WO1994020453A1

WO1994020453A1 - Taxol polyalkylene oxide conjugates of taxol and taxol intermediates

Info

Publication number: WO1994020453A1
Application number: PCT/US1994/002530
Authority: WO
Inventors: Richard B. Greenwald; Durgadas Bolikal
Original assignee: Enzon, Inc.
Priority date: 1993-03-09
Filing date: 1994-03-09
Publication date: 1994-09-15
Also published as: AU6400094A

Abstract

Polyalkylene oxide conjugated β-amides are disclosed, as well as the polyalkylene oxide conjugates of taxol, taxol analogues and intermediates prepared therefrom. Processes for preparing the polyalkylene oxide conjugated β-amides and forming the polyalkylene oxide conjugates of taxol, taxol analogues and intermediates therefrom are also disclosed.

Description

TAYOT, OLYALKYLENE OXIDE CONJUGATES OF TAXOL

AND TAXOL INTERMEDIATES

BACKGROUND OF THE INVENTION

The present invention relates to polyalkylene oxide conjugated 3-amides for the preparation of polyalkylene oxide conjugated taxols and taxol intermediates, processes for the preparation of polyalkylene oxide conjugated taxols and taxol intermediates, and polyalkylene oxide conjugated taxols and taxol intermediates prepared by such processes utilizing the /3-amides of the present invention.

The taxane family of terpenes, of which taxol is a member, has attracted considerable interest. Taxol is a promising cancer chemotherapeutic agent with a broad spectrum of antileukemic and tumor-inhibiting activity, having the following structure:

The bark from several species of yew currently supplies taxol. However, taxol is found only in minute quantities in the bark of these slow-growing evergreens. The isolation procedure is difficult, low-yielding and fatal to the trees, causing considerable concern that the supply of taxol will prove to be limited, and never meet the demand. Thus, the need exists for a viable synthetic route for the preparation of taxol or taxol analogs possessing antileukemic and tumor-inhibiting activity. Denis et al., J. Amer. Chem. Soc, 110(17), 5917-9 (1988) disclose the preparation of taxol from 10-deacetyl baccatin III, which has the structure shown below:

10-deacetyl baccatin III is easily extracted in large quantities from the leaves of Taxus Baccata L. The leaves are quickly regenerated and through prudent harvesting, large amounts of 10-deacetyl baccatin III can be continuously supplied with negligible affect on the yew population. The 10-deacetyl baccatin III is converted to taxol by attachment of the C-0 acetyl group and by attachment of the C13 /3-amide ester side chain through the esterification of the C13 alcohol with a β-amido carboxylic acid unit. The C13 /3-amide side chain has the following structure:

The synthesis of this side chain is reported by Denis et al., J. Org. Chem., 51(1), 46—50 (1986). ani et al., J. Amer. Chem. Soc, 93, 2325 (1971) disclose that the presence of the β-amide side chain at C13 is required for anti-tumor activity. Denis et al., J. Org.

Chem. 55(6), 1957-9 (1990) disclose streamlined , synthesis of the side chain. U.S. Patent Nos. 5,015,744 and 5,136,060 represent another taxol side chain , attachment method using an oxazinone. Georg et al., J.

Med. Chem., 35, 4230-7 (1992) discloses that 5 p—substitution of the N—benzoyl and 3'—phenyl rings of taxol produced derivatives having taxol-like activity. This article discloses p—chloro-derivatives and also reviews prior art disclosures of other taxol derivatives having significant activity, such as N—tiglyl, N—tosyl,

10 N—hexanoyl and N—glutaryl analogues.

Because taxol is obtained either from a limited resource or through a complex synthesis route, the supply of taxol is limited and costly. Therefore, a need exists for taxol derivatives having the physiological activity

15 of taxol but increased bloodstream clearance times. U.S.

Patent No. 4,179,337 to Davis et al. discloses polyalkylene oxide modified macromolecules exhibiting dramatically reduced immunogenicity and antigenicity, while retaining a substantial portion of the compound's

20 physiological activity. Because of the reduced immunogenicity and antigenicity, the polyalkylene oxide conjugates, when injected into a living organism, remain in the bloodstream considerably longer than the corresponding non-conjugated products.

25 Polyalkylene oxide conjugated taxols are unreported.

Therefore, a need exists for such conjugated taxols having an increased blood circulating time without a loss of physiological activity. At the same time, there

_* remains a need for a readily available side chain unit

30 that can be easily attached to the C13 alcohol of a taxane such as 10—deacetyl baccatin III. SUMMARY OF THE INVENTION

Both needs are met by the present invention. It has now been discovered that the C13 β—amide taxol side chain can be polyalkylene oxide conjugated without affecting the compound's physiological activity. In addition, the resulting polyalkylene oxide conjugated taxol possesses enhanced water solubility. Furthermore, this polyalkylene oxide conjugation can be ideally performed prior to attachment of the side chain to the C13 alcohol of a tetracyclic taxane nucleus such as 10—deacetyl baccatin III without affecting the attachment of the side chain to the taxane. It is preferable not to include the scarce taxane in an additional conjugation step in order to avoid even the most minimal process loss of the material. Instead, the /3-amide side chain precursor is polyalkylene oxide conjugated in a reaction that does not include the taxane.

