HK1022314B - Epothilone d, e and f, production process,and their use as cytostatic as well as phytosanitary agents - Google Patents

Description

Epothilone D, E and F, method for the production thereof, and use thereof as cytostatic and plant-protective agents

no marking

The present invention relates to epothilones C, D, E and F, processes for their preparation, and their use in the preparation of therapeutic compositions and compositions for plant protection.

Epothilone C and D

According to one embodiment, the present invention is directed to epothilones (C and D) which are obtained by:

(a) sorangiumcellulosum DSM6773 is cultured in the presence of an adsorbent resin in a manner known in the art,

(b) the adsorbent resin was removed from the culture and washed with a water/methanol mixture,

(c) eluting the washed adsorbent resin with methanol, concentrating the eluate to obtain crude extract,

(d) extracting the concentrate with ethyl acetate, concentrating the extract, and partitioning between methanol and hexane,

(e) concentrating the methanol phase to obtain a residual liquid, performing fractional extraction on the concentrate on a Sephadex column,

(f) obtaining an extracted fraction comprising metabolites of the microorganism used,

(g) the fractions obtained above were chromatographed on a C18 reverse phase column with a methanol/water mixture and gave, in succession:

-a first part comprising Epothilone A,

-a second part comprising Epothilone B,

-a third part comprising a first further Epothilone,

-a fourth portion comprising a second other Epothilone;

then separating

(h1) The first other Epothilone in the third section above; and

(h2) the second other Epothilone in the fourth section above.

The invention also relates to compounds of the empirical formula C₂₆H₃₉NO₅Epothilone (C) of S, characterized by having the structure shown in Table 1¹H-and¹³C-NMR spectrum.

The present invention also relates to Epothilone C of the formula:

Epothilone C：R＝H。

the invention also relates to compounds of the empirical formula C₂₇H₄₁NO₅Epothilone (D) of S, characterized by having the structure shown in Table 1¹H-and¹³C-NMR spectrum.

The present invention also relates to Epothilone D of the following formula:

Epothilone D：R＝CH₃。

epothilone C and D can be used for preparing the compound of the following formulA 1, and the derivatization process can refer to the derivatization method described in WO-A-97/19086.

In formula 1 above:

R＝H，C_1-4-an alkyl group;

R¹、R²、R³、R⁴、R⁵＝H，C_1-6-alkyl radical, C_1-6-acylbenzyloxy, C_1-4Trialkylsilyl, benzyl, phenyl, C_1-6-alkoxy radical, C₆-alkyl-, hydroxy-and halogen-substituted benzyl or phenyl;

R¹-R⁵two of the radicals may also be combined to form the radical- (CH)₂)_n- (wherein n ═ 1-6), the alkyl or acyl group contained in the group is a linear or branched group;

y and Z, which may be identical or different, are each hydrogen, halogen, such as F, Cl, Br or I, pseudohalogen, such as-NCO, -NCS, or-N₃，OH，O-(C_1-6) -acyl, O- (C)_1-6) -alkyl, O-benzyloxy; y and Z may also be O atoms in epoxides, but are not required to protect epothilones a and B or to form one of the C — C bonds in a C ═ C double bond.

Thus, the 12, 13-double bond can be selectively carried out

Hydrogenation, for example catalytically or with diimine, to give compounds of formula 1 in which Y ═ Z ═ H; or

Epoxidation, e.g. with dimethyldioxirane or a peracid, to give compounds of formula 1 in which Y and Z are ═ O-, or

Conversion into dihalides, ditallohalides or diazides to give compounds of formula 1 in which Y and Z are halogen, pseudohalogen or N₃。

Epothilone E and F

According to another embodiment, the present invention relates to a bioconverter of Epothilone a, obtained by:

(a) incubating Sorangiumcellulosum DSM6773 in a manner known in the art in the presence of an adsorbent resin, removing the adsorbent resin, and if desired, treating all or part of the isolated culture with a solution of Epothilone A in methanol;

(b) incubation with Epothilone a treated cultures followed by treatment with adsorbent resin;

