WO2003057896A1

WO2003057896A1 - Methods for preparing anthracyclinone derivatives and anthracyclinone derivatives per se

Info

Publication number: WO2003057896A1
Application number: PCT/US2002/041603
Authority: WO
Inventors: Peter C. Michels; Joseph O. Rich; Harold Meckler; Ian Cotterill
Original assignee: Albany Molecular Research, Inc.
Priority date: 2001-12-27
Filing date: 2002-12-23
Publication date: 2003-07-17
Also published as: AU2002360813A1

Abstract

The present invention relates to a process for preparation of a product compound of the Formula (I), where R1 is an acyl group, R2 is H, an N-alkylated amino sugar, or a non-basic sugar moiety, and R3 is H, OH, or OCH3. The process involves reacting a starting compound of the Formula (II) with an acyl donor compound in the presence of a non-chemically modified lipase, under conditions effective to produce the product compound. Another aspect of the present invention relates to a compound of the following Formula (III), where: R1 is an acyl radical of a carboxylic acid selected from the group consisting of: polyethylene glycol acetic acid and polyunsaturated fatty acid; and R2 = 2,6-dideoxy-2-fluoro-α-talopyranosyl; 3-trifluoroacetylamino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl; or 3-deamino-3-(2'-pyrroline-1'-yl)-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl; or a pharmaceutically acceptable salt thereof. The present invention also relates to a compound of the Formula (IV) where n is 4 or 5, or a pharmaceutically acceptable salt thereof. Another aspect of the present invention relates to a compound of the Formula (V) where: R1 is an acyl group, R2 is 3-substituted allyloxycarbonylamino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl, R3 is H, OH, or OCH3.

Description

METHODS FORPREPARINGANTHRACYCLINONEDERIVATIVES AND ANTHRACYCLINONEDERIVATIVES PERSE

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/344,280, filed December 27, 2001 , which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for preparing derivatives of the anti-cancer compounds of the anthracyclinone class that are site-selectively acylated at the C-14 position as well as anthracyclinone derivatives per se.

BACKGROUND OF THE INVENTION

[0003] Over thirty years ago, the anthracycline doxorubicin was first isolated from Streptomyces peucetius var. Caesius and recognized for its antitumor properties (Arcamone et al., Biotechnology and Bioengineering, 11:1101-1110 (1969)). While doxorubicin has remained one of the most potent and extensively-used chemotherapeutics in cancer treatment (Hortobagyi, Drugs, 54:1-7 (1997); Lown, Pharmacol. Ther.. 60:185-214(1993)), it has anumber of therapeutic drawbacks including dose-limiting cardiotoxicity and susceptibility to multidrug resistance (Hortobagyi, Drugs, 54:1-7 (1997); Gewirtz, Biochem. Pharmacol.. 57:727-741 (1999); Muggia et al., Crit. Rev. Oncol. Hematol.. 11:43- 64 (1991); Weiss, Semin. Oncol. 19:670-686 (1992)). In the intervening decades, thousands of doxorubicin analogs have been developed in the largely unsuccessful search for an improved pharmaceutical (Weiss, Semin. Oncol., 19:670-686 (1992)). As with many other natural compounds, the chemical synthesis of doxorubicin is a challenging process. Several extensive procedures exist for generating doxorubicin, but none is convenient for rapid generation of diverse analogs (Broadhurst et al., Tetrahedron. 40:4649-4656^1 84)). Most efforts, therefore, to modify doxorubicin take the more practical approach of starting from the complete drug. Unfortunately, the polyfunctional structure (including two phenolic hydroxyls, and one each of a quinone, ketone, primary hydroxyl, secondary hydroxyl, tertiary hydroxyl, primary amine, methyl ether, and glycosidic linkage) and the sensitivity to pH, heat, metal ions, and light complicate conventional efforts at controlled derivatization (Bouma et al., Pharm, Weekbl fSci], 8:109-133 (1986)).

Doxorubicin

[0004] The abundant literature on doxorubicin structure-activity relationships (Farquhar et al., Journal of Medicinal Chemistry. 41:965-972 (1998); Gabbay et al., Biochemistry, 15:2062-2070 (1976); Priebe et al., Pharmacol. Ther., 60:215-234 (1993); Chaires et al, Biochemistry, 35:2047-2053 (1996); Capranico et al., Molecular Pharmacology, 45:908-915 (1994)) provides some guidance for focusing analog design. Most notably, DNA intercalation is central to doxorubicin's primary mode of action: the inhibition of topoisomerase II and subsequent DNA strand breakage. When doxorubicin is intercalated, the C-9 ketone element and the daunosamine sugar project into the minor groove and stabilize the DNA complex by hydrogen bonding and by interacting with topoisomerase II. Although promising doxorubicin derivatives to date predominately involve alterations to the sugar structure (Farquhar et al., Journal of Medicinal Chemistry. 41:965-972 (1998); Capranico et al., Molecular Pharmacology. 45:908-915 (1994); Arcamone et al., Pharmacology &

Therapeutics. 76:117-124 (1997)), such as the attachment of alkylating agents, a relatively small number of studies advocate the C-9 site as a promising target for activity-enhancing modifications (Scott et al., British Journal of Cancer. 53:595- 600 (1986); Israel et al, Cancer Res., 35:1365-1368 (1975); Adams et al., Journal of Medicinal Chemistry, 33:2380-2384 (1990); Coley et al, Anti-Cancer Drug Design, 7:471-481 (1992); Povarov et al., Bioorganicheskaya Khimiya. 21:925- 932 (1995)). [0005] The chemical fragility and functional diversity of doxorubicin provide an excellent opportunity to take advantage of biocatalysis for regioselective reaction under mild reaction conditions. Many of the original, and ongoing, efforts to develop anthracycline analogs have utilized microbial transformations (Marshall et al., Biochemistry, 15:4139-4145 (1976); Grafe et al., Biotechnol. Adv.. 7:215-239 (1989); Johdo et al., J. Antibiot (Tokyo . 49:669-675 (1996)). But none of these allow selective modification of the 14-position. In addition, the heterogeneous reaction environment, significant potential for side reactions catalyzed by the mixture of enzymes present within the cell, and difficulties for isolation and purification of small molecules from aqueous microbial fermentation broths make the use of isolated enzymes in non-aqueous solvents more likely to be practical for a commercially- viable process. [0006] Nonaqueous enzymology is an increasingly valuable tool for synthetic chemistry (Khmelnitsky et al., Curr. Opin. Chem. Biol., 3:47-53 (1999)). Within the unnatural environment of organic solvents, enzymes catalyze regioselective and enantioselective reactions under mild conditions with a broad range of substrates (Wong et al., Enzymes in Synthetic Organic Chemistry, 1st ed.; Tarrytown, N.Y., Oxford, U.K.:Pergamon (1994)); Drauz et al., Enzyme Catalysis in Organic Synthesis: A Comprehensive Handbook, Vch: Weinheim:New York (1995)). The derivatization of natural products and synthetic multifunctional substrates presents an especially rich opportunity for exploiting biocatalyst selectivity. By employing enzymatic techniques, chemists can often circumvent the challenges of protective chemistries that might be required to perform identical transformations using traditional synthetic methods. Hydrolytic enzymes can be used in organic solvents or biphasic environments to biocatalytically acylate nucleophilic groups. This procedure is most often applied to small compounds with high structural similarity to the enzymes' natural substrates. Biotransformation of novel complex structures has been a subject of increasing interest, especially as part of a biocatalytic approach to combinatorial chemistry (Altreuter et al., Current Opinion in Biotechnolo y. 10: 130-136 (1999); Krstenansky et al., Bioorgan. Med. Chem.. 7:2157-2162 (1999); Michels et al. Trends Biotech.. 16:210-215 (1998); Mozhaev et al., Tetrahedron. 54:3971-3982 (1998); Khmelnitsky et al., J. Amer. Chem. Soc, 119:11554-11555 (1997)). [0007] Previously, the use of one modified lipase for the acylation of doxorubicin has been presented (Altreuter et al., "Combinatorial Biocatalytic Derivatization of Doxorubicin," (abstract) American Chemical Society Meeting (1998)). Enzyme modifications found to allow catalytic activity with doxorubicin included solubilization of the enzyme in a enzyme-surfactant complex, and co- lyophilization of enzyme in a salt solution. The selectivity of the reaction relied on the type of modification applied to the enzyme, with different types of enzyme modification affording from one to three different acylated doxorubicin products upon reaction. In addition, these modification and solubilization techniques require a separate modification step to prepare the enzyme catalyst as well as a complicated purification step to remove either the modified enzyme or modification agent (e.g. surfactant) from the product stream. [0008] The present invention is directed to overcoming these deficiencies in the art.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a process for preparation of a product compound of the formula:

where R is an acyl group, R is H, an N-alkylated amino sugar, or a non-basic sugar moiety, and R₃ is H, OH, or OCH₃. The process involves reacting a starting compound of the formula:

with an acyl donor compound in the presence of a non-chemically modified lipase, under conditions effective to produce the product compound. [0010] Another aspect of the present invention relates to a compound of the following formula:

where:

Ri. is an acyl radical of a carboxylic acid selected from the group consisting of: polyethylene glycol acetic acid and polyunsaturated fatty acid; and

R₂ = 2,6-dideoxy-2-fluoro-α-talopyranosyl;

3 -trifluoroacetylamino-2,3 ,6-trideoxy-α-L-j o-hexopyranosyl; or

3 -deamino-3-(2 ' -pyrroline- 1 '-yl)-2,3 ,6-trideoxy-α-L-b/ o-hexopyranosyl; or a pharmaceutically acceptable salt thereof.