Therefore, in accordance with the present invention, there is provided a water—soluble polyalkylene oxide conjugated /3-amide for the preparation of polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof, having a structure represented by Formula I:

wherein R is a water-soluble polyalkylene oxide;

X is a terminal moiety of the polyalkylene oxide;

I^ is a moiety covalently linking the polyalkylene oxide to the /3-amide;

T is hydrogen or a hydroxyl protecting moiety; and

R_! is a moiety selected from hydrogen, alkyl, aryl and arylalkyl moieties.

The polyalkylene oxide conjugates of taxol and taxol intermediates of the present invention are obtained by attaching the polyalkylene oxide conjugated /3-amides of Formula I to a taxane tetracyclic nucleus at C13. Therefore, in accordance with the present invention, there is also provided water-soluble, polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof, having a structure represented by Formula II:

wherein R, L_H T and X are the same as described above with respect to Formula I and R₃ is a taxane moiety. R₃ is preferably a baccatin III derivative having a structure represented by Formula III:

wherein R, is hydrogen, —COCH₃ or a hydroxyl protecting group and R₅ is hydrogen or a hydroxyl protecting group. R₄ is —COCH₃ for taxol and hydrogen for 10— eacetyl baccatin III. Thus, referring to Formula II, when L, has a structure represented by —OR₂— in which R₂ is a phenyl ring and R₃ has a structure represented by Formula III in which R is —COCH₃, the compound is a /3-amide polyalkylene oxide conjugate of taxol. The other compounds of Formula II are intermediates in the synthesis of polyalkylene oxide conjugates of taxol and taxol analogues, and can be converted to taxol or compounds having taxol—like activity by methods well understood to those of ordinary skill in the art. In order to conjugate the polyalkylene oxides to the /3-amide side chain precursor, the hydroxyl end—groups of the polymer must first be substituted with reactive functional groups. This process is frequently referred to "activation" and the product is called an "activated polyalkylene oxide." A particularly advantageous feature of the present invention is that the polyalkylene oxide conjugates of taxol and taxol analogues and intermediates thereof are formed from polyalkylene oxide-conjugated β- amide side chain precursors that are prepared by a method which directly reacts activated polyalkylene oxides with the azide disclosed by Denis et al., J. Org. Chem., 55(6), 1957—9 (1990). In other words, the polyalkylene oxide conjugation is performed by substituting activated polyalkylene oxides for the reagents disclosed by Denis et al. (1990) , rather than by performing an additional polyalkylene oxide conjugation process step.

Therefore, in accordance with the present invention, there is provided a process for the preparation of a polyalkylene oxide conjugated /3-amide having a structure corresponding to Formula I, which process includes the steps of: providing an azide having a structure represented by Formula IV:

wherein T is the same as described above with respect to Formula I and Rg is a moiety selected from alkyl, aryl and arylalkyl moieties; and reacting the azide with an activated polyalkylene oxide having a structure represented by Formula V:

(V) X-R-L₂

wherein X and R are the same as described above with respect to Formula I and L_j is a moiety capable of reacting with the azide to form a linkage corresponding to -L_j-CO- of Formula I, so that a polyalkylene oxide conjugated /3-amide having a structure represented by Formula I is formed.

In order to attach the polyalkylene oxide conjugated b-amides of Formula I to a taxane tetracyclic nucleus at C13 to obtain the polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof of the present invention, the polyalkylene oxide conjugated b-amides are reacted with a taxane tetracyclic nucleus having a hydroxyl group at C13 by the method disclosed by Denis et al., J. Amer. Chem. Soc, 110(17), 5917-9 (1988) . As indicated in that reference, the α-hydroxyl group should be suitably protected, and the conjugate, if in the form of an ester, should be converted to an acid. Therefore, for attachment of the polyalkylene oxide conjugated β- a ides prepared by the above-described process in which T is hydrogen to a 13—hydroxy taxane, processes in accordance with the present invention further include the step of substituting the α-hydroxyl moiety of the polyalkylene oxide conjugated /3-amide with a hydroxyl protecting moiety, so that T is a hydroxyl protecting group.

When the polyalkylene oxide conjugated /3-amides of the above-described processes include an ester group, —COOR,, in which R, is an alkyl, aryl or arylalkyl moiety, the ester group must first be hydrolyzed to a carboxylic acid before the /3-amide can be attached to the 13—hydroxy taxane. The hydrolysis should be performed under mild conditions so that the α-hydroxyl protecting group is not disturbed. Therefore, processes in accordance with the present invention in which R-- is alkyl, aryl or arylalkyl may further include the step of hydrolyzing the —COOR_! ester group of the polyalkylene oxide conjugated /3-amide under mild conditions so as not to disturb the α-hydroxyl protecting group, so that R_x is hydrogen.