(c) separating the adsorbent resin from the culture, eluting with methanol, and concentrating the eluate to obtain crude extract;

(d) partitioning the crude extract between ethyl acetate and water, separating ethyl acetate phase, and concentrating to oil;

(e) the above oil was subjected to reverse phase chromatographic separation under the following conditions:

column material: nucleosil 100C-187 μm

Column size: 250 x 16mm

Eluent: methanol/water ratio of 60: 40

Flow rate: 10ml/min

And isolating a fraction containing the biotransformants, which fraction is detectable by UV extinction at 254nm, the R of which_tThe value was 20min, and the biotransformants were then isolated.

The invention also relates to a biotransformation of Epothilone A of this type which is obtained by isolating the culture when the culture is cultivated in step (a) for 3 or 4 or more days.

The invention also relates to bioconverters of Epothilone A of this type, obtained by incubation for 1 or 2 or more days in step (b).

The invention also relates to compounds of the empirical formula C₂₆N₃₉NO₇A compound of S characterized by having¹H-NMR Spectroscopy (300MHz, CDCl)₃)：δ＝2.38(2-H_a)，2.51(2-H_b)，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-H_a)，2.07(14-H_b)，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-H₃)，2.04(27-H₃)。

The invention also relates to a compound of the formula (Epothilone E):

Epothilone E：R＝H。

according to yet another embodiment, the present invention relates to a bioconverter of Epothilone B, obtained by:

(a) incubating Sorangiumcellulosum DSM6773 in a manner known in the art in the presence of an adsorbent resin, separating from the adsorbent resin, and if desired, treating all or part of the separated culture with a solution of Epothilone B in methanol;

(b) incubation with Epothilone B-treated cultures followed by treatment with adsorbent resin;

(c) separating the adsorbent resin from the culture, eluting with methanol, and concentrating the eluate to obtain crude extract;

(d) partitioning the crude extract between ethyl acetate and water to separate an ethyl acetate phase, and concentrating to an oil;

(e) the above oil was subjected to reverse phase chromatographic separation under the following conditions:

column material: nucleosil 100C-187 μm

Column size: 250 x 16mm

Eluent: methanol/water ratio of 60: 40

Flow rate: 10ml/min

And isolating a fraction containing the biotransformants, which fraction is detectable by UV extinction at 254nm, the R of which_tThe value was 24.5min, and the biotransformants were then isolated.

The invention also relates to the biotransformants of Epothilone B of this type, which are obtained by isolating the culture when the culture is cultured in step (a) for 3 or 4 or more days.

The invention also relates to bioconverters of Epothilone B of this type, obtained by incubation for 1 or 2 or more days in step (B).

The invention also relates to compounds of the empirical formula C₂₇N₄₁NO₇A compound of S characterized by having¹H-NMR Spectroscopy (300MHz, CDCl)₃)：δ＝2.37(2-H_a)，2.52(2-H_b)，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-H_b)，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-H₃)，1.35(26-H₃)，2.06(27-H₃)。

The invention also relates to a compound of the formula (Epothilone F):

Epothilone F：R＝CH₃。

preparation and composition

The compounds or epothilones according to the invention can be obtained by the methods described above.

The present invention also relates to compositions for protecting plants in agriculture, forestry and/or horticulture consisting of one or more of the aforementioned epothilones C, D, E and F or consisting of one or more of the aforementioned epothilones together with one or more conventional carriers and/or diluents.

The invention finally relates to therapeutic compositions consisting of one or more of the above-mentioned compounds or one or more of the above-mentioned compounds together with one or more conventional carriers and/or diluents. In particular, these compositions have cytotoxic activity and/or produce an immunosuppressive effect and/or are used for controlling malignant tumors, and they are particularly preferably used as cytostatics.

The invention will be illustrated and described in more detail below by means of a description of some specific embodiments.