[0011] The present invention also relates to a compound of the following formula:

where n is 4 or 5, or a pharmaceutically acceptable salt thereof. [0012] Another aspect of the present invention relates to a compound of the following formula:

where: Ri_. is an acyl group,

R₂ is 3-substituted allyloxycarbonylamino-2,3,6-trideoxy-α-L-/j;;cø- hexopyranosyl, R₃ is H, OH, or OCH₃.

[0013] The present invention describes a process for the use of enzymes for selective 14-O-acylation of anthracyclinone derivatives using acyl donor compounds such as vinyl esters, trihaloethyl esters, vinyl carbonates, or carboxylic acids. Alternative chemical syntheses already known for the production of 14-acyl anthracyclinone derivatives have involved an alkyl halide intermediate converted to the ester by a nucleophilic displacement. Both of these chemical routes are multistep, and give relatively low (<40%) overall yields. Therefore, the scheme of the present invention represents an improved process over alternative chemical routes.

[0014] Furthermore, in comparison to the solubilized or salt-activated enzymes, the use of non-chemically modified enzymes removes the need for a modification step to activate the enzyme catalyst and simplifies product recovery by eliminating the need to remove either the enzyme or activating agent such as Aerosol OT (AOT) from the resultant product stream.

[0015] In addition, the scheme of the present invention could be applied for the more efficient production of 14-O-acyl anthracyclinone derivative compounds of economic value.

[0016] Accordingly, the regioselective acylation of anthracyclinone derivatives using non-chemically modified lipases in a nonaqueous environment represents a unique pathway to achieve potent cytotoxic anthracyclinone analogs, and expands the repertoire of biocatalytic techniques available for a combinatorial lead-development program.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to a process for preparation of a product compound of the formula:

where Ri is an acyl group, R₂ is H, an N-alkylated amino sugar, or a non-basic sugar moiety, and R₃ is H, OH, or OCH₃. The process involves reacting a starting compound of the formula:

with an acyl donor compound in the presence of a non-chemically modified lipase, under conditions effective to produce the product compound. [0018] In order to obtain complete conversion of the starting compound to the acylated product compound, the above process can also be carried out under conditions effective to exclusively acylate the starting compound at its C-14 position. [0019] The reaction of the present invention can be carried out in an organic solvent. Possible organic solvents include but are not limited to toluene, methyl-tert-butyl ether, pyridine, chloroform, acetonitrile, N,N-dimethyl formamide ("DMF"), tetrahydrofuran ("THF"), isooctane, and mixtures of these solvents. [0020] The reaction of the present invention can be carried out at temperature of 0 to 150°C, more preferably at 25 to 110°C. [0021] Acyl donor compounds of the present invention can be either non- activated acyl donors or activated acyl donors. Non-activated acyl donors are defined as reagents that contribute an acyl group to the reaction with water or ammonia as the sole leaving group; these include the classes of molecules containing free carbonyl groups (free acids), free amides, carbonates, and carbamates. Activated acyl donors are defined as reagents that contribute an acyl group to the reaction with a more reactive leaving group; these include, but are not limited to, simple esters of acids, trihaloethyl esters, thioethyl esters, oxime esters, vinylic and enol esters (e.g. vinyl acetate and diketene), vinyl carbonates, and anhydrides.

[0022] Examples of non-activated acyl donors include carboxylic acids such as enanthic acid, caprylic acid, fumaric acid, maleic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, lauric acid, myristic acid, palmitic acid, stearic acid, glyoxylic acid diethylacetal, acrylic acid, crotonic acid, isocrotonic acid, butenoic acid, pentenoic acid, oleic acid, retinoic acid, linoleic acid, linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, c*s-5,8, 11,14,17- eicosapentaenoic acid, cts-4,7,10,13,16,19-docosahexaenoic acid, malonic acid, succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, undecanedioic acid, dodecanedioic acid, 1,12-dodecanedicarboxylic acid, benzoic acid, picolinic acid, nicotinic acid, isonicotinic acid, piperazine-2-carboxylic acid, pyrrole-2-carboxylic acid, pyrrole-3-carboxylic acid, furan-2-carboxylic acid, furan-3 -carboxylic acid, thiophene-2-carboxylic acid, thiophene-3 -carboxylic acid, imidazole-2-carboxylic acid, imidazole-4(5)-carboxylic acid, phenylacetic acid, 3,5-dibromo-4-hydroxybenzoic acid, 3-(2-furyl) acrylic acid, 3,4- (methylenedioxy) phenyl acetic acid, norbornane acetic acid, 2-thiophene acetic acid, 2,6-dimethoxy nicotinic acid, 3-indol butyric acid, 3,4-(methylenedioxy) cinnamic acid, 4-formylcinnamic acid, N-CBZ-isonipecotic acid, N-CBZ-L- proline acid, N-CBZ-tyrosine acid, N-CBZ-alanine acid, Fmoc-sarcosine acid, N- CBZ-glycine acid, N-CBZ-phenylalanine acid, bromoacetic acid, chloroacetic acid, chlorobenzoic acid, cinnamic acid, formic acid, iodoacetic acid, methacrylic acid, pivalic acid, sorbic acid, 2-ethyl hexanoic acid, salicylic acid, 3-amino-6- thiophen-2-yl-4-trifluoromethyl-thieno [2,3-b] pyridine carboxylic acid, indole-2- carboxylic acid, indole-3 -carboxylic acid, indoline-2-carboxylic acid, indoline-3- carboxylic acid, 3-(S-glutathionyl)propionic acid, polyethylene glycol acetic acid, methyl polyethylene glycol acetic acid, N-protected leucine, N-protected isoleucine, N-protected tert-leucine, and N-protected cycloleucine.

[0023] Activated acyl donors such as vinyl esters, trihaloethyl esters, or vinyl carbonates can also be used for the present invention. [0024] Examples of vinyl esters include 3,3-diphenyl propionic acid vinyl ester; 3,5-dibromo-4-hydroxybenzoic acid vinyl ester; 3-(2-furyl) acrylic acid vinyl ester; 3,4-(methylenedioxy) phenyl acetic acid vinyl ester; norbomane acetic acid vinyl ester; 2-thiophene acetic acid vinyl ester; 2,6-dimethoxy nicotinic acid vinyl ester; 3-indol butyric acid vinyl ester; pyrrole-2-carboxylic acid vinyl ester; 3,4-(methylenedioxy) cinnamic acid vinyl ester; 4-formylcinnamic acid vinyl ester; N-CBZ-isonipecotic acid vinyl ester; N-CBZ-L-proline vinyl ester; N-CBZ- tyrosine vinyl ester; N-CBZ-alanine vinyl ester; Fmoc-sarcosine vinyl ester (Fmoc-N-Me-Gly-OH vinyl ester); N-CBZ-Glycine vinyl ester; N-CBZ- phenylalanine vinyl ester; 2-furoic acid vinyl ester; acrylic acid vinyl ester; adipic acid divinyl ester; benzoic acid vinyl ester; bromoacetic acid vinyl ester; butyric acid vinyl ester (vinyl butyrate); caproic acid vinyl ester; enanthic acid vinyl ester; caprylic acid vinyl ester; chloroacetic acid vinyl ester; chloroformic acid vinyl ester; chlorobenzoic acid vinyl ester; cinnamic acid vinyl ester; crotonic acid vinyl ester; formic acid vinyl ester; iodoacetic acid vinyl ester; lauric acid vinyl ester; methacrylic acid vinyl ester; myristic acid vinyl ester; n-capric acid vinyl ester; n- caprylic acid vinyl ester; palmitic acid vinyl ester; phenyl acetic acid vinyl ester; pivalic acid vinyl ester; propionic acid vinyl ester; sorbic acid vinyl ester; stearic acid vinyl ester; acetic acid vinyl ester; 2-ethyl hexane vinyl ester; vinyl salicylate; trimethyl-vinyloxycarbonylmethyl-ammonium bromide; 3 -amino-4,6-dimethyl- thieno [2,3-b] pyridine-2-carboxylic acid vinyl ester (1S-27086); 3-amino-6- thiophen-2-yl-4-trifluoromethyl-thieno [2,3-b] pyridine carboxylic acid vinyl ester (1S-21501); divinyl succinate; divinyl glutarate; and divinyl subarate.

