Epothilone c, d, e and f, receipt and tools based on them

 

(57) Abstract:

The invention relates to new epothilone formula 1, where R=CH3N. Also, these compounds are used for plant protection in agriculture and forestry and/or horticulture, and also as a therapeutic agent, as cytostatic funds. 5 C. and 12 C.p. f-crystals, 5 Il., table 4.

The invention relates to epothilone C, D, E and F, to receive them and to apply for a therapeutic means and means for protection of plants.

Epothilone C and D

One of the objects of the present invention relates to epothilone C and D, which receive

a) cultivating a known microorganism Sorangium cellulosum DSM 6773 in the presence of adsorbing resin

b) Department of adsorbing resin from culture and leaching of water-methanol mixture,

C) elution washed adsorbent resin with methanol and concentration of the eluate to obtain a crude extract,

d) extraction concentrate the ethyl acetate, the concentration of the extract and split it with methanol and hexane,

d) concentrating the methanol phase to the raffinate and fractionation of the resulting concentrate on the column is/BR> W) chromatographytandem the fractions obtained on C18-reversed phase using methanol/water in the following temporary sequence:

after the first fraction epothilones and

the second fraction with epothilones

a third faction with one first subsequent epothilones and

- fourth fraction one second subsequent epothilones and

ç1) allocation epothilone first subsequent fraction and/or

Z2) allocation epothilone second subsequent fraction.

Further, the invention relates to epothilone With total formula C26H39NO5S different1H and13C-NMR spectra according to the table 1.

Further, the invention relates to epothilone With formulas

< / BR>
epothilone S, where R=H.

Further, the invention relates to epothilone [D] the total formula C27H41NO5S different1H and13C-NMR spectra according to the table 1.

Further, the invention relates to epothilone D formula

< / BR>
epothilone D, where R=CH3.

Epothilone C and D can be used to obtain compounds of the following formula 1, with regard to their derivatization, it is possible to invoke methods of derivatization description is H, C1-6-alkyl, C1-6-allantoin, C1-4-trialkylsilyl, unsubstituted or substituted C1-6-alkoxygroup,6-alkyl, actigraphy or halogen benzyl or phenyl, and two of the residues R1-R5may together form a group -(CH2)n- where n=1-6, and contained in the residues of alkyl and acyl groups are linear or branched residues;

Y and Z are the same or different and mean hydrogen, halogen, such as F, C1, Br or J, pseudohalogen, such as-NCO, -NCS or-N3HE, O-(C1-6)-acyl, O-(C1-6)-alkyl, O-benzoyl, Y and Z can represent a and the oxygen atom of the epoxide, and epothilone a and b are not discussed, or to form one of the C-C-bonds in the double bond C=C.

For example, 12,13-double bond can be selectively

to gidrirovanii, for example, by catalytic or using diamine, resulting in the receive connection of formula 1, where Y=Z=H,

to epoxidizing, for example, dimethyldioxirane or nagkalat, resulting in the receive connection of formula 1, where Y and Z = or

- to make dihalogenide, disengagement or diazide, resulting in the receive connection of formula 1, where Y and Z denote halogen, pseudohalogen atilon And, get the

a) cultivating a microorganism Sorangium cellulosum DSM 6773 in the presence of adsorbing resin in a known manner, a separation of culture from absorbent resin and, where necessary, by adding to the whole number or to a part of the separated culture methanolic solution epothilone AND,

b) incubating the culture with the added epothilones And then adding absorbent resin

the Department absorbent resin from culture, elution with methanol and concentration of the eluate to obtain a crude extract,

g) separation of the crude extract with ethyl acetate and water, separating an ethyl acetate phase with the subsequent concentration to obtain oil,

d) chromatography of the oil on reversed phase under the following conditions:

the column material: Nucleosil 100 C-18, 7 μm

the size of the column 250 x 16 mm

solvent: methanol/water = 60:40

the rate of flow of solvent 10 ml/min

with the subsequent compartment containing biotransformed fractions, detected by fluorescence quenching at 254 nm, a value of Rt20 min, and the selection biotransformed.

Further, the invention relates to such biotransformed epothilone And that obtaining such biotransformed epothilone And, given the fact that at the stage (b) incubated for 1-2 nights or more.

