EP1976505A1

EP1976505A1 - Combretastatin derivatives and related therapeutic methods

Info

Publication number: EP1976505A1
Application number: EP06837549A
Authority: EP
Inventors: Chunlin Tao; Qinwei Wang; Tapas De; Neil P. Desai; Patrick Soon-Shiong
Original assignee: Abraxis Bioscience Inc USA
Current assignee: Abraxis Bioscience LLC
Priority date: 2005-11-14
Filing date: 2006-11-14
Publication date: 2008-10-08
Also published as: WO2007059118A1

Abstract

Compounds of the formula (I) wherein R1, R5, R6, R7 and R10 are H, wherein R2, R3, R4 and R8 are OMe, and wherein R9 is -X-CO-R', R' is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 5-21 carbon atoms and does not include N, and X is O, S or N, compounds related thereto, and methods for their use, particularly for modulating tumor growth or metastasis in an animal.

Description

COMBRETASTATIN DERIVATIVES AND RELATED THERAPEUTIC METHODS

FIELD OF THE INVENTION

[0001] The present invention relates the derivatives of combretastatin and related therapeutic methods.

BACKGROUND OF THE INVENTION

[0002] There exist many strategies for treating cancer. Of these, two are directed toward reducing the supply of nutrients to a tumor: antiangiogenesis and vascular targeting. Antiangiogenesis is aimed at preventing the growth of new blood vessels in a tumor. In contrast, vascular targeting utilizes agents which destroy the existing neo vasculature of a tumor in an effort to cause the selective shutdown of tumor blood vessels and extensive ischemic necrosis. See Thorpe et al. Cancer Res. 2003, 63, 1144; U.S. Patent No. 6,538,038.

[0003] Studies have identified angiogenesis and blood supply as necessary requirements for primary tumor growth (e.g., invasiveness and metastasis), and that adequate vasculature is important to the growth of tumors. Since one blood vessel nourishes thousands of tumor cells, targeting tumor vasculature as a molecular approach to cancer chemotherapy is becoming an important area of research. From this research, two drug models are emerging: a first model which prevents the formation of new blood vessels in the tumor (antiangiogenesis), and a second which targets vascular destruction as a means of limiting tumor nourishment and/or the impermeability of the luminal surface of vessel endothelial cells to cancer drugs such as immunotherapies. See New England Journal of Medicine 1998, 339, 473. The antiangiogenic model is basically a cytostatic approach wherein angiogenic factors generally produced by tumors, such as vascular endothelial growth factor (VEGF) and platelet derived endothelial cell growth factor, are blocked by antiangiogenic compounds, such as the natural polypeptides angiostatin and endostatin, thereby preventing new blood vessel growth. See The Cancer Journal Scientific American 1998, 4(4), 209; Cell, 1997, 88, 277. In contrast, the vascular destruction model is a cytotoxic approach wherein tumor vessels are targeted in order to enhance tumor cell cytotoxicity by hypoxia or direct acting chemotherapy. [0004] The selective destruction of tumor vasculature may have a significant impact on the clinical treatment of cancer. Reversal of neovascularization by damaging newly- formed vascular endothelium is expected to have a beneficial therapeutic effect. Therefore, it is desired to identity drugs to specifically damage newly-formed vasculature without affecting the normal, established vascular endothelium of the host species. TWs property is of value in the treatment of disease states associated with angiogenesis such as cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi's sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, endometriosis, dysfunctional uterine bleeding and ocular diseases with retinal vessel proliferation.

[0005] The combretastatins are a group of anti-mitotic agents isolated from the bark of the South African tree Comhretum cqffrum. The most potent member is combretastatin A-4, referred to herein as CA-4. See the Figure. CA-4 was found to be a potent inhibitor of micro tubulin assembly; significantly active against murine L1210 and P338 lymphocytic leukemia as well as multidrug resistant (MDR) cancer cell lines, and displaying potent and selective toxicity toward tumor vasculature. See U.S. Patent No. 4,996,237; Pettit et al., Experimentia, 1989, 45, 209; Lin et al., MoI. Pharmacol, 1988, 34, 200; Grosios et al., Brit. J. Cancer, 1999, 81, 1318; Lin et al., Biochemistiγ, 1989, 28, 6984; Woods et al., Brit. J. Cancer, 1995, 71, 705; McGown et al., Cancer Chemother. Pharmacol, 1990, 26, 79; El-Zayat et al., Anti-Cancer Drugs, 1993, 4, 19; Dark et al., Cancer Research, 1997, 57, 1829.

[0006] CA-4 has been demonstrated to elicit selective and irreversible shutdown of blood flow to neoplastic cells while permitting the blood supply to reach healthy cells. CA-4 is able to inhibit cell growth at low concentrations (IC5₀, P388 murine leukaemia cell line 2.6 nM), thereby destroying tumor blood vessels and effectively starving the tumors of nutrients.

[0007] Despite its very potent cytotoxic and antitubulin activities in vitro, CA-4 as an antimitotic agent showed poor antitumor effects in in vivo models. Thus, the use of CA-4 as a clinically useful anticancer agent has been limited. See Brown et al, J Chem. Soc, Perkin Tram. 1995, 1, 577.

[0008] In order to increase its efficacy, efforts have been directed to create prodrug derivatives of combretastatin A-4 that will regenerate combretastatin A-4 when exposed to physiological conditions. For example, Ohsumi et al. describe the synthesis of amino acid HCl prodrugs of a combretastatin analog, wherein an amino acid salt is attached to the amino group of a combretastatin derivative containing a basic amino group. See Ohsumi et al., Anti-Cancer Drug Design, 1999, 14, 539. Although such prodrugs may have an increased solubility compared to CA-4, they possess an inherent limitation in that the regeneration of CA-4 is dependent upon endogenous aminopeptidase in the blood of the subject to whom the prodrug is administered.

[0009] U.S. Patent No. 5,561,122 and U.S. Published Patent Application No. 20020072507A1 also describe CA-4 water-soluble prodrugs. Among them, a disodium phosphate prodrug (CA-4P) displayed potent antivascular and antitumor effects in a wide range of preclinical tumor models. CA-4P is currently in Phase II clinical trials. See Pettit et al., Anti-Cancer DrugDes., 1995, 10, 299; Hadimani et al., Bioorg. Med. Chem. Lett. 2003, 13(9), 1505.

[0010] Due to the dual antivascular/antitumor features possessed by CA-4P, a number of diverse ligands designed to mimic CA-4 have been synthesized. In vivo, CA- 4P is rapidly enzymatically converted to CA-4, a potent inhibitor of tubulin polymerization (IC_5O = 1-2 μM), and rapidly causes a decrease in blood flow in tumor tissues. Wildiers et al. evaluated the effect of different intervals and sequences of the vascular targeting agent CA-4P and CPT-11 administration on tumor growth delay and intratumoral uptake of CPT-11 using a syngeneic rhabdomyosarcoma tumor mode. See Wildiers et al., Eur. J. Cancer 2004, 40(2), 284). It was found in this study that CA-4P significantly enhances the antitumor effect of CPT-11, which is not greatly influenced by the administration sequence.

[0011] At the molecular level, both CA-4 and CA-4P target tubulin, binding strongly at or close to the colchicine binding site, preventing polymerization of α,β-tubulin heterodimer to microtubules. The inhibition of microtubule formation prevents mitosis and is important in disrupting the growth of new vascular epithelial cells. In addition, disruption of the intracellular microtubule networks by CA-4 leads to the destruction of microvessels within the tumor. This antivascular activity offers therapeutic possibilities, as the destruction of microvessels results in the death of all tumor cells which depend on the vessel for nutrients and oxygen. The multi-functional role of tubulin in both healthy and cancer cells highlights the need for selectively targeted drugs. [0012] Dark et al. showed that CA-4P causes rapid, extensive and irreversible vascular shutdown in experimental tumor models following the administration of a single dose at 1/lOth the maximum tolerated dose (MTD). See Dark, et al., Cancer Res. 1997, 57, 1829. A reduction in vascular volume of 93% was measured 6 h following drug administration. In another study, a significant increase in the hypoxic fraction of a mouse mammary carcinoma was observed 1 h after the injection of CA-4P in tumor-bearing animals. See Horsman et al., Int. J. Radia. Oncol. Biol. Phys. 1998, 42, 895. [0013] Many tubulin binding agents, such as the combretastatins and taxane analogs, however, are water-insoluble and require formulation before evaluation in the clinic. One approach that has been used successfully to overcome this clinical development problem is the formulation of biolabile water-soluble prodrugs, such as the phosphate salt derivatives of CA-4 and taxane, which allow metabolic conversion back into the water in soluble form. See Anticancer Drug Des. 1998, 13(3), 183; U.S. Patent No. 5,561,122; Bioorganic Med. Chem. Lett. 1993, 3, 1766; Bioorganic Med. Chem. Lett. 1993, 3, 1357. [0014] When the CA-4P prodrug was used in vitro and in vivo cell and animal models, it displayed a remarkable specificity for vascular toxicity. See Int. J. Radiat. Oncol. Biol. Phys. 1998, 42(4), 895; Cancer Res. 1997, 57(10), 1829). [0015] Several reports have disclosed the preparation of combretastatins as well as derivatives thereof. The synthesis of stilbene derivatives or combretastatins, which may be in the form of a pharmaceutically acceptable salt, and the pharmaceutical compositions which include them, are disclosed in U.S. Patent Nos. 4,996,237, 5,525,632, 5,731,353, 5,674,906, 6,743,937 and 6,759,555. These patents disclose combretastatins and their metabolites and disclose their in vitro oncologic activity. For example, CA-4 may be prepared from (3,4,5-trimethoxybenzyl)triphenylphosphonium salts which are condensed with a 3-hydroxy-4-methoxybenzaldehyde (the hydroxy 1 group of which is protected) in the presence of sodium hydride or of lithium derivatives, after which the desired product may be purified with standard chemical protocol. A novel synthetic method of CA-4 analogs also has been reported. See Gaukroger et al, J. Org.

Chem. 2001, 66, 8135.

[0016] There are reports that CA-4 and its derivatives can be used effectively to enhance immune responsiveness without causing vascular destruction (WO02058535A2) and in the treatment of diabetic retinopathy. See Cirla et al., Nat. Prod. Rep. 2003, 20(6),

558.

[0017] The prodrug is non-toxic even though the parent combretastatin compound may be substantially more toxic. The prodrug is hydrolyzed to the parent combretastatin compound after administration, thereby reducing the overall toxicity experienced by the patient during combretastatin therapy.

[0018] In view of the foregoing, there exists a need for compounds that provide for an enhanced biological life span and bioavailability of combretastatins, while also exhibiting anti-cancer activity and relatively low toxicity.

SUMMARY OF THE INVENTION

[0019] In one aspect, the present invention provides a compound of the foπnula:

wherein R), R₅, R₆, R₇ and Rio are H, wherein R₂, R₃, R₄ and R₈ are OMe, and wherein R₉ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 5-21 carbon atoms and does not include N, and X is O, S or NH, with the proviso that when X is O, R" is not an optionally substituted 9-fluorenyl or 9- xanthenyl. [0020] Another aspect of the present invention provides a compound of the formula:

wherein R₁, R₅, R₆, R₇ and R]₀ are H; wherein R₃, R₄, Rs, R₉ are OMe, and wherein R₂ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms, and X is O, S or NH.

[0021] A further aspect of the present invention provides a compound of the formula:

wherein Ri, R₅, R₆, R₇ and Rio are H; wherein R₄ and Rs are OMe; wherein R₂ and R₃ form -OCH2-O; and wherein R₉ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms and does not include N, and X is O₅ S or NH.

[0022] In another aspect, the present invention provides a compound of the formula:

wherein Ri, R₅, R₆ and R₇ are H; wherein R₂, R₃, R₄ and R₈ are OMe; and wherein R₉ and R_]0 are -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms, and X is O, S or NH.

[0023] Yet another aspect of the present invention provides a compound of the formula:

wherein R₁, R₅, R₆, R₇ and Rio are H; wherein R₃, R₄ and R₈ are OMe; and wherein R₂ and R₉ are -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms, and X is O, S or NH.

[0024] In a related aspect, the present invention contemplates a method of modulating tumor growth or metastasis in an animal comprising identifying an animal afflicted with a tumor growth or metastasis and administering at least one of the aforesaid compounds to the afflicted animal.