[0025] The trihaloethyl esters of the present invention can be trifluoroethyl esters or trichloroethyl esters, preferably trifluoroethyl esters. Examples of trifluoroethyl esters include 3,3-diphenyl propionic acid trifluoroethyl ester; 3,6- dioxaheptanoic acid trifluoroethyl ester; oxalic acid trifluoroethyl ester; malonic acid trifluoroethyl ester; (-)-2-oxo-4-thiazolidine-2-carboxylic acid trifluoroethyl ester; pyrazine-2-carboxylic acid trifluoroethyl ester; nicotinic acid trifluoroethyl ester; 1 ,4-cyclohexane dicarboxylic acid di trifluoroethyl ester; terephtalic acid di trifluoroethyl ester; 4-(dimethylamino) benzoic acid trifluoroethyl ester; 4- (bromomethyl) phenylacetic acid trifluoroethyl ester; benzimidazole propionic acid trifluoroethyl ester; Fmoc-L-thiazolidine-4-carboxylic acid trifluoroethyl ester; glutaric acid ditrifluoroethyl ester; 2-formylphenoxy acetic acid trifluoroethyl ester; 4-carboxybenzaldehyde trifluoroethyl ester; 4- (dimethylamino)phenyl acetic acid trifluoroethyl ester; isonitinic acid trifluoroethyl ester; and picolinic acid trifluoroethyl ester. [0026] Examples of vinyl carbonates include butyl vinyl carbonate; 1- methyl-3-piperidine methanol vinyl carbonate; 3,3'-diethoxypropanol vinyl carbonate; 4-tert-butylphenethyl vinyl carbonate; benzyl vinyl carbonate; 4- methyl-5-thiazole ethanol vinyl carbonate; glycidol vinyl carbonate; 1,3 -propylene divinyl carbonate; 1,4-cyclohexane dimethanol di(vinyl carbonate); 1,6- hexanediol di(vinyl carbonate); 4-hydroxybenzyl alcohol di(vinyl carbonate); 2,3- O-benzylidene thrietol di(vinylcarbonate); 2,5-furan-dimethanol di(vinylcarbonate); 2,6-pyridine dimethanol di(vinylcarbonate); acetone oxime vinyl carbonate; l,4-but-2-ene-diol di(vinylcarbonate); 3-thiophene methanol vinyl carbonate; 2-methylsulfonyl ethanol vinyl carbonate; 4-(2-hydroxyethyl) morpholine vinyl carbonate; and 3-methyl-2-norbornane methanol vinyl carbonate. [0027] Non-chemically modified lipases of the present invention can either be native (wild-type) lipases or genetically-modified lipases engineered using standard methods of molecular biology (Berglund, "Controlling Lipase Enantioselectivity for Organic Synthesis," Biomol. Eng„ 18(1): 13-22 (2001); Brocca et al, "Novel Lipases Having Altered Substrate Specificity, Methods for Their Preparation, and Their Use in Biocatalytic Applications," Eur. Pat. Appl. pp.33 (2001); Svendsen, "Lipase Protein Engineering," Biochim. Biophys. Acta, 1543(2): 223-238 (2000); Wong et al., "Lipase Engineering: A Window into Structure-Function Relationships," Methods in Enzymology, 284: 171-84 (1997), which are hereby incorporated in their entirety). [0028] When the acyl donor compound is a non-activated acyl donor, non- chemically modified lipases that can be used for the selective 14-O-acylation of the starting compound include lipases from microbial sources such as Alcaligenes sp., Candida antartica, Mucor miehei, Pseudomonas cepacia, Pseudomonas stutzeri, and Rhizopus oryzae.

[0029] When the acyl donor compound is an activated acyl donor, non- chemically modified lipases that can be used for the selective 14-O-acylation of the starting compound include lipases from microbial sources such as Alcaligenes sp., Candida antartica, Mucor miehei, Pseudomonas cepacia, Pseudomonas stutzeri, Rhizopus oryzae, Candida cylindracea, Candida rugosa, and Thermomyces sp.

[0030] In one embodiment, the nonaqueous biocatalysis of the present invention can be carried out where the non-chemically modified lipase is immobilized to a solid support. Examples of solid support include diatomaceous earth, polypropylene, or acrylic resins. Such solid-phase synthesis has a number of advantages such as simplifying purification, enabling excess reagents to drive reactions, avoiding limitations of substrate solubility, and facilitating handling of reactions on a small scale. [0031] In a typical reaction, the appropriate anthracyclinone substrate is dissolved in a suitable solvent, containing 10-30 equivalents of the desired acid, or alternatively an activated ester, carbonate, or similar suitable activated acyl donor. To this solvent, ca. 10-50 grams of a non-chemically modified lipase (either suitable wild type, or engineered using standard methods of molecular biology) per liter of reaction mixture is added as a lyophilized powder or "immobilized preparation." The most suitable solvent and lipase is determined by a rapid parallel screening of miniaturized reactions in multiwell polypropylene plates, with rapid mass spectral analysis of reaction aliquots. The reaction flask can be incubated at ca. 25-50°C, or, alternatively, if an accumulation of the reaction product water inhibits the progress of the reaction, fitted with a Dean-Stark and condenser assembly and stirred at the reflxix temperature of the solvent, from

5 hours up to 2 days. Reaction progress is followed by thin layer chromatography ("TLC") and/or liquid chromatography/mass spectroscopy ("LC/MS"). Upon depletion of the substrate the reaction mixture is filtered to remove the insoluble enzyme powder. The enzyme is washed with an appropriate solvent and the combined solvent is washed with saturated aqueous sodium bicarbonate, dried over MgSO₄, and evaporated under reduced pressure. The product is purified using a suitable method, most routinely silica gel chromatography, liquid chromatography on other solid adsorbants, or preparative high performance liquid chromatography ("HPLC").

[0032] In another embodiment of the present invention, R₃ is OCH₃.

[0033] In yet another embodiment, R₂ is 2,6-dideoxy-2-fluoro-α- talopyranosyl and R₃ is OH or OCH₃. Rn can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy-naphthoic, cyclopentylpropionic, 2'- pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis- 4,7,10,13,16,19-docosahexanoic, or ethylcarbonic acid. Preferably, Ri is an acyl radical of a c .s-4,7,10,13,16,19-docosahexanoic acid, a methylpolyethylene oxide acetic acid which has a molecular weight from about 2,000 to about 40,000, or a polyethylene oxide diacetic acid which has a molecular weight from about 2,000 to about 40,000.

[0034] In another embodiment of the present invention, the product compound has the formula:

where:

R_t is an acyl radical of a carboxylic acid; and

R₂ = H

2,6-dideoxy-2-fluoro-α-talopyranosyl;

3-trifluoroacetylamino-2,3,6-trideoxy-α-L-/jαo-hexopyranosyl; or 3-deamino-3-(2'-pyrroline- -yl)-2,3,6-trideoxy-α-L-/j o-hexopyranosyl; or a pharmaceutically acceptable salt thereof.

[0035] Preferably, R_\ is an acyl radical of hexanoic acid, heptanoic acid, polyethylene glycol acetic acid, or polyunsaturated fatty acid. [0036] The polyethylene glycol acetic acid can be methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, or polyethylene glycol diacetic acid which has a molecular weight of about 35,000. [0037] The polyunsarurated fatty acid can be linoleic, linolenic, arachidonic, dihomo-γ-linolenic, cw-5,8,l l,14,17-eicosapentaenoic, or cis- 4,7,10,13,16, 19-docosahexanoic acid. [0038] In another embodiment, R₂ is 3-trifluoroacetylamino-2,3,6- trideoxy-α-L-/j;χø-hexopyranosyl and R₃ is OCH₃.

[0039] In yet another embodiment, R₂ is 3- t-butoxycarbonylamino-2,3,6- trideoxy-α-L-fy o-hexopyranosyl and R₃ is OCH₃.

[0040] In another embodiment, R₂ is 3-acetylamino-2,3,6-trideoxy-α-L-

/yxσ-hexopyranosyl and R₃ is OCH₃. [0041] In yet another embodiment, R₂ is 3-(9- fluorenylmethyloxycarbonylamino-2,3 ,6-trideoxy-α-L-/j o-hexopyranosyl and R is OCH₃.

[0042] In another embodiment, R₂ is 3-[(S)-2-amino-4-methylvaleramido]

-2,3,6-trideoxy-α-L- μxø-hexopyranosyl and R₃ is OCH₃. [0043] In yet another embodiment, R₂ is 2-pyrrolino and R₃ is OCH₃.

can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, heptanoic, octanoic, alanic, leucinic, valeric, cz_?-4,7,10,13,16,19- docosahexanoic, or ethylcarbonic acid. Preferably, R_\ is an acyl radical of a succinic acid.

[0044] In another embodiment, R₂ is morpholino and R₃ is OCH₃. Ri can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4, 7, 10, 13, 16,19-docosahexanoic, or ethylcarbonic acids. /057896

- 14 -

[0045] In yet another embodiment, R₂ is 3-(2-methoxy-4-morpholinyl)-

2,3,6-trideoxy-a-L-/y o-hexopyranosyl and R₃ is H, OH, or OCH₃. R] can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy-naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, or ethylcarbonic acid. [0046] In another embodiment, R is 3-(3-cyano-4-morpholinyl)-2,3,6- trideoxy-α-L-/ o-hexopyranosyl and R₃ is H, OH, or OCH₃. R_\ can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy-naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, or ethylcarbonic acid. [0047] In yet another embodiment, R₂ is 3-trifluoroacetylamino-2,3,6- trideoxy- -L-arabino-hexopyranosyl and R₃ is H, OH, or OCH₃. [0048] In another embodiment, R is 4-trifluoroacetylamino-6-methyl-2H- ρyran-2-yl and R₃ is H, OH, or OCH₃.