When T is a hydroxyl protecting group and R, is hydrogen, processes in accordance with the present invention further includes the step of contacting the polyalkylene oxide conjugated /3-amide with a 13—hydroxy taxane, so that a polyalkylene oxide conjugate of taxol, a taxol analogue or intermediate thereof is formed. When Rj is hydrogen, the step of converting the ester to a carboxylic acid is omitted. When T is a hydroxyl protecting group, the step of substituting the α-hydroxyl group with a hydroxyl protecting group is omitted. The process steps of contacting polyalkylene oxide conjugated /3-amides with 13—hydroxy taxanes may be performed independently of the process steps for the preparation of polyalkylene oxide conjugated /3-amides. Therefore, processes in accordance with the present invention in which polyalkylene oxide conjugated /3-amides are contacted with 13—hydroxy taxanes may utilize polyalkylene oxide conjugated /3-amides from any source. Preferred 13—hydroxy taxanes are baccatin III or derivatives thereof having a structure represented by Formula IIIA in which R₄ and R₅ are the same as described above with respect to Formula III:

When the polyalkylene oxide conjugate formed is a taxol intermediate, processes in accordance with the present invention may further include the step of converting the polyalkylene oxide conjugated taxol intermediate to a polyalkylene oxide conjugate of taxol. Likewise, when the polyalkylene oxide conjugate formed is a taxol analogue intermediate, processes in accordance with the present invention may further include the step of converting the polyalkylene oxide conjugated taxol analogue intermediate to a polyalkylene oxide conjugated taxol analogue. The present invention also includes polyalkylene oxide conjugates of taxol, polyalkylene oxide conjugated taxol analogues and polyalkylene oxide conjugated taxol and taxol analogue intermediates prepared according to the process of the present invention. The polyalkylene oxide conjugated taxols and taxol analogues of the present invention are promising broad spectrum chemotherapeutic agents with varying antileukemic and tumor inhibiting activity. The present invention therefore also provides a method of treatment in which a mammal in need thereof is administered a chemotherapeutically effective amount of the polyalkylene oxide conjugated taxols or taxol analogues of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof of the present invention are prepared by attaching a polyalkylene oxide-conjugated /3-amide taxol side chain to the C13 hydroxyl of a taxane such as 10—deacetyl baccatin III. The polyalkylene oxide conjugated /3-amide taxol side chain precursors have a structure represented by Formula I depicted above.

The polyalkylene oxides (R of Formula I) are soluble in water at room temperature. Polyalkylene oxides meeting this requirement are polyethylene glycol (PEG) and block copolymers thereof. Block copolymers of PEG with polypropylene glycol or polypropylene oxide are suitable for use with the present invention, provided that the degree of block copolymerization is not so great as to render the polymer insoluble in water at room temperature. A molecular weight range suitable for use with the present invention is a number average molecular weight range between about 200 and about 100,000 daltons, and preferably between about 2,000 and about 20,000 daltons. Polyalkylene oxides having a number average molecular weight of about 5,000 are most preferred. The terminal moiety of the polyalkylene oxide (X of

Formula I) can be a group into which a terminal hydroxyl group may be converted, including the reactive derivatives of the prior art disclosed in U.S. Patent Nos. 4,179,337, 4,847,325, 5,122,614 and in copending and commonly owned U.S. Patent Application Serial No.

626,696, filed March 18, 1991, the disclosures of all of which are hereby incorporated herein by reference thereto. The heterobifunctional polymers can be prepared by methods known to those skilled in the art, without undue experimentation.

The polyalkylene oxide can also be a pre-blocked polymer having only one labile hydroxyl group per polymer moiety. In such polymers, X would represent a blocking group such as an alkyl group of one to four carbon atoms or an arylalkyl group. The preferred blocking group is a methyl group. A commercially available alkyl group blocked polyalkylene oxide is poly(ethylene glycol monomethyl ether) . The moieties represented by L, in Formula I are linking groups that form a covalently bonded linkage between a polyalkylene oxide and the β-amido group and are well-known to those of ordinary skill in the art. A wide variety of linking groups may be employed, a number of which are prepared from commercially available activated polyalkylene oxides extensively used for linking macromolecules. j is preferably —O—, —NH— or —OR₂—, in which R₂ is a moiety selected from lower alkyl, cycloalkyl, alkenyl, alkenoxy and alkoxy moieties containing up to six carbon atoms, phenyl and phenoxy moieties and alkylphenyl, alkylphenoxy and phenylalkoxy moieties, the alkyl portions of which contain up to six carbon atoms. For phenyl, alkylphenyl, alkylphenoxy and phenylalkoxy moieties, R is preferably substituted para to the β- amide. L_j is more preferably —O—, —NH—, —CH₂— or —OR₂—, with R₂ preferably being a phenyl ring. For the phenyl, alkylphenyl, alkylphenoxy and phenylalkoxy moieties of R₂, R is preferably substituted para to the /3-amide. Ri of Formula I is a moiety selected from hydrogen, alkyl, aryl and arylalkyl moieties. R_! is preferably selected from hydrogen, methyl, ethyl, phenyl and benzyl moieties. T is hydrogen or a hydroxyl protecting group such as triethylsilyl, benzyloxycarbonyl, 2,2,2—trichloroethoxycarbonyl, and the like. Of the hydroxyl protecting groups, triethylsilyl and benzyloxy carbonyl are most preferred.