Examples

Example 1: epothilone C and D

A. Preparation of strains and culture conditions according to the epothilone basic patent DE-B-4138042

B. Preparation with DSM6773

The culture of 75 liters of culture was carried out as described in the basic patent and used for inoculation of a preparation fermenter containing 700 liters of preparation medium consisting of 0.8% starch, 0.2% glucose, 0.2% soya flour, 0.2% yeast extract, 0.1% CaCl₂·2H₂O、0.1％MgSO₄·7H₂O, 8mg/l Fe-EDTA, pH 7.4, and optionally 15 l Amberlite XAD-16 adsorbent resin. The fermentation was continued at 30 ℃ for 7-10 days with an aeration of 0.1NL/m³. Controlling the rotation speed to pO₂The concentration was maintained at 30%.

C. Separation of

Using 0.7m²A100 mesh process filter separates the adsorbent resin from the culture and washes with 3 bed volumes of water/methanol 2: 1, thereby removing polar co-species. Elution with 4 bed volumes of methanol gave a crude extract which was evaporated in vacuo until an aqueous phase appeared. The material was extracted three times with the same volume of ethyl acetate. Evaporating the organic phase to obtain240g of crude extract, which was partitioned between methanol and hexane to separate off lipophilic concomitant substances. Evaporation in vacuo gave 180g of a residue from the methanol phase. The raffinate was fractionated on Sephadex LH-20 (column 20X 100cm, 20ml/min methanol) to give three fractions. Epothilone was contained in a total of 72g fractions with an elution retention time of 240- & ltwbr/& gtfor 300 min. To isolate epothilones, the fractions were chromatographed on Lichrosorb RP-18(15 μm, column 10X 40cm, eluent 180ml/min methanol/water 65: 35) in three fractions. After epothilones A and B, R elutes_t90-95min epothilones C and R_tAfter evaporation in vacuo, 100-.

D. Physical Properties

Epothilone C：R＝H

Epothilone C：R＝CH₃

Epothilone C

C₂₆H₃₉NO₅S(477)

ESI-MS (positive ion): 478.5[ M + H]⁺

¹H and¹³c is shown in an NMR chart

TLC：R_f＝0.82

TLC aluminum foil 60F 254 Merck, eluent: dichloromethane/methanol ratio 9: 1

And (3) detection: UV extinction at 254 nm. Sprayed with a vanillin-sulfuric acid reagent and appears bluish gray when heated to 120 ℃.

HPLC：R_t＝11.5min

Column: nucleosil 100C-187 μm, 125X 4mm

Eluent: methanol/water ratio of 65: 35

Flow rate: 1ml/min

And (3) detection: diode array

Epothilone D

C₂₇H₄₁NO₅S(491)

ESI-MS (positive ion): 492.5[ M + H]⁺

¹H and¹³c is shown in an NMR chart

TLC：R_f＝0.82

TLC aluminum foil 60F 254 Merck, eluent: dichloromethane/methanol ratio 9: 1

And (3) detection: UV extinction at 254 nm. Sprayed with a vanillin-sulfuric acid reagent and appears bluish gray when heated to 120 ℃.

HPLC：R_t＝15.3min

Column: nucleosil 100C-187 μm, 125X 4mm

Eluent: methanol/water ratio of 65: 35

Flow rate: 1ml/min

And (3) detection: diode array

Table 1: epothilone C and epothilone D at [ D₆]At 300MHz in DMSO¹H-and¹³C-NMR data

