Method for isolating epotilons from reaction mixture and desorption from synthetic resin (variants), using low-polar or nonpolar solvent for realization of method

FIELD: organic chemistry, chemical technology, medicine, oncology.

SUBSTANCE: invention relates to a method for isolation of epotilons used in medicine in treatment of cancer diseases. Method for desorption of epotilons A, B, D and/or E from synthetic resin is based on using low-polar or nonpolar solvent chosen from the group comprising (lower)-alkyl halides and aromatic solvents, or mixture of two or more amount of such solvents wherein prefix "(lower)" means that radical comprises up to 7 carbon atoms. Used aromatic solvent is chosen from the group including naphthalene, benzene or naphthalene and benzene substituted with one or some substitutes chosen from the following group: (lower)-alkyl, (lower)-alkoxy-group, halogen atom, nitro-group and (lower)-alkoxy-(lower)-alkyl wherein prefix "(lower)" means that radical comprises up to 7 carbon atoms. Solvent is removed to the required level but up to preparing a dry residue preferably. If necessary, residue is dissolved in mixture alcohol/hydrocarbon in the corresponding volume ratio. Alcoholic phase is evaporated until dry and then alcoholic extract is crystallized from mixture alcohol and hydrocarbon. Then formed crystallized product is dissolved in mixture nitrile/water but preferably in mixture acetonitrile/water taken in the ratio = 2:3 (vol./vol.). Formed solution is applied on column (if necessary, after separation for some distillates) for preparative chromatography in reversed phase followed by elution with mixture nitrile/water, removing nitrile and extraction of an aqueous phase with ester. Ester extract is evaporated and formed product is subjected for crystallization. Method for preparing epotilons A, B, D and/or E from resin or reaction mixture involves the following steps: (a) desorption of epotilons with low-polar or nonpolar solvent chosen from the group including (lower)-alkyl halides and aromatic solvents, or mixture of two or more amount of such solvents being the desorption step can be repeated up to achievement of the more complete desorption; (b) removal of solvent used in desorption from formed solutions by evaporation; (c) optional crystallization of epotilon(s) after desorption and first of all for crystallization of epotilon B by addition of mixture of alcohol with hydrocarbon and the following evaporation of alcoholic phase until dry and crystallization of epotilon B from the corresponding mixture of solvents; (d) (obligatory step) separation of epotilons by method of chromatography in reversed phase and the following dissolving a residue obtained in previous step in suitable solvent, elution with mixture nitrile/water and removing nitrile from epotilon-containing fractions by evaporation. If necessary, water remained with epotilon is extracted with ester followed by evaporation of epotilon-containing ester phase until dry; (e) optional purification by adsorption chromatography method, and final recrystallization of purified epotilon from corresponding solvents or mixture of solvents. If necessary, in this process between each step formed solutions or suspensions are concentrated, and/or liquid or solid components are separated of one another. Separation of epotilons A and B is carried out by chromatography method based on a mobile layer modeling. Invention provides simplifying methods for preparing large amounts of epotilons for satisfying requirement in these agents.

EFFECT: improved isolating method.

12 cl, 2 ex

The invention relates to a new method of desorption epothilones primarily epothilone and/or epothilone, from resins and to new methods of obtaining, processing and purification or production epothilones, including the specified desorption process, and the use of some solvent for desorption epothilones of resin.

Background of invention

Epothilone a and b represent a new class of stabilizing microtubules cytotoxic active substances (see Gerth, K., and others, J. Antibiot. 49, 560-3 (1966)) of the formula:

where R denotes a hydrogen (epothilone A) or methyl (epothilone).

Since the description of these epothilones (see WO 93/10121) suggested several methods of synthesis and preparation of both of these epothilones and especially the numerous derivatives epothilones (hereinafter in the context of the present description have the group name "epothilone"), for example, epothilones described in WO 99/03848, WO 00/49020, WO 00/49021, WO 00/47584, WO 00/00485, WO 00/23452, WO 99/03848, WO 00/49019, WO 99/07692, WO 98/22461, WO 99/65913, WO 98/38192, WO 00/50423, WO 00/22139, WO 99/58534, WO 97/19086, WO 98/25929, WO 99/67252, WO 99/67253, WO 00/31247, WO 99/42602, WO 99/28324, WO 00/50423, WO 00/39276, WO 99/27890, WO 99/54319, WO 99/54318, WO 99/02514, WO 99/59985, WO 00/37473, WO 98/08849, patent US 6043372, patent US 5969145, WO 99/40047, WO 99/01124 and WO 99/43653.

In addition epothilone and epothilone interesting properties have primarily epothilone D and E, described in WO 97/19086 and WO 98/22461, and e is botilony E and F, described in WO 98/22461 and epothilone described in WO 99/02514.

As an example of therapeutic use in the international application WO 99/43320 described numerous ways to use epothilones as agents against proliferative diseases, primarily associated with tumor diseases, which, due to the similarity of their mechanism of action with well-known and marketed as an anticancer agent Taxol (Taxol^®), have a comparable effect. In WO 99/39694 describes some specific compositions epothilones, first of all, a and B.