Further, the invention relates to the connection of the total formula26H39NO7S, differing in the following1H-NMR spectrum (300 MHz, CDC13): 2.38 (2-Ha), 2.51 (2-Nb), 4.17 (3-H), 3.19 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H2, 10-H2, 11-H2), 2.89 (12-H), 3.00 (13-H), 1.88 (14-Hand), 2.07 (14-Hb), 5.40 (15-H), 6.57 (17-H), 7.08 (19-H), 4.85 (21-H2), 1.05 (22-H3), 1.32 (23-H3), 1.17 (24-H3), 0.97 (25-N3), 2.04 (27-N3).

Further, the invention relates to the compound (epothilone E) formula

< / BR>
epothilone E, where R=H.

Another object of the invention relates to biotransformed epothilone In, get the

a) cultivating a microorganism Sorangium cellulosum DSM 6773 in the presence of adsorbing resin in a known manner, a separation of culture from absorbent resin and, if necessary, by adding to the whole number or to a part of the separated culture methanolic solution epothilone IN,

b) incubating the culture with the added epothilones B and then adding absorbent resin

the Department absorbent resin from the culture by elution with methanol and concentration of the eluate to poluchasa with subsequent concentration to obtain oil,

d) chromatography of the oil on reversed phase under the following conditions:

the column material: Nucleosil 100 C-18, 7 μm

the size of the column 250 x 4 mm

solvent: methanol/water = 60:40

the rate of flow of solvent 10 ml/min

with the subsequent compartment containing biotransformed fractions, detected by fluorescence quenching at 254 nm, a value of Rt24,5 min, and the selection biotransformed.

Further, the invention relates to such biotransformed epothilone, which is obtained by the fact that at the stage of (a) separate culture in the age of 3-4 or more days.

Further, the invention relates to such biotransformed epothilone, which is obtained by the fact that at the stage (b) incubated for 1-2 nights or more.

Further, the invention relates to the connection of the total formula C27H41NO7S, differing in the following 1H-NMR spectrum (300 MHz, D13): 2.37 (2-Nand), 2.52 (2 Hb), 4.20 (3-H), 3.27 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H2, 10-H2, 11-H2), 2.78 (13-H), 1.91 (14-H), 2.06 (14-Hb), 5.42 (15-H), 6.58 (17-H), 7.10 (19-H), 4.89 (21-H2), 1.05 (22-H3), 1.26 (23-H3), 1.14 (24-H3), 0.98 (25-N3), 1.35 (26-H3), 2.06 (27-N3).

Further, the invention against the most epothilones and tools containing

Offer epothilone get the above ways.

Further, the invention relates to tools used for plant protection in agriculture, in forestry and/or horticulture and consisting of one or more of the above epothilone C, D, E and F or one or more of the above epothilones along with one or more conventional carriers and/or diluents.

Finally, the invention relates to therapeutic means consisting of one or more of the above compounds together with one or more conventional carriers and/or diluents. In particular, these tools can have cytotoxic activity and/or cause immunosuppression and/or they can be used for the treatment of malignant tumors, preferably they are used as drugs.

The invention is illustrated by the following examples.

Example 1. Epothilone C and D.

A. producing Strains and culturing conditions, appropriate epothilone, the basic patent DE-B-4138042.

B. Obtaining epothilones using strain DSM 6773.

In accordance with the main patent is grown is th 0.8% starch, of 0.2% glucose, 0.2% of soybean flour, 0.2% of yeast extract, 0.1% of CaCl22H20, 0,1% MgSO47H2O, 8 mg/l Fe-EDTA (pH 7.4) and, if necessary, 15 l adsorbing resin Amberlite XAD-16. The fermentation process is carried out in the course of 7-10 days at 30oWith aeration of 0.1 nl/m3. The speed regulation is Rho2support at the level of 30%.