Epothilone C				Epothilone D
							H atom	δ(ppm)	C atom	δ(ppm)	δ(ppm)	C atom	δ(ppm)
		1	170.3		1	170.1
							2-Ha	2.38	2	38.4	2.35	2	39.0
2-Hb	2.50	3	71.2	2.38	3	70.8
							3-H	3.97	4	53.1	4.10	4	53.2
3-OH	5.12	5	217.1	5.08	5	217.4
							6-H	3.07	6	45.4	3.11	6	44.4
7-H	3.49	7	75.9	3.48	7	75.5
							7-OH	4.46	8	35.4	4.46	8	36.3
8-H	1.34	9	27.6	1.29	9	29.9
							9-Ha	1.15	10	30.0	1.14	10	25.9
9-Hb	1.40	11	27.6	1.38	11	31.8*
							10-Ha	1.15*	12	124.6	1.14*	12	138.3
10-Hb	1.35*	13	133.1	1.35*	13	120.3
							11-Ha	1.90	14	31.1	1.75	14	31.6*
11-Hb	2.18	15	76.3	2.10	15	76.6
							12-H	5.38**	16	137.3		16	137.2
13-H	5.44**	17	119.1	5.08	17	119.2
							14-Ha	2.35	18	152.1	2.30	18	152.1
14-Hb	2.70	19	117.7	2.65	19	117.7
							15-H	5.27	20	164.2	5.29	20	164.3
17-H	6.50	21	18.8	6.51	21	18.9
							19-H	7.35	22	20.8	7.35	22	19.7
21-H3	2.65	23	22.6	2.65	23	22.5
							22-H3	0.94	24	16.7	0.90	24	16.4
23-H3	1.21	25	18.4	1.19	25	18.4
							24-H3	1.06	27	14.2	1.07	26	22.9
25-H3	0.90			0.91	27	14.1
							26-H3				1.63
27-H3	2.10			2.11

^*、^**Can be interchanged

Example 2：

Epothilone A and 12, 13-diepoxy-Epothilone A from Epothilone C

50mg of epothilone A were dissolved in 1.5ml of acetone and treated with 1.5ml of a 0.07mol solution of dimethyl-dioxirane in acetone. After standing at room temperature for 6 hours, the mixture is evaporated in vacuo and the residue is separated by preparative HPLC on silica gel (eluent: tert-butyl methyl ether/petroleum ether/methanol 33: 66: 1).

Yield:

25mg of epothilone A, R_t3.5min (analytical HPLC, 7 μm, column 4X 250mm, eluent as above, flow rate 1.5ml/min), and

20mg of 12, 13-diepoxy-epothilone A, R_t3.7min, ESI-MS (positive ion): 494[ M + H ] M/e]⁺，¹H-NMR([D₄]Methanol) selection signal:

δ＝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-H)

12, 13-diepoxy-epothilone a: r is H

Example 3: novel biotransformation products of epothilones E and F, epothilones A and B

Preparation of the Strain

GBF isolated Sorangiumcelluslose So 90 strain from soil samples from Zambesi river banks at 7 months 1985 and deposited at 28 days 1991 in the German Collection of Microorganisms (German Collection for Microorganisms) with the deposit number DSM 6773.

Product characteristics and culture conditions are described in: h  lfe, g.; bedorf, K.Gerth & HReichenbach: epothilones, processes for their preparation and compositions containing them. DE 4138042A 1 was published at 27.5.1993.

Formation of epothilones E and F during fermentation

A typical fermentation reaction is carried out as follows: a100 liter bioreactor was charged with 60 liters of medium (0.8% starch,0.2% glucose, 0.2% soybean powder, 0.2% yeast extract, 0.1% CaCl₂·2H₂O、0.1％MgSO₄·7H₂O, 8mg/l Fe-EDTA, pH 7.4). Adding 2% of adsorption resin (XAD-16, Rohm)&Haas). The medium was autoclaved (2 hours, 120 ℃). The inoculation was carried out with 10 l of a preculture which had been cultivated in the same medium (additionally 50mM HEPES buffer, pH 7.4) in a shaking flask (160rpm, 30 ℃). The fermentation was carried out at 32 ℃ with a stirrer speed of 500rpm, 0.2Nl of air per cubic meter per hour being introduced and the pH being maintained at 7.4 by addition of potassium hydroxide. The fermentation lasts for 7-10 days. During fermentation, the epothilones formed are continuously bound to the adsorbent resin. After removal of the broth (e.g., sieving with a process filter), the resin is washed with 3 bed volumes of water and then rinsed with 4 bed volumes of methanol. The eluate was concentrated to dryness and taken up in 700ml of methanol.