Treatment epothilone, primarily epothilones and most preferably by epothilones In, has a number of advantages compared with the accepted treatment systems, especially in those cases when tumors become resistant to Taxol. Thus, there is an urgent need to develop methods for the synthesis of large quantities of epothilones to meet the potential needs of these agents.

The most effective methods of obtaining includes at least several stages of biosynthesis and excretion epothilones from the culture media or other

The previously described extraction of natural substances using myxobacteria primarily extraction epothilones from cells of strain Sorangium cellulosum Soce90 (deposited on the number 6773 in German is the second collection of microorganisms, see WO 93/10121). To obtain a sufficient concentration of natural substances, primarily epothilones previously always in the culture medium was added as an adsorbing agent environment polystyrene resin, for example Amberlite XAD-1180 (firm Rohm & Haas, Frankfurt, Germany).

However, the disadvantage of this method is that when using it for krupnomasshtabnogo production raises a number of problems. The valves are damaged by globules of resin pipe can be blocked and the device may be subjected to greater wear due to mechanical friction. Globule resins are porous and therefore have a large area of the inner surface (about 825 m²/g resin). There are problems with sterilization, because the air inside the resin, not autoclaved. Thus, a method involving adding the resin in the process of cultivation producing epothilone microorganisms, it is impossible to implement on a large scale.

Was discovered and described an improved method of obtaining epothilones primarily epothilones a and b (WO 99/42602). This method consists in education including epothilone complexes in the culture media producing epothilone microorganisms, where these environments contain cyclodextrins or other complexing agents, mixing with the purpose of absorption apoti the ones cell-free culture medium (for example, the filtrate or centrifugate this culture medium) with a synthetic resin, such as resin, the basis matrices which are copolymers of styrene/divinylbenzene, such as Amberlite XAD-16 (firm Rohm & Haas Germany GmbH, Frankfurt, Germany) or Diaion HP-20 (Resindion S.R.L., Mitsubishi Chemical Co., Milan, Italy), and desorption, primarily alcohol, most preferably isopropanol. Then in the alcohol phase, water is added, remove the phase solvent (preferably by evaporation), split phase obtained residue in the presence of ester, primarily ethyl acetate or isopropylacetate, normally carried out by molecular filtration (gel chromatography) dried ester phase, divide the mixture epothilones using GHUR with reversed phase (preferably, by elution with a mixture of nitrile/water, for example, acetonitrile/water), and optionally perform additional purification by phase separation in the presence of a mixture of water/simple ether, preferably with subsequent adsorption chromatography on silica gel to further remove impurities and carry out the crystallization/recrystallization.

Despite its progressive character and suitability for large-scale industrial production, this method suffers from some drawbacks.

For example, to obtain the demand is purity, it is advisable to use either stage molecular filtration, either phase adsorption chromatography on silica gel, or both processes. Difficulties associated with the separation of the phases in the presence of ester, such as ethyl acetate that (especially because of the long time required for the implementation of phase separation water/ester phase with krupnomasshtabno production) is time-consuming, as well as with the subsequent phase evaporation, the implementation of which also troubled because of foaming and splashing.

This task is solved by the present invention to avoid these disadvantages and to find new and most appropriate way of allocating epothilones primarily epothilones and after their adsorption on the resin.

Summary of the invention

In the claimed invention unexpectedly found that by simply replacing the alcohol used as solvent for desorption, certain other solvents (which are represented as polar or apolar solvents as listed below), you can achieve a real breakthrough in solving the above-mentioned problems with additional benefits, such as increased desorption and higher final yield of the product. Of the added benefits it should be noted (I) higher election is entrusted desorption; (II) a higher number of desorbed epothilones, indicating a more complete desorption; (III) no need to re-extraction of the complex ester (e.g. ethyl acetate), which is associated with major problems associated with the separation of water/ester phase and does not require evaporation of the ether; (IV) the absence of molecular stage filtration and, as a rule, adsorption chromatography; (V) a significant reduction in time spent on desorption; (VI) the reduction in the number of process steps, (VII) reducing the risk of contamination (which is important for highly epothilones) and/or (VIII) for easier and safer production (depending on the solvent used for desorption = extraction); and (IX) are also depending on the solvent unexpectedly even lower amounts of by-products or impurities having a polarity similar to the polarity of epothilones primarily epothilone In that you want to delete after chromatography with reversed phase. These and other advantages should become apparent after reading the following detailed description of the invention:

Detailed description of the invention

One of the objects of the invention is a method of desorption epothilones primarily epothilone and/or, in particular epothilone In, from the resin, especially a synthetic resin, with the help of kabobo is popular or apolar solvent.

The next object of the invention is a method of treatment (or treatment) epothilones, especially after their receipt in a standard environment for chemical synthesis or preferably in the culture medium, which contains microorganisms, primarily this type of bacteria, in particular of the genus Sorangium, which can be used for production epothilones primarily epothilone a and/or b, and a complexing component, and the process involves the application of polar or apolar solvent for desorption epothilones of resin.

The third object of the invention is the use of polar or apolar solvent for desorption epothilones primarily epothilone a and/or b, especially epothilone In, from the resin, especially from sinteticheski resin.

