds; And provide the preparation method of this compounds: this method is used for preparing 4-demethylation epothilone d or the B and the corresponding azoles counterpart of the novel epothilone compounds of this class, and the method that adopts chemically modified or bio-transformation makes other novel epothilone compounds of the present invention from epothilone d or B and 4-demethylation epothilone d or B; It is antitumor in preparation to have the present invention further provides the novel epothilone compounds of this class, the application in the pharmaceutical composition that the inhibition cell transition increases and the termination cell is grown.
Esperamicin derivatives provided by the invention has following general formula (I)
Formula (I)
Wherein,
A-D is the carbon-carbon double bond of following formula (a) or is the epoxide group of formula (b):
Q and R1 are independently selected from H, C
1-4Alkyl or NH
2In any one,
R4 does not exist, or when A-D was the C-C singly-bound, R4 was an oh group;
R2, R3 are selected from H, hydroxyl, NH independently of one another
2And CH
3
X is O or N-R, and wherein R is H, NH
2, aryl, or CH
3
W is S or O.
Described esperamicin derivatives with general formula (I) can be to be 4-demethylation esperamicin, formula (II) as follows.
Formula (II)
Wherein,
A-D is the carbon-carbon double bond of following formula (a) or is the epoxide group of formula (b):
Q is independently selected from H, C
1-4Alkyl or NH
2In any one,
R4 does not exist, or when A-D was the C-C singly-bound, R4 was an oh group;
R2, R3 are selected from H, hydroxyl, NH independently of one another
2And CH
3
X is O or N-R, and wherein R is H, NH
2, aryl, or CH
3
W is S or O.
Wherein, 4-demethylation epothilone d [seeing Figure 11 Chinese style 19] or B[see Figure 11 Chinese style 17] fermentation of recombination engineering strain obtains can to pass through to send out of the present invention.The present invention than suitable embodiment in, adopt the Myxococcus xanthus or the Sorangium cellulosum engineering strain of reorganization to prepare 4-demethylation epothilone d or B.
Recombination engineering strain provided by the invention, comprised the esperamicin PKS gene after a kind of modify, the gene of being modified is the transmethylase functional group (methyl-transferase that makes the 8th assembly (module 8) in the esperamicin PKS gene in the host cell by the DNA recombinant technology, MTdomain) dna sequence dna is by sudden change, disappearance or replacement and inactivation, thus make the adorned esperamicin PKS of esperamicin PKS gene gene substitution in the host cell.Wherein the host to organize born of the same parents are any cells that comprise esperamicin PKS gene and produce esperamicin.Described host cell can be Myxococcus xanthus cell or Sorangium cellulosum cell.
Engineering strain provided by the invention obtains by recombinant technology, this technology utilizes the method for homologous recombination that the adjacent area of required reformed gene or functional group is cloned in the suicide vector, the homologous gene of examining in the contained genome by gene in the suicide vector and host cell carries out the double cross reorganization, cause participating in biosynthetic one or more genes of esperamicin or functional group inactivation or be substituted, and the genetically engineered that obtains recombinating is produced bacterial strain.For example; replace the special AT functional group of methylmalonyl CoA (methylmalonyl-CoA) or make MT functional group inactivation with an acylase special (acyl-transferase, AT domain) functional group malonyl coenzyme A (malonyl-CoA).This deactivation can pass through at random or the point gene sudden change, and disappearance or replacement are finished.The reorganization PKS that obtains thus is different from natural PKS, and it can be produced in recombinant host cell with the esperamicin derivatives is primary product, and its derivative lacks one or two methyl group at esperamicin corresponding C-4 position.For example, the host cell M.xanthus that includes esperamicin PKS synthetic gene, can produce epothilone B is primary product, disappearance takes place and causes inactivation in the dna sequence dna of the MT functional group of the 8th functional unit (module 8) in esperamicin PKS wherein, thereby synthetic 4-demethylation-epothilone B of this recombinant bacterial strain and D are primary product.
The further preferred W=O of described general formula (II) compound just the azoles counterpart (oxazole) of 4-demethylation-esperamicin [for example formula 18,20], it is Myxococcusxanthus cell or the Sorangium cellulosum cell that utilizes reorganization, obtains by changing fermentation condition.
Other compound of the present invention can be by to the natural production bacterial strain of for example S.cellulosum, or the compound of other genetic engineering bacterial strain fermentative preparation, or the compound of chemosynthesis carries out further chemically modified or microbial transformation and makes.
The further preferred 7-O-methyl isophthalic acid 2-hydroxyl esperamicin of general formula (I) compound (BGE8[formula 5]), BGE5[formula 21].This compounds can make by the microbial conversion process of describing among the embodiment 5.The BGE5 structure is as follows:
The further preferred 4-demethylation of general formula (I) compound, 21-hydroxyl epothilone B [formula 14].This compounds can make by the microbial conversion process of describing among the embodiment 6.
The further preferred 4-demethylation of general formula (I) compound, 14-hydroxyl epothilone d.Use a kind of hydroxylase that derives from microorganism to make the C-14 hydroxylation of 4-demethylation-epothilone d, can obtain this compound.The exemplary method of this type of conversion is described with epothilone d in embodiment 7 and is prepared 14-hydroxyl epothilone d, and structural formula is as follows:
The epoxide of the further preferred compound of general formula (I) compound (12, the 13-epoxy derivative).This compounds can make by the epoxidizing method of the chemical equation of description among the embodiment 8.
The further preferred 7-O-methyl esperamicin derivatives of general formula (I) compound.This compounds can make by the universal method that chemical equation among the embodiment 11 is described.
The further preferred 12-hydroxyl esperamicin derivatives of general formula (I) compound.This compounds can make by the universal method that chemical equation among the embodiment 12 is described.
The further preferred 7-O-methyl esperamicin derivatives of general formula (I) compound.This compounds can make by the universal method that chemical equation among the embodiment 11 is described.
The further preferred 4-demethylation of general formula (I) compound, 21-hydroxyl esperamicin derivatives and 4-demethylation, the amino esperamicin derivatives of 21-.This compounds can make by the universal method that chemical equation among the embodiment 13 is described.In addition, use the hydroxylase that comes from microorganism to make the methyl end groups group generation hydroxylation of thiazole part (thiozole), also can obtain the 4-demethylation, 21-hydroxyl esperamicin derivatives.
It is antitumor in preparation to have the present invention further provides the esperamicin derivatives with general formula (I), the application in the pharmaceutical composition that the inhibition cell transition increases and the termination cell is grown.
Pharmaceutical composition of the present invention generally includes the esperamicin derivatives provided by the invention and the pharmaceutically acceptable carrier of some amount.Compound of the present invention can be free form or medicine acceptable carrier, as the salt and the ester of prodrug (prodrugs) and The compounds of this invention.Compound can be any form, as solid-state, and semi-solid state or liquid state.
Compound of the present invention can adopt other formulation method for preparing drugs of low aqueous solubility as is known to make.For example, compound can form emulsion (referring to document WO 00/71163 and US6458373 B1) with vitamin-E and/or PEG polyacrylic acid derivative.Usually, earlier compound of the present invention is dissolved in ethanol, adds vitamin-E and/or PEG polyacrylic acid derivative then and form treatment agent solution.Ethanol is removed, formed the aqueous solution that precursor emulsion or adding contain tensio-active agent (stablizer) then and form precursor emulsion.Be used for intravenous administering mode, precursor emulsion can be disperseed to form uniform emulsion.Be used for medicinal preparation for oral administration, precursor emulsion generally places gel capsule.
The mechanism of action of esperamicin family and cancer drug taxol (paclitaxel, Taxol) very identical, mainly be by induce tubulin polymerization and stabilize microtubules the assembling (microtubule assembly), thereby the function of interference cell microtubule.This has just caused the interior signal transmission of pair cell division and cell migration and born of the same parents and the inhibition of protein excretion, because these behaviors all depend on the quick and depolymerization efficiently of microtubule.
