WO2010014254A1

WO2010014254A1 - Oxabicycloheptanes and oxabicycloheptenes, their preparation and use

Info

Publication number: WO2010014254A1
Application number: PCT/US2009/004430
Authority: WO
Inventors: John S. Kovach; Francis Johnson
Original assignee: Lixte Biotechnology, Inc.
Priority date: 2008-08-01
Filing date: 2009-07-30
Publication date: 2010-02-04
Also published as: CA2730489A1; US8541458B2; EA022311B1; JP2011529883A; EA201170288A1; EP2307344B1; BRPI0911717A2; AU2009277031B2; CN102333752A; US8227473B2; JP2015044816A; CA2730489C; HK1151791A1; EP2307344A4; US20120264764A1; EP2307344A1; AU2009277031A1; JP5666443B2; MX2011001007A; US20100029484A1

Abstract

This invention provides compounds having the structure (I) which may be used for the treatment of tumors.

Description

OXABICYCLOHEPTANES AND OXABICYLCOHEPTENES, THEIR PREPARATION AND USE

This application claims the benefit of U.S. Provisional Application No. 61/137,691, filed August 1, 2008, the content of which in its entirety is hereby incorporated by reference.

Throughout this application, certain publications are referenced. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state-of-the art to which this invention relates.

Background of the Invention

Retinoids, metabolites of vitamin A, have been examined therapeutically against a variety of tumors, including gliomas.

(Yung et al. (1996)) Nuclear receptor co-repressor (N-CoR) is closely associated with the retinoid receptor and is released upon ligand binding to the receptor. (Bastien et al. (2004)) By preventing the action of protein phosphatase- 1 and protein phosphatase-2A, anti-phosphatases increase the phosphorylated form of N-CoR and promotes its subsequent cytoplasmic translocation. (Hermanson et al. (2002))

The phosphatase inhibitor, Cantharidin, has anti-tumor activity against human cancers of the liver (hepatomas) and of the upper gastrointestinal tract but is toxic to the urinary tract (Wang, 1989) . The publication of a report that cantharidin acts as a protein phosphatase inhibitor prompted a more general interest in compounds with this type of chemical structure (Li and Casida, 1992) . Previously, it had been found that the simpler congener and its hydrolysis product (commercially available as the herbicide, Endothall) are hepatotoxic (Graziani and Casida, 1997) . Binding studies have shown that the action of certain cantharidin homologs is direct on protein phosphatase-2A and indirect on protein phosphatase-1 (Honkanen et al. , 1993; Li et al., 1993) .

Despite these successes, few compounds of this type have been screened for anti-tumor or cytotoxic activity. Currently, there is a significant need to develop inhibitors of protein phosphatases that are more active, less toxic and more specific in action than the known substances mentioned above.

Summary of the Invention

This invention provides a compound having the structure

wherein bond α is present or absent; Ri and R₂ is each independently H, O^" or OR₉, where R₉ is H, alkyl, substituted alkyl, alkenyl, alkynyl or aryl, or Ri and R₂ together are =0; R₃ and R4 are each different, and each is 0 (CH₂) 1-6R9 or ORi₀, or

where X is 0, S, NRn, or N⁺RnRn, where each Rn is independently H, alkyl, hydroxyalkyl , substituted C₂-Ci₂ alkyl, alkenyl, substituted C₄-Ci₂ alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where the substituent is other than chloro when Ri and R₂ are =0 ,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHR₁₂ or -NH⁺ (R₁₂ ) ₂,where each Ri₂ is independently alkyl, alkenyl or alkynyl, each of which is substituted or unsubstituted, or H; where Rio is substituted alkyl, substituted alkenyl, substituted alkynyl, or substituted aryl, or R₃ and R₄ are each different and each is OH or

R₅ and Re is each independently H, OH, or R₅ and R₆ taken together are =0; and R₇ and Rs is each independently H, F, Cl, Br, SC^Ph, CO₂CH₃, or SRi₃, where R₁₃ is H, aryl or a substituted or unsubstituted alkyl , alkenyl or alkynyl,or a salt, enantiomer or zwitterion of the compound.

This invention provides a process for preparing the above compound comprising (a) reacting compounds of the structure

to form an anhydride the structure

(b) reacting the anhydride having the above structure with at least one nucleophile to form compounds having the structure

where R3 and R4 are each dif ferent , and each is O (CH₂ ) 1-6^9 or OR10, or

where X is 0, S, NRn, or N⁺RnRn, where each Rn is independently H, alkyl, hydroxyalkyl , substituted C₂-C₁₂ alkyl, alkenyl , substituted C₄-C₁₂ alkenyl, alkynyl, substituted alkynyl, aryl , substituted aryl where the substituent is other than chloro when Ri and R₂ are =0,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHRi₂ or -NH⁺ (R₁₂) 2,where each R₁₂ is independently alkyl, alkenyl or alkynyl, each of which is substituted or unsubstituted, or H,-where Rio is substituted alkyl, substituted alkenyl, substituted alkynyl, or substituted aryl, or R3 and R4 are each different and each is OH or

R₇ and R₈ is each independently- H, F, Cl, Br, SO₂Ph, CO₂CH₃, or SR₁₃, where Ri3 is H, aryl or a substituted or unsubstituted alkyl, alkenyl or alkynyl.

This invention provides a method of controlling undesired vegetation comprising contacting the vegetation or its environment with a herbicidally effective amount of the compounds of this invention. This invention provides a method of inhibiting plant phosphatase activity comprising contacting the plant or its environment with a herbicidally effective amount of the compounds of this invention.

The invention provides a method of preventing or treating a fungal infection in a subject comprising administering to the subject an effective amount of the compounds of this invention.

This invention provides a method of treating a subject with a neurodegenerative disease comprising administering to the subject an effective amount any of the compounds of this invention, thereby treating the subject.

This invention provides a method for reducing the amount of GSK- 3β in a cell comprising contacting the cell with an effective amount of any of the compounds of this invention so as to thereby reduce the amount of GSK-3β in the cell.

This invention provides a method for increasing the amount of phosphorylated Akt in a cell comprising contacting the neural cell with an effective amount of any of the compounds of this invention, so as to thereby increase the amount of phosphorylated Akt in the cell.

This invention provides a method for reducing the phosphorylation of Tau in cell, comprising contacting the cell with an effective amount of any of the compounds of this invention, so as to thereby reduce the phosphorylation of Tau in the cell. This invention provides a method for reducing the aggregation of

Tau in a cell, comprising contacting the cell with an effective amount of any of the compounds of this invention, so as to thereby reduce the phosphorylation of Tau in the cell.

