CN1208465C - Preparation technology of geldanamycin - Google Patents

Abstract

The present invention relates to a preparation process of broad-spectrum anti-cancer antibiotic geldanamycin and derivatives thereof. Moist streptomycete of gear denece is cultured in the liquid culture media of the present invention, and the geldanamycin is obtained through purifying culture solutions by using chromatographic media with large apertures. A large amount of geldanamycin with high purity can be obtained by the process so that the process sets a basis for the large-scale industrial production of the derivatives of geldanamycin.

Description

The preparation technology of geldanamycin

Technical field

The present invention relates to a kind of broad spectrum anticancer microbiotic---the preparation technology of geldanamycin, relate in particular to from the nutrient solution of cultivating the moist streptomycete of Ge Erdenasi (Streptomyces hygroscopicus var Geldanus) and utilize the wide aperture chromatography media to prepare the technology of highly purified geldanamycin.

The invention still further relates to the liquid nutrient medium of cultivating the moist streptomycete of Ge Erdenasi.

Background technology

Antitumor antibiotic is the small molecules chemical substance or derivatives thereof with antitumour activity that is produced by microbial metabolism.Antitumor antibiotic is of a great variety, be applied to clinical treating malignant tumor have ten surplus kind, as the antitumor antibiotic of natural origins such as mitomycin, Zorubicin, daunorubicin, bleomycin, Zhengguangmycin A5, gengshengmeisu and through the congener of chemically modified, they have become all kinds of malignant tumours of treatment medicine commonly used.In the last few years, countries in the world had been reported the new type anticancer microbiotic that acts on different target position successively, and its main target position comprises tumor vessel, dna profiling, matrix metalloproteinase and heat shock protein 90 (HSP90) etc.This wherein, the inhibitor geldanamycin of HSP90 (geldanamycin, GA) and the anticancer effect of some derivative especially noticeable.Geldanamycin is the compound that a kind of moist streptomycete excretory contains the benzoquinones structure, belongs to benzoquinones ansamycin (benzoquinone ansamycin) class microbiotic.1971, people found that in the nutrient solution of moist streptomycete this kind has the microbiotic of anti-tumor activity (U.S. Pat 3,595,955), and the solubleness of this microbiotic in the aqueous solution is lower, and experimentation on animals finds that it is toxic to liver etc.Experiment in vitro in recent years shows that GA has good inhibition activity to the growth of kinds of tumor cells, has wide spectrum antiproliferative and antitumor action.

Geldanamycin causes the unstable and degraded of carcinogenic protein by to the specific inhibition of HSP90.Geldanamycin has realized inhibition to multiple cancer target spot and approach to the inhibition of HSP90 function, and this multipath combination restraining effect makes geldanamycin have the broad-spectrum anti-tumor cytoactive.HSP90 belongs to stress response protein, and the content in healthy tissues is extremely low, and the content in malignant tumor tissue is higher, so geldanamycin class medicine can be brought into play optionally restraining effect to tumour cell.The advantage of geldanamycin also is the popularity of its effect, and HSP90 follows proteic conformation to keeping some crucial cancer cells, it is extremely important to stablize its function etc.The albumen of being responsible for maintaining by HSP90 comprises: the p53 of receptor tyrosine kinase, transmembrane conductance regulator, serine/threonine kinase, sudden change, c-erbB, Bcr-Ab1, Raf1, Akt and hypoxic inducing factor-1 etc., they cause breeding, the cell cycle advances and the signal transduction pathway of apoptosis in work; In addition, they are also very important for the characteristics of keeping such as malignant tumour phenotypes such as infiltration, angiogenic growth and transfers.GA by HSP90 to multiple cancer target spot the time restraining effect also have the another one advantage, be exactly that tumour cell is difficult for it is produced resistance, because a tumour cell is difficult to change simultaneously the multiple signal pathway of keeping its growth, to escape the lethal effect of medicine.