[0025] The present invention further provides pharmaceutical formulations which include one or more of the aforesaid compounds and a phaπnaceutically-acceptable carrier, as well as formulations in which the one or more compounds, when introduced into the earner, possess an average particle size of no more than about 500 nm.

[0026] The compounds and pharmaceutical formulations of the present invention may administered simultaneously with other pharmaceuticals, separately or at intervals so as to obtain the maximum efficacy of the combination; it being possible for each administration to vary in its duration from a rapid administration to a continuous perfusion.

[0027] The present invention also contemplates combination therapy, wherein the compounds of the present invention and at least one other anticancer agent are prepared as two separate pharmaceutical preparations and administered to a patient in need thereof, simultaneously, semi-simultaneously, separately or at intervals .

BRIEF DESCRIPTION OF THE DRAWING

[0028] The Figure is a graph of the percentage of CA4 concentration produced over time, for Compounds 3, 4, 5, and 6.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The compounds of the present invention are those combretastatin derivatives of formula (I):

Formula (I) wherein:

Ri, R₂, R₃, R₄, R₅, R_δ, R7, Rs, R₉ and Rio are respectively hydrogen, halogen atom, OH, OMe, OEt, OCH₂CH₂CH₃, and one or more of them are: X-R', wherein,

X is O, NH or S;

R' represents a group of formula R"-CO-, where R" represents: a hydrogen atom, alkyl, alkoxy, alkylthiol, alkylamino, alkenyl, alkynyl, having from 1 to 30 carbon atoms and which are unsubstituted or which is substituted by at least one of the substitutes (a), as defined below; an aryl group which has from 6 to 14 ring atoms and which is unsubstituted or which is substituted by at least one of substituents (b), as defined below; a heterocyclic group which has 5 or 6 ring atoms, of wliich from 1 to 3 are nitrogen and/or oxygen and/or sulphur hetero-atoms, said heterocyclic group being unsubstituted or being substituted by at least one of substituents (c), defined below; wherein the substituents (a) are: hydroxy groups; alkoxy groups wliich have from 1 to 20 carbon atoms and which are unsubstituted or are substituted by at least one alkoxy group having from 1 to 4 carbon atoms and/or alkylthio group having from 1 to 4 carbon atoms; aryl groups which have from 6 to 14 ring atoms and which are unsubstituted or wliich are substituted by at least one of substituents (b), defined below; groups of formula -NR¹R², -CONR¹R² and - OCON R¹R², wherein R¹ and R² are the same or different and each represents: a hydrogen atom; alkyl, alkoxy, alkenyl, alkynyl, having from 1 to 20 carbon atoms, which group is unsubstituted or is substituted by at least one of substituents (a) other than said groups of formula -NR¹R², -CONR¹R² and -OCON R¹R² or an aryl group in which the aryl part has from 6 to 10 ring carbon atoms and is unsubstituted or is substituted by at least one of substituents (b), defined below; or heterocyclic group being unsubstituted or being substituted by at least one of substituents (c), defined below; wherein the substituents (b) are: hydroxy groups; cyano groups; mercapto groups; halogen atoms; alkyl groups having from 1 to 8 carbon atoms; groups of formula -NR¹R², -CONR¹R² and -OCON R¹R², where R¹ and R² are as defined above; alkylthio groups having from 1 to 20 carbon atoms; heterocyclic groups which have 5 or 6 ring atoms, of which from 1 to 3 are nitrogen and/or oxygen and/or sulphur hetero-atoms, said heterocyclic group being unsubstituted or being substituted by at least one of substituents (c), defined below; and wherein the substituents (c) are: hydroxy groups; halogen atoms, cyano groups; alkyl groups having from 1 to 8 carbon atoms; aryl groups which have from 6 to 10 ring atoms and which are unsubstituted or which are substituted by at least one of substituents (b), defined above; groups of formula -NR¹R², -CONR¹R² and -OCON R¹R², where R¹ and R² are as defined above; aryl groups in which the aryl part has from 6 to 10 ring carbon atoms and is unsubstituted or is substituted by at least one of substituents (b), defined above; aliphatic carboxylic acyl groups having from 1 to 20 carbon atoms; aromatic carboxylic acyl groups in which the aryl part has from 6 to 10 ring carbon atoms and is unsubstituted or is substituted by at least one of substituents (b), defined above; provided that R₁, R₂, R₃, R₄, R₅, R₆, R₇, Rg, R₉ and Rio do not all represent hydrogen atoms and that R₁, R₂, R₃, R₄, R₅, R₆, R₇, Rs, R₉ and R₁₀ do not all represent phosphate and its pharmaceutically acceptable salts thereof.

[0030] In the compounds of the present invention, wherein R" is an alkyl group, this group desirably has from 1 to 30 carbon atoms, and may be a straight or branched chain group, or natural or unnatural fatty acid. Examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, 3-methylbutyl, 2,2- dimethylpropyl, 1,1-dimethylpropyl, hexyl, 1-methylpentyl, 4-methylpentyl, heptyl, 1- methylhexyl, 2-methylhexyl, 5-methylhexyl, 3-ethylpentyl, octyl, 2-methylheptyl, 6- methylheptyl, 2-ethylhexyl, 2-ethyl-3-methylpentyl, 3-ethyl-2-methylpentyl, nonyl, 2- methyloctyl, 7-methyloctyl, 4-ethylheptyl, 3-ethyl-2-methylhexyl, 2-ethyl-l-methylhexyl, decyl, 2-methylnonyl, 8-methylnonyl, 5-ethyloctyl, 3-ethyl-2-methylheptyl, 3,3- diethylhexyl, undecyl, 2-methyldecyl, 9-methyldecyl, 4-ethylnonyl, 3,5-dimethylnonyl, 3-propyloctyl, 5-ethyl-4-methyloctyl, 1-pentylhexyl, dodecyl, 1 -methylundecyl, 10- methylundecyl, 3-ethyldecyl, 5-propylnonyl, 3,5-diethyloctyl, tridecyl, 11- methyldodecyl, 7-ethylundecyl, 4-propyldecyl, 5-ethyl-3-methyldecyl, 3-pentyloctyl, tetradecyl, 12-methyltiidecyl, 8-ethyldodecyl, 6-propylundecyl, 4-butyldecyl, 2- pentylnonyl, pentadecyl, 13-methyltetradecyl, 10-ethyltridecyl, 7-propyldodecyl, 5-ethyl- 3-methyldodecyl, 4-pentyldecyl, 1-hexylnonyl, hexadecyl, 14-methylpentadecyl, 6- ethyltetradecyl, 4-propyltridecyl, 2-butyldodecyl, heptadecyl, 15-methylhexadecyl, 7- ethylpentadecyl, 3-propyltetradecyl, 5-pentyldodecyl, octadecyl, 16-methylheptadecyl, 5- propylpentadecyl, nonadecyl, 17-methyloctadecyl, 4-ethylheptadecyl, icosyl, 18- methylnonadecyl, 3-ethyloctadecyl, henicosyl, docosinyl, tricosinyl, tetracosinyl and pentacosinyl groups.

[0031] Substituents (a) may desirably include aryl groups, the latter which may be as defined above and exemplified below. Wherein R" is an alkyl group, the latter may be substituted to provide aralkyl groups, these groups preferably having from 1 to 4 carbon atoms in the alkyl part and from 6 to 10 carbon atoms in the aryl part. The aryl part may be substituted or unsubstituted. Examples of such aralkyl groups include the benzyl, phenethyl, l-phenylethyl, 1-, 2- and 3-phenylpropyI, 1-, 2-, 3-, 4- and 5-ρhenylpentyl and the 1- and 2-naphthylmethyl groups.

[0032] When R" is an alkoxy group, this group may desirably comprise from 1 to 20 carbon atoms, and further may be a straight or branched chain group. Examples of such groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t- butoxy, pentoxy, 3-methylbutoxy, 2,2-dimethylpropoxy, 1,1-dimethylpropoxy, hexyloxy, 1-methylpentyloxy, 4-methylpentyloxy, heptyloxy, l-methylhexyloxy₅ 2- methylhexyloxy, 5-methylhexyloxy, 3-ethylpentyloxy, octyloxy, 2-methylheptyloxy, 6- methylheptyloxy, 2-ethylhexyloxy, 2-ethyl-3-methylpentyloxy, 3-ethyl-2- methylpentyloxy, nonyloxy, 2-methyloctyloxy, 7-methyloctyloxy, 4-ethylheptyloxy, 3- ethyl-2-methylhexyloxy, 2-ethyl-l-methylhexyloxy, decyloxy, 2-methylnonyloxy, 8- methylnonyloxy, 5-ethyloctyloxy, 3-ethyl-2-methylheptyloxy, 3,3-diethylhexyloxy, undecyloxy, 2-methyldecyloxy, 9-methyldecyloxy, 4-ethylnonyloxy, 3,5- dimethylnonyloxy, 3-propyloctyloxy, 5-ethyl-4-methyloctyloxy, 1 -pentylhexyloxy, dodecyloxy, 1-methylundecyloxy, 10-methylundecyloxy, 3-ethyldecyloxy, 5- propymonyloxy, 3,5-diethyloctyloxy, tridecyloxy, 11-methyldodecyloxy, 7- ethylundecyloxy, 4-propyldecyloxy, 5-ethyl-3-methyldecyloxy, 3-pentyloctyloxy, tetradecyloxy, 12-methyltridecyloxy, 8-ethyldodecyloxy, 6-propylundecyloxy, 4- butyldecyloxy, 2-pentykionyloxy, pentadecyloxy, 13-methyltetradecyloxy, 10- ethyltridecyloxy, 7-propyldodecyloxy, 5-ethyl-3-methyldodecyloxy, 4-pentyldecyloxy, 1- hexylnonyloxy, hexadecyloxy, 14-methylpentadecyloxy, 6-ethyltetradecyloxy, 4- propyltridecyloxy, 2-butyldodecyloxy, heptadecyloxy, 15-methylhexadecyloxy, 7- ethylpentadecyloxy, 3-propyltetradecyloxy, 5-pentyldodecyloxy, octadecyloxy, 16- methylheptadecyloxy, 5-propylpentadecyloxy, nonadecyloxy, 17-methyloctadecyloxy, 4- ethylheptadecyloxy, icosyloxy, 18-methylnonadecyloxy, and 3-ethyloctadecyloxy groups.

[0033] When R" is an alkenyl group, this group desirably includes from 2 to 30 carbon atoms, and further may be a straight or branched chain group and, preferably, is a natural or unnatural fatty acid. It may have 1 or more, and preferably from 2 to 6, double bonds. Examples of such groups include vinyl, allyl, 1-propenyl, isopropenyl, 2-methyl- 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 8-nonenyl, 1-nonenyl, 1-decenyl, 9-decenyl, 8-tridecenyl, cis-8- pentadecenyl, frαrø-8-pentadecenyl, 8-heptadecenyl, 8-heptadecenyl, 8,11- heptadecadienyl, 8,11,14-heptadecatrienyl, 4,7,11,14-nonadecatetraenyl and 2,6- dimethy l-8-(2 ,6,6-trimethyl- 1 -cyclohexen- 1 -yl)- 1 ,3 ,5 ,7-nonatetraen- 1 -yl, cis- 10- nonadecaenyl, 10, 13-nonadecadienyl, cis-1, 10, 13-nonadecatrienyl, 5,8,11,14- nonadecatetraenyl, nonadecapentaenyl, henecosatetraenyl, henecosapentaenyl, and henecosahexaenyl.

[0034] When R" is an alkynyl group, it desirably possesses from 2 to 10 carbon atoms, and may be a straight or branched chain group. Examples of such groups include ethynyl, 1-propynyl, propargyl, 1-heptynyl, 1-octynyl and 1-decynyl groups. [0035] When R" represents an aryl group, it desirably has from 6 to 14 ring atoms, in a single ring or in multiple rings. Examples include phenyl, 1-naphthyl, 2-naphthyl, fluorenyl, 1-anthryl and 1-phenanthryl groups. Such groups may be substituted or unsubstituted, and, if substituted, the substituents may be selected from substituents (a), as defined above. Examples include phenoxy, 1-naphthyloxy, 2-naphthyloxy, fluorenyloxy, 1-anthryloxy and 1-phenanthryloxy groups.