[0049] In yet another embodiment, R is 3-trifluoroacetylamino-2,3,6- trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-α-L-hexopyranosyl and R₃ is OCH₃. can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4, 7, 10, 13, 16,19-docosahexanoic, or ethylcarbonic acid. [0050] In another embodiment, R₂ is 3-butoxycarbonylamino-2,3,6- trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-α-L-hexopyranosyl and R₃ is OCH₃. Ri can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, or ethylcarbonic acids.

[0051] In yet another embodiment, R₂ is 3-acetylamino-2,3,6-trideoxy-4-

O-(tetrahydro-2H-pyran-2-yl)-a-L- )xo-hexopyranosyl and R₃ is OCH . R_\ can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, or ethylcarbonic acids.

[0052] In another embodiment, R₂ is 7-O-[4-O-(3-amino-2,3,6-trideoxy-α-

L-mannopyranosyl)-2,6-dideoxy-α-L-galactopyranosyl] and R₃ is H. Ri can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy-naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cω-4,7,10,13,16,19-docosahexanoic, or ethylcarbonic acids. [0053] In yet another embodiment, R₂ is 2,6-dideoxy-2-iodo-α-L- mannohexopyranosyl and R₃ is H. Ri can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy-naphthoic, cyclopentylpropionic, 2'- pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis- 4,7,10,13, 16,19-docosahexanoic, or ethylcarbonic acid. [0054] In another embodiment, R₂ is 3-[(S)-2-amino-4-methyl valeramido]-2,3,6-trideoxy-α-L-/jχø-hexopyranosyl and R₃ is OCH₃. Ri can be an acyl radical of an acid such as acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, or ethylcarbonic acids. [0055] An alternative method could be used in order to improve substrate solubility, product recovery and production efficiency when anthracycline derivatives containing a free amine are targeted.

[0056] In a typical method, doxorubicin HCI salt is suspended in DMF and reacted with allyl chloroformate to produce N-alloc doxorubicin. The N-alloc doxorubicin precipitate is highly soluble in less polar organic solvents suitable for synthesis, and is added to suitable solvents containing 5-10 equivalents of an organic acid, or alternatively an activated ester, carbonate or similar activated acyl donor. To this solvent, ca. 10-50 g of a non-chemically modified lipase (either suitable wild type, or engineered using standard methods of molecular biology) per liter of reaction mixture is added as a lyophilized powder or "immobilized preparation." Upon depletion of the substrate, the solvent is removed under vacuum and the excess acyl donor is removed by washing the resulting solid with a suitable organic solvent to afford 14-O-acylated doxorubicin-3'-N-alloc- derivative. The 14-O-acylated doxorubicin-3'-N-alloc-derivative is dissolved in THF containing dimedone, sodium carbonate and tetrakis(triphenylphosphine)palladium(0) - uniquely allowing to selectively remove the Alloc group without altering any other chemical functionality on the product anthracyclinone . The resulting 14-O-acyl-doxorubicin-free base precipitates and is further purified by repeated washings of the resulting solid with EtOAc. The free base is then dissolved in l,4-dioxane/CH₂Cl₂ containing 1 equivalent of HCI. The resulting solid is dried under vacuum to afford the doxorubicin- 14-O-acyl-doxorubicin HCI salt.

[0057] Thus, in another embodiment of the present invention, the product compound has the formula:

wherein: R_! is an acyl group,

R₂ is 3 -substituted allyloxycarbonylamino-2,3,6-trideoxy-α-L-/ ' ø- hexopyranosyl, R₃ is H, OH, or OCH₃.

[0058] The substituted allyloxycarbonylamino substituent can be an Alloc group. Ri can be an acyl radical of an acid selected from the group consisting of formic, acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids.

Preferably, R_\ is an acyl radical of a hexanoic or heptanoic acid.

[0059] In another embodiment of the present invention, the product compound has the formula:

where n is 4 or 5, or a pharmaceutically acceptable salt thereof.

EXAMPLES

[0060] ^* The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Example 1 - Synthesis of Doxorubicin-14-Butyrate-3'-Trifluoroacetamide

[0061] Doxorubicm-3'-trifluoroacetamide (80 mg, 0.125 mmol) was dissolved in MTBE (40 mL), and vinyl butyrate (1 mL, 7.9 mmol) was added, followed by PS 30 lipase (2 g, 68 kU). The reaction mixture was incubated at 45 °C (290 rpm) for 12 h. The enzyme was then removed by filtration and washed with cliloroform (3 x 20 mL). The combined organic phases were evaporated and the red oil was washed with 10 mL of hexane to give a red solid. Silica gel chromatography (load in CHC1₃ - eluent, EtOAc/hexanes 2:1, followed by neat EtOAc) gave doxorubicin- 14-butyrate-3'-frifluoroacetamide as a red solid (62 mg, 70%).

Example 2 - Synthesis of Doxorubicin-14-Valerate-3'-Trifluoroacetamide

[0062] Doxorubicin-3'-trifluoroacetamide (50 mg, 0.078 mmol) was dissolved in MTBE (30 mL) and valeric acid (99%, 2 mL, 18.4 mmol, Aldrich, Milwaukee, WI) was added followed by anhydrous sodium sulfate (1 g) and Chirazyme L2, C3 (2 g, 20,000 U, Biocatalytics, Pasadena, CA). The reaction /057896

- 18 -

mixture was incubated at 45°C (290 rpm) for 18 h. LC/MS analysis indicated a 60% conversion to the desired product.

Example 3 - Alternative Method of Doxorubicin-14-Valerate-3'- Trifluoroacetamide Synthesis

[0063] Doxorubicin-3 '-trifluoroacetamide (60 mg) was dissolved in 60 mL MTBE then 1 mL of valeric acid was added followed by 1.5 g of Chirazyme L2, C3 lipase (Biocatalytics, Pasadena, CA). The flask was fitted with a Dean- Stark (Aldrich, Milwaukee, WI) apparatus and the reaction mixture was heated to the reflux temperature of MTBE (~55^°C) for 5h. LC/MS analysis shows >90% conversion to the desired 14-valerate with ~4% starting material remaining.

Example 4 - Synthesis of Doxorubicin-14-c s-4,7,10,13,16,19- Docosahexaenoate-3 '-Trifluoroacetamide

[0064] Doxorubicin-3 '-trifluoroacetamide (30 mg, 0.047 mmol) was dissolved in 30 mL dry MTBE. To this was added cis-4,1, 10,13,16,19- Docosahexaenoic acid (430 μL, 1.22 mmol) followed by 0.5 g Chirazyme L2, C3. The reaction was heated to 55°C in a 100-mL round bottom flask fitted with a 15- mL Dean Stark apparatus and a condenser. The Dean Stark apparatus contained molecular sieves. After six hours, LC/MS analysis showed 85% conversion to the desired product with 9% unreacted starting material. The reaction mixture was chilled to room temperature and filtered with filter paper. The enzyme was rinsed with EtOAc (2 x 10 mL); the extracts were combined with the filtrate, washed with saturated NaHCO₃ (3 x 30mL) and dried over MgSO₄. The product was purified by silica gel chromatography eluting with EtOAc/Hexane 1.5: 1. The solvent was removed under reduced pressure to give doxorubicin- 14-cis- 4,7, 10, 13,16, 19-docosahexaenoate-3 '-trifluoroacetamide (21.6 mg, 49% yield) as a red solid.

Example 5 - Synthesis of Doxorubicin-14-Acetoxy-Polyethylene Glycol (2000 mw)-Oxyacetate-3'-Trifluoroacetamide [0065] Doxorubicin-3 '-trifluoroacetamide (15 mg, 0.023 mmol) was dissolved in dry toluene (40 mL) and to this was added polyethylene glycol diaceticacid 2000 mw (500 mg, 0.25 mmol) and lipase L2, C3 (0.9 g, 9,000 U). The flask was fitted with a Dean-Stark apparatus and condenser and the reaction mixture was heated to the reflux temperature of toluene (~110°C) for 20 h. LC/MS analysis shows -60% conversion to the desired product with ~ 40% starting material remaining.