HPLC analysis of XAD eluents

The eluate was concentrated to 100: 1 relative to the initial volume of the reactor (70 l). The analysis was performed using a 1090HPLC apparatus from Hewlett Packard. For separation of the components, a microporous column (125/2 Nuclear 120-5C) from Machere-Nagel (Duren) was used₁₈). Elution was performed with a gradient of water/acetonitrile from 75: 25 at the start to 50: 50 after 5.5 minutes. This ratio was maintained to 7 minutes and then increased to 100% acetonitrile at 10 minutes.

The measurements were performed at a wavelength of 250nm, with a bandwidth of 4 nm. The diode array spectra were measured in the wavelength range of 200-400 nm. In XAD eluates, two novel substances, R, were found_t5.29 and 5.91, respectively, and their absorption spectra are identical to those of epothilones a and B (fig. 1, E corresponds to a and F corresponds to B). These substances are formed only in very small amounts under the given conditions.

Bioconversion of Epothilone A and B to Epothilone E and F

500ml of the So ce90 culture, which had been cultured for 4 days, were subjected to specific biotransformation with the adsorbent resin. 250ml of this culture was transferred to a sterile 1 liter Erlenmeyer flask, leaving XAD behind. A total of 36mg of epothilone A +14mg of epothilone B in methanol was then added and the flask incubated on a shaking rack at 30 ℃ and 200rpm for 2 days. The formation of epothilones E and F was analyzed directly from the centrifugation supernatant of 10 μ l of culture (fig. 2). Transformation occurs only in the presence of cells and depends on the density and time of the cells used. The kinetics of transformation of Epothilone A are shown in FIG. 3.

Isolation of Epothilone E and F

To isolate epothilones E and F, the three bioconversion shake flasks were combined (see above) and shaken with 20ml XAD-16 for 1 hour. XAD was obtained by filtration and rinsed with 200ml methanol. The eluent was evaporated in vacuo to give 1.7g of crude extract. The crude extract was partitioned between 30ml ethyl acetate and 100ml water. Evaporation in vacuo gave 330mg of an oily residue from the ethyl acetate phase, which was subjected to 5 cycles of chromatography on a 250X 20mm RP-18 column (eluent methanol/water 58: 42, detection at 254 nm).

Yield: epothilone E: 50mg of

F：10mg

Biological Effect of Epothilone E

In cell culture, the concentration at which growth is reduced by 50% (IC) is determined₅₀) And compared to epothilone A.

Cell line	IC50(ng/ml)
			Epothilone E	Epothilone A
	HeLa.KB-3.1 (human)	5			1
Murine fibroblasts, L929			20	4

Epothilone E

C₂₆H₃₉NO₇S(509)

ESI-MS (positive ion): 510.3[ M + H]⁺

TLC：R_f＝0.58

TLC aluminum foil 60F 254 Merck, eluent: dichloromethane/methanol ratio 9: 1

And (3) detection: UV extinction at 254 nm. Sprayed with a vanillin-sulfuric acid reagent and appears bluish gray when heated to 120 ℃.

HPLC：R_t＝5.0min

Column: nucleosil 100C-187 μm, 250X 4mm

Eluent: methanol/water ratio of 60: 40

Flow rate: 1.2ml/min

And (3) detection: diode array

¹H-NMR(300MHz，CDCl₃)：δ＝2.38(2-H_a)，2.51(2-H_b)，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-H_a)，2.07(14-H_b)，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-H₃)，2.04(27-H₃)

Epothilone F

C₂₇H₄₁NO₇S(523)

ESI-MS (positive ion): 524.5[ M + H]⁺

TLC：R_f＝0.58

TLC aluminum foil 60F 254 Merck, eluent: dichloromethane/methanol ratio 9: 1

And (3) detection: UV extinction at 254 nm. Sprayed with a vanillin-sulfuric acid reagent and appears bluish gray when heated to 120 ℃.