Used in the context of the present description General definitions have the following meanings:

The concept of "epothilone" preferably refers to any epothilone or derived epothilone referred to in patent applications WO 99/03848, WO 00/49020, WO 00/49021, WO 00/47584, WO 00/00485, WO 00/23452, WO 99/03848, WO 00/49019, WO 99/07692, WO 98/22461, WO 99/65913, WO 98/38192, WO 00/50423, WO 00/22139, WO 99/58534, WO 97/19086, WO 98/25929, WO 99/67252, WO 99/67253, WO 00/31247, WO 99/42602, WO 99/28324, WO 00/50423, WO 00/39276, WO 99/27890, WO 99/54319, WO 99/54318, WO 99/02514, WO 99/59985, WO 00/37473, WO 98/08849, patent US 6043372, patent US 5969145, WO 99/40047, WO 99/01124 and/or WO 99/43653, more preferably is to epothilone And primarily epothilone, but in a broader object of the invention to epothilone D and E, described in WO 97/19086 and WO 98/22461 to epothilones E and F, described in WO 98/22461, or to epothilones described in WO 99/02514. All of these documents primarily related to derivatives epothilones and the above-mentioned preferred derivatives epothilones included in the present description by reference.

Polar or apolar solvent preferably has the following features:

Preferred is a solvent for which allotropy a number determined according to Snyder and others, J. Chromatogr. Sci. 16, 223 (1978), has the following characteristics, where X_ddenotes the parameter of the proton acceptor (an indicator of the tendency to form hydrogen bonds as an acceptor of hydrogen), X_ddenotes the parameter of the proton donor (an indicator of the tendency to form hydrogen bonds as a donor of hydrogen), and x_nindicates the parameter dipole (indicator characteristics of the dipole), provided that x_e+x_d+x_n=1, or a mixture of such solvents are:

x_e=0,20-0,40; x_d=0,15-0,36; and x_n=0,38-0,60; preferably,

x_e=0,22-0,32; x_d=0,17-0,34; and x_n=0,39-0,54.

Most preferred among polar or apolar solvents are (ness.)alkyl-(ness.)alkylene or cyclic ketones, such as ACET is h, methyl ethyl ketone, 2-pentanone, methyl isobutyl ketone or cyclohexanone, more preferably ethers, especially cyclic ethers, in particular tetrahydrofuran or dioxane; even more preferably halogenated aliphatic compounds, especially (ness.)alkylhalogenide, primarily methylenechloride (= methylene chloride or ethylene dichloride; or most preferably aromatic solvents, primarily naphthalene, or preferably benzene or naphthalene, or (preferably) benzene, substituted by one or more, preferably 1-3 substituents selected from the group comprising (ness.)alkyl, especially methyl, ethyl or isopropyl, (ness.)alkoxygroup, especially methoxy or ethoxypropan, halogen, especially fluorine, chlorine, bromine or iodine, a nitro-group, and (ness.)alkoxy-(ness.)alkyl, especially ethoxymethyl; primarily toluene, ethylbenzene, xylene, primarily ortho-, meta - or paraxylene, mesitylene, pseudocumene, hamiliton, cumin, isopropyltoluene, phenylalanine, primarily torbenson, chlorobenzene, bramasol or iadanza, (ness.)alkoxybenzyl, primarily ethoxybenzoyl or methoxybenzoyl, or (ness.)alkoxy-(ness.)the alkyl benzenes, especially ethoxymethylene (simple benzyl ether); or any mixture of two or more, for example, 2-4 of these solvents; Nai is more preferred are (ness.)the alkyl benzenes, first of all, ethylbenzene, xylene, primarily ortho-, meta - or paraxylene, mesitylene, pseudocumene, hamalian, cumin, isopropyltoluene and most preferably toluene.

The term "polar or apolar solvent" includes mixtures of two or more of the solvents described above and below, for example 2-4 of such solvents.

Console "(ness.)" in all cases suggests that marked a radical preferably contains up to 7 carbon atoms, in particular up to 4 carbon atoms and is branched or unbranched. (Ness.)alkyl may, for example, can be unbranched or to have one or more side chains and denotes, for example, methyl, ethyl, propyl, such as the isopropyl or n-propyl, butyl, such as isobutyl, sec-butyl, tert-butyl or n-butyl and pentyl, such as amyl or n-pentyl.

Halogen preferably represents iodine, bromine, chlorine or fluorine.

The hydrocarbon preferably is an organic compound consisting of 4-32, more preferably from 4 to 20, more preferably from 6 to 16 carbon atoms, which may be an aliphatic, for example, linear, branched or cyclic saturated alkane, for example, cyclohexane, linear, branched or cyclic (non-aromatic) 'or is anicescu connection with one or more double and/or triple bonds or aromatic hydrocarbon, which may be unsubstituted or may be substituted by one or more, e.g. up to 3 substituents selected from the group comprising (ness.)alkyl, especially methyl, ethyl or isopropyl, (ness.)alkoxygroup, especially methoxy or ethoxypropan, halogen, especially fluorine, chlorine, bromine or iodine, a nitro-group, and (ness.)alkoxy-(ness.)alkyl, especially ethoxymethyl; preferably 1-3 (ness.)alkyl groups.