[0036] When R" is a heterocyclic group, it desirably possesses 5 or 6 ring atoms. Of these atoms, it is preferable that 1 to 3 are nitrogen and/or oxygen and/or sulphur hetero- atoms. Where there are 3 hetero-atoms, it is desirable that at least one (and more desirably 2) be a nitrogen atom and one or two should be nitrogen, oxygen or sulphur atoms (and, where there are two, they may be the same or different). When it is desired to include two hetero-atoms, these may be the same or different and they are nitrogen and/or oxygen and/or sulphur atoms; however, more preferably one is a nitrogen atom and the other is a nitrogen, oxygen or sulphur atom. Such groups may be unsubstituted or they may be substituted by at least one (preferably from 1 to 3) of substituents (c), as defined and exemplified above. Examples of such unsubstituted groups include furyl, thienyl, pyrrolyl, pyridyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, pyranyl, pyrazinyl, pyridazinyl, pyrimidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, thiazolidinyl, thiazolinyl, isothiazolinyl, imidazolidinyl, imidazolinyl, oxazolinyl, isoxazolinyl, oxazolidinyl, pyrazolidinyl, piperazinyl, dioxopiperazinyl, tetrahydropyrimidinyl, dihydropyridazinyl, morpholinyl, tliiomorpholinyl, pyrrolidonyl, piperidonyl, pyridonyl, 2H-pyrrolyl, furazanyl and pyrazolinyl groups, especially the furyl, pyrrolyl, pyridyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyridazinyl, pyrrolidinyl, thiazolinyl, isothiazolinyl, imidazolyl, piperazinyl, dioxopiperazinyl, morpholinyl, pyrrolidonyl and piperidonyl groups. Such groups may be unsubstituted or they may be substituted by at least one of substituents (c), as defined above and exemplified below.

[0037] When R" is a cycloalkyl group, the latter desirably possesses from 3 to 8 carbon atoms, and may be unsubstituted or substituted. If substituted, it may be substituted by at least one of substituents (c), as defined above and exemplified below. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl groups and substituted analogues thereof. [003S] When R" represents a cycloalkenyl group, the latter desirably has from 5 to 8 carbon atoms, and also may be unsubstituted or substituted. If substituted, it may be substituted by at least one of substituents (c), as defined above and exemplified below. More desirably, it may have one or more, preferably 1 or 2, and more preferably 1, carbon-carbon double bond or bonds. Examples of such groups include 1-cyclopenten-l- yl, 2-cyclopenten-l-yl, 1-cyclohexen-l-yl, 2-cyclohexen-l-yl, 3-cyclohexen-l-yl, 1- cylohepten-1-yl, 2-cyclohepten-l-yl, 1-cycloocten-l-yl and 3-cycloocten-l-yl groups and substituted analogues thereof.

[0039] Examples of groups and atoms which may be included in substituents (a) include: Hydroxy groups; chlorine, fluorine, bromine and iodine atoms; cyano group; mercapto groups; carboxy groups; alkoxy groups which have from 1 to 20 carbon atoms and which are unsubstituted or are substituted by at least one alkoxy group having from 1 to 4 carbon atoms and/or alkylthio group having from 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentyloxy, methoxymethoxy, 1-methoxyethoxy, 2-methoxy ethoxy, butoxymethoxy, 2- butoxyethoxy, 3-ethoxypropoxy, 3 -methyl butoxy, 2,2-dimethylpropoxy, 1,1- dimethylpropoxy, hexyloxy, 1-methylpentyloxy, 4-methylpentyloxy, heptyloxy, 1- methylhexyloxy, 2-methylhexyloxy, 5-methylhexyloxy, 3-ethylpentyloxy, octyloxy, 2- methylheptyloxy, 6-methylheptyloxy, 2-ethylhexyloxy, 2-ethyl-3-methylpentyloxy, 3- ethyl-2-methylpentyloxy, nonyloxy, 2-methyloctyloxy, 7-methyloctyloxy, 4- ethylheptyloxy, 3-ethyl-2-methylhexyloxy, 2-ethyl-l-methylhexyloxy, decyloxy, 2- methybionyloxy, 8-methylnonyloxy, 5-ethyloctyloxy, 3-ethyl-2-methylheptyloxy, 3,3- diethylhexyloxy, undecyloxy, 2-methyldecyloxy, 9-methyldecyloxy, 4-ethylnonyloxy, 3,5-dimethylnonyloxy, 3-propyloctyloxy, 5-ethyl-4-methyloctyloxy, 1-pentylhexyloxy, dodecyloxy, 1-methylundecyloxy, 10-methylundecyloxy, 3-ethyldecyloxy, 5- propylnonyloxy, 3,5-diethyloctyloxy, tridecyloxy, 11-methyldodecyloxy, 7- ethylundecyloxy, 4-propyldecyloxy, 5-ethyl-3-methyldecyloxy, 3-pentyloctyloxy, tetradecyloxy, 12-methyltridecyloxy, 8-ethyldodecyloxy, 6-propylundecyloxy, 4- butyldecyloxy, 2-pentylnonyloxy, pentadecyloxy, 13-methyltetradecyloxy, 10- ethyltridecyloxy, 7-propyldodecyloxy, 5-ethyl-3-methyldodecyloxy, 4-pentyldecyloxy, 1- hexylnonyloxy, hexadecyloxy, 14-methylpentadecyloxy, 6-ethyltetradecyloxy, A- propyltridecyloxy, 2-butyldodecyloxy, heptadecyloxy, 15-methylhexadecyloxy, 7- ethylpentadecyloxy, 3-propyltetradecyloxy, 5-pentyldodecyloxy, octadecyloxy, 16- methylheptadecyloxy, 5-propylpentadecyloxy, nonadecyloxy, 17-methyloctadecyloxy, 4- ethylheptadecyloxy, icosyloxy and 18-methylnonadecyloxy, 3-ethyloctadecyloxy groups; aryl groups which have from 6 to 14 ring atoms and which are unsubstituted or which are substituted by at least one of substituents (b), defined above and exemplified below, such as the phenyl, 1-naphthyl, 2-naphthyl, fluorenyl, 1-anthryl and 1-phenanthryl groups and substituted analogues thereof; alkylthio groups having from 1 to 20 carbon atoms, such as the methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, t-butyithio, pentylthio, 3-methylbutyltliio, 2,2-dimethylpropylthio, 1,1-dimethylpropylthio, hexylthio, 1-methylpentyltlτιio, 4-methylpentylthio, heptylthio, 1-methylhexylthio, 2- methylhexylthio, 5-methylhexylthio, 3-ethylpentylthio, octylthio, 2-methylheptylthio, 6- methylheptyltliio, 2-ethylhexylthio, 2-ethyl-3-methylpentylthio, 3-ethyl-2- methylpentylthio, nonylthio, 2-methyloctylthio, 7-methyloctylthio, 4-ethylheptylthio, 3- ethyl-2-methylhexylthio, 2-ethyl- 1-methylhexylthio, decylthio, 2~methylnonylthio, 8- methylnonylthio, 5-ethyloctyltliio, 3-ethyl-2-methylheptylthio, 3,3-diethylhexyltliio, undecylthio, 2-methyldecylthio, 9-methyldecylylthio, 4-ethylnonylthio, 3,5- dimethylnonylthio, 3-propyloctylthio, 5-ethyl-4-methyloctylthio, 1-pentylhexylthio, dodecylthio, 1-methylundecylthio, 10-methylundecylthio, 3-ethyldecylthio, 5- propylnonylthio, 3,5-diethyloctylthio, tridecylthio, 11-methyldodecylthio, 7- ethylundecylthio, 4-propyldecylthio, 5-ethyl-3-methyldecylthio, 3-pent)doctyltliio, tetradecylthio, 12-methyltridecylthio, 8-ethyldodecylthio, 6-propylundecyltliio, 4- butyldecylthio, 2-pentylnonyltlτio, pentadecyltliio, 13-methyltetradecylthio, 10- eth}dtridecylthio, 7-propyldodecylthio, 5-ethyl-3-methyldodecylthio, 4-pentyldecylthio, 1-hexylnonylthio, hexadecylthio, 14-methylpentadecylthio, 6-ethyltetradecylthio, 4- propyltridecylthio, 2-butyldodecylthio, heptadecylthio, 15-methylhexadecylthio, 7- ethylpentadecyltliio, 3-propyltetradecylthio, 5-pentyldodecylthio, octadecyltlαio, 16- methylheptadecylthio, 5-propylpentadecyltliio, nonadecylthio, 17-methyloctadecylthio, 4- ethylheptadecyltliio, icosyltliio, 18-methylnonadecylthio and 3-ethyloctadecyltliio groups; groups of formula -NR¹R², -CONR¹R² and -OCON R¹R², where R¹ and R² are the same or different and each represents, a hydrogen atom; an alkyl group having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and t- butyl groups. [0040] Examples of the groups and atoms which may be included in substituents (b) include: alkyl groups having from 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, 3-methylbutyl, 2,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl, 1-methylpentyl, 4-methylpentyl, heptyl, 1 -methylhexyl, 2- methylhexyl, 5 -methylhexyl, 3-ethylpentyl, octyl, 2-methylheptyl, 6-methylheptyl, 2- ethylhexyl, 2-ethyl-3-methylpentyl and 3-ethyl-2-methylpentyl groups; and hydroxy, alkoxy, aryl, aryloxy, aliphatic acyloxy, aromatic acyloxy, carboxy, alkoxycai'bonyl, aryloxycarbonyl, mercapto, alkylthio, arylthio, aralkylthio, aralkyldithio, aiyldithio, alkyldithio, alkylsulphinyl, arylsulphinyl, alkylsulphonyl, arylsulphonyl, cyano, aliphatic and aromatic acyl and heterocyclic groups and groups of formula -NR¹R², -CONR¹R² and -OCON R¹R², wherein R¹ and R² are as defined above, and halogen atoms, as exemplified in relation to substituents (a).

[0041] Substituent (c) desirably may be an oxygen atom, forrn an oxo group (>C=O) with the carbon atom to which it is attached, or it may be various other groups and atoms as exemplified above in relation to substituents (a) and (b). [0042] Preferred classes of compounds of the present invention include those compounds of formula (I) in which among of Ri, R₂, R₃, R₄, R₅, R^ R7, Rg, R9 and Rio at least one of them represents a group of formula R"-CO-, wherein the group represented by R" is selected from: unsubstituted alkyl, alkenyl, or alkynyl groups having from 6 to 22 carbon atoms; substituted alkyl^", alkenyl, or alkynyl groups having from 6 to 22 carbon atoms and substituted by at least one of substituents: such as hydroxy groups, protected hydroxy groups, amino groups, protected amino groups, groups of formula -NR³R⁴, wherein:

R³ and R⁴ are the same or different and each represents an alkyl group having from 1 to 4 carbon atoms, an alkylcarbonyl group having a total of from 2 to 5 carbon atoms, a benzoyl group, a substituted benzoyl group in which the substituents are selected from substituents (b) as defined above; mercapto groups, protected mercapto groups, alkoxy groups having from 1 to 8 carbon atoms, alkylthio groups having 1 or 2 carbon atoms, phenyl groups, substituted phenyl groups in which the substituents are selected from substituents (b) as defined above, phenoxy groups, substituted phenoxy groups in which the substituents are selected from substituents (b) as defined above, cyano groups, alkylcarbonyl groups having from 2 to 9 carbon atoms, benzoyl groups, carbamoyloxy groups, heterocyclic groups having 5 or 6 ring atoms, of which 1 is a nitrogen and/or oxygen and/or sulphur hetero-atom, and halogen atoms.

[0043] More preferred classes of compounds of the present invention include those compounds of formula (I), R₁, R₂, R₃, R_t, R₅, R_O, R7, R_S5 R₉ and R₁₀ that are hydrogen or methoxy and at least one of them represents a group of formula R" -CO-, wherein the group represented by R" is selected from: unsubstituted alkyl, alkenyl, or alkynyl groups having from 3 to 25 carbon atoms; substituted alkyl, alkenyl, or alkynyl groups having from 3 to 25 carbon atoms and substituted by at least one of substituents: hydroxy groups, protected hydroxy groups, amino groups, protected amino groups, carboxy groups, protected carboxy groups, mercapto groups, protected mercapto groups, alkoxy groups having from 1 to 8 carbon atoms and alkylthio groups having 1 or 2 carbon atoms.

[0044] Various embodiments of the present invention include compounds of formula 1, wherein R_b R₅, R₆, and R₇ are H; R₂ is OMe, -X-CO-R" (wherein X and R" are, independently, as defined herein) or, together with R₃, forms -OCH₂-O; R₃ is OMe or, together with R₂, forms -OCH₂-O; R₄ and Rs are OMe; R₉ is OMe, or -X-CO-R" (wherein X and R" are, independently, as defined herein); and Rio is H or -X-CO-R" (wherein X and R" are, independently, as defined herein).