On the one hand, The compounds of this invention can be used for treating cancer, comprises head and neck; The cancer of liver and courage portion; Mammary cancer, ovarian cancer, lung cancer, the cancer of the brain, intestinal cancer, leukemia, malignant tumour, and lymphoma.This method comprises the The compounds of this invention of taking the treatment significant quantity to the cancer patients.If necessary, this method can be reused, to prevent the metastasis of cancer or radical cure cancer.On the other hand, compound of the present invention and composition can use separately, also can use jointly with other cancer therapy drug or therapy.Multiple drug resistance makes the conventional chemotherapy medicine inoperative to patient, and this drug-fast generation is because medicine is discharged the extracellular by P-glycoprotein.Esperamicin can be united use with other cancer therapy drug and treatment means, is used for the treatment of cancer and other proliferative disease, strengthening the drug effect of other chemotherapeutics, and very useful to the control multiple drug resistance.For example, can can be selected from metabolic antagonist with other chemotherapeutic that compound one of the present invention is used from combination therapy, as 5 FU 5 fluorouracil and strong pool (Gemcitabine), perhaps be selected from alkylating agent such as carboplatin (Carboplatin and cis-platinum (Cisplatin), perhaps be selected from signal conduction depressant drug such as Iressa, Herceptin and geldanamycin derivative.Conjoint therapy can adopt the mode of administration successively, promptly earlier with using second kind of medicament behind first kind of pharmaceutical treatment again; Also can be that two kinds of medicaments use simultaneously.
On the other hand, to can be used for treating with the cell hyperplasia be the non-cancer class disease of feature to compound of the present invention.In one aspect of the invention, the compound that the present invention describes is used for covered stent, is similar to line-webmaster, is used to end the cell growth, and the obstruction again that prevents restenosis or artery.In the clinical trial, compound of the present invention can cause one or more following phenomenons: (i) increase artery blood flow; The clinical symptom that (ii) palliates a disease; (iii) reduce the restenosis speed behind the cardiac valve procedure; Or (iv) stop/alleviate the process of CAS disease.
Description of drawings
Figure 1A is the HPLC figure of the product that obtains from QTB5 culture XAD elutriant among the embodiment 1.
Figure 1B is the mass spectrum of 4-demethylation epothilone B
Fig. 1 C is the mass spectrum of 4-demethylation epothilone d-2
Fig. 2 A is the color atlas of 4-demethylation epothilone d-1
Fig. 2 B is the LC/MS figure of 4-demethylation epothilone d-1
Fig. 3 is a 4-demethylation epothilone d-1
1H-NMR figure
Fig. 4 A is the color atlas of 4-demethylation epothilone d-2
Fig. 4 B is the LC/MS figure of 4-demethylation epothilone d-2
Fig. 5 is a 4-demethylation epothilone d-2
1H-NMR figure
Fig. 6 A is the color atlas of 4-demethylation epothilone B
Fig. 6 B is the LC/MS figure of 4-demethylation epothilone B
Fig. 7 A is the color atlas of 7-O-methyl isophthalic acid 2-hydroxyl esperamicin (BGE8)
Fig. 7 B is the LC/MS figure of 7-O-methyl isophthalic acid 2-hydroxyl esperamicin
Fig. 8 is a 7-O-methyl isophthalic acid 2-hydroxyl esperamicin
1H-NMR figure
Fig. 9 A is the color atlas of compd B GE5
Fig. 9 B is the LC/MS figure of compd B GE5
Figure 10 is compd B GE5's
1H-NMR figure
Figure 11 is the structural formula of compound formula 1 to formula 24
Embodiment
Prepare demethylation esperamicin and analogue thereof from the M.xanthus genetic engineering bacterium
Present embodiment has been described and has been made up the M.xanthus production bacterial strain that produces 4-demethylation-esperamicin.This makes up the MT functional group disappearance that the method that adopts homologous recombination makes the 8th functional unit in the esperamicin PKS gene (module 8).The 8th functional unit of esperamicin PKS has comprised a ketoreductase domain (KS functional group), the AT functional group that methylmalonyl CoA is special, one causes forming dimethylated MT functional group (methyltransgerase functional group) on 4 of Epothilone Cs.
The method of present embodiment uses the M.xanthus host bacterium that can produce esperamicin to carry out genetic manipulation.Being used for esperamicin of the present invention biosynthetic esperamicin PKS gene and dna sequence dna can obtain as producing the bacterial strain S.cellulosum from esperamicin from natural resource.Prepare total DNA from the natural production bacterial strain of esperamicin Sorangium cellulosum So ce90, method is referring to document Jaona etal., 1992, Plasmid 28:157-165.
(disappearance takes place the dna sequence dna of the MT functional group of module 8 causes the MT functionally inactive to make up bacterial strain by making the 8th functional unit among the esperamicin PKS.The dna sequence dna of MT functional group adjacent area is cloned by PCR method, and its Oligonucleolide primers is: OBG-1,5 '-TCCCATGGCGAAGCCGTGTTGC; And OBG-2,5 '-TTCCATGGACCGGAGGGCATCC.The PCR fragment of resulting 2.94 kb is through the NcoI enzymolysis, is cloned among the pLitmus28 that NcoI handled and obtains plasmid pBG101.With EcoRV part enzymolysis pBG101, the big fragment connection of postdigestive 2.78kb is obtained pBG102 then, introduce the disappearance of MT functional group dna sequence dna therefrom.PBG102 uses earlier the HindIII-AvrII enzymolysis, the dna fragmentation of 2.78kb is cloned into the SpeI-HindIII site of a pLitmus28 derivative vector, wherein this derivative vector contains kalamycin resistance gene and galK gene (the KG gene of 3 kb in the DraI site, Ueki et.al.1996, Gene 183:153-157), and obtain transforming plasmid pBG103, utilize electroporation technology (electroporation) plasmid pBG103 to be transferred to bacterial strain M.xanthus K111-32 (the Jelien ﹠amp that produces epothilone B; Shah, Antimicrobial.Agents Chemother.46:2772-2778).The screening transformant is picked out the kantlex sensitivity and the survival semi-lactosi resistance, therefrom identifies the clone strain of removing the KG gene.Recombinant bacterial strain is inoculated in the seed culture medium that contains 5ml CYE in the glass test tube, and this substratum comprises 10g/l casein Digestive system (MarcorType M), 5g/l yeast extract (Difco), 2g/l MgSO
47H
2O, 50mM HEPES damping fluid, pH7.6.Cell was cultivated 3 days in the shaking table of 200rpm at 30 ℃, and seed culture fluid is used to inoculate the CTS substratum of 50ml then, cultivates 7 days, and this substratum comprises 5g/l casein Digestive system (Marcor Type M), 2g/l MgSO
47H
2O and 50mM HEPES damping fluid, pH7.6, and 2% XAD-16.Collect XAD-16 from nutrient solution, and with the methyl alcohol of 10ml metabolism product such as wash-out esperamicin from XAD.With HPLC and mass spectroscopy methanol extract.The engineering bacteria that obtains a strain called after QTB5 thus can produce 4-demethylation esperamicin.
Figure 1A is under following HPLC condition, the total chromatography of ions of HPLC of the product that obtains from QTB5 culture XAD elutriant.Figure 1B and Fig. 1 C are the mass spectrums of compound 4-demethylation epothilone B [formula 17] and 4-demethylation epothilone d [formula 19].
The HPLC stratographic analysis of compound: carry out small-scale fermentation (50ml scale) earlier, identify the new product that the genetic engineering bacterial strain is produced with LC/MS and HPLC.Sample passes through preposition guard column (the guard column of a 4.6 * 10mm earlier; Inertsil; ODS-3,5 μ m), then by separator column (a 4.6 * 150mm; Inertsil; ODS-3,5 μ m), with 35%-100% acetonitrile concentration tonsure; flow velocity 1ml/min reaches tens minutes and monitors under UV250nm through carrying out wash-out.With this understanding, epothilone d comes out at 9.9 minutes wash-outs, and two isomer of 4-demethylation epothilone d come out at 8.8 minutes and 9.0 minutes wash-outs, and 4-demethylation epothilone B comes out at 6.5 minutes wash-outs.
