WO2006029081A2 - Nucleoside-lipid conjugates, their method of preparation and uses thereof - Google Patents

Abstract

The invention provides methods for synthesizing nucleoside-lipid conjugates having varying fatty acid and alkyl chain lengths with or without unsaturation and their use in the treatment of cancer and viral diseases. More particularly, the invention provides methods for preparing gemcitabine-cardiolipin conjugates, and analogues thereof, cytarabine-cardiolipin conjugates, and analogues thereof. Additionally, the methods of the invention comprise administering a compound of invention as prodrug or a pharmaceutical preparation to combat mammalian diseases, preferably cancer, viral infections and bone disorders.

Description

NUCLEOSIDE-LIPID CONJUGATES, THEIR METHOD OF PREPARATION AND USES

THEREOF

CROSSREFERENCETORELATEDAPPLICTIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/405378 filed on September 2, 2004, the disclosure of which is incorporated herein.

FIELD OF THE INVENTION

[0002] This invention is related to novel nucleoside-lipid conjugates, the methods of preparing them and the compositions that contain them. The novel nucleoside-lipid conjugates comprise nucleosides and nucleoside analogues conjugated with phospholipids such as cardiolipin and cardiolipin analogues. In addition, the invention relates to the methods of preparing gemcitabine-cardiolipin conjugates, cytarabine- cardiolipin conjugates and analogues thereof. The invention also relates to the use of such nucleoside-lipid conjugates for the treatment of mammalian diseases, such as cancer and viral infections.

BACKGROUND OF THE INVENTION

[0003] Nucleoside analogues are compounds which mimic naturally occurring nucleosides by participating in nucleic acid metabolism. To date, several nucleoside analogues have been developed for the treatment of cancer and viral infections. Some examples include dideoxynucleoside analogues, such as 3'-Azido-3'deoxythymidine (AZT) (Mitsuya et al. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7096-7100), dideoxyinsosine (ddl), dideoxycytidine (ddC) (Mitsuya and Border. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 1911-1915), 2'3'-didehydro-2^>dideoxythymidine (d4T) and 3'-azido-2',3'-dideoxyuridine (AZddU) (Balzarini et al. MoI. Pharmacol. 1987, 32, 162-167). Another example is cytarabine (1-β-D- arabinofuranosylcytosine, ara-C) which is a pyrimidine nucleoside analogue used for the treatment of hematological malignancies, such as acute myelogenous leukemia and non-Hodgkin's lymphoma. Another example is fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine) which is a purine nucleoside analog used to treat low-grade lymphomas and chronic lymphocytic leukemia (Chun et al, J. Clin. Oncol., 1991, 9, 175-188). Cladribine (2-chloro-2'-deoxyadenosine, 2-CdA) is another nucleoside analogue that has been shown to be active in low-grade lymphomas and chronic lymphocytic leukemia (Damaraju et al, Oncogene, 2003, 22, 7524-7536). Another example is capecitabine (5'-deoxy-5-N-[(pentoxy)carbonyl]cytidine) which is a pyrimidine nucleoside analogue and a prodrug of 5'-fluorouracil. Capecitabine, after oral administration, is metabolized by carboxyesterases to 5'-deoxy-5-fluorocytidine which, in turn, is deaminated by cytidine deaminase to 5'deoxy-5-fluorouridine. Another example is gemcitabine (2',2'- difluorodeoxyribofuranosylcytosine) which is a difluorinated analogue of deoxycytidine currently marketed as Gemzar^®. Gemcitabine is used for the treatment of non-small cell lung and pancreatic cancer. It is a potent antitumor agent in murine and human xenograft solid tumor models (Myhren et al.TJS 6,384,019 Bl, Johnson, P. G. et al, Cancer Chromatography and Biological Response Modifiers, Annual 16, Chap. 1, ed. Vwsάo et al. 1996).

[0004] The limitations of these drugs include anticancer drug resistance, inadequate delivery of drugs to the brain and central nervous system (CNS), inadequate uptake of drugs by lymphoid and hematopoietic tissues, toxicity, oral bioavailability, short-drug half-life, and extracellular drug metabolism (Kucera et.al. US 2002/0082242 Al).

[0005] Methods, that have been employed to circumvent these problems, include the development of lipid-nucleosides conjugates, prodrugs, and liposome preparations. A series of anti-HIV nucleosides conjugates of ether (1-O-alkyl) and tbioether (1-5-alkyl) lipids, linked by a pyrophosphate diester (diphosphate) bond, has been synthesized as micelle-forming prodrugs of the nucleosides to improve their therapeutic efficacy. These include AZT-5'-diphosphate-røc-l-^octadecyl-2-0-palmitoyl-l-thioglycerol, 3'-azido-2',3'-dideoxyuridine-5'-diphosphate-rac-l-5'-octadecyl-2-0-palmitoyl-l-thioglycerol, 2',3'- dideoxycytidine-5 ' diphosphate-rac- 1 -S-octadecyl-2-ø-palmitoyl- 1 -thioglycerol and AZT 5 ' -diphosphate- rac-l-O-tetradecyl-2-O-palmitoylglycerol (Hong et alJ. Med. Chem., 1996, 39, Ylll-Ylll; Hong, et al. US Patent 5,484,911); Hong, et. al. J. Lipid Mediators Cell Signalling,(\99A), 10, 159-161. In addition, nucleoside phosphonate analogues, covalently linked to a substituted or unsubstituted alkylglycerol, alkylpropanediol, alkylethanediol such as Adefovir hexadecyloxypropyl ester, AZT-phosphonate hexadecyloxypropyl ester, l-O-hexadecyloxypropane-3-Alendronate, Cidofovir hexadecyloxypropyl, Cidofir octadecyloxypropyl, Cidofϊr octadecyloxyethyl ester etc., were developed to target viral and cancer diseases (Hostetler et α/ US Patent No: 6,716,825 B2). Furthermore, the synthesis of gemcitabine derivatives such as elaidic acid (5')-gemcitabine ester and elaidic acid (N⁴)-gemcitabine amide (Myhren et al. US 6,384,019 Bl) have improved the half-life of gemcitabine.

[0006] Most of the nucleoside-lipid conjugates exhibited improved anticancer and antiviral efficacies compared to their respective parent compounds. Among them, the most effective conjugates includes ara- CDP-rac- 1 -S-tetradecyl-2-ø-palmitoyl- 1 -thioglycerol, ara-CDP-rac- 1 -S-octadecyl-2-ø-ρalmitoyl- 1 - thioglycerol, ara-CDP-rac- 1 -S-octadecyl-2-ø-linoleoyl- 1 -thioglycerol, AZTDP-rac- 1 -S-octadecyW-O- palmitoyl-1 -thioglycerol and 3'-azido-2',3'-dideoxyuridine (AZddU) analogue (Hong, et al, J. Lipid Mediators Cell Signalling, 1994, 10, 159-161). BRIEF SUMMARY OF THE INVENTION

[0007] The invention provides methods for synthesizing nucleoside and nucleoside analogues conjugated with cardiolipin and cardiolipin analogues such as gemcitabine-cardiolipin conjugates, cytarabine-cardiolipin conjugates and analogues thereof. Additionally, the invention provides for a method of administering such compositions as a prodrug or administering a pharmaceutical composition comprising the invention as a prodrug to in order to combat mammalian diseases.

BRIEF DESCRIPTION OF THE INVENTION

[0008] Figure 1 depicts the general structure for the 5-membered cyclic sugars.

[0009] Figure 2 depicts a scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0010] Figure 3 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0011] Figure 4 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0012] Figure 5 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0013] Figure 6 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0014] Figure 7 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0015] Figure 8 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0016] Figure 9 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0017] Figure 10 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0018] Figure 11 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0019] Figure 12 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0020] Figure 13 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0021] Figure 14 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0022] Figure 15 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0023] Figure 16 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates. [0024]

[0025] Figure 17 depicts an alternate scheme for synthesizing gemcitabine-cardiolipin conjugates.

[0026] Figure 18 depicts the structure for a gemcitabine-cardiolipin conjugate.

[0027] Figure 19(a) depicts gemcitabine-cardiolipin conjugate and gemcitabine induced dose- dependent cytotoxicity in cancer cells.

[0028] Figure 19(b) depicts gemcitabine-cardiolipin conjugate and gemcitabine induced time- dependent cytotoxicity in cancer cells.

[0029] Figure 20 depicts gemcitabine-cardiolipin conjugate and gemcitabine induced caspase 3/7 expression. [0030] Figure 21 depicts the effect of dipyridamole on gemcitabine-cardiolipin conjugate-induced cytotoxicity.

[0031] Figure 22 depicts the percent survival rate after administration of gemcitabine-cardiolipin conjugates or gemcitabine.

[0032] Figure 23 depicts the effect on tumor growth after administration of gemcitabine-cardiolipin conjugates or gemcitabine.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention describes methods for synthesizing nucleoside-cardiolipin conjugates having general formula I.

Wherein Yi and Y₂ are the same or different and are -O-C(O)-, -O-, -S-, -NH-C(O)- or the like;

Ri and R₂ are the same or different and are selected from a group consisting of H, saturated alkyl group and unsaturated alkyl group;

X is selected from a group consisting of H, alkyl group ranging from Ci to Cio, and a cation (preferably a non-toxic cation such as ammonium, sodium, potassium, calcium and barium);

R₃ is selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, hypoxanthine and xanthine, wherein R₃ is optionally substituted with one, two, three or four substituents selected from a group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, triflurormethyl, and N(R^a)(R^b) wherein R^a and

R^b are independently selected from the group consisting of H and (C]-C₈) alkyl;

R_t and R₅ are the same or different and are selected from a group consisting of a halo group (H, F, Cl, Br,

I), nitro, hydroxyl, substituted alkyl, an alkyl group (C_rCi₅) and an alkoxy group (such as methoxy, ethoxy, propoxy, butoxy, and polyalkoxy); and

Re is selected from a group consisting of OH, azido group, amino group, substituted amino, alkyl group, and a halo group (H, F, Cl, Br, I). [0034] The present invention also describes methods for the preparation of nucleoside-cardiolipin conjugates having general formula II.

Il

Wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -0-, -S-, -NH-C(O)- or the like;

R₁ and R₂ are the same or different and are selected from a group consisting of hydrogen, saturated alkyl group and unsaturated alkyl group;

R₃ and R₇ are the same or different and are selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, hypoxanthine and xanthine, wherein R₃ and/or R₇ are optionally substituted with one, two, three or four substituents selected from a group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, triflurormethyl, and N(R^a)(R^b) wherein R^a and R^b are independently selected from the group consisting of H and (C₁-C₈) alkyl;

R) and R₅ are the same or different and are selected from a group consisting of a halo group (H, F, Cl, Br,

I), nitro, hydroxyl, substituted alkyl, alkyl group (C₁-C_1S), alkoxy group (such as methoxy, ethoxy, propoxy, butoxy, polyalkoxy group); and

R₆ is selected from a group consisting of OH, azido group, amino group, substituted amino, alkyl group, and a halo group (H, F, Cl, Br, I).

[0035] The present invention also describes methods for the preparation of nucleoside-cardiolipin conjugates having general formula III.

III

Wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -O-, -S-, -NH-C(O)- or the like;

Ri and R₂ are the same or different and are selected from a group consisting of H, saturated and unsaturated alkyl group;

X is selected from a group consisting of H, alkyl group ranging from Ci to C₁₀, and a cation (preferably a non-toxic cation such as ammonium, sodium, potassium, calcium and barium);

R₃ is selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, hypoxanthine and xanthine, wherein R₃ is optionally substituted with one, two, three or four substituents selected from a group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, rrifluoromethyl, and N(R^a)(R^b) wherein R^a and

R are independently selected from a group consisting of H and (CpCs) alkyl groups;

R₄ and R₅ are the same or different and are selected from a group consisting of a halo group (H, F, Cl, Br,

I), nitro, hydroxyl, substituted alkyl, alkyl group (C_rCi₅), alkoxy group (such as methoxy, ethoxy, propoxy, butoxy and polyalkoxy).

[0036] The present invention also describes methods for the preparation of nucleoside-cardiolipin conjugates having general formula IV.

IV

Wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -0-, -S-, -NH-C(O)- or the like; Ri and R₂ are the same or different and are selected from a group consisting of H, saturated alkyl group and unsaturated alkyl group;

R₃ and R₇ are the same or different and are selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, hypoxanthine and xanthine, wherein R₃ and/or R₇ is optionally substituted with one, two, three or four substituents selected from a group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, trifluoromethyl, and N(R^a)(R^b) wherein R^a and R^b are independently selected from a group consisting of H and (C₁-C₈) alkyl; and

R₄ and R₅ are the same or different and are selected from a group consisting of a halo group (H, F, Cl, Br,

I), nitro, hydroxyl, substituted alkyl, alkyl group (C_rC[₅), alkoxy group (such as methoxy, ethoxy, propoxy, butoxy and polyalkoxy).

[0037] The term "linker" is defined herein as a group or chain containing one or more functional group for covalent binding with the lipid carrier and biologically active nucleoside. Preferred embodiments comprise a linker having at least two functional groups, wherein the linker has a first end and a second end and wherein the lipid is attached to the first end of the linker through a first linker functional group and the nucleoside is attached the second end of the linker through a second linker functional group. These groups can be designated either as weak or strong, based on the stability of the covalent bond which the linker functional group will form between the linker and either the lipid carrier or the biologically-active nucleoside. The weak functionalities include, but are not limited to, phosphoramidite, phosphoesters (such as phosphodiester, phosphotriester and phosphonate), carbonate, amide, carboxyl-phosphoryl anhydride, ester and thioester. The strong functionalities include, but are not limited to, ether, thioether, amine, amide, and ester. The use of a strong linker functional group between the linker and the nucleoside will tend to decrease the rate at which the compound will be released in vivo, whereas the use of a weak linker functional group between the linker and the nucleoside may act to facilitate in vivo release of the compound. In preferred embodiments, each of the first and second functional linker groups is a hydroxyl group, a primary or secondary amino group, phosphate group or substituted derivative thereof, a carboxylic acid, carbonate, carbamate or carbonyl group. The "linker" herein also comprises, in addition to the functional groups at either end, (CH₂)_n groups (where n = 0-20) in the center optionally substituted with functional groups, such as alkyl, alkoxy, hydroxyl, carbonyl, carboxyl, carbamate, aldehyde, amino, halo, polyalkoxy, PEG groups, phosphate, phosphonate and pyrophosphate groups.

[0038] The term "lipid" herein includes cardiolipin and cardiolipin derivatives or analogues having varying fatty acid and or alkyl chain with or without unsaturation.