Brief Description of the Figures

Figure 1: Compound 110 inhibition of DAOY xenografts

Medulloblastoma DAOY cells were implanted subcutaneousIy in the flanks of SCID mice. After 7 days when the implanted tumor cells reached a mass with the average diameter of 6mm, 6 animals received 0.12 mg of Compound 110, 6 animals received 0.18 mg of Compound 110, and 6 animals, received vehicle (PBS) only. After two weeks of treatment all animals were sacrificed, the subcutaneous tumor masses resected, and their volumes calculated. Both doses of drugs led to significant inhibition of tumor growth.

Figure 2. Jn vitro activity of Compound 109

Inhibition of growth of glioblastoma multiforme cells of line U373 by exposure for 7 days to increasing concentrations of compound 109 compared to lOuM compound 100.

Figure 3. In vitro activity of compound 110

Inhibition of growth of glioblastoma multiforme cells of line U373 by exposure for 7 days to increasing concentrations of compound 110 compared to lOuM compound 100.

Figure 4. In vitro activity of compound 112 Inhibition of growth of glioblastoma multiforme cells of line U373 by exposure for 7 days to increasing concentrations of compound 112 compared to compound 205, a compound known to inhibit this cell line. Figure 5. In vitro activity of compound 113

Inhibition of growth of glioblastoma multiforme cells of line U373 by exposure for 7 days to increasing concentrations of compound 113 compared to 10 uM compound 100.

Detailed Description of the Invention

This invention provides a compound having the structure

wherein

bond α is present or absent; Ri and R₂ is each independently H, 0^" or ORg, where R₉ is H, alkyl, substituted alkyl , alkenyl, alkynyl or aryl , or Ri and R₂ together are =0;

R₃ and R4 are each different, and each is O(CH₂)i-6R9 or OR10, or

where X is 0, S, NRn, or N⁺RnRu, where each Rn is independently H, alkyl, hydroxyalkyl , substituted C₂-Ci₂ alkyl, alkenyl, substituted C₄-Ci₂ alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where the substituent is other than chloro when Ri and R₂ are =0,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHR_i2 or -NH⁺(Ri₂J₂, where each Ri₂ is independently alkyl, alkenyl or alkynyl, each of which is substituted or unsubstituted, or H; where Ri₀ is substituted alkyl, substituted alkenyl, substituted alkynyl, or substituted aryl, or R3 and R4 are each different and each is OH or

R₅ and Re is each independently H, OH, or R₅ and Re taken together are =0; and

R₇ and Rs is each independently H, F, Cl, Br, SO₂Ph, CO₂CH₃, or SR₁₃, where Ri₃ is H, aryl or a substituted or unsubstituted alkyl, alkenyl or alkynyl,

or a salt, enantiomer or zwitterion of the compound.

one embodiment, the above compound has the structure

In one embodiment bond oc is present. In another embodiment bond α is absent.

In one embodiment of the above compound R₃ is OR₉ or O (CH₂) l-eRio, where Rg is aryl or substituted ethyl; where Rio is substituted phenyl, wherein the substituent is in the para position;

R₄ is

where X is O, S, NRn, or N⁺RnRn, where each Rn is independently H, alkyl, hydroxyalkyl , substituted C₂-Ci₂ alkyl, alkenyl, substituted C₄-C₁₂ alkenyl, alkynyl , substituted alkynyl , aryl, substituted aryl where the substituent is other than chloro,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂COR₁₂, -NHR_i2 or -NH⁺(Ri₂) 2, where R_i2 is alkyl, alkenyl or alkynyl, each of which is substituted or unsubstituted, or H;

In another embodiment of the above invention R₄

where Rn is alkyl or hydroxylalkyl; or R₄ is

when R₃ is OH.

In another embodiment of the above compound, Ri and R₂ together are =0;

R₃ is OR₉ or ORio or 0 (CH₂) 1-2R9, where Rg is aryl or substituted ethyl; where Rio is substituted phenyl, wherein the substituent is in the para position; or R₃ is OH and R₄ is

R₄ IS

where Rn is alkyl or hydroxyl alkyl;

R₅ and R₆ together are =0; and

R₇ and Rs are each independently H. nother embodiment of the above compounds, Ri and R2 together are =0;

R₃ is OH, 0(CH₂)Rg, Or ORi₀, where R9 is phenyl; where Ri₀ is CH₂CCl₃,

R₄ is

where Rn is CH₃ or CH₃CH₂OH;

R.5 and R_& together are =0; and

R₇ and R₈ are each independently H.

In one embodiment, R₃ is ORi₀, where Ri₀ is (CH₂) i-β (CHNHBOC)CO₂H, (CH₂) i-₆ (CHNH₂) CO₂H, or (CH₂) i-₆CCl_3.

In another embodiment, R_i0 is CH₂(CHNHBOC)CO₂H. In a further embodiment, Rio is CH₂CCl₃.

In one embodiment of the above compounds, R₃ is O(CH₂)i-6Rg where R₉ is phenyl.

In another embodiment of the above compounds, R₃ is 0(CH₂)Rg where R9 is phenyl.

In an embodiment of the above compounds R₃ is OH and R₄ is

In another embodiment of the above compounds, R₄ is

wherein Rn is hydroxyalkyl .

In another embodiment of the above compound, Rn is -CH₂CH₂OH.

In an embodiment of the above compound, R₄ is

wherein Rn is alkyl . In further embodiment, Rn is -CH3 In another embodiment of the above compounds R₄ is wherein R₄ is

In an embodiment, the compound has the structure

(compound 109) ,

(compound 110) , (compound 112) ,

compound 113), or

(compound 114]

In another embodiment, the compound has the structure

(compound 109E) ,

10

(compound 11 OE) , (compound 112E) ,

(compound 113E) , or

(compound 114E)

This invention provides a pharmaceutical composition comprising any of the above described compounds and a pharmaceutically acceptable carrier.

This invention provides a process for preparing any of the above compounds comprising

(a) reacting compounds of the structure

to form an anhydride the structure

where

R3 and R4 are each different, and each is 0(CH₂) 1-6^9 or OR10, or

where X is O, S, NRn, OrN⁺RnR₁₁, where each R₁₁ is independently H, alkyl, hydroxyalkyl , substituted C₂-C₁₂ alkyl, alkenyl, substituted C₄-C₁₂ alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where the substituent is other than chloro when R₁ and R₂ are =0,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHR_i2 or -MH⁺(Ri₂) ₂, where each R_i2 is independently alkyl, alkenyl or alkynyl , each of which is substituted or unsubstituted, or H; where Ri₀ is substituted alkyl, substituted alkenyl, substituted alkynyl, or substituted aryl, or R₃ and R4 are each different and each is OH or

R₇ and Rs is each independently H, F, Cl, Br, SO₂Ph, CO₂CH₃, or SR₁₃ , where Ri₃ is H, aryl or a substituted or unsubstituted alkyl, alkenyl or alkynyl.