The structure explanation of compound:
1The data of H NMR (400MHz) are with Bruker DRX400 spectrometer CDCl under 300K of QNP zaxis gradient detection head equipment
3Record under the solution environmental.CDCl
3The chemical transformation of solution refers to
1δ 7.26 in the H spectrum.By manual peak match(ing) (manual peaking-matching), adopting negative ion mode turbine-ion injection source (trubo-ionspray source) (ejection end voltage, 5500V with Applied Biosystems MarinerTOF spectrometer; 400 ℃ of spray chamber temperature; Spray nozzle voltage 110V), obtains the HRMS spectrum by FIA.
From preparation of M.xanthus QTB5 bacterial strain and purification 4-demethylation esperamicin
Present embodiment has been described from genetic engineering bacterial strain M.xanthus and has been separated and the fermentation The compounds of this invention.The host bacterium M.xanthus that includes esperamicin PKS synthetic gene, can produce primary product is epothilone B, disappearance takes place and causes inactivation in the dna sequence dna of the MT functional group of the 8th functional unit (module 8) in esperamicin PKS wherein, thereby this recombinant production bacterial strain has been produced 4-demethylation-epothilone B and D is a primary product.
Fermentation and purification process by M.xanthus QTB5 bacterial strain production 4-demethylation epothilone d and B that present embodiment is described are as follows.From fresh CYE culture medium flat plate, thalline is transferred to the glass culture test tube that 5mlCYE and 1 oil dripping acid methyl esters (10 μ l) are housed, cell cultures 2-5 days (30 ℃, 175rpm), until examining under a microscope the nutrient solution retrogradation.Then whole invisible spectro nutrient solution is transferred in the flask that contains 45ml CYE and 100 μ l Witconol 2301s, through the growth of 48+/-12 hour (30 ℃, 175rpm) after, nutrient solution can be used for preparing the cell bank that contains 20% glycerine of refrigerated storage.If be used for the seed liquor of fermentor tank inoculation, can carry out further seed amplification as required.The 0.45L seed liquor (inoculum size is 10%) that is used in the Feng Mahe bottle (Fembach flask) is inoculated the 5L seed fermentation jar that the 4.0L seed culture medium is housed.When the 100L fermentation cylinder for fermentation, the production substratum that uses in the fermentor tank is the CTS substratum, and 2%XAD-16, adds trace element solution 4ml/l and Witconol 2301 2ml/l.Trace element solution: dense H
2SO
4, 10ml/l; FeCl
3.6H
2O, 14.6g/l; ZnCl
2, 2g/l; MnCl
2.4H2O, 1g/l; CuCl
2.2H
2O, 0.42g/l; H
3BO
3, 0.31g/l; CaCl
2.6H2O, 0.24g/l and Na
2MoO
4.2H2O, 0.24g/l.Adding casein Digestive system (Macro Type M) and Witconol 2301 in the 100L fermentor tank, to carry out the main operating process of fed-batch fermentation as follows: carry out autoclaving in the fermentor tank (B.Bruan) to XAD-16 (Rohm and Haas) that 1.6kg is housed and 65L water.With the 0.3L trace metal solution behind the filtration sterilization (0.2 micron polyethersulfone membrane vesicle by sterilization in advance filters) and the MgSO of capacity
4Solution (to ultimate density 2g/l) directly adds in the fermenting container.After in the charging stock tank of 10L, the mixed solution 5L of the Witconol 2301 of the casein Digestive system of 15g/L and 175ml/L being sterilized, the mixed solution of 3.2L is added in the 100L fermentation container, make ultimate density respectively be 5g/L and 2ml/L respectively.Inject container with (using same capsule strainer) after the water filtration, the final volume that makes fermenting container is 70L.The stirring velocity of setting in the fermentor tank is 200rpm, and jet velocity remains on 0.1v/v/m.Pressure tank remains on 100-300 mbar.Control pH makes pH remain on pH7.4 by adding 2.5N potassium hydroxide and 2.5N sulfuric acid.Temperature is set at 30 ℃, by regulation and control stir speed (S.S.) 200-400rpm and air-flow 23-100Lpm, makes dissolved oxygen remain on 50% or a little more than 50% saturation ratio.4L inoculum from 5L fermentor tank inoculation (inoculum size is 5%) is in the fermentor tank of 100L then.Inoculate after one day, join in the fermentor tank every 1 hour mixture with casein Digestive system (150g/L) and Witconol 2301, rate of feeding is casein Digestive system 2g/L/ days, Witconol 2301 3ml/L/ days.About 12 days of inoculation back, fermentation are ended to collect XAD-16 and are obtained meta-bolites.The primary product that this method obtains is a 4-demethylation epothilone d, and two kinds of isomer products are arranged, and respectively account for 10-15mg/L, and 4-demethylation epothilone B is 2-3mg/L.
The evaluation of 4-demethylation epothilone analogs with separate:
Separation from fermenting culture and method of purification comprise following material and step: (1) collects XAD-16 with filter basket (150 μ m) from fermenting culture, and heap is gone in the post, with the water washing (1L/ minute) of 3 times of column volumes.17 liters of eluted product of methanol aqueous solution with 75% (last flow velocity remained on 1L/ minute) (may need to pile again post during hypertonia).(2) with 50% methanol aqueous solution diluted product storehouse, and be loaded in the post (Amicon VA180), the HP20SS resin (Mitsubishi) that 0.5L is crossed by 50% methanol aqueous solution balance of 5 times of column volumes in advance is housed in the post, load back (loading flow velocity is 1L/ minute), with the methanol wash of 1.3L 60%, use the methanol-eluted fractions (flow velocity 325ml/ minute) of 8L 75% then.With UV250nm monitor monitoring cut, the part that contains meta-bolites that obtains with product library simmer down to oily, in case of necessity, adds ethanol to reduce foaming under the vacuum condition on the Buchi-Rotavapaor rotatory evaporator.(3) with the 100ml methyl alcohol dried matter that suspends again, and be diluted to 45% methanol solution, the solution that obtains is loaded into (55 * 4.8cm on the 0.1L C18 reverse-phase chromatographic column, Bakerbond, 40 μ m), this chromatographic column is crossed (loading the flow velocity average out to 100ml/ minute) in advance by the methanol solution balance of 3 volumes 45%.With the methanol wash of 0.2L 45%, and use 0.5L 50% successively, 1.1L 55%, and 1.3L 60%, and the methyl alcohol of 0.5L 65% (flow velocity is 100mL/ minute) wash-out loads post.The product library that obtains is partly carried out stratographic analysis, and obtain 4-demethylation epothilone B in turn, 4-demethylation Epothilone C 1, C2 and 4-demethylation epothilone d 1, D2. further separates each product library in case of necessity.Each product library is diluted to 40%, squeeze into pump on the C18 reverse-phase chromatographic column of 70ml (9 * 10cm), flow velocity average out to 360ml/ minute.With the methanol wash of 0.1L 40%, and load post with the ethanol elution of 350ml 100%.With rotatory evaporator elutriant is evaporated to drying.(4) resuspension solid in 10ml acetone is removed insolubles with No. 2 filter paper filterings of Whatman.After the acetone extracting, in filtrate, add the 0.2g decolorizing carbon.Stir this mixture and reach 1 hour, use No. 50 filter paper filterings of Whatman then.Merging filtrate and rotary evaporation are to dry. and (4B, optional).Exsiccant material (4-demethylation epothilone d or B) is suspended in 5L 50% methyl alcohol again, and (55 * 4.8cm) go up (loading the flow velocity average out to 80ml/ minute) to be loaded into the 0.1L C18 post of being crossed by 3 volumes, 50% methanol solution balance in advance.Then load post with methanol wash, and with 65% methyl alcohol isocratic elution with 0.1L 50%.(5) under good stirring, in beaker,, and slowly add the water of 17ml with the dry thing of the ethanol resuspension of 15ml 100%.Adding a small amount of (1mg) crystal seed in case of necessity forms to promote crystalline.