[0039] The term "prodrug" is defined as a pharmacologically inactive compound that is converted into an active agent by metabolic transformation. The objective is to chemically modify the antitumor agent (into its prodrug) in order to render it temporarily inactive. In vivo, via the action of enzyme(s), this prodrug then decomposes thereby liberating the active principle. In most cases, a judiciously selected chemical group is bound covalently to the active principle. This group will often govern the solubility of the prodrug, its stability, the rate at which it liberates the active principle and the particular enzyme(s) required for its transformation (Malet-Martino et al. Curr. Med. Chem. -Anti-Cancer Agents, 2002, 2, 267- 310)

[0040] The term "therapeutic agent" herein means any compound or composition, which upon entering a mammalian cell, is capable of contributing to the alleviation or treatment of a mammalian disease. The mammalian cell can be any type of mammalian cell, including both cancerous and non¬ cancerous cells. The cancer can be any type of cancer in a mammal. By way of example and not by limitation, "therapeutic agents" include small organic molecules, peptides, nucleoside analogues, anticancer agents, antiviral agents, ribozymes, protease inhibitors, polymerase inhibitors, reverse transcriptase inhibitors, antisense oligonucleotides and other drugs. As used herein, the term "anticancer agent" means a therapeutic agent capable of exhibiting efficacy at combating a cancer in a mammal or in a mammalian cell, or any compound which is capable of being converted intracellularly to a compound which is capable of exhibiting efficacy at combating a cancer in a mammal or in a mammalian cell. As used herein, the term, "combating a cancer" means any one or more of the following: to increase survival of a mammal, to arrest or decrease tumor size in a mammal, or to increase the time period of remission of cancer regrowth in a mammal.

[0041] The term "alkyl" encompasses saturated or unsaturated straight chain and branched-chain hydrocarbon moieties. The term "substituted alkyl" comprises alkyl groups further bearing one or more substituents selected from hydroxyl, alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group), cycloalkyl, substituted cycloalkyl, halogen, cyano, nitro, amino, amido, irnino, thio, -C(O)H, acyl, oxyacyl, carboxyl and the like.

[0042] In a preferred embodiment, for formulae I-IV, the fϊve-membered cyclic sugar is ribofuranose, arabinofuranose, deoxyribofuranose or xylofuranose. The nomenclature is based on the specific orientation or absence of the hydroxyl groups at C2'and C3' position and the attachment of the heterocylic base at Cl '(Figure 1).

[0043] It must be noted that the nucleoside moiety, in the formulae above (I-IV), does not indicate the stereochemistry of the compounds of the present invention, and the stereochemistry is not a critical aspect of the invention. Accordingly, it will be understood that the present invention refers to a nucleoside- lipid conjugate of all possible stereochemical orientations, while recognizing that certain stereochemical species will be found to be more effective than other orientations. For example, it has been found that nucleosides with β configuration (attachment of base to the 5-membered cyclic sugar at Cl') are more efficacious than the α-nucleosides. The β-nucleosides are therefore preferred compounds for making the present nucleoside-lipid conjugates.

[0044] In a preferred embodiment, for formulae I-IV, Ri and R₂ are the same or different and are selected from a group comprising C₁-C₃₄ saturated and/or unsaturated alkyl group, more preferably between Ci and C₂₄ carbon atoms.

[0045] In a preferred embodiment, R₃ is cytosine, guanine, adenine, thymine, uracil, inosine, hypoxanthine, xanthine, wherein R₃ is optionally substituted with one, two, three or four substituents selected from a group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, trifluoromethyl, and N(R^a)(R ), wherein R^a and R are independently selected from a group consisting of H and (Ci-Cg) alkyl.

[0046] In a preferred embodiment, R₄ and R₅ are the same or different and are selected from a group consisting of a halo (H, F, Cl, Br, I), nitro, hydroxyl, alkyl, substituted alkyl, and alkoxy group (such as methoxy, ethoxy, propoxy, butoxy group).

[0047] In a preferred embodiment, R₆ is preferably a hydroxyl group, azido, amino, substituted amino, or halo (H, F, Cl, Br, I) group.

[0048] In a preferred embodiment, the linker comprises an alkyl, substituted alkyl, dicarbonyl alkyl (for example, succinimidoyl group), alkoxy, polyalkoxy, PEGylated (PEG) group, phosphate, phosphonate, diphosphate, triphosphate, phosphodiester, phosphotriester, phosphoramidite, a peptide, dipeptide, polypeptide and the like.

[0049] In a more preferred embodiment, the compound, according to general formula I, is a gemcitabine-cardiolipin conjugate having structures V and VI, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, Rg is hydroxyl group, X is methyl or ammonium, Y₁, Y₂ are oxo (-O-) groups, the linker is succinimidoyl group and

©

Vl, X = NH4

Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

[0050] In a more preferred embodiment, the compound, according to general formula I, is a gemcitabine-cardiolipin conjugate having structures VII and VIII, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, R₆ is hydroxyl group, X is methyl or ammonium, Yi, Y₂ are -O-C(O)-, the linker is a succinimidoyl group, and

© VIII, X = NH4

Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

[0051] In a more preferred embodiment, the compound, according to general formula I,is a gemcitabine-cardiolipin conjugate having structures IX and X, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, Rβ is hydroxyl group, X is methyl or ammonium, Yi, Y₂ are oxo (-0-) groups, the linker is a succinimidoyl group, and

©

X, X = NH4

Ri is H, CpC₃₄ saturated or unsaturated alkyl groups, and R₂ is a methyl group.

[0052] In a more preferred embodiment, the compound, according to general formula III, is a gemcitabine-cardiolipin conjugate having structures XI and Xπ, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, X is methyl or ammonium, Yj, Y₂ are oxo (-0-) groups, the linker is succinimidoyl group, and

XII_I X = NH4

Ri and R₂ are the same or different and are H, C₁-Ca₄ saturated or unsaturated alkyl groups.

[0053] In a more preferred embodiment, the compound, according to general formula HI, is a gemcitabine-cardiolipin conjugate having structures Xπi and XIV, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, X is methyl or ammonium, Y₁, Y₂ are -0-(CO)- groups, the linker is a succinimidoyl group, and

© X1V, X = NH4

Ri and R₂ are the same or different and are H, CpC₃₄ saturated or unsaturated alkyl groups.

[0054] In a more preferred embodiment, the compound, according to general formula III, is a gemcitabine-cardiolipin conjugate having structures XV and XVI, wherein R₃ is cytosine, R₄ and R₅ are fiuoro groups, X is methyl or ammonium, Y_b Y₂ are oxo (-O-) groups, the linker is succinimidoyl group,

Ri is H, Ci-C₃₄ saturated or unsaturated alkyl groups and R₂ is a methyl group.

[0055] In a more preferred embodiment, the compound, according to general formula I, is a gemcitabine-cardiolipin conjugate having structures XVII and XVIII, wherein R₃ is cytosine, R₄ and R₅ are fiuoro groups, R₆ is hydroxyl group, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-O-) groups, and the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl) and

©

XVIII, X = NH4

R[ and R₂ are the same or different and are H, Ci-C₃₄ saturated or unsaturated alkyl groups.

[0056] In a more preferred embodiment, the compound, according to general formula I, is a gemcitabine-cardiolipin conjugate having structures XIX and XX, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, R₆ is hydroxyl group, X and R₈ are methyl or ammonium, Y_b Y₂ are -0-(CO)- groups, and the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl), and

© XX, X = NH4

Ri and R₂ are the same or different and are H, Q-C₃₄ saturated or unsaturated alkyl groups.

[0057] In a more preferred embodiment, the compound, according to general formula I, is a gemcitabine-cardiolipin conjugate having structures XXI and XXII, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, R₆ is hydroxyl group, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-0-) groups, the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl),

Ri is H, Ci-C₃₄ saturated or unsaturated alkyl groups, and R₂ is methyl group.

[0058] In a more preferred embodiment, the compound, according to general formula III, is a gemcitabine-cardiolipin conjugate having structures XXIII and XXIV, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, X and R₈ are methyl or ammonium, Yi, Y₂ are oxo (-0-) groups, the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl),

© XXIV, X = NH4

and Ri and R₂ are the same or different and are H, Q-C₃₄ saturated or unsaturated alkyl groups.

[0059] In a more preferred embodiment, the compound, according to general formula III, is a gemcitabine-cardiolipin conjugate having structures XXV and XXVI, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, X and R₈ are methyl or ammonium, Yi, Y₂ are -0-(CO)- groups, and the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl),

©

XXVI, X = NH4

Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

[0060] In a more preferred embodiment, the compound, according to general formula III, is a gemcitabine-cardiolipin conjugate having structures XXVII and XXVIII, wherein R₃ is cytosine, R₄ and R₅ are fiuoro groups, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-O-) groups, the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl),

©

XXVIII, X = NH4

Ri is H, C₁-C₃₄ is saturated or unsaturated alkyl groups, and R₂ is a methyl group.

[0061] In a more preferred embodiment, the compound, according to general formula II, is a gemcitabine-cardiolipin conjugate having structure XXIX, wherein R₃ and R₇ are cytosine, R₄ and R₅ are fiuoro groups, R₆ is hydroxyl, Y₁, Y₂ are oxo (-O-) or -OC(O)- groups, the linker is - CH₂CH₂CH₂NHCOCH₂CH₂C(0)-group, and

XXIX

Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups

[0062] In a more preferred embodiment, the compound, according to general formula IV, is a gemcitabine-cardiolipin conjugate having structure XXX, wherein R₃ and R₇ are cytosine, R₄ and R₅ are fluoro groups, Y₁, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is -CH₂CH₂CH₂NHCOCH₂CH₂C(O)- group,

XXX

Ri and R₂ are the same or different and are H, Q-C₃₄ saturated or unsaturated alkyl groups [0063] In a more preferred embodiment, the compound, according to general formula I, is a cytarabine-cardiolipin conjugate having structures XXXI and XXXII, wherein R₃ is cytosine, R₄ and Re are hydroxyl, R₅ is hydrogen, X is methyl or ammonium, Y_b Y₂ are oxo (-O-) or -0-C(O)- groups, and the linker is succinimidoyl group,

©

XXXII, X = NH4

R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

[0064] In a more preferred embodiment, the compound, according to general formula III, is cytarabine-cardiolipin conjugate having structures XXXIII and XXXIV, wherein R₃ is cytosine, R₄ is hydroxyl, R₅ is hydrogen, X is methyl or ammonium, Yi, Y₂ are oxo (-O-) or -O-C(O)- groups, the linker is succinimidoyl group, and

©

XXXIV, X = NH4

Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups. [0065] In a more preferred embodiment, the compound, according to general formula I, is a cytarabine-cardiolipin conjugate having structures XXXV and XXXVI, wherein R₃ is cytosine, R₄ and Rg are hydroxyl, R₅ is hydrogen, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl), and R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

© XXXVI, X = NH4

[0066] In a more preferred embodiment, the compound, according to general formula III, is a cytarabine-cardiolipin conjugate having structures XXXVII and XXXVIII, wherein R₃ is cytosine, R₄ is hydroxy^ and R₅ is hydrogen, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is a phosphodiester (R₈ is ammonium) or phosphotriester (R₈ is a methyl), and

₃

©

XXXVIII, X = NH4

R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

[0067] In a more preferred embodiment, the compound, according to general formula II, is a cytarabine-cardiolipin conjugate having structure XXXIX, wherein R₃ and R₇ are cytosine, R₄ and R₆ are hydroxyl, R₅ is hydrogen, Y₁, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is - CH₂CH₂CH₂NHCOCH₂CH₂C(O)-group,

XXXIX

and Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

[0068] In a more preferred embodiment, the compound, according to general formula IV, is a gemcitabine-cardiolipin conjugate having structure XXXX, wherein R₃ and R₇ are cytosine, R₄ and R₆ are hydroxyl, R₅ is hydrogen, Y₁, Y₂ are oxo (-0-) or -0-C(O)- groups, the linker is - CH₂CH₂CH₂NHCOCH₂CH₂C(0)-group, and

XXXX

R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups. [0069] Nucleoside-lipid conjugates can be prepared by any desired method. One preferred method of the present invention is set forth in Figure 2, which depicts a general approach to the synthesis of a gemcitabine-cardiolipin conjugate. In this method, a cardiolipin derivative 6 is synthesized by reacting 1,2- disubsituted glycerol 1 with ^Λζ-diisopropylmethylphosphonamidic chloride 2 in the presence of a base (for example, N, N, diisopropylethylamine (DIPEA) and the like) and subsequently with 2-substituted glycerol, (for example, 2-benzylglycerol and the like) in the presence of lH-tetrazole, followed by oxidation with an mild oxidizing agent such as m-chloroperoxybenzoic acid (mCPBA) or hydrogen peroxide (H₂O₂) (see Ahmad et al PCT/US2003/16412). Intermediate 6 is then reacted with succinic anhydride in an inert solvent (for example, 1,2-dichloroethane and the like) in the presence of a base (for example, rriethylamine and the like) to provide intermediate 7. Intermediate 7 is then reacted with 4-N-3 '- O-Bis(tert-butoxycarbonyl)gemcitabine 8 (Guo and Gallo, J. Org. Chem., 1999, 64, 8319-8322) in the presence of dicyclohexylcarbodimide (DCC), NTV-dimethyaminopyridine (DMAP) and an inert solvent (such as dichloromethane and the like). The protecting groups are removed in an acidic medium (such as trifluoroacetic acid (TFA) and the like) in dichloromethane to provide gemcitabine-cardiolipin-conjugate 10.

[0070] Figure 3 describes another embodiment for synthesizing gemcitabine-cardiolipin conjugates as diammonium salts in which 1,2-disubstituted glycerol 1 is reacted with a phosphoramidite reagent of the general formula 11 (X = phosphate protecting group, preferably a benzyl, 2-cyanoethyl or methyl) in the presence of lH-tetrazole in dichloromethane and subsequently with 2-substituted glycerol 12 followed by oxidation with mCPBA to give intermediate 14. The protecting group can be removed by any suitable agent depending the nature of protecting group (for example, tert-butyldimethylsilyl (TBDMS) group can be removed with an acid, benzyl group by hydrogenation) to give intermediate 15. Intermediate 15, on reacting with succinic anhydride and later with 4-N-3'-O-Bis(tert-butoxycarbonyl)gemcitabine 8, gives 17. The protecting groups (tert-butoxy carbonyl, t-BOC) are removed with trifluoroacetic acid in dichloromethane. The phosphate protecting groups can be removed with any suitable reagent depending on the nature of group (for example, a benzyl group can be removed by catalytic hydrogenation, and a methyl group can be removed by heating with sodium iodide in the presence of 2-butanone or acetone). The removal of protecting groups is followed by a treatment with dilute ammonium hydroxide to furnish gemcitabine-cardiolipin conjugate 18 as a diammonium salt.

[0071] Another embodiment for synthesizing gemcitabine-cardiolipin conjugates as diammonium salts is depicted in Figure 4. In this method, an optically pure 1,2-disubstituted glycerol 1 can be phosphorylated using phosphoramidite reagent 19 to yield phosphate triesters which are then reacted with any suitable 2-0-protected glycerol, such as, for example, 2-(fert-butyldimethylsily)glycerol 20 or 2- levulinoyl-l,3-ρropane diol, using pyridinium perbromide and phosphonium salt methodology, to get cardiolipin 22 (see Ahmad et al PCT/US2003/027806). Reacting 22 with succinic anhydride followed by coupling with 4-N-3'-C-Bis(tert-butoxycarbonyl)gemcitabine 8 gives 24. Removal of the tert- butoxycarbonyl group can be done using an acid catalyst (such as TFA or the like) and the benzyl groups can be removed by catalytic hydrogenation. This is followed by treatment with dilute ammonium hydroxide.