C. Selection epothilones.

Adsorbing resin is separated from the culture by means of technical filter (suction) 0.7 m2hole size 100 mesh, after which it is freed from polar impurities by washing 3 by volume mixture of water and methanol (2:1). By elution with methanol (volume 4) get the crude extract, which is evaporated in vacuum until the aqueous phase. The last three times extracted with equal volumes of ethyl acetate. In the evaporation of the organic phase obtain 240 g of a crude extract, which share methanol and heptane to separate lipophilic impurities. Then by evaporating the methanol phase in vacuum get 180 grams of raffinate, which three portions fractionary on a column of Sephadex LH-20 (column 20 x 100 cm, 20 ml/min of methanol). Get epothilone see elyuirovaniya fraction (retention time Rt240-300 min) number 72 is 10 x 40 cm the solvent is methanol/water 65:35, 180 ml/min). After epothilones and elute aptilon With (Rt=90-95 min) and epothilone D (Rt=100-110 min), the output of each after evaporation in vacuum is 0.3 g in the form of a colorless oil.

G. Physical properties.

< / BR>
epothilone S, where R=H,

epothilone D, where R=CH3.

Epothilone

WITH26H39NO5S [477].

ESI-MS (positive ions): 478.5 for [M+H]+.

1H and13C-NMR spectra are indicated in the table with the data of NMR spectroscopy.

TCX:Rf= 0,82.

Aluminum foil for thin-layer chromatography type 60 F 254 company Merck, the solvent is dichloromethane/methanol = 9:1.

Detection: fluorescence quenching at 254 nm. Sprinkle based reagent vanillin and sulfuric acid, when heated to 120oWith - staining in blue-gray color.

HPLC: Rt= 11,5 minutes

Column: Nucleosil 100 C-18, particle size 7 μm, column size 125 x 4 mm

Solvent: methanol/water = 65:35.

The rate of flow of solvent 1 ml/min

Detection: led system.

Epothilone D

C27H41NO5S [491].

ESI-MS (positive Copie.

TCX:Rf= 0,82.

Aluminum foil for thin-layer chromatography type 60 F 254 company Merck, the solvent is dichloromethane/methanol = 9:1.

Detection: fluorescence quenching at 254 nm. Sprinkle based reagent vanillin and sulfuric acid, when heated to 120oWith - staining in blue-gray color.

HPLC: Rt= 15,3 minutes

Column: Nucleosil 100 C-18, particle size 7 μm, column size 125 x 4 mm

Solvent: methanol/water = 65:35.

The rate of flow of solvent 1 ml/min

Detection: led system.

Example 2. Epothilone and 12,13-ISAPI-epothilone And epothilone C.

50 mg epothilone And dissolved in 1.5 ml of acetone, then add 1.5 ml of 0.07-molar solution of dimethyldioxirane in acetone. After settling in for 6 hours at room temperature the solution is evaporated in vacuum and separated epothilone from each other preparative HPLC on silica gel (solvent - tert-butyl methyl ether/petroleum ether/methanol, 33:66:1).

Exit 25 mg epothilone A, Rt= 3.5 minutes (analyte. HPLC, particle size 7 μm, column size 4 x 250 mm, solvent - as mentioned above, the flow velocity of the solvent of 1.5 ml/min) and

20 m is ctroscopy in methanol [D4] selected signals: 4.32 (3-H), 3.79 (7-H), 3.06 (12-H), 3.16 (13-H), 5.54 (15-H), 6.69 (17-H), 1.20 (22-H), 1.45 (23-N).

< / BR>
12,13-ISAPI-epothilone A, where R=H.

Example 3. Epothilone E and F - new biotransformation products epothilones a and b, respectively.

Producing strains of

In July 1985, the company "Gesellschaft für Biotechnologische Forschung" was able to extract from soil samples taken on the shore R. Zambezi, producing strains of Sorangium cellulosum So ce 90. October 28, 1991, he was deposited under the number DSM 6773 in the German collection of microorganisms.

Producing strains and cultivation described by the authors G. Hofle, N. Bedorf, K. Gerth, H. Reichenbach in "Erothilone, deren Herstellungsverfahren compounds sie enthaltende Mittel", DE 4138042 Al, wylo. may 27, 1993.