The feature of epothilone analogs:
4-demethylation epothilone d-1[formula 19]: HRESITOFMS m/z; C
26H
40NO
5S calculated value: 478.2638; [M+H]
+478.2622.The color atlas of 4-demethylation epothilone d-1, LC/MS and
1The data of H-NMR are seen Fig. 2 A respectively, Fig. 2 B and shown in Figure 3.
4-demethylation epothilone d-2[formula 19]: HRESITOFMS m/z; C
26H
40NO
5S calculated value: 478.2638; [M+H]
+478.2622.The color atlas of 4-demethylation epothilone d-2, LC/MS and
1The data of H-NMR are seen Fig. 4 A respectively, Fig. 4 B and shown in Figure 5.
4-demethylation epothilone B [formula 17]: color atlas and LC/MS data are seen respectively shown in Fig. 6 A and Fig. 6 B.
Utilize the S.cellulosum genetic engineering bacterium to produce 4-demethylation epothilone analogs
Present embodiment has been described from esperamicin and has been produced bacterial strain S.cellulosum, by gene recombination technology the dna sequence dna of its MT functional group of the 8th functional unit (module 8) in the PKS of esperamicin being lacked, is primary product thereby this production bacterial strain produces 4-demethylation-esperamicin.As describing in the M.xanthus recombinant bacterial strain, the MT functional group of the 8th functional unit (module 8) is undergone mutation or is lacked in the esperamicin PKS gene of S.cellulosum, makes up the bacterial strain that produces 4-demethylation-esperamicin or its analogue.The fermentation condition of S.cellulosum is with reference among the PCT:WO 93/101021 and Gerth et al., and 1996, the flow process described in the J.ofAntibiotics, 49:560-563.
The genetic engineering bacterial strain method that present embodiment makes up the S.cellulosum So ce90 that produces 4-demethylation esperamicin is as follows.Similar with the QTB5 strain construction, the 2.78kb segment that contains among the plasmid pBG102 of MT functional group disappearance is cloned among the plasmid pBG105, its pBG105 include from the kantlex of Tn5 and bleomycin resistance marker and in conjunction with source RP4-oriT to form plasmid pBG106.Plasmid pBG106 is transferred and imports among the E.coli ED8767, it contains helper plasmid pUZ8 (Hedges and Matthew, 1979, Plasmid 2:269-278), this helper plasmid provides the function of allosome transfer for the transfer of plasmid, referring to Jaoua et al.1992, Plasmid 28:157-165.The reorganization E.coli bacterium that obtains with carry out mating as the combining in the test (conjugate) of S.cellulosum So ce90 bacterium of the anti-streptomycin of donor sudden change, this S.cellulosum So ce90 utilizes the feature of anti-streptomycin can carry out selective screening and draws and eliminate as the E.coli bacterium of acceptor and screen anti-streptomycin, the zygote of kantlex and bleomycin.For the mating combination, about 5 * 10
9The S.cellulosum cell mix with 1: 1 cell proportion and reorganization E.coli bacterium, this S.cellulosum cell is from the nutrient solution that is grown in 30 ℃ stationary phase morning.Then, nutrient solution centrifugal 10 minutes in the speed of 4000rpm is suspended in 0.5ml G51b substratum (0.2% glucose, 0.5% starch, 0.2% peptone, 0.1%probion, 0.05%CaCl again
2.2H
2O, 0.05%MgSO
4.7H2O, 1.2%HEPES, Ph7.4) in, and point drops in the dull and stereotyped center of the SolE nutrient agar that contains the 50mg/l kantlex.SolE nutrient agar: 0.35% glucose, 0.05% pancreas albumen, 0.15%MgSO
4.7H
2O, 0.05% ammonium sulfate, 0.1%CaCl
2, 0.006%K
2HPO
4, 0.01% V-Brite B, 0.0008%Fe-EDTA, 1.2%HEPES, the aseptic stable state S.cellulosum culture of 3.5% (v/v) suspension, pH regulator to 7.4.Be collected in 30 ℃ of cells of cultivating after 24 hours, and be suspended in again in the 0.8mlG51B substratum, the suspension of 0.1-0.3ml is tiled in to contain selects medicine [phleomycin (30mg/l), Streptomycin sulphate (300mg/l), kantlex (50mg/l)] the solid medium flat board on,, separate with plastic hoop at the bacterium colony of selecting to grow on the flat board after 8-12 days through 30 ℃ of cultivations, line is carried out second and is taken turns selection and purification on same selection substratum.So the bacterium colony that obtains after the nutrient agar of selecting is cultivated 7 days should be by homologous recombination the plasmid integration that changes over to be gone into zygote in the karyomit(e).After the non-selective incubation growth of several generations in the liquid medium within, culture is tiled on the SolE agar through after the suitable dilution, to separate the reorganization bacterium colony of phleomycin-sensitivity, its reorganization bacterium colony has passed through the homologous recombination second time of the plasmid disappearance that makes integration.In the responsive bacterium colony of isolated three kinds of phleomycin, wherein two to be returned to wild type be primary product with production Epothilones A and B, yet a remaining recombination mutation with expectation is attended by the disappearance of MT at its chromosomal esperamicin gene.Mutant bacteria is designated as QTBG2.Analyze with HPLC, to produce 4-demethylation Epothilones A, B, C and D are primary product in the affirmation QTBG2 shake-flask culture thing.
Following method is used for separating the plain analogue in dust slope from S.cellulosum recombinant bacterial strain QTBG2 fermentation.
Middle substratum G52: less salt yeast extract (BioSpringer, Maison Alfort, France) 0.2%, MgSO
4.7H
2O 0.1%, CaCl
2.2H
2O 1g, defatted soyflour Soyamine 50T (LucasMeyer, Hamburg, Germany) 0.2%, yam starch Noredux A-150 (Blattnann, Eaedenswil, Swizerland) 0.8%, dextrose anhydrous 0.2%, EDTA-Fe (III)-Na salt (8g/l) 1ml regulates PH to 7.4 with KOH.
Main medium 1B12: yam starch Noredux A-150 (Blattmann, Waedenswil, Swizerland) 2%, defatted soyflour Soyamine 50T (Lucas Meyer, Hamburg, Germany) 1.1%, EDTA-Fe (III)-Na salt (8g/l) 1ml regulates PH to 7.8 with KOH.
Fermentation: keep culture: the frozen cell preface of taking out 1ml from liquid nitrogen is inoculated among the G52 of 10ml, and at 30 ℃, 180rpm cultivated 3 days under the condition of 25mm displacement.The inoculum of 5ml was added among the G52 of 45ml growth 3 days, and continued among the G52 that culture with 50ml adds 450ml growth 3 days, 50mm displacement (displacement)).Later every 3-4 days, the 50ml inoculum is inoculated among the G52 of 450ml with 10% inoculum size, to carry out the successive seed culture.
The main fermenting culture of 100L: sterilize inoculum in the middle of the inoculation 15L after the 100L fermentor tank added the 1B12 substratum of 60L and 2%XAD-16.Fermentation culture 6-7 days, condition was: 30 ℃, 200rpm, 0.5L air/L/ minute, overvoltage was 0.5bars, with H2SO4/KOH control pH value to 7.6+/--0.5.
Inoculum in the middle of the 20L: will contain 18L G52 substratum and inoculate with seed culture fluid before the 2L at the fermentor tank of 30L, and continue to cultivate 3-4 days, condition is: 30 ℃, and 250rpm, 0.5L air/L/ minute, overvoltage was 0.5bars, does not control pH value.(Tegosipon, Goldschmidt AG is Essen) to prevent that foam from forming to add 10ml silicone resin defoamer.
Product analysis: from fermentor tank, take out 50ml and contain polystyrene resin AmberliteXAD-16 (Rohm+Hass, Frankfurt, sample Germany).Nylon mesh with 150mm is filtered resin, after the less water washing, adds 10ml methyl alcohol and also at room temperature shakes 30 minutes.Get the 2ml supernatant liquor and be used for the HPLC analysis.