[0072] Another embodiment for synthesizing gemcitabine-cardiolipin conjugates as diammonium salts is depicted in Figure 5. This embodiment involves reaction of cardiolipin intermediate 7 with 4-N-5'- <9-Bis(tert-butoxycarbonyl)gemcitabine 26 in the presence of DCC and DMAP in an inert solvent (for example, dichloromethane and the like) followed by deprotection using TFA to give gemcitabine- cardiolipin conjugate 28.

[0073] Another embodiment for synthesizing gemcitabine-cardiolipin conjugates as diammonium salts is depicted in Figure 6. Cardiolipin derivative 16, on reacting with 4-7V-5'-O-Bis(tert- butoxycarbonyi)gemcitabine 26, gives 29. The protecting groups (te?t-butoxycarbonyl) are removed with trifluoroacetic acid in dichloromethane. The protecting groups on the phosphates can be removed with any suitable reagent depending on the nature of group (for example, a benzyl group can be removed by catalytic hydrogenation, and a methyl group can be removed by heating with sodium iodide in the presence of 2- butanone or acetone). This removal is followed by a treatment with dilute ammonium hydroxide to furnish gemcitabine-cardiolipin conjugate 30 as a diammonium salt.

[0074] Another embodiment for synthesizing gemcitabine-cardiolipin conjugates as diammonium salts 33 is depicted in Figure 7. In this method, coupling cardiolipin with 4-N-5'-O-bis(tert- butoxycarbonyl)gemcitabine 26 gives 31. Removal of tert-butoxycarbonyl group can be done using an acid catalyst (such as TFA or the like) and the benzyl groups can be removed by catalytic hydrogenation. This is followed by treatment with dilute ammonium hydroxide.

[0075] Another embodiment of the present invention is depicted in Figure 8. In this method, cardiolipin analogue 6 is reacted with Λζ7V,-diisopropylmethylρhosphonamidic chloride 2 in the presence of a base (for example, DIPEA or the like) in an inert solvent (for example dichloromethane or the like) and then reacted with 4-N-3'-O-bis(tert-butoxycarbonyl)gemcitabine 8 to give fully protected gemcitabine- cardiolipin conjugate 34. Removal of the t-BOC groups with a suitable acid (like TFA or the like) affords gemcitabine-cardiolipin conjugate 35.

[0076] Another embodiment of the present invention is set forth in Figure 9. In this method, cardiolipin analogue 6 is reacted with benzyl tetraisopropylphosphoramidite 36 in the presence of IH- tetrazole and subsequently with 4-N-3'-O-bis(tert-butoxycarbonyl)gemcitabine followed by oxidation with a suitable oxidizing agent (for example, mCPBA or the like). Removal of t-BOC groups with TFA and benzyl groups by catalytic hydrogenation, followed by subsequent treatment with dilute ammonium hydroxide, provides conjugate 39 as an ammonium salt.

[0077] Another embodiment of the present invention is outlined in Figure 10, which illustrates the synthesis of a gemicitabine-cardiolipin conjugate as a diammnoium salt. In this method, cardiolipin analogue 22 is reacted with ΛζN, -diisopropylmethylphosphonamidic chloride 2 in the presence of a base (for example, DIPEA or the like) in an inert solvent (for example, dichloromethane or the like) and later with 4-N-3'-O-bis(tert-butoxycarbonyl)gemcitabine 8 in presence of lH-tetrazole followed by oxidation with a suitable oxidizing agent (for example, mCPBA or the like) to give protected gemcitabine-cardiolipin conjugate 40. Removal of t-BOC groups with TFA, and removal of the benzyl groups by catalytic hydrogenation followed by treatment with dilute ammonium hydroxide gives conjugate 42 as a diammonium salt.

[0078] Another embodiment of the present invention, represented in Figure 11, leads to the synthesis of a gemcitabine-cardiolipin conjugate as a triammonium salt, wherein cardiolipin intermediate 22 is first reacted with benzyl tetraisopropyl phosphoramidite 36 in the presence of lH-tetrazole and then subsequently reacted with 4-N-3'-0-bis(fert-butoxycarbonyl)gemcitabine 8 to give protected conjugate 43. Removal of t-BOC groups with TFA and benzyl groups by catalytic hydrogenation followed by treatment with dilute ammonium hydroxide provides gemcitabine-cardiolipin conjugate 45 as a triammoniun salt.

[0079] Another embodiment of the present invention is represented in Figure 12. In this method, cardiolipin analogue 6 is first reacted with ΛζΛζ-diisopropylmethylphosphonamidic chloride 2 in the presence of a base (for example, DIPEA or the like) in an inert solvent (for example dichloromethane or the like) and then reacted with 4-N-5'-0-bis(tert-butoxycarbonyl)gemcitabine 26 to give fully protected gemcitabine-cardiolipin conjugate 46. Removal of the t-BOC groups with a suitable acid (like TFA or the like) affords gemcitabine-cardiolipin conjugate 47.

[0080] Another embodiment of the present invention is set forth in Figure 13, which depicts an approach for synthesizing a gemcitabine-cardiolipin conjugate as an ammonium salt. In this method, cardiolipin analogue 6 is reacted with benzyl tetraisopropylphosphoramidite 36 in the presence of IH- tetrazole and subsequently with 4-N-5'-O-bis(fert-butoxycarbonyl)gemcitabine 26 followed by oxidation with suitable oxidizing agent (for example, mCPBA or the like). Removal of t-BOC groups with TFA and benzyl groups by catalytic hydrogenation, followed by subsequent treatment with dilute ammonium hydroxide, provides conjugate 50 as an ammonium salt. [0081] Another embodiment of the present invention is outlined in Figure 14, which illustrates the synthesis of gemicitabine-cardiolipin conjugate as a diammnoium salt. In this method, cardiolipin analogue 22 is reacted with N, 7V,-diisoρropylmethylρhosρhonamidic chloride 2 in the presence of a base (for example, DIPEA or the like) in an inert solvent (for example, dichloromethane or the like) and later with 4- N-5'-0-bis(tert-butoxycarbonyi)gemcitabine 26 in the presence of lH-tetrazole followed by oxidation with a suitable oxidizing agent (for example, mCPBA or the like) to give protected gemcitabine-cardiolipin conjugate 51. Removal of the t-BOC groups with TFA furnishes 52. The benzyl groups on phosphates can be removed by catalytic hydrogenation. Treatment with dilute ammonium hydroxide gives conjugate 53 as a diammonium salt.

[0082] Another embodiment of the present invention is represented in Figure 15. Cardiolipin intermediate 22 is first reacted with benzyl tetraisopropylphosphoramidite 36 in the presence of IH- tetrazole and then subsequently reacted with 4-//-5'-O-bis(tert-butoxycarbonyl)gemcitabine 26 to give protected conjugate 54. Removal of the t-BOC groups with TFA and benzyl groups by catalytic hydrogenation followed by treatment with dilute ammonium hydroxide provides gemcitabine-cardiolipin conjugate 56 as a triammoniun salt.

[0083] Another embodiment of the present invention is represented in Figure 16. In this scheme, optically pure 1,2-disubstituted glycerol 1 is reacted with 2-cyanoethyl-ΛζN- diisopropylchlorophosphoramidite 57, in the presence of a base (for example, DIPEA or the like) in an inert solvent (for example, dichloromethane or the like), and subsequently reacted with 2-O-protected glycerol (for example, 2-O-ter-butyldimethylsilyl glycerol 20) in the presence of lH-tetrazole followed by oxidation with mCPBA which provides cardiolipin derivative 59. Conversion of cyano to amine functionalities can be achieved by hydrogenation using a suitable catalyst (for example, Pd/C or Raney Ni). The resulting amine on reaction with succinic anhydride furnishes 61. Intermediate 61 on coupling with 4-JV-3'-O- bis(tert-butoxycarbonyl)gemcitabine 8, followed by deprotection in an acidic condition (for example, TFA or the like), provides gemcitabine-cardiolipin conjugate 63.

[0084] Another embodiment of the present invention is shown in Figure 17, in which cardiolipin derivative 61 is first reacted with 4-Λ^r-5'-0-bis(fe/t-butoxycarbonyl)gemcitabine 26 and then deprotected with TFA. This reaction results in gemcitabine-cardiolipin conjugate 65.

[0085] The synthetic methods described herein can be modified in any suitable manner. For example, the protection of gemcitabine is not limited to t-BOC groups but also includes benzyloxycarbonyl (Cbz), trityl benzyloxymethyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, acetyl, pivaloyl, levulinoyl and the like. The deprotection can be acheived by a method depending on the protecting group. For example, removal of t-BOC groups is not limited to TFA but also includes HCl and the like. A benzyl group can be removed by catalytic hydrogenolysis or by treatment with sodium iodide, and a silyl group can be deprotected with fluoride or an acidic medium. In addition, the protecting group of a cardiolipin derivative is not limited to only benzyl and methyl but also includes phosphate, 2-cyanoethyl, ethyl, ter- butyldimethylsilyl, levulinoyl, pivaloyl, methoxymethyl, benzyloxymethyl and the like.

100861 ^^e described methods can be used to prepare a variety of novel nucleoside-cardiolipin conjugates. For example, the methods can be used to prepare a nucleoside-cardiolipin conjugate in a pure form containing any fatty acid chains. Preferred fatty acids range from carbon chain lengths of about Qto C_34, preferably between about C₄ and about C₂₄, and include tetranoic acid (C_{4 0}), pentanoic acid (C₅ o)_; hexanoic acid (Cg o)_> heptanoic acid (C_{7 0}), octanoic acid (Cg ₀), nonanoic acid (C_{9 0}), decanoic acid (Cm 0), undecanoic acid (Cn ₀), dodecanoic acid (C_{12 0}), tridecanoic acid (C_{13 0}), tetradecanoic (myristic) acid (CM ₀), pentadecanoic acid (Ci_{5 0}), hexadecanoic (palmatic) acid (Ci₆.o), heptadecanoic acid (Ci₇₀), octadecanoic (stearic) acid (Ci_{8 0}), nonadecanoic acid (Ci_9:0), eicosanoic (arachidic) acid (C₂o ₀). heneicosanoic acid (C₂₁ o)_> docosanoic (behenic) acid (C_{22 O})_J tricosanoic acid (C_{23 0}), tetracosanoic acid (C₂₄o)) 10-undecenoic acid (Cu ₁), 11-dodecenoic acid (Ci_{2 1}), 12-tridecenoic acid (C_{13 1}), myristoleic acid (CM 0, 10-pentadecenoic acid (C_{15 1}), palmitoleic acid (C_{16 1}), oleic acid (Qg ₁), linoleic acid (Qs ₂)₁ linolenic acid (Qs ₃), eicosenoic acid (C_2O i), eicosdienoic acid (C_{2O 2})_J eicosatrienoic acid (C₂₀.₃), arachidonic acid (cw-5,8,l l,14-eicosatetraenoic acid), and cz^'s-5,8,ll,14,17-eicosapentaenoic acid, among others. For ether analogs, the alkyl chain will also range from Ci to C₃₄ preferably between about C₄ and about C₂₄. Other fatty acid chains also can be employed as Ri and/or R₂ substituents. Examples of such include saturated fatty acids such as ethanoic (or acetic) acid, propanoic (or propionic) acid, butanoic (or butyric) acid, hexacosanoic (or cerotic) acid, octacosanoic (or montanic) acid, triacontanoic (or melissic) acid, dotriacontanoic (or lacceroic) acid, tetratriacontanoic (or gheddic) acid, pentatriacontanoic (or ceroplastic) acid, and the like; monoethenoic unsaturated fatty acids such as irans-2-butenoic (or crotonic) acid, cM-2-butenoic (or isocrotonoic) acid, 2-hexenoic (or isohydrosorbic) acid, 4-decanoic (or obtusilic) acid, 9-decanoic (or caproleic) acid, 4-dodecenoic ( or linderic) acid, 5-dodecenoic (or denticetic) acid, 9- dodecenoic (or lauroleic) acid, 4-tetradecenoic (or tsuzuic) acid, 5-tetradecenoic (or physeteric) acid, 6- octadecenoic (or petroselenic) acid,

( or elaidic) acid, fraω-l l-octadecenoic ( or vaccinic) acid, 9-eicosenoic ( or gadoleic) acid, 11-eicosenoic ( or gondoic) acid, 11-docosenoic ( or cetoleic) acid, 13-decosenoic ( or erucic) acid, 15-tetracosenoic ( or nervonic) acid, 17-hexacosenoic ( or ximenic) acid, 21-triacontenoic ( or lumequeic) acid, and the like; dienoic unsaturated fatty acids such as 2,4-pentadienoic (or β-vinylacrylic) acid, 2,4-hexadienoic (or sorbic) acid, 2,4-decadienoic (or stillingic) acid, 2,4-dodecadienoic acid, 9,12-hexadecadienoic acid, cis-9, cw-12-octadecadienoic (or α-linoleic) acid, trans-9, brans'- 12-octadecadienoic (or linlolelaidic) acid, trans-lO,trans-l2-octadecaάienoic acid, 11,14- eicosadienoic acid, 13,16-docosadienoic acid, 17,20-hexacosadienoic acid and the like; trienoic unsaturated fatty acids such as 6,10,14-hexadecatrienoic (or hiragonic) acid, 7,10,13-hexadecatrienoic acid, cis-6, cis-9- ds-12-octadecatrienoic (or γ-linoleic) acid, trans-S, trans-10- fr-α«.ϊ-12-octadecatrienoic (or β-calendic) acid, cis-S, trωιs-10- cw-12-octadecatrienoic acid, cis-9, cis-12- m-15-octadecatrienoic (or α-linolenic) acid, trans-9, trans-Yl- frcrø-15-octadecatrienoic (or α-linolenelaidic) acid, cis-9, trans-11- trans-13- octadecatrienoic (or α-eleostearic) acid, trans-9, trans-ll- i7"αrø-13-octadecatrienoic (or β-eleostearic) acid, cis-9, trans-11- cw-13-octadecatrienoic (or punicic) acid, 5,8,11-eicosatrienoic acid, 8,11,14- eicosatrienoic acid and the like; tetraenoic unsaturated fatty acids such as 4,8,11,14-hexadecatetraenoic acid, 6,9,12,15- hexadecatetraenoic acid, 4,8,12,15-octadecatetraenoic (or moroctic) acid, 6,9,12,15- octadecatetraenoic acid, 9,11,13,15- octadecatetraenoic ( or α -or β-parinaric) acid, 9,12,15,18- octadecatetraenoic acid, 4,8,12,16-eicosatetraenoic acid, 6,10,14,18-eicosatetraenoic acid, 4,7,10,13- docasatetraenoic acid, 7,10,13,16-docosatetraenoic acid, 8,12,16,19-docosatetraenoic acid and the like; penta- and hexa-enoic unsaturated fatty acids such as 4,8,12,15,18-eicosapentaenoic (or timnodonic) acid, 4,7,10,13,16-docosapentaenoic acid, 4,8,12,15, 19-docosapentaenoic (or clupanodonic) acid, 7,10,13,16,19- docosapentaenoic, 4,7,10, 13,16,19-docosahexaenoic acid, 4,8,12,15,18,21-tetracosahexaenoic (or nisinic) acid and the like; branched-chain fatty acids such as 3-methylbutanoic (or isovaleric) acid, 8- methyldodecanoic acid, 10-methylundecanoic (or isolauric) acid, 11-methyldodecanoic (or isoundecylic) acid, 12-methyltridecanoic (or isomyristic) acid, 13-methyltetradecanoic (or isopentadecylic) acid, 14- methylpentadecanoic (or isopalmitic) acid, 15-methylhexadecanoic, 10-methylheptadecanoic acid, 16- methylheptadecanoic (or isostearic) acid, 18-methylnonadecanoic (or isoarachidic) acid, 20- methylheneicosanoic (or isobehenic) acid, 22-methyltricosanoic (or isolignoceric) acid, 24- methylpentacosanoic (or isocerotic) acid, 26-methylheptacosanoic (or isomonatonic) acid, 2,4,6- trimethyloctacosanoic (or mycoceranic or mycoserosic) acid, 2-methyl-cώ-2-butenoic(angelic)acid, 2- methyl-/ra«5-2-butenoic (or tiglic) acid, 4-methyl-3-ρentenoic (or pyroterebic) acid and the like. For ether analogues, the alkyl chain also will range from carbon chain lengths of Ci to C₃₄.