[0045] Preferred compounds useful in the embodiments of the present invention as described herein include:

(a) compounds of the foπnula:

wherein R₁, R₅, R₆, R₇ and Rio are H, wherein R₂, R₃, R₄ and Rs are OMe₅ and wherein R₉ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 5-21 carbon atoms (with the alky and alkenyl being most preferred) and, desirably, does not include N, and X is O, S or NH, with the proviso that when X is O₅ R" is not an optionally substituted 9-fluorenyl or 9-xanthenyl. Of these compounds, it is more preferable if X is O and/or R" is one of

-CH₂(CHs)₃CH₃, -(CH₂)₇CH=CH(CH₂)₇CH₃ or -CH₂CH₂(CH₂=CHCH₂)₆CH₃; (b) compounds of the formula:

wherein R_h R₅, R₆₅ R₇ and Ri₀ are H; wherein R₃, R₄, Rg, R₉ are OMe, and wherein R₂ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21, and more desirably 5-21, carbon atoms(with the alky and alkenyl being most preferred), and X is O₅ S or NH. Of these compounds, it is more preferable if X is O and/or R" is one of-CH₂(CH₂)₃CH₃,

-(CH₂)₇CH=CH(CH₂)₇CH₃ or-CH₂CH₂(CH₂=CHCH₂)₆CH₃. Preferred compounds include those in which R" comprises 2-21, and more preferably 5-21, carbon atoms and/or does not include N;

(c) compounds of the formula:

wherein R₁, R₅, R₆, R₇ and Rio are H; wherein R₄ and Rs are OMe; wherein R₂ and R₃ form -0CH2-0; and wherein Rg is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21, and desirably 5-21, carbon atoms (with the alkyl and alkenyl being most preferred) and does not include N, and X is O, S or NH. Of these compounds, it is more preferable if X is O and/or R" is one of - CH₂(CH₂)₃CH₃, -(CH₂)₇CH=CH(CH₂)₇CH₃ or

-CH₂CH₂(CH₂=CHCH₂)₆CH₃. Preferred compounds include those in which R" comprises 2-21, and more preferably 5-21, carbon atoms and/or does not include N; (d) compounds of the formula:

wherein R₁, R₅, R₆ and R₇ are H; wherein R₂, R₃, R₄ and R₈ are OMe; and wherein Rg and Rio are -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21, and desirably 5-12, carbon atoms (with the alky and alkenyl being most preferred), and X is O, S or NH. Of these compounds, it is more preferable if X is O and/or R" is one of-CH₂(CH₂)₃CH₃,

-(CH₂)₇CH=CH(CH₂)₇CH₃ or -CH₂CH₂(CH₂=CHCH₂)₆CH₃. Preferred compounds include those in which R" comprises 2-21, and more preferably 5-21, carbon atoms and/or does not include N; and (e) compounds of the formula:

wherein Ri, Rs, R₆, R₇ and R₁₀ are H; wherein R3, R₄ and R₈ are OMe; and wherein R₂ and R₉ are -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21, and desirably 5-21, carbon atoms (with the alky and alkenyl being most preferred), and X is O, S or NH. Of these compounds, it is more preferable if X is O or N and/or R" is one of -CH₂(CH₂)_SCH₃, -(CH₂)₇CH=CH(CH₂)₇CH₃ or- CH₂CH₂(CH₂=CH-CH₂)₆CH₃. Preferred compounds include those in which R" comprises 2-21, and more preferably 5-21, carbon atoms and/or does not include N. [0046] In any of the compounds of the present invention which include a hydroxy, amino, mercapto or carboxy group, any of these groups may be protected by a suitable protecting group. Where the protecting group is on a compound intended for use merely as a chemical intermediate, its nature is not critical to the invention; and any of the well known protecting groups may be employed. Where the resulting compound is intended for therapeutic use, the protecting group should be pharmaceutically acceptable. [0047] Examples of hydroxy-protecting groups include: formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl. benzoyl, 4-nitrobenzoyl, 2-nitrobenzoyl, tetrahydropyran- 2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran- 2-yl and 4-methoxytetrahydrothiopyran-4-yl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t- butylsilyl, triisopropylsilyl, diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl, methoxymethyl, 1-ethoxyethyl, 1- (isopropoxy)ethyl, 2,2,2-trichloroethyl, benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphlhyldiphenylmethyl and 9-anthrylmethyl, 4- methylbenzyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4- chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl. [0048] The most preferred classes of compounds of the present invention include those compounds of formula (I), wherein Ri, R₂, R₃, R₄, Rs, R_<5, R7_> Rs₅ Rg and R₁O are hydrogen or methoxy and at least one of them represents a group of formula R"-CO-, wherein the group represented by R" is selected from: unsubstituted alkyl, alkenyl, or alkynyl groups having from 7 to 21 carbon atoms; preferably the natural or unnatural fatty acids. substituted alkyl, alkenyl, or alkynyl groups having from 7 to 21 carbon atoms and substituted by at least one of substituents: hydroxy groups, alkylamino groups, alkoxy groups having from 1 to 8 carbon atoms.

[0049] The compounds of the invention may contain asymmetric carbon atoms and/or carbon-carbon double bonds and can, therefore, form optical and/or cisl trans isomers. Although these are all referred to herein by a single formula, the present invention envisages both mixtures of the isomers as well as the individual isolated isomers. [0050] Many of the compounds of the present invention have anti -tumor activity, as referred to herein. Others may be of value as intermediates in the preparation of other compounds of the present invention, which may have greater relative activity. [0051] Examples of specific compounds of the present invention are those compounds of formula (I), as described above, in which R of formula (II) are defined in the following Table 1.

(II) Table 1

[0052] Among the compounds in this invention, the lipophilic combretastatin analogs are preferred:

Undecanoylcombretastatin -A4; lauroylcombretastatin -A4; tridecanoylcombretastatin -A4; myristoylcombretastatin -A4; pentadecanoy lcombretastatin -A4 ; palmitoyl combretastatin -A4; heptadecanoyl combretastatin -A4; stearoylcombretastatin -A4; palmitoleoylcombretastatin -A4; oleoylcombretastatin -A4; linolenoylcombretastatin -A4; eicosanoylcombretastatin -A4; eicosatetraenoylcombretastatin -A4 ; docosahexacnoylcombretastatin -A4.

[0053] Fatty acids, as constituents of phospholipids, make up the bulk of cell membranes. Due to their lipophilic nature, fatty acids can easily partition into and interact with the cell membrane in a non-toxic way. In general, increased lipophilicity has been suggested as a mechanism for enhancing intestinal uptake of drugs into the lymphatic system, thereby enhancing the entry of the conjugate into the brain and also thereby avoiding first-pass metabolism of the conjugate in the liver. The type of lipophilic molecules employed have included phospholipids, non-naturally occurring branched and unbranched fatty acids, and naturally occurring branched and unbranched fatty acids ranging from as few as 4 carbon atoms to more than 30 carbon atoms. [0054] Fatty acids of specific types are of considerable interest both for the maintenance of healthy tissue and in the treatment of various diseases. Some fatty acids are of interest in themselves, some because they give rise to particular metabolites such as prostaglandins or other oxygenated derivatives, and some for both reasons. Among them, are the essential fatty acids (EFAs) not made by the body and therefore constituting essential nutrients. Among the EFAs of particular interest for both reasons are gamma- linolenic acid (GLA), dihomogammalinolenic acid (DGLA) and eicosapentaenoic acid (EPA). DGLA is an important component of cell membranes and is also the precursor of prostaglandin Ei (PGEi); PGEi has many desirable effects being an antithrombotic, antiinflammatory, vasodilator, immunomodulating and cholesterol lowering agent. GLA is an immediate precursor of DGLA and is rapidly converted to DGLA within the body. EPA is also a component of cell membranes and is a precursor of PGE₃ which has actions similar to those of PGEi. A further fatty acid which is of particular interest as a component of cell membranes is docosahexaenoic acid (DHA), which is also an important constituent of the brain. U.S. Patent No. 6,602,902 disclosed DHA- pharmaceutical agent conjugates to improve tissue selectivity relative to the unconjugated pharmaceutical agents. The conjugates, in general, render the activity of these compounds selective for colon tissue, breast tissue and central nervous system tissue. Fatty acids previously have been conjugated with drugs to help the drugs as conjugates cross the blood brain barrier.

[0055] It is known that nervous tissues are very rich in essential polyunsaturated fatty acids and polyunsaturated fatty acids play an important role in normal cerebral development. Thus, dietary deficiency of essential unsaturated fatty acids or the disruption of their metabolism during a period of cerebral development might affect myelinization in man. U.S. Patent Nos. 5,604,216 and 5,654,290 disclosed the fatty acid based drugs, especially polyunsaturated fatty acid derivatives.

[0056] Recently, Menendez et al. reported that in laboratory experiments, oleic acid, the main monounsaturated fatty acid in olive oil, suppressed one of the most important genes, Her-2/neu (erb B-2) expression, involved in breast cancer. They found that oleic acid also synergistically enhances the growth inhibitory effects of trastuzumab (Herceptine) in breast cancer cells with Her-2/neu oncogene amplification. See Menendez et al., Annals of Oncology, Advance Access, published January 10, 2005. [0057] A major goal in the pharmacological arts has been the development of methods and compositions to facilitate the specific delivery of therapeutic and other agents to the appropriate cells and tissues that would benefit from such treatment, and the avoidance of the general physiological effects of the inappropriate delivery of such agents to other cells or tissues of the body.

[0058] It is preferred that the conjugate be of a single fatty acid molecule and the covalent attachment be directly to the combretastatin derivatives through a linkage or directly conjugating to the combretastatin skeleton. It was postulated that the lipid molecule interacted with the phospholipid membrane to act as a distill anchor for the receptor ligand in the membrane micro environment of the receptor. [0059] The preparation of combretastatin A-4 is illustrated in Scheme 1. The synthesis of the stilbenes using the Perkin condensation (Perkin, W. H. J. Chem. Soc. 1868, 21, 181; Perkin, W. H. J. Chem. Soc, 1877, 31, 388.), described by Letcher et al. (Letcher et al., J. Chem. Soc, Perkin Trans. 1 1972, 206) and Gaukroger et al., Org. Chem., 2001, 66 (24) 8135. The mixture of 3,4,5-trimethoxyphenylacetic acid , 3- hydroxy-4-methoxybenzaldehyde, triethylamine, and acetic anhydride was heated at reflux. Upon cooling, the reaction mix was acidified with concentrated hydrochloric acid, and the cinnamic acid was precipitated from the solution. TWs acid was isolated and recrystallized from in ethyl acetate/hexanes in about 41% yield. Decarboxylation of the acid was achieved using copper chromite and quinoline at about 190-200⁰C for about 15 minutes. The pure cis combretastatin A-4 acetate was purified in good yield (ca. 70- 80%) using flash column chromatography, following by recrystallization from ethyl acetate/hexanes.

[0060] Alternatively, the mixture was further treated with sodium hydroxide to provide the desired product CA-4, after twice recrystallization from toluene, in 65-70% yield. HPLC analysis of the crude reaction mix showed a Z:E ratio of 90:10, but following recrystallization this changed to 99.5:0.5. [0061] Scheme 1

NaOH DMF

[0062] Other cis combretastatin A-4 analog could be prepared in the similar procedures.

[0063] The compounds of the present invention may be prepared by a variety of methods well known for preparing acylated compounds from the corresponding hydroxy compound.

[0064] For example, the acylation products as in formula (I) of the present invention are prepared by reaction of a compound of foπnula (II):

Formula (III)

with a compound of formula (FV): R₃' -COOH (IV)

in which R₃' represents any of the groups or atoms represented by R" or any such group in which any active group or atom is protected, and R" is as defined above or with a reactive derivative thereof, and, if required, removing any protecting group, to give said compound of formula (I).