From the 100L fermenting culture, separate meta-bolites: the XAD that collects about 8L with strainer.After washing with water, add 30L methyl alcohol and stir 30 minutes wash-outs.Remove methyl alcohol by the vaporizer concentrate eluant, the water of usefulness EtOAc extracting remainder 3 times.Extract is concentrate drying in rotatory evaporator, is dissolved in then in the 500ml methyl alcohol, with folding strainer elimination insolubles.Add solution in the Sephadex LH20 post and use methanol-eluted fractions.To contain carrying of meta-bolites and heat up in a steamer partial concentration, and be loaded in the C18 chromatographic column and separate to dry and be dissolved in once more in 50% the methyl alcohol.Following step will be according to the carrying out of (3) description among the embodiment 2.
Contain the production of azoles esperamicin
Present embodiment has been described azoles (oxazole) counterpart for preparing 4-demethylation epothilone d by the fermentation condition of adjusting the M.xanthus host cell, and the formation of its azoles comes from Serine and combines the non-Yeast Nucleic Acid polypeptide of epoB gene product synthetic enzyme (NRPS) with the competition of halfcystine.
In the present embodiment, produce bacterium to esperamicin,, in the fermentation of preparation epothilone compounds, adjust fermentation condition for example by S.cellulosum bacterial strain provided by the invention or M.xanthus bacterial strain by in fermentation, replenishing superfluous L-Serine.For example, when the condition of describing is carried out fermentation strain QTB5, in fermention medium, prepare esperamicin in adopting embodiment 2, with HPLC and mass spectroscopy fermentation broth sample with the Serine that replenishes 75mM.To the analysis revealed of QTB5 tunning, Serine additional caused containing in the 4-demethylation esperamicin azoles counterpart to be increased.
The production of esperamicin derivatives BGE8 and BGE5
Present embodiment has been described the novel epothilone analogs that epothilone d or derivative obtain through Saccharopolyspora erythraeaeryA mutant strain QTB14 bio-transformation.
R2YE substratum with refrigerated one bottle S.erythraea eryA mutant strain QTB14 (no erythromycin, erythramycin produces mutant bacteria) inoculation 5ml.Culture was cultivated 24 hours on 30 ℃ shaking table, and the epothilone d of adding 10mg or suitable The compounds of this invention continue to cultivate 2-3 days in flask.The Separation and Recovery esperamicin derivatives.Add XAD-16 at bio-transformation liquid and stirred 30 minutes, with 100 ml methanol wash-outs, by the vaporizer concentrate eluant, the water of usefulness EtOAc extracting remainder 3 times.Extract is concentrate drying in rotatory evaporator, will contain carrying of meta-bolites and heat up in a steamer part and utilize HPLC to carry out separation and purification.
For example, from reclaim the derivative of several epothilone ds that obtain as the initial compounds epothilone d.
A.7-O-methyl isophthalic acid 2-hydroxyl esperamicin (BGE8) [formula 5]: HRESITORMS m/z524.3019; C
28H
46NO
6S[M+H]
+Calculated value, 524.3040.The color atlas of 7-O-methyl isophthalic acid 2-hydroxyl esperamicin, LC/MS and
1The H-NMR data are seen Fig. 7 A respectively, Fig. 7 B and shown in Figure 8.
B.BGE5[formula 21]: HRESITORMS m/z 508.2736; C
27H
42NO
6S[M+H]
+Calculated value, 508.2727.The color atlas of compd B GE5, LC/MS and
1The H-NMR data are seen Fig. 9 A respectively, Fig. 9 B and shown in Figure 10.
C.11-hydroxyl epothilone d [formula 22]: HRESITORMS m/z 508.2703; C
27H
42NO
6S[M+H]
+Calculated value, 508.2727.
1H-NMR(CDCl3，400MHz)δ6.97(s，H-19)，6.56(s，H-17)，5.28(t，J＝8Hz，H-13)，5.22(dd，J＝6.0，3.0Hz，H-15)，4.74(t，J＝8.0Hz，H-11)，4.42(dd，J＝11.0，2.0Hz，H-3)，3.75(dd，J＝6.5，1.0Hz，H-7)，3.24(qd，J＝7.0，1.0Hz，H-6)，2.69(s，H-21)，2.52(2H，m，H2-14)，2.44(dd，J＝14.0，11.0，H-2a)，2.19(dd，J＝14.0，2.0，h-2B)，2.01(d，J＝1.0Hz，H-27)，1.90(m，H-8)，1.72(m，H-10a)，1.70(s，H-26)，1.58(H-10b)，1.55(m，H-9a)，1.37(s，H-23)，1.29(m，H-9b)，1.14(s，H-22)，1.03(d，J＝7.0，H-25)。
D.26-hydroxyl epothilone d [formula 23]: HRESITORMS m/z 508.2723;
C
27H
42NO
6S[M+H]
+Calculated value, 508.2727.
1H-NMR (CDCl3,400MHz) δ 6.98 (s, 1H), 6.66 (s, 1H), 5.45 (dd, J=9.4,5.8Hz, 1H), 5.24 (dd, J=8.8,2.0Hz, 1H), 4.33 (dd, J=11.2,2.4Hz, 1H), 4.09 (d, J=13.2Hz, 1H), 4.01 (d, J=13.2Hz, 1H), 3.69 (dd, J=4.4,2.0Hz, 1H), 3.18 (qd, J=6.8,2.4Hz, 1H), 2.71 (s, 3H), 2.63 (dt, J=15.2,9.2Hz, 1H), 2.46 (dd, J=14.8,11.2Hz, 1H), 2.37 (m, 1H), 2.25 (overlapping, 1H), 2.24 (dd, J=14.8,2.4AHz, 1H), 2.12 (m, 1H), 2.05 (d, J=1.2Hz, 3H), 1.76 (m, 1H), 1.67 (m, 1H), 1.36 (s, 3H), 1.34 (m, 3H), 1.18 (d, J=6.8Hz, 3H), 1.05 (s, 3H), 0.98 (d, J=9.6Hz, 3H).
E.21-hydroxyl epothilone d [formula 24]: HRESITORMS m/z 508.2713;
C
27H
42NO
6S[M+H]
+Calculated value, 508.2727.
1H-NMR (CDCl3,400MHz) δ 7.01 (s, 1H), 6.59 (s, 1H), 5.34 (m, 1H), 5.24 (d, J=8.6Hz, 1H), 5.13 (dd, J=10.0,4.9Hz, 1H), 4.90 (s, 2H), 4.30 (dd, J=11.2,2.7Hz, 1H), 3.70 (dd, J=4.0,2.4Hz, 1H), 3.14 (qd, J=6.8,2.1Hz, 1H), 2.62 (dt, J=15.0,9.8Hz, 1H), 2.47 (dd, J=14.7,11.1Hz, 1H), 2.30 (overlapping, 1H), 2.27 (dd, J=14.7,2.7Hz, 1H), 2.06 (s, 3H), 2.00 (m, 1H), 1.89 (m, 1H), 1.75 (m, 1H), 1.68 (m, 1H), 1.66 (s, 3H), 1.33 (s, 3H), 1.26 (overlapping, 3H), 1.19 (d, J=6.9Hz, 3H), 1.05 (s, 3H), 1.01 (d, J=7.0Hz, 3H).
The bio-transformation of preparation 21-hydroxyl esperamicin derivatives
1. by 4-demethylation epothilone d [formula 19] preparation 4-demethylation, the bio-transformation of 21-hydroxyl 4-demethylation epothilone B [formula 14]
Under 30 ℃, in the 1L substratum, cultivate the S.cellulosum esperamicin and produce bacterium or esperamicin PKS mutant strain (no esperamicin is produced bacterium) or QTBG2, its substratum contains yam starch (0.8%), glucose (0.8%), defatted soyflour (0.2%), yeast extract (0.2%), CaCl
2.2H
2O (0.1%), MgSO
4.7H
2O (0.1%), Fe-EDTA (8mg/L), 2%XAD-16 regulates PH7.4 with KOH.With 150rpm stir culture thing, and with 0.1 volume/minute speed spray sterilised air.Cultivate after 4 days, separate XAD, nutrient solution by 0.3 micron membranes cross flow filter to be concentrated to 0.3 liter of volume.The methanol solution that in cell concentration liquid, adds 4-demethylation epothilone d (0.5g/L).Continue to cultivate at 30 ℃, stir, spray sterilised air with 6 liters/minute speed simultaneously with 450rpm speed.After 24 hours, add 10mlXAD-16 and in culture, also continue to stir half an hour.In filter basket, collect XAD-16, and wash with water to remove liquid nutrient medium and cell.Then with the XAD methanol-eluted fractions.Concentrate eluant is also used ethyl acetate extraction.Use Na
2SO
4Dry extract filters and evaporation, obtains thick esperamicin.Use SiO
2Chromatogram (1: 2 hexane/ethyl acetate) separation obtains 21-hydroxyl, 4-demethylation epothilone d [formula 15] and B[formula 14].