F00871 ^^e described methods can be used to prepare a variety of novel nucleoside-cardiolipin conjugates in a pure form containing any alkyl or acyl chain ranging from C_rC₃₄ chain lengths. Preferred alkyl chains range from carbon chain lengths of about C₁TO C₂₄. The term "alkyl" encompasses saturated or unsaturated straight-chain and branched chain hydrocarbon moieties. The term "substituted alkyl" or substituted "alkoxy" and the like includes alkyl or alkoxy groups further bearing one or more substitutent selected from hydroxyl, alkoxy (of lower alkyl group), halogen, cyano, and the like.

T00881 Nucleoside-lipid conjugates which are compatible with the present invention include nucleosides which act on the peripheral nerves, adrenergic receptors, cholinergic receptors, the skeletal muscles, the cardiovascular system, smooth muscles, the blood circulatory system, synaptic sites, neuroeffector junctional sites, endocrine and hormone systems, the immunological system, the reproductive system, the skeletal system, the alimentary and excretory systems, the histamine system and the central nervous system. Active nucleosides of nucleoside-lipid conjugates of the present invention can be analgesics, anesthetics, anti-arrythmic, antiallergic, antifungal, anticancer (e.g. gemcitabine, cytarabine) antihypertensive, anticoagulants; antidepressants; antidiabetic, anti-epilepsy, anti-inflammatory, antiulcer, anti-protozoal, antivirals, or nucleoside-lipid conjugates for treating demyelinating diseases including multiple sclerosis; ophthalmic agents; vaccines (e.g., against influenza virus, pneumonia, hepatitis A, hepatitis B, hepatitis C, cholera toxin B-subunit, typhoid, Plasmodium falciparum, diptheria, tetanus, herpes simplex virus, tuberculosis, HIV, bordetela pertusis, measles, mumps, rubella, bacterial toxoids, vaccinea virus, adenovirus, SARS virus, canary virus, bacillus calmette Guerin, klebsiella pneumonia vaccine, etc.); histamine receptor antagonists, hypnotics, kidney protective agents, lipid regulating agents, muscle relaxants, neuroleptics, neurotropic agents, opioid agonists and antagonists, parasympathomimetics, protease inhibitors, prostglandins, sedatives, sex hormones (e.g., androgens, estrogens, etc.), stimulants, sympathomimetics, vasodilators and xanthine and synthetic analogs of these species.

[0089] The new nucleoside-lipid conjugates of this invention are potentially useful for the treatment of cancer. The cancer can be any type of cancer in a mammal. Examples include, but are not limited to, cancers of the head, neck, brain, blood (e.g. leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, lymphoma, myeloma), breast, lung, pancreas, bone, spleen, bladder, prostate, testes, colon, kidney ovary and skin (e.g. Kaposi's sarcoma). In addition, the nucleoside-lipid conjugates of the present invention are useful in reducing the tendency of cancers cells to develop a resistance to other therapeutic agents such as anti-cancer agents, chemotherapy and radiation.. Thus, other therapeutic agents can be advantageously employed with the present invention in the formation of an active combination or by separate administration.

[0090] The new nucleoside-lipid conjugates of this invention can also be potentially useful in the treatment of viral diseases such as HIV, herpes simplex viruses (HSVl and HSV2), human herpes virus 6, human herpes virus 7, human herpes virus 8, orthopoxviruses (e.g. variola major and minor, small pox), ebola virus, influenza virus, tuberculosis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, parainfluenza virus, respiratory syncytial virus, cholera, pneumonia, SARS virus, canary virus, West Nile virus (WNV), respiratory syncytial virus (RSV), dengue virus, vericella zoster virus, corona viruses, vaccinia virus, cytomegalovirus (CMV), human rhinovirus (HRV), papilloma virus (PV) and Epstein Barr virus.

[0091] It should be noted that, where the inventive nucleoside-lipid conjugates are employed to treat or alleviate diseases (e.g., cancer, viral) in human or animal patients, they need not result in a complete cure or remission of the disease to be shown to be successfully employed. In addition, as used herein, "alleviating a disease" means reducing the severity of a symptom of the disease. As used herein, "treating a disease" means reducing the frequency with which a symptom of the disease is experienced by a mammal. Thus, for example, the disease may be treated by using the inventive nucleoside-lipid conjugate, the progress of the disease is slowed or retarded in the patient. Alternatively, the disease may be considered to be treated if, for adjunctive uses, the inventive nucleoside-lipid conjugate renders the disease more amenable to other treatments or demonstrates additive, but not necessarily synergistic, therapeutic potential as compared to monotherapy using other treatment regimens. In some embodiments, the use of the nucleoside-lipid conjugates, in accordance with the present invention, can lead to remission of cancer or other diseases.

[0092] The new nucleoside-lipid conjugates of this invention are potentially useful in the treatment or alleviation of bone disorders. These conjugates can be used in methods for inhibiting bone resorption, methods for increasing bone formation by preventing osteoblast and osteocyte apoptosis and methods for increasing bone mass and strength. Indications for their use include the treatment or alleviation of osteoporosis, Paget's disease, metastatic bone cancers, hyperparathyroidism, rheumatoid arthritis, algodistrophy, sterno-costo-clavicular hyperostosis, Gaucher's disease, Engleman's disease and certain non-skeletal disorders.

[0093] The nucleoside-lipid conjugates of the present invention can be administered intravenously, subcutaneously, locally, orally, parenterally, intraperitoneally, and/or rectally, nasally, vaginally, lingually or by direct injection into tumors or sites in need of treatment by such as are known or developed. The present pharmaceutical preparations can contain the nucleoside-lipid conjugate alone or can contain further substances of pharmaceutical importance. They can further comprise a pharmaceutically acceptable carrier.

[0094] Tablets, dragees, capsules, pills, granules, suppositories, solutions suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays can be suitable pharmaceutical preparations. Suppositories can contain, in addition to the nucleoside-lipid conjugate, suitable water soluble or water- insoluble excipients. Suitable excipients are those in which the inventive nucleoside-lipid conjugate is sufficiently stable to allow for therapeutic use, for example polyethylene glycols, certain fats, and esters or mixture of substances. Ointments, pastes, creams and gels can also contain suitable expcipients in which the lipid-nucleoside conjugate is stable.

[0095] Another use for the inventive nucleoside-lipid conjugate is in the preparation of liposomes and other lipid-containing formulations. In addition to the nucleoside-lipid conjugate, the invention includes a composition, wherein the liposome further comprises phospholipids and/or lysophospholipids selected from a group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, cholesterol, α-tocopherol, natural or synthetic cardiolipin and analogues thereof, synthetic cationic cardiolipin analogues and derivatives thereof, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphotidylglycerol, lysophosphatidic acid, lysophosphotidylcholine, lysophosphatidylserine and PEG modified lipids.

[0096] Liposomes, according to the present invention, can be multilamellar vesicles, unilamellar vesicles, or a mixture thereof. Moreover, the liposomes can be of varying size or can be substantially uniform in size. For example, the liposomes can have a size range of about 1 mm or less, and more preferably are in the micron or sub-micron range. For example, the liposomes can have a diameter of about 5μm or less, such as about lμm or less, or even about 0.5μm or less, such as about 0.2μm or less or even about 0.1 μm or less.

[0097] The inventive nucleoside-lipid conjugates can be employed in the form of a combination with other therapeutic agents. The therapeutic agent can become complexed with a portion of the lipid (such as the inventive nucleoside-lipid conjugate) or the therapeutic agent can become entrapped within the liposomes. Alternatively, the therapeutic agent can be administered separately. In addition, a second therapeutic agent can be administered adjunctively, prior to, concurrently with, or after the first therapeutic agent. Preferred agents include antineoplastic, antifungal, antibiotic and other therapeutic agents, particularly cisplatin, antisense oligonucleotides, siRNA, oxaliplatin, paclitaxel, vinorelbine and epirubicin.

[0098] In addition to synthetic nucleoside-lipid conjugates, liposomes can include stabilizers, absorption enhancers, antioxidants, phospholipids, biodegradable polymers, and medicinally active agents among other ingredients. The amount of nucleoside-lipid conjugates in liposomes can be controlled by varying the composition of lipids and/or other components in it.

[0099] In some embodiments, it is preferable for liposomes to also include targeting agents such as a carbohydrate, a protein, ligands that bind to a specific substrate (such as antibodies or fragments thereof), or ligands that recognize cellular receptors. The inclusion of such agents (such as a carbohydrate, or one or more proteins selected from a group of proteins consisting of antibodies, antibody fragments, peptide, peptide hormones, receptor ligands, such as an antibody to a cellular receptor, and mixtures thereof) can facilitate targeting the liposome to a predetermined tissue or cell type.

[00100] Liposomes, according to the present invention, can be prepared by any suitable technique. Lipophilic liposome-forming ingredients, such as phosphatidylcholine, a nucleoside-lipid conjugate prepared by the methods described above, cholesterol and α-tocopherol can be dissolved or dispersed in a suitable solvent or combination of solvents and dried. Suitable solvents include any non-polar or slightly polar solvent, such as ϊ-butanol, ethanol, methanol, chloroform, or acetone that can be evaporated without leaving a pharmaceutically unacceptable residue. Drying can be by any suitable means such as by lyophilization. Hydrophilic ingredients can be dissolved in polar solvents, including water.

[00101] Liposomes can be formed by mixing the dried lipophilic ingredients with the hydrophilic mixture. Mixing the polar solution with the dried lipid film can be by any means that strongly homogenizes the mixture. The homogenization can be effected by vortexing, magnetic stirring and/or sonicating. Once formed, the liposomes can be filtered through suitable filters to control their size distribution. Suitable filters include these that can be used to obtain the desired size range of liposomes from a filtrate. For examples, the liposomes can be formed and thereafter filtered through a 5 micron filter to obtain liposomes of about 5 microns or less. Alternatively, 1 μm, 500 nm, 100 nm or other suitable filters can be used to obtain liposomes of a desired size. The present inventive liposomes can also be filtered through microbial retentative filters to obtain a sterile pharmaceutical product.

[00102] The invention also includes a kit for administering the composition of the present invention to a mammal for the treatment or alleviation of a disease. The disease can be any one or more of the diseases described herein. The kit comprises the composition of the invention and an instructional manual which describes the administration of the composition to a mammal by any of the routes of administration described herein. In another embodiment, this kit comprises a solvent, preferably sterile solvent, suitable for dissolving or suspending the composition of the invention prior to administering the composition to the mammal.

[00103] All references, including publications, patent applications, and patents cited herein, including those in the preceding list and otherwise cited in this specification, are hereby incorporated by reference to the same extent as if each reference is were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [00104] The use the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. AU methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specifications should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00105] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments can become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the inventions to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00106] The following specific examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

Example 1 Synthesis of a Gemcitabine Cardiolipin Conjugate

[00107] First, commercially available (i?)-(+)-3-benzyloxy-l,2-propanediol was reacted with 1- bromohexane in the presence of sodium hydride (60% in oil dispersion) in dimethyl formamide (DMF). This reaction was followed by debenzylation via catalytic hydrogenolysis (H₂/Pd-C) which provided 1,2-di- O-hexyl-sw-glycerol in a 89% yield. The next step involved reacting 1,2-di-O-hexyl-in-glycerol with a bifunctional phosphitylating reagent ΛζTV-diisopropylmethylphosphonamidic chloride in the presence of ΛζΛf-diisopropylethylamine (DIPEA) in dichloromethane at room temperature to give l,2-di-0-hexyl-.s7z- glycero-ΛζiV-diisopropyl methylphosphoramidite. 1 ,2-di-O-hexyl-j/z-glycero-NN-diisopropyl methylphosphoramidite was then reacted with 2-benzyloxy-l,3-propanediol in the presence of l//-tetrazole to provide a phosphite triester. In situ oxidation of the phosphite trimester, with m-chloroperbenzoic acid (mCPBA) at -40 ⁰C, afforded 2-0-benzyl-l,3-bis-(l,2-di-0-hexyl-j«-glycero-3-phosplioryl)glycerol dimethyl ether in the form of a colorless oil. A yield of 79% was achieved after purification of the 2-0- benzyl-l,3-bis-(l,2-di-O-hexyl-JH-glycero-3-phosphoryl)glycerol dimethyl ether on a silica gel column (hexane-ethyl acetate, 8:2-6:4). Purification was followed by hydrogenolysis by reacting 2-0-benzyl-l,3- bis-(l,2-di-0-hexyl-jn-glycero-3-phosphoryl)glycerol dimethyl ether with H₂/Pd-C at 50 psi for 2 hours. This reaction furnished a 96% yield of l,3-bis-(l,2-di-O-hexyl-.57J-glycero-3-phosphoryl)glycerol dimethyl ether in the form of a colorless oil. The central hydroxyl functionality of l,3-bis-(l,2-di-O-hexyl-.57z- glycero-3-phosphoryl)glycerol dimethyl ether (cardiolipin analogue) was then reacted with succinic anhydride in the presence of triethylamine and 4-dimethylamino pyridine (DMAP) in 1,2-dichloroethane to provide a 79% yield of l,3-bis-(l,2-di-0-hexyl-sκ-glycero-3-phosphoryl)-2-succinylglycerol dimethyl ether. l,3-bis-(l,2-di-0-hexyl-j7z-glycero-3-phosphoryl)-2-succinylglycerol dimethyl ether was then reacted with 4-N-3'-O-bis(fert-butoxycarbonyl)gemcitabine in the presence of //.JV-dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 8 hours. This reaction was followed by purification on a silica gel column (hexane-ethyl acetate, 7:3-4:6) which afforded a 84% yield of 4-N-3'-(9-di(tert-butoxycarbonyl)-5'-C'-succinyl-[2-0-l,3-bis-(l,2-di-0-hexyl-5ⁱw-glycero)-3-phosphoryl glycerol dimethyl ether] gemcitabine. The protecting groups of 4-7V-3'-O-di(tert-butoxycarbonyl)-5'-O- succinyl-[2-0-l,3-bis-(l,2-di-C>-hexyl-₁sⁱH-glycero)-3-phosphoryl glycerol dimethyl ether] gemcitabine were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and then concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-96:4) afforded an 80% yield of pure 5'-0-succinyl-[2-0-l,3-bis-(l,2-di-0-hexyl-«z-glycero)-3- phosphorylglycerol dimethyl ether] gemcitabine in the form of a colorless viscous oil. This final product was characterized by ¹H NMR, ¹³C NMR, IR and Mass Spectroscopy. The purity was checked by HPLC and elemental analysis.