The alcohol is preferably a hydroxy(ness.)alkane, especially methanol, ethanol or n - or isopropanol.

The resin is preferably a synthetic resin, preferably a resin based on a copolymer of styrene and divinylbenzene, more preferably Amberlite XAD-4, or preferably Amberlite XAD-16 [the company Rohm & Haas Germany GmbH, Frankfurt] or Diaion HP-20 [Resindion S.R.L., Mitsubishi Chemical Co., Milan]. It should be noted that epothilone are non-covalent contact with the resin, from which epothilone or epothilone you want to decarbonate according to the invention, (e.g., reversibly bound or sorbed on it). In other words, when you use the term "resin", it should be understood that the resin contains one or more epothilones, which are in contact with it, especially reversibly bound or adsorbed on her."

Preferred embodiments of the Soberania.

The preferred embodiment of the invention relates to a method involving the desorption from the resin with polar or apolar solvent, above or below, and any additional purification stages, desirable or necessary to obtain in pure form respective epothilones primarily epothilone and most preferably epothilone, preferably described in this invention as preferred.

Processing epothilones precedes the reaction or process leading to the containing epothilone of the reaction product to be processed, they emit (I) or mixtures of chemical reactions after dissolution in an appropriate polar aqueous medium, or (II) is more preferable from the supernatant (e.g., containing epothilone cyclodextrin, according to the method described in WO 99/42602) culture medium containing producing epothilone microorganisms, by the division of culture into the liquid phase (for example, centrifugal or filtrate) and solid phase (cells), primarily through filtration or centrifugation (tubular centrifuge or the separator).

After this pre-treatment is preferably carried out direct mixing of a solution of ((I)) or liquid phase ((II)) with resin primarily synthetic grass is the first resin, first of all resins based on copolymers of styrene and divinylbenzene as a matrix (hereinafter designated as polystyrene), such as Amberlite XAD-16 or Diaion HP-20 (preferably in a volume ratio of centrifugal: resin from about 10:1 to 100:1, preferably about 50:1). After contact with a duration of preferably 0.25 to 50 hours, preferably from 0.8 to 22 h, the resin is separated, for example by filtration, sedimentation or centrifugation. If necessary, after the adsorption of the resin is washed with a highly polar solvent, preferably water.

Then begin a preferred process of the present invention. Desorption epothilones (according to the most preferred object of the invention) perform slightly polar or apolar solvent, in particular (ness.)alkylhalogenide, primarily methylenechloride or ethylene dichloride, or more preferably aromatic solvents, primarily naphthalene, or preferably benzene or naphthalene, or (preferably) benzene, substituted by one or more, preferably 1 to 3 radicals selected from the group comprising (ness.)alkyl, especially methyl, ethyl or isopropyl, (ness.)alkoxygroup, especially methoxy or ethoxypropan, halogen, especially fluorine, chlorine, bromine or iodine, a nitro-group, and (ness.)alkoxy-(or the sh.)alkyl, first of all ethoxymethyl; most preferably toluene. Stage extraction can be repeated one or more times, preferably 0-3 times before only 1 time, in order to gain a more complete desorption if it is necessary or desirable.

After processing the thus obtained solutions desorbed epothilones (which unite in the case of re-extraction) preferably carry out the removal of used for desorption of the solvent from the resulting solution by evaporation (distillation), preferably with concentration in the reactor and then using a rotary evaporator under vacuum.

Then perform additional processing using the following stages, with stage purification using chromatography with reversed phase elution nitrile is mandatory, while other stages are not mandatory:

- crystallization epothilone(s) after desorption; primarily for crystallization epothilone In, add a mixture of alcohol and hydrocarbon, primarily (ness.)alkanol and cyclic aliphatic hydrocarbon with 3-10 ring atoms, most preferably methanol and cyclohexane (preferably in the ratio (vol./about.) from 1:10 to 10:1, especially 1:3 to 3:1. The addition of water leads to a rapid separation of the phases and alcohol f is zu evaporated to dryness, for example, using a rotary evaporator under vacuum. The resulting extract, which contains epothilone In, then crystallized from the appropriate solvent mixture, first from a mixture of alcohol/cyclic aliphatic hydrocarbon, most preferably from a mixture of isopropanol/cyclohexane, preferably in the ratio (vol./about.) from 1:10 to 10:1, more preferably from 1:6 to 6:1, most preferably from 1:6 to 1:4;