Polyalkylene oxide conjugated /3-amide side chain precursors of Formula I of the present invention are formed by catalytic hydrogenation of the azide of Formula IV in ethyl acetate in the presence of an excess of an activated polyalkylene oxide of Formula V. The azide is prepared as described in Denis et al., J. Org. Chem., 55(6), 1957-9 (1990).

For the azide, T is hydrogen or a hydroxyl protecting group and R_<* is a moiety selected from alkyl, aryl and arylalkyl moieties. R_e is preferably selected from methyl, ethyl, phenyl and benzyl moieties. X and R of the activated polyalkylene oxide of Formula V are the same as described with respect to Formula I. I»* is a moiety capable of reacting with the azide to form a linkage corresponding to -L-^CO- of Formula I. Activated polyalkylene oxides capable of forming the above-described linking groups are well known and essentially conventional. Many are commercially available.

The polyalkylene oxide conjugate of Formula I in which Li is —CH_?— is prepared using the activated polyalkylene oxide of Formula V in which L₂ is -CH₂—CO-Cl. This activated polyalkylene oxide is the acid chloride derivative of a polyalkylene oxide carboxylic acid, the preparation of which is well known and essentially conventional. Polyalkylene oxide carboxylic acids are prepared by the method disclosed by Buckmann et al., Makromol. Chem., 182(5), 1379-84 (1981). T h e polyalkylene oxide conjugate of Formula I in which L, is —O— may be prepared using the activated polyalkylene oxide of Formula V in which Lj is —O—CO-C1. This activated polyalkylene oxide is a polyalkylene oxide chloroformate. The preparation of polyalkylene oxide chloroformates is disclosed by U.S. Patent No. 5,122,614. The polyalkylene oxide conjugate of Formula I in which Lj is —O- may also be prepared using the activated polyalkylene oxide of Formula V in which 1^ is the oxycarbonyl N— icarboximide moiety disclosed by U.S. Patent No. 5,122,614. The preferred oxycarbonyl N— icarboximide moiety is a succinimidyl carbonate moiety.

The polyalkylene oxide conjugate of Formula I in which L_j is —NH- is prepared using the activated polyalkylene oxide of Formula VI in which Lj is -N=C=0. This activated polyalkylene oxide is a polyalkylene oxide isocyanate.

The polyalkylene oxide conjugate of Formula I in which Li is —OR₂—, wherein R₂ is a phenyl moiety is prepared using the activated polyalkylene oxides of Formula V in which L2 is -OR₂—CO-Cl. This activated polyalkylene oxide is prepared by reacting a hydroxy benzoic acid with a polyalkylene oxide substituted with a moiety capable of undergoing nucleophilic displacement in the presence of a base. Polyalkylene oxide tosylates are preferred, which are prepared by reacting polyalkylene oxides with toluenesulfonyl chloride in a well—known reaction. See, e.g., the procedure of Mutter, Tetrahedron Lett., 31, 2839-42 (1978). Other suitable substituted polyalkylene oxides are polyalkylene oxide mesylates and polyalkylene oxide triflates, which are prepared similarly. The resulting carboxylic acid is converted to the acid chloride by well-known methods. Meta- and para-substituted hydroxy benzoic acids are suitable for use with the present invention, and para-substituted acids are preferred.

The stoichiometry and reaction conditions for forming the activated polyalkylene oxides of Formula V are well understood and essentially conventional. The reactions are carried out using inert solvents in which the reactants are soluble, such as toluene. Reaction temperatures will vary from 0°C and 100°C according to the reactants. All materials must be essentially free of water. Scrupulous care must be taken not to contaminate the reaction mixture with water. The activated polyalkylene oxides are purified from low molecular weight materials by conventional methods. The activated polyalkylene oxides of Formula V can then be reacted as described above with the azide of Formula IV to form the polyalkylene oxide conjugated jS-amide of Formula I. The resulting product represents an intermediate in the synthesis of polyalkylene oxide-conjugates of taxol, taxol analogues and intermediates thereof.