Example 2

[00108] Alkylation of commercially available (R)-(+)-2,2-dimethyl- 1 ,3-dioxolane-methanol with 1 - bromohexane in the presence of sodium hydride (60% in an oil dispersion) in tetrahydrofuran (THF) followed by deprotection of the isopropylidene group with 2N HCl in methanol provided 1-O-hexyl- glycerol. Tritylation of l-O-hexyl-glycerol,using trityl chloride in the presence of triethylamine and DMAP in dichloromethane, gave l-O-hexyl-3-O-trityl glycerol. Methylation of the trityl derivative with iodomethane, in the presence of sodium hydride in THF, followed by detritylation with p-toluenesulfonic acid monohydrate, in THF, provided l-O-hexyl-2-O-methylglycerol with a 63% yield. The next step involved the reaction of l-O-hexyl-2-O-methyl-.sⁱH-glycerol with N,N-diisopropylmethylphosphonamidic chloride in the presence of N, jV-diisopropylethylamine (DIPEA) in dichloromethane at room temperature. This reaction resulted in l-O-hexyl-2-O-methyl-sw-glycero-ΛζN-diisopropyl methylphosphoramidite which was then reacted with 2-benzyloxy-l,3-propanediol, in the presence of liϊ-tetrazole, to provide a phosphite triester. In situ oxidation of the phosphite trimester, with 7«-chloroperbenzoic acid (mCPBA) at -40 ⁰C, afforded 2-0-benzyl-l,3-bis-(l-0-hexyl-2-0-methyl-j«-glycero-3-phosphoryl)glycerol dimethyl ether. Purification of this product on a silica gel column (hexane-ethyl acetate, 8:2-4:6) resulted in 2-O-benzyl- l,3-bis-(l-0-hexyl-2-O-methyl--SM-glycero-3-phosphoryl)glycerol dimethyl ether as a colorless oil in 87% yield. Hydrogenolysis of 2-0-benzyl- 1 ,3 -bis-( 1 -0-hexyl-2-O-methyl--.s7i-glycero-3 -phosphoryl)glycerol dimethyl ether, with H₂/Pd-C at 50 psi for 2 hours, resulted in l,3-bis-(l-O-hexyl-2-O-rnethyl--.57z-glycero- 3-phosphoryl)glycerol dimethyl ether as a colorless oil in 96% yield. The central hydroxyl functionality of l,3-bis-(l-0-hexyl-2-0-methyl~5'«-glycero-3-phosphoryl)glycerol dimethyl ether (cardiolipin analogue) was reacted with succinic anhydride in the presence of triethylamine and 4-dimethylamino pyridine (DMAP) in 1,2-dichloroethane which provided l,3-bis-(l-0-hexyl-2-0-methyl--s«-glycero-3-phosphoryl)- 2-succinyl glycerol dimethyl ether in a 68% yield. l,3-bis-(l-O-hexyl-2-(9-methyl~i'«-glycero-3- phosphoryl)-2-succinyl glycerol dimethyl ether was then coupled with 4-N-3'-O-bis(tert- butoxycarbonyl)gemcitabine in the presence of Af N-dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 8 hours. This was followed by purification on a silica gel column (dichloromethane-acetone, 8:2-6:4) which afforded 4-7V-3'-O-di(tert-butoxycarbonyl)-5'-O-succinyl-[2-O- l,3-bis-(l-O-hexyl-2-O-methyl -5ⁱκ-glycero)-3-phosphoryl glycerol dimethyl ether] gemcitabine in a 60% yield. The protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-9:1) afforded pure 5'-O-succinyl-[2-O-l,3-bis-(l-O-hexyl-2-O-methyl -5^r«- glycero)-3-phosphorylglycerol dimethyl ether] gemcitabine as a viscous oil in a 70% yield. The product was characterized by ¹H NMR, ¹³C NMR, IR and HRMS. The purity was checked by HPLC.

Example 3

[00109] l,2-di-O-hexyl-s7z-glycerol was treated with dibenzyl diisopropylphosphoramidite and tetrazole in anhydrous dichloromethane and then reacted with 2-0-te/t-butyldimethylsilyl-l,3-propanediol and pyridinium tribromide in the presence of triethyl amine and pyridine in anhydrous dichloromethane. This reaction was followed by purification on a silica gel column (hexane: ethyl acetate , 9:1 to 7:3) to give 2-O-tetrabutyldimethyl silyl-l,3-bis [(1, 2-di-0-hexyl-_7z-glycero-3)-phosphoryl] glycerol dibenzyl ether in a 60% yield. The silyl protecting group was removed using 7:3 mixture of ethanol: HCL to give 1, 3 -bis [(1, 2-di-0-hexyl-«z-glycero-3)-phosphoryl] glycerol dibenzyl ether. The central hydroxyl functionality of 1, 3-bis [(1, 2-di-O-hexyl-.s7z-glycero-3)-phosphoryl] glycerol dibenzyl ether (cardiolipin analogue) was reacted with succinic anhydride in the presence of triethylamine and 4-dimethylamino pyridine (DMAP) in 1,2-dichloroethane to provide l,3-bis-(l, 2-di-0-hexyl-w-glycero-3-ρhosphoryi)-2-succinyl glycerol dibenzyl ether with a 78% yield. l,3-bis-(l, 2-di-O-hexyl-5^r«-glycero-3-phosphoryl)-2-succinyl glycerol dibenzyl ether was coupled with 4-Λf-3'-<9-bis(fer£-butoxycarbonyl)gemcitabine in the presence of N₁N- dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 8 hours. This was followed by purification on a silica gel column (dichloromethane-methanol, 9:5-0:5) which afforded 4- Λ^f-3'-0-di(tert-butoxycarbonyl)-5'-0-succinyl-[2-0-l,3-bis-(l-0-hexyl-i'«-glycero)-3-phosphoryl glycerol dimethyl ether] gemcitabine in a 71% yield. The t-BOC protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-9:1) afforded pure 5'-O-succinyl-[2-O-l,3-bis-(l,2-di-(9-hexyl-5«-glycero)-3-phosphorylglycerol dibenzyl ether] gemcitabine as a colorless viscous oil with a 64% yield. The benzyl groups were removed by hydrogenation using Pd/C in ethanol at 30 psi for 3 hrs. The catalyst was removed by filtration on celite. A few drops of ammonium hydroxide were added to the filtrate and concentrated. The crude product was crystallized with methylene chloride and acetone to provide 5'-O-succinyl-[2-O-l,3-bis-(l,2-di-O-hexyl-5»-glycero)-3- phosphorylglycerol diammonium salfjgemcitabine. The product was then characterized by ¹H NMR.

Example 4

[00110] l,3-bis-(l,2-di-0-hexyl-jn-glycero-3-phosphoryl)-2-succinylglycerol dimethyl ether was coupled with 4-N-5'-O-bis(tert-butoxycarbonyl)gemcitabine in the presence of ΛζiV-dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 8 hours. This reaction was followed by purification on a silica gel column (hexane-ethyl acetate, 7:3-4:6) which afforded 4-N-5'-O- di(tert-butoxycarbonyl)-3'-0-succinyl-[2-<9-l,3-bis-(l,2-di-0-hexyl-5«-glycero)-3-phosphoryl glycerol dimethyl ether] gemcitabine in a 84% yield. The protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was then neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-96:4) afforded pure 3'-O-succinyl-[2- O-l,3-bis-(l,2-di-O-hexyl-j«-glycero)-3-phosρhorylglycerol dimethyl ether]gemcitabine as a viscous oil with a 80% yield. The product was characterized by ¹H ΝMR, ¹³C ΝMR, IR and Mass Spectroscopy. The purity was checked by HPLC and elemental analysis.

Example 5

[00111] l,3-bis-(l,2-di-0-hexyl-£M-glycero-3-phosphoryl)glycerol dimethyl ether was reacted with ΛζN-diisopropylmethylphosphonamidic chloride in the presence of Λζ/V-diisopropylethylamine (DIPEA) in dichloromethane at room temperature. The resulting intermediate was subsequently reacted with 4-N-3'-O- bis(tert-butoxycarbonyl)gemcitabine, in the presence of l#-tetrazole, to provide a phosphite triester. In situ oxidation of the phosphite trimester, with m-chloroperbenzoic acid (mCPBA) at -40 ⁰C, afforded 4-iV- 3'-<9-bis(tert-butoxycarbonyl)-5'C>-methyl phosphoryl-[l,3-bis-(l,2-di-0-hexyl-sn-glycero-3- phosphoryl)glycerol dimethyl ether] gemcitabine which was then purified on a silica gel column (methylene chloride:methanol 9.8:0.2). This purification resulted in 4-N-3'-O-bis(fert-butoxycarbonyl)-5O-methyl phosphoryl-[l,3-bis-(l,2-di-0-hexyl-jH-glycero-3-phosphoryl)glycerol dimethyl ether] gemcitabine as a colorless oil with a 60% yield. The protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-9.5:0.5) afforded pure 5'-O-methyl phosphoryl-[2-O- l,3-bis-(l-O-hexyl-2-O-methyl -j/j-glycero)-3-phosphorylglycerol dimethyl ether] gemcitabine as a viscous oil in a 51% yield. The product was characterized by ¹H NMR, and HRMS. The purity was checked by HPLC.

Example 6

[00112] l,2-O-dimvristyl-5κ-glycerol was reacted with Λζ Λf-diisopropylmethylphosphonarnidic chloride in the presence of ΛζN-diisopropylethylamrne (DIPEA) in dichloromethane at room temperature to give 1,2-O-dimyristyl-όΗ-glycero-ΛζTV-diisopropyl methylphosphoramidite which was subsequently reacted with 2-benzyloxy-l,3-propanediol, in the presence of liZ-tetrazole, to provide a phosphite triester. In situ oxidation of the phosphite trimester, with »z-chloroperbenzoic acid (OTCPBA) at -40 ⁰C, afforded 2-0- benzyl-l,3-bis-(l,2- O-dimyristyl -,s72-glycero-3-phosphoryl)glycerol dimethyl ether. l,3-bis-(l,2- O- dimyristyl -,yrø-glycero-3-phosphoryl)glycerol dimethyl ether was then purified on a silica gel column (hexane-ethyl acetate, 1:0-1:1), resulting in a colorless oil with a 90% yield. Hydrogenolysis of 2-0- benzyl-l,3-bis-(l,2- O-dimyristyl -sn-glycero-3-phosphoryl)glycerol dimethyl ether, with H₂/Pd-C at 50 psi for 5 hours, furnished l,3-bis-(l,2- O-dimyristyl -."/j-glycero-S-phosphory^glycerol dimethyl ether as a colorless oil in a 98% yield. The central hydroxyl functionality of l,3-bis-(l,2-O-dimyristyl-iΗ-glycero-3- phosphoryl)glycerol dimethyl ether (cardiolipin analogue) was reacted with succinic anhydride in the presence of triethylamine and 4-dimethylamino pyridine (DMAP) in 1,2-dichloroethane to provide 1,3-bis- (1,2- O-dimyristyl -.sra-glycero-3-phosρhoryl)-2-succinylglycerol dimethyl ether in a 88% yield. 1,3-bis- (l,2-0-dimyristyl-jK-glycero-3-phosphoryl)-2-succinylglycerol dimethyl ether was coupled with 4-N-3'-O- bis(fert-butoxycarbonyi)gemcitabine in the presence of ΛζN-dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 8 hours. This reaction was followed by purification on a silica gel column (hexane-ethyl acetate, 7:3-1:1) which afforded 4-7V-3'-0-di(tert-butoxycarbonyl)-5'-0-succinyl- [2-O-l,3-bis-(l,2- O-dimyristyl -j»-glycero)-3-ρhosρhoryl glycerol dimethyl ether] gemcitabine with a 73% yield. The protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-96:4) afforded pure 5'-O-succinyl-[2-O-l,3-bis-(l,2- 0-dimyristyl -sn- glycero)-3-phosphorylglycerol dimethyl etherjgemcitabine as a white crystalline solid with a 69% yield. The product was characterized by ¹H NMR. The purity was checked by HPLC and elemental analysis.