In one embodiment of the above process, the nuclephile comprises at least one hydroxyl group. In another embodiment, the nucleophile is 0 (CH₂) i_₆R₉ or OR₁₀, wherein R₉ and Rio are as described above.

In another embodiment, the nucleophile comprises at least one free amine group. In a further embodiment the nucleophile is

or

where X is as described herein.

In another embodiment, the above process further comprises (c) reacting the product of step (b) with hydrogen in the presence of a catalyst to form a compound having the structure

The compounds disclosed hereinabove may be used in a a method of controlling undesired vegetation comprising contacting the vegetation or its environment with a herbicidally effective amount of the compounds of any one of invention.

The compounds disclosed hereinabove may also be used in method of inhibiting plant phosphatase activity comprising contacting the plant or its environment with a herbicidally effective amount of the compounds of any one of the invention.

The compounds disclosed herein above may be used in a method of preventing or treating a fungal infection in a subject comprising administering to the subject an effective amount of the compounds of the invention to treat the fungal infection, thereby treating the fungal infection. The compounds disclosed herein maybe used in a method of treating a subject afflicted with breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, or acute lymphocytic leukemia, comprising administering to the subject a therapeutically effective amount of the compounds of the invention, thereby treating the subject.

The compounds disclosed herein may be used in a method of treating a subject with a neurodegenerative disease comprising administering to the subject an effective amount any of the compounds of the invention, thereby treating the subject.

The compounds disclosed herein may be used in a method for reducing the amount of GSK-3β in a cell comprising contacting the cell with an effective amount of any of the compounds of the invention so as to thereby reduce the amount of GSK-3β in the cell.

The compounds disclosed herein may be used in a method for increasing the amount of phosphorylated Akt in a cell comprising contacting the neural cell with an effective amount of any of the compounds of the invention, so as to thereby increase the amount of phosphorylated Akt in the cell.

The compounds disclosed herein may be used in a method for reducing the phosphorylation of Tau in cell, comprising contacting the cell with an effective amount of any of the compounds of the invention, so as to thereby reduce the phosphorylation of Tau in the cell. The compounds disclosed herein may be used in a method for reducing the aggregation of Tau in a cell, comprising contacting the cell with an effective amount of any of the compounds of the invention, so as to thereby reduce the phosphorylation of Tau in the cell.

The compounds of the invention may also be used in a method of inhibiting proliferation of a cancer cell which does not overexpress N-CoR comprising administering to the subject any of the compounds of the invention in an amount to inhibit proliferation of the cancer cell.

The compounds of the invention may also be used in a method of inhibiting proliferation of a cancer cell which overexpresses TCTP comprising administering to the subject any of the compound of the invention in an amount effective to inhibit proliferation of the cancer cell.

In the above described methods, the cancer may be adrenocortical cancer, bladder cancer, osteosarcoma, cervical cancer, esophageal, gallbladder, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, renal cancer, melanoma, pancreatic cancer, rectal cancer, thyroid cancer and throat cancer.

In the method of the invention, the histone deacetylase ligand may be an inhibitor, e.g. the histone deacetylase inhibitor of HDAC-3 (histone deacetylase-3 ) . The histone deacetylase ligand may also be selected from the group consisting of 2-amino-8-oxo- 9, 10-epoxy-decanoyl, 3- (4-aroyl-lH-pyrrol-2-yl) -N-hydroxy-2- propenamide, APHA Compound 8, apicidin, arginine butyrate, butyric acid, depsipeptide, depudecin, HDAC-3, m- carboxycinnamic acid bis-hydroxamide, N- (2-aminophenyl) -4- [N- (pyridin-3-ylmethoxycarbonyl) aminomethyl] benzamide, MS 275, oxamfiatin, phenylbutyrate, pyroxamide, scriptaid, sirtinol, sodium butyrate, suberic bishydroxamic acid, suberoylanilide hydroxamic acid, trichostatin A, trapoxin A, trapoxin B and valproic acid.

The compounds of this invention may be used in combination with compounds which inhibit the enzyme histone deacetylase (HDAC) . These HDAC enzymes post-translationally modify histones (U.S. Patent Publication No. 2004/0197888, Armour et al.) Histones are groups of proteins which associate with DNA in eukaryotic cells to form compacted structures called chromatin. This compaction allows an enormous amount of DNA to be located within the nucleus of a eukaryotic cell, but the compact structure of chromatin restricts the access of transcription factors to the DNA. Acetylation of the histones decreases the compaction of the chromatin allowing transcription factors to bind to the DNA. Deacetylation, catalysed by histone deacetylases (HDACs) , increases the compaction of chromatin, thereby reducing transcription factor accessibility to DNA. Therefore, inhibitors of histone deacetylases prevent the compaction of chromatin, allowing transcription factors to bind to DNA and increase expression of the genes.

The invention further contemplates the use of prodrugs which are converted in vivo to the compounds of the invention (see, e.g., R. B. Silverman, 1992, "The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chapter 8, the entire contents of which are hereby incorporated by reference) . Such prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically enter a reactive site) or the pharmacokinetics of the compound. The compounds described in the present invention are in racemic form or as individual enantiomers . The enantiomers can be separated using known techniques, such as those described, for example, in Pure and Applied Chemistry 69, 1469-1474, (1997) IUPAC .

As used herein, "zwitterion" means a compound that is electrically neutral but carries formal positive and negative charges on different atoms. Zwitterions are polar, have high solubility in water and have poor solubility in most organic solvents.

The compounds disclosed herein may also form zwitterions For example, a compound having the structure

may also for the following zwitterionic structure

where X is as defined throughout the disclosures herein.

"Solvent" as used herein is intended to include compounds such as, hexanes, benzene, toluene, diethyl ether, chloroform, methylene chloride, ethyl acetate, 1,4-dioxane, water, THF, acetone, acetonitrile, DMF, DMSO, acetic acid, n-butanol, isopropanol, n-propanol, ethanol, methanol, formic acid, carbon tetrachloride, benzenethiol, chlorobenzene, cyclohexanethiol , 1- diethylaminoethanol, ethylene dichloride, ethylene glycol, xylene, 1, 1 , 2, 2-tetrachloroethane, phenol, acetic • acid, 1- butanol, 2-butanol, 2-butanone, diglyme, dimethylether, dioxane, petroleum ether, (NMP) iV-methyl-2-pyrrolidinone, heptane, glycerin, HMPA (Hexamethylphosphorus triamide) , MTBE (methyl t-butyl ether), nitromethane, pyridine , 1-propanol, 2-propanol, and triethylamine.

Certain embodiments of the disclosed compounds can contain a basic functional group, such as amino or alkylamino, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids, or contain an acidic functional group and are thus capable of forming pharmaceutically acceptable salts with bases. The instant compounds therefore may be in a salt form. As used herein, a "salt" is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. The salt may be pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. For a description of possible salts, see, e.g., Berge et al . (1977) "Pharmaceutical Salts", J^". Pharm. Sci . 66:1-19.