[0065] If a carboxylic acid of formula (IV) is used in the form of the free acid, the reaction is preferably effected in the presence of a suitable condensation agent, for example l,r-oxalyldiimidazole, 2,2'-dipyridyl disulphide, N₉N'- dicyclohexylcarbodiimide, N,N'-disuccinimidyl carbonate, N,N'-bis(2-oxo-3- oxazolidinyl)phosphinic chloride, N,N'-carbodiimidazole, N,N'-disuccinimidyl oxalate, N,N'-diphthalimide oxalate, N,N'-bis(norbornenylsuccinimidyl) oxalate, 1,1'- bis(benzotriazolyl)oxalate, l,r-bis(6-chlorobenzotriazolyl) oxalate or l,l'-bis(6- trifluoromethylbenzotriazoly 1) oxalate .

[0066] When a condensation agent is employed, the reaction is preferably conducted in the presence of an inert solvent. There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: aliphatic hydrocarbons, such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons, such as benzene, toluene and xylene; halogenated hydrocarbons, especially aromatic and aliphatic hydrocarbons, such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and the dichlorobenzenes; esters, such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane. dimethoxyethane and diethylene glycol dimethyl ether; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; nitro compounds, which may be nitroalkanes or nitroaranes, such as nitroethane and nitrobenzene; nitriles, such as acetonitrile and isobutyronitrile; amides, which may be fatty acid amides, such as formamide, dimethylformamide, dimethylacetamide and hexamethylphosphoric triamide; and sulphoxides, such as dimethyl sulphoxide and sulpholane.

[0067] The reaction may take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. In general, it was found convenient to cany out the reaction at a temperature of from 0⁰C to 100⁰C.

[0068] The compound of the present invention may be obtained by reaction of a compound of formula (III) as defined above with one and more equivalents of a carboxylic acid and a condensation agent or of a reactive derivative of the carboxylic acid.

[0069] Any acid can be used for the invention to obtain the desired product, such as aliphatic acids, aromatic carboxylic acids, and heterocyclic carboxylic acids, preferably, the fatty acids. These acids may contain one or more unsaturated bonds in the molecule and carry one or more substituents such as halogen atoms, amino groups and hydroxyl groups, such as various kinds of fart)' acid and amino acids. The illustrative examples are fatty acids with 5 to 30 carbons and their derivatives.

[0070] Unbranched, naturally occurring fatty acids embraced by aspects of the invention include propionic acid (C3), butyric acid (C4), valeric acid (C5), hexanoic acid

(C6), 4-methylhexanoic acid (C6), octanoic acid (C8), decanoic acid (ClO), lauric acid

(C 12), myristic acid (C 14), palmitic acid (C 16), palmitoleic acid (C 16), stearic acid

(CIS), oleic acid (CIS), vaccenic acid (CIS), linoleic acid (CIS), α-linolenic acid (CIS), eleostearic acid (C 18), 6-linolenic acid (C 18), eicosanoi acid (C20), cis-l 1-eicosenoic acid (C20), 11,14-eicosadienoic acid (C20), cis-S,l 1,14-eicosatrienoic acid (20),

5,8,11,14-eicosatetraenoic acid (20), eicosapentaenoic acid (C20), docosenoic acid (C22), erucic acid (C22), docosatetraenoic acid (C22), docosapentaenoic acid (C22), docosahexacnoic acid (C22), tetracosanoic acid (C24), nervonic (C24), octacosanoic acid

(C28), and triacontanoic acid (C30). Highly preferred unbranched, naturally occurring fatty acids are those with between 12 and 22 carbon atoms.

[0071] Examples of reactive derivatives of the carboxylic acid of formula (IV) include: acid halides, such as the acid chloride and the acid bromide; and acid anhydrides. [0072] When an acid anhydride is employed, the reaction is preferably carried out in the presence of an inert organic solvent, or in the absence of an inert organic solvent by using an excess of the acid anhydride. There is no particular restriction on the nature of the solvent which may be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: aromatic hydrocarbons, such as benzene, toluene and xylene; and ethers, such as dioxane, tetrahydrofuran and diethylene glycol dimethyl ether.

[0073] The reaction may be carried out with a compound in Formula (III), 2 to 10 molar equivalent of the acid anhydride, and a base (such as tetraethyl amine and pyridine). The mixture is stirred at room temperature to about 0-100 ⁰C under nitrogen for 5-48 h. After cooling to room temperature, the mixture was concentrated and purified with silica gel column to afford the desired product.

[0074] In this way, any anhydrides can be used for the invention to obtain the desired product in Formula (I), such as aliphatic, aromatic carboxylic, and heterocyclic carboxylic anhydride. Generally, the anhydride can be commercial available, or the anhydride can be prepared from the corresponding acid with the general synthetic method in the prior art.

[0075] When an acid halide is employed, the reaction is preferably conducted in an inert organic solvent in the presence of an acid binding agent. The acid binding agent may be any such compound which binds to an acid and does not interfere with the reaction, and examples include: alkali metal hydroxides, such as sodium hydroxide and potassium hydroxide; alkali metal carbonates, such as sodium carbonate and potassium carbonate; and organic bases, such as triethylamine, pyridine, 4-dimethylaminopyridine and 1-methylimidazole. The reaction is carried out with the corresponding acid chloride in the presence of acid binding agent in anhydrous solvent. There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: aromatic hydrocarbons, such as benzene, toluene and xylene; halogenated hydrocarbons, especially halogenated aliphatic hydrocarbons, such as chloroform, methylene chloride and trichloroethane; ethers, such as diethyl ether, tetrahydrofuran and dioxane; aliphatic dialkylamides, which may be fatty acid amides, such as dimethylformamide and dimethylacetamide; nitriles, such as acetonitrile; ketones, such as acetone; dimethyl sulphoxide; and pyridine. [0076] The reaction can take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. In general, we find it convenient to carry out the reaction at a temperature of from -10⁰C to 100⁰C. [0077] Thus, any acid chloride can be used for the invention to obtain the desired product in Formula (III), such as aliphatic, aromatic carboxylic, and heterocyclic carboxylic acid chloride. Generally, the acid chloride can be commercial available or, the acid chloride can be prepared from the corresponding acid with the general synthetic method in the prior art. For example, the acid chloride derivative of the fatty acid can be prepared by reacting the pure fatty acid with thionyl chloride. Then the combretastatin fatty acid ester can be made by mixing the fatty acid chloride with combretastatin in the presence of dichloromethane and pyridine. After workup, the residue can then be subjected to column chromatography. Other methods of preparation may be used by those skilled in the art.

[0078] When the compound of formula (III) contains a protecting group, the desired compound of formula (I) can be obtained by its removal, if necessary. [0079] When the removal of a protecting group is required, the reaction employed will, as is well known in the art. For example, if a silyl group is employed as the hydroxy-protecting group, it can generally be removed by treatment with a compound capable of forming fluorine anions, such as tetrabutylammonium fluoride. The reaction is preferably carried out in the presence of a solvent. There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include ethers, such as tetrahydrofuran and dioxane. When the reaction substrate contains a sulphur atom, aluminium chloride- sodium iodide is preferably employed. [0080] The compounds of the present invention may be prepared by a variety of methods well known for preparing acylated compounds from the corresponding hydroxy compound. For example, compounds of formula (I) may be prepared by reacting a compound of formula (III):

with a compound of formula (V):

R₃' -NCO (V)

or with a compound of formula (VI):

R₃' -OCOCl (VI)

[0081] The reaction of a compound of formula (III) with a compound of formula (V) can be carried out by methods known in the art. The reaction is preferably conducted in the presence of an inert solvent. There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: aliphatic hydrocarbons, such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons, such as benzene, toluene and xylene; halogenated hydrocarbons, especially aromatic and aliphatic hydrocarbons, such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene, ethyl acetate, diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane. Dimethoxyethane, diethylene glycol dimethyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetonitrile, formamide, dimethylformamide, dimethylacetamide, dimethyl and sulphoxide.

[0082] The reaction may take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. In general, it was found convenient to cany out the reaction at a temperature of from O⁰C to 100⁰C. [0083] The reaction of a compound of foπnula (III) with a compound of formula (VI) may be canned out by methods known in the art. The reaction is preferably conducted in an inert organic solvent in the presence of an acid binding agent. There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: aromatic hydrocarbons, such as benzene, toluene and xylene; halogenated hydrocarbons, especially halogenated aliphatic hydrocarbons, such as chloroform, methylene chloride and trichloroethane; ethers, such as diethyl ether, tetrahydrofuran and dioxane; aliphatic dialkylamides, which may be fatty acid amides, such as dimethylformamide and dimethylacetamide; nitriles, such as acetonitrile; ketones, such as acetone; dimethyl sulphoxide; and pyridine. The acid binding agent may be any such compound which binds to an acid and does not interfere with the reaction, and examples include: alkali metal hydroxides, such as sodium hydroxide and potassium hydroxide; alkali metal carbonates, such as sodium carbonate and potassium carbonate; and organic bases, such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine and 1-methylimidazole. [0084] The reaction may take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. In general, it was found convenient to carry out the reaction at a temperature of from -10 ⁰C to 100 ⁰C. [0085] Alternatively, the compound of the formula (I) may be prepared in the following two step procedure: a) preparing the intermediates in formula (VII) from the compounds of the formula (III), b) treatment of VII with the compounds in Formula (VIII).

(VII)

R3'-Y (VIII)

[0086] In compound (VII), X' is chloride, imidazole, or 4-nitrophenyloxy, and in compound (VIII), Y is OH, NH₂, or SH, with R₃' representing any of the groups or atoms represented by R" or any such group in which any active group or atom is protected, and R" is as defined above or with a reactive derivative thereof, and, if required, removing any protecting group, to give said compound of formula (I). [0087] The intermediates of Formula (VII) can be prepared by the reaction of a compound in Formula (III) with phosgene, triphosgen, diimidazolecabonate, or A- nitrophenyl chlorformate in the presence of a base, preferably an organic base such as triethylamine, in an inert organic solvent such as dichloromethane. The reaction is usually carried out in a temperature range of -60° C to 90° C, most commonly at around ambient temperature.

[0088] The reaction of a compound of formula (VII) with a compound of formula (VIII) can be earned out by methods known in the art. The reaction is preferably conducted in an inert organic solvent, or in the presence of an acid binding agent. There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: aromatic hydrocarbons, such as benzene, toluene and xylene; halogenated hydrocarbons, especially halogenated aliphatic hydrocarbons, such as chloroform, methylene chloride and trichloroethane; ethers, such as diethyl ether, tetrahydrofuran and dioxane; aliphatic dialkylamides, which may be fatty acid amides, such as dimethylformamide and dimethylacetamide; nitriles, such as acetonitrile; ketones, such as acetone; dimethyl sulphoxide; and pyridine. The acid binding agent may be any such compound which binds to an acid and does not interfere with the reaction, and examples include: alkali metal hydroxides, such as sodium hydroxide and potassium hydroxide; alkali metal carbonates, such as sodium carbonate and potassium carbonate; and organic bases, such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine and 1- methylimidazole.

[0089] The reaction can take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. Ln general, we find it convenient to carry out the reaction at a temperature of from -10⁰C to 100 ⁰C. [0090] If the hydroxy-protecting, amino-protecting group, mercapto-protecting or carboxy-protecting group need to be removed to generate the desired products in Formula (I), it can be carried out with the methods in the prior art. See Greene et al., Protective Groups in Organic Synthesis, 3^rd edition, John Wiley & Sons, 1999. [0091] There is no particular restriction on the nature of the solvent to be employed, provided that it has no adverse effect on the reaction or on the reagents involved and that it can dissolve the reagents, at least to some extent, and any solvent commonly used for hydrolysis reactions may equally be used here. Examples of suitable solvents include: water; and mixtures of water with an alcohol, such as methanol, ethanol or propanol, or with an ether, such as tetrahydrofuran or dioxane. The reaction can take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. Ln general, we find it convenient to carry out the reaction at a temperature of from 0⁰C to 150⁰C, depending on the starting materials and the base employed, in order to inhibit any side reaction. The time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents. [0092] There is no particular limitation to the form of administration of the antitumor agents. Anticancer agents are routinely administered intravenously, parenterally, and orally.

[0093] The compounds of the present invention may be administered as a pharmaceutical composition containing the compounds and a pharmaceutically acceptable earner or diluent. The active materials can also be mixed with other active materials which do not impair the desired action and/or supplement the desired action. The active materials according to the present invention can be administered by any acceptable route including, but not limited to, orally, parenterally, intravenously, intradermal Iy, subcutaneously, through an inhaler or topically, in liquid or solid form. [0094] Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the aforesaid compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.