The polyalkylene oxide conjugated /3-amides of Formula I are attached to a taxane tetracyclic nucleus at C13 to obtain the polyalkylene oxide conjugates of taxol, taxol analogues and intermediates of Formula II. Specifically, the polyalkylene oxide conjugated /3-amides are reacted with a taxane tetracyclic nucleus having a hydroxyl group at C13 by the method disclosed by Denis et al., J. Amer. Chem. Soc, 110(17), 5917-9 (1988). In particular, the α-hydroxyl groups of the polyalkylene oxide conjugated /3-amides are first protected by substitution with hydroxyl protecting groups such as acetals. The —COOR_! ester groups of the polyalkylene oxide conjugated /3-amides are then hydrolyzed to carboxylic acids under mild conditions so as not to disturb the α-hydroxyl protecting groups. The polyalkylene oxide conjugated /3-amides and taxanes are then reacted in toluene solution in the presence of an activating agent, for example, one or more tertiary amines preferably selected from 4-(dimethylamino) pyridine (DMAP) , triethyl amine, diisopropyl ethyl a ine, pyridine, N-methyl imidate, 1,4—diazo-bicyclo[2,2,2]—octane (DABCO) , and the like. Basic activating agents such as di—2—pyridyl carbonate (DPC) are preferred, which maintains the stability of the hydroxyl protecting group. The reaction thus provides the polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof of Formula II having a taxane tetracyclic nucleus substituted at C13 with a polyalkylene oxide conjugated /3-amide side chain. The 7—position of the taxane moiety may also be carbamate conjugated by the method disclosed by cc— ending and commonly owned U.S. Patent Application Serial No.

07/934,131, filed August 21, 1992, the disclosure of which is hereby incorporated herein by reference thereto.

The 13-hydroxy taxane is preferably a baccatin III derivative having a structure corresponding to Formula

IIIA. When R₅ is hydroxyl and R, is acetyl, the compound having the structure represented by Formula IIIA is baccatin III, the taxane nucleus of taxol. When R₄ is hydrogen, the compound having a structure represented by Formula IIIA is 10—deacetyl baccatin III. As noted above, 10-deacetyl baccatin III is naturally occurring and also has utility as a starting material in the preparation of baccatin III and taxol.

When reacting the baccatin III derivatives of Formula IIIA with the polyalkylene oxide conjugated b-amides of Formula I to obtain the polyalkylene oxide conjugates of taxol and taxol intermediates of Formula II, R₄ and R₅ of Formula IIIA are preferably hydroxyl protecting groups. Therefore, the preferred baccatin III derivative of Formula IIIA for the preparation of the polyalkylene oxide conjugates of taxol of the present invention is 7— -triethylsilyl baccatin III, which has a structure represented by Formula IIIA in which R, is —COCH_j and R₅ is —Si(C₂H₅)₃. 7-0—triethylsilyl baccatin III may be obtained as described by Denis et al., J. Amer. Chem. Soc, 110(17), 5917-9 (1988) or by other routes.

As shown in the following reaction scheme, 7—O-triethylsilyl baccatin III may be reacted with an equivalent amount of a polyalkylene oxide conjugated β- amide of Formula I. The reaction provides a polyalkylene oxide conjugated taxol intermediate in which the C7 hydroxyl group is protected with a triethylsilyl group. This protecting group, and the α-hydroxyl protecting group of the polyalkylene oxide conjugated side chain are then removed by hydrolysis under mild conditions so as not to disturb the ester linkage or the taxane substituents, yielding a polyalkylene oxide conjugate of taxol.

The stoichio etry and reaction conditions are well understood and essentially conventional. The reactions are carried out in a common solvent in which the reactants are soluble, such as toluene, and mixtures thereof. Reaction temperatures between 50°C and 150°C are suitable, and temperatures between 60°C and 100°C are preferred. The reaction is carried out in the presence of an activating agent, preferably between about 3 and about 10 mmol of one or more of the above-listed tertiary amines to provide a pH at which the hydroxyl protecting groups are stable. The synthesis of the polyalkylene oxide conjugate of taxol is carried out as follows:

L_j is preferably -OR₂-, wherein R₂ is a phenyl group, so that the reaction product is equivalent to a polyalkylene oxide conjugate of the natural product taxol. Although the present invention is directed to the synthesis of compounds corresponding to polyalkylene oxide conjugates of taxol, it can be used with modifications in either the polyalkylene oxide conjugated /3-amide of Formula I, or the baccatin III derivative of Formula IVA, which can be obtained from natural or unnatural sources, to prepare taxol analogues or intermediates of such taxol analogues contemplated within the present invention. Synthesis of polyalkylene oxide conjugates of taxol and taxol analogues from the intermediates may be performed under an appropriate reaction scheme well known to those of ordinary skill in the art.

Essentially any 13—hydroxy taxane can be reacted in accordance with the above-depicted reaction scheme to obtain other taxol analogues, taxol intermediates and taxol analogue intermediates. Furthermore, other precursors of the taxol side chain may be similarly polyalkylene oxide conjugated at the /3-amide position and then reacted in accordance with the above—depicted reaction scheme to obtain polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof, including the oxazinone of U.S. Patent Nos. 5,015,744 and 5,136,060.

