CN102120730A - 19-S-methyl geldanamycin and 4,5-dihydrogen-19-S-methyl geldanamycin and preparation method thereof - Google Patents
19-S-methyl geldanamycin and 4,5-dihydrogen-19-S-methyl geldanamycin and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a group of benzoquinone ansamycin biologically synthesized analogs, in particular to a geldanamycin biologically synthesized analog. Experimental results show that: the biological activity of analogs 19-S-methyl geldanamycin and 4,5-dihydrogen-19-S-methyl geldanamycin is lower than that of the geldanamycin, but the water solubility and the photostability of the analogs 19-S-methyl geldanamycin and 4,5-dihydrogen-19-S-methyl geldanamycin are higher than those of the geldanamycin, so that the analogs 19-S-methyl geldanamycin and 4,5-dihydrogen-19-S-methyl geldanamycin are expected to be developed into clinically available antineoplastic medicines.
Description
Technical field:
The present invention relates to one group of benzoquinones type AMSA microbiotic biosynthesizing analogue, in particular to geldanamycin biosynthesizing analogue and its production and use.
Background technology:
Streptomyces hygroscopicus 17997 (Streptomyces hygrocopicus 17997) is that the geldanamycin (chemical structure is seen formula 1 for geldanamycin, GDM) that Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences is separated to from China's soil produces bacterium.Known GDM is a kind of benzoquinones type AMSA microbiotic, has various biological activity [Sasaki K et al.J Antibiot (Tokyo) 1979,32 (8): 849-51] such as antitumor, protozoacide, antiviral and immunosuppression; [Chinese patent ZL97100523.0; Tao Peizhen etc., Chinese microbiotic magazine, 1997,22:368-372].The king of Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences with light etc. in to GDM biosynthesizing research process, cloned the GDM biological synthesis gene cluster, some GDM biosynthesis gene blocked mutants have been obtained, gdmP gene wherein (Codocyte cytochrome p 450 monooxygenase) blocked mutant no longer produces GDM, but produce 4, the two hydrogen geldanamycin (DHGDM) of 5-.
The specific effect target spot of GDM and DHGDM is that (heat shock protein90, Hsp90), Hsp90 is the mate molecule of many signal proteins to HUMAN HEAT SHOCK PROTEINS 90.GDM is by to the inhibition of Hsp90, can the remote effect human body cell in the function of signal protein, therefore be expected to become a kind of antitumor drug that has much potentiality.But GDM has shortcomings such as big, water-soluble little, the light stability difference of toxicity, and these defectives will limit its exploitation becomes clinical active drug.Therefore, seek that a kind of toxicity is low, good water solubility, GDM derivative that light stability is high, become association area scientific research personnel's goal in research.
As is generally known the streptomycete secondary metabolite has complicated biosynthesizing mechanism usually, cause the microbial secondary meta-bolites to have complicated chemical structure, it is complete synthesis or chemical complete synthesis with high costs to be difficult to carry out chemistry.Meanwhile, the enzyme of subparticipation synthesis step is not strong to the specificity of substrate in the streptomycete secondary metabolite biosynthetic pathway, or biosynthetic pathway itself has branch or intersection, causes the microbial secondary meta-bolites to be generally the compound of one group of similar.This situation is commonplace in microbiotic (streptomycete secondary metabolite), forms so-called antibiotic polycomponent phenomenon.For example, ten quaternary macrolide antibiotics erythromycin have A, B, C, D, E and six analogs of F (component), and wherein A is a main ingredient, and its anti-microbial activity is higher and toxicity is less.Different components in the polycomponent microbiotic often presents different biological activity of degree and toxicity.Therefore, antibiotic different components (being the biosynthesizing analogue) is studied, not only can deep understanding be arranged, and development has important practical significance with exploitation to new antibiotic antibiotic biosynthesizing mechanism.