Example 7

[00113] Alkylation of commercially available (R)-(-)-2,2-dimethyl-l,3-dioxolane-methanol with 1- bromotetradecane in the presence of sodium hydride (60% in oil dispersion) in #,7V-dimethylformamide (DMF) followed by deprotection of isopropylidene group, with 2N HCl in methanol, provided 1-0- tetradecyl-glycerol. Tritylation of 1-0-tetradecyl-glycerol was accomplished by using trityl chloride in the presence of triethylamine and DMAP in dichloromethane. This resulted in l-0-tetradecyl-3-0-trityl glycerol. The trityl derivative was methylated with iodomethane, in the presence of sodium hydride in THF. This reaction was followed by detritylation with p-toluenesulfonic acid monohydrate in THF which provided l-0-tetradecyl-2-0-methylglycerol with a 65% yield. The next step involved the reaction of l-O- tetradecyl-2-0-methyl-J7J-glycerol with ΛζΛf-diisopropylmethylphosphonamidic chloride, in the presence of ΛζN-diisopropylethylamine (DIPEA) in dichloromethane at room temperature, to give l-O-tetradecyl-2-0- methyl-^z-glycero-ΛζiV-diisopropyl methylphosphoramidite which was subsequently reacted with 2- benzyloxy-l,3-propanediol in the presence of 1/7-tetrazole to provide a phosphite triester. In situ oxidation of the phosphite trimester, with m-chloroperbenzoic acid (mCPBA) at -40 ⁰C, afforded 2-0-benzyl-l,3-bis- (l-0-tetradecyl-2-0-methyl-5w-glycero-3-phosphoryl)glycerol dimethyl ether which was then purified on a silica gel column (methylene chloride-ethyl acetate, 8:2-6:4). This resulted in 2-0-benzyl-l,3-bis-(l-0- tetradecyl-2-0-methyl-_;κ-glycero-3-phosphoryl)glycerol dimethyl ether as a colorless oil with a 68% yield. Hydrogenolysis of2-0-benzyl-l,3-bis-(l-0-tetradecyl-2-0-methyl-i'M-glycero-3-phosphoryl)glycerol dimethyl ether, with H₂/Pd-C at 50 psi for 2 hours, furnished l,3-bis-(l-0-tetradecyl-2-0-methyl-5n- glycero-3-phosphoryl)glycerol dimethyl ether as a colorless oil with a 96% yield. The central hydroxyl functionality of l,3-bis-(l-0-tetradecyl-2-0-methyl-57z-glycero-3-phosphoryl)glycerol dimethyl ether (cardiolipin analogue) was reacted with succinic anhydride in the presence of triethylamine and 4- dimethylamino pyridine (DMAP) in 1,2-dichloroethane to provide l,3-bis-(l-0-tetradecyl-2-0-methyl-.ϊ«- glycero-3-ρhosρhoryl)-2-succinyl glycerol dimethyl ether with a 79% yield. l,3-bis-(l-0- tetradecyl -2-O- methyl--.yra-glycero-3-phosphoryi)-2-succinyl glycerol dimethyl ether was coupled with 4-N-3 '-0-bis(fert- butoxycarbonyl)gemcitabine in the presence of N,N-dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 8 hours. This reaction was followed by purification on a silica gel column (hexane-acetone, 8.5:1.5-7:3) which afforded 4-N-3'-O-di(tert-butoxycarbonyl)-5'-O-succinyl-[2-0- l,3-bis-(l-0-tetradecyl-2-0-methyl-5κ-glycero)-3-phosphoryl glycerol dimethyl ether] gemcitabine with a 60% yield. The protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (chloroform-methanol, 98:2-9.5:0.5) afforded pure 5'-O-succinyl-[2-O-l,3-bis-(l-0-tetradecyl-2-O- methyl-jn-glycero)-3-ρhosphorylglycerol dimethyl ether] gemcitabine as a viscous oil with a 70% yield. The product was characterized by ¹H NMR, ¹³C NMR, IR and HRMS. The purity was checked by HPLC.

Example 8

[00114] l,2-O-dimyristoyl-.s7z-glycerol was reacted with ΛζΛ'-diisopropylmethylphosphonamidic chloride in the presence of ΛζN-diisopropylethylamine (DIPEA) in dichloromethane at room temperature to give 1,2- (9-dimyristoyl -5n-glycero-ΛζΛ^r-diisopropyl methylphosphoramidite which was subsequently reacted with 2-benzyloxy-l,3-propanediol, in the presence of l//-tetrazole, to provide a phosphite triester. In situ oxidation of the phosphite trimester, with m-chloroperbenzoic acid (mCPBA) at -40 ⁰C, afforded 2- O-benzyl-l,3-bis-(l,2-O-dimyristoyl -5«-glycero-3-phosphoryl)glycerol dimethyl ester which was then purified on a silica gel column (hexane-ethyl acetate, 2:1-1:1) resulting in 2-O-benzyl-l,3-bis-( 1,2-0- dimyristoyl -sn-glycero-3-phosphoryl)glycerol dimethyl ester as a colorless oil with a 90% yield. Hydrogenolysis of 2-O-benzyl-l,3-bis-(l,2-O-dimyristoyl -5κ-glycero-3-ρhosphoryl)glycerol dimethyl ester, with H₂/Pd-C in ethanol at 40 psi for 3 hours, furnished l,3-bis-(l,2-0-dimyristoyl-i'n-glycero-3- phosphoryl)glycerol dimethyl ester as a colorless oil with a 96% yield. The central hydroxyl functionality of l,3-bis-(l,2-0-dimyristoyl-5«-glycero-3-phosρhoryl)glycerol dimethyl ester (cardiolipin analogue) was reacted with succinic anhydride in the presence of triethylamine and 4-dimethylamino pyridine (DMAP) in 1 ,2-dichloroethane to provide 1 ,3-bis-( 1 ,2-O-dimyristoyl-572-glycero-3 -phosphoryl)-2-succinylglycerol dimethyl ester with a 78% yield. l,3-bis-(l,2-O-dimyristoyl-i^r«-glycero-3-phosphoryl)-2-succinylglycerol dimethyl ester was coupled with 4-^-3 '-O-bis(tert-butoxycarbonyl)gemcitabine in the presence of N₁N- dicyclohexyl carbodimide (DCC) and DMAP in dichloromethane at room temperature for 5 hours. This reaction was followed by purification on a silica gel column (hexane-ethyl acetate, 7:3-4: 1 and hexane:ethylacetate: methanol, 1:1:0.1) which afforded 4-iV-3'-0-di(tert-butoxycarbonyl)-5'-0-succinyl-[2- 0-l,3-bis-(l,2-0-dimyristoyl-src-glycero)-3-phosphoryl glycerol dimethyl ester] gemcitabine in a 72% yield. The protecting groups were removed using trifluororacetic acid (TFA) in dichloromethane at room temperature. The reaction solution was neutralized with 5% aqueous sodium bicarbonate at 0 ⁰C, extracted with dichloromethane and concentrated. Purification of the crude compound on a silica gel column (methylene chloride-ethyl acetate, 9.8:0.2-9.6:0.4) afforded pure 5'-O-succinyl-[2-O-l,3-bis-(l,2- O- dimyristoyl -57z-glycero)-3-phosphorylglycerol dimethyl ester]gemcitabine as a viscous oil in a 50% yield. The product was characterized by ¹H NMR. The purity was checked by HPLC.

Example 9

Efficacy and Toxicity Studies for Gemcitabine Cardiolipin Conjugate

A. Materials 1. Chemicals

[00115] Trichloroacetic acid (TCA), sulforhodamine B (SRB), 6-[(4-Nitrobenzyl)thio]-9-/3-D- ribofuranosylpurine (NBMPR), dipyridamole, 100% ethanol and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). Dextrose (5%) and saline was purchased from Abbott Laboratories (Abbott Park, IL).

2. Formulation preparations

[00116] Gemcitabine hydrochloride (Gemzar, Eli Lilly, Indianapolis, IN) was used within 24 hours after reconstitution. The gemcitabine-cardiolipin conjugate (Figure 18, XXXXIII, R & D Facility, NeoPharm Inc.,Waukegan, IL) was dispersed into 5% dextrose from an ethanol stock solution followed by 30 second vortex. The dispersion was diluted into cell culture medium for in vitro use or administered to animals directly. The final formulation of the gemcitabine-cardiolipin conjugate had a mean particle size of 286 nm.

3. Cell culture

[00117] Human lung A549, breast MX-I, colon HT-29 and murine leukemia P388 cell lines were obtained from the National Cancer Institute (Fredrick, MD). These cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (HI-FBS). Human pancreatic BxPC-3 cancer cells were purchased from American Type Culture Center (Manassas, VA) and maintained in RPMI 1640 medium containing 10% HI-FBS, 10 mM HEPES, 1 mM sodium pyruvate and 4.5 g/L glucose. All culture media contained penicillin 100 unit/mL and streptomycin 100 μg/mL. All cells were incubated at 37 ⁰C in a 5% CO₂ incubator. The culture media and reagents were purchased from Invitrogen Co (Carlsbad, CA).

B. Methods

1. Cytotoxicity assay

[00118] The growth inhibition was determined based on SRB assay (Chien P. et. al. Cancer Gene Therapy in press, 2004). In brief, different cancer cells (10,000 - 25,000 cells per well) were pre-incubated on 96-well microliter plates overnight. Freshly prepared gemcitabine-cardiolipin conjugate suspensions or gemcitabine dilutions were added to the culture medium at final concentrations of 5x10^"2 to 5><10⁴ nM. Cells were co-cultured with the drugs for 48 hours before fixing with TCA at 4⁰C for 1 hour and cytotoxicity was estimated based on total cellular protein levels as previously described (Skehan P, et. al. J Natl Cancer Inst, 1990, 82(13): 1107-1112). The 50% growth inhibition (GI₅₀) was estimated as the concentration of the drug that gives 50% growth inhibition. For NT inhibitor experiment, NBMPR and dipyridamole were first dissolved in DMSO as stock solutions and further diluted in medium before use. A549 (10,000 cells/well) or BxPC-3 (20,000 cells/well) were first incubated with 1 μM NBMPR, or 10 μM dipyridamole for 1 hour. After incubating with NT inhibitors or control vehicle (DMSO), cells were further co-cultured with serial diluted gemcitabine-cardiolipin conjugate or gemcitabine for additional 48 hours. The cytotoxicity of gemcitabine-cardiolipin conjugate or gemcitabine in the presence of NT inhibitors was determined by SRB assay and compared with those without NT inhibitors. The final DMSO concentration in each well was 0.1% (v/v).

2. Apoptosis assay

[00119] A549 cells (10,000/well) were treated with gemcitabine-cardiolipin conjugate or gemcitabine for 48 hours as described in previous section. The cells were washed with PBS and the caspase 3/7 expression was quantified using a Caspase-Glo™ 3/7 assay kit (Promega, Madison, WI) according to the manufacturer's protocol.

3. In vivo toxicity

[00120] Multiple-dose toxicity studies were conducted on normal female CD2F1 mice (5-6 weeks old). Mice were randomized into groups of 5 based on body weight and were injected with gemcitabine- cardiolipin conjugate or gemcitabine at doses of 18, 27 and 36 μmol/kg for 5 consecutive days. Mice in control group were given equal dose volume of vehicle (5% dextrose and 5% ethanol). The toxicities were evaluated by the mortality, body weight loss and by obtaining the peripheral blood cell counts and plasma levels of alanine transaminase (ALT), aspartate transaminase (AST) at the end of the study. Blood samples were collected from the orbital sinus into either K₂-DETA-coated tubes for CBC or plasma separating tubes for plasma enzyme and creatinine analysis. All laboratory examinations were conducted at Antech Diagnostics (Chicago, IL). Percentage of body weight change was calculated as 100 x (W_t-W₀)/W_t; where W₁ represents the body weight at any time point and Wo represent initial point.

4. Tumor models

[00121] A mouse leukemic model was established with female CD2F1 mice (5-6 weeks old). Each mouse was injected with log-phase P388 cells (1 x 10⁵ cells in 0.2 ml PBS) via lateral tail veins on day 0. Next day, mice were randomly divided into groups of 5 based on their body weight and treated with gemcitabine-cardiolipin conjugate or gemcitabine at doses of 9, 18 and 27 μmol/kg. Mice, in the control group, were administrated with a control vehicle (5% dextrose and 5% ethanol). Mice were observed, once a day, for signs of moribundity and mortality for 60 days. Body weights were recorded during dosing and twice a week thereafter. [00122] A human pancreatic tumor xenograft was established with female CB-17 SCID mice (4-5 weeks old) by s.c. injection of 2 x 10⁶ BxPC-3 cells at right flank region. Mice were treated with gemcitabine-cardiolipin conjugate, gemcitabine or control vehicle intravenously when the tumor reached a volume of 80-160 mm³. Tumor length (L) and width (W) were measured with a digital caliper twice a week. The tumor volume (V) and tumor growth (% of initial tumor volume) were calculated as follows: V (mm³) = length x (width/2)² x π; where π = 3.14; Tumor growth or % initial tumor volume = 100 x (VyVo); where Vi represents the tumor volume at any given day and Vo is the tumor volume on the day of treatment initiation.

5. Statistical comparison

[00123] Data were expressed as mean ± SD. Statistical comparisons were made by use of the Student t test (P < 0.05).

C. Results

1. Gemcitabine-cardiolipin conjugate-induced cytotoxicity in cancer cells

[00124] Both the gemcitabine-cardiolipin conjugate and gemcitabine showed dose-dependent cytotoxicity against human pancreatic (BxPC-3), lung (A549), breast (MX-I), colon (HT-29) cancer cells and murine leukemia (P388) after 48 hrs treatment (Figure 19). At higher concentration (> 5 μM), cytolysis was found in cells treated with the gemcitabine-cardiolipin conjugate. A similar phenomenon did not occur with cells treated with gemcitabine. GI₅₀ was estimated from the dose curves in Figure 19a and summarized in Table 1. Among the cell lines tested, P388 showed the highest sensitivity to the gemcitabine-cardiolipin conjugate with a GI₅₀ of 44 nM followed by HT-29 and BxPC-3 with GI₅₀ of 69 and 182 nM, respectively. BxPC-3 and P388 cell lines were chosen for further in vivo efficacy evaluation.

Change in Cell lines Gemcitabine-cardiolipin conjugate (nM) Gemcitabine (nM)

Sensitivity GI₅₀ (fold)

BxPC-3 182 19 10

A549 589 95 6

MX-I 575 205 3

HT-29 69 4 17

P388 44 8 6

Table 1 GI₅₀ of gemcitabine-cardiolipin conjugate and Gemcitabine on Different Cancer Cell

Lines. Cells were incubated with serial diluted gemcitabine-cardiolipin conjugate or gemcitabine for 48 hrs before measurement of total cellular level with SRB assay. [00125] The time-dependent cytotoxicity of gemcitabine-cardiolipin conjugate and gemcitabine was also investigated. The onset of gemcitabine-cardiolipin conjugate effects, on cancer cell lines, was faster compared to that of gemcitabine. The cytotoxicity could be detected as early as 5 hours after the treatment with 50 μM of gemcitabine-cardiolipin conjugate in A549 cells (Figure 19b). In contrast, gemcitabine- induced cytotoxicity did not reach significant levels until 24 hrs after the treatment. These results indicate that the gemcitabine-cardiolipin conjugate enters the cells and exerts its cytotoxicity more rapidly than gemcitabine. At 5 μM, both the gemcitabine-cardiolipin conjugate and gemcitabine showed similar time- dependent cytotoxicity against A549 cells (Figure 19b).

[00126] Apoptosis has been shown to be one of the major mechanisms of gemcitabine-induced cytotoxicity (Nabhan C. et. al. , MoI Cancer Ther., 2001, 1(13): 1221-1227). To investigate whether the conjugation of cardiolipin has modified the cytotoxicity pathway of gemcitabine, apoptosis was determined in A549 cells treated with gemcitabine-cardiolipin conjugate and gemcitabine by detecting the increase of caspase 3/7 expression. As shown in Figure 20, both the gemcitabine-cardiolipin conjugate and gemcitabine enhanced caspase 3/7 expression in A549 cells as compared to untreated cells. The signal of caspase 3/7 increased as the concentration of either the gemcitabine-cardiolipin conjugate or gemcitabine increased. This indicated that both drugs induced apoptosis by activating caspase cascade.

2. Equilibrative NT inhibitor study

[00127] NT is one main factor in determining the gemcitabine sensitivity in cancer chemotherapy. Among all the members from NT family, equilibrative NT is most important because of its broad tissue expression (Cass C.E., et. al Pharm Biotechnol, 1999, 12: 313-352).