Example 6 - Identification of Enzymes for Selective 14-O-Acylation of

Anthracyclinones Containing an N-AIkylated Amino Sugar or Non-Basic Sugar Moiety

[0066] Doxorubicin-3 '-trifluoroacetamide (2 mM) was dissolved in toluene containing 50 mM of valeric acid (Aldrich, Milwaukee, WI), and 0.5 mL was added to a each well of a 96-well plate (2 mL total volume of each well), each well prefilled with ca. 100 mg of a different lipase as a dry solid. The plate was sealed and incubated at 45°C and rotated at 10 rpm in an incubator for up to 5d. Samples were periodically withdrawn and analyzed by high-throughput LC/MS to determine the product identity and best conversion to doxorubicin- 14- valerate-3'- trifluoroacetamide. [0067] With valeric acid as the acyl donor and doxorubicin-3 '- trifluoroacetamide as the non-basic anthracycline, the lipases from Mucor miehei (Chirazyme L-9, Biocatalytics, Pasadena CA) and Candida antarctica (Chirazyme L-2, Biocatalytics, Pasadena CA) were identified as the most efficient catalysts. Five lipases, including Lipase PL from. Alcaligenes sp. (Meito Sangyo, Tokyo, Japan), lipase from Mucor miehei (Fluka, Milwaukee, WI), Lipase AH from Pseudomonas cepacia (Amano, Lombard, IL), Lipase TL from Pseudomonas stutzeri (Meito Sangyo), and Lipase A-10FG from Rhizopus oryzae (Nagase, Kyoto, Japan), were identified as having some activity for the selective 14-O- acylation of doxorubicin-3 '-trifluoroacetamide with at least one free acid acyl donor.

Example 7 - Selection and Use of Immobilization Support

[0068] For improving the handling and recoverability of suitable enzyme catalysts for the selective acylation of non-basic anthracyclines, immobilization supports could be used with the lipases in organic reaction solvents. Accurel (polypropylene beads, Akzo Nobel Fazer AG, Obernburg, Germany), and Celite (diatomaceous earth, Hyflo Super Cel, Sigma, St. Louis, MO) represent examples that proved to be acceptable immobilization supports for the successful selective synthesis, in addition to free lipase powders prepared by lyophilization or precipitation of the enzymes.

[0069] Anthracyclines containing a basic moiety (free amine) typically absorbed strongly to these immobilization supports, significantly reducing recovered product yields. This highlights another advantage of the use of the current invention for the production of selectively acylated anthrcyclines, using non-basic anthracyclines as starting reactants.

Example 8 - Removal of Excess Acyl Donor

[0070] A simple hexane wash of the filtered reaction mixture was found to be capable of removing most of the excess acyl donor. A second or third wash may be performed if desired to completely remove the acyl donor. Depending on the chemical nature of the synthesized monoester, little or no anthracycline product dissolved in hexane.

Example 9 —Purification of Anthracycline Monoesters

Doxorubicin-3 '-trifluoroacetamide- 14-esters

[0071] The isolation of selectively monoacylated esters of non-basic anthrcyclines could be easily accomplished from the enzymatic reaction mixtures using silica gel chromatography. For example, the purification of doxorubicin-3 '- trifluoroacetamide- 14-esters could typically be achieved as follows: the crude reaction mixture was dried under vacuum and reconstituted in chloroform and loaded onto a 25 mm ID glass column containing a ca. 6 in bed of silica (63- 200 μm, Selecto Scientific, Suwanee, GA). A suitable mobile phase (e.g., EtOAc/hexanes 2:1) eluted product-related compounds and ca. 5 mL fractions were collected. Fractions were pooled based upon TLC analysis and the resulting product-containing solution was dried under vacuum to give the isolated purified product as a red solid. Example 10 - Identification of Product Structure

LC/MS analysis of Doxorubicin Derivatives

[0072] Samples were analyzed on a PE-Sciex API 2000 LC/MS/MS system equipped with an HP 1000 photodiode array detector. A Luna C8 (5μ, 50 x 2 mm, Phenomenex, Torrance, CA), column was used with a mobile phase linear gradient of water (+ 0.4% acetic acid) and acetonitrile (+ 0.4% acetic acid). [0073] Anthracycline derivatives exhibit ionization as M+H (positive mode) and M-H (negative mode). Mass fragmentation analysis using a electrospray ionization interface allowed a quick confirmation of product structure, in comparison with authentic standards or computer predicted models of fragmentation.

NMR Analysis of Doxorubicin Derivatives [0074] The formation of selectively acylated anthracycline derivatives is clearly demonstrated in the NMR spectrum by a downfield shift of the acylated methylene protons. For instance, with doxorubicin-3 '-trifluoroacetamide-14- esters, the methylene protons appear as two doublets between δ 5 and 5.5 ppm in deuterated acetone or methanol. Samples were run on a WM-360 MHz or a DRX- 400 MHz (Bruker, Billerica, MA) spectrophotometer. As appropriate for the specific derivatives, deuterated acetone or deuterated methanol were used for the analysis.

Example 11 - Synthesis of Doxorubicin-14-O-Hexanoate HCI via N- Alloc Doxorubicin-14-O-Hexanoate

[0075] This example shows use of the subject selective enzymatic procedure to cleanly acylate the 14-position of anthracyclines that have been modified at the 3' amine to improve solubility, product recovery, and production efficiency. It also illustrates novel methods for improving production of doxorubicin derivatives with a free 3' amine by modification and removal of a suitable group to the 3' amine without altering the final chemical functionality of the sugar moiety, quinine, or any other functionality on doxorubicin. [0076] Doxorubicin HCI salt (5.0 g, 8.62 mmol) was suspended in dry DMF (250 mL) under a nitrogen atmosphere. To this was added DIPEA (6 mL, 34.5 mmol) and the mixture was stirred for 15 min at room temperature. The solution was cooled to -10°C and allyl chloroformate (0.92 mL, 8.67 mmol) in dry DMF (30 mL) was added dropwise over 5 min. Stirring was continued for 10 min at -10°C, then the reaction mixture was allowed to warm to room temperature over a 30 min period. Water (3 mL) was added and stirring was continued for 5 min. The solvent was evaporated under reduced pressure to ca. 25 mL volume, then ethyl acetate (3 x 100 mL) was added to precipitate any unreacted doxorubicin. The solid was removed by filtration and the solvent was evaporated under reduced pressure. Toluene (100 mL) was added and the solvent was evaporated under reduced pressure to give N-alloc doxorubicin (4.6 g, 85%) as a red solid.

[0077] N- Alloc doxorubicin (1.3 g, 2.07 mmol) was dissolved in dry 2- butanone (250 mL) under a nitrogen atmosphere and to this was added Chirazyme L2, C3 (6.5 g, 20442 U) and caproic acid vinyl ester (21 mmol, 3.35 L). The reaction mixture was stirred vigorously and heated at 50°C for 24 h. The enzyme was removed by filtration and washed with 2-butanone (2 x 100 mL). The solvent was evaporated under reduced pressure and the resulting solid was washed with hexane (3 x 100 mL). Residual hexane was removed by evaporation under reduced pressure to afford N-alloc doxorubicin- 14-O-hexanoate (1.3 g, 86%) as a red solid. [0078] N-alloc doxorubicin- 14-O-hexanoate (750 mg, 1.03 mmol) was dissolved in dry THF (20 mL) under a nitrogen atmosphere. To this was added dimedone (1.44 g, 10.3 mmol), sodium carbonate (20 mg, 0.19 inmol) and tetrakis(triphenylphosphine)palladium(0) (115 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 45 min. Hexane (60 mL) was added and the solid was removed by filtration. The solid was washed with hexane (2 x 50 mL) and dried by evaporation of excess solvent under reduced pressure to give doxorubicin- 14-O-hexanoate (530 mg, 0.82 mmol) as an orange solid. The solid was dissolved in a 1,4 dioxane/methylene chloride 1:1 mixture (200 mL) and 4M HCI in 1,4 dioxane (0.205 mL, 0.82 mmol) was added. The mixture was stirred at room temperature for 10 min, then the solvent was evaporated under reduced pressure. The remaining solid was washed with ethyl acetate (3 x 20 mL) and dried under vacuum to afford doxorubicin- 14-O-hexanoate HCI salt (461 mg, 70%) as an orange solid. Example 12 - Synthesis of Doxorubicin-14-O-Heptanoate HCI via N- Alloc Doxorubicin-14-O-Heptanoate [0079] This example shows use of the subject selective enzymatic procedure to cleanly acylate the 14-position of anthracyclines that have been modified at the 3' amine to improve solubility, product recovery, and production efficiency. It also illustrates novel methods for improving production of doxorubicin derivatives with a free 3' amine by modification and removal of a suitable group to the 3 ' amine without altering the final chemical functionality of the sugar moiety, quinone, or any other functionality on doxorubicin. [0080] Doxorubicin HCI salt (5.0 g, 8.62 mmol) was suspended in dry

DMF (250 mL) under a nitrogen atmosphere. To this was added DIPEA (6 mL, 34.5 mmol) and the mixture was stirred for 15 min at room temperature. The solution was cooled to -10°C and allyl chloroformate (0.92 mL, 8.67 mmol) in dry DMF (30 mL) was added drop wise over 5 min. Stirring was continued for 10 min at -10°C, then the reaction mixture was allowed to warm to room temperature over a 30 min period. Water (3 mL) was added and stirring was continued for 5 min. The solvent was evaporated under reduced pressure to ca. 25 mL volume, then ethyl acetate (3 x 100 mL) was added to precipitate any unreacted doxorubicin.