- (mandatory stage) Department epothilones using chromatography with reversed phase, followed by dissolving in a suitable solvent, especially in a mixture of nitrile and water, preferably acetonitrile/water, in the preferred ratio (vol./about.) from 1:10 to 10:1, especially 1:3 to 1:1, and elution with a mixture of nitrile and water, preferably this stage differs in that the chromatography is carried out on a column of material with reversed phase, loaded with hydrocarbons, such as hydrocarbons containing 18 carbon atoms, especially material RP-18, eluent used, containing nitrile, primarily (ness.)alkynylaryl, in particular acetonitrile, in particular a mixture of nitrile/water, primarily a mixture of acetonitrile/water, preferably in the ratio of nitrile and water from about 1:99 to 99:1, especially from 1:9 to 9:1, for example, from 2:8 to 7:3, for example, 3:7 or 4:6; and remove either the Rhyl collected containing epothilone (primarily epothilone And most preferably epothilone) fractions by evaporation (distillation; if necessary, remaining with epothilones water then extracted with ether complex, primarily (ness.)alkyl-(ness.)alkanoates, preferably by isopropylacetate, followed by evaporation to dryness (preferably initially in the reactor, and then in a rotary evaporator under vacuum) containing epothilone ester phase; (if necessary, the original solution epothilone can be divided into parts and for the Department to use more than one ring on reversed phase);

- only when necessary (for example, as an alternative crystallization after desorption) adsorption chromatography, in particular, using a column of silica gel and elution with appropriate solvent or mixture of solvents, primarily a mixture of ester /hydrocarbon, for example, (ness.)alkylalcohol /S₄-C₁₀alkane, especially ethyl or isopropylacetate/ n-hexane, and the ratio of ester and hydrocarbon is preferably from 99:1 to 1:99, preferably from 10:1 to 1:10, for example 4:1;

and the final recrystallization, for example, from suitable solvents or mixtures of solvents, such as esters, mixtures of esters/hydrocarbons or alcohols, especially ethyl or isopropylacetate: toluene in a ratio of from 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or e is of racette (epothilone);

in this process, if necessary, and/or, if desired, applied between each of the stages of the resulting solution or suspension concentrate and/or liquid or solid components are separated from each other, in particular, by sedimentation, filtration or centrifugation of the solutions/suspensions. More precise recommendations mentioned above and below can preferably be used in carrying out the above individual steps.

The preferred embodiment of the invention is a method of separation epothilones adsorbed on a synthetic resin, especially epothilone And most preferably epothilone In which envisages

(I) desorption epothilones of synthetic resin polar or apolar solvent, in particular (ness.)alkylhalogenide, primarily methylenechloride or ethylene dichloride, or more preferably aromatic solvents, primarily naphthalene, or preferably benzene or naphthalene, or (preferably) benzene, substituted by one or more, preferably 1 to 3 radicals selected from the group comprising (ness.)alkyl, especially methyl, ethyl or isopropyl, (ness.)alkoxygroup, especially methoxy or ethoxypropan, halogen, especially fluorine, chlorine, bromine or iodine, nitro-group and (ness.)is laxi-(ness.)the alkyl, first of all ethoxymethyl; most preferably toluene; or in a broader object of the invention a mixture of two or more such solvents; and

(II) the Department epothilones using chromatography with reversed phase, followed by dissolving in an appropriate solvent, especially in a mixture of nitrile and water, preferably acetonitrile/water, in the preferred ratio (vol./about.) from 1:10 to 10:1, especially 1:3 to 1:1, and elution with a mixture of nitrile and water, preferably this stage differs in that the chromatography is carried out on a column of material with reversed phase, primarily material RP-18, which is loaded with hydrocarbons, for example, hydrocarbons containing 18 carbon atoms, and use eluent containing nitrile, primarily (ness.)alkynylaryl, in particular acetonitrile, in particular a mixture of nitrile/water, primarily a mixture of acetonitrile/water, preferably in the ratio of nitrile and water from about 1:99 to 99:1, especially from 1:9 to 9:1, for example, from 2:8 to 7:3, for example, 3:7 or 4:6.

As a preferred object of the invention is the method described in the previous paragraph, which begins with stage (I) and contains stage (II), and which additionally includes any additional purification stages, desirable or necessary to obtain in pure form with the relevant epothilones, first of all epothilone and most preferably epothilone, preferably described as preferred according to the present invention.

Preparations for the treatment is preferably carried out as follows: adsorption epothilones primarily from chemical reaction mixtures, or more preferably from supernatant cultures of microorganisms can be carried out according to the method described in WO 99/4260, or a similar method; in General, the method consists in the following: containing epothilone centrifugal directly mixed with a synthetic resin, especially a copolymer of styrene/divinylbenzene, such as Amberlite XAD-16 or Diaion HP-20 (preferably in a volume ratio of centrifugate:resin from about 10:1 to 100:1, preferably about 50:1) and stirred the mixer. At this stage epothilone transferred to the resin. After a period of time sufficient for adsorption, for example after a period of contact of about 0.2 to 10 h, the resin is separated by centrifugation or filtration. Adsorption epothilones resin can be carried out on a chromatographic column, introducing the resin into the column and disperse centrifugal on the resin. After adsorption of the resin washed with water.