[00128] Tumor cells with non-functioning NT are resistant to gemcitabine (Achiwa H. et. al. Cancer Sd, 2004, 95(9): 753-757; Mackey J.R. et. al, Cancer Res, 1998, 58(19): 4349-4357). In order to explore the potential application of gemcitabine-cardiolipin conjugate on NT-deficient and gemcitabine-resistant tumors, we investigated the cytotoxicity of gemcitabine-cardiolipin conjugates against A549 cells in the presence of dipyridamole. By incubating with 10 μM of dipyridamole, A549 cells with non-functioning NT were obtained (Mackey J.R. et. al, Cancer Res, 1998, 58(19): 4349-4357). Blocking the normal function of NT, by dipyridamole, had no impact on gemcitabine-cardiolipin conjugate -induced cytotoxicity against A549 cells as indicated by insignificant changes in its growth inhibition curves (Figure 21a). In contrast, when NT was blocked with dipyridamole, the growth curve was shifted toward right, an indication of decreased cytotoxicity of gemcitabine against A549 cells (Figure 21b). The same experiment was also conducted with BxPC-3 cells. The potency of dipyridamole on gemcitabine-cardiolipin conjugate or gemcitabine-induced cytotoxicity against A549 and BxPC-3 cells was expressed with a ratio Of GI₅₀ in the presence and absence of dipyridamole (Table 2).

Gemcitabine-cardiolipin conjugate Gemcitabine

Dipyridamole Ratio Ratio

(-) (+) (-) (+)

(10 μU) (+) / (-) (+) / (-)

A549 1190 273 0.23 123 6262 51

BxPC-3 649 429 0.66 128 3880 30

Table 2 Effect of Dipyridamole on GI₅₀ (nM) of Gemcitabine-cardiolipin conjugate or

Gemcitabine against A549 and BxPC-3 Cells. Cells were treated with serial diluted gemcitabine-cardiolipin conjugate or gemcitabine for 48 hrs in the presence or absence of 10 /JM. of dipyridamole before SRB assay.

3. In vivo toxicity study

[00129] Based on our preliminary results, three doses of 18, 27 and 36 μmol/kg were used to find the maximum tolerable toxicity of gemcitabine and the gemcitabine-cardiolipin conjugate in CD2F1 mice. After 6 daily injections, all mice treated with gemcitabine at 27 and 36 μmol/kg were found moribund and had to be sacrificed on day 7. Mice treated with gemcitabine at 18 and gemcitabine-cardiolipin conjugate at 36 μmol/kg developed dehydration and rough coat but were not considered moribund. Therefore, the maximum tolerable dose of gemcitabine, at the current schedule (6 daily injections) on CD2F1 mice, was considered to be 18 μmol/kg for gemcitabine and 36 μmol/kg for the gemcitabine-cardiolipin conjugate. [00130] The toxicity of gemcitabine-cardiolipin conjugate at 18 μmol/kg after 6 daily treatments and the body weight loss on Day 7 was significantly less compared to gemcitabine. When mice were treated with gemcitabine-cardiolipin conjugate at 18 μmol/kg for 5 days, the maximum body weight loss was only 3% compare to 22% for gemcitabine. In addition, a significant decrease in WBC was found in mice treated with gemcitabine (Table 3) but not in mice treated with the gemcitabine-cardiolipin conjugate. Both the gemcitabine-cardiolipin conjugate and gemcitabine-treated mice developed neutropenia. There were no significant differences in RBC, HGB, platelet counts, plasma ALT, AST and creatine levels in mice treated with the gemcitabine-cardiolipin conjugate as compared to the control vehicle, indicating no significant liver or kidney toxicity of gemcitabine-cardiolipin conjugate. These results were confirmed by histopathologic examination where the gemcitabine-cardiolipin conjugate -treated mice did not induce any clinically significant changes in morphology of lung, liver or kidney.

Table 3 Body Weight Changes and Hematology Test in Mice Treated with 6 Daily Doses of

Gemcitabine-cardiolipin conjugate or Gemcitabine

4. In vivo efficacy studies

[00131] A systemic leukemia model was established by i.v. injection of P388 murine leukemia cells (1 x 10⁵) to female CD2F1 mice. Without treatment, the mice died after an average of 12 days. Treatment with the gemcitabine-cardiolipin conjugate at doses of 18 or 27 μmol/kg body weight for 5 days increased median survival time by 55 to 73% (Figure 22), suggesting a strong anti-leukemic activity from gemcitabine-cardiolipin conjugate. A dose of 18 μmol/kg, gemcitabine resulted in the toxic death of all treated mice. At 9 μmol/kg, gemcitabine exhibited comparable survival increase to the gemcitabine- cardiolipin conjugate at 18 μmol/kg. It was noted that 25% of mice treated with the gemcitabine-cardiolipin conjugate at 18 or 27 μmol/kg survived for more than 60 days.

[00132] For the human pancreatic (BxPC-3) xenograft model, the mice were initially treated with 4 weekly injections and 3 twice a week injections of the gemcitabine-cardiolipin conjugate at doses of 18 and 36 μmol/kg or gemcitabine at a dose of 18 μmol/kg. As indicated in Figure 23 , the growth of established BxPC-3 tumor cells was significantly inhibited by treatment with the gemcitabine-cardiolipin conjugate or gemcitabine at a dose of 18 μmol/kg. On day 50, the growth inhibition by the gemcitabine-cardiolipin conjugate was 52% (P = 0.0001) and 32% (P = 0.0002) by gemcitabine. At 36 μmol/kg, the gemcitabine- cardiolipin conjugate inhibited the growth by 64% (P = 0.0001) (Figure 23).

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Claims

WHAT IS CLAIMED IS:

1. A nucleoside-cardiolipin conjugate having the following structure:

I

wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -O-, -S-, -NH-C(O)- or the like; R₁ and R₂ are the same or different are selected from a group consisting of H, saturated alkyl group and unsaturated alkyl group;

X is selected from a group consisting of H, alkyl group and a cation;

R₃ is a nucleoside selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, xanthine and hypoxanthine;

R₄ and R₅ are the same or different and are selected from a group consisting of hydrogen, hydroxyl, halo group, nitro, alkyl group, substituted alkyl and alkoxy group; R₆ is selected from a group consisting of hydrogen, hydroxyl group, azido group, amino group, alkyl group, halo group and substituted amino;

Five membered cyclic sugar is selected from a group consisting of ribofuranose, arabinofuranose, deoxyribofuranose and xylofuranose. 2. A nucleoside-cardiolipin conjugate having the following structure:

Il wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -O-, -S-, -NH-C(O)- or the like;

Ri and R₂ are the same or different are selected from a group consisting of H, saturated alkyl group and unsaturated alkyl group;

R₃ and R₇ are nucleosides selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, xanthine and hypoxanthine;

R₄ and R₅ are the same or different and are selected from a group consisting of hydrogen, hydroxyl, halo group, nitro, alkyl group, substituted alkyl and alkoxy group;

R₆ is selected from a group consisting of hydrogen, hydroxyl group, azido group, amino group, alkyl group, halo group and substituted amino;

Five membered cyclic sugar is selected from a group consisting of ribofuranose, arabinofuranose, deoxyribofuranose and xylofuranose.

3. A nucleoside-cardiolipin conjugate having the following structure:

wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -O-, -S-, -NH-C(O)- or the like; R₁ and R₂ are the same or different are selected from a group consisting of H, saturated alkyl group and unsaturated alkyl group;

X is selected from a group consisting of H, alkyl group and a cation;

R₃ is a nucleoside selected from a group consisting of cytosine, guanine, adenine, thymine, uracil, inosine, xanthine and hypoxanthine;

R₄ and R₅ are the same or different and are selected from a group consisting of hydrogen, hydroxyl, halo group, nitro, alkyl group substituted alkyl and alkoxy group; Five membered cyclic sugar is selected from a group consisting of ribofuranose, arabinofuranose, deoxyribofuranose and xylofuranose. 4. The nucleoside-cardiolipin conjugate having the following structure:

IV

wherein Y₁ and Y₂ are the same or different and are -O-C(O)-, -0-, -S-, -NH-C(O)- or the like;

R₁ and R₂ are the same or different are selected from a group consisting of H, saturated alkyl group and unsaturated alkyl group;

R₃ and R₇ are nucleosides selected from a group consisting of cytosine, guanine, adenine, thymine, uracil inosine, xanthine and hypoxanthine;

R₄ and R₅ are the same or different and are selected from a group consisting of hydrogen, hydroxyl, halo group, nitro, alkyl group, substituted alkyl and alkoxy group;

Five membered cyclic sugar is selected from a group consisting of ribofuranose, arabinofuranose, deoxyribofuranose and xylofuranose.

5. The composition of claim 1 or 3, wherein the X is an alkyl group ranging from C₁ - C₁₀.

6. The composition of claim 1 or 3, wherein X is a non-toxic cation.

7. The composition of claim 6, wherein the non-toxic cation is selected from a group consisting of ammonium, sodium, potassium, calcium and barium.

8. The composition of any of claims 1-4, wherein R₃ is optionally substituted with substituents selected from the group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, trifiuoromethyl and N(R^a)(R^b) wherein R^a and R^b are independently selected from a group consisting of H and alky] groups.

9. The composition of claim 2 or 4, wherein R₇ is optionally substituted with substituents selected from the group consisting of halo, nitro, alkyl, alkenyl, alkoxy, aryl, trifiuoromethyl and N(R^a)(R^b) wherein R^aand R^b are independently selected from a group consisting of H and alkyl groups.

10. The composition of any of claims 1-9, wherein the nucleoside is a β-nucleoside.

11. The composition of any of claims 1-9, wherein the nucleoside is an α-nucleoside.

12. The composition of any of claims 1-11, wherein the linker comprises a first end and a second end wherein said first end is attached to a lipid through a first linker functional group and said second end is attached to a nucleoside through a second linker functional group. 13. The composition of claim 12, wherein the first and second linker functional groups are selected from a group consisting of hydroxyl group, primary amino group, secondary amino group, phosphate group or substituted derivative thereof, carboxylic acid, carbonate, carbonyl and carbamate group.

14. The composition of any of claims 12-13, wherein the linker further comprises (CH₂)_n, wherein n=0-20.

15. The composition of claim 14, wherein the (CH₂),, is substituted with functional groups selected from a group consisting of alkyl, alkoxy, hydroxyl, carbonyl, carboxyl, carbamate, aldehyde, amino, halo, polyalkoxy, PEG group, phosphate, phosphonate and pyrophosphate.

16. The composition of any of claims 12-13, wherein the linker is selected from a group consisting of an alkyl, substituted alkyl, dicarbonyl alkyl, alkoxy, polyalkoxy, PEGylated group, phosphate, phosphonate, diphosphate, triphosphate, phosphodiester, phosphotriester, phosphoramidite, peptide, dipeptide and polypeptide.

17. The composition of claim 1, wherein R₃ is cytosine; R₄ and R₅ are fluoro; R₆ is hydroxyl group; X is selected from a group consisting of methyl or ammonium; Yi and Y₂ are oxo (-O-) groups; the linker is succinimidoyl group and Rj and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

18. The composition of claim 17, wherein Ri is H, CpC₃₄ saturated or unsaturated alkyl groups and R₂ is a methyl group.

19. The composition of claim 1 wherein R₃ is cytosine; R₄ and R₅ are fluoro; Rg is hydroxyl group; X is methyl or ammonium; Yi, Y₂ are -O-C(O)-; the linker is a succinimidoyl group;

Ri and R₂ are the same or different and are H, C_rC₃₄ saturated or unsaturated alkyl groups.

20. The composition of claim 3, wherein R₃ is cytosine; R₄ and R₅ are fluoro groups; X is methyl or ammonium; Y₁, Y₂ are oxo (-O-) groups;the linker is succinimidoyl group, and

Ri and R₂ are the same or different and are H, CpC₃₄ saturated or unsaturated alkyl groups.

21. The composition of claim 20, wherein Ri is H, CpC₃₄ saturated or unsaturated alkyl groups and R₂ is a methyl group.

22. The composition of claim 3, wherein R₃ is cytosine, R₄ and R₅ are fluoro group, X is methyl or ammonium, Y₁, Y₂ are -0-(CO)- groups, the linker is a succinimidoyl group, and Ri and R₂ are the same or different and are H, CpC₃₄ saturated or unsaturated alkyl groups.

23. The composition of claim 1, wherein R₃ is cytosine, R₄ and R₅ are fluoro, R₆ is hydroxyl group, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-O-) groups, and the linker is a phosphodiester or phosphotriester group and Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

24. The composition of claim 23, wherein R₁ is H, C₁-C₃₄ saturated or unsaturated alkyl groups, and R₂ is methyl group. 25. The composition of claim 1, wherein R₃ is cytosine, R₄ and R₅ are fluoro, R₆ is hydroxyl group, X and R₈ are methyl or ammonium, Yi, Y₂ are -0-(CO)- groups, and the linker is a phosphodiester or phosphotriester group, and R₁ and R₂ are the same or different and are H, Ci-C₃₄ saturated or unsaturated alkyl groups.

26. The composition of claim 3, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, X and Rs are methyl or ammonium, Y₁, Y₂ are oxo (-O-) groups, the linker is a phosphodiester or phosphotriester group and R₁ and R₂ are the same or different and are H, Cj-C₃₄ saturated or unsaturated alkyl groups.

27. The composition of claim 26, wherein R₁ is H, C₁-C₃₄ is saturated or unsaturated alkyl groups, and R₂ is a methyl group.

28. The composition of claim 3, wherein R₃ is cytosine, R₄ and R₅ are fluoro groups, X and Rg are methyl or ammonium, Yi, Y₂ are -0-(CO)- groups, and the linker is a phosphodiester or phosphotriester group and R₁ and R₂ are the same or different and are H, CpC₃₄ saturated or unsaturated alkyl groups.

29. The composition of claim 2, wherein R₃ and R₇ are cytosine, R₄ and R₅ are fluoro, R₅ is hydroxyl, Y₁, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is -CH₂CH₂CH₂NHCOCH₂CH₂C(O)-group, and R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups

30. The composition of claim 4, wherein R₃ and R₇ are cytosine, R₄ and R₅ are fluoro, Y₁, Y₂ are oxo (-O-) or -O-C(O)- groups, the linker is -CH₂CH₂CH₂NHCOCH₂CH₂C(O)-group, R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

31. The composition of claim 1, wherein R₃ is cytosine, R₄ and R₆ are hydroxyl, R₅ is hydrogen, X is methyl or ammonium, Yi, Y₂ are oxo (-O-) or— O-C(O)- groups, and the linker is succinimidoyl group, R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

32. The composition of claim 3, wherein R₃ is cytosine, R₄ is hydroxyl, R₅ is hydrogen, X is methyl or ammonium, Yi, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is succinimidoyl group, and R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

33. The composition of claim 1, wherein R₃ is cytosine, R₄ and Re are hydroxyl, R₅ is hydrogen, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is a phosphodiester or phosphotriester group, and R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

34. The composition of claim 3, wherein R₃ is cytosine, R₄ is hydroxyl and R₅ is hydrogen, X and R₈ are methyl or ammonium, Y₁, Y₂ are oxo (-0-) or -0-C(O)- groups, the linker is a phosphodiester or phosphotriester group, and R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups.