Though GA shows good inhibition tumor promotion on human tumor cells and tumor animal model,, fail to be applied to the treatment of clinical tumor because it produces difficulty and bigger to the toxicity of liver.Through screening experiment to multiple geldanamycin derivant, find 17-allyl amido geldanamycin (17-allylamino, 17-demethoxygeldanamycin, 17AAG) kept the restraining effect of GA to HSP90, in cancer cell in vitro cultivation and tumour transplatation model, shown tangible anti-tumor activity, solubleness in the aqueous solution is higher than GA, and hepatotoxicity is lower than GA.The cell in vitro experiment shows, geldanamycin, and 17-allyl amido geldanamycin can be used for multiple treatment for cancer such as leukemia, lung cancer, cancer of the stomach, colorectal carcinoma, the rectum cancer, liver cancer, mammary cancer, prostate cancer.

Geldanamycin is the raw material of synthetic multiple antitumor drug, owing to extract comparatively difficulty of highly purified geldanamycin in moist streptomycete fermentation liquid, the rate of recovery is lower, becomes the antibiotic obstacle of research benzoquinones ansamycins for a long time.Composition in the moist streptomycete fermentation product is very complicated, the technology of therefrom extracting geldanamycin is loaded down with trivial details, the rapid organic solvent extraction of multistep is the major cause that causes the geldanamycin rate of recovery low, therefore in report in the past, do not see that geldanamycin output is above the report of 100mg in every liter of fermented liquid.

In the report in the past, the prescription of moist streptomycete fermentation liquid of preparation geldanamycin is without optimization, employing be mostly general streptomycete culture medium prescription, fermentation density is not high, causes geldanamycin unit volume output lower.

The loaded down with trivial details liquid phase allocative decision of the many employings of the purifying of geldanamycin, what mainly utilize is the difference of geldanamycin solubleness in different solvents, the rapid extraction repeatedly of multistep causes the overall rate of recovery lower.

At the deficiency that exists in the above preparation geldanamycin, we have improved the prescription of substratum, make its growth that is fit to moist streptomycete more, have improved the output of moist streptomycete fermentation liquid unit volume geldanamycin.Cooperate the use of highly selective wide aperture chromatography media, make that the purifying of geldanamycin is easier.For preparation geldanamycin and derivative thereof provide possibility in a large number.

Summary of the invention

The object of the present invention is to provide a kind of novel process for preparing geldanamycin, adopt this technology can obtain highly purified antitumor antibiotic---geldanamycin in a large number, for the highly purified geldanamycin derivant of a large amount of preparations provides high quality raw material.Such antitumor antibiotic has higher selectivity to multiple malignant cell, is applicable to the chemotherapy of multiple malignant tumour.

According to an aspect of the present invention, provide a kind of liquid nutrient medium of suitable moist streptomycete growth, comprised in every liter of substratum:

Glucose 0.1～100 gram

Tryptones 0.5～15 gram

Rolled oats 0.5～15 gram

1～50 milliliter of refining Trisun Oil R 80

Proteolysate 0.1～25 gram

Yeast extract 0.1～20 gram

Yeast nitrogen base 0.1～50 gram

Zulkovsky starch 0.1～15 gram

0.1～10 milliliter in molasses; And

An amount of inorganic microelement

The pH of substratum is 5.0～8.0.

The preferred substratum of the present invention consists of:

Every liter contains:

Glucose 40 grams

Tryptones 2.5 grams

Rolled oats 1 gram

Zulkovsky starch 5 grams

Yeast extract 2.5 grams

10 milliliters of refining Trisun Oil R 80s

Proteolysate 2.5 grams

Yeast nitrogen base 1 gram

10 milliliters in molasses

Dipotassium hydrogen phosphate 0.015 gram

SODIUM PHOSPHATE, MONOBASIC 0.005 gram

Calcium chloride 0.03 gram

1 milliliter of trace element mixed solution

Described micro-mixed solution sulfur acid copper, manganous sulfate, iron(ic) chloride, zinc sulfate, cobalt chloride, sodium manganate, Sodium Tetraborate and boric acid, concentration separately is 0.1～10mmol/l; PH is 7.0～7.2.