As used herein, "therapeutically effective amount" means an amount sufficient to treat a subject afflicted with a disease (e.g. cancer or a neurodegenerative disease) or to alleviate a symptom or a complication associated with the disease.

As used herein, "herbicidally effective" means an amount sufficient to adversely affect plant growth, particularly through inhibition of plant phosphatase 2 A activity.

As used herein, "treating" means slowing, stopping or reversing the progression of a disease, particularly cancer or a neurodegenerative disease.

As used herein, a "neurodegenerative disease" refers to a disease in which degeneration occurs of either gray or white matter, or both, of the nervous system. Thus, such a disease can be diabetic neuropathy, senile dementias, Alzheimer's disease,

Mild Cognitive Impairment (MCI) , dementia, Lewy Body Dementia, Frontal Temporal Lobe dementia, Parkinson's Disease, facial nerve (Bell's) palsy, glaucoma, Huntington's chorea, amyotrophic lateral sclerosis (ALS), status epilepticus, non-arteritic optic neuropathy, intervertebral disc herniation, vitamin deficiency, prion diseases such as Creutzfeldt-Jakob disease, carpal tunnel syndrome, peripheral neuropathies associated with various diseases, including but not limited to, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acute sensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis, primary amyloidosis, obstructive lung diseases, acromegaly, malabsorption syndromes, polycythemia vera, IgA and IgG gammapathies, complications of various drugs (e.g., metronidazole) and toxins (e.g., alcohol or organophosphates) , Charcot-Marie-Tooth disease, ataxia telangectasia, Friedreich's ataxia, amyloid polyneuropathies, adrenomyeloneuropathy, Giant axonal neuropathy, Refsum's disease, Fabry's disease and lipoproteinemia.

As used herein, "tauopathies" refers to a class of neurodegenerative diseases which result from aggregation of tau protein in neurofibrillary tangles. Examples of tauopathies include, but are not limited to, Alzheimer's disease, Frontotemporal dementia (Pick's disease), Progressive Supranuclear Palsy, and Corticobasal degeneration.

As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, Ci-C_n as in "Ci-C_n alkyl" is defined to include groups having 1, 2, ...., n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, and so on. An embodiment can be Ci-Ci₂ alkyl. "Alkoxy" represents an alkyl group as described above attached through an oxygen bridge.

"Hydroxyalkyl" represents an alkyl group as described aboved with a hydroxyl group. Hydroxyalky groups include, for example, (CH₂)i-ioOH and includes CH₂OH, CH₂CH₂OH, CH₂CH₂CH₂OH and so forth.

The term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non- aromatic carbon-carbon double bonds may be present. Thus, C₂-C_n alkenyl is defined to include groups having 1, 2, .... , n-1 or n carbons. For example, "C₂-Ce alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon- carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C₆ alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl . As described above with respect to alkyl , the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C₂-Ci₂ alkenyl .

The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon- carbon triple bonds may be present. Thus, C₂-C_n alkynyl is defined to include groups having 1, 2, ...., n-1 or n carbons. For example, "C2-C6 alkynyl" means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl , propynyl and butynyl . As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C₂-C_n alkynyl . As used herein, "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydro-naphthyl , indanyl, biphenyl, phenanthryl , anthryl or acenaphthyl . In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring. The substituted aryls included in this invention include substitution at any suitable position with amines, substituted amines, alkylamines, hydroxys and alkylhydroxys , wherein the "alkyl" portion of the alkylamines and alkylhydroxys is a C2-C_n alkyl as defined hereinabove. The substituted amines may be substituted with alkyl, alkenyl , alkynl, or aryl groups as hereinabove defined.

The alkyl, alkenyl, alkynyl, and aryl substituents may be unsubstituted or unsubstituted, unless specifically defined otherwise. For example, a (Ci-Cδ) alkyl may be substituted with one or more substituents selected from OH, oxo, halogen, which includes F, Cl, Br, and I, alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl, piperidinyl, and so on.

In the compounds of the present invention, alkyl, alkenyl, and alkynyl groups can be further substituted by replacing one or more hydrogen atoms by non-hydrogen groups described herein to the extent possible. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

The term "substituted" as used herein means that a given structure has a substituent which can be an alkyl, alkenyl, or aryl group as defined above. The term shall be deemed to include multiple degrees of substitution by a named substitutent . Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By iadependently substituted, it is meant that the (two or more) substituents can be the same or different.

As used herein, "administering" an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. The administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymalIy .

The following delivery systems, which employ a number of routinely used pharmaceutical carriers, may be used but are only representative of the many possible systems envisioned for administering compositions in accordance with the invention.

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA' s).

Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.

Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc) .

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) .

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) . In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens , vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA). It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results .

Discussion

Cantharidin has anti-tumor activity against human cancers of the liver (hepatomas) and of the upper gastrointestinal tract but is toxic to the urinary tract (Wang, 1989) . Norcantharidin, a demethylated cantharidin, maintains antitumor activity of cantharidin against hepatomas and cancers of the stomach and esophagus, but has little or no urinary tract toxicity. Norcantharidin also stimulates white blood cell production in patients and mice, a phenomenon not understood mechanistically, but a pharmacological effect of potential benefit as an anticancer agent (Wang et al . , 1986; Wang, 1989).

The publication of a report that cantharidin acts as a protein phosphatase inhibitor prompted a more general interest in compounds with this type of chemical structure (Li and Casida, 1992) . Previously, it had been found that the simpler congener and its hydrolysis product (commercially available as the herbicide, Endothall) are hepatoxic (Graziano and Casida, 1997). The primary targets in liver appear to be the protein phosphatases PP2A and PPl, all of the compounds showing ED₅₀ values at the micromolar level. Binding studies have shown that the action of certain cantharidin homologs is direct on PP2A and indirect on PPl (Honkanen et al . , 1993; Li et al . , 1993). Phosphatase PPlB is affected only at millimolar levels of these compounds, whereas the enzyme PP2C is not influenced at all.

In the past, several cantharidin analogues had been synthesized and evaluated for anti-phosphatase activity and for their ability to inhibit the growth of cancer cells in culture (Sakoff and McClusky, 2004; Hart et al . , 2004). Some of the previously evaluated modified norcantharidin molecules inhibited the growth of several human tumor cell lines. The activity of norcantharidin analogues against cells of tumors overexpressing N-CoR or the activity of norcantharidins combined with other potential anti-tumor agents was not analyzed. Further studies included 16 "modified norcantharidins" evaluated for activity against four human tumor cell lines including ovarian, kidney, colorectal and lung as well as a mouse leukemia line. None were as active as single agents as cantharidin or norcantharidin and none were evaluated for activity in combination with another antitumor agent (McCluskey et al . , US Patent Application Publication No. 2006/0030616, 2006).