The AMSA microbiotic is the important microbial secondary meta-bolites of a class.According to the difference of aromatic nucleus in the molecule, the AMSA microbiotic can be divided into benzene AMSA microbiotic and naphthalene AMSA microbiotic.Belong to that the benzene AMSA is antibiotic geldanamycin (geldanamycin), Da Bei rhzomorph (macbecin), herbimycin (herbimyicn) and an ansamitocin (ansamitocin) etc.Belong to that the naphthalene AMSA is antibiotic rifomycin (rifamycin), naphthomycin (naphthomycin) and an Ah watt's mycin (awamycin) etc.No matter be benzene AMSA microbiotic or naphthalene AMSA microbiotic, generally be polycomponent microbiotic, particularly ansamitocin and rifomycin, nearly more than ten of the component quantity of having found.Produce in the secondary metabolite of bacterium at GDM, reported 4, GDM biosynthesizing analogues such as the two hydrogen geldanamycin of 5-, hydroquinone type geldanamycin, they are the small component of GDM.
Yet up to now, Shang Weijian has discovery 19-S-methyl geldanamycin or 4 from GDM generation bacterium or GDM biosynthesis gene blocked mutant both at home and abroad, the relevant report of the two hydrogen of 5--19-S-methyl geldanamycin.
The objective of the invention is to, prepare that a kind of toxicity is low, the geldanamycin novel derivative of good water solubility, good light stability, in the hope of becoming clinical useful medicine.
Summary of the invention:
The invention provides from GDM and produce a kind of new GDM biosynthesizing analogue 19-S-methyl geldanamycin of acquisition the bacterium streptomyces hygroscopicus 17997 (19-S-methylgeldanamycin is called for short compound 1, and chemical structure is seen formula 1).
The present invention also provides from the gdmP gene disruption of streptomyces hygroscopicus 17997 and becomes a kind of new GDM biosynthesizing analogue 4 of acquisition the strain, the two hydrogen of 5--19-S-methyl geldanamycin (4,5-dihydro-19-S-methylgeldanamycin is called for short compound 2, and chemical structure is seen formula 1).
The invention provides the preparation method of compound 1 and 2, mainly may further comprise the steps:
1. the gdmP gene disruption of streptomyces hygroscopicus 17997 becomes the structure of strain;
2. streptomyces hygroscopicus 17997 and gdmP gene disruption become the fermentation of strain;
3. compound 1 and 2 detection;
4. compound 1 and 2 preparation.
The chemical structure that the invention provides compound 1 and 2 is resolved.
The present invention also provides the biologic activity of compound 1 and 2, water-soluble and light stability experimental study.
1. streptomyces hygroscopicus 17997gdmP gene disruption becomes the structure of strain
With homologous gene double exchange principle, according to gdmP gene order (GenBank DQ914285) design primer, with streptomyces hygroscopicus 17997 genomic dnas is that template is carried out PCR, obtain two fragments respectively with the gdmP dna homolog, insert selected marker with corresponding restriction enzyme site therebetween, as kantlex (kanamycin) resistant gene, make up the recombinant vectors that obtains to be used to block goal gene by intestinal bacteria/streptomycete shuttle plasmid; Recombinant vectors is imported in the streptomyces hygroscopicus 17997.According to selected marker screening transformant.Utilize PCR method to identify and confirm to have inserted the selected marker in the gdmP gene order, the gdmP gene disruption that obtains streptomyces hygroscopicus 17997 becomes strain.
2. streptomyces hygroscopicus 17997 or gdmP gene disruption become the fermentation of strain
With fresh Chinese microbiotic magazines such as streptomyces hygroscopicus 17997[Tao Peizhen, 1997,22 (5): 368], [ZL97100523.0] or gdmP gene disruption become the strain culture, digs piece to be inoculated in the liquid fermentation medium, ferment through shaking culture.
3. compound 1 and 2 detection
It is a small amount of to get streptomyces hygroscopicus 17997 or gdmP gene disruption change strain fermented liquid, the centrifugal precipitation of abandoning, supernatant liquor concentrate after with ethyl acetate extraction, carry out silica-gel plate TLC and separate and analyze, can detect red compound 1 or 2 respectively, they all show blueness (Fig. 1) with 2.0mol/LNaOH spraying back.