2.4-the demethylation epothilone B is to the 4-demethylation, the bio-transformation of 21-hydroxyl epothilone B
Refrigerated one bottle Amycolata autotrophica ATCC35203 bacterial strain (1ml), described in PCT publication No.WO00/39276, be used to inoculate the BGA substratum of 50ml, this substratum comprises the dextrose of 10g/L, the maltose extract of 5g/L, the yeast extract of 5g/L and the peptone of 1g/L.Culture was cultivated 1 day on shaking table under 30 ℃.The 4-demethylation epothilone B of interpolation 10mg or suitable The compounds of this invention continued 2-3 days on 28 ℃ of shaking tables in flask.From culture, isolate converted product and reclaim product, confirm as the 21-hydroxyl counterpart of initial compounds with HPLC and mass spectroscopy.
Produce the bio-transformation of 14-hydroxyl esperamicin derivatives
With 1 bottle (1ml) refrigerated Streptomyce sp.ATCC55098 inoculation 5ml FM-6 substratum.Culture was cultivated 2 days on 30 ℃ of shaking tables.The culture of 2.5ml is transferred in the FM-6 substratum of 50ml in the flask, cultivated 24 hours down at 30 ℃ then, the epothilone d of adding 10mg or suitable The compounds of this invention continue to cultivate 2 to 3 days in flask.From culture, isolate converted product and reclaim esperamicin derivatives.For example, 14-hydroxyl epothilone d is separated from the epothilone d as initial compounds as primary product.
14-hydroxyl epothilone d: HRESITORMS m/z 508.2728; C
27H
42NO
6S[M+H]
+Calculated value, 508.2727.
1H-NMR(CDCl3，400MHz)δ7.02(s，1H)，6.67(s，1H)，5.22(d，J＝9.4Hz，1H)，4.98(d，J＝9.4Hz，1H)，4.57(t，J＝9.2Hz，1H)，4.26(bro?d，J＝10.1Hz?1H)，3.70(m，1H)，3.14(qd，J＝6.7，1.7Hz，1H)，2.70(s，3H)，2.45(m，1H)，2.44(dd，J＝14.3，11.2Hz，1H)，2.22(bro?d，J＝14.3Hz?1H)，2.17(s，3H)，1.96(m，1H)，1.76(m，1H)，1.73(m，1H)，1.72(s，3H)，1.29(m，3H)，1.34(s，3H)，1.20(d，J＝6.8Hz，3H)，1.06(s，3H)，1.03(d，J＝7.0Hz，3H)。
The chemistry epoxidation
Present embodiment has been described when A-Q connects together C=C pair of key (deoxy compound of formula I compound) of formation, the epoxidation reaction of formula I compound.At-78 ℃, (0.1M in the acetone is 17ml) to the 10ml CH of deoxy compound of the present invention (505mg) to drip dimethyl ethylene oxide solution
2Cl
2In the solution.Mixture heating up to-50 ℃, was kept 1 hour, add another part dimethyl ethylene oxide solution (5ml) afterwards, be reflected at-50 ℃ and continued 1.5 hours down.N at-50 ℃
2Dry reagent contained within the steam.This universal method is applicable to from other compound manufacturing of the present invention corresponding 12, the 13-epoxy derivative.Use SiO
2The chromatogram purified product.
The preparation of 4-demethylation epothilone B lactan
Step 1: in argon gas, handle the tetrahydrofuran (THF)/water (10: 1v/v) 4-demethylation epothilone B (2.62g) in the solution and sodiumazide (0.49g) that is dissolved in the 55ml degassingization with 4 (triphenyl phosphorus) palladium (0.58g).Be reflected at 45 ℃ and kept 1 hour,, and use ethyl acetate extraction then with the dilution of 50ml water.With saturated NaCl liquid washing extract, use Na
2SO
4Drying is filtered the back evaporation.Use SiO
2The chromatogram purified product.
Step 2: under the envrionment temperature, in argon gas, be dissolved in above-mentioned steps 1 product (565mg, 15-trinitride) 2 hours of 15ml THF/ water (10: 1) solution with toluene (3ml) solution-treated of trimethylammonium phosphorus 1.0M.Enriched mixture is used SiO
2The chromatogram purified product.
Step 3: with above-mentioned steps 2 product (540mg, 15-ammonia) (20: 1v/v) solution is cooled to 0 ℃, uses I-hydroxybenzotriazole (0.135g) and 1-(3-dimethyl aminopropyl)-3-ethyl-carbodiimide hydrochloride (0.5g) to handle then to be dissolved in 15ml acetonitrile/dimethyl formamide.With mixture heating up under envrionment temperature and kept 12 hours, dilute with water and use ethyl acetate extraction then.Then water, saturated NaHCO
3, and saturated NaCl liquid washing extract, use Na then
2SO
4Drying is filtered and evaporation.Use SiO
2The chromatogram refined product is to obtain 4-demethylation epothilone B lactam form [formula 11] (product of the step 3 in the reaction formula).
The preparation of 4-demethylation epothilone d lactan
Tetrahydrofuran (THF) (20ml) solution of tungsten hexachloride (0.76g) is at-78 ℃, with 1.6M just-hexane solution (2.5ml) of butyllithium handles.Mixture is heated to envrionment temperature with the time more than 20 minutes.The solution 13.8ml that obtains is added in the 4-demethylation epothilone B lactan (360mg) that is dissolved in the 2ml tetrahydrofuran solution at ambient temperature.After 30 minutes, reactant is cooled to 0 ℃, and uses saturated NaHCO
3(10ml) handle, filter the back evaporation.Use SiO
2The chromatogram purified product obtains 4-demethylation epothilone d lactan [formula 10] and (sees and implement the step 4) in the reaction formula in 9.
Preparation 7-methoxyl group esperamicin
The a.TESCl/ imidazoles; The esperamicin of 210mg can be with TESCl and 0 ℃ of 5mlCH that contains the 2eq. imidazoles of 1.5eq.
2Cl
2The solution protection;
B. deprotection: with the NH of 0.4ml
2Cl/NaH
2PO
4CH with 0.4ml
3The CN deprotection.
C. at THF: make with MeBr and K-O-t-Bu among the DMSO (1: 1) to methylate.
D. use AcOH (ACN: H
2O) come deprotection.
Present embodiment has been described the chemical modification of the C-7 hydroxyl of formula I compound to the C-7 methoxyl group.
The preparation of preparation 12-hydroxyl esperamicin
Present embodiment is described and is known clearly by the C-12 oxy-compound of chemical modification preparation formula I.
Embodiment 13:
4-demethylation-epothilone B is converted into 21-OH, 4-demethylation-epothilone B and D and 21-NH2-4-demethylation-epothilone B and D.
(a) 4-demethylation-epothilone B of 1.98g (3.9mmol) is dissolved in the anhydrous CH of 60ml under argon gas
2Cl
2In.The m-chlorine peroxybenzoic acid (4.17mmol, 1.07 equivalents) that adds 0.72g in solution, 25 ℃ were stirred 25.5 hours down.This reaction mixture NaHCO of 60ml
3End, use the CHCl of 3 * 75ml then
3Extraction.Then water, saturated NaHCO
3, and saturated NaCl liquid washing extract, use Na then
2SO
4Drying is filtered and evaporation.Use SiO
2The chromatogram refined product is to obtain the N-oxide compound of 4-demethylation epothilone B.