HPLC：R_t＝5.4min

Column: nucleosil 100C-187 μm, 250X 4mm

Eluent: methanol/water ratio of 60: 40

Flow rate: 1.2ml/min

And (3) detection: diode array

¹H-NMR Spectroscopy (300MHz, CDCl)₃)：δ＝2.37(2-H_a)，2.52(2-H_b)，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-H_b)，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-H₃)，1.35(26-H₃)，2.06(27-H₃)。

Example 4

Preparation of epothilones E and F by bioconversion with Sorangium cellulosum So ce90

1) Carrying out biotransformation

For the biotransformation, a culture of Sorangium cellulosum So ce90, which had been grown on a 2% XAD 16 adsorbent tree, was usedFat (Rohm)&Haas, Frankfurt/M.) was shaken at 160rpm and 30 ℃ for 4 days. The medium had the following composition (g/liter distilled water): potato starch (Maizena), 8; glucose (Maizena), 8; defatted soy flour, 2; yeast extract (Marcor), 2; ethylenediaminetetraacetic acid, iron (III) sodium salt, 0.008; MgSO (MgSO)₄·7H₂O，1；CaCl₂·2H₂O, 1; HEPES, 11.5. The pH was adjusted to 7.4 with potassium hydroxide prior to autoclaving. XAD was separated from the culture by sieving with a stainless steel sieve (mesh width 200 μm). The bacteria were pelleted by centrifugation at 10000rpm for 10 minutes and the pellet was resuspended in 1/5 culture supernatant. Epothilone A or epothilone B in methanol solution was then added to the concentrated bacterial suspension at a concentration of 0.5 g/liter. The culture was further cultured as described above. To analyze the biotransformation, 1ml of sample was taken at the desired time, 0.1ml of XAD was added, and the sample was shaken for 30 minutes at 30 ℃. XAD was rinsed with methanol. The eluate was concentrated to dryness and then redissolved in 0.2ml of methanol. The sample was analyzed by HPLC.

FIG. 4 is the kinetics of epothilone A conversion to epothilone E.

FIG. 5 is the kinetics of epothilone B conversion to epothilone F.

2) Preparation of epothilone E by bioconversion of 1g epothilone A

In a 10 liter bioreactor, the Soranging cell cellulose So Ce90 strain was cultured at 30 ℃ in 8.5 liters of the above medium (but without XAD) for 4 days, at a rotation speed of 150rpm, and 0.1vvm of air was introduced.

The culture was concentrated to 3 liters by cross-flow filtration. For this purpose, 0.6m is used²The membrane of (4), having a pore diameter of 0.3. mu.m.

The concentrated culture was transferred to a 4 liter bioreactor and a solution of 1 gepothialone a in 10ml methanol was added. The culture was further cultured for 21.5 hours. The temperature was 32 ℃, the stirrer speed was 455rpm and the air introduction rate was 6 l/min. At the time of collection, 100ml of XAD was added and the mixture was incubated for 1 hour. XAD was isolated from the cells by sieving and eluted completely with methanol. The concentrated eluate was analyzed by HPLC.

Equilibrium of biotransformation:

epothilone A used: 1000 mg-100%

Epothilone A recovered after 21.5 hours: 53.7mg ═ 5.4%

Epothilone E formed after 21.5 hours: 661.4 mg%

Fully decomposed epothilone A: 28.5 percent

Example 5

The epothilones of the present invention were tested for their effect on cell culture (Table 2) and for promoting polymerization (Table 3).

Table 2: epothilone experiments with cell cultures

Epothilone	A493	B507	C477	D491	E509	F523
							IC-50(ng/ml)
Mouse fibroblast L929	4	1	100	20	20	1.5
							Human tumor cells
HL-60 (leukemia)	0.2	0.2	10	3	1	0.3
							K-562 (leukemia)	0.3	0.3	20	10	2	0.5
U-937 (lymphoma)	0.2	0.2	10	3	1	0.2
							KB-3.1 (cervical cancer)	1	0.6	20	12	5	0.5
KB-V1 (cervical cancer)	0.3	0.3	15	3	5	0.6
							A-498 (renal carcinoma)	-	1.5	150	20	20	3
A-549 (Lung cancer)	0.7	0.1	30	10	3	0.1

Table 3: polymerization experiments with epothilones

Parameters are as follows: time to half maximum polymerization reaction compared to control