A different series of cantharidin analogues had been previously synthesized and evaluated as pesticides and for antitumor activity against cancer cell lines . Forty-three analogues of endothal and cantharidin have been developed and assessed for their activity as herbicides and their lethality to mice (Matsuzawa et al . , 1987). Endothal thioanhydride was shown to be a more potent herbicide than endothal but was toxic to the liver of mice (Matsuzawa et al . , 1987; Kawamura et al . , 1990).

More recently, it has been found that endothal thioanhydride is an active agent against PP2A and PPl in vivo (Erdodi et al. , 1995) . Endothal and endothal thioanhydride, like cantharidin, inhibit the activity of PP2A and to some extent, the activity of PPl (Erdodi et al . , 1995). In the liver, the principal target appears to be PPl. In fibroblasts, only endothal thioanhydride caused marked morphological changes whereas cantharidin and endothal did not (Erdodi et al . , 1995) . The enhanced activity of endothal thioanhydride in vivo is thought to be related to its enhanced lipophilicity resulting in increased diffusion across the plasmalemma (Essers et al., 2001). A more recent publication has described the synthesis of the mono-, and the di-fluoro analogues of Endothal and also the corresponding anhydrides, however no pharmacological data accompanied this synthetic work (Essers et al . , 2001) . In pursuing the development of new drug substances in this area, we have found it essential to develop inhibitors that have greater specificity, especially towards those enzymes which display high activity against the replication processes of cancer cells. High specificity also holds out the possibility of avoiding targets important to normal cell function. From the point of view of the physical characteristics of any newly- developed drug substance, it must preeminently have good membrane permeability (i.e., has a log P value of between 2 and 4 units) .

The compounds described herein have an antagonistic effect on phosphatase-2A and phosphatase 1. In addition, compounds 110, 112, 113 and 114 each have properties that enhance their entry into the brain.

Endothal is also known as an active defoliant and potent contact herbicide used in many agricultural situations. It is considered effective as a pre-harvest desiccant and as a selective pre- emergence herbicide (Crafts, 1953) .

Endothal, norcantharidins and cantharidin are all well known inhibitors of mammalian protein phosphatase as well as potent herbicides (Matsuzawa et al . , 1987). The mechanism by which endothal and other homologs exert their potent herbicidal activity has not been studied extensively despite the widespread use of endothal internationally in agriculture. It should be noted that endothal is water soluble where cantharidin and norcantharidin are not.

It was assumed that the activity of endothal as a contact herbicide and defoliant is related to the known irritating toxicity of its parent compound, norcantharidin. However, more recent studies suggest that the herbicidal activity of endothal may be a function primarily of its anti-plant protein phosphatase (PP2A) activity. Li et al . (1993) showed that cantharidin and endothal inhibit spinach leaf PP2A and PPl and inhibit the activation of nitrate reductase by light in the intact spinach leaf, a process mediated by PP2A. Smith et al.

(1994) demonstrate that the structurally unrelated protein phosphatase inhibitors okadaic acid and calyculin-A are potent inhibitors at nanomolar concentrations of the growth of certain plants. The activity of okadaic acid and calyculin-A strongly suggest that the activity of endothal as an herbicide is due to its anti-phosphatase activity.

Baskin and Wilson (1997) showed inhibitors of serine-threonine protein phosphatases including cantharidin inhibit organization of plant microtubules. Ayaydin et al. (2000) show that endothal inhibited PP2A activity causing alteration of cell division in cultured alfalfa cells. They noted that endothal was cell permeable.

The compounds herein, therefore, are useful, commercially feasible, and safer herbicides both with respect human exposure and to the environment.

The compounds disclosed herein are also useful for the treatment of tumors. In one embodiment, the compounds are useful for the treatment of tumors which overexpress N-CoR, TCTP, or both.

The compounds disclosed herein are also useful for the treatment of fungal infections. In one embodiment, the compounds are useful for the treatment of a fungal infections of T. rubruπi. The compounds disclosed herein can be obtained by methods described herein and as described in PCT International Application PCT/US08/01549.

The human medulloblastoma cell line DAOY is available from the American Type Culture Collection (ATCC), P.O. Box 1549, Manassa, Virginia, 20108, as ATCC No. HTB-186.

Experimental Details

Methods and Materials

4 .3- [4- (2-Hydroxyethyl ) -pipβrazine-1-carbonyl] -7 -oxa- bicyclo [2 .2.1]heptane-2-carboxylic acid benzyl ester ( 12 , Compound- 109 ) :

Step 1 : Synthesis of 7-oxa-bicyclo [2 .2.1]heptane-2 , 3-dicarboxylic acid monobenzyl ester (10 ) :

dloxauβ,80° C, 16 h

^Md. ^WL: «⁸.¹⁵ i_fcftfift_ll

A mixture of 4 , 10-dioxa-tricyclo [5.2.1.02 , 6] decane-3, 5-dione

(8, 3.7 g, 22.0 mmol) and benzyl alcohol (9) (4.5 mL, 44.0 mmol) in dioxane was heated at 80 ⁰C for 16 h. Cooled to room temperature and evaporated to remove solvent. Residue obtained was triturated with diisopropyl ether (20 mL) to give white solid of 10 which was filtered, washed with diisopropyl ether (10 mL) and dried. Yield: 3.6 g (59%). ¹H NMR (300 MHz, CDCl₃) δ 1.45-1.55 (m, 2H); 1.78-1.81 (m, 2H); 3.05 (s, 2H); 4.91 (d, J=9.4Hz, 2H) ; 5.02 (d, J=6.3Hz, IH) ; 5.13 (d, J=6.3Hz, IH) ; 7.28-7.40 (m, 5H) .

Step 2 : 3- [4- ( 2-Hydroxyethyl ) -piperazine-1-carbonyl] -7-oxa- bicyclo[2.2.1] heptane-2-carboxylic acid benzyl ester (12, Compound 109) :

CjiHjglMϊOs

MoI. Wi; 388.46

To a solution of compound 10 (3.0O g, 11.6 inmol) in CH₂Cl₂ at 0 ⁰C (60 mL) was added piperazine-1-ethanol (11) (1.82 g, 14.0 mmol) , EDC (3.12 g, 16.3 mmol), HOBt (0.20 g) and DIPEA (5.8 mL, 34.9 mmol). The mixture was allowed to warm to RT over 16 h. TLC (5% MeOHZCH₂Cl₂) showed no starting material. The reaction mixture was diluted with CH₂Cl₂ (50 mL) , washed with water (2 x 40 mL) and dried. Evaporation of organic layer gave a residue. The residue was triturated with diisopropyl ether (20 mL) to get 3- [4- (2-hydroxy-ethyl) - piperazine-1 -carbonyl] -7-oxa-bicyclo[2.2.1] heptane-2- carboxylic acid benzyl ester (12) as a white solid. Yield: 3.24 g (72 %) . Mp 72-75 C. ¹H NMR (300 MHz, CDCl₃) δ 1.42- 1.56 (m, 2H); 1.76-1.82 (m, 2H); 2.02 (s, 2H); 2.29-2.51 (m, 4H); 2.90 (d, J=6.4 Hz, IH); 3.08 (d, J=6.4 Hz, IH) ; 3.18- 3.24 (m, 2H); 3.41 (bs , IH); 3.60 (t, J=2.3 Hz, 2H); 3.69 (bs, IH); 4.90 (dd, J=6.8 Hz, 2.3 Hz, 2H); 5.08 (s, 2H); 7.28-7.40 (m, 5H) . 1.3-(4-Methylpiperazinc-l-carbonyl)-7-oxa-bicydo|2,2,l lheptane-2-carboxylic acid 4-(2-/«rf-butoxycarbonylamino-2- carboxyethyl ) -phenyl ester (4, Compound 112):

Step 1: 3- (4-Methylpiperazine-l-carbonyl)-7-oxa- bicyclo[2,2,l]heptane-2-carbonyl chloride (1):

C₁₃HMNJO₄ C₁₃H₁₉CIN₂O₃ Mβl. Wt.: 26831 MoLWt: 286.75

To an ice-cold solution of 3- (4-methylpiperazine-l carbonyl) -7-oxa-bicyclo [2, 2 , 1] -heptane-2-carboxylie acid (938 mg, 3.5 mmole) in methylene chloride (30 mL) was added thionyl chloride (1 mL) followed by a few drops of DMF. After stirring at ice-cold temperature for 30 min, the ice-bath was removed and stirring continued at room temperature overnight. The excess thionyl chloride was removed using oil-free vacuum pump at -50° C and to the residue was added methylene chloride (10 mL) . The resulted thin slurry of 1 was used as such in the next reaction.

Step 2:3- (4-Methylpiperazine-l-carbonyl)-7-oxa- bicyclo |2,2,l]heptane-2-carboxylic acid 4- (2-benzyloxycarbonyl-2- tert-butoxycarbonylamino-2-carboxyethyl) -phenyl ester (3):

MaL Wi.: 611.72

To an ice-cold solution, of Boc-L-tyrosine benzyl ester (2, 780 mg, 2.1 mmole) and DMAP (100 mg) in methylene chloride (10 mL) and TEA (2.9 mmole, 21 mmole) was added the above suspension of acid chloride (1,1.0 g, 3.5 mmole) in methylene chloride (10 mL) . After stirring for 10 minutes at ice bath temperature, ice- bath was removed and stirred at room temperature for 1 h. At this point the TLC (95:5 ::

showed the disappearance of starting material 2. The reaction mixture was diluted with methylene chloride (30 mL) and washed with water (30 mL) followed by brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude residue was purified by column chromatography using 5% methanol in methylene chloride to give pure required compound 3 (1.040 g, 76%). ¹H NMR (CDCl₃) δ 1.48 (s, 9H), 1.64 (d, 2H), 1.96 (m, 2H), 2.34 (s, 3H), 2.54 (m, 4H), 3.14 (m, 2H), 3.15 (d, J= 9.00 Hz, IH), 3.35 (d, J=9.00 Hz, IH), 3.67 (m, 4H), 4.62 (m, IH), 4.91 (d, IH), 5.0 (m, IH), 5.20 (m, 3H), 7.06 (m, 4H), 7.40 (m, 5H). EIMS: 621 (M⁺).

Step 3 :3- (4-Methylpiperazine-l-carbonyl) -7-oxa-bicyclo[2 ,2,1] heptane-2-carboxylic acid 4-(2-tert-butoxycarbon.ylaitιliio-2- carboxyethyl) -phenyl ester (4, Compound 112):

MoI.WM 531.60

A solution of above coupled product 3 (600 mg, 0.965 mmole) in methanol (40 ml) was hydrogenated using hydrogen balloon and Pd(OH)₂ (100 mg, 20% Pd on C) as a catalyst overnight. The catalyst was filtered through celite, the filtrate was concentrated to dryness and the residue was triturated with ethyl acetate (15 mL) . Separated solid was filtered to give pure title compound 4 as a white solid (460 mg, 89%). Mp 165° C (decomp) . ¹H NMR (CDCl₃) δ 1.44 (s, 9H), 1.60 (m, 2H), 1.81 (m, 2H), 2.48 (s, 3H), 2.93 (m, 5H), 3.01 (m, IH), 3.22 (m, IH), 3.32 (m, IH), 3.35 (m, IH), 3.68 (m, 4H), 4.41 (m, IH), 4.79 (d, IH), 5.07 (d, IH), 5.32 (m, IH), 6.95 (d, J = 7.00 Hz, 2H), 7.16 (d, J = 7.00 Hz, 2H) . EST: 530 (M⁺-H) .

2.3-(4-Methylpiperazine-l-carbonyl) -7-oxa-bicyclo [2,2,1]heptane- 2-carboxylie acid 4- (2-amino-2-carboxyethyl) -phenyl ester hydrochloride salt (5, Compound 110):

Cj₇IIwNjOg CJJHJOCΪNJOJ

MoI. Wu 531.60 MoI. Wt.: 467.94

To an ice-cold solution of BOC derivative (4,150 mg, 0.28 mmole) in methylene chloride (10 mL) was added a solution of 2M HCl in ether (1 mL) . As the addition started to the reaction mixture, the solid started separating out. The suspension was stirred over-night at room temperature. The reaction mixture was concentrated to dryness and co-evaporated with hexane . It was triturated with hexane to give solid which on filtration gave pure title compound 5 as an off white solid (5,130 mg, 99%). Mp 110° C (decomp) . ¹HNMR (NaOD/D₂O) δ 1.37 (m, 2H), 1.49 (m, 2H), 2.03 (s, 3H), 2.21 (m, 3H), 2.54 (m, 6H), 3.01 (m, IH), 3.21 (rn, IH), 3.38 (m, IH), 4.53 (m, 2H), 4.75 (m, 2H), 6.38 (d, 2H), 6.79 (d, 2H) . ESMS: 432 (M-HH) .