[0095] The tablets, pills, capsules and the like may contain the following ingredients: a binder such as macrocrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, corn starch and the like; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added. When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically or veterinarially pure and non-toxic in the amounts used. [0096] For the puiposes of parenteral therapeutic administration, the active ingredient may be incorporated into a solution or suspension. The solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0097] The pharmaceutical forms suitable for injectable use include sterile solutions, dispersions, emulsions, and sterile powders. The final form must be stable under conditions of manufacture and storage. Furthermore, the final pharmaceutical form must be protected against contamination and must, therefore, be able to inhibit the growth of microorganisms such as bacteria or fungi. A single intravenous or intraperitoneal dose can be administered. Alternatively, a slow long teπn infusion or multiple short term daily infusions may be utilized, typically lasting from 1 to 8 days. Alternate day or dosing once every several days may also be utilized.

[0098] Sterile, injectable solutions are prepared by incorporating a compound in the required amount into one or more appropriate solvents to which other ingredients, listed above or known to those skilled in the art, may be added as required. Sterile injectable solutions are prepared by incorporating the compound in the required amount in the appropriate solvent with various other ingredients as required. Sterilizing procedures, such as filtration, then follow. Typically, dispersions are made by incorporating the compound into a sterile vehicle which also contains the dispersion medium and the required other ingredients as indicated above. In the case of a sterile powder, the preferred methods include vacuum drying or freeze drying to which any required ingredients are added.

[0099] Suitable pharmaceutical earners include sterile water; saline, dextrose; dextrose in water or saline; condensation products of castor oil and ethylene oxide combining about 30 to about 35 moles of ethylene oxide per mole of castor oil; liquid acid; lower alkanols; oils such as com oil; peanut oil, sesame oil and the like, with emulsifiers such as mono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin, and the like; glycols; polyalkylene glycols; aqueous media in the presence of a suspending agent, for example, sodium carboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and the like, alone, or with suitable dispensing agents such as lecithin; polyoxyethylene stearate; and the like. The carrier may also contain adjuvants such as preserving stabilizing, wetting, emulsifying agents and the like together with the penetration enhancer. In all cases the final form, as noted, must be sterile and must also be able to pass readily through an injection device such as a hollow needle. The proper viscosity may be achieved and maintained by the proper choice of solvents or excipients. Moreover, the use of molecular or particulate coatings such as lecithin, the proper selection of particle size in dispersions, or the use of materials with surfactant properties may be utilized.

[00100] In accordance with the present invention, there are provided compositions of combretastatin derivative and methods useful for the in vivo delivery of combretastatin derivatives in the form of nanoparticles, which are suitable for aforesaid any route administrations.

[00101] U. S. Patent Nos. 5,916,596, 6,506,405 and 6,537,579 teach the preparation of nanoparticles from the biocompatible polymers, such as albumin. These are incorporated by reference in there entirety. Thus, in accordance with the present invention, there are provided methods for the formation of nanoparticles of present invention by a solvent evaporation technique from an oil-in- water emulsion prepared under conditions of high shear forces (e.g., sonication, high pressure homogenization, or the like). [0100] Thus, in accordance with the present invention, combretastatin derivative is dissolved in a water miscible organic solvent (e.g., a solvent having greater than about 10% solubility in water, such as, for example, ethanol) is added to the oil phase at a final concentration in the range of about l%-99% v/v, more preferably in the range of about 5%-25% v/v of the total organic phase. The water miscible organic solvent can be selected from such solvents as ethyl acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethyl formamide, methyl pyrrolidinone, and the like. Alternatively, the mixture of water immiscible solvent with the water miscible solvent is prepared first, followed by dissolution of the pharmaceutically active agent in the mixture.

[0101] Next, a protein (e.g., human serum albumin) is added (into the aqueous phase) to act as a stabilizing agent for the formation of stable nanodroplets. Protein is added at a concentration in the range of about 0.05 to 25% (w/v), more preferably in the range of about 0.5%-5% (w/v). Unlike conventional methods for nanoparticle formation, no surfactant (e.g. sodium lauryl sulfate, lecithin, tween 80, pluronic F-68 and the like) is added to the mixture. Optionally, a sufficient amount of the first organic solvent (e.g. chloroform) is dissolved in the aqueous phase to bring it close to the saturation concentration. A separate, measured amount of the organic phase (which now contains the pharmacologically active agent, the first organic solvent and the second organic solvent) is added to the saturated aqueous phase, so that the phase fraction of the organic phase is between about 0.5%- 15% v/v, and more preferably between 1% and 8% v/v. An emulsion is formed by homogenization under high pressure and high shear forces. Such homogenization is conveniently carried out in a high pressure homogenizer, typically operated at pressures in the range of about 3,000 up to 30,000 psi. Preferably, such processes are earned out at pressures in the range of about 6,000 up to 25,000 psi. The resulting emulsion comprises very small nanodroplets of the nonaqueous solvent (containing the dissolved pharmacologically active agent) and very small nanodroplets of the protein stabilizing agent. Acceptable methods of homogenization include processes imparting high shear and cavitation such as high pressure homogenization, high shear mixers, sonication, high shear impellers, and the like.

[0102] Finally, the solvent is evaporated under reduced pressure to yield a colloidal system composed of protein coated nanoparticles of pharmacologically active combretastatin derivative and protein. Acceptable methods of evaporation include the use of rotary evaporators, falling film evaporators, spray driers, freeze driers, and the like. Thus, a colloidal dispersion system (pharmacologically active agent and protein) in the form of nanoparticles of about 10-500 nm is obtained. In addition, under particular conditions, extremely small nanoparticles, i.e., particles in the range of about 10 nm-200 nm diameter are obtained that can be sterile-filtered. The preferred size range of the particles is between about 50 nm-150 nm, depending on the formulation and operational parameters.

[0103] Colloidal systems prepared in accordance with the present invention may be further converted into powder form by removal of the water, e.g., by lyophilization at a suitable temperature-time profile. The protein (e.g., human serum albumin) itself acts as a cryoprotectant, and the powder is easily reconstituted by addition of water, saline or buffer, without the need to use such conventional cryoprotectants as mannitol, sucrose, glycine, and the like. While not required, it is of course understood that conventional cryoprotectants may be added to invention formulations if so desired. [0104] The polymeric shell containing solid or liquid cores of pharmaco logically active agent allows for the delivery of high doses of the pharmacologically active agent in relatively small volumes. This minimizes patient discomfort at receiving large volumes of fluid and minimizes hospital stay. In addition, the walls of the polymeric shell or coating are generally completely degradable in vivo by proteolytic enzymes (e.g., when the polymer is a protein), resulting in no side effects from the delivery system as is the case with current formulations.

[0105] A number of biocompatible materials may be employed in the practice of the present invention for the formation of a polymeric shell. Several biocompatible materials may be employed in the practice of the present invention for the formation of a polymeric shell. For example, naturally occurring biocompatible materials such as proteins, polypeptides, oligopeptides, polynucleotides, polysaccharides (e.g., starch, cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, and the like), lipids, and so on, are candidates for such modification.

[0106] Examples of suitable biocompatible materials, naturally occurring or synthetic proteins, that may be employed include albumin, insulin, hemoglobin, lysozyme, immunoglobulins, a-2-macroglobulin, fibronectin, vitronectin, fibrinogen, casein and the like, as well as combinations of any two or more thereof. Similarly, synthetic polymers are also good candidates for preparation of the drug formulation. In this regard, polyalkylene glycols (e.g., linear or branched chain), polyvinyl alcohol, polyacrylates, polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamides, polyisopropyl acrylamides, polyvinyl pyrrolidinone, polylactide/glycolide and the like, and combinations thereof, are good candidates for the biocompatible polymer in the invention formulation.

[0107] These biocompatible materials may also be employed in several physical forms such as gels, crosslinked or uncrosslinked to provide matrices from which the pharmacologically active ingredient, for example paclitaxel, may be released by diffusion and/or degradation of the matrix. Temperature sensitive materials may also be utilized as the dispersing matrix for the invention formulation. Thus for example, the camptothecin diester may be injected in a liquid formulation of the temperature sensitive material (e.g., copolymers of polyacrylamides or copolymers of polyalkylene glycols and polylactide/glycolides) which gel at the tumor site and provide slow release of combretastatin derivatives.

[0108] Particles of biologic substantially completely contained within a polymeric shell, or associated therewith, prepared as described herein, are delivered neat, or optionally as a suspension in a biocompatible medium. This medium may be selected from water, buffered aqueous media, saline, buffered saline, optionally buffered solutions of amino acids, optionally buffered solutions of proteins, optionally buffered solutions of sugars, optionally buffered solutions of carbohydrates, optionally buffered solutions of vitamins, optionally buffered solutions of synthetic polymers, lipid-containing emulsions, and the like.

[0109] In addition, the polymeric shell can optionally be modified by a suitable agent, wherein the agent is associated with the polymeric shell through an optional covalent bond. Covalent bonds contemplated for such linkages include ester, ether, urethane, diester, amide, secondary or tertiary amine, phosphate ester, sulfate ester, and the like bonds. Suitable agents contemplated for this optional modification of the polymeric shell include synthetic polymers (polyalkylene glycols (e.g., linear or branched chain polyethylene gfycol), polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamide, polyvinyl pyrrolidinone, and the like), phospholipids (such as phosphatidyl choline (PC)₅ phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI)₅ sphingomyelin, and the like), proteins (such as enzymes, antibodies, and the like), polysaccharides (such as starch, cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, and the like), chemical modifying agents (such as pyridoxal 5'-phosphate, derivatives of pyridoxal, dialdehydes, diaspirin esters, and the like), or combinations of any two or more thereof.

[0110] The prepared nanoparticle with this invention can be administered by any acceptable route including, but not limited to, orally, intramuscularly, transdermally, intravenously, through an inhaler or other air bome delivery systems, and the like. When preparing the composition for injection, particularly for intravenous delivery, the continuous phase preferably comprises an aqueous solution of tonicity modifiers, buffered to a pH below 7, more preferably below 6.

[0111] The prepared nanoparticles of this invention may be enclosed in a hard or soft capsule, may be compressed into tablets, or may be incorporated with beverages, food or otherwise into the diet. Capsules may be formulated by mixing the nanoparticle with a pharmaceutical diluent which is inert and inserting this mixture into a hard gelatin capsule having the appropriate size. If soft capsules are desired a slurry of the compound with an acceptable vegetable, light petroleum, or other inert oil can be encapsulated by machine into a gelatin capsule. The percentage of the final composition and the preparations may, of course, be varied and may conveniently range between 1 and 90% of the weight of the final foπn, e.g., tablet. The amount in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions according to the current invention are prepared so that an oral dosage unit form contains between about 5 to about 50% by weight (% w) in dosage units weighing between 50 and 1000 mg.

[0112] The lipophilic compounds in this invention will be more easily passed through the cell membranes and distributed tissues and cross the blood brain barrier. The tissue can be tissue of the Blood and Blood Forming system: including platelets, blood vessel wall, and bone marrow; Cardiovascular system: including heart and vascular system; Digestive and excretory system: including alimentary tract, biliary tract, kidney, liver, pancreas and urinary tract; Endocrine system: including adrenal gland, kidney, ovary, pituitary gland, renal gland, salivary gland, sebaceous gland, testis, thymus gland and thyroid gland; Muscular system: including muscles that move the body. Reproductive System: including breast, ovary, penis and uterus; Respiratory system: including bronchus, lung and trachea; Skeletal system: including bones and joints; Tissue, fiber, and integumentary system: including adipose tissue, cartilage, connective tissue, cuticle, dermis, epidermis, epithelium, fascia, hair follicle, ligament, bone marrow, melanin, melanocyte, mucous membrane, skin, soft tissue, synovial capsule and tendon. [0113] Accordingly, the combretastatin derivatives of the present invention can be used in warm-blooded animals, including humans, as an anti-tumor agent against these kinds of tumoral diseases. The compounds may be administered by any convenient route, for example by parenteral administration methods, such as intravenous injection, subcutaneous injection, intramuscular injection or by suppositories; or oral administration by using, for example, capsules, powders or granules.

[0114] The dosage to an adult human may vary depending on the nature of the disease, the route of administration and the administration frequency and period. However, a daily dosage of from 1 to 100 mg in a single dose or in divided doses may be given.