Measuring	w	x	y	z	Reagent [ s ]]	Reagent [% ]]
							Control	20	170	180	210	190	100
Epothilone A	95	60	70	70	74	39
							Epothilone B		23	25	30	26	14
Epothilone C	125	76	95	80	94	49
							Epothilone D	125	73	120		106	56
Epothilone E	80	60	50	45	59	31
							Epothilone F	80	40	30	50	50	26

Standard experiments used tubulin/ml of 0.9mg and sample concentration of 1 μm

The polymerization experiment was an in vitro experiment using tubulin purified from pig brain. Evaluation was performed with a spectrophotometer. The polymerization-promoting substance, such as epothilone, reduces the time required to reach half-maximal polymerization, i.e., the shorter the time, the higher the activity of the compound. w, x, y and z are four independent experiments and the relative activity is expressed as a percentage of the control in the last column; the lowest value indicates the best activity. The ranking table is in good agreement with the values found in cell cultures.

Claims (12)

1. Epothilone D of the formula:

Epothilone D：R＝CH₃。

2. a compound of the formula Epothilone E:

Epothilone E：R＝H。

3. a process for recovering the compound Epothilone E of claim 2 wherein:

(a1) culturing Sorangiumcellulosum DSM6773 in the presence of an adsorbent resin and separating from the adsorbent resin, or

(a2) Treating all or a portion of the isolated culture from step (a1) with a methanolic solution of Epothilone A, incubating the culture treated with Epothilone A, and then treating with an adsorbent resin;

(b) separating the adsorbent resin from the culture, eluting with methanol, and concentrating the eluate to obtain crude extract;

(c) partitioning the crude extract between ethyl acetate and water, separating ethyl acetate phase, and concentrating to oil;

(d) the above oil was subjected to reverse phase chromatographic separation under the following conditions:

column material: nucleosil 100C-187 μm

Column size: 250 x 4mm

Eluent: methanol/water ratio of 60: 40

Flow rate: 1.2ml/min

And isolating a fraction containing the biotransformants, which fraction is detectable by UV extinction at 254nm, the R of which_tThe value was 5.0min, and the biotransformants were then isolated.

4. A method according to claim 3, wherein in step (a1) cultures are isolated which have been cultured for 3 or 4 or more days.

5. The method of claim 3 or 4, wherein the incubation in step (a2) is carried out for 1 or 2 or more days.

6. A compound of the formula Epothilone F:

Epothilone F：R＝CH₃。

7. a process for recovering the compound of claim 6, Epothilone F, wherein:

(a1) culturing Sorangiumcellulosum DSM6773 in the presence of an adsorbent resin and separating from the adsorbent resin, or

(a2) Treating all or a portion of the isolated culture from step (a1) with a methanolic solution of Epothilone B, incubating the culture treated with Epothilone B, and then treating with an adsorbent resin;

(b) separating the adsorbent resin from the culture, eluting with methanol, and concentrating the eluate to obtain crude extract;

(c) partitioning the crude extract between ethyl acetate and water, separating ethyl acetate phase, and concentrating to oil;

(d) the above oil was subjected to reverse phase chromatographic separation under the following conditions:

column material: nucleosil 100C-187 μm

Column size: 250 x 4mm

Eluent: methanol/water ratio of 60: 40

Flow rate: 1.2ml/min

And isolating a fraction containing the biotransformants, which fraction is detectable by UV extinction at 254nm, the R of which_tThe value was 5.4min, and the biotransformants were then isolated.

8. The method of claim 7, wherein in step (a1) a culture is isolated that has been cultured for 3 or 4 or more days.

9. The method of claim 7 or 8, wherein the culturing in step (a2) is performed for 1 or 2 or more days.

10. A therapeutic composition consisting of at least two of Epothilone D, Epothilone E and Epothilone F, or one or more of these compounds and one or more conventional carriers and/or diluents.

11. Use of a composition according to claim 10 for the manufacture of a therapeutic agent with cytotoxic activity, an immunosuppressant or a therapeutic agent for the treatment of cancer.

12. The use of claim 11, wherein the composition is used as a cytostatic agent.