The above-disclosed polyalkylene oxide conjugates of taxol and taxol analogues are promising broad spectrum chemotherapeutic agents with varying antileukemic and tumor-inhibiting activity, of which the polyalkylene oxide conjugate of taxol is most promising. Mammals in need thereof may be treated by administering chemotherapeutically effective amounts of the polyalkylene oxide conjugates of taxol and taxol analogues of the present invention. The polyalkylene oxide conjugated taxol intermediates and taxol analogue intermediates possess utility in the synthesis of polyalkylene oxide conjugates of taxol and taxol analogues. The polyalkylene oxide conjugated intermediates of taxol analogues possess additional utility by being useful in the investigation of taxol analogues. The polyalkylene oxide conjugated b-amides of the present invention also possess utility as intermediates in the synthesis of polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof. The compounds which can be used in the formation of taxol analogues and taxol analogue intermediates likewise possess additional utility by being useful in the investigation of taxol analogues.

The taxol and taxol analogues in the form of polyalkylene oxide conjugates remain in the bloodstream considerably longer than the corresponding non-conjugated products without a significant loss of antileukemic and tumor-inhibiting activity. This represents an advancement in the cancer chemotherapeutic use of taxol and analogues thereof.

The following non-limiting examples illustrate certain aspects of the invention. All parts and percentages are by weight unless otherwise noted, and all temperatures are in degrees Celsius.

EXPERIMENTAL

Example 1

(2R,3S)-(-)-N-carbonyloxy—PEG—3-phenyliso-serine methyl ester, which corresponds to the /3-amide of Formula I in which X is methyl, R is PEG, L_j is —O—, j is methyl and T is hydrogen, was prepared by first adding to 50 mL of ethyl acetate 0.22 g (1.0 mmol) of (2R, 3S)-(+)—methyl 3—azido—2—hydroxy—3—phenylpropionate (prepared according to Denis et al., J. Org. Chem., 55, 1957 (1990)), 4.9 g

(0.97 mmol) of the chloroformate of poly(ethylene glycol monomethyl ether)—5000 (prepared as described in U.S. Patent No. 5,122,614), 3.0 g (3.0 mmol) of triethylamine, and 5.0 mg (0.041 mmol) of dimethylamino pyridine. This mixture is stirred at 20°C for four hours. To this is then added 0.2 mL of methanol and after three hours of stirring, 50 mg of 10% palladium on charcoal is added. The resulting mixture is stirred under hydrogen for four days. The product is isolated by stripping the solvent and extracting the residue with methylene chloride. The crude product obtained after solvent evaporation is purified by recrystallization from 2-propanol. The pure product (approx. 90% yield) is characterized by specific rotation, IR, and NMR.

Example 2

The α-hydroxyl moiety of the polyalkylene oxide conjugated /3-amide of Example 1 is substituted with a hydroxyl protecting group, and the ester is converted to an acid by adding to 20 mL of methylene chloride 3.9 g (0.75 mmol) of the polyalkylene oxide conjugated β-amide of Example 1, 0.11 g (1.5 mmol) of ethyl vinyl ether, and 7.6 mg (0.040 mmol) of p-toluenesulfonic acid. The resulting solution is stirred at room temperature for three hours and then evaporated to dryness. 20 mL of 1% aqueous NaHC0₃ is added to the residue and stirred for one hour. The product is extracted into methylene chloride, dried over MgS0₄ and isolated by solvent removal and drying under high vacuum. The product is characterized by specific rotation, IR and NMR. Example 3

The polyalkylene oxide conjugated /3-amide is then attached to a taxane tetracyclic nucleus by dissolving in 20 mL of toluene 34 mg (0.05 mmol) of 7—triethylsilyl baccatin III (prepared as described by Denis et al., J. Amer. Chem. Soc, 110(17), 5917-9 (1988)), 2.6 g (0.5 mmol) of the polyalkylene oxide conjugated /3-amide of Example 2, 108 mg (0.5 mmol) of di—2—pyridylcarbonate, and 18.3 mg (0.15 mmol) of dimethylamino pyridine. The resulting solution is heated at 80°C for five days. The reaction mixture is evaporated to dryness and then stirred with 20 mL of 0.5% HC1 for one hour at 0°C. The product is isolated by extraction with methylene chloride followed by the usual work up. The crude product obtained above is purified by HPLC using a preparative C8 reverse phase column (methanol—water eluent with step gradient) . The purified product is characterized by HPLC, IR, and NMR and these data were consistent with taxanes functionalized with PEG.

As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED:

1. A water-soluble polyalkylene oxide conjugated /3-amide having a structure represented by:

X-

wherein R is a water-soluble polyalkylene oxide;

X is a terminal moiety of said polyalkylene oxide; L. is a moiety covalently linking said polyalkylene oxide to said b-amides;

T is hydrogen or a hydroxyl protecting moiety; and R_t is a moiety selected from the group consisting of hydrogen, alkyl, aryl and arylalkyl moieties.

2. The polyalkylene oxide conjugated /3-amide of claim 1, wherein said polyalkylene oxide is selected from the group consisting of polyethylene glycol and block copolymers of polyethylene glycol and polypropylene glycol.