[0115] For example, compositions for injection can be provided in the form of ampoules, each containing a unit dose amount, or in the form of a container containing multiple doses. The composition may sometimes contain additives such as emulsifiers, stabilizers and/or dispersants, and may often be in the form of a powder which is intended to be dissolved by the pharmacist in a suitable solvent, such as a pyrogen-free sterilized aqueous solvent, just before use. Such a preparation can be prepared, for example, as follows: the radicicol derivative is dissolved in acetone, and the acetone solution is poured into vials, water is added, and then the mixture is lyophilized. On the other hand, compositions for oral administration can be provided by means of capsules, powders, granules or syrups each containing a suitable amount of one or more of the radicicol derivatives of the present invention. [0116] In accordance with the present invention, the compounds of the present invention are used to treat cancers which include but are not limited to tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas. The compounds of the present invention are used to treat cancers of the liver and biliary tree (particularly hepatocellular carcinoma), intestinal cancers, particularly colorectal cancer, ovarian cancer, small cell and non-small cell lung cancer, breast cancer, sarcomas (including fibrosarcoma, malignant fibrous histiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma, neuro-fibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part sarcoma), neoplasms of the central nervous systems (particularly brain cancer), lymphomas (including Hodgkin's lymphoma, lymphoplasmacytoid lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, B- lineage large cell lymphoma, Burkitt's lymphoma, and T-cell anaplastic large cell lymphoma).

[0117] The antiangiogenic treatment defined hereinbefore may be applied as a sole therapy or may involve, in addition to a compound of the invention, one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. In the field of medical oncology it is normal practice to use a combination of different forms of treatment to treat each patient with cancer. In medical oncology the other component(s) of such conjoint treatment in addition to the antiangiogenic treatment defined hereinbefore may be: surgery, radiotherapy or chemotherapy. Such chemotherapy may include the following categories of therapeutic agent:

(a) other antiangiogenic agents that work by different mechanisms from those defined hereinbefore (for example linomide, inhibitors of integrin .alpha, function, angiostatin, endostatin, razoxin, thalidomide) and including vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors (RTKIs) (for example those described in International Patent Applications Publication Nos. WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354); (b) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene), progestogens (for example megestrol acetate), aromatase inhibitors (for example anastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (for example flutamide, nilulamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (for example goserelin acetate, luprolide), inlαibitors of testosterone 5.alpha.-dihydroreductase (for example finasteride), anti-invasion agents (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function) and inhibitors of growth factor function, (such growth factors include for example epidermal growth factor (EGF), platelet derived growth factor and hepatocyte growth factor such inhibitors include growth factor antibodies, growth factor receptor antibodies, tyrosine kinase inhibitors and serine/threonine kinase inhibitors);

(c) biological response modifiers (for example interferon);

(d) antibodies (for example edrecolomab); and

(e) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as antimetabolites (for example antifolates like methotrexate, fluoropyrimidines like 5-fluorouracil, purine and adenosine analogues, cytosine arabinoside); antirumour antibiotics (for example anthracyclines like doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (for example cisplatin, carboplatin); alkylating agents (for example nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic agents (for example vinca alkaloids like vincristine and taxoids like taxol, taxotere); enzymes (for example asparaginase); thymidylate synthase inhibitors (for example raltitrexed); topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan, irinotecan). [0118] As stated above the compounds defined in the present invention are of interest for their vascular damaging effects. Such compounds of the invention are expected to be useful in the prophylaxis and treatment of a wide range of disease states where inappropriate angiogenesis occurs including cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi's sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, endometriosis, dysfunctional uterine bleeding and ocular diseases with retinal vessel proliferation. In particular such compounds of the invention are expected to slow advantageously the growth of primary and recurrent solid tumors of, for example, the colon, breast, prostate, lungs and skin.

[0119] In addition to their use in therapeutic medicine, the compounds of formula (I) and their pharmaceutically acceptable salts, solvates or pro-drugs are also useful as pharmacological tools in the development and standardization of in vitro and in vivo test systems for the evaluation of the effects of vascular damaging agents in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.

[0120] Another aspect of the present invention contemplates the use of kits for the administration of the compounds, nanoparticles and compositions described herein. [0121] In certain embodiments the kits may include a unit dosage amount of at least one of the therapeutic compounds or composition as disclosed herein. Kits may further comprise suitable packaging and/or instructions for use of the compound or composition. Kits may also comprise a means for the delivery of the inventive compounds or compositions, such as a syringe for injection or other device as described herein and known to those of skill in the art.

[0122] Additionally, the compounds and compositions thereof may be assembled in the form of kits. The kit may provide the compounds or compositions thereof and reagents to prepare a pharmaceutical composition for administration. The composition may be in a dry or lyophilized form, or in a solution, particularly a sterile solution. When the composition is in a dry form, the reagent may comprise a pharmaceutically acceptable diluent for preparing a liquid formulation. Such diluents include those known to those of skill in the art and are additionally described herein.

[0123] The kit may also contain a device for administration or for dispensing the compositions, including, but not limited to syringe, pipette, or other device known to those of skill. When in a wet form, the composition may be stored in an ampoule or other sterile sealed container, including those known to persons of skill in the art. [0124] The kits may include other therapeutic compounds for use in conjunction with the compounds described herein. In one embodiment, the therapeutic agents are other anticancer agents. These agents may be provided in a separate form, or mixed with the compounds of the present invention, provided such mixing does not reduce the effectiveness of either the additional therapeutic agent of the compositions and formulations described herein. Similarly the kits may include additional agents for adjunctive therapy. For example, agents to reduce the adverse effects of the drug {e.g., anti-nausea agents, anti-alopecia agents, immuno-enhancing agents, etc.). [0125] The kits will include appropriate instructions for preparation and administration of the compound/composition, side effects of the compound/compositions, and any other relevant information. The instructions may be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, or optical disc.

[0126] In a particular embodiment of the invention, kits for treating an individual who suffers from or is susceptible to the conditions described herein are provided, comprising a first container comprising a dosage amount of the inventive compounds or compositions as disclosed herein, and instructions for use. The container may be any of those known in the art and appropriate for storage and delivery of intravenous formulations. In certain embodiments the kit further comprises a second container comprising a pharmaceutically acceptable earner, diluent, adjuvant, etc. for preparation of the composition to be administered to the individual.

[0127] Kits may also be provided that contain sufficient dosages of the compounds or compositions as disclosed herein to provide effective treatment for an individual for an extended period, such as a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, S weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. [0128] Kits may also include multiple unit doses of the inventive compounds and compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding phaπnacies. [0129] In certain embodiments, the kit may comprise appropriately packaged oral dosage forms and instructions for use. [0130] In particular embodiments of the kits described herein, the kits comprise a at last one inventive compound or a composition thereof, wherein the compound is an inventive compound listed in Tables 1 or 2.

[0131] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE l

[0132] This example illustrates the preparation of Compound 1. A mixture of 3- hydroxy-4-methoxybenzaldehyde (9.0Og, 59.15 mmol), 3,4,5-trimethoxyphenyl acetic acid (6.0Og, 26.52 mmol), acetic anhydride (12.0 mL), and triethylamine (6.0 mL) was heated under reflux for 5 hours. After acidification with concentrated hydrochloric acid (18.0 mL), water (100 mL) was added and the mixture was extracted by ethyl acetate. The organic layer was dried by sodium sulfate and concentrated to leave a gum. The crude product was purified by column chromatography on silica gel, eluted by 20-75% ethyl acetate in hexanes (~0.5% acetic acid was used for the later potions). The desired product was collected, concentrated, and crystallized from ethyl acetate-hexanes to give a product of 1 as white solid (3.7Og, 35%): ¹H NMR (500 MHz, CDCl₃) δ 7.81 (IH, S, HIa'), 7.00 (IH, dd, J = 8.62, J = 2.12 H6'), 6.79 (IH, d, J = 8.72, H5'), 6.74 (IH, d, J = 2.24, H2'), 6.46 (2H, s, H2,6), 3.89 (3H, s, 4-OCH₃), 3.80 (3H, s, 4'-OCH₃), 3.78 (6H, s, 3,5-OCH₃), 2.24 (3H, s, CH₃CO); MS calculated for (C₂₁H₂₂O₈Na) 425, found 425 (MNa⁺).

EXAMPLE 2

[0133] This example illustrates the preparation of Compound 2. To a solution of Compound 1 (1.0Og, 2.49 mmol) in dioxane (1OmL) and ethanol (ImL), there was added a solution of NaOH (0.50Og) in water (3 mL) and the mixture was stirred at room temperature overnight. The solution was acidified with dilute HCl (IN, 50 mL) and a

( precipitate was formed. After filtration, the solid was recrystallized from EtOAc-EtOH-

Hexane to give Compound 2 as white crystals (0.83 g, 89%): see Table 2 for the ¹H NMR data; MS calculated for (Ci₉H₂₀O₇Na) 383, found 383 (MNa⁺). EXAMPLE 3

[0134] This example illustrates the preparation of CA-4. Compound 1 (1.61 g, 3.99 mmol) was treated with acetic anhydride (15.0 mL)-pyridine (15 mL) for 2 hours at 90⁰C and the solvents were evaporated to give a gum. The resulting gum was mixed with quinoline (15 mL) and copper chromite (0.41 g, 1.30 mmol), and heated under reflux for 15 minutes under argon. Ethyl acetate was added and the product was filtered off through Celite. The filtrate was washed with dilute hydrochloric acid until pH 1~ 2, water, dried (Na₂SO4) and concentrated to leave a residue. To a solution of this residue in DMF (18 mL) there was added a solution of NaOH (1.20 g) in water (7.50 mL) and the mixture was stirred at room temperature for 1 hour. Water was added and the mixture was extracted by ethyl acetate. The combined organic layer was washed by water, dried (Na₂SO₄), and concentrated to leave a residue. The residue was purified by column chromatography on silica gel (eluting solvent: 0-40% ethyl acetate in hexanes) yielding a mixture of cis /trans isomers as an oil. The oil was crystallized with ethyl ether-ethyl acetate-hexanes to give CA-4 as white crystals (0.75 g, 59%): see Table 2 for the ¹H NMR data; MS calcd for (Ci₈H₂₀O₅Na) 339, found 339 (MNa⁺); HPLC retention time 8.181 minute, 98.95%.

EXAMPLE 4

[0135] TMs example illustrates the preparation of Compound 3. To a solution of CA- 4 (0.19 g, 0.60 mmol) in anhydrous dichloromethane (10 mL) there was added thiethylamine (0.21 mL, 1.51 mmol) and the mixture was cooled to 0 ⁰C. Hexanoly chloride (0.13 mL, 0.91 mmol) was added and the mixture was stirred at 0 ⁰C to room temperature overnight. Ethyl acetate was added and the mixture was washed by 5% NaHCO₃, water, dried (Na₂SO₄) and concentrated to leave a residue. The residue was purified by column chromatography on silica gel (eluting solvent: 0-20% ethyl acetate in hexanes) yielding Compound 3 as an oil (0.24 g, 97%): ¹H NMR (500 MHz, CDCl₃) δ 7.11 (IH₅ dd, J = 8.51, J = 2.13, H6')_s 7.00 (IH, d, J = 2.09, H2'), 6.84 (IH, d, J = 8.51, H5'), 6.51 (2H₃ s, H2, 6), 6.47 (IH, d, .T - 12.23, HIa), 6.44 (IH, d, J = 12.22, HIa'), 3.83 (3H, s, 4-OCH₃), 3.80 (3H, s, 4'-OCH₃), 3.71 (6H, s, 3,5-OCH₃), 2.52 (2H, t, J =7.49, CH₂CO)₅ 1.73 (2H, m, CH₂CH₂CO), 1.37 (4H, m, 2xCH₂), 0.91 (3H₅ 1, J = 6.94, CH₃); MS calculated for (C₂₄H₃₀O₆Na) 437, found 437 (MNa⁺); HPLC retention time 28.512 minute, 99.63%.