4. compound 1 and 2 preparation
Streptomyces hygroscopicus 17997 or gdmP gene disruption become the fermenting culture supernatant liquor of strain, use ethyl acetate extraction, extraction liquid concentrates through rotary evaporation and obtains crude product, utilize silica gel column chromatography, adopt mixed liquid of petroleum ether-ethyl acetate or the mixed liquid of methylene chloride-methanol to carry out wash-out, the TLC silica-gel plate detects fraction collection liquid, compile the collection liquid that contains compound 1 or 2 respectively, after utilizing the Sephadex-LH20 column chromatography to be further purified, refining through preparation HPLC, obtain red compound 1 or 2 pure product respectively.
5. compound 1 and 2 chemical structure are resolved
The present invention adopts liver cancer HepG2 cell strain, by the sulphonyl rhodamine B (Sulforhodamine B, SRB) method studied compound 1 and 2 anti-tumor activity, and through the dissolving and the illumination experimental study their water-soluble and light stability.The result shows that compound 1 and 2 all has the antitumor cell activity, its IC
50Be respectively 19.32 μ M and 23.18 μ M, the water-soluble of them all improved more than 1000 times than GDM.
The invention effect:
The new analogue of from the fermenting culture of streptomyces hygroscopicus 17997 (its tunning C4, C5 position are the GDM of two keys) and cytochrome P 450 monooxygenases gene (gdmP) blocked mutant (its tunning C4, C5 are single bonded GDM) thereof, having found the modification of GDM C19 position respectively involved in the present invention, through separation and purification, structure elucidation, proving conclusively them is and carries out the compound that S-methylates and modifies in the C19 position of GDM benzoquinones ring, be respectively 19-S-methyl geldanamycin (compound 1) and 4, the two hydrogen of 5--19-S-methyl geldanamycin (compound 2). Compound 1 and 2 has the antitumor cell activity; Particularly, compound 1 and 2 water-soluble and light stability are significantly improved with respect to GDM and improve; Compound 1 and 2 is expected to develop and becomes clinical useful antitumor drug or its lead compound.
Description of drawings:
The silica-gel plate TLC that Fig. 1 streptomyces hygroscopicus 17997 and gdmP gene disruption become the strain fermented product extract analyzes wherein: 1-compound 1; 2-compound 2
The GDM-geldanamycin; DHGDM-4, the two hydrogen geldanamycin of 5-;
Before left figure-usefulness NaOH solution-treated; After right figure-usefulness NaOH solution-treated.
Fig. 2 compound 1
1The H-NMR collection of illustrative plates
Fig. 3 compound 1
13The C-NMR collection of illustrative plates
Fig. 4 compound 2
1The H-NMR collection of illustrative plates
Fig. 5 compound 2
13The C-NMR collection of illustrative plates
Fig. 6 compound 1 compares with the light stability of GDM
---be compound 1 ... be GDM
Fig. 7 compound 2 compares with the light stability of GDM
---be compound 2 ... be GDM
Embodiment:
Below listed embodiment only for helping those skilled in the art to understand the present invention better, but do not limit the present invention in any way.