3.3- (4-Mβthyl-pipβrazine-l-carbonyl) -7-oxa- bicylco[2_#2, l]heptane-2-carboxylic acid 2,2,2-trichloroethyl ester (7, Compound 113):

CiJH₂₀N]O₄ CiJH₂₁CIjN₂O₄ 1 MoI. Wt: 268.31 MoL WL: 399.70

To a suspension of acid (6, 536 mg, 2 mmole) in methylene chloride (15 mL) was added SOCl₂ (1 mL) followed by 2 drops of DMF. The reaction mixture was stirred at room temperature overnight. It was still a suspension. To this suspension added trichloroethanol (6 mL) . After the addition of trichloroethanol, the reaction mixture became homogeneous . Stirring was continued for 1.5 h followed by evaporation of the solvent. Added ethyl acetate (30 mL) to the residue and extracted with water (2x 25 mL) . Water layer neutralized with NaHCO₃ to pH 5-6 and evaporated to dryness. The residue dissolved in acetonitrile (30 mL) on heating and the separated NaCl was removed by filtration. The filtrate was treated with charcoal, evaporated to dryness, triturated with hot ethyl acetate and filtered the solid to give pure title ester 7 as colorless crystals. (416 mg, 52%). MP 229- 232° C. ¹H NMR (D₂O) 5 1.68 (m, 4H), 2.88 (s, 3H), 3.10 (m, 3H), 3.48 (m, 5H), 4.25 (m, 2H), 4.76 (m, 5H). EIMS: 399 (M⁺).

Preparation of 3- [2- (2,5-Dioxo-4,4-diphenyl-imidazolidin-l-yl) - ethylcarbamoyl] -7-oxa-bicyclo(2.2.1] heptane-2-carboxylic acid (3_# Compound 114)

MoI. VW.: 168.15 IMoI. Wt: 295.34 MoI.WL:483.48

To a mixture of exo-7-oxabicγclo [ 2.2. l]heptane-2 , 3-dicarboxylic acid anhydride (504 mg, 3 mmol) and N³- (2-aminoethyl ) -5, 5- diphenylhydantoin (2.0 g, 6.8 mmol) (prepared according to the procedure reported by Shaffer et . al . J^". Med. Cham. 1967, W, 739) was added dry toluene (10 mL) and the mixture was heated at 1OO°C for 5 h. The solvent was evaporated on rotary evaporator and added water (10 mL) and ethyl acetate (20 mL) to the residue. The solution was made acidic to pH 2 with aq. citric acid (10%) and the organic layer was separated. Aqueous layer was extracted again with EtOAc (2 x 30 mL) . Combined organic layers was washed with water (10 mL) , dried (Na₂SU₄) and evaporated. The residue was recrystallized from EtOAc to give colorless crystalline desired product. Yield: 700 mg (50%). M.p. : 208-210⁰C; ¹NMR (CDCl₃, 300 MHz): δ 1.43-1.48 (m, 2 H), 1.66-1.77 (m, 2H), 2.48 (s, 2H), 3.72-3.82 (m, 4H), 4.69-4.71 (m, 2H); 6.47 (bs, IH), 7.34-7.39 (m, I OH); ESI-MS: (m/z) 445 (M⁺-18) .

Example 1; Effect of Compound 110 and related analogues on Medulloblastoma DAOY cells In vivo experiments

Human Medulloblastoma DAOY cells were implanted subcutaneousIy in the flanks of SCID mice. After 7 days when the implanted tumor cells reached a mass with the average diameter of 6mm, 6 animals received 0.12mg of Compound 110, 6 animals received 0.18mg of Compound 110, and 6 animals received vehicle (PBS) only. After two weeks of treatment, all animals were sacrificed, the subcutaneous tumor masses resected, and their volumes calculated. As shown in Figure 1, both doses of Compound 110 led to signigicant inhibition of tumor growth.

Example 2: Inhibition of growth of glioblastoma multiforme cells of line U373 by exposure for 7 days to increasing concentrations of compound 109, 110, 112, and 113

At the highest concentrations of compound 109, there is slight inhibition of cell growth after 3 day. At lower concentrations, compound 109 has slight stimulatory activity, increasing over 7 days (Figure 1) . Other compounds of the compound 100 series at very low concentrations have mild to modest stimulator activity on cells in culture that is lost at higher concentrations when the drugs are inhibitory in a dose dependent manner (see Figures 2-4). Compounds 110, 112, and 113 inhibited cell growth in a dose dependent manner.

Discussion:

The compounds described herein increase the phosphorylation of several regulatory proteins including Akt . At low doses that are non-toxic to mice, these compounds slightly stimulate cell proliferation and increase phosphorylation of Akt in human cancer cells lines tested, including SH-SY5Y. When given intraperitoneally to normal mice, compounds 110, 113 and 114 also increased Akt phosphorylation in the cell lines tested, as set forth in the examples herein.

Because the compounds increase cellular Akt at low non-toxic doses and also increase acetylation of histones in neurons of the intact animal, these compounds are useful for the treatment of neurodegenerative diseases, particulary Alzheimer's disease and other tauopathies. While each of the compounds increase Akt phosphorylation of multiple tumor cell lines, they also increase Akt phosphorylation of the neuroblastoma cell line SH-SY5Y.

The results with compounds 110, 113 and 114 show that each of these has properties that enhance their entry into the brain.

The mechanism by which the compounds described herein exert their neuroprotective effect may be by increasing the intra- neuronal cell activity of Akt-1 and enhancing the acetylation of neuronal histones. Each of these compounds when given by intraperitoneal injection increase Akt phosphorylation in mouse neurons. This increase in Aky phosphorylation is associated with an increase in the phosphorylation of GSK-3β. Because increased phosphorylation of GSK-3β is known to decrease its activity, chronic suppression of GSK 3β by the compounds described herein may reduce tau phosphorylation. Reduction in tau phosphorylation reduces the formation of paired helical filaments, an intervention that should lessen the progression of tauopathies, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other rarer neurodegenerative diseases characterized by abnormal depositions of tau molecules. References

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Claims

What is claimed is :

1. A compound having the structure

wherein

bond α is present or absent;

Ri and R₂ is each independently H, O^" or ORg, where Rg is H, alkyl, substituted alkyl, alkenyl, alkynyl or aryl , or Ri and R₂ together are =0;

R₃ and R₄ are each different, and each is 0 (CH₂) 1-6^9 or OR10, or

where X is O, S, NRu, OrN⁺RnRn, where each Rn is independently H, alkyl, hydroxyalkyl , substituted C₂-Ci₂ alkyl, alkenyl, substituted C₄-Ci₂ alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where the substituent is other than chloro when Ri and R₂ are =0,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHR₁₂ or -NH⁺(Ri₂J₂, where each R₁₂ is independently alkyl, alkenyl or alkynyl, each of which is substituted or unsubstituted, or H; where Rio is substituted alkyl, substituted alkenyl, substituted alkynyl, or substituted aryl , or R₃ and R₄ are each different and each is OH or

R₇ and R₈ is each independently H, F, Cl, Br, SO₂Ph, CO₂CH₃, or

SR13, where R1₃ is H, aryl or a substituted or unsubstituted alkyl , alkenyl or alkynyl ,

or a salt, enantiomer or zwitterion of the compound.