The solid was removed by filtration and the solvent was evaporated under reduced pressure. Toluene (100 mL) was added and the solvent was evaporated under reduced pressure to give N-alloc doxorubicin (4.6 g, 85%) as a red solid. [0081] N- Alloc doxorubicin (2.5 g, 3.98 mmol) was dissolved in dry 2- butanone (300 mL) under a nitrogen atmosphere and to this was added Chirazyme L2, C3 (12 g, 37739 U) and heptanoic acid (70.6 mmol, 10 mL). The reaction flask was fitted with a Dean-Stark and condenser assembly in which the receiver was filled with a mixture of 4A molecular sieves (ca. 15 g) and the reaction solvent. The reaction mixture was stirred vigorously and heated at relux temperature for ca. 50 h. After this time, the conversion to desired product as seen by LC/MS analysis was estimated to be -60%.

[0082] The enzyme was removed by filtration and washed with 2- butanone (3 x 100 mL). The solvent was evaporated under reduced pressure to ca. 200 mL volume, then ethyl acetate (200 mL) was added. The solvent was washed with saturated aqueous sodium bicarbonate (4 x 100 mL), saturated aqueous NaCl (100 mL), then dried with MgSO₄ and evaporated under reduced pressure to give a sticky, red solid (3 g). Silica gel chromatography (eluent, ethyl acetate) to remove any unreacted N-alloc doxorubicin afforded N-alloc doxorubicin-14-O- heptanoate (1.6 g) as a sticky, red solid. Purity as seen by LC/MS analysis was estimated to be ~80%.

[0083] N-alloc doxorubicin- 14-O-hexanoate (1.6 g, 2.16 mmol) was dissolved in dry THF (30 mL) under a nitrogen atmosphere. To this was added dimedone (3 g, 21.4 mmol), sodium carbonate (20 mg, 0.19 mmol) and tetrakis(triphenylphosphine)palladium(0) (250 mg, 0.216 mmol). The reaction mixture was stirred at room temperature for 45 min. Hexane (60 mL) was added and the solid was removed by filtration. The solid was washed with hexane (2 x 50 mL) and dried by evaporation of excess solvent under reduced pressure to give crude doxorubicin- 14-O-hexanoate (2.5 g) as a red solid. The solid was dissolved in a 1,4 dioxane/methylene chloride 1:1 mixture (400 mL) and 4M HCI in 1,4 dioxane (0.450 mL, 1.8 mmol) was added. The mixture was stirred at room temperature for 10 min, then the solvent was evaporated under reduced pressure. The remaining solid was washed with ethyl acetate (6 x 30 mL) and dried under vacuum to afford doxorubicin- 14-O-hexanoate HCI salt (612 mg, 41%) as an orange solid.

[0084] Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

A process for preparation of a product compound of the formula:

wherein:

R_\ is an acyl group,

R₂ is H, an N-alkylated amino sugar, or a non-basic sugar moiety,

R₃ is H, OH, or OCH₃, said process comprising: reacting a starting compound of the formula:

with an acyl donor compound in the presence of a non-chemically modified lipase, under conditions effective to produce the product compound.

2. The process of claim 1, wherein said reacting is carried out under conditions effective to exclusively acylate the starting compound at its C-14 position.

3. The process of claim 1, wherein said reacting is carried out in an organic solvent.

4. The process of claim 3, wherein the organic solvent is toluene, methyl-tert-butyl ether, pyridine, or mixtures thereof, or mixtures with N,N-dimethyl formamide.

5. The process of claim 3, wherein said reacting is carried out at a temperature of 0 to 150°C.

6. The process of claim 5, wherein said reacting is carried out at a temperature of 25 to 110°C.

7. The process of claim 1, wherein the acyl donor compound is a non-activated acyl donor.

8. The process of claim 7, wherein the non-activated acyl donor is a carboxylic acid selected from the group consisting of: enanthic acid, caprylic acid, fumaric acid, maleic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, lauric acid, myristic acid, palmitic acid, stearic acid, glyoxylic acid diethylacetal, acrylic acid, crotonic acid, isocrotonic acid, butenoic acid, pentenoic acid, oleic acid, retinoic acid, linoleic acid, linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, czs'-5,8,ll,14,17-eicosapentaenoic acid, cώ-4,7,10,13,16,19-docosahexaenoic acid, malonic acid, succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, undecanedioic acid, dodecanedioic acid, 1,12-dodecanedicarboxylic acid, benzoic acid, picolinic acid, nicotinic acid, isonicotinic acid, piperazine-2-carboxylic acid, pyrrole-2- carboxylic acid, pyrrole-3 -carboxylic acid, furan-2-carboxylic acid, furan-3- carboxylic acid, thiophene-2-carboxylic acid, thiophene-3 -carboxylic acid, imidazole-2-carboxylic acid, imidazole-4(5)-carboxylic acid, phenylacetic acid, 3,5-dibromo-4-hydroxybenzoic acid, 3-(2-furyl) acrylic acid, 3,4- (methylenedioxy) phenyl acetic acid, norbomane acetic acid, 2-thiophene acetic acid, 2,6-dimethoxy nicotinic acid, 3-indol butyric acid, 3,4-(methylenedioxy) cinnamic acid, 4-formylcinnamic acid, N-CBZ-isonipecotic acid, N-CBZ-L- proline acid, N-CBZ-tyrosine acid, N-CBZ-alanine acid, Fmoc-sarcosine acid, N- CBZ-glycine acid, N-CBZ-phenylalanine acid, bromoacetic acid, chloroacetic acid, chlorobenzoic acid, cinnamic acid, formic acid, iodoacetic acid, methacrylic acid, pivalic acid, sorbic acid, 2-ethyl hexanoic acid, salicylic acid, 3-amino-6- thiophen-2-yl-4-trifluoromethyl-thieno [2,3-b] pyridine carboxylic acid, indole-2- carboxylic acid, indole-3 -carboxylic acid, indoline-2-carboxylic acid, indoline₇3- carboxylic acid, 3-(S-glutathionyl)propionic acid, polyethylene glycol acetic acid, methyl polyethylene glycol acetic acid, N-protected leucine, N-protected isoleucine, N-protected tert-leucine, and N-protected cycloleucine.

9. The process of claim 7, wherein the non-chemically modified lipase is from a microbial source selected from the group consisting of Alcaligenes sp., Candida antartica, Mucor miehei, Pseudomonas cepacia, Pseudomonas stutzeri, and Rhizopus oryzae.

10. The process of claim 1 , wherein the acyl donor compound is an activated acyl donor.

11. The process of claim 10, wherein the activated acyl donor is a vinyl ester selected from the group consisting of: 3,3-diphenyl propionic acid vinyl ester; 3,5-dibromo-4-hydroxybenzoic acid vinyl ester; 3-(2-furyl) acrylic acid vinyl ester; 3,4-(methylenedioxy) phenyl acetic acid vinyl ester; norbomane acetic acid vinyl ester; 2-thiophene acetic acid vinyl ester; 2,6-dimethoxy nicotinic acid vinyl ester; 3-indol butyric acid vinyl ester; pyrrole-2-carboxylic acid vinyl ester; 3,4-(methylenedioxy) cinnamic acid vinyl ester; 4-formylcinnamic acid vinyl ester; N-CBZ-isonipecotic acid vinyl ester; N-CBZ-L-proline vinyl ester; N- CBZ-tyrosine vinyl ester; N-CBZ-alanine vinyl ester; Fmoc-sarcosine vinyl ester (Fmoc-N-Me-Gly-OH vinyl ester); N-CBZ-Glycine vinyl ester; N-CBZ- phenylalanine vinyl ester; 2-furoic acid vinyl ester; acrylic acid vinyl ester; adipic acid divinyl ester; benzoic acid vinyl ester; bromoacetic acid vinyl ester; butyric acid vinyl ester (vinyl butyrate); caproic acid vinyl ester; enanthic acid vinyl ester; caprylic acid vinyl ester; chloroacetic acid vinyl ester; chloroformic acid vinyl ester; chlorobenz ic acid vinyl ester; cinnamic acid vinyl ester; crotonic acid vinyl ester; formic acid vinyl ester; iodoacetic acid vinyl ester; lauric acid vinyl ester; methacrylic acid vinyl ester; myristic acid vinyl ester; n-capric acid vinyl ester; n- caprylic acid vinyl ester; palmitic acid vinyl ester; phenyl acetic acid vinyl ester; pivalic acid vinyl ester; propionic acid vinyl ester; sorbic acid vinyl ester; stearic acid vinyl ester; acetic acid vinyl ester; 2-ethyl hexane vinyl ester; vinyl salicylate; trimethyl-vinyloxycarbonylmethyl-ammonium bromide; 3-amino-4,6-dimethyl- thieno [2,3-b] pyridine-2-carboxylic acid vinyl ester (1S-27086); 3-amino-6- thiophen-2-yl-4-trifluoromethyl-thieno [2,3-b] pyridine carboxylic acid vinyl ester (1S-21501); divinyl succinate; divinyl glutarate; and divinyl subarate.