3. The polyalkylene oxide conjugated /3-amide of claim 2, wherein said polyalkylene oxide comprises polyethylene glycol.

4. The polyalkylene oxide conjugated /3-amide of claim 1, wherein said polyalkylene oxide has a number average molecular weight between about 200 and about 100,000 daltons.

5. The polyalkylene oxide conjugated j3-amide of claim 4, wherein said polyalkylene oxide has a number average molecular weight between about 2,000 and about 20,000 daltons.

6. The polyalkylene oxide conjugated /3-amide of claim 5, wherein said polyalkylene oxide has a number average molecular weight of about 5,000 daltons.

7. The polyalkylene oxide conjugated /3-amide of claim 1, wherein L_j is a moiety selected from the group consisting of -0-, -NH-, -CH₂-, -N(CH₃)- and -0R₂-; and wherein R₂ is a moiety selected from the group consisting of lower alkyl, cycloalkyl, alkenyl, alkenoxy and alkoxy moieties containing up to six carbon atoms, phenyl and phenoxy moieties and alkylphenyl, alkylphenoxy and phenylalkoxy moieties, the alkyl portions of which contain up to six carbon atoms.

8. The polyalkylene oxide conjugated /3-amide of claim 7, wherein R₂ is a phenyl moiety.

9. The polyalkylene oxide conjugated /3-amide of claim 1, wherein X is a moiety selected from the group consisting of alkyl moieties containing up to four carbon atoms and arylalkyl moieties.

10. The polyalkylene oxide conjugated /3-amide of claim 9, wherein X is a methyl moiety.

11. Water-soluble, polyalkylene oxide conjugates of taxol, taxol analogues and intermediates thereof, having a structure represented by:

wherein R is a water-soluble polyalkylene oxide; X is a terminal moiety of said polyalkylene oxide; L, is a moiety covalently linking said polyalkylene oxide to said _/3-amide;

T is hydrogen or a hydroxyl protecting moiety; and R₃ is a taxane moiety.

12. The polyalkylene oxide conjugate of claim 11, wherein said polyalkylene oxide is selected from the group consisting of polyethylene glycol and block copolymers of polyethylene glycol and polypropylene glycol.

13. The polyalkylene oxide conjugate of claim 11, wherein said polyalkylene oxide has a number average molecular weight between about 200 and about 100,000 daltons.

14. The polyalkylene oxide conjugate of claim 13, wherein said polyalkylene oxide has a number average molecular weight of about 5,000 daltons.

15. The polyalkylene oxide conjugate of claim 11, wherein Lj is a moiety selected from the group consisting of —O-, —NH—, —CH₂—, —N(CH₃)— and —OR₂—, wherein R₂ is a moiety selected from the group consisting of lower alkyl, cycloalkyl, alkenyl, alkenoxy and alkoxy moieties containing up to six carbon atoms, phenyl and phenoxy moieties and alkylphenyl, alkylphenoxy and phenylalkoxy moieties, the alkyl portions of which contain up to six carbon atoms.

16. The polyalkylene oxide conjugate of claim 15, wherein R₂ is a phenyl moiety.

17. The polyalkylene oxide conjugate of claim 11, wherein R₃ is a baccatin III moiety having a structure represented by:

wherein R,, is a moiety selected from the group consisting of hydrogen, —COCH₃ and hydroxyl protecting moieties and R₅ is a moiety selected from the group consisting of hydrogen and hydroxyl protecting moieties.

18. The polyalkylene oxide conjugate of claim 17, wherein R₂ is a phenyl moiety, and R, is —COCH₃.

19. The polyalkylene oxide conjugate of claim 11, wherein said taxane moiety is carbamate conjugated at the 7—position.

20. A process for the preparation of a polyalkylene oxide conjugated /3-amide, said process comprising the steps of: providing an azide having a structure represented by:

wherein T is hydrogen or a hydroxyl protecting group and Re is a moiety selected from the group consisting of alkyl, aryl and arylalkyl moieties; reacting said azide with an activated polyalkylene oxide having a structure represented by: X-R-Lj so that a polyalkylene oxide conjugated /3-amide is formed having a structure represented by:

wherein L*, is a moiety capable of reacting with said azide under reducing conditions to form a linkage corresponding to —L,—CO-, R is a water-soluble polyalkylene oxide; L_j is a moiety covalently linking said polyalkylene oxide to said /3-amide; R_{ is a moiety selected from the group consisting of hydrogen, alkyl, aryl and arylalkyl moieties; T is hydrogen or a hydroxyl protecting group and X is a terminal moiety of said polyalkylene oxide; and recovering said polyalkylene oxide conjugated β- amide.