＜embodiment 1〉the gdmP gene disruption of streptomyces hygroscopicus 17997 becomes the structure of strain
Nucleotide sequence (GenBank DQ914285) design PCR primer (primer P1,5 '-CCG according to gdmP gene and upstream and downstream thereof
GAATTCCGACGAGCGAGGGCACTTT-3 ' contains the EcoRI restriction enzyme site; Primer P2,5 '-CGG
GGTACCCGAGACCACGGCCAACATG-3 ' contains the KpnI restriction enzyme site; Primer P3,5 '-AAAA
CTGCAGGCCGTTGAGGCTGGAGTT-3 ' contains the PstI restriction enzyme site; Primer P4,5 '-CTAG
TCTAGAGGTATCTGCGTTACTGGGTG-3 ' contains the XbaI enzyme cutting site.P1 and P2 pairing are used for pcr amplification homologous gene double exchange upstream dna fragmentation, the about 1.0kb of size; P3 and P4 pairing, be used for pcr amplification homologous gene double exchange downstream DNA fragment, the about 1.2kb of size), with streptomyces hygroscopicus 17997 genomic dnas is that template is carried out PCR, acquisition is used for two dna fragmentations of homologous gene double exchange, after restriction enzyme is cut, with kantlex (kanamycin, Kan) resistant gene is (from plasmid pUC119, with KpnI and PstI double digestion, obtain the Kan resistant gene of about 0.8kb size) connect, and be connected with intestinal bacteria/streptomycete shuttle vector such as pGH112 carrier (through EcoRI and XbaI double digestion), transformed into escherichia coli DH5 α, acquisition is used for the recombinant vectors of gdmP gene disruption; This recombinant vectors is imported in the streptomyces hygroscopicus 17997 by conjugal transfer.According to kantlex selected marker screening zygote, utilize PCR method (P5 primer, 5 '-CCCTCGGGCAGATTCAC-3 '; The P6 primer, 5 '-GTCGCACTCCAACCATTTC-3 ') screening and differentiate that the gene disruption that has inserted kalamycin resistance gene in the gdmP gene of streptomyces hygroscopicus 17997 genomic dnas becomes strain (the pcr amplification product size that gene disruption becomes strain is 3.3kb for 3.8kb, former strain size), obtains the gdmP gene disruption change strain of streptomyces hygroscopicus 17997.
＜embodiment 2〉streptomyces hygroscopicus 17997 and gdmP gene disruption become the fermentation culture of strain
Get fresh streptomyces hygroscopicus 17997 or its gdmP gene disruption and become strain cultivation inclined-plane or flat board (yeast powder 0.4%, malt extract 1.0%, glucose 0.4%, agar powder 1.5%.Annotate: in cultivating gdmP gene disruption change strain substratum, add kantlex to final concentration 50 μ g/ml), dig piece and be inoculated in liquid seed culture medium (glucose 0.5%, yeast extract 0.4%, corn steep liquor 0.5%, cotton seed powder cake 0.25%, KH
2PO
40.05%, MgSO
40.05%, CaCO
30.3%) in, 24-48h is cultivated in 28 ℃ of vibrations (100-300r/min); 5% transferred species amount is inoculated in liquid fermentation medium (starch 2%, cotton seed powder cake 0.5%, glucose 0.5%, corn steep liquor 1.0%, yeast powder 0.5%, CaCO
30.2%), 28 ℃ are continued shaking culture, to 120-144h, obtain the fermentation culture that streptomyces hygroscopicus 17997 or its gdmP gene disruption become strain.
＜embodiment 3〉streptomyces hygroscopicus 17997 and its gdmP gene disruption become the detection of compound 1 in strain fermenting culture and 2
Get the about 10ml of fermentation culture supernatant liquor that streptomyces hygroscopicus among the embodiment 2 17997 and its gdmP gene disruption become strain, use the equal-volume ethyl acetate extraction; Inclining acetic acid ethyl acetate extract, after drying up in air, redissolves in 100 μ l ethyl acetate, get 10-20 μ l point sample on the TLC silica-gel plate, in ethyl acetate: methylene dichloride: normal hexane: (9: 6: 6: 1) exhibition layer in the solvent system, compound 1 and 2 was redness, R to methyl alcohol
fValue is the (R of GDM about 0.30
fValue is 0.54), behind small amount of N aOH (2.0mol/L) solution spraying, show blue.