Then begin the implementation of the preferred process according to the invention, which is carried out as follows: desorption epothilones from the Ola preferably carried out using a polar or apolar solvent according to the invention, first of all solvent, the above or below as the preferred, preferably methylene chloride or most preferably toluene. The solvent is then removed to the desired extent, preferably to obtain a dry residue. If necessary, the residue is dissolved in a relatively small volume in a mixture of alcohol/hydrocarbon, primarily in methanol/cyclohexane, preferably taken in the above-described ratio. The alcohol phase is evaporated, preferably to dryness, and the alcoholic extract is then crystallized from a mixture of alcohol, especially isopropanol, hydrocarbons, primarily cyclohexane, preferably taken in the above-described ratios. The resulting solid crystallized product is then dissolved in a mixture of nitrile/water, preferably as described above, first of all in a mixture of 2:3 (about./about.) acetonitrile/water, and the resulting mother liquor make, if necessary, after separation on a few straps, in a column for preparative chromatography with reversed phase. Then carry out the elution nitrile/water, primarily according to the above-described method. (Aceto)nitrile obtained fractions containing epothilone primarily epothilone and most preferably epothilone To remove by evaporation (distillation), and the resulting aqueous phase is extracted with SL is mportant ether, first of all isopropylacetate. Then ester extract evaporated, preferably to dryness, and then the resulting product is subjected to recrystallization, for example, the fraction epothilone And crystallized directly from a mixture of ester/hydrocarbon, for example, ethyl acetate:toluene = 2:3, and the fraction epothilone In crystallized from ether complex, especially ethyl acetate, or preferably from alcohol, especially methanol.

Was opened eluent with high electoral action (desorbent), primarily toluene, the use of which allows to achieve approximately 100%output for half of the period of time required for the implementation of desorption using isopropanol, which was applied in WO 99/42602. With the invention it has been unexpectedly found that the amount of desorbed epothilones increases, for example, up to 130%after desorption with toluene compared to desorption using isopropanol. (Although at first glance it seems to be impossible from a theoretical point of view, this fact is most fully illustrates the main advantage of the present invention: the results associated with the analysis of loaded resin. Since this result is obtained by using isopropanol for desorption, was based on incomplete desorption process, the source led to C is low the results of the analysis, after the creation of the present invention should be considered as false). The mixture epothilones can (without crystallization or pre-crystallization) to contribute directly to the column with reversed phase. This process largely is not dependent on the amount of the solvent, the stirring speed and temperature. When using alcohols (e.g. ethanol or isopropanol) under stirring may be biphasic desorption, when the first period of time, the desorption of the first number (the first portion) containing epothilone product (which can be explained by the distribution of pore size in polystyrene XAD-16, which is characterized by two maxima in the distribution), and after any additional period of time, the desorption of the second portion of the product, this unwanted behavior is not observed when using toluene or dichloromethane, when the desorption of the product occurs during the first period of time. Under comparable conditions formed after evaporation the residue epothilones a and b, obtained from supernatants containing cyclodextrins cultures described in WO 99/42602, when using isopropanol, is, according to one example, 40 g, using methylene chloride and 3.3 g, as in the case of toluene only 0.9 g, CPA is to for example, the fact that the rest epothilone In is 17-18 g - % after desorption from resins based on styrene/divinylbenzene, these results suggest a higher purity product. Despite the fact that toluene has the most pronounced advantages, the advantage of dichloromethane, is the ease of removal due to low boiling point.

Division epothilones a and b can be realized also by means of chromatography described in the present description, using the approach of modeling the moving layer (SMB). SMB chromatography is widely used for the separation of binary mixtures, for example, separation of a racemate by chiral stationary phases, for example, in SORBEX processes in petrochemical production, type Parex or Molex, or SAREX process for sugar production. Compared with chromatography periodic action advantage of SMB chromatography is a continuous countercurrent process, which leads to increased productivity and reduced absorption of the mobile phase. Some systematic procedure for implementing the method of SMB chromatography known to specialists in this field. Such procedures are described, for example, in R.-M.Nicoud, M.Bailly, J.Kinkel, R.M.Devant, T.R.E.Hampe and EKüsters in: Proceedings of the 1^stEuropean Meeting on Simulated Moving Bed Chromatography, (1993), ISBN 2-905-267-21-6, cc.65-88; E.Küsters, G.Gerber and F.D.Antia, Chromatographia,40 (1995) 387; TRGöll and EKüsters, J.Chromatogr. A, 800 (1998) 135; or Sneag, EKüsters, T.Plattner and A.Seidel-Morgenstern, J.Chromatogr. A, 827 (1998) 175. The basic parameters required for the separation epothilone a and b using SMB chromatography, can be obtained directly from the conventional separation using LC. Preferably, the silica gel reversed-phase (RP 18) is used as the stationary phase and a mixture of water/acetonitrile as mobile phases. A finite range of flow velocities (for individual zones of the SMB, and time, respectively) can be selected either on the basis of a simple schematic flow proposed EKüsters, and others: Chromatographia, 40 (1995) 387, or after careful determination of adsorption isotherms according to the method described in J.Chromatogr. And, 800 (1998) 135 and J.Chromatogr. A, 827 (1998) 175. Processing of the extract and purified flows can be made according to the method used for normal separation using standard LC.

The invention is most preferably applied to processes and methods, which are described below in the examples.

Examples.

Below the invention is illustrated in the examples, not limiting its scope.

Precautions: When working with epothilone need to apply appropriate remedies due to their high toxicity.

Example 1. The process epothilone

JASS is Blu 591,7 kg loaded resin (resin, representing a copolymer of styrene/divinylbenzene XAD-16, bearing epothilone a and b from the culture medium) is carried out by mixing the resin with two portions of toluene at 720 l each for about 8 hours Department toluene phase from the resin is carried out using a vacuum filter. The combined toluene phases are washed with two portions of water 250 l each. After separation of the phases in toluene extract was concentrated in a 1000 liter reactor to approximately 20-40 l and then concentrated to dryness using a rotary evaporator under vacuum. The result toluene extract weight 4,095 kg, containing 209 g epothilone Century Toluene extract is dissolved in 16.5 l of methanol and 24.5 l of cyclohexane. After adding 0.8 l of water immediately there is a separation of phases. The methanol fraction is evaporated to dryness using a rotary evaporator under vacuum, obtaining 1,025 kg one stripped off the resin, containing 194 g epothilone Century Methanol extract is then crystallized in a mixture of solvents containing 2,05 l of isopropanol and of 10.25 liters of cyclohexane, gaining 0.4 kg crystallized product, which contains 184 g epothilone C. the Crystals are dissolved in 3.2 liters of a mixture of acetonitrile/water = 2/3 (about./about.) and the resulting mother liquor separated using 3 different straps on the column for preparative chromatography with reversed phase (25 kg sherice is anyone silica gel RP-18, YMC-Gel ODS-A 120; 5-15 μm; Waters Corp., Milford, Massachusetts, USA.) Elution is carried out by using as mobile phase a mixture of acetonitrile/water = 2/3 (about./about.) a flow rate of 2.3 l/min;

retention time epothilone And is 77-96 min, retention time epothilone In - 96-119 minutes Monitoring fractionation is carried out using UV detector at a wavelength of 250 nm. Acetonitrile together with the fractions containing epothilone (three Poonam)is distilled off and the remaining aqueous phase is extracted with 504 l isopropylacetate. Isopropylacetate the extract was concentrated in the 630-liter reactor to approximately 20-40 l and then concentrated to dryness using a rotary evaporator under vacuum. The mass of one stripped off the rest of the factions epothilone In 170 g, which corresponds to the content of 98.4 per cent according to GHUR (external standard). The resulting product is finally crystallized in 2,89 l of methanol at 0-5°getting 150 grams of pure kristalliset epothilone C. melting point: 124-125°C;

¹H-NMR data for epothilone In (500 MHz NMR, solvent: DMSO-d6. The chemical shift 6 in part./million relative to TMS (tetramethylsilane was). S = singlet, d = doublet, m = multiplet):

Example 2. Comparison of different desorption for drugs epothilone

In 360 ml of each aqueous suspension of the resin constituting the copolymer of styrene/divinylbenzene type XAD-16 uploaded by epothilones a and b from the cultures of mycobacteria, using the method described in WO 99/42602 (which corresponds to 194 g of wet resin Amberlite® XAD-16), extracted with solvents and conditions specified in the table below, mixed (ordinary laboratory anchor stirrer) in a glass p is the actor with Frit, stainless fabric on the bottom (hand-made solid-phase batch reactor, the, 10 cm (inner diameter) ×20 cm (length), (hereinafter designated as "mixer /Frit").

From these experiments and other data we can conclude that compared with isopropanol extraction with methylene chloride has a greater selectivity, shortens the duration of the extraction (about 2 times), accelerates the distillation of the solvent (boiling point of methylene chloride is about 40°and isopropanol 81-83° (C)in this case does not require the implementation of a long and fraught with many problems of phase separation of the ethyl acetate/water, is no longer required to perform the second distillation of the solvent, resulting in a smaller number of stages reduces the risk of contamination, improves and becomes more secure handling; the possibility of implementation of the process in half the volume (for example, in a 1000 liter instead of a 2000-liter reactor); the product epothilone has improved purity profile (fewer by-products having a polarity similar to the polarity of epothilone), and the residue after evaporation is not sprayed and does not form a foam, as in the case of extraction with ethyl acetate. When desorption of toluene receive higher the th output compared to extraction with isopropanol (about 100 instead of 80%), thus it is possible to achieve improved selectivity when using isopropanol approximately 10-fold the number of products subjected to desorption), the duration of extraction is reduced significantly (about 3 times), simplifies a complex filtering process after desorption with isopropanol (extraction with isopropanol difficult when krupnomasshtabno production), no longer require a second distillation of the solvent, the processing can be performed in reactors of smaller size (and in this case, for example, you can use a 1000 liter reactor instead of the 2,000-liter), you can refuse chromatography on silica gel (one stripped off the remainder after desorption already contains about 40% of the mixture epothilones A/b), and one stripped off the remainder after desorption has no ability to foaming and splashing that occurs in the case of residue after extraction with ethyl acetate.

1. The way desorption epothilones a, b, D and/or E of the resin, based on the use of polar or apolar solvent selected from the group comprising (ness.)alkylhalogenide and aromatic solvents or a mixture of two or more such solvents, where the prefix "(ness.)" means that the radical contains up to 7 carbon atoms.

2. The method according to claim 1, where epothilone that you want to decarbonate, are the battle epothilone or epothilone Century

3. The method according to any of claim 1 or 2, where the resin is a synthetic resin.

4. The method according to claim 3, where the resin is a synthetic resin based on a copolymer of styrene/divinylbenzene.

5. The method according to any one of claims 1 to 4, where the use of an aromatic solvent selected from the group comprising naphthalene, benzene or naphthalene and benzene substituted by one or more substituents selected from the group comprising (ness.)alkyl, (ness.)alkoxygroup, halogen, the nitro-group and (ness.)alkoxy-(ness.)alkyl, where the prefix "(ness.)" means that the radical contains up to 7 carbon atoms.

6. The allocation method epothilones a, b, D and/or E adsorbed on synthetic resin, providing (III) desorption epothilones of synthetic resin with polar or apolar solvent selected from the group comprising (ness.)alkylhalogenide and aromatic solvents or a mixture of two or more such solvents, where the prefix "(ness.)" means that the radical contains up to 7 carbon atoms, and (IV) separation epothilones using chromatography with reversed phase after their dissolution in an acceptable solvent and elution with a mixture of nitrile and water.

7. The method of obtaining epothilones a, b, D and/or E from the reaction mixture, which desorption epothilones poorly Olamim or apolar solvent, selected from the group comprising (ness.)alkylhalogenide and aromatic solvents or a mixture of two or more such solvents, and the stage of desorption can be repeated to achieve a more complete desorption; removing used for desorption of the solvent from the resulting solution by evaporation; optional crystallization epothilone(s) after desorption; primarily for crystallization epothilone In by adding a mixture of alcohol and hydrocarbon, and the process of evaporation of the alcohol phase to dryness and crystallization epothilone In from the appropriate solvent mixture; (mandatory stage) Department epothilones using chromatography with reversed phase, followed by dissolving the residue obtained in the previous stage, in a suitable solvent and elution with a mixture of nitrile and water, and the removal of the nitrile from the collected containing epothilone fractions by evaporation; if necessary, remaining with epothilones water then extracted with ether complex with subsequent evaporation to dryness containing epothilone ester phase; optional purification adsorption chromatography; and a final recrystallization cleaned epothilone from suitable solvents or mixtures of solvents; in this process if necessary, and/or, if desired, between each applicable stage of the formed rastv the market or suspension concentrate and/or liquid or solid components are separated from each other; moreover, the prefix "(ness.)" means that the radical contains up to 7 carbon atoms.

8. The method according to claim 7, where the desorption epothilones from the resin exercise polar or apolar solvent selected from the group comprising (ness.)alkylhalogenide and aromatic solvents or a mixture of two or more such solvents, and then the solvent is removed to the desired level, preferably to obtain a dry residue, if necessary, the residue is dissolved in a mixture of alcohol/hydrocarbon in a respective volume ratio; alcohol phase is preferably evaporated to dryness and then alcohol extract is crystallized from a mixture of alcohol and hydrocarbon, then the resulting solid crystallized product is dissolved in a mixture of nitrile/water according to claim 7, primarily in a mixture of acetonitrile/water in the ratio 2:3 (about./vol.), and formed the original solution contribute, if necessary, after separation on a few straps and columns for preparative chromatography with reversed phase; then carry out elution nitrile/water; then remove the nitrile obtained from fractions containing epothilone, and the resulting aqueous phase is extracted with ether complex; then ester extract is evaporated and then the resulting product is subjected to recrystallization; where the prefix "(ness.)" means, is that the radical contains up to 7 carbon atoms.

9. The method according to any of p, 7 or 8, where the separation potelov a and b is carried out using chromatography-based simulation of a moving layer (SMB).

10. The method according to any one of claims 1, 6, 7 or 8, wherein the polar or apolar solvent selected from the group comprising (ness.)alkylhalogenide and aromatic solvents, where the prefix "(ness.)" means that the radical contains up to 7 carbon atoms, or a mixture of two or more such solvents, or a mixture of such solvents has characteristics allotropes number determined according to Snyder and others:

x_e-0,22-0,32; x_d=0,17-0,34; x_n=0,39-0,54,

where x_edenotes the parameter of the proton acceptor, x_ddenotes the parameter of the proton donor and x_ndenotes the parameter of the dipole, provided that x_e+x_d+x_n=1.

11. The use of polar or apolar solvent for desorption of a, b, D and/or E epothilones from the resin chosen from the group comprising (ness.)alkylhalogenide and aromatic solvents or a mixture of two or more such solvents, where the prefix "(ness.)" means that the radical contains up to 7 carbon atoms.

12. The application of claim 11, where polar or apolar solvent, which is selected from a range including (ness.)alkylhalogenide and aromatic RA the founders, first of all, naphthalene, benzene or naphthalene or benzene substituted by one or more substituents selected from the group comprising (ness.)alkyl, (ness.)alkoxygroup, halogen, the nitro-group and (ness.)alkoxy-(ness.)alkyl; or mixtures of two or more such solvents; and the resin is a synthetic resin, where the prefix "(ness.)" means that the radical contains up to 7 carbon atoms.