According to a further aspect in the invention, provide and from the liquid nutrient medium of cultivating moist streptomycete, adopted chromatography to prepare the technology of geldanamycin, comprise and adopt liquid culture of the present invention based on the moist streptomycete of fermentation culture in the bio-reactor of different volumes, centrifugal or remove by filter insoluble substance such as thalline.Utilize geldanamycin under low pH condition, to combine than stable properties with macroporous resin that XAD-7 etc. contains the similar functions group, wash chromatography column with pure water, methyl alcohol stream successively, obtaining cut is placed the vacuum drying oven dried overnight, and the solid substance of acquisition is geldanamycin.

The geldanamycin of prepared is measured through HPLC according to the present invention, and purity is greater than 98.5%, and therefore, technology of the present invention can obtain highly purified geldanamycin.Lay a good foundation in follow-up technology, obtaining highly purified geldanamycin derivant.Every liter of fermented liquid can obtain more than the high purity geldanamycin 250mg.

Brief description of drawings

Fig. 1 is a C18 reversed-phase column high performance liquid chromatography (HPLC), 50% acetonitrile moving phase color atlas;

Fig. 2 is a C18 reversed-phase column high performance liquid chromatography (HPLC), methyl alcohol color atlas, moving phase: 50% acetonitrile;

Fig. 3 is a C18 reversed-phase column high performance liquid chromatography (HPLC), geldanamycin (methanol solvate) color atlas, moving phase: 50% acetonitrile;

Fig. 4 is a C18 reversed-phase column high performance liquid chromatography (HPLC), 17-allyl amido geldanamycin (methanol solvate) color atlas, moving phase: 50% acetonitrile.

Embodiment

Below by description, describe the present invention in detail to better embodiment of the present invention:

Embodiment 1 The extraction of geldanamycin and purifying

Geldanamycin is extracted the nutrient solution from the moist streptomycete of Ge Erdenasi (Streptomyces hygroscopicus varGeldanus).Streptomycete is cultivated, product extracts and purifying process is as follows:

(1) substratum is formed

Contain organism such as glucose, Tryptones, rolled oats, proteolysate, yeast extract, yeast nitrogen base, Zulkovsky starch, plant molasses in the liquid nutrient medium, each main component content is between 0.01～5%, inorganic microelements such as an amount of simultaneously adding dipotassium hydrogen phosphate, biphosphate are received, calcium chloride are regulated pH between 5.0～8.0.

The composition of preferred a kind of liquid nutrient medium following (every liter):

Glucose 40 grams

Tryptones 2.5 grams

Rolled oats 1 gram

Zulkovsky starch 5 grams

Yeast extract 2.5 grams

10 milliliters of refining Trisun Oil R 80s

Proteolysate 2.5 grams

Yeast nitrogen base 1 gram

10 milliliters in molasses

Dipotassium hydrogen phosphate 0.015 gram

SODIUM PHOSPHATE, MONOBASIC 0.005 gram

Calcium chloride 0.03 gram

1 milliliter of trace element mixed solution

Described micro-mixed solution sulfur acid copper, manganous sulfate, iron(ic) chloride, zinc sulfate, cobalt chloride, sodium manganate, Sodium Tetraborate and boric acid, concentration separately is 0.1～10mmol/l;

Adjust pH to 7.0～7.2.Standby after autoclaving or the filtration sterilization.

(2) plant daughter bacteria inoculation and fermentation:

Moist streptomycete (the Streptomyces hygroscopicus varGeldanus) bacterial classification of the used Ge Erdenasi of the present invention derives from USDA agricultural research institute preservation center (Northern Utilizationand Research Division, Agricultural Research, U.S.Department of Agriculture, Peoria, Ill., U.S.A.), this bacterial classification was submitted permanent preservation in 1969, preserving number is: NRRL 3602, can not have any restrictedly granting at present and give the public.