＜embodiment 4〉separation and purification and the preparation of compound 1 and compound 2
Get the fermenting culture supernatant liquor that streptomyces hygroscopicus 17997 among the embodiment 2 or gdmP gene disruption become strain, use the equal-volume ethyl acetate extraction, rotary evaporation concentrates the back and goes up silica gel column chromatography, carry out wash-out with the mixed liquid (volume ratio is 9: 1) of methylene chloride-methanol, perhaps the mixed liquid of petroleum ether-ethyl acetate carries out gradient elution (initial volume ratio is 50: 50, with 5% is that a gradient progressively improves the ethyl acetate ratio, to sherwood oil: ethyl acetate=35: 65 o'clock obtains the purpose elutriant), the fraction collection elutriant, after the TLC silica-gel plate detects, merge the fraction collection elutriant (redness) that contains compound 1 or compound 2, rotary evaporation redissolves in methyl alcohol after concentrating, through Sephadex LH-20 column chromatography for separation, the fraction collection elutriant, merge the fraction collection elutriant (redness) that contains compound 1 or compound 2, rotary evaporation carries out reversed-phase HPLC (ODS-C after concentrating
18Post, 150mm * 20mm, mobile phase methanol-water are 14: 11, flow velocity is 2-5ml/min), the retention time (30-40 minute) that goes out the peak according to HPLC merges the elutriant that contains compound 1 or compound 2, and rotary evaporation concentrates, and obtains red compound 1 or compound 2 pure product respectively.
＜embodiment 5〉 compound 1 and 2 chemical structure identify
Compound 1 and 2 has been carried out the high resolution mass spectrum analysis: the actual measurement molecular mass of compound 1 is 629.25688 ([M+Na]
+), the actual measurement molecular mass of compound 2 is 631.26481 ([M+Na]
+).Binding compounds 1 and 2 element sulphur analytical results, content is respectively 5.16% and 5.00%, thereby the molecular formula of compound 1 and 2 is determined: compound 1 molecular formula is C
30H
42N
2O
9S (theoretical molecular mass, [M+Na]
+, 629.25087); Compound 2 molecular formula are C
30H
44N
2O
9S (theoretical molecular mass, [M+Na]
+, 631.26652).Compound 1 and 2 has been carried out nuclear magnetic resonance spectroscopy.According to nuclear magnetic resonance data [Hong YS, Lee D, Kim W, the et al.J Am Chem Soc.2004 Sep 15 of compound 1 with geldanamycin (GDM); 126 (36): 11142-3] relatively, how the difference of it and GDM has been on the C19 position one-SCH
3Signal, and all the other
13C-,
1The H-NMR signal is identical substantially, so the chemical structure of compound 1 is defined as 19-S-methyl geldanamycin.There were significant differences for compound 2 and C4, the C5 of compound 1 and the nuclear magnetic resonance data of the hydrogen atom that they were connected, all the other
13C-,
1The H-NMR signal is identical substantially; In addition, compound 2 and 4, nuclear magnetic resonance data [Lee D, Lee K, Cai XF, the et al.Chembiochem.2006Feb of the two hydrogen geldanamycin of 5-; 7 (2): 246-8] compare, it and 4, how the difference of the two hydrogen geldanamycin of 5-has been on the C19 position one-SCH
3Signal, and all the other
13C-,
1The H-NMR signal is identical substantially; In view of the above, can clearly judge between C4, the C5 of compound 2 is that carbon-carbon single bond connects (being carbon-carbon double bond between the C4 of compound 1, the C5), and the chemical structure of compound 2 is 4, the two hydrogen of 5--19-S-methyl geldanamycin.Compound 1 and 2
13C-,
1H-nucleus magnetic resonance (NMR) signal ownership sees Table 1.
Table 119-S-methyl geldanamycin (1) and 4, the NMR data of the two hydrogen of 5--19-S-methyl geldanamycin (2) (are dissolved in CD
3OD)
＜embodiment 6〉the antitumor cell activity of compound 1 and compound 2
Liver cancer HepG2 cell strain, 96 orifice plates, sulphonyl rhodamine B (Sulforhodamine B, SRB) method are adopted in this experiment.