2. The compound of claim 1, wherein the compound has the structure

3. The compound of claim 1 or 2, wherein bond α is present

4. The compound of claim 1 or 2, wherein bond α is absent.

5. The compound of any of claims 1-4, wherein

R₃ is OR₉ or 0 (CH₂) i-₆Rio, where Rg is aryl or substituted ethyl; where Rio is substituted phenyl, wherein the substituent is in the para position;

R₄ is

where X is 0, S, NRu, or N⁺R₁₁Rn, where each R₁₁ is independently H, alkyl, hydroxyalkyl , substituted C₂-C₁₂ alkyl, alkenyl, substituted C₄-Ci₂ alkenyl , alkynyl, substituted alkynyl, aryl , substituted aryl where the substituent is other than chloro,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHRi₂ or -NH⁺(Ri₂J₂, where R₁₂ is alkyl , alkenyl or alkynyl , each of which is substituted or unsubstituted, or H;

6. The compound of any of claims 1-5, wherein R₄ is

where Rn is alkyl or hydroxylalkyl or R₄ is

when R₃ is OH .

7. The compound of any of claims 1-6, wherein

Ri and R₂ together are =0;

R₃ is OR₉ or ORio or 0 (CH₂) 1-2R9, where R₉ is aryl or substituted ethyl; where Ri₀ is substituted phenyl, wherein the substituent is in the para position; or R₃ is OH and R₄ is

R₄ is

where Rn is alkyl or hydroxyl alkyl;

R₅ and Re together are =0; and

R₇ and R₈ are each independently H.

8. The compound of any of claims 1-7, wherein

Ri and R₂ together are =0;

R₃ is OH, 0(CH₂)R₉, or OR10, where R₉ is phenyl ; where R_i0 is CH₂CCl₃,

R₄ is

where Rn is CH₃ or CH₃CH₂OH;

R₅ and Re together are =0; and

R₇ and Rs are each independently H.

9. The compound of any of claims 1-8, wherein R₃ is ORio wherein Rio is (CH₂) i-₆ (CHNHBOC)CO₂H, (CH₂)^₆(CHNH₂)CO₂H, or

(CH₂) i-₆CCl₃.

10. The compound of claim 9, wherein Ri₀ is CH₂(CHNHBOC)CO₂H

11. The compound of claim 9, wherein Ri₀ is CH₂(CHNH₂)CO₂H.

12. The compound of claim 9, wherein Ri₀ is CH₂CCl₃.

13. The compound of any of claims 1-9, wherein R₃ is O(CH₂)].-6R9 where Rg is phenyl.

14. The compound of any of claims 1-9, wherein R₃ is 0(CH₂)Rg where R9 is phenyl.

15. The compound of any of claims 1-9, wherein R₃ is OH and R₄ is

16. The compound of any of claims 1-14, wherein R₄ is

wherein Rn is hydroxyalkyl .

17. The compound of claim 16, wherein Rn is -CH₂CH₂OH.

18. The compound of claim 1-14, wherein R₄ is

wherein Rn is alkyl .

19. The compound of claim 18, wherein Rn is -CH3.

20. The compound of any of claims 1-14, wherein R₄ is

21. The compound of claim 1, having the structure

22. The compound of claim 1, having the structure

23. A pharmaceutical composition comprising the compound of any one of claims 1-22 and a pharmaceutically acceptable carrier .

24. A process for preparing the compounds of any of claims 1-22 comprising (a) reacting compounds of the structure

to form an anhydride the structure

where

R₃ and R₄ are each different, and each is O(CH₂)i-6R9 or ORχo, or

where X is 0, S, NRn, or N⁺RnR₁₁, where each R₁₁ is independently H, alkyl, hydroxyalkyl , substituted C₂-C₁₂ alkyl, alkenyl, substituted C4-C₁₂ alkenyl, alkynyl , substituted alkynyl, aryl , substituted aryl where the substituent is other than chloro when Ri and R₂ are =0,

-CH₂CN, -CH₂CO₂Ri₂, -CH₂CORi₂, -NHR_i2 or -NH⁺(Ri₂J₂, where each R₁₂ is independently alkyl, alkenyl or alkynyl, each of which is substituted or unsubstituted, or H; where Rio is substituted alkyl, substituted alkenyl, substituted alkynyl, or substituted aryl, or R₃ and R₄ are each different and each is OH or

R₇ and R₈ is each independently H, F, Cl, Br, SO₂Ph, CO₂CH₃, or SRi₃, where R13 is H, aryl or a substituted or unsubstituted alkyl, alkenyl or alkynyl.

25. The process of claim 24, wherein the nucleophile comprises at least one hydroxyl group.

26. The process of claim 25, wherein the nucleophile is 0 (CH₂) i_ 6R9 or OR10.

27. The process of claim 24, wherein the nucleophile comprises at least one free amine group.

28. The process of claim 27, wherein the nucleophile is

29 The process of claim 24, further comprising

(c) reacting the product of step (b) with hydrogen in the presence of a catalyst to form a compound having the structure

30 A method of controlling undesired vegetation comprising contacting the vegetation or its environment with a herbicidally effective amount of the compounds of any one of claims 1-22.

31. A method of inhibiting plant phosphatase activity comprising contacting the plant or its environment with a herbicidally effective amount of the compounds of any one of claims 1-22.

32. A method of preventing or treating a fungal infection in a subject comprising administering to the subject an effective amount of the compounds of any one of claims 1-22 to treat the fungal infection, thereby treating the fungal infection.

33. A method of treating a subject afflicted with breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, or acute lymphocytic leukemia, comprising administering to the subject a therapeutically effective amount of the compounds of any one of claims 1- 22, thereby treating the subject.

34. A method of treating a subject with a neurodegenerative disease comprising administering to the subject an effective amount any of the compounds of claims 1-22, thereby treating the subject.

35. A method for reducing the amount of GSK-3β in a cell comprising contacting the cell with an effective amount of any of the compounds of claims 1-22 so as to thereby reduce the amount of GSK-3β in the cell.

36. A method for increasing the amount of phosphorylated Akt in a cell comprising contacting the neural cell with an effective amount of any of the compounds of claims 1-22, so as to thereby increase the amount of phosphorylated Akt in the cell.

37. A method for reducing the phosphorylation of Tau in cell, comprising contacting the cell with an effective amount of any of the compounds of claims 1-22, so as to thereby reduce the phosphorylation of Tau in the cell .

38. A method for reducing the aggregation of Tau in a cell, comprising contacting the cell with an effective amount of any of the compounds of claims 1-22, so as to thereby reduce the phosphorylation of Tau in the cell.