12. The process of claim 10, wherein the activated acyl donor is a trihaloethyl ester.

13. The process of claim 12, wherein the activated acyl donor is a trifluoroethyl ester selected from the group consisting of: 3,3-diphenyl propionic acid trifluoroethyl ester; 3,6-dioxaheptanoic acid trifluoroethyl ester; oxalic acid trifluoroethyl ester; malonic acid trifluoroethyl ester; (-)-2-oxo-4- thiazolidine-2-carboxylic acid trifluoroethyl ester; pyrazine-2-carboxylic acid trifluoroethyl ester; nicotinic acid trifluoroethyl ester; 1,4-cyclohexane dicarboxylic acid di trifluoroethyl ester; terephtalic acid di trifluoroethyl ester; 4- (dimethylamino) benzoic acid trifluoroethyl ester; 4-(bromomethyl) phenylacetic acid trifluoroethyl ester; benzimidazole propionic acid trifluoroethyl ester; Fmoc- L-thiazolidine-4-carboxylic acid trifluoroethyl ester; glutaric acid ditrifluoroethyl ester; 2-formylphenoxy acetic acid trifluoroethyl ester; 4-carboxybenzaldehyde trifluoroethyl ester; 4-(dimethylamino)phenyl acetic acid trifluoroethyl ester; isonitinic acid trifluoroethyl ester; and picolinic acid trifluoroethyl ester.

14. The process of claim 10, wherein the activated acyl donor is a vinyl carbonate selected from the group consisting of: butyl vinyl carbonate; 1-methyl- 3-piperidine methanol vinyl carbonate; 3,3'-diethoxypropanol vinyl carbonate; 4- tert-butylphenethyl vinyl carbonate; benzyl vinyl carbonate; 4-methyl-5-thiazole ethanol vinyl carbonate; glycidol vinyl carbonate; 1,3 -propylene divinyl carbonate; 1,4-cyclohexane dimethanol di(vinyl carbonate); 1,6-hexanediol di(vinyl carbonate); 4-hydroxybenzyl alcohol di(vinyl carbonate); 2,3-0- benzylidene thrietol di(vinylcarbonate); 2,5-furan-dimethanol di(vinylcarbonate); 2,6-pyridine dimethanol di(vinylcarbonate); acetone oxime vinyl carbonate; 1,4- but-2-ene-diol di(vinylcarbonate); 3-thiophene methanol vinyl carbonate; 2- methylsulfonyl ethanol vinyl carbonate; 4-(2-hydroxyethyl) morpholine vinyl carbonate; and 3-methyl-2-norbornane methanol vinyl carbonate.

15. The process of claim 10, wherein the non-chemically modified lipase is from a microbial source selected from the group consisting of Alcaligenes sp., Candida antartica, Mucor miehei, Pseudomonas cepacia, Pseudomonas stutzeri, Rhizopus oryzae, Candida cylindracea, Candida rugosa, and Thermomyces sp.

16. The process of claim 1 , wherein the non-chemically modified lipase is a native lipase.

17. The process of claim 1 , wherein the non-chemically modified lipase is a genetically modified lipase.

18. The process of claim 1 , wherein the non-chemically modified lipase is immobilized to a solid support.

19. The process of claim 1, wherein R₃ is OCH₃.

20. The process of claim 1, wherein R₂=2,6-dideoxy-2-fluoro- α-talopyranosyl and R₃ is OH or OCH₃.

21. The process of claim 20, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

22. The process of claim 21 , wherein Ri is an acyl radical of a methylpolyethylene oxide acetic acid which has a molecular weight from about 2,000 to about 40,000.

23. The process of claim 21 , wherein R_\ is an acyl radical of a polyethylene oxide diacetic acid which has a molecular weight from about 2,000 to about 40,000.

24. The process of claim 21 , wherein Ri is an acyl radical of a cis-4,1, 10, 13 , 16, 19-docosahexanoic acid.

25. The process of claim 1 , wherein the product compound has the formula:

wherein:

Ri is an acyl radical of a carboxylic acid; and

R₂ = H;

2,6-dideoxy-2-fluoro-α-talopyranosyl;

3-trifluoroacetylamino-2,3,6-trideoxy-α-L- μxo-hexopyranosyl; or 3-deamino-3-(2'-pyrroline- -yl)-2,3,6-trideoxy-α-L-/jχo-hexopyranosyl; or a pharmaceutically acceptable salt thereof.

26. The process of claim 25, wherein R₂ is H.

27. The process of claim 25, wherein

Ri is an acyl radical of a carboxylic acid selected from the group consisting of: hexanoic acid; heptanoic acid; polyethylene glycol acetic acid; and polyunsaturated fatty acid; and

R₂=2,6-dideoxy-2-fluoro-α-talopyranosyl.

28. The process of claim 27, wherein Ri is an acyl radical of hexanoic acid.

29. The process of claim 27, wherein Ri is an acyl radical of polyethylene glycol acetic acid.

30. The process of claim 29, wherein the polyethylene glycol acetic acid is selected from the group consisting of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, and polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

31. The process of claim 27, wherein Ri is an acyl radical of polyunsaturated fatty acid.

32. The process of claim 31 , wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic, linolenic, arachidonic, dihomo-γ-linolenic, cω-5,8,ll,14,17-eicosapentaenoic, and cis-4,1, 10, 13, 16,19- docosahexanoic acids.

33. The process of claim 25 , wherein

R₂=3 -trifluoroacetylamino-2,3 ,6-trideoxy-α-L-fy o-hexopyranosyl.

34. The process of claim 33, wherein Ri is an acyl radical of hexanoic acid.

35. The process of claim 33, wherein Ri is an acyl radical of polyethylene glycol acetic acid.

36. The process of claim 35, wherein the polyethylene glycol acetic acid is selected from the group consisting of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, and polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

37. The process of claim 33, wherein Ri is an acyl radical of polyunsaturated fatty acid.

38. The process of claim 37, wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic, linolenic, arachidonic, dihomo-γ-linolenic, cis-5, 8,11,14,17-eicosapentaenoic, and cz^'s-4,7,10,13,16,19- docosahexanoic acids.

39. The process of claim 25, wherein

R₂ is 3 -deamino-3 -(2' -pyrroline- 1 -yl)-2,3,6-trideoxy-a-L-/yxo-hexopyranosyl.

40. The process of claim 39, wherein Ri is an acyl radical of hexanoic acid.

41. The process of claim 39, wherein Ri is an acyl radical of polyethylene glycol acetic acid.

42. The process of claim 41 , wherein the polyethylene glycol acetic acid is selected from the group consisting of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl polyethylene glycol acetic acid wliich has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, and polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

43. The process of claim 39, wherein Ri is an acyl radical of polyunsaturated fatty acid.

44. The process of claim 43, wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic, linolenic, arachidonic, dihomo-γ-linolenic, czs-SjSjl ljMjπ-eicosapentaenoic, and cis-4,1 ,10,13,16,19- docosahexanoic acids.

45. The process of claim 1 , wherein the product compound has the formula:

wherein n is 4 or 5, or a pharmaceutically acceptable salt thereof.

46. The process of claim 45, wherein n is 4.

47. The process of claim 45, wherein n is 5.

48. The process of claim 1 , wherein R₂=3-frifluoroacetylamino- 2,3,6-trideoxy-α-L-/yxo-hexopyranosyl and R₃= CH₃.

49. The process of claim 1 , wherein R₂=3- t- butoxycarbonylamino-2,3 ,6-trideoxy-α-L- y >-hexopyranosyl and R₃=OCH₃.

50. The process of claim 1 , wherein R₂=3-acetylamino-2,3,6- trideoxy-α-L-/yco-hexopyranosyl and R₃=OCH₃.

51. The process of claim 1 , wherein R₂=3 -(9- fluorenylmethyloxycarbonylamino-2,3,6-trideoxy-a-L-fyxo-hexopyranosyl and R₃=OCH₃.

52. The process of claim 1, wherein R₂=3-[(S)-2-amino-4- methylvaleramido] -2,3,6-trideoxy-α-L-fyxø-hexopyranosyl and R₃=OCH₃.

53. The process of claim 1, wherein R₂=2-pyrrolino and R₃=OCH₃.

54. The process of claim 53, wherein _\ is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, heptanoic, octanoic, alanic, leucinic, valeric, cis-4,1, 10,13, 16,19- docosahexanoic, and ethylcarbonic acids.

55. The process of claim 54, wherein Ri is an acyl radical of a succinic acid.

56. The process of claim 1 , wherein R₂=morpholino and R₃=OCH₃.

57. The process of claim 56, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

58. The process of claim 1 , wherein R₂=3-(2-methoxy-4- moφholinyl)-2,3,6-trideoxy-α-L-/ ø-hexopyranosyl and R₃= H, OH, or OCH₃.

59. The process of claim 58, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

60. The process of claim 1 , wherein R =3-(3-cyano-4- morpholinyl)-2,3,6-trideoxy-α-L-/yj o-hexopyranosyl and R₃= H, OH, or OCH₃.

61. The process of claim 60, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succimc, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

62. The process of claim 1 , wherein R₂=3-trifluoroacetylamino- 2,3,6-trideoxy-α-L-arabino-hexopyranosyl and R₃= H, OH, or OCH₃.