A certain amount of compound 1 or 2 (being dissolved in DMSO) adding, 96 well culture plates (are contained 5 * 10
3Individual cells/well) in, obtain to contain the cell culture fluid of different final concentrations (concentration range is 2-300 μ M) compound 1 or 2, each concentration adds 3 holes, in 37 ℃, 5%CO
2, cultivated 48 hours in the 100% humidity incubator, inclining supernatant liquor; Fix in 96 well culture plates behind the cell with the physiological saline that contains 10% trichoroacetic acid(TCA), rinse well, add sulphonyl rhodamine B solution (0.4%) staining cell with deionized water; Inclining staining fluid, rinses the back well with 1% acetate and adds Tris alkali lye (10mM, pH10.5), the mixing that slowly vibrates is measured the OD of solution in every hole
570nm, calculate the cell inhibitory rate [inhibiting rate (%)=(the average OD of 1-experimental port under the different compound concentrations
570nmThe average OD of value/control wells
570nmValue) * 100%].The IC of computerized compound
50[calculation formula is value: IC
50=lg
-1[X
m-i (∑ P-0.5)]; Wherein, X
mBe the logarithmic value of peak concentration of design, i is the logarithmic value of each concentration multiple proportions concentration; ∑ P is each group growth inhibition ratio sum; 0.5 be empirical constant, at first the value of experimental port and control wells is deducted zeroing hole value, the IC of acquisition compound 1 or compound 2 during calculating
50Value (table 2).
The IC of table 2GDM
50Be 0.062 μ M.
| GDM concentration (μ M) | 30 | 6 | 1.2 | 0.24 | 0.048 | 0.0096 | 0.00192 |
| Cell inhibitory rate (%) | 97.42 | 79.70 | 72.26 | 70.37 | 65.62 | 28.52 | 12.26 |
The IC of compound 1
50Be 19.32 μ M
| |
300 | 100 | 50 | 10 | 3 |
| Cell inhibitory rate (%) | 97.47 | 93.29 | 81.97 | 40.19 | 3.71 |
The IC of compound 2
50Be 23.18 μ M
| |
110 | 90 | 45 | 20 | 2 |
| Cell inhibitory rate (%) | 94.80 | 93.24 | 64.02 | 22.53 | 3.44 |
＜embodiment 7〉compound 1 and the experiment of 2 water-soluble and light stability
Water-soluble experiment: respectively with excessive compound 1 and 2, and be contrast with GDM, be dissolved in the sodium chloride injection (containing 0.9%NaCl), under room temperature (25 ℃), black out condition, stir 12h, behind the high speed centrifugation, content with compound 1,2 and GDM in the HPLC method mensuration supernatant liquor calculates its solubleness.The result shows: the solubleness of compound 1 is 2019 μ g/ml, and the solubleness of compound 2 is 2132 μ mg/ml, and the solubleness of GDM is 2.0 μ g/ml.Therefore, compound 1 and 2 all exceeds more than 1000 times than the water-soluble of GDM.
Light stability experiment: with compound 1 (0.10mg/ml, be dissolved in methyl alcohol), compound 2 (0.10mg/ml, be dissolved in methyl alcohol) and contrast GDM (0.10mg/ml, be dissolved in methyl alcohol) place identical vial to accept illumination (intensity of illumination is 3500lx) irradiation respectively, take a sample respectively at different time (20,40,60,80,100 and 120 minutes), with the content of compound 1,2 and GDM in the HPLC working sample.GDM has degraded about 80% in the time of 120 minutes in illumination, about 20% (Fig. 6 and 7) and compound 1 and 2 has only been degraded under the same conditions.
＜embodiment 8〉reduction of compound 1 and compound 2
Take by weighing a certain amount of compound 1 and 2 respectively, be dissolved in the ethyl acetate, 10% V-Brite B aqueous solution with the proper amount of fresh preparation, under room temperature (about the 25 ℃) condition slowly the about 10-30 of jolting minute, it is colourless that red compound 1 and 2 ethyl acetate organic solution become mutually, shows and carried out reduction reaction; Inclining organic phase (upper strata), removes ethyl acetate organic solution through vacuum rotation volatilization, obtains the hydroquinone type goods of compound 1 and 2.These goods are placed in air, can slow oxidation be compound 1 and 2 again.
Claims (9)
1. the derivative of group geldanamycin biosynthesizing benzoquinones ring modification is characterized in that described derivative comprises 19-S-methyl geldanamycin (compound 1) and 4, the two hydrogen of 5--19-S-methyl geldanamycin (compound
2), chemical structure is as follows:
2. prepare the method for the described derivative of claim 1, it is characterized in that, compound 1 is by streptomyces hygroscopicus 17997 bacterial strain direct fermentations; Compound 2 becomes the strain fermentation by streptomyces hygroscopicus 17997P450 cytopigment mono-oxygenase gdmP gene disruption and produces.
3. the described preparation method of claim 2, it is characterized in that, the structure that produces compound 2 used change strains is, according to gdmP gene order (GenBank DQ914285) design primer, with streptomyces hygroscopicus 17997 genomes is template, carries out PCR, obtains two fragments with the gdmP dna homolog respectively, insert the selected marker with corresponding restriction enzyme site therebetween, and by making up the recombinant vectors that intestinal bacteria/streptomycete shuttle plasmid obtains to be used for the gdmP gene disruption; Recombinant vectors is imported in the streptomyces hygroscopicus 17997, according to selected marker screening transformant; Utilize PCR method to identify and confirm, inserted the selected marker in the gdmP gene order, thereby the gdmP gene disruption that obtains streptomyces hygroscopicus 17997 becomes strain.
4. the described preparation method of claim 2 is characterized in that, streptomyces hygroscopicus 17997 or its gdmP gene disruption change strain inclined-plane or flat board with fresh culture dig piece and be inoculated in liquid seed culture medium, cultivate 24-48h; 5% transferred species amount is inoculated in liquid fermentation medium, and 28 ℃ are continued shaking culture, to 120-144h, obtains the fermentation culture that streptomyces hygroscopicus 17997 or its gdmP gene disruption become strain.
5. the described preparation method of claim 2, it is characterized in that, streptomyces hygroscopicus 17997 or gdmP gene disruption become the fermentation culture supernatant liquor of strain, use the equal-volume ethyl acetate extraction, rotary evaporation concentrates the back and goes up silica gel column chromatography, carry out wash-out with the mixed liquid of the methylene chloride-methanol of 9: 1 volume ratios, be that 50: the 50 mixed liquid of petroleum ether-ethyl acetate carries out gradient elution perhaps with initial volume ratio, with 5% is that a gradient progressively improves the ethyl acetate ratio, to sherwood oil: ethyl acetate is to obtain the purpose elutriant at 35: 65 o'clock, the fraction collection elutriant, after the TLC silica-gel plate detects, merge the red elutriant of the fraction collection that contains compound 1 or compound 2, rotary evaporation redissolves in methyl alcohol after concentrating, through Sephadex LH-20 column chromatography for separation, the fraction collection elutriant, merge the red elutriant of the fraction collection that contains compound 1 or compound 2, after rotary evaporation concentrates, carry out reversed-phase HPLC, go out the retention time at peak according to HPLC, merge the elutriant that contains compound 1 or compound 2, rotary evaporation concentrates, and obtains red compound 1 or compound 2 pure product respectively.
6. the application of the described derivative of claim 1 in the preparation antitumor drug.
7. the pharmaceutical composition of the described derivative of claim 1 is to be effective constituent with described derivative, forms with pharmaceutically acceptable one or more carriers.
8. the described composition of claim 7 is meant to be fit to medicinal any dosage form.
9. claim 7 or the 8 described compositions application in the preparation antitumor drug.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57163369A (en) * | 1981-03-31 | 1982-10-07 | Kaken Pharmaceut Co Ltd | Novel geldanamycin derivative, its preparation, and drug comprising it as active ingredient |
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2010
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57163369A (en) * | 1981-03-31 | 1982-10-07 | Kaken Pharmaceut Co Ltd | Novel geldanamycin derivative, its preparation, and drug comprising it as active ingredient |
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Application publication date: 20110713 |