(b) the N-oxide compound of 0.97g is dissolved in the anhydrous CH that contains 35ml under argon gas
2Cl
2, 2, and the 6-lutidine (1.73ml, 14.8mmol, 8equ) and (CF3CO)
2O (1.84ml, 13mmol, 7equ.) in the pipe, sealing then, 70 ℃ were heated 25 minutes.With the mixed solution cooling, go down to desolventize at argon gas, then vacuum concentration.Reaction is added the NH of 2.9ml 28% by the MeOH of 25ml dilution
4OH (solution), 45 ℃ were heated 20 minutes down then, again cool to room temperature.Thick product concentrates and makes 21-OH-4-demethylation-epothilone B with the silica gel chromatography chromatography and becomes [formula 14]. and adopt currently known methods,, make 21-OH-4-demethylation-epothilone d [formula 15] 21-OH-4-demethylation-epothilone B epoxide deoxidation.
(c) under 0 ℃ of argon gas, and stirring 21-OH-4-demethylation-epothilone B in the tetrahydrofuran (THF) of 20ml (957mg, 1.83mmol); (604mg, 2.19mmol 1.2equ.), stirred 3 minutes to add the biphenyl phosphoryl trinitride of 0.47ml again; Adding 1,8-diazabicyclo[5.4.0] (1.83mmol 1equ.), stirred 2 hours at 0 ℃ undec-7ene, was warming up to 25 ℃ and kept 20 hours for 0.27ml, 278mg.With 150ml ethyl acetate dilution and wash with water, water is used ethyl acetate extraction more then.Then organic phase is used saturated Na
2SO
4Drying is filtered and evaporation.Use SiO
2The chromatogram refined product is to obtain the 4-demethylation epothilone B of 21-trinitride.
(d) under argon gas, (0.163g, 2.145mmol 1.1equ.) join among the THF of the 30ml that contains the 21-trinitride with 0.22ml trimethylammonium phosphuret-(t)ed hydrogen.The H that adds 5.5ml again
2O, 25 ℃ of stirrings.After 3 hours, add the liquid NH of 3ml 28%
4OH finishes the conversion from the phosphoryl imines to amine.Stir after 1 hour, go down to desolventize in vacuum environment.Thick product concentrates and makes 21-amino-4-demethylation-epothilone B [formula 13] with the silica gel chromatography chromatography.
(e) 21-amino-4-demethylation-epothilone B (126mg, in ethanol 0.24mmol) (4ml) solution, add triethylamine (67ul, 0.48mmol, 2equ.) and 2-t-butyl-sodium bicarbonate (65mg, 0.3mmol, 1.25equ.).Reactant stirred 2 hours.Thick product concentrates and makes 21-N-BOC-amino-4-demethylation-epothilone B with the silica gel chromatography chromatography.
(f) anhydrous THF (3ml) places the flask of oven dry under argon gas, is cooled to-78 ℃.Under argon gas stream, with WCl
6(206mg, 0.52mmol 2equ.) are added among the refrigerative THF, just adding-butyllithium (the 1.6M solution of the 0.65ml in hexane, 1.04mmol, 4equ.).Reaction is shifted out from-78 ℃ cooling pool, at room temperature stirred 15 minutes, be put into 0 ℃ of restir 5 minutes, add 21-N-BOC-amino-4-demethylation-epothilone B solution.Being reflected at 0 ℃ kept 45 minutes.With saturated liquid NaHCO3 (5ml) stopped reaction, reactant is at saturated liquid NaHCO
3(25ml) and CH
2Cl
2(50ml) form two-phase.Liquid phase CH
2Cl
2Extract 3 times.Then organic phase is used saturated Na
2SO
4Drying is filtered and evaporation.Use SiO
2The chromatogram refined product is to obtain 21-N-BOC-amino-4-demethylation-epothilone d.
G. at 0 ℃, (98mg is 0.16mmol) with the CH that contains 10% 3 fluoro alkanoic acid of precooling for 21-N-BOC-amino-4-demethylation-epothilone d
2Cl
2(4ml) solution-treated.After 40 minutes, reaction rises to normal temperature, after 20 minutes, adds three pure fluoro alkanoic acids of 0.6ml.After 50 minutes, add the three fluoro alkanoic acids of 0.5ml again.After 2 hours, reaction is fast near 50% o'clock that finishes, and vacuum is removed solvent.Residue ethyl acetate (50ml) and saturated NH
4Ethyl acetate (3 * 50ml) extractions are used in the dissolving of OH (50ml) aqueous solution again.Then organic phase is used saturated Na
2SO
4Drying is filtered and evaporation.Use SiO
2The chromatogram refined product is to obtain 21-amino-4-demethylation-epothilone d [formula 12].
Bioactive mensuration
Adopt sulphur rhodamine B (SRB) test (referring to Skehan et al., J.Natl.Cancer Inst.82:1107-1112 (1990) lists in this with for referencial use), screen the antitumour activity of alternative cpd of the present invention four kinds of different tumour cell strains.Cultured cells is counted then by trypsinized, and is diluted to suitable concentration (5000-7500 cell/100 μ l) with growth medium.The microtiter plate that the cell suspending liquid in 100 μ l/ holes is added the 96-hole.After 20 hours, the 100 μ l compounds that will be diluted to 2 * 1000nM to 2 * 0.001nM in growth medium are added in each hole.After 3 days cultivation,,, at room temperature dyeed 20 minutes with 0.2%SRB/1% acetate then 4 ℃ of following fixed cells 1 hour with the trichoroacetic acid(TCA) of 100 μ l10%.The unconjugated dyestuff of acetate rinsing with 1%, with 200 μ l, the Tris alkali dissolution bonded SRB of 10mM.The amount of combination dye is calculated by the OD value of measuring wavelength 515nm., the amount of combination dye is directly proportional with total cell protein amount.Data KaleidaGraph programanalysis and calculation of half inhibitory concentration (IC50).Replicate(determination) epothilone d and B are to make comparisons.The cell toxicity test result of the selection compound of testing among the present invention is as follows.The also available similar methods of other compound of the present invention is measured.Novel epothilone compounds of the present invention sees the following form shown in the table 1 for the anti-proliferative effect of the mankind's cancerous cell line.The result shows them not only to general non-resistant tumors cell, and the tumour cell of multi-drug resistant is had powerful antibiosis long-term job.
(cell-based) tubulin polymerization test determination mechanism of action with cell levels.In the test, under 37 ℃, the MCF-7 cell that is incubated in the 35mm culture dish was handled 1 hour with every kind of compound of 1 μ M.Do not contain the PBS washed cell twice of Ca and Mg with 2mL, at room temperature use lysate (20Mm Tris, ph6.8, the 1mM MgCl of 300 μ L
2, 2mM EDTA, 1% Triton X-100 adds proteinase inhibitor) handle cell 5-10 minute and make lysis.Lysate is transferred in the Eppendorf pipe of 1.5-mL.Under the room temperature, lysate centrifugal 12 minutes at 18000g.To contain the supernatant liquor of solubility or unpolymerized tubulin and contain the granular precipitate and separate of insoluble or polymeric tubulin, and transfer in the new pipe.Then with the lysate of the 300 μ L granular precipitation that suspends again.Carry out each sample of immunoblotting assay by SDS-PAGE with 'beta '-tubulin antibody (Sigma),, use the amount of 'beta '-tubulin signal on the NIHImage programanalysis trace then, thereby measure the variation of tubulin polymerization in the cell.
Tubulin polymerization test explanation 4-demethylation esperamicin, BGE8, BGE5 have the mechanism of action identical with epothilone d, and they all can promote the polymerization of tubulin strongly under test condition, to epothilone d similar kinetics and effectiveness are arranged.Other compound of the present invention also same methods is measured.