63. The process of claim 1 , wherein R₂=4-trifluoroacetylamino- 6-methyl-2H-pyran-2-yl and R₃= H, OH, or OCH₃.

64. The process of claim 1 , wherein R₂=3-trifluoroacetylamino- 2,3 ,6-trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-α-L-hexopyranosyl and R₃=OCH₃.

65. The process of claim 64, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

66. The process of claim 1 , wherein R₂=3- butoxycarbonylamino-2,3,6-trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-α-L- hexopyranosyl and R₃=OCH₃.

67. The process of claim 66, wherein R_t is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

68. The process of claim 1, wherein R =3-acetylamino-2,3,6- trideoxy-4-O-(tetrahydro-2H-pyran-2-yl )-α-L-/y o-hexopyranosyl and R₃=OCH₃.

69. The process of claim 68, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

70. The process of claim 1 , wherein R₂=7-O-[4-O-(3 -amino- 2,3,6-trideoxy-α-L-mannopyranosyl)-2,6-dideoxy-α-L-galactopyranosyl] and R₃-H.

71. The process of claim 70, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1, 10, 13, 16,19-docosahexanoic, and ethylcarbonic acids.

72. The process of claim 1, wherein R₂=2,6-dideoxy-2-iodo-α- L-mannohexopyranosyl and R₃=H.

73. The process of claim 72, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cis-4,1,10,13, 16,19-docosahexanoic, and ethylcarbonic acids.

74. The process of claim 1, wherein R₂ = 3-[(S)-2-amino-4- methyl valeramido]-2,3,6-trideoxy-α-L-/j;χo-hexopyranosyl and R₃=OCH₃.

75. The process of claim 74, wherein Ri is an acyl radical of an acid selected from the group consisting of acetic, octanoic, benzoic, propionic, phenylacetic, nicotinic, formic, butyric, glycolic, glycinic, succinic, 2'-hydroxy- naphthoic, cyclopentylpropionic, 2'-pyrrolcarboxylic, carbamic, subaric, hexanoic, alanic, leucinic, valeric, cz -4,7,l 0,13, 16,19-docosahexanoic, and ethylcarbonic acids.

76. The process of claim 1 , wherein R₂=3-substituted allyloxycarbonylamino-2,3,6-trideoxy-α-L-fy o-hexopyranosyl.

77. The process of claim 76, wherein the substituted allyloxycarbonylamino substituent is an Alloc group.

78. A compound of the following formula:

wherein:

Ri is an acyl radical of a carboxylic acid selected from the group consisting of: polyethylene glycol acetic acid and polyunsaturated fatty acid; and R₂ = 2,6-dideoxy-2-fluoro-α-talopyranosyl;

3-trifluoroacetylamino-2,3,6-trideoxy-α-L-/3 o-hexopyranosyl; or

3-deamino-3-(2'-pyrroline- -yl)-2,3,6-trideoxy-α-L-/ αo-hexopyranosyl; or a pharmaceutically acceptable salt thereof.

79. The compound according to claim 78, wherein R₂ is 2,6- dideoxy-2-fluoro-α-talopyranosyl.

80. The compound according to claim 79, wherein R_\ is an acyl radical of polyethylene glycol acetic acid.

81. The compound according to claim 80, wherein the polyethylene glycol acetic acid is selected from the group consisting of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, and polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

82. The compound according to claim 81, wherein Ri is an acyl radical of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500.

83. The compound according to claim 81, wherein R_\ is an acyl radical of methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000.

84. The compound according to claim 81, wherein Ri is an acyl radical of polyethylene glycol diacetic acid which has a molecular weight of about 2,000.

85. The compound according to claim 81, wherein Ri is an acyl radical of polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

86. The compound according to claim 79, wherein Ri is an acyl radical of polyunsaturated fatty acid.

87. The compound according to claim 86, wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic, linolenic, arachidonic, dihomo-γ-linolenic, cz^'s-5,8,l l,14,17-eicosapentaenoic, and cis-4,1, 10, 13 , 16, 19-docosahexanoic acids.

88. The compound according to claim 87, wherein Ri is an acyl radical of linoleic acid.

89. The compound according to claim 87, wherein Ri is an acyl radical of linolenic acid.

90. The compound according to claim 87, wherein Ry is an acyl radical of arachidonic acid.

91. The compound according to claim 87, wherein Ri is an acyl radical of dihomo-γ-linolenic acid.

92. The compound according to claim 87, wherein Ri is an acyl radical of cts-5,8,l l,14,17-eicosapentaenoic acid.

93. The compound according to claim 87, wherein Ri is an acyl radical of cis-4,1,10,13, 16,19-docosahexanoic acid.

94. The compound according to claim 78, wherein R₂ is 3- trifluoroacetylamino-2,3 ,6-trideoxy-a-L-/jx0 hexopyranosyl.

95. The compound according to claim 94, wherein Ri is an acyl radical of polyethylene glycol acetic acid.

96. The compound according to claim 95, wherein the polyethylene glycol acetic acid is selected from the group consisting of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, and polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

97. The compound according to claim 96, wherein Ri is an acyl radical of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500.

98. The compound according to claim 96, wherein Ri is an acyl radical of methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000.

99. The compound according to claim 96, wherein Ri is an acyl radical of polyethylene glycol diacetic acid which has a molecular weight of about 2,000.

100. The compound according to claim 96, wherein Ri is an acyl radical of polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

101. The compound according to claim 94, wherein Ri is an acyl radical of polyunsaturated fatty acid.

102. The compound according to claim 101, wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic, linolenic, arachidonic, dihomo-γ-linolenic, cts-5,8,l l,14,17-eicosapentaenoic, and cis-4,1,10,13, 16,19-docosahexanoic acids.

103. The compound according to claim 102, wherein Ri is an acyl radical of linoleic acid.

104. The compound according to claim 102, wherein Ri is an acyl radical of linolenic acid.

105. The compound according to claim 102, wherein Ri is an acyl radical of arachidonic acid.

106. The compound according to claim 102, wherein R_t is an acyl radical of dihomo-γ-linolenic acid.

107. The compound according to claim 102, wherein Ri is an acyl radical of czs-5,8,ll,14,17-eicosapentaenoic acid.

108. The compound according to claim 102, wherein Ri is an acyl radical of cis-4,1,10,13, 16,19-docosahexanoic acid.

109. The compound according to claim 78, wherein R₂ is 3- deamino-3 -(2 '-pyrroline- 1 -yl)-2,3 ,6-trideoxy-α-L-/ ιxo-hexopyranosyl.

110. The compound according to claim 109, wherein Ri is an acyl radical of polyethylene glycol acetic acid.

111. The compound according to claim 110, wherein the polyethylene glycol acetic acid is selected from the group consisting of methylpolyethylene glycol acetic acid which has a molecular weight of about 2,500, methyl-polyethylene glycol acetic acid which has a molecular weight of about 20,000, polyethylene glycol diacetic acid which has a molecular weight of about 2,000, and polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

112. The compound according to claim 111, wherein Ri is an acyl radical of methyl polyethylene glycol acetic acid which has a molecular weight of about 2,500.

113. The compound according to claim 111, wherein R is an acyl radical of methyl polyethylene glycol acetic acid which has a molecular weight of about 20,000.

114. The compound according to claim 111, wherein Ri; is an acyl radical of polyethylene glycol diacetic acid which has a molecular weight of about 2,000.

115. The compound according to claim 111, wherein Ri is an acyl radical of polyethylene glycol diacetic acid which has a molecular weight of about 35,000.

116. The compound according to claim 109, wherein Ri is an acyl radical of polyunsaturated fatty acid.

117. The compound according to claim 116, wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic, linolenic, arachidonic, dihomo-γ-linolenic, cz^'s-5,8,l l,14,17-eicosapentaenoic, and cis-4,1, 10, 13 , 16, 19-docosahexanoic acids.

118. The compound according to claim 117, wherein Ri is an acyl radical of linoleic acid.

119. The compound according to claim 117, wherein Ri is an acyl radical of linolenic acid.

120. The compound according to claim 117, wherein Ri is an acyl radical of arachidonic acid.

121. The compound according to claim 117, wherein Ri is an acyl radical of dihomo-γ-linolenic acid.

122. The compound according to claim 117, wherein _\ is an acyl radical of cz^'s-S^l l_j^π-eicosapentaenoic acid.

123. The compound according to claim 117, wherein Ri is an acyl radical of cis-4,1,10,13, 16,19-docosahexanoic acid.

124. A compound of the following formula:

wherein n is 4 or 5, or a pharmaceutically acceptable salt thereof.

125. The compound according to claim 124, wherein n is 4.

126. The compound according to claim 124, wherein n is 5.

127. A compound of the following formula:

wherein:

Ri is an acyl group,

R₂ is 3-substituted allyloxycarbonylamino-2,3,6-trideoxy-α-L-/yxo- hexopyranosyl, R₃ is H, OH, or OCH₃.

128. The compound according to claim 127, wherein the substituted allyloxycarbonylamino substituent is an Alloc group.

129. The compound according to claim 128, wherein Ri is an acyl radical of an acid selected from the group consisting of formic, acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids.

130. The compound according to claim 129, wherein Ri is an acyl radical of a hexanoic or heptanoic acid.