In-70 ℃ of refrigerators or liquid nitrogen container, take out in frozen bacterial classification inoculation to the 2 50ml flask of low temperature, 28 ℃, cultivate after 60 hours for 220 rev/mins, 4 200ml flasks are gone in switching, 28 ℃, 200 rev/mins are continued to cultivate after 36 hours, be inoculated into 6 2000ml flasks, 28 ℃, 220 rev/mins of cultivations, be inoculated into after 36 hours in 150 liters the seed fermentation jar, cultured continuously 30 hours, took a sample to check once in per 6 hours and (control dissolved oxygen, pH and foam), be inoculated in afterwards in the fermentor tank of 1500 liters (or bigger volumes), (add-on of every liter of nutrient solution Trisun Oil R 80 is 1-50ml to the adding speed of the refining Trisun Oil R 80 of culturing process adjustment to eliminate foam, optic vesicle foam control situation and decide), control dissolved oxygen simultaneously, jar inner air pressure (can reach 8psi) and pH, the content (high performance liquid chromatography) of per 6 hours sampling one-time detection geldanamycin, when no longer obviously increasing, geldanamycin content stops to cultivate, the last incubation time is about 120～170 hours (volume of looking fermentor tank is different), and the content of geldanamycin can reach more than the 300mg/L when stopping fermentation.Also can adopt other defoamer control foam in the fermenting process.

(3) extraction of geldanamycin

Adopt and filter or centrifuging clarification nutrient solution, in filtered liquid or centrifuged supernatant, add the pH to 4.5 that hydrochloric acid or sulfuric acid etc. are adjusted liquid, sample flow (or is contained the macroporous resin of similar functions group through prepackage XAD-7 macroporous resin, as HP2MG resin of Mitsubishi chemical production etc.), UV-detector (254nm/304nm) detects the flowing liquid of chromatography column, after application of sample is finished, pure water stream with 10～60 times of column volumes is washed chromatography column, stream be washed till baseline steadily after, use methanol-eluted fractions, collect elution peak, obtaining cut is placed vacuum drying oven or rotatory evaporator drying, the solid substance that obtains is geldanamycin, and HPLC measures purity greater than 98%.

The mass spectrograph determining molecular weight is 560, fusing point 252-254 ℃, be soluble in organic solvents such as chloroform, methylene dichloride, dimethyl sulfoxide (DMSO), and be dissolved in acetone, methyl alcohol etc. slightly, be slightly soluble in water, room temperature preservation is stable after the freeze-drying.

The results are shown in Figure 1, Fig. 2 and Fig. 3.

Embodiment 2 The chemosynthesis of 17-allyl amido geldanamycin

In the ground glass flask that fills the 100ml trichloromethane, add 600mg geldanamycin (purity is not less than 98%), shake well 10 minutes after the dissolving, adds 2ml allylamine (SIGMA/FLUKA/ALDRICH fully, purity is not less than 99%), stirring at low speed reaction 20～24 hours.After this in reactor, add 100ml in the ultrapure water of 2～4 ℃ of precoolings, transfer pH to 3.0 with 6M hydrochloric acid, high-speed stirring at least 30 minutes moves into liquid in the 250ml glass separating funnel after stopping to stir and leaves standstill, treat the right layering of its order, collect the trichloromethane phase of lower floor.Add the 100ml trichloromethane once more to aqueous phase, fully stir, treat to collect the trichloromethane phase after its layering, the trichloromethane of twice collection is merged mutually, pour in the sintered glass funnel that contains anhydrous sodium sulphate, stir with glass stick, suction filtration is removed solid sodium sulfate, collect filtered solution, with getting orange solid substance behind the rotatory evaporator concentrating under reduced pressure.This solid substance can obtain 617mg 17-allyl amido geldanamycin (rate of recovery 98.1%) with acetone-n-hexane recrystallization behind the drying under reduced pressure, fusing point is 212～214 ℃, and the mass spectrograph determining molecular weight is about 586.The results are shown in Figure 4.