Table 1
| Compound | Cell growth half-inhibition concentration (IC 50，nM) | |||
| MCF-7 (mammary cancer) | NCI/ADR-RES (MDR mammary cancer) | SF-268 (neurospongioma) | NCl-H460 (lung cancer) | |
| Epothilone d | ????9 | ???34 | ??17 | ??12 |
| Epothilone B | ????0.5 | ???2 | ??0.8 | ??0.7 |
| 4-demethylation-epothilone d-1 | ????40 | ???500 | ??50 | ??52 |
| 4-demethylation-epothilone d-2 | ????7 | ???28 | ??11 | ??10 |
| ????BGE8 | ????10 | ???54 | ??30 | ??19 |
| The 14-OH-epothilone d | ????35 | ???360 | ??42 | ??45 |
| The 11-OH-epothilone d | ????21 | ???169 | ??42 | ??29 |
| The 21-OH-epothilone d | ????37 | ???257 | ??46 | ??49 |
| 4-demethylation-epothilone B | ????1 | ???3.5 | ??1 | ??1 |
| ????BGE5 | ????29 | ???281 | ??35 | ??32 |
| Taxol (Taxol) | ????2 | ???＞3000 | ??8 | ??9 |
Claims (19)
1. the gene of the esperamicin PKS that modifies of encoding is characterized in that MT functional group dna sequence dna in the 8th assembly of PKS gene by sudden change, disappearance or replacement and inactivation.
2. recombinant host cell that comprises the esperamicin PKS gene of the described modification of claim 1, this host cell is any cell that comprises esperamicin PKS gene, makes esperamicin PKS gene in the host cell by the esperamicin PKS gene substitution of described modification by the dna homology recombinant technology.
3. host cell as claimed in claim 2 is characterized in that this host cell is Myxococcusxanthus cell or Sorangium cellulosum cell.
4. esperamicin derivatives, this compound has following general formula (I)
Wherein,
A-D is the carbon-carbon double bond of following formula (a) or is the epoxide group of formula (b):
Q and R1 are independently selected from H, C
1-4Alkyl or NH
2In any one,
R4 does not exist, or when A-D was the C-C singly-bound, R4 was an oh group;
R2, R3 are selected from H, hydroxyl, NH independently of one another
2And CH
3
X is O or N-R, and wherein R is H, NH
2, aryl, or CH
3
W is S or O.
5. esperamicin derivatives as claimed in claim 4 is characterized in that described compound is 4-demethylation epothilone d or 4-demethylation epothilone B.
6. a method for preparing the described esperamicin derivatives of claim 5 is characterized in that this method adopts the described recombinant host cell of cultivation claim 2.
7. the method for preparing esperamicin derivatives as claimed in claim 6 is characterized in that this method adopts the described recombinant host cell of cultivation claim 3.
8. esperamicin derivatives, this compd B GE5 has following structure:
9. esperamicin derivatives as claimed in claim 4 is characterized in that described compound Q in general formula (I) is CH
3, as shown in the formula:
10. esperamicin derivatives as claimed in claim 9, it is following various to it is characterized in that described compound is selected from:
11. esperamicin derivatives as claimed in claim 4 is characterized in that described compound is that R1 is H in the general formula (I), i.e. following formula general formula (II)
Formula (II)
12. esperamicin derivatives as claimed in claim 11, it is following various to it is characterized in that described compound is selected from:
13. esperamicin derivatives as claimed in claim 11 is characterized in that described compound W in general formula (II) is O.
14. esperamicin derivatives as claimed in claim 13, it is following various to it is characterized in that described compound is selected from:
15. esperamicin derivatives as claimed in claim 4 is characterized in that described compound is that R4 was an oh group when A-D was the C-C singly-bound in the general formula (I), as shown in the formula general formula:
16. esperamicin derivatives as claimed in claim 15, it is following various to it is characterized in that described compound is selected from:
17. esperamicin derivatives as claimed in claim 4 is characterized in that described compound is an epoxy compounds, promptly A-D is epoxide group formula (b) in the general formula (I).
18. claim 4,5 and claim 8-17 in each described esperamicin derivatives antitumor in preparation, suppress cell transition and increase and end application in the pharmaceutical composition of cell growth.
19. one kind antitumor, suppress the pharmaceutical composition that cell transition increases and end the cell growth, it is characterized in that this pharmaceutical composition comprises at least a claim 4,5 and claim 8-17 in each described esperamicin derivatives and at least a conventional carrier and/or thinner.
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|---|---|---|---|
| CN2007101995604A CN101177425B (en) | 2003-01-28 | 2003-01-28 | Novel epothilones compound as well as preparation method and use thereof |
| CNB031030319A CN100359014C (en) | 2003-01-28 | 2003-01-28 | Novel epothilones compound and its preparation method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519404A (en) * | 2008-02-29 | 2009-09-02 | 唐莉 | 15-cyclothiaketone derivative, preparation method and application thereof |
| WO2010040252A1 (en) * | 2008-10-06 | 2010-04-15 | 山东大学 | Epothilone glycoside compounds, compositions containing epothilone glycoside compounds as active components and uses thereof |
| WO2011072496A1 (en) | 2009-12-17 | 2011-06-23 | Tang Li | Epothilone compounds, preparation method and use thereof |
| WO2021204188A1 (en) * | 2020-04-08 | 2021-10-14 | 北京华昊中天生物医药股份有限公司 | Utidelone semi-hydrated single crystal and preparation method therefor and use thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE408612T1 (en) * | 1996-11-18 | 2008-10-15 | Biotechnolog Forschung Gmbh | EPOTHILONES E AND F |
| US6605599B1 (en) * | 1997-07-08 | 2003-08-12 | Bristol-Myers Squibb Company | Epothilone derivatives |
| EP1135470A2 (en) * | 1998-11-20 | 2001-09-26 | Kosan Biosciences, Inc. | Recombinant methods and materials for producing epothilone and epothilone derivatives |
| CN1086389C (en) * | 1999-11-12 | 2002-06-19 | 中国科学院上海有机化学研究所 | Isoesperamicin and its synthesizing process and usage |
| US20020045609A1 (en) * | 2000-05-26 | 2002-04-18 | Gary Ashley | Epothilone derivatives and methods for making and using the same |
-
2003
- 2003-01-28 CN CNB031030319A patent/CN100359014C/en not_active Expired - Lifetime
- 2003-01-28 CN CN2007101995604A patent/CN101177425B/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519404A (en) * | 2008-02-29 | 2009-09-02 | 唐莉 | 15-cyclothiaketone derivative, preparation method and application thereof |
| WO2009105969A1 (en) | 2008-02-29 | 2009-09-03 | 唐莉 | Epothilone analogues, their pharmaceutical compositions, their use and their preparations |
| CN101519404B (en) * | 2008-02-29 | 2016-01-20 | 唐莉 | 15 ring thiophene ketone derivatives and preparation method thereof and application |
| WO2010040252A1 (en) * | 2008-10-06 | 2010-04-15 | 山东大学 | Epothilone glycoside compounds, compositions containing epothilone glycoside compounds as active components and uses thereof |
| WO2011072496A1 (en) | 2009-12-17 | 2011-06-23 | Tang Li | Epothilone compounds, preparation method and use thereof |
| JP2013514281A (en) * | 2009-12-17 | 2013-04-25 | 唐莉 | EPOSILON COMPOUND AND METHOD FOR PREPARING THE SAME |
| US8895590B2 (en) | 2009-12-17 | 2014-11-25 | Li Tang | Epothilone compounds, preparation method and use thereof |
| WO2021204188A1 (en) * | 2020-04-08 | 2021-10-14 | 北京华昊中天生物医药股份有限公司 | Utidelone semi-hydrated single crystal and preparation method therefor and use thereof |
| JP2023509871A (en) * | 2020-04-08 | 2023-03-10 | 北京華昊中天生物医薬股▲ふん▼有限公司 | Uchideron hemihydrate single crystal, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101177425B (en) | 2012-07-18 |
| CN101177425A (en) | 2008-05-14 |
| CN100359014C (en) | 2008-01-02 |
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Address after: Room 310, floor 3, building 3, No. 88, Kechuang 6th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing 100176 Patentee after: Beijing Huahao Zhongtian biomedical Co.,Ltd. Address before: 100000, Beijing, Chaoyang District agricultural light 117 building, Jinsong building, 10 floor Patentee before: BEIJING BIOSTAR TECHNOLOGIES, Ltd. |
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