21. The process of claim 20, wherein l^ is one of

—O-CO—Cl, so that Lj is —O—; an oxycarbonyl N—dicarboximide moiety, so that L_! is —O—; —N=C=0, so that L, is —NH—; —CH₂—CO-C1, so that L, is -CH₂- and —OR₂—CO—Cl, so that L_j is —OR₂—, wherein R₂ is a moiety selected from the group consisting of lower alkyl, cycloalkyl, alkenyl, alkenoxy and alkoxy moieties containing up to six carbon atoms, phenyl and phenoxy moieties and alkylphenyl, alkylphenoxy and phenylalkoxy moieties, the alkyl portions of which contain up to six carbon atoms.

22. The process of claim 21, wherein said oxycarbonyl N—dicarboximide moiety is a succinimidyl carbonate moiety.

23. The process of claim 20, wherein T is a hydroxyl protecting group and R_j is hydrogen, and said process further comprises the step of: contacting said polyalkylene oxide conjugated β- amide with a 13—hydroxy taxane so that a polyalkylene oxide conjugate of taxol, a taxol analogue or an intermediate thereof is formed.

24. The process of claim 23, wherein said

13—hydroxy taxane has a structure represented by:

wherein ,, is a moiety selected from the group consisting of hydrogen, —C0CH₃ and hydroxyl protecting moieties and R₅ is a moiety selected from the group consisting of hydrogen and hydroxyl protecting moieties.

25. A process for the preparation of a polyalkylene oxide conjugate of a taxol, a taxol analogue or intermediates thereof, comprising the steps of:

providing a polyalkylene oxide conjugated α- hydroxyl, /3-amide having a structure represented by:

wherein R is a water-soluble polyalkylene oxide; L, is a moiety covalently linking said polyalkylene oxide to said /3-amide; Rj is a moiety selected from the group consisting of alkyl, aryl and arylalkyl moieties; and X is a terminal moiety of said polyalkylene oxide; substituting said α-hydroxyl group of said polyalkylene oxide conjugated /3-amide with a hydroxyl protecting group; hydrolyzing said —COOR_! group of said polyalkylene oxide conjugated /3-amide to a carboxylic acid under mild conditions, so as not to disturb said hydroxyl protecting group, so that Rj is hydrogen; and contacting said polyalkylene oxide conjugated β- amide with a 13—hydroxy taxane so that a polyalkylene oxide conjugate of taxol, a taxol analogue or an intermediate thereof is formed.

26. The process of claim 25, wherein said 13-hydroxy taxane has a structure represented by:

wherein R,

from the group consisting of hydrogen, —COCH₃ and hydroxyl protecting moieties and R₅ is a moiety selected from the group consisting of hydrogen and hydroxyl protecting moieties.

27. The conjugate prepared according to the process of claim 25.

28. A process for the preparation of a polyalkylene oxide conjugate of taxol, a taxol analogue, or intermediates thereof, comprising the steps of:

providing a polyalkylene oxide conjugated /3-amide having a structure represented by:

wherein R is a water-soluble polyalkylene oxide; L_j is a moiety covalently linking said polyalkylene oxide to said /3-amide; R, is a moiety selected from the group consisting of alkyl, aryl and arylalkyl moieties; T is a α-hydroxyl protecting group and X is a terminal moiety of said polyalkylene oxide; hydrolyzing said —COOR,_, group of said polyalkylene oxide conjugated /3-amide to a carboxylic acid under mild conditions, so as not to disturb said α-hydroxyl protecting group, so that Rj is hydrogen; and contacting said polyalkylene oxide conjugated β- amide with a 13—hydroxy taxane so that a polyalkylene oxide conjugate of taxol, a taxol analogue or an intermediate thereof is formed.

29. The process of claim 28, wherein said 13—hydroxy taxane has a structure represented by:

wherein R« is a moiety selected from the group consisting of hydrogen, —C0CH₃ and hydroxyl protecting moieties and R₅ is a moiety selected from the group consisting of hydrogen and hydroxyl protecting moieties.

30. The conjugate prepared according to the process of claim 28.

31. A process for the preparation of a polyalkylene oxide conjugate of taxol, a taxol analogue or intermediates thereof comprising the steps of: contacting a polyalkylene oxide conjugated /3-amide having a structure represented by:

wherein R is a water-soluble polyalkylene oxide; L_j is a moiety covalently linking said polyalkylene oxide to said 3-amide; T is a hydroxyl protecting group and X is a terminal moiety of said polyalkylene oxide; and contacting said polyalkylene oxide conjugated /3-amide with a 13—hydroxy taxane so that a polyalkylene oxide conjugate of taxol, a taxol analogue or an intermediate thereof is formed.

32. The process of claim 31, wherein said 13—hydroxy taxane has a structure represented by:

wherein R₄ is a moiety selected from the group consisting of hydrogen, —COCH₃ and hydroxyl protecting moieties and R₅ is a moiety selected from the group consisting of hydrogen and hydroxyl protecting moieties.

33. The conjugate prepared according to the process of claim 31.

34. A method of treatment comprising administering to a mammal in need thereof a chemotherapeutically effective amount of said polyalkylene oxide conjugate of taxol or a taxol analogue of claim 11.