Education epothilones E and F in the fermentation of

A typical fermentation process carried out as follows. In the bioreactor with a volume of 100 l load 60 l of a medium (0.8% starch, 0.2% of glucose, 0.2% of soybean flour, 0.2% of yeast extract, 0.1% of CaCl22H2O, 0,1% MgS47H2O, 8 mg/l Fe-EDTA; pH 7.4). Then add 2% absorbent resin (XAD-16, f-s Rohm & Haas). The medium is sterilized in an autoclave for 2 hours, 120oC). Sow 10 l pre-culture grown in the same environment (with the addition of 50 mm HEPES buffer, pH 7.4). the purpose of 0.2 nl/m3per hour, and the pH is maintained at 7.4 by addition of sodium hydroxide solution. The fermentation process carried out within 7-10 days. Formed within permantaly epothilone continuously contacted with an adsorbent resin. After separation of the culture fluid, for example, by screening using technical filter the resin washed with water (3 volumes) and elute with methanol (4 volumes). The eluate concentrated to dryness and dissolved in 700 ml of methanol.

HPLC-analysis of the eluate XAD

Compared with the original volume of the reactor (70 l) the eluate is concentrated in the ratio of 100: 1. Analysis of the eluate performed by setting HPLC 1090 Hewlett Packard. To separate contained in the eluate substances use column type Microbore (125/2 nucleosil 120-5 C18firms Machery-Nagel (, düren). Elute with a mixture of water and acetonitrile with a gradient starting with a ratio of 75: 25 and ending in 5.5 min ratio 50:50. The last ratio is maintained until the seventh minute, with the consequent increase the ratio until the tenth minute to 100% acetonitrile.

Spectra measured at a wavelength of 250 nm and bandwidth of 4 nm. Spectra led system is measured in the wavelength range 200 - 400 nm. The eluate resin XAD contains two n the spectra epothilones a and b, respectively (see Fig.1), and epothilone E meets epothilone And, as epothilone F - epothilone Century, These substances are formed in these conditions, fermentation only in trace.

Biotransformation epothilones a and b to epothilone E and F

For targeted biotransformation epothilones a and b using 4-daily fixed on absorbent resin culture (500 ml) microorganism So CE 90. Leaving XAD in place, transfer 250 ml of this culture in one litre of sterile Erlenmeyer flask. Then add a methanol solution of a mixture of 36 mg epothilone and 14 mg epothilone In, and then incubate the flask in a rocking chair for two days at 30oAnd with the speed of rotation 200 rpm Education epothilones E and F define the analysis directly 10 μl of centrifuged acculturating fluid (see Fig.2). Turning epothilones only occurs in the presence of cells and depends on the density of cells and time. The kinetics of the transformation process for epothilone As shown in Fig. 3.

Selection epothilones E and F

To highlight epothilones E and F collect initial mixture of the three flasks used in the above process of biotransformation, and shake them for one hour with 20 ml of resin XAD-th extract, shared by 30 ml of ethyl acetate and 100 ml of water. From an ethyl acetate phase by evaporation in a vacuum get 330 mg of oily residue that 5 threads chromatographic on the type column RP-18 size 250 x 20 mm (a solvent mixture of methanol and water in the ratio of 58:42, detection at 254 nm).

Output epothilone E 50 mg, epothilone F 10 mg.

Biological effects epothilone E

Determine the concentration of epothilone E, reducing the growth of cell cultures by 50% (IC50), and compare it with the corresponding concentration epothilone (see table 2).

Epothilone E

C26H39NO7S [509].

ESI-MS (positive ions): 510.3 for [M+H]+.

TCX: Rf= 0,58.

Aluminum foil for thin-layer chromatography type 60 F 254 company Merck, the solvent is dichloromethane/methanol = 9:1.

Detection: fluorescence quenching at 254 nm. Sprinkle based reagent vanillin and sulfuric acid, when heated to 120oWith - staining in blue-gray color.

HPLC: Rt= 5,0 minutes

Column: Nucleosil 100 C-18, particle size 7 μm, column size 250 x 4 mm

Solvent: methanol/water = 60:40.

The rate of flow of solvent 1, D13): 2.38 (2-Nand), 2.51 (2-Nb), 4.17 (3-H), 3.19 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H2, 10-H2, 11-H2), 2.89 (12-H), 3.00 (13-H), 1.88 (14-Hand), 2.07 (14-Hb), 5.40 (15-H), 6.57 (17-H), 7.08 (19-H), 4.85 (21-H2), 1.05 (22-H3), 1.32 (23-H3), 1.17 (24-H3), 0.97 (25-N3), 2.04 (27-N3).

Epothilone F

C21H41NO7S [523].

ESI-MS (positive ions): 524.5 for [M+H]+.

TCX: Rf= 0,58.

Aluminum foil for thin-layer chromatography type 60 F 254 company Merck, the solvent is dichloromethane/methanol = 9:1.

Detection: fluorescence quenching at 254 nm. Sprinkle based reagent vanillin and sulfuric acid, when heated to 120oWith - staining in blue-gray color.

HPLC: Rt= 5,4 minutes

Column: Nucleosil 100 C-18, particle size 7 μm, column size 250 x 4 mm

Solvent: methanol/water = 60:40.

The rate of flow of solvent 1.2 ml/min

Detection: led system.

Results1H-NMR (300 MHz, D13): 2.37 (2-Nand), 2.52 (2 Hb), 4.20 (3-H), 3.27 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H2, 10-H2, 11-H2), 2.78 (13-H), 1.91 (14-H), 2.06 (14-Hb), 5.42 (15-H), 6.58 (17-H), 7.10 (19-H), 4.89 (21-H2), 1.05 (22-HNie epothilones E and F by biotransformation using microorganism Sorangium cellulosum So ce 90.

1) the process of biotransformation.

For the process of biotransformation using a culture of the microorganism Sorangium cellulosum So ce 90 that 4 days was shaken in the presence of 2% of the adsorber resin XAD-16 company Rohm und Haas, Frankfurt am main, 30oC and 160 rpm In the composition of the culture medium consisted of the following components (g/l distilled water: potato starch (Maizena) 8, glucose (Maizena) 8, soy flour defatted 2, yeast extract (firm Mgsog) 2, Fe(III)-sodium salt of ethylenediaminetetraacetic acid 0,008, MgSO47H2About 1, CaCl22H2ABOUT 1, HEPES 11,5. Before processing environment in an autoclave with a solution of caustic potash is set to pH 7.4. Resin XAD Tsevaot from culture, sifting it through a sieve of stainless steel (hole diameter 200 μm). Bacteria precipitated by centrifugation at 10,000 rpm for 10 min, and the resulting spherical precipitate re-resuspended in 1/5 acculturating fluid. In a concentrated suspension of bacteria added respectively epothilone and epothilone In the form of a methanol solution with a concentration of 0.5 g/l Culture continues to be cultivated as described above. To analyze the extent of biotransformation at the right time is given to using methanol. The eluate is evaporated to dryness, and then dissolved in 0.2 ml of methanol. The obtained sample was subjected to HPLC.

Fig. 4 illustrates the kinetics of biotransformation epothilone And to epothilone E; Fig.5 - kinetics of biotransformation epothilone to epothilone F.

2) Obtaining epothilone E. biotransformation of 1 g epothilone A.

Strain Sorangium cellulosum So CE 90 for 4 days grown in 8.5 l of the above medium (but without the addition of XAD resin) in a 10-liter fermenter at 30oWith the mixer rotation speed of 150 rpm and the aeration of 0.1.about. in minutes

Then the culture method for the tangential filtration of concentrate to 3 L. For this purpose, 0.6 m2membranes with a pore size of 0.3 microns.

Concentrated culture was transferred to a 4-liter bioreactor, and then add a methanol solution of 1 g epothilone And 10 ml of methanol. Then continue to cultivate a culture within a 21.5 h, and the temperature is 32oWith the speed of rotation of the agitator 455 rpm and the aeration is carried out with a speed of 6 l/min During the collection of cells, add 100 ml of XAD resin and continue to incubate for 1 h Then the resin chaff is separated from the cells and elute sufficient quantity of methanol. Finally concentrated the womb And 100 mg = 100%

The number epothilone And detected through a 21.5 h of 53.7 mg = 5,4%

The number epothilone E, formed through a 21.5 h of 661.4 mg = 66,1%

The number of completely decomposed epothilone AND 28.5% OF

Example 5.

Proposed according to the invention epothilone tested on cell cultures (see table 3), as well as activation of polymerization (see table 4).

Parameter: period of time to achieve premaxillae polymerization degree of control

Conditions standard test: 0.9 mg tubulin/ml, the concentration of the sample 1 micron.

A test of the ability epothilones to activate the polymerization process carried out in vitro using purified tubulin from brain pigs. The test results are analyzed by means of photometry. Such activating polymerization substances as epothilone, reduce the time required to achieve premaxillae degree of polymerization, i.e. the smaller the time period, the better the connection. The symbols w, x, y, z represent the four independent experience, and the relative effectiveness of the compounds listed in the last column. It is expressed in percent with respect to the action of a control substance. And here the lowest value indicates * * the cell cultures.

1. Epothilone General formula

< / BR>
where R=H, CH3.

2. Epothilone under item 1, representing aptilon With R=N.

3. Epothilone under item 1, representing epothilone D with R=CH3.

4. Epothilone on p. 2 total formula C26H39NO5S different1H and13C-NMR spectra according to the table.1.

5. Epothilone on p. 3 total formula C27H41NO5S different1H and13C-NMR spectra according to the table.1.

6. Epothilone General formula

< / BR>
where R=H, CH3.

7. Epothilone on p. 6, representing epothilone E when R=N

8. Epothilone on p. 6, representing epothilone F with R=CH3< / BR>
9. Epothilone on p. 7 total formula C26H39NO7S, differing in the following1H-NMR spectrum (300 MHz, CDCl3): =2.38 (2-Ha), 2.51 (2-Nb), 4.17 (3-H), 3.19 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H2, 10-H2, 11-H2), 2.89 (12-H), 3.00 (13-H), 1.88 (14-Ha), 2.07 (14-Hb), 5.40 (15-H), 6.57 (17-H), 7.08 (19-H), 4.85 (21-H2), 1.05 (22-H3), 1.32 (23-H3), 1.17 (24-H3), 0.97 (25-N3), 2.04 (27-N3).

10. Epothilone on p. 8 total formula C27H41NO7S, differing in the following1H-NMR spectrum (300 MHz, 13-H), 1.91 (14-H), 2.06 (14-Hb), 5.42 (15-H), 6.58 (17-H), 7.10 (19-H), 4.89 (21-H2), 1.05 (22-H3), 1.26 (23-H3), 1.14 (24-H3), 0.98 (25-N3), 1.35 (26-H3), 2.06 (27-N3).

11. Epothilone E or F, is obtained by microbiological transformation of the original epothilone a or b, namely (a) cultivating a microorganism Soranqium cellulosum DSM 6773 in the presence of adsorbing resin in a known manner, a separation of culture from absorbent resin and, if necessary, by adding to the whole number or to a part of the separated culture methanolic solution of the original epothilone, b) incubating the culture with the added epothilones then adding absorbent resin, the Department absorbent resin from the culture by elution with methanol and concentration of the eluate to obtain a crude extract, g) separation of the crude extract with ethyl acetate and water, Department of an ethyl acetate phase with the subsequent concentration to obtain oil, d) chromatography of the oil on reversed phase under the following conditions: column material: nucleosil 100 C-18, 7 μm; the size of the column: h mm; solvent: methanol/water = 60:40; flow velocity of the solvent: 1.2 ml/min; followed by the separation of containing epothilone E or F of fractions, d is obtained, however, what stage d) are separated fraction with a value of Rtequal to 5.0 minutes

13. Epothilone on p. 14 obtained by the fact that at the stage d) are separated fraction with a value of Rtequal to 5.4 minutes

14. Epothilone on PP.11-13, obtained by the fact that at the stage a) is separated culture in the age of 3-4 or more days.

15. Epothilone on PP.11-14, obtained by the fact that at the stage b) is carried out incubation for 1-2 nights or more.

16. Cytotoxic agent comprising one or more compounds according to one or more paragraphs.1-15.

17. Cytotoxic agent comprising one or more compounds according to one or more paragraphs.1-15 along with one or more conventional carriers and/or diluents.

Priority points:

18.11.1996 on PP.1-5, 17;

25.02.1997 on PP.6-10;

18.11.1997 on PP.11-17.

 

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