EXAMPLE S

[0136] This example illustrates the preparation of Compound 4. To a solution of CA- 4 (0.16 g, 0.51 mmol) in anhydrous dichloromethane (10 mL) there was added thiethylamine (0.18 mL, 1.26 mmol) and the mixture was cooled to 0 ⁰C. Lauroyl chloride (0.18 mL, 0.76 mmol) was added and the mixture was stirred at room temperature for 2.5 hours. Ethyl acetate was added and the mixture was washed by 5% NaHCO₃, water, dried (Na₂SO₄) and concentrated to leave a residue. The residue was purified by column chromatography on silica gel (eluting solvent: 0-10% ethyl acetate in hexanes) yielding Compound 4 as an oil (0.24 g, 93%): ¹H NMR (500 MHz, CDCl₃) δ 7.11 (IH, dd, J - 8.43, J = 2.01, H6'), 7.00 (IH, d, J = 2.15, H2'), 6.84 (IH, d, J = 8.46, H5'), 6.50 (2H, s, H2, 6), 6.47 (IH, d, J = 12.18, HIa), 6.43 (IH, d, J = 12.27, HIa'), 3.83 (3H, s, 4-OCH₃), 3.79 (3H, s, 4'-OCH₃), 3.70 (6H, s, 3,5-OCH₃), 2.52 (2H, t, J =7.49, CH₂CO), 1.71 (2H, m, CH₂CH₂CO), 1.38 (2H, m, CH₂), 1.30 (14H, m 7xCH₂), 0.88 (3H, t, J = 6.87, CH₃); MS calculated for (C₃₀H₄₂O₆Na) 521, found 521 (MNa⁺); HPLC retention time 41.611 minute, 99.02%.

EXAMPLE 6

[0137] This example illustrates the preparation of Compound 5. To a solution of CA- 4 (0.20 g, 0.63 mmol) in anhydrous dichloromethane (10 mL) there was added linoleic acid (0.25 mL, 0.79 mmol) and DMAP (catalytic amount). A solution of DCC (0.16 g, 0.79 mmol) in anhydrous dichloromethane (2 mL) was added to the above mixture and the resulting solution was stirred at room temperature for 2.5 hours. Hexane was added and the product was filtered off by gravity filtration. After removal of the solvents, the residue was purified by column chromatography on silica gel (eluting solvent: 0-15% ethyl acetate in hexanes) yielding Compound 5 as an oil (0.22g, 60%): ¹H NMR (500 MHz, CDCl₃) δ 7.11 (IH, dd, J = 8.45, J = 2.07, H6'), 6.99 (IH, d, J = 2.09, H2'), 6.83 (IH, d, J = 8.57, H5'), 6.50 (2H, s, H2, 6), 6.45 (IH, d, J = 12.22, HIa), 6.43 (IH, d, J = 12.15, HIa'), 5.36 (4H₅ m, 4xCH), 3.83 (3H, s, 4-OCH₃), 3.79 (3H, s, 4'-OCH₃), 3.70 (6H, s, 3,5-OCH₃), 2.772H, t, J = 7.49, CHCH₂CH), 2.52 (2H, t, J = 6.73, CH₂CO), 2.05 (4H, m, 2xCH₂CH), 1.71 (2H, m, CH₂CH₂CO), 1.32 (14H, m 7xCH₂), 0.88 (3H, t, J = 6.79, CH₃); MS calculated for (C₃₆H₅₀O₆Na) 601, found 601 (MNa⁺); HPLC retention time 51.08 minute, 93.52% (the impurity peak at 29.83 min (3.50%) may be from the HPLC system).

EXAMPLE 7

[0138] This example illustrates the preparation of Compound 6. To a solution of CA- 4 (0.20 g, 0.63 mmol) in anhydrous dichloromethane (10 mL) there was added oleic acid (0.21 mL, 0.66 mmol) and DMAP (catalytic amount). A solution of EDC (N-(3- dimethylaminopiOpyl)-N'-ethylcarbodiimide hydrochloride) (0.14 g, 0.66 mmol) in anhydrous dichloromethane (2 mL) was added to the above mixture and the resulting solution was stirred at room temperature overnight. The mixture was diluted by dichloromethane, washed by IN HCl, 5% NaHCO₃, water, dried (NaSO₄) and concentrated. The residue was purified by column chromatography on silica gel (eluting solvent: 0-20% ethyl acetate in hexanes) yielding Compound 6 as an oil (0.27g, 74%): ¹H NMR (500 MHz, CDCl₃) δ 7.11 (IH, dd, J = 8.45, J = 2.07, H6'), 6.99 (IH, d. J = 2.09, H2'), 6.83 (IH, d, J = 8.57, H5'), 6.50 (2H, s, H2, 6), 6.45 (IH, d, J = 12.22, HIa), 6.43 (IH, d, J = 12.15, HIa'), 5.35 (2H, m, 2xCH), 3.83 (3H, s, 4-OCH₃), 3.79 (3H, s, 4'- OCH₃), 3.70 (6H₅ s, 3,5-OCH₃), 2.52 (2H, t, J = 7.50, CH₂CO), 2.01 (4H₅ m, 2xCH₂CH), 1.71 (2H, m, CH₂CH₂CO), 1.32 (2OH, m 1OxCH₂), 0.88 (3H, t, J - 6.85, CH₃); MS calculated for (C₃₆H₅₂O₆Na) 603, found 603 (MNa⁺).

EXAMPLE 8

[0139] This example illustrates the preparation of albumin nanoparticle compositions from Compound 6. 30 Mg of Compound 6 (as prepared in Example 7) was dissolved in 3.0 ml methylene chloride/methanol (9/1). The solution was then added into 27.0 ml of human serum albumin solution (3% w/v). The mixture was homogenized for 5 minutes at low RPM (Vitris homogenizer model: Tempest LQ.) in order to form a crude emulsion, and then transferred into a high-pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while recycling the emulsion for at least 5 cycles. The resulting system was transferred into a Rotavap and solvent was removed at reduced pressure for 20-30 minutes. The resulting dispersion was translucent and the typical average diameter of the resulting particles was in the range 50-200 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The resulting cake could be easily reconstituted to the original dispersion by the addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and proportions of drug, solvents, proteins used in this example are not limiting in any way. EXAMPLE 9: IN MTRO EVALUATION

[0140] This example showed the in vitro growth inhibition experiments for several compounds described herein on MX-I (human breast carcinoma) cells. The cytotoxicity assay was quantitated using the Promega CellTiter Blue Cell Viability Assay. Briefly, cells (5000 cells/well) were plated onto 96-well microliter plates in RPMI 1640 medium supplemented with 10% FBS and incubated at 37°C in a humidified 5% CO₂ atmosphere. After 24 h, cells were exposed to various concentrations of compound in DMSO and cultured for another 72 h. 10OuI of media were removed and 20ul of Promega CellTiter Blue reagent were added to each well and shaken to mix. After 4 hours of incubation at 37⁰C in a humidified 5% CO₂ atmosphere, the plates were read at 544ex/620em. The fluorescence produced is proportional to the number of viable cells. After plotting fluorescence produced against drug concentration, the IC₅₀ was calculated as the half-life of the resulting non-linear regression. The data is set forth in Table 2.

Table 2. Cytotoxic Activities

EXAMPLE 10

[0141] This example demonstrates the percentage of CA4 produced via hydrolysis of CA4 analogs.

[0142] A standard solution of each of Compounds 3, 4, 5, and 6 was prepared at 6 mg/mL in acetonitrile. Sample solutions of each compound were then prepared by spiking the standard solution into human serum or human liver microsome to obtain a solution at 50 μg/mL. Each sample solution was aliquoted and then incubated at 37 ⁰C for 48 hours. The samples were retrieved as the indicated time and extracted with acetonitrile at a ratio of 1 :2, then centrifuged at 14,000 rpm for 10 min. The supernatant was injected to HPLC to obtain the percent CA4 generated from each sample with reference to time (see the Figure). The compounds in the invention can be converted to CA4 in the presence of microsome and can used as its prodrugs.

[0143] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0144] The use of 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 teπns (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. All 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 specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0145] 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 may 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 invention 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.

Claims

What is claimed is:

1. A compound of the formula:

wherein Ri, R₅, R$, R₇ and Rio are H, wherein R₂, R3, R₄ and Rs are OMe₅ and wherein R₉ is -X-CO-R", R" is an unsubstiruted or a substituted alkyl, alkenyl or aryl group comprising 5-21 carbon atoms and does not include N₅ X is O₅ S or NH, and when X is O, R" is not an optionally substituted 9-fluorenyl or 9- xanthenyl.

2. The compound according to claim 1, wherein X is O.

3. The compound according to claim 2, wherein R" is -CH₂(CHa)₃CH₃.

4. The compound according to claim 2, wherein R" is -(CH₂)₇CH=CH(CH₂)₇CH₃.

5. The compound according to claim 2, wherein R" is -CH₂CH₂(CH₂=CHCH₂)_OCH₃.

6. A compound of the formula:

wherein R₁, R₅, Rg, R₇ and Rio are H; wherein R₃, R₄, R₈, R₉ are OMe, and wherein R₂ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms and X is O, S or NH.

7. The compound according to claim 6, wherein X is O.

8. The compound according to claim 7, wherein R" is -CH₂(CH₂)₃CH₃.

9. The compound according to claim 7, wherein R" is -(CH₂)₇CH=CH(CH₂)₇CH₃.

10. The compound according to claim 7, wherein R" is -CH₂CH₂(CH₂=CHCH₂)₆CH₃.

11. A compound of the formula:

wherein R₁, R₅, R₆, R₇ and Ri₀ are H; wherein R₄ and R₈ are OMe; wherein R₂ and R₃ form O-CH2-O; and wherein R₉ is -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms and does not include N, and X is O₅ S or NH.

12. The compound according to claim 11, wherein X is O.

13. The compound according to claim 12, wherein R" is -CH₂(CH₂)₃CH₃.

14. The compound according to claim 13, wherein R" is -(CH₂^CH=CH(CHa)₇CH₃.

15. The compound according to claim 14, wherein R" is -CH₂CH₂(CH₂=CHCH₂)₆CH₃.

16. A compound of the formula:

wherein R₁, R₅, R₆ and R₇ are H; wherein R₂, R₃, R₄ and R₈ are OMe; and wherein R₉ and R₁₀ are -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl giOup comprising 2-21 carbon atoms, and X is O, S or NH.

17. The compound according to claim 16, wherein X is O.

18. The compound according to claim 17, wherein R" is -CH₂(CH₂)_JCH₃.

19. The compound according to claim 17, wherein R" is -(CH₂)_VCH=CH(CH₂)_VCH₃.

20. The compound according to claim 17, wherein R" is -CH₂CH₂(CH₂=CHCHO₆CH₃.

21. A compound of the formula:

wherein R₁, R₅, R₆, R₇ and Rio are H; wherein R₃, R₄ and R₈ are OMe; and wherein R₂ and R₉ are -X-CO-R", R" is an unsubstituted or a substituted alkyl, alkenyl or aryl group comprising 2-21 carbon atoms and X is O, S or NH.

22. The compound according to claim 21 , wherein X is O.

23. The compound according to claim 22, wherein R" is -CH₂(CH?)₃CH₃.

24. The compound according to claim 22, wherein R" is

25. The compound according to claim 22, wherein R" is -CH₂CH₂(CH₂=CHCHa)₆CH₃.

26. The compound according to claim 1, wherein X is NH.

27. The compound according to claim 26, wherein R" is -CH₂(CH₂)₃CH₃.

28. The compound according to claim 26, wherein R" is -(CH₂)TCH-CH(CH₂)VCH₃.

29. The compound according to claim 26, wherein R" is -CH₂CH₂(CH₂=CHCH₂)₆CH₃.

30. A method of modulating tumor growth or metastasis in an animal comprising identifying an animal afflicted with a tumor growth or metastasis and administering the compound of claim 1 to the afflicted animal.

31. A method of treating cancer in a patient comprising administering a composition comprising a compound of claim 1 to said patient in an amount effective to treat said cancer.

32. The method of claim 31, wherein said cancer is lung, breast, colon, prostate, melanoma, pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries, urinary tract, gastrointestinal, or leukemia.

33. The method of claim 31, wherein said cancer is in the form of a solid tumor or blood bom tumor.

34. The method of claim 30, wherein said composition is administered orally, parenterally, intramuscularly, transdermally or by an airborne delivery system.

35. A composition comprising a plurality of particles of the compound of claim 1, wherein the average size of the particles is no greater than about 500 nm.

36. The composition according to claim 35, wherein the average size of the particles is no greater than about 200 nm

37. A pharmaceutical formulation comprising the compound of claim 1 and a pharmaceutically-acceptable carrier.

38. The use of a compound of any of claims 1-29 in the manufacture of a medicament for the treatment of cancer.

39. The use of claim 38, wherein said cancer is lung, breast, colon, prostate, melanoma, pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries, urinary tract, gastrointestinal, or leukemia.

40. The use of claim 38, wherein said cancer is in the form of a solid tumor or blood born tumor.

41. The use of claim 38, wherein the average size of particles of said compound is no greater than about 500 nm.