Embodiment 3 Be used for treatment for cancer

Experimental studies have found that, 17AAG can bring into play restraining effect from many aspects to the growth of lung carcinoma cell, except as to other tumour cells, bring into play antiproliferative and make tumour cell to effect such as chemotherapy drug susceptibility enhancing, 17AAG can also obviously suppress the expression of erB1/erB2, suppress tumor cell secretion MMP-9 and VEGF, strengthen the expression of E-cadherins, thereby suppress the transfer of tumour cell.Experiment shows that the chemotherapy regimen that contains 17AAG can obviously dwindle the volume of nude mice lotus knurl than other schemes in the animal body, prolongs survival time of mice, and 17AAG treatment group has the tumour completely dissolve of 50% nuclear knurl mouse approximately.

The above description of this invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, has only not break away from spirit of the present invention and content, all should belong to the scope of claims of the present invention.

Claims (4)

1, the preparation technology of geldanamycin may further comprise the steps:

1) cultivate the moist streptomycete (Streptomyces hygroscopicusvar Geldanus) of Ge Erdenasi with liquid nutrient medium, this humidity streptomycete preserving number is NRRL 3602, and described substratum contains for every liter:

Glucose 40 grams

Tryptones 2.5 grams

Rolled oats 1 gram

Zulkovsky starch 5 grams

Yeast extract 2.5 grams

10 milliliters of refining Trisun Oil R 80s

Proteolysate 2.5 grams

Yeast nitrogen base 1 gram

10 milliliters in molasses

Dipotassium hydrogen phosphate 0.015 gram

SODIUM PHOSPHATE, MONOBASIC 0.005 gram

Calcium chloride 0.03 gram

1 milliliter of trace element mixed solution

Described micro-mixed solution sulfur acid copper, manganous sulfate, iron(ic) chloride, zinc sulfate, cobalt chloride, sodium manganate, Sodium Tetraborate and boric acid, concentration separately is 0.1～10mmol/l

Adjust pH to 7.0～7.2 of substratum;

Cultivate the moist streptomycete of Ge Erdenasi at 25～30 ℃, obtain to contain the nutrient solution of geldanamycin;

2) purifying geldanamycin from above-mentioned nutrient solution may further comprise the steps:

A) centrifugal or filtration nutrient solution obtains supernatant liquor;

B) in described supernatant liquor, add hydrochloric acid or sulfuric acid, adjust the pH to 4.5 of solution;

C) with solution stream through containing the chromatography column of separating filler, wash and wash-out with flowing lotion, elutriant stream successively, can obtain geldanamycin after concentrating.

2, technology according to claim 1, wherein the described separating filler of step c) is XAD-7 macroporous resin or HP2MG macroporous resin.

3, technology according to claim 1, wherein the described flowing lotion of step c) is a pure water, elutriant is a methyl alcohol.

4, be used to cultivate the substratum of the moist streptomycete of Ge Erdenasi, it is characterized in that every liter of described substratum contains following composition:

Glucose 40 grams

Tryptones 2.5 grams

Rolled oats 1 gram

Zulkovsky starch 5 grams

Yeast extract 2.5 grams

10 milliliters of refining Trisun Oil R 80s

Proteolysate 2.5 grams

Yeast nitrogen base 1 gram

10 milliliters in molasses

Dipotassium hydrogen phosphate 0.015 gram

SODIUM PHOSPHATE, MONOBASIC 0.005 gram

Calcium chloride 0.03 gram

1 milliliter of trace element mixed solution

Described micro-mixed solution sulfur acid copper, manganous sulfate, iron(ic) chloride, zinc sulfate, cobalt chloride, sodium manganate, Sodium Tetraborate and boric acid, concentration separately is 0.1～10mmol/l;

Medium pH is 7.0～7.2.

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