35. The composition of claim 2, wherein R₃ and R₇ are cytosine, R₄ and R^ are hydroxyl, R₅ is hydrogen, Y₁, Y₂ are oxo (-0-) or -0-C(O)- groups, the linker is - CH₂CH₂CH₂NHCOCH₂CH₂C(O)-group, and R₁ and R₂ are the same or different and are H, C₁- C₃₄ saturated or unsaturated alkyl groups.

36. The composition of claim 4, wherein wherein R₃ and R₇ are cytosine, R₄ and R₆ are hydroxyl, R₅ is hydrogen,Y_b Y₂ are oxo (-O-) or -0-C(O)- groups, the linker is -

CH₂CH₂CH₂NHCOCH₂CH₂C(O)-group, and R₁ and R₂ are the same or different and are H, CpC₃₄ saturated or unsaturated alkyl groups.

37. A composition of any of claims 1-36, wherein the nucleoside-cardiolipin conjugate is a prodrug.

38. A pharmaceutical composition comprising the nucleoside-cardiolipin conjugate of any of claims 1-37 and a pharmaceutically acceptable carrier.

39. A method of preparing a nucleoside-cardiolipin conjugate comprising reacting a cardiolipin derivative XXXXI with succinic anhydride in an inert solvent in the presence of base.

XXXXI

Wherein X^a is a protecting group;

R₁ and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups;

Y₁ and Y₂ are oxo(-O-) or -0-C(O)- groups.

40. The method of claim 39, wherein X^a is selected from a group consisting of a phosphate, methyl group, benzyl group, 2-cyanoethyl, ethyl.

41. The method of claim 39-40, further comprising the addition of a gemcitabine derivative in the presence of dicyclohexylcarbodimide (DCC), NW'-dimethyaminopyridine (DMAP) and an inert solvent.

42. The method of claim 41, wherein the gemcitabine derivative is selected from a group consisting of 4-iV-3'-O-Bis(X^b)gemcitabine _and 4-//-5'-O-Bis(X^b)gemcitabine, wherein X^b is a protecting group.

43. The method of claim 42, wherein X^b is selected from a group consisting of benzyloxycarbonyl, trityl, tert-butoxycarbonyl, benzyloxymethyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, acetyl, pivaloyl and levulinoyl.

44. The method of claim 39-43, further comprising the removal of the protecting groups.

45. The method of claim 44, wherein the removal of the protecting groups comprises the addition of an acidic medium in an inert solvent.

46. The method of claim 45, wherein the acidic medium is TFA and the inert solvent is dichloromethane. 47. The method of claim 44, wherein the removal of the protecting groups comprises catalytic hydrogenation.

48. The method of claim 44, wherein the removal of protecting groups comprises heating sodium iodide in the presence of 2-butanone or acetone.

49. The method of any of claims 39-48, further comprising the addition of dilute ammonium hydroxide.

50. The method of any of claims 39-40, further comprising the addition of N₁N,- diisopropylmethylphosphonamidic chloride in the presence of a base in an inert solvent.

51. The method of claim 50, further comprising the addition of a gemcitabine derivative in the presence of dicyclohexylcarbodimide (DCC), TVW-dimethyaminopyridine (DMAP) and an inert solvent.

52. The method of claim 51, wherein the gemcitabine derivative is selected from a group consisting of 4-Λf-3'-O-Bis(X^b)gemcitabine and 4-N-5'-O-Bis(X^b)gemcitabine, wherein X^b is a protecting group.

53. The method of claim 52, wherein X^b is selected from a group consisting of benzyloxycarbonyl, trityl, tert-butoxycarbonyl, benzyloxymethyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, acetyl, pivaloyl and levulinoyl.

54. The method of claim 50-53, further comprising removal of the protecting groups.

55. The method of claim 54, wherein the removal of the protecting groups comprises the addition of an acidic medium in an inert solvent.

56. The method of claim 55, wherein the acidic medium is TFA and the inert solvent is dichloromethane.

57. The method of claim 54, wherein the removal of the protecting groups comprises catalytic hydrogenation.

58. The method of claim 54, wherein the removal of protecting groups comprises heating sodium iodide in the presence of 2-butanone or acetone.

59. The method of any of claims 50-58, further comprising the addition of dilute ammonium hydroxide.

60. The method of any of claims 39-40, further comprising the addition of benzyl tetraisopropylphosphoramidite in the presence of lH-tetrazole.

61. The method of claim 60, further comprising the addition of a gemcitabine derivative in the presence of dicyclohexylcarbodimide (DCC), iVW-dimethyaminopyridine (DMAP) and an inert solvent.

62. The method of claim 61, wherein the gemcitabine derivative is selected from a group consisting of 4-7V-3'-O-Bis(X^b)gemcitabine and 4-N-5'-OBis(X^b)gemcitabine, wherein X^b is a protecting group. 63. The method of claim 62, wherein X^b is selected from a group consisting of benzyloxycarbonyl, trityl and tert-butoxycarbonyl, benzyloxymethyl, trimethylsilyl, tert-butyldirnethylsilyl, benzoyl, acetyl, pivaloyl, levulinoyl.

64. The method of any of claims 60-63, further comprising oxidation with a suitable oxidizing agent.

65. The method of any of claims 60-64, further comprising removal of the protecting groups.

66. The method of claim 65, wherein the removal of the protecting groups comprises the addition of an acidic medium in an inert solvent.

67. The method of claim 66, wherein the acidic medium is TFA and the inert solvent is dichloromethane.

68. The method of claim 65, wherein the removal of the protecting groups comprises catalytic hydrogenation.

69. The method of claim 65, wherein the removal of protecting groups comprises heating sodium iodide in the presence of 2-butanone or acetone.

70. The method of any of claims 60-69 further comprising the addition of dilute ammonium hydroxide.

71. A method of preparing a nucleoside-cardiolipin conjugate comprising hydrogenation of a cardiolipin derivative XXXXII.

xxxxπ

Wherein X^a is a protecting group;

Ri and R₂ are the same or different and are H, C₁-C₃₄ saturated or unsaturated alkyl groups;

Y₁ and Y₂ are oxo(-O-) or -0-C(O)- groups.

72. The method of claim 71, wherein X^a is selected from a group consisting of benzyl, ter- butyldimethylsilyl, levulinoyl, pivaloyl, methoxymethyl and benzyloxymethyl.

73. The method of any of claims 71-72, further comprising the addition of succinic anhydride.

74. The method of any of claims 71-73, further comprising the addition of a gemcitabine derivative.

75. The method of claim 74, wherein the gemcitabine derivative is selected from a group consisting of 4-N-3'-O-Bis(X^b)gemcitabine and 4-N-5'-O-Bis(X^b)gemcitabine, wherein X^b is a protecting group. 76. The method of claim 75, wherein X^b is selected from a group consisting of benzyloxycarbonyl, trityl, tert-butoxycarbonyl, benzyloxymethyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, acetyl, pivaloyl and levulinoyl.

77. The method of any of claims 71-76, further comprising the removal of protecting groups.

78. The method of claim 77, wherein the removal of the protecting groups comprises the addition of an acidic medium in an inert solvent.

79. The method of claim 78, wherein the acidic medium is TFA and the inert solvent is dichloromethane.

80. A nucleoside-cardiolipin conjugate prepared in accordance with the method of any of claims 39- 49.

81. A nucleoside-cardiolipin conjugate prepared in accordance with the method of any of claims 50- 59.

82. A nucleoside-cardiolipin conjugate prepared in accordance with the method of any of claims 60- 70.

83. A nucleoside-cardiolipin conjugate prepared in accordance with the method of any of claims 71- 79.

84. A method for preparing a liposome, comprising preparing a nucleoside-cardiolipin by any of the methods of claims 39-79 and then including said nucleoside-cardiolipin in a liposome.

85. A method of retaining an active agent in a liposome, comprising preparing a nucleoside- cardiolipin or a cardiolipin analogue by any of the methods of claims 39-79 and then including said nucleoside-cardiolipin analogue and at least one active agent in a liposome.

86. The method of claim 85, wherein the active agent is a drug.

87. The method of claim 85, wherein the active agent is selected from a group consisting of antineoplastic, antifungal and antibiotic agents.

88. The method of claim 85, wherein the active agent includes an oligonucleotide.

89. The method of any of claims 85-88, wherein the active agent becomes entrapped within the liposomes.

90. The method of any of claims 85-88, wherein the active agent becomes complexed with the nucleoside-cardiolipin analogue.

91. The method of any of claims 84-90, further comprising lyophilizing the liposomes.

92. The method of any of claims 84-91, further comprising the addition of a hydrophilic mixture.

93. A liposomal composition prepared in accordance with the method of any of claims 84-92.

94. A composition comprising the nucleoside-cardiolipin conjugate of any claims 1-37.

95. The composition of claim 94, further comprising a phosphatidylcholine.

96. The composition of any of claims 94-95, further comprising a sterol.

97. The composition of any of claims 94-96, further comprising a tocopherol. 98. The composition of claim 94, further comprising phosphatidylcholine, sterol and α-tocopherol.

99. The composition of any of claims 93-98, further comprising at least one lipid selected from a group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, cholesterol, α-tocopherol, natural or synthetic cardiolipin and analogues thereof, synthetic cationic cardiolipin analogues and derivatives thereof, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphotidylglycerol, lysophosphatidic acid, lysophosphotidylcholine, and lysophosphatidylserine. lOO.The composition of any of claims 93-99, wherein the lipids are pegylated.

101. The composition any of claims 93-99, wherein the lipids are linked to polyethylene glycol derivatives.

102. The composition of any of claims 93-101, further comprising a targeting agent. 103. The composition of claim 102, wherein the targeting agent is a protein. 104.The composition of claim 103, wherein the protein is selected from a group consisting of antibodies, antibody fragments, peptides, peptide hormones, receptor ligands and mixtures thereof. 105. The composition of any of claims 93-104, further comprising a pharmaceutically acceptable carrier. 106. The composition of any of claims 93-105, wherein said liposomes comprise vesicles having a size of about 5 μm or less. 107. The composition of claim 106, wherein said liposomes comprise vesicles having a size of about 1 μm or less. 108. The composition of claim 107, wherein said liposomes comprise vesicles having a size of about

0.5 μm or less. 109.The composition of claim 108, wherein said liposomes comprise vesicles having a size of about

0.1 μm or less. HO.The composition of any of claims 93-109, wherein said liposome is a mixture of multilamellar vesicles and unilamellar vesicles. 111. The composition of any of claims 93-109, wherein said liposome is comprised of multilamellar vesicles. 112.The composition of any of claims 93-109, wherein said liposome is comprised of unilamellar vesicles.

113. A method for the treatment of a cellular proliferative disease comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof. 114.A method for the alleviation of a cellular proliferative disease comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof. 115. A method for the treatment of a cellular proliferative disease comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof.

116. A method for the alleviation of a cellular proliferative disease comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof.

117. The method of any of claims 113-116, wherein said patient is human.

118. The method of any of claims 113-117, wherein said cellular proliferative disease is cancer.

119. The method of claim 118, wherein the cancer is selected from a group consisting of cancers of the head, neck, brain, blood, breast, lung, pancreas, bone, spleen, bladder, prostate, testes, colon, kidney ovary and skin. 120. The method of any of claims 113-119, wherein the composition is administered adjunctively with a second antineoplastic agent. 121. The method of any of claims 113-119, wherein the composition is administered prior to, concurrently with, or after the second antineoplastic agent.

122. The method of any of claim 120-121, wherein the second antineoplastic agent is radiation.

123. The method of any of claims 120-121, wherein the second antineoplastic agent is chemotherapy. 124.A method for the treatment of a viral disease comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof.

125. A method for the alleviation of a viral disease comprising administering a therapeutically effective amount of the composition of any of claims 1 -38 to a patient in need thereof.

126.A method for the treatment of a viral disease comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof.

127.A method for the alleviation of a viral disease comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof.

128. The method of any of claims 124-127, wherein said patient is human.

129. The method of any of claims 124-128, wherein the viral disease is selected from a group consisting of HIV, herpes simplex viruses, human herpes virus 6, human herpes virus 7, human herpes virus 8, orthopoxviruses, ebola virus, influenza virus, tuberculosis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, parainfluenza virus, respiratory syncytial virus,cholera, pneumonia, SARS virus, canary virus, West Nile virus (WNV), respiratory syncytial virus (RSV), dengue virus, vericella zoster virus, corona viruses, vaccinia virus, cytomegalovirus (CMV), human rhinovirus (HRV), papilloma virus (PV) and Epstein Barr virus.

130.A method for the alleviation of a bone disorder comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof.

13 LA method for the treatment of a bone disorder comprising administering a therapeutically effective amount of the composition of any of claims 1 -38 to a patient in need thereof. 132.A method for the alleviation of bone resorption comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof. 133. A method for increasing bone formation comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof. 134.A method for increasing bone mass comprising administering a therapeutically effective amount of the composition of any of claims 1-38 to a patient in need thereof. 135.A method for the alleviation of a bone disorder comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof. 136. A method for the treatment of a bone disorder comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof. 137. A method for the alleviation of bone resorption comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof. 138. A method for increasing bone formation comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof. 139. A method for increasing bone mass comprising administering a therapeutically effective amount of the composition of any of claims 93-112 to a patient in need thereof. 14O.The method of any of claims 130-139, wherein said patient is human. 141. The method of any of claims 130-140, wherein the bone disorder is selected from a group consisting of osteoporosis, Paget's disease, metastatic bone cancers, hyperparathyroidism, rheumatoid arthritis, algodistrophy, sterno-costo-clavicular hyperostosis, Gaucher's disease,

Engleman's disease. 142.The method of any of claims 130-141, wherein the composition is administered adjunctively with a second therapeutic agent. 143.The method of any of claims 130-141, wherein the composition is administered prior to, concurrently with, or after the second therapeutic agent.

144.The method of any of claims 113-143, wherein the administration is intravaneously. 145. The method of any of claims 113-143, wherein the administration is dermally. 146.The method of any of claims 113-143, wherein the administration is directly to a tumor. 147. The method of any of claims 113-143, wherein the administration is orally.

148. A kit for administering the liposomal composition of any of claims 1-38 to a mammal.

149. A kit for administering the liposomal composition of any of claims 93-112 to a mammal.

150. The kit of claim 148, wherein the kit comprises the composition of any of claims 1-38 and an instructional manual which describes the administration of the composition to the mammal.

151. The kit of claim 149, wherein the kit comprises the composition of any of claims 93-112 and an instructional manual which describes the administration of the composition to the mammal.

152. The kit of any of claims 148-151, further comprising a sterile solvent suitable for dissolving or suspending the composition prior to administering the composition to the mammal.