WO2017132775A1 - Novel compositions and methods for the treatment of leiomyoma - Google Patents

Abstract

This invention provides novel compositions and methods for the treatment of leiomyoma. In some other aspects, this invention provides novel compositions and methods comprising prodrugs of (-)-epigallocatechin-3-gallate (EGCG) and prodrugs of (-)-EGCG analogs, for the treatment of leiomyoma and conditions with common or related mechanisms as described. A novel method of synthesis is also provided for(-)- EGC 3-hydroxybenzoate (compound 2). (-)-EGC 3-hydroxybenzoate hexaacetate (compound 2a), (-)-EGC 3, 5 -dil ly droxybenzoate (compound 4), and (-)-EGC 3,5- dihydroxybcnzoate heptaacetate (compound 4a), each in some embodiments of the invention may act as dual inhibitors of proteasomal chymotrypsin-like activity and Akt signaling.

Description

NOVEL COMPOSITIONS AND METHODS

FOR THE TREATMENT OF LEIOMYOMA

FIELD OF THE INVENTION [0001] This invention relates to novel compositions and methods for making same. In some other aspects the invention relates but is not limited to the treatment of leiomyomas.

BACKGROUND OF THE INVENTION

[0002] A leiomyoma (also known as fibroid) is a benign smooth muscle tumor that can occur in any organ but most commonly in the uterus, small bowel and esophagus. Uterine fibroids (or uterine leiomyomas) are monoclonal tumors of the smooth muscle (myometrial) cells of the uterus. They are considered to be the most common benign tumor of the female genital tract. They are highly prevalent with 70-80% of women affected by the end of their reproductive years (Baird, D. D. et al., Am. J. Obstet. Gynecol. (2003) 188, 100). Uterine fibroids are associated with multiple clinical complications including heavy bleeding, pain, urinary incontinence and infertility. The only cure at present is hysterectomy. Over 200,000 such procedures are performed annually in the U.S. alone, as a treatment for women with symptomatic uterine fibroids. All other therapeutic interventions have numerous limitations to their widespread use. For example, pharmaceuticals in current use to treat uterine fibroids have limitations regarding efficacy or safety for long-term use. All of these pharmaceuticals alter hormone levels or affect hormone receptors. It is believed that uterine fibroid formation and growth involves dysregulated cellular proliferation, angiogenesis and fibrosis (Islam, M.S. et al, Mol. Nutr. Food. Res. (2014) 58, 1667). [0003] A recent pilot clinical study with a green tea extract (containing 45% (-)-

EGCG) reported biological responses in treating symptomatic women with uterine fibroids (Roshdy, E. et al., Int. J. Women 's Health (2013) 5, 477). However, there was a large inter-patient variability in those clinical responses, potentially resulting from several contributing factors discussed below. [0004] Biologically active components of green tea include polyphenols catechins. Several studies indicate that (-)-EGCG is the most abundant catechin in green tea, and the most biologically active among the green tea polyphenols (Liao, S. et al. Cancer Lett. (1995) 96, 239; Paschka, A. G. et al. Cancer Lett. (1998) 130, 1 ; Hao, X. et al, Nutr. Cancer (2007) 59, 62; Chen, D. et al, Histol. Histopathol. (2008) 23, 487; Han, D. H. et al. Arch. Pharm. Res. (2009) 32, 543; Du, G. J. et al. Nutrients (2012) 4, 1679). The activity of purified (-)-EGCG has been demonstrated in numerous animal models including in vivo models of leiomyoma (Ozercan, I. H. et al, Nutr. Res. (2008) 28, 92).

[0005] Overall, the therapeutic use of green tea extracts ("GTE") and (-)-EGCG has several limitations including poor bioavailability. In the fasted state, the estimated oral bioavailability of (-)-EGCG in humans is approximately 0.2-2.0% of the ingested amount (Nakagawa, K. et al, Biosci. Biotechnol Biochem. (1997) 61, 1981). The poor bioavailability of (-)-EGCG can be attributed to: (a) its ease of degradation in alkaline or neutral conditions (i.e. physiologic pH); (b) poor cellular uptake because of its high aqueous solubility and low hydrophobicity; and (c) metabolic transformations such as methylation, glucuronidation and sulfation resulting in reduced activity after absorption (Okushio, K. et al, Biosci. Biotechnol. Biochem. (1999) 63, 430; Yoshino, K. et al, J. Nutr. Biochem. (1999) 10, 223; Kohri, T. et al, J. Agric. Food Chem. (2001) 49, 4102; Li, C. et al, Chem. Res. Toxicol. (2001 ) 14, 702; Kohri, T. et al, J. Agric. Food Chem. (2003) 51, 5561 : Lambert, .1. D. et al, ./. Nutr. (2003) 133, 4172; Lu, H. et al. Drug Metab. Disp. (2003) 31, 452; Lu, H. et al. Drug. Metab. Disp. (2003) 31, 572).

[0006] In order to improve bioavailability, an (-)-EGCG prodrug (hereinafter "(-)

-EGCG-octaacetate") was designed, where acetate groups were added to the (-)-EGCG backbone (Lam, W. H. et al, Bioorg. Med. Chem. (2004) 12, 5587; US 7,544,816, Figure 1). (-)-EGCG-octaacetate is converted to (-)-EGCG in mouse plasma (Lambert, J. D. et al. Drug. Metab. Disp. (2006) 34, 21 1 1). In human breast cancer MDA-MB-231 cells, (- )-EGCG-octaacetate is converted to its active metabolite, (-)-EGCG, and is better absorbed (Landis-Piwowar, K. R. et al. Cancer Res. (2007) 67, 4303). It has better bioavailability than (-)-EGCG in esophageal and colon cancer cells (Lambert, J. D. et al, Drug. Metab. Disp. (2006) 34, 2111). Lambert et al, (2006) further showed that intragastric administration of (-)-EGCG-octaacetate to mice resulted in higher bioavailability compared to administration of equimolar doses of (-)-EGCG. The biological efficacy of (-)-EGCG-octaacetate and its enhanced potency compared to (-)- EGCG has been established in mouse models of breast cancer (Landis-Piwowar, K. R. et al., Cancer Res. (2007) 67, 4303 ), prostate cancer (Lee, S. K. et al, Nutr. Cancer (2008) 60, 483), skin cancer (Vyas. S. et al., J. Agric. Food Chem. (2007) 55, 6319; Chiou, Y. S. et al., Carcinogenesis (2013) 34, 1315), colon cancer (Chiou, Y. S. et al., J. Agric. Food Chem. (2012) 60, 3441 ), colitis (Chiou, Y. S. et al, J. Agric. Food Chem. (2012) 60, 3441) and endometriosis (Wang, C. C. et al, Angiogenesis (2013) 16, 59).

[0007] (-)-EGCG is methylated by the enzyme catechol-O-methyltransferase (COMT) to 0-methylated derivatives (Zhu, B. T. et al. Drug Metab. Dispos. (2000) 28, 1024; Meng, X. et al, Chem. Res. Toxicol. (2002) 15, 1042). Inhibition of COMT activity has been shown to enhance the antiproliferative and proapoptotic activity of (-)-EGCG in human lung cancer and breast cancer cell lines, respectively (Forester, S. C. and Lambert, J. D, Carcinogenesis (2014) 35, 365; Landis-Piwowar, K. et al, Oncol. Rep. (2010) 24, 563).

[0008] In humans, a single gene for COMT encodes both a soluble COMT (S-

COMT) and a membrane-bound COMT (MB -COMT). A single nucleotide polymorphism (G to A) in codon 108 (S-COMT) or 158 (MB-COMT) results in a valine to methionine (Val to Met) substitution, leading to a high- (Val/Val [H/H]), intermediate- (Val/Met [H/L]), or low-activity (Met/Met [L/LJ) form of COMT. There is a three-to-four-fold difference in enzyme activity between the high- and low-activity expressed genes (Lachman, H. M. et al. Pharmacogenetics. (1996) 6, 243). A case-control study of breast cancer in Asian-American women revealed that women who consumed green tea and who also carried the low activity COMT polymorphism had a reduced risk of breast cancer (Wu, A. H. et al. Cancer Res. (2003) 63, 7526). In contrast, among those who were homozygous for the high activity COMT allele, breast cancer risk did not differ between tea drinkers and non-tea drinkers. A possible explanation for this difference is that in women with the high activity COMT allele, (-)-EGCG may have been converted by COMT into methylated (-)-EGCG metabolites, and that (-)-EGCG and other tea polyphenols may be less cancer-protective upon methylation. Previous studies have shown that methylated (-)-EGCGs have reduced biological activity (Landis-Piwowar, K. R. et al, J. Cell. Physiol. (2007) 213, 252). Tanaka, H. et al., (Chem. Asian J. (2010) 5, 2231 ), provide further support for this hypothesis. The COMT genotype may be a contributing factor to the variable response to GTE and (-)-EGCG in the human population. Investigators have studied potential correlations of COMT genotype and EGCG metabolism and pharmacokinetics in humans. However, no studies to date have correlated EGCG biological response (efficacy) with COMT genotype.

[0009] Therefore there is a need for a new medical treatment for uterine fibroids

(or uterine leiomyomas). Further, there is a need for novel treatment methodologies with non-hormonal mechanisms of action, providing antiproliferative, antiangiogenic and antifibrotic properties, ideally from a single agent.

[0010] There is also a need for novel compositions that affect similar molecular pathways as (-)-EGCG but are less susceptible to methylation and hence have enhanced potency and/or broader use in the human population.

SUMMARY OF THE INVENTION

[0011] This invention relates to novel compositions and methods for the treatment of leiomyomas, in some embodiments, uterine leiomyomas (also known as uterine fibroids). In some aspects, this invention provides novel compositions and methods incorporating prodrugs of (-)-epigallocatechin-3-gallate (EGCG) and prodrugs of (-)- EGCG analogs, for the treatment of leiomyoma. In some other aspects, the invention provides methods of synthesis for compounds of formula 1 and Compounds 2 and 2a of Figure 3.

[0012] The present inventors have demonstrated novel biological activity and properties of (-)-EGCG-octaacetate, and novel analog compounds, such as compounds (- )-2a and (-)-4a, also herein referred to as compounds 2a and 4a as designated in Figure 3 in leiomyoma cells, such as uterine leiomyoma cells. Said analogs are (-)-EGC 3- hydroxybenzoate hexaacetate (compound (-)-2a, otherwise referred to herein as Compound 2a of Figure 3) and (-)-EGC 3,5-dihydroxybenzoate heptaacetate (compound (-)-4a). otherwise referred to herein as Compound 4a of Figure 3). [0013] In some aspects, the invention provides a method for treating leiomyoma or uterine leiomyomas otherwise referred to herein as uterine fibroids comprising administering to a patient in need thereof (-)-EGCG-octaacetate, (-)-EGC 3- hydroxybenzoate hexaacetate (compound 2a of Figure 3) and (-)-EGC 3,5- dihydroxybenzoate heptaacetate (compound 4a of Figure 3). In some embodiments (-)- EGCG-octaacetate together with a COMT activity inhibitor is administered which is surprisingly is more effective than (-)-EGCG-octaacetate alone.

[0014] In some embodiments, analogs of (-)-EGCG, where one or two hydroxy! groups have been removed from the gallate ester moiety (D ring), such as compounds (-)- 2a and (-)-4a, which are less prone to variations in biological activity based on COMT genotype, useful in treating leiomyoma or uterine fibroids, are provided. In some embodiments, these analogs are used in the treatment of leiomyomas, such as uterine leiomyomas.

[0015] In some other embodiments, the invention provides a method for design and synthesis of novel analogs of (-)-EGCG and their acetylated prodrugs, such as compounds (-)-2a and (-)4a, that are less prone to COMT-mediated methylation than (-)-

EGCG and in certain embodiments of the invention are useful in the treatment of leiomyoma (uterine fibroids).

[0016] In yet some other embodiments, the invention provides novel compositions and methods for the treatment of leiomyoma. In particular, in one embodiment, the invention provides novel compositions and methods incorporating prodrugs of (-)-EGCG and prodrugs of (-)-EGCG analogs for the treatment of leiomyoma, such as uterine leiomyoma.

[0017] In some other embodiments, the invention provides the use of the novel analogs of (-)-EGCG-octaacetate as an antiproliferative, antiangiogenic and/or antifibrotic in human leiomyoma cells.

[0018] Some embodiments of the present invention provides novel compositions of (-) -EGCG analogs and their acetylated prodrugs and methods for their synthesis. [0019] In some further embodiments the present invention includes novel compositions for the treatment of leiomyoma comprised of (-)-EGCG-octaacetate, in combination with a COMT inhibitor.

[0020] In some other embodiments, the present invention provides novel methodologies for the treatment of leiomyoma. In one embodiment, these methodologies involve novel acetylated prodrugs of (-)-EGCG analogs. In a second embodiment, these methodologies involve the administration of (-)-EGCG octaacetate in patients with the low activity COMT genotype.

[0021] In yet some other embodiments of the invention a method for the treatment of a condition requiring antiproliferative, antiangiogenic. and/or antifibrotic therapy through the administration of a formulation containing an acetylated prodrug of an (-)- EGCG analog as the active component is provided.

[0022] Some other embodiments of the present invention include novel methodologies for the treatment of conditions requiring the use of an inhibitor of proteasomal chymotrypsin-like activity, or a dual inhibitor of proteasomal chymotrypsin- like activity and protein kinase B (Akt) signaling, whereby such methodologies involve the use of a novel acetylated prodrug of an (-)-EGCG analog.

[0023] In some other embodiments of the invention, suitable formulations comprising the compositions of this invention may be administered by a variety of routes, such as oral, rectal, nasal, topical, vaginal, parenteral, and pulmonary administration.

[0024] Accordingly, in some embodiments, the invention provides a method for treating a condition requiring an induction of cell apoptosis and/or a condition requiring a reduction in proteasomal chymotrypsin-like activity using a compound of formula I, or in a preferred embodiment, a compound of 4a as per Figure 3. In another embodiment a compound 4 or pharmaceutically acceptable salt thereof or a compound that metabolizes to a compound 4 of Figure 3 once administered to a patient (such as a human patient or subject) is used. In some embodiments, the method comprises administering to a subject in need thereof, a therapeutically effective amount of compound of formula I (such as compound 4a of Figure 3) or a pharmaceutical composition comprising said compound. [0025] In some other embodiments, the invention provides a method for treating a condition requiring: (a) a reduction in cell proliferation; (b) a reduction in angiogensis; (c) a reduction in fibrosis; (d) a reduction in Akt pathway signaling; (e) a reduction in Akt pathway signaling and a reduction in proteasomal chymotrypsin-like activity, using a compound of formula I, (-)-EGC 3 -hydroxybenzoate, or (-)-EGC 3 -hydroxy benzoate hexaacetate, in another embodiment compound 4a or 2a of Figure 3, or in another embodiment a compound 4a of Figure 3. In another embodiment compound 4 or 2 of Figure 3 or a compound that metabolizes to compound 4 or 2 once administered or a pharmaceutically acceptable salt of compound 4 or 2 is used. In some embodiments, the method comprising administering to a subject (such as a human) in need said compounds or a therapeutically effective amount of a said compounds or a pharmaceutical composition comprising same.

[0026] In some other embodiments, the invention provides a method for treating leiomyoma, such as uterine leiomyoma, or endometriosis, the method comprising administering to a subject (such as a human) in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3 -hydroxybenzoate hexaacetate, in another embodiment compound 4a or 2a of Figure 3, or in another embodiment compound 4a of Figure 3. In another embodiment compound 4 or 2 of Figure 3 or a compound that metabolizes to compound 4 or 2 once administered or a pharmaceutically acceptable salt of compound 4 or 2 is used. In some embodiments, the method comprising administering to a subject in need said compounds or a therapeutically effective amount of a said compounds or a pharmaceutical composition comprising same.

[0027] In some other embodiments, the invention provides a method for treating leiomyoma (such as uterine leiomyoma) in patients with the low activity COM Γ genotype, using a therapeutically effective amount of (-)-EGCG octaacetate, the method comprising administering to a subject (such as a human) in need said compound or, a pharmaceutical composition comprising a therapeutically effective amount of (-)-EGCG octaacetate. [0028] In some other embodiment, the invention provides a method for treating leiomyoma (such as uterine leiomyoma), such as in patients with high or intermediate activity COMT genotype, using an effective amount of (-)-EGCG octaacetate and a COMT inhibitor. In some embodiments the method comprises administering to a subject (such as a human) in need, a pharmaceutical composition comprising a therapeutically effective amount of a combination of (-)-EGCG octaacetate and a COMT inhibitor in the same or different pharmaceutical compositions. In some other aspects, patients with high and intermediate activity COMT genotype, a dose of up to four-fold higher dosage of (-)- EGCG octaacetate is used than used in patients with low activity COMT genotype. The method may comprise measuring surrogate markers of efficacy.

[0029] In some embodiments, the COMT inhibitor is selected from the group consisting of: dinitrocatechol, entacapone (DNC), tolcapone, and nitecapone, or in one embodiment, DNC.

[0030] In some other embodiments, the invention provides a method for treating leiomyoma, using an inhibitor of proteasomal ehymotrypsin-like activity or a dual inhibitor of proteasomal chymotrypsin-like activity and Akt signaling. In some aspects the method comprises administering to a subject (such as a human) in need, a pharmaceutical composition comprising a therapeutically effective amount of said inhibitor. [0031 J Additional aspects and advantages of the present invention will be apparent in view of the description which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Particular embodiments of the present invention will now be explained by way of example and with reference to the accompanying drawings, in which: [0033] Figure 1 shows the structures of (-)-EGCG, (-)-EGCG analogs 2 and 4, (-)-

EGCG-octaacetate (Pro-EGCG or compound 1), and (-)-EGCG-oetaacetate analogs 2a and 4a.

[0034] Figure 2 shows the structures of the racemic trans-isomers of 2a and 4a. [0035] Figure 3 shows the synthetic scheme for compounds 2, 4, 2a and 4a starting from (-)-EGCG, where a, b, c, d, and e are as described in Examples 1 - 8.

[0036] Figure 4 shows the inhibition of human leiomyoma cell growth by (-)-

EGCG-octaacetate and its analogs as compared to (-)-EGCG in UtLM-ht cells (transformed cells) [Figure 4A], and UtLM cells (non-transformed cells) [ Figure 4B]. [0037] Figure 5 shows the inhibition of proteasomal chymotrypsin-like activity by

(-)-EGCG, (-)-EGCG-octaaeetate and its analogs in human leiomyoma UTLM-ht cells.

[0038] Figure 6 shows the effect of (-)-EGCG, (-)-EGCG-octaacetate and its analogs on protein biomarkers of cell proliferation, apoptosis and angiogenesis, and Akt phosphorylation in human leiomyoma cells. Figure 6A is in UtLM-ht cells (transformed cell) and Figure 6B is in UtLM cell (non-transformed cells).

[0039] Figure 7 shows the inhibition of RMA expression of fibrosis biomarkers by

(-)-EGCG-octaacetate analogs in human leiomyoma UTLM-ht cells.

[0040] Figure 8 shows how COMT inhibition preferentially increases the antiproliferative activity of (-)-EGCG and (-)-EGCG-octaacetate in human leiomyoma UTLM-ht cells.

DETAILED DISCLOSURE OF THE INVENTION

[0041 ] The present invention is directed to novel compositions of (-)-EGCG analogs, their acetylated prodrugs, and methods for treating leiomyomas, such as uterine leiomyoma. [0042] In some aspects, this invention provides novel compositions and methods for the treatment of leiomyomas, such as uterine leiomyomas. In some embodiments, the invention provides novel compositions and methods comprising prodrugs of (-)- epigallocatechin-3-gallate (EGCG) and prodrugs of (-)-EGCG analogs, for the treatment of said leiomyomas.

[0043] In some aspects, these compounds provide a novel mechanism of action for treating leiomyomas, with antiproliferative, antiangiogenic and antifibrotic properties. [0044] The novel (-)-EGCG analogs and acetylated prodrugs of the present invention may be synthesized using methods disclosed herein. In some aspects of the invention, the invention provides a novel method of synthesis for (-)-EGC 3- hydroxybenzoate hexaacetate (compound (-)-2a) and (-)-EGC 3,5-dihydroxybenzoate heptaacetate (compound (-)-4a). In some embodiments of the invention, these compounds act as dual inhibitors of proteasomal chymotrypsin-like activity and Akt signaling. It is herein shown that the antiproliferative activity of compounds (-)-2a and (-)-4a are less sensitive to catechol-Q-methyltransferase activity (COMT) than (-)-EGCG, which in some aspects may reduce the biological activity of (-)-EGCG. The present invention has also shown that compounds (-)-2a and (-)-4a have greater biological activity than (-)- EGCG and (-)-EGCG-octaacetate in human leiomyoma cell lines.

DEFINITIONS

[0045] Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purpose of the present invention the following terms are defined below:

[0046] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more. [0047] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0048] The term "analog" is intended to mean a compound that is similar or comparable in structure, but not identical, to a reference compound. As used herein, an analog is a chemical compound that may be structurally related to another but differs in composition (for example as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group). An analog may be derived from a natural source or be prepared using chemical synthesis.

[0049] Pharmaceutical compositions according to the present invention may be comprised of a combination of analogs of the present invention, as described herein.

[0050] As used herein, the term "pharmaceutically acceptable carrier" is a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and non-toxic and neither biologically or otherwise undesirable. It may include any and all solvents such as phosphate buffered saline, water, saline, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. For a review, such as taught in Remington's Pharmaceutical Sciences, 17^th ed., Mack Publishing Company, Easton Pa., 1985 or Remington's: The Science and Practice of Pharmacy, edited by David B. Troy, Paul Beringer, Lippincott Williams & Wilkins, 2006, incorporated herein by reference.

[00511 As used herein, the term "pharmaceutically acceptable salt" includes those salts of compounds of the invention that are safe and effective for the respective intended use and that possess the desired biological activity. For a review, such as taught in Remington's Pharmaceutical Sciences, 17* ed., Mack Publishing Company, Easton Pa., 1985 or Remington's: The Science and Practice of Pharmacy, edited by David B. Troy, Paul Beringer, Lippincott Williams & Wilkins, 2006. incorporated herein by reference.

[0052] "Administration" is used to describe the process in which the compositions of the present invention are delivered to the subject. The compositions may be administered in various ways including oral, rectal, nasal, topical, vaginal, parenteral and pulmonary routes, among others.

[0053] The terms "treatment" or "treating" are intended to mean obtaining a desired pharmacologic and/or physiologic effect, or an improvement in a disease condition in a subject or improvement of a symptom associated with a disease or a medical condition in a subject. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom associated therewith and/or may be therapeutic in terms of a partial or complete cure for a disease and/or the pathophysiologic effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal and includes: (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, (e.g., arresting its development or progression); (c) relieving the disease (e.g., reducing symptoms associated with the disease); or (d) maintaining the subject in a disease- or symptom-free state following other therapeutic intervention. [0054] The term "subject" is used to describe an animal, preferably a human, to whom treatment is administered with the compositions of the present invention.

[0055] The term "therapeutically effective" is intended to mean an amount of a composition sufficient to substantially improve a symptom associated with a disease or a medical condition or to improve, ameliorate, reduce or prevent the underlying disease or medical condition, for example as measured with a biological marker. A therapeutically effective amount of a composition may provide a treatment for a disease such that the onset of the disease is delayed, hindered, or prevented, or the disease symptoms are ameliorated, or the term of the disease is altered.

[0056] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency. Abbreviations: a-SMA: a-smooth muscle actin;

Ac: acetyl; Alct: protein kinase B; AMC: 7-amino-4-methylcoumarin; Coll: collagen type

I; COMT: catechol- O-methyltransferase; CT: chymotrypsin; DCM: dichloromethane;

DMF: dimethylformamide; DMSO: dimethyl sulfoxide; DNC: dinitrocatechol; EGC: epigallocatechin; EGCG: epigallocatechin gallate; GAPDH: glyceraldehyde-3 -phosphate dehydrogenase; GTE: green tea extract; MW: molecular weight; MTT: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD: optical density; pAkt: phosphorylated Akt; PARP: poly ADP ribose polymerase; PCNA: proliferation cell nuclear antigen; PGR: polymerase chain reaction; RT: reverse transcriptase; SMA: smooth muscle actin; THF: tetrahydrofuran; TLC: thin layer chromatography; VEGF: vascular endothelial growth factor; VEGF-R2: VEGF receptor 2.

Novel Compounds and Methods of Producing Same

[0057] In some embodiments, the invention provides compounds having the formula:

wherein R is either I I or Ac, and R' is either OH or OAc, with the proviso that when R is H, then R' is OH, and when R is Ac, then R' is OAc.

[0058] In some other embodiments, the invention provides a method for the synthesis of the above noted compounds of formula I, said method comprising the steps of: a. reacting (-)-EGCG with benzyl bromide under conditions to produce perbenzyl-

(-)-EGCG;

b. reacting perbenzyl-(-)-EGCG under conditions suitable to produce 5, 7, 3', 4', 5'-pentabenzyl-(-)-EGC;

c. reacting 5, 7, 3", 4', 5'-pentabenzyl-(-)-EGC with 3, 5-dibenzyloxybenzoic acid under conditions to produce 5, 7, 3 ', 4', 5 ' -pentabenzy 1 -(-)-EGC 3,5, dibenzyloxybenzoate; and d. reacting 5, 7, 3'. 4\ 5 ' -pentabenzyl-(-)-EGC 3,5-dibenzyloxybenzoate with palladium on carbon in a mixture of tetrahydrofuran and methanol under conditions to produce (-)-EGC 3,5, dihydroxybenzoate (Compound 4 of Figure 3). Said method further comprising step e when making Compound 4a of Figure 3 : e. reacting (-)-EGC 3.5, dihydroxybenzoate with pyridine and acetic anhydride under conditions to produce (-)-EGC 3,5, dihydroxybenzoate heptaacetate.

[0059] In some other embodiments, the invention provides a method for the synthesis of the compound of formula I, wherein R is either H or Ac and R' is H resulting in compounds (-)-EGC 3,5 dihydrobenzoate (compound 2 of Figure 3) and (-)-EGC 3,5, hydroxybenzoate hexaacetate (compound 2a of Figure 3), said method comprising the steps of: a. reacting (-)EGCG with benzyl bromide under conditions to produce perbenzyl-

(-)-EGCG;

b. reacting perbenzyl-(-)-BGCG under conditions suitable to produce 5, 7, 3', 4', 5'-pentabenzyl-(-)-EGC;

c. reacting 5, 7, 3 ', 4', 5'-pentabenzyl-(-)-EGC with 3-benzyloxybenzoic acid under conditions to produce 5, 7, 3', 4', 5¹ -pentabenzy l-(-)-EGC 3- benzyloxybenzoate; and

d. reacting 5, 7, 3'. 4', 5'-pentabenzyl-(-)-EGC 3-benzyloxybenzoate with a palladium on carbon in a mixture of tetrahydrofuran and methanol under conditions to produce EGC 3.5, dihydroxybenzoate (Compound 2 of Figure 3). Said method further comprising step e when making Compound 2a of Figure 3 : e. reacting EGC 3 -hydroxybenzoate with pyridine and acetic anhydride under conditions to produce (-)-EGC 3 -hydroxybenzoate hexaacetate.

[0060] Suitable conditions as referred to herein would be known to a person of skill in the art upon reading this description and the examples.

Methods and Uses

[0061 ] A method for treating a condition requiring a reduction in proteasomal chymotrypsin-like activity, such as cancer the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula 1 . In a preferred embodiment, the compound 4a of Figure 3, is used in said treatment. In another embodiment, compound 4a of Figure 3 is used in the methods of the invention. 100621 A method for treating a condition requiring an induction of cell apoptosis, such as cancer, leiomyoma (such as uterine leiomyoma), and/or endometriosis, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I.

[0063 j In one embodiment, the invention provides a method for treating a condition requiring a reduction in cell proliferation, such as cancer, leiomyoma (such as uterine leiomyoma) and/or endometriosis, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, (-)-EGC 3 -hydroxybenzoate (compound 2 of Figure 3), or (-)-EGC 3 -hydroxybenzoate hexaacetate (compound 2a of Figure 3). [0064J In another embodiment, the invention provides a method for treating a condition requiring a reduction in angiogenesis, such as cancer or leiomyoma (such as uterine leiomyomas), the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, (-)-EGC 3 -hydroxybenzoate (compound 2 of Figure 3), or (-)- EGC 3 -hydroxy benzoate hexaacetate (compound 2a of Figure 3).

[0065] In another aspect, the invention provides a method for treating a condition requiring a reduction in Fibrosis, such as endometriosis or leiomyoma (such as uterine leiomyoma), the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula 1, (- )-EGC 3 -hydroxybenzoate, or (-)-EGC 3 -hydroxybenzoate hexaacetate.

[0066] In one embodiment, the invention provides a method for treating a condition requiring a reduction in Akt pathway signaling, such as cancer, leiomyoma and/or endometriosis, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula 1, (-)-EGC 3-hydroxybenzoate, or (-)-EGC 3 -hydroxy benzoate hexaacetate.

[0067] The invention also provides a method for treating leiomyoma, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula 1, (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3-hydroxybenzoate hexaacetate.

[0068] In one aspect, the invention provides a method for treating leiomyoma

(such as uterine leiomyoma), in patients with the low activity COMT genotype, the method comprising administering to a subject, such as a human, in need, a pharmaceutical composition comprising a therapeutically effective amount of (-)-EGCG octaacetate.

[0069] In another aspect the invention provides a method for treating leiomyoma

(such as uterine leiomyoma), the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a combination of (-)-EGCG octaacetate and a COMT inhibitor. In one embodiment, the COMT inhibitor is selected from the group consisting of: dinitrocatechol ("DNC"), entacapone, toleapone, and nitecapone.

[0070] In a further embodiment, the invention provides a method for treating leiomyoma (such as uterine leiomyoma), in patients with the high and intermediate activity COMT genotypes, the method comprising administering to a subject in need, a pharmaceutical composition comprising up to a four-fold higher dose of (-)-EGCG octaacetate than used in patients with the low activity COMT genotype, and measuring surrogate markers of efficacy.

[0071] In some aspects, the invention provides a method for treating leiomyoma

(such as uterine leiomyoma), the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of proteasomal chymotrypsin-like activity. In another embodiment, the method for treating leiomyoma comprises administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a dual inhibitor of proteasomal chymotrypsin-like activity and Akt signaling, such as those comprising compounds 2a and/or 4a.

[0072] To the extent that compounds of Figure 1, (-)-ECGC octaacetate, (-)-2a and (-)-4a form salts, the use of said pharmaceutically acceptable salts of said compounds may also be used in said methods.

PHARMACEUTICAL COMPOSITIONS

[0073] In one aspect, the compounds and compositions of the invention comprise: a compound of formula I, (-)-EGCG octaacetate, (-)-EGC 3 -hydroxybenzoate (Compound 2 of Figure 3), (-)-EGC 3 -hydroxybenzoate hexaacetate (Compound 2a of Figure 3), (-)- EGC 3,5-dihydroxybenzoate (Compound 4 of Figure 3), or (-)-EGC 3,5- dihydroxybenzoate heptaacetate (Compound 4a of Figure 3 ). In another embodiment, the invention compositions of the invention comprise one or more compounds of formula I, pharmaceutical acceptable salts thereof or mixtures thereof. Pharmaceutically acceptable salts are known in the art and it should be understood that to the extent that the aforementioned compounds form pharmaceutically acceptable salts, said salts of the compounds described herein are encompassed by the present invention.

[0074] In one embodiment, the invention provides pharmaceutical compositions of the invention comprising at least one compound as noted above or in other embodiments at least one of said compounds and one or more pharmaceutically acceptable carriers.

[0075] In another embodiment, the invention provides pharmaceutical compositions comprising a compound of the invention as per formula I, (-)-EGC 3- hydroxybenzoate (Compound 2), or (-)-EGC 3 -hydroxybenzoate hexaacetate (Compound 2a) and one or more pharmaceutically acceptable carriers. [0076] In another embodiment, the invention comprises compositions comprising compound 2a. In another embodiment, the invention comprises compositions comprising compound 4a. In another embodiment the invention comprises compositions of compound 1 ((-)-EGCG octaacetate), pharmaceutical acceptable salts or mixtures thereof. In another embodiment the invention comprises compositions of compound 1 ((-)-EGCG octaacetate) pharmaceutical acceptable salts or mixtures thereof and a COMT inhibitor. In another embodiment the compositions comprise one or more pharmaceutically acceptable carriers.

[0077] In another embodiment, the invention provides a pharmaceutical composition useful in treating or inhibiting leiomyoma (such as uterine leiomyoma) in a subject, such as a human, in need thereof such composition comprising: (-)-EGCG octaacetate and one or more pharmaceutically acceptable carriers; (-)-EGCG octaacetate and a COMT inhibitor and one or more pharmaceutically acceptable carriers; Compound 2a and one or more acceptable carriers; Compound 4a and one or more acceptable carriers. In another embodiment, the compositions comprise pharmaceutically acceptable salts of said compounds (-)EGCG octaacetate, compound 2a and compound 4a. In one embodiment, the leiomyoma is a fibroid, in another embodiment a uterine fibroid.

[0078] Compositions of the invention include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration.

[0079] Compositions of the present invention suitable for oral administration can be presented for example as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; or as a liquid solution, suspension or emulsion. The active ingredient can also be presented as bolus, electuary, or paste. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. For example, in an embodiment each tablet may contain from about 2.5 mg to about 500 mg of the active ingredient and each cachet or capsule may contain from about 2.5 to about 500 mg of the active ingredient.

[0080] Formulations suitable for topical administration in the mouth include lozenges or pastilles comprising the active ingredient. Pharmaceutical compositions for topical administration according to the present invention can be formulated for example as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients, and optionally one or more excipients or diluents. Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier. Formulations for rectal administration may be provided as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held to the nose. Suitable formulations wherein the carrier is a liquid for administration by a nebulizer, include for example aqueous or oily solutions of the active agent. Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain preservatives, buffers, bacteriostatic agents and solutes which render the formulation isotonic with the blood of the patient; and aqueous and nonaqueous sterile suspensions which can include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. [0081] It should be understood that in addition to the ingredients particularly mentioned above, the compositions and formulations of this invention can include other agents conventional in the art regarding the type of formulation in question. For example, formulations suitable for oral administration can include such further agents as sweeteners, thickeners, and flavoring agents.

[0082] The compositions can be administered by any of a variety of routes, such as orally, intranasally, parenterally or by inhalation, and can take the form, for example, of tablets, lozenges, granules, capsules, pills, ampoule, suppositories or aerosol form. They can also be in the form of suspensions, solutions, and emulsions of the active ingredient(s) in aqueous or nonaqueous diluents, syrups, granulates or powders. Methods of delivery include, but are not limited to, intraarterial, intramuscular, intravenous, intranasal, and oral routes. In a specific embodiment, the compositions of the invention can be administered locally to the area in need of treatment; such local administration can be achieved, for example, by local infusion during surgery, by injection, or by means of a catheter.

[0083] The magnitude of prophylactic or therapeutic dose of a composition of the invention will, of course, vary with the nature of the severity of the condition to be treated and with the particular composition of the invention and its route of administration. It will also vary according to the age, weight and response of the individual patient, and the efficacy and toxicity of the composition. Suitable dosage formulations and methods of administering the agents can be readily determined by those of skill in the art. For example, a daily dosage can be divided into one, two or more doses in a suitable form to be administered at one, two or more times throughout a time period.

[0084] Ideally, the composition of the invention should be administered to achieve peak concentrations of the active compound at sites of the disease. Peak concentrations at disease sites can be achieved, for example, by orally administering, for example, a tablet, capsule or syrup containing the active ingredient.

[0085] The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. EXAMPLES

[0086] Compound (-)-2a, the peracetate derivative of (-)-epigallocatechin (EGC)

3-hydroxybenzoate, was previously synthesized using a semi-synthesis approach starting from (-)-EGC (Huo, C. et al., Can. ./. Chem. (2008) 86, 495). Because (-)-EGC is not readily available, the invention provides an alternative semi-synthesis starting from the more readily available (-)-EGCG according to the scheme presented in Figure 3. This approach has also been applied to the synthesis of compound (-)-4a, the peracetate derivative of (-)-EGC 3,5-dihyd roxy benzoate . Further in the examples below where indicated, Human uterine leiomyoma (UtLM) cells (GM 10964) were purchased from Coriell Institute and transformed human uterine leiomyoma cells (UtLM-ht) transfected with human telomerase reverse transcriptase were provided as a kind gift from Dr. Darlene Dixon (NIEHS) [Carney et al., 2002].

EXAMPLE 1

Synthesis of perbenzyl-(-)-EGCG (compound 3)

[0087] (-)-EGCG (5.00 g; 10.6 mmol: 1.0 eq) was dissolved in dry dimethylformamide (DMF) (200 mL) at room temperature under argon. Potassium carbonate (30.63 g; 222 mmol; 21.4 eq) and benzyl bromide (24.5 mL; 206 mmol; 19.4 eq) were added in succession. The mixture was stirred at room temperature for 72 hours. The mixture was slowly poured into ice water (6 L) under vigorous stirring. The mixture was extracted with ethyl acetate (2 x 600 mL). The organic layers were combined, dried over MgSCu and concentrated at reduced pressure. Toluene (400 mL) was added to the crude mixture to form an azeotrope with the remaining DMF, then the solvent was evaporated at reduced pressure. After evaporation, the residue was still a liquid. The product crystallized after addition of 100 mL of diethyl ether and storage at room temperature overnight. The precipitate was filtered over a fritted glass funnel of fine porosity, washed with diethyl ether and dried under vacuum to afford compound 3 (Figure 3). EXAMPLE 2

Synthesis of 5,7,3',4',5'-pentabenzyl-(-)-EGC (compound 5)

[0088] Compound 3 (6.71 g; 5.69 mmol; 1.0 eq) was dissolved in 1,2- dimethoxyethane ( 130 mL) and methanol (130 mL), then potassium carbonate (2.845 g; 19.9 mmol; 3.5 eq) was added. The mixture was stirred at room temperature for 8 hours. The solvent was evaporated at reduced pressure. The crude mixture was taken with dichloromethane (DCM) (300 mL) and filtered over a fritted glass funnel of fine porosity to remove the K2CO3. Purification of the crude mixture by flash chromatography on silica gel (eluent: from pure hexane to hexane/ethyl acetate = 5:5) afforded compound 5 (Figure 3).

EXAMPLE 3

Synthesis of 5,7,3',4',5'-pentabeozyl-(-)-EGC 3,5-dibenzyloxybenzoate

(compound 7)

[0089] To a solution of 3.5 -di benzy loxybenzoic acid (367 mg. 1.10 mmol, 1.0 eq) in DCM (30 mL) under argon, was added 4-dimethylaminopyridine (134 mg, 1.10 mmol, 1.0 eq), compound 5 (821 mg, 1.10 mmol, 1.0 eq), and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (231 mg, 1.19 mmol, 1.1 eq). The solution was stirred at room temperature for 48 hours. The solution was extracted with saturated aqueous KHSO4 (1 x 15 ml.), saturated aqueous NaHCCb (2 x 15 mL), and saturated aqueous NaCl (1 x 15 mL). The organic layer was concentrated at reduced pressure, then purified by automatic flash chromatography on silica gel (eluent: hexanes → hexanes/ethyl acetate = 8/2) to afford compound 7 (Figure 3).

EXAMPLE 4

Synthesis of 5,7,3',4',5'-pentabenzyl-(-)-EGC 3-benzyloxybenzoate (compound 6) [0090] To a solution of 3-benzyloxybenzoic acid (259 mg, 1.13 mmol, 1.0 eq) in

DCM (30 mL) under argon, was added 4-dimethylaminopyridine (138 mg, 1.13 mmol, 1.0 eq), compound 5 (858 mg, 1.13 mmol, 1.0 eq), and l-ethyl-3-(3- dimethylaminopropyl)earbodiimide hydrochloride (239 mg, 1.25 mmol, 1.1 eq). The solution was stirred at room temperature for 48 hours. The solution was extracted with saturated aqueous KHSO4 (1 x 15 mL), saturated aqueous NaHCCb (2 x 15 mL), and saturated aqueous NaCl (1 x 15 mL). The organic layer was concentrated at reduced pressure, then purified by automatic flash chromatography on silica gel (eluent: hexanes → hexanes/ethyl acetate = 8/2) to afford compound 6 (Figure 3).

EXAMPLE 5

Synthesis of (-)-EGC 3,5-dihydroxybenzoate (compound 4)

[0091] Palladium on carbon 10% w/w (26 mg; 45.6 μιηοΐ Pd; 0.2 eq) was added to a solution of compound 7 (242 mg; 225 μιηοΙ; 1.0 eq) in a mixture of tetrahydrofuran (THF) (5 mL) and methanol (7 mL). The suspension was stirred in the presence of hydrogen gas (10 bar) at room temperature for 48 hours. The reaction mixture was filtered over celite and concentrated at reduced pressure, then purified by preparative thin layer chromatography (TLC) (silica gel, eluent: hexane/ethyl acetate = 4/6) to afford compound 4 (Figure 3).

EXAMPLE 6

Synthesis of (-)-EGC 3-hydroxybenzoate (compound 2)

[0092] Palladium on carbon 10% w/w (55 mg; 96.5 μιηοΐ Pd; 0.2 eq) was added to a solution of compound 6 (463 mg; 479 μηιοΐ; 1.0 eq) in a mixture of THF (5 mL) and methanol (7 mL). The suspension was stirred in the presence of hydrogen gas (10 bar) at room temperature for 48 hours. The reaction mixture was filtered over celite and concentrated at reduced pressure, then purified by preparative TLC (silica gel, eluent: hexane/ethyl acetate = 4/6). Crystallization with a mixture of Et20/hexanes [1/5 (v/v)] afforded compound 2 (Figure 3). EXAMPLE 7

Synthesis of (-)-EGC 3,5-dihydroxybenzoate hepiaacetate (compound 4a, also referred to herein as compound (-)-4a) [0093] Compound 4 (42 mg; 57.0 μηιοΐ; 1.0 eq) was added to a mixture of pyridine (710 μί; 8.8 mmol; 80 eq) and acetic anhydride (725 μί; 7.7 mmol; 70 eq) under argon. The mixture was stirred at room temperature for 18 hours, then ethyl acetate (50 mL) and 1 M HC1 (1 mL) were added. The mixture was washed with a saturated aqueous solution of CuSCU (3 x 10 mL), water (2 x 10 mL), brine (1 x 10 mL), and NaHCOj (2 x 10 mL). The organic layer was dried over MgSC¼ and concentrated at reduced pressure. Purification by preparative TLC on silica gel (eluent: hexane/ethyl acetate = 4/6), followed by crystallization with a mixture of DCM/hexanes [1/6 (v/v)] afforded compound 4a (Figure 3).

EXAMPLE 8

Synthesis of (-)-EGC 3-hydroxybenzoate hexaacetate (compound 2a, also referred to herein as compound (-)-2a)

[0094] Compound 2 (126 mg; 296 μηιοΐ; 1.0 eq) was added to a mixture of pyridine (1904 μί; 23.6 mmol; 80 eq) and acetic anhydride (1949 μ!>; 20.7 mmol; 70 eq) under argon. The mixture was stirred at room temperature for 18 hours, then ethyl acetate (50 mL) and 1 M HCl (1 mL) were added. The mixture was washed with a saturated aqueous solution of CUSCM (3 x 10 mL), water (2 x 10 mL), brine (1 x 10 mL), and NaHCCb (2 x 10 mL). The organic layer was dried over MgSCU and concentrated at reduced pressure. Purification by preparative TLC on silica gel (eluent: hexane/ethyl acetate = 4/6), followed by crystallization with a mixture of DCM/hexanes [1/6 (v/v)] afforded compound 2a (Figure 3).

EXAMPLE 9

Inhibition of leiomyoma cell proliferation [0095] Human leiomyoma UtLM-ht (A) or UtLM (B) cells were treated with (-)-

EGCG, (-)-EGCG-octaacetate, (-)-2a or (-)-4a (at 5, 10, or 25 μΜ) daily for 3 days (with each compound repeatedly added every 24 hours), followed by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (Nam, S. et al, J. Biol. Chem. (2001) 276, 13322). Briefly, cells were plated at a density of 2* 10³ cells/well in 96-well plates. After 24 hours, the cells (in triplicate wells) were treated daily with the control solvent dimethyl sulfoxide (DMSO) or the indicated compounds and concentrations. At the end of the experiment, the cells were incubated with 100 μΐ/well of 0.5% MTT solution for 4 hours at 37°C. The M i l solution was removed and the dye was solubilized with 100 μΐ/well of DMSO for 10 minutes. The optical density (OD) of each well was measured with a spectrophotometer at 570 nm. Mean values of OD for each concentration were calculated.

[0096] It is shown herein that (-)-EGCG-octaacetate and compounds (-)-2a and (-

)-4a can inhibit the proliferation of leiomyoma cells in a concentration-dependent fashion (Figure 4). It is also shown that (-)-EGCG octaacetate and compounds (-)-2a and (-)-4a are more potent than (-)-EGCG in inhibiting the proliferation of leiomyoma cells (Figure 4). The present invention also shows that compounds (-)-2a and (-)-4a have antiproliferative activity. The racemic trans-substituted isomers of 2a and 4a (Figure 2) were previously observed to reduce the proliferation of human breast cancer MCF-7 cells (Kuhn. D. et al., Front. Biosci. (2005) 10, 1010), but not these compounds.

EXAMPLE 10

Inhibition of proteasomal chymotrypsin-like activity in leiomyoma cells [0097] Human leiomyoma UTLM-ht cells were treated with the control solvent

DMSO, not treated (NT), or treated with (-)-EGCG, (-)-EGCG-octaacetate, (-)-2a or (-)- 4a (at 25 μΜ) for 24 hours, followed by preparation of whole cell extracts and performance of a cell-free proteasomal chymotrypsin (CT)-like activity assay. The cells were harvested by a scraper, centriftiged at 1,500 rpm and suspended in ice-cold cell lysis buffer (50 mM Tris-Cl, pH 7.4/ 150 mM NaCl/ 0.5% NP-40) for 30 minutes, followed by centrifugation at 12,000 rpm for 15 min. The clear supernatant was used as a whole cell protein extract. Whole-cell extracts (10 μ&) were incubated with Suc-Leu-Leu-Val-Tyr- AMC (40 μχηοΙ/L) fluorogenic substrate at 37°C in 100 uL of assay buffer (50 mmol/L Tris-HCl, pH 8) for 2 hours. After incubation, production of hydrolyzed 7-amino-4- methyleoumarin (AMC) groups was measured using a Victor3 Multilabel Counter with an excitation filter of 380 nra and an emission filter of 460 nm.

[0098] It is herein shown that (-)-EGCG, (-)-EGCG octaacetate and compounds (-

)-2a and (-)-4a, are able to inhibit proteasomal chymotrypsin-like activity in leiomyoma cells (Figure 5). It is also shown that (-)-EGCG octaacetate and compounds ( )-2a and (-)- 4a, are more potent than (-)-EGCG in inhibiting proteasomal chymotrypsin-like activity in leiomyoma cells (Figure 5). The results also demonstrate the inhibition of proteasomal chymotrypsin-like activity by compound (-)-4a. It was previously shown that the racemic trans 4a (Figure 2) can inhibit proteasomal chymotrypsin-like activity in human T cell leukemia Jurkat cells (Kuhn, D. et al., Front. Biosci. (2005) 10, 1010). Previous studies with (-)-2a in Jurkat T cells showed that it can also inhibit proteasomal chymotrypsin-like activity in that cell line (Huo, C. et al., Can. J. Chem. (2008) 86, 495).

EXAMPLE 11

Inhibition of protein biomarkers of proliferation, apoptosis, and angiogenesis, and

Akt signaling in leiomyoma cells

[0099] Human leiomyoma UtLM-ht (A) or UtLM (B) cells were either untreated

(NT) or treated with the control solvent DMSO, (-)-EGCG, (-)-EGCG-octaacetate, (-)-2a or (-)-4a (at 1 5, 25 or 35 μΜ) for 48 hours, followed by Western blotting using specific antibodies to proliferation cell nuclear antigen (PCNA) (MW 37 kDa), cyclin A (MW 54 kDa), caspase-3 (MW 32 kDa), poly A DP ribose polymerase (PARP) (MW 116 kDa), vascular endothelial receptor 2 (VEGF-R2) (MW 152 kDa), vascular endothelial growth factor C (VEGF-C) (MW 46 kDa), phosphorylated Akt (pAkt) and total Akt (MW 64 kDa) and actin (MW 46 kDa). The cells were grown to -80% confluency in 100 mm dishes, and treated as described followed by harvesting and preparing whole cell extracts as described in Example 10. Equivalent amounts of protein extracts (30-50 μg) were separated by NuPAGE No vex 10% Bis- I ris Gel under reducing conditions using 1 16 V for 120 minutes. The proteins were then electrophoretically transferred onto PVDF membranes using the XCell 11 Blot Module. After blocking nonspecific binding sites by incubation for 1 hour with PBS-containing 5% fat-free milk and 0.1% Tween 20, the membranes were incubated with the primary antibodies overnight at 4°C. Immunologic detection was performed using the following primary antibodies: PCNA (at 1 :500 dilution), eye I in A ( 1 :500), VEGF-C (1 :300), VEGF-R2 ( 1 :500), PARP (1 :500), caspase 3 (1 :500), Akt (1 :500) and pAkt (1 :500). The membranes were then incubated for 1 hour with horseradish peroxidase conjugated secondary antibodies diluted at 1 :5000 with blocking buffer. The antigen-antibody complexes were detected with the ECL chemiluminescence detection system. The membranes were reprobed with a monoclonal antibody raised against β-actin (diluted 1 :1000 to 5000) as an internal control for protein loading and normalization between samples. Films exposed to blots were scanned and the optical densities of the positive signals were quantified.

[00100] To provide further molecular evidence for the antiproliferative activity of (-)-EGCG octaacetate and compounds (-)-2a and (-)-4a (Figure 4), the effects of these agents were measured on expression of cell cycle-specific proteins. PCNA and cyclin A are two S-phase markers (Dou, Q. P. and Pardee, A. B. Prog. Nucl. Acid Res. Mol. Biol.

( 1996) 53, 197). We show for the first time that (-)-EGCG octaacetate and compounds (-

)-2a and (-)-4a can reduce the levels of protein biomarkers of proliferation of leiomyoma cells in a concentration-dependent fashion (Figure 6). This is the first assessment of the effect of (-)-EGCG octaacetate, (-)-2a and (-)-4a on cellular PCNA and cyclin A protein levels.

[00101 ] The inventors observed that compounds (-)-2a and (-)-4a induced apoptosis-related morphological changes in leiomyoma cells (data not shown). [00102] In order to confirm these observations, the effect of these compounds on PARP protein levels and caspase activation (which is associated with decreased pro- caspase-3 protein levels) (Yang, H. and Dou, Q. P., Curr. Drug Targets (2010) 11, 733) were measured. The present studies show that compounds (-)-2a and (-)-4a can reduce the levels of PARP and caspase-3 protein in leiomyoma cells, associated with an induction of apoptosis (Figure 6). This effect is more potent than that observed with (-)-EGCG or (-)- EGCG octaacetate. Surprisingly, a previous study with (-)-2a in Jurkat T cells indicated that it was less potent than (-)-EGCG octaacetate in reducing PARP protein levels and inducing cell death, and equally potent in reducing caspase-3 protein levels (Huo, C. et al., Can. J. Chem. (2008) 86, 495). This implies that the biological activity of (-)-2a and/or (-)-EGCG octaacetate, is dependent on the cellular environment (e.g. type of cell). The data relating to the biologic activity of (-)-4a in relation to apoptosis biomarkers in leiomyoma cells, is comparable with previous observations using the racemic trans 4a (Figure 2) in human T cell leukemia Jurkat cells (Kuhn, D. et al., Front. Biosci. (2005) 10, 1010). [00103] Angiogenesis is necessary for the development of uterine fibroids (Islam, M.S. et al., Mol. Nutr. Food. Res. (2014) 58, 1667). Shown herein is the effect of (-)- EGCG, (-)-EGCG octaacetate, (-)-2a and (-)-4a on two angiogenesis biomarkers - VEGF- R2 and VEGF-C (Ebos, J. M. L. and Kerbel, R. S. Not. Rev. Clin. Oncol. (201 1 ) 8, 210). Results indicate that compound (-)-4a can reduce the protein levels of VEGF-R2 and VEGF-C in leiomyoma cells, suggesting antiangiogenic properties (Figure 6). It is also shown that compound (-)-2a can reduce the protein levels of VEGF-R2 in leiomyoma cells while its effect on VEGF-C levels may relate to the cellular environment (Figure 6). Similarly, the effect of (-)-EGCG octaacetate on VEGF-C protein levels may also depend on the cellular environment (Figure 6). This is supported by the lack of effect of (-)- EGCG on these biomarkers in leiomyoma cells, unlike previous observations in endometrial tissue (Xu, H. et al., Fertil. Steril. (2011) 96, 1021 ).

[00104] Akt is a serine-threonine kinase that is activated by phosphoinositide-3- kinase (PI3K). Phosphorylated Akt triggers a number of downstream signals, regulating multiple cellular processes including proliferation, apoptosis, angiogenesis and fibrosis (Naumann, R. W. Gynecol Oncol. (201 1 ) 123, 41 1 ; Makker, A. et al. Gynecol. Endocrinol. (2012) 28, 175). (-)-EGCG octaacetate has previously been shown to inhibit Akt signaling in leukemia cell lines and in vivo models (Lam, W. H. et al. Bioorg. Med. Chem. (2004) 12, 5587; Chiou, Y. S. et al, J. Agric. Food Chem. (2012) 60, 3441 ; Chiou, Y. S. et al. Carcinogenesis (2013) 34, 131 5). It was assessed whether (-)-EGCG octaacetate and its analogs could inhibit Akt activation in human leiomyoma cells. The results indicate that (-)-EGCG octaacetate, (-)-2a and (-)-4a all inhibit Akt phosphorylation (Akt signaling) in leiomyoma cells (Figure 6).

EXAMPLE 12

Inhibition of biomarkers of fibrosis

[00105] Human leiomyoma UtLM-ht cells were either untreated (NT) or treated with 25 μΜ of (-)-EGCG, (-)-EGCG-octaacetate, (-)-2a or (-)-4a for 48 hours, followed by preparation of RNAs and reverse transcriptase (RT) - polymerase chain reaction (PGR) analysis for levels of a-smooth muscle actin (aSMA) and collagen type I (CoLl) with glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) as a loading control. Total RNA was extracted from cells using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. One microgram of total RNA was used for reverse transcription using the RT first-strand kit. The PGR reactions were carried out in a final volume of 20 μί. The standard cycling condition was 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The products were visualized by agarose gel electrophoresis followed by ethidium bromide staining.

[00106] Fibrosis plays an important role in uterine leiomyoma formation and growth

(Islam, M.S. et al, Mol. Nutr. Food. Res. (2014) 58, 1667). It is herein demonstrated that compounds (-)-2a and (-)-4a can reduce the gene expression of two fibrosis biomarkers - aSMA and Coll and in this activity, are more effective than (-)-EGCG and (-)-EGCG octaacetate (Figure 7). (-)-EGCG has been shown to reduce these fibrosis markers in other models of a mouse liver injury model (Tipoe, G. L. et al., Toxicol. (2010) 273, 45), again implying a relationship of this activity to cellular environment. EXAMPLE 13

Inhibition of COMT enhances the antiproliferative activity of

(-)-EGCG and (-)-EGCG octaacetate

[00107] The data herein indicates that compounds (-)-2a and (-)-4a have properties consistent with more potent antiproliferative, antifibrotic, and antiangiogenic activity than

(-)-EGCG octaacetate and (-)-EGCG in human leiomyoma cell lines. One plausible mechanism for this increased activity might be related to a reduced susceptibility to methylation in the presence of COMT, due to the absence of the catechol structure in the ester D ring. To test this hypothesis, a known inhibitor of COMT, dinitrocatechol (DNC) (Perez, R. A. et al., Biochem. Pharmacol. (1993) 45, 1973) was used. Human leiomyoma UtLM-ht cells were pre-treated with 10 μΜ DNC or control solvent DMSO for 2 hours, followed by co-treatment with DMSO, (-)-EGCG, (-)-EGCG-octaacetate, (-)-2a or (-)-4a at the indicated concentrations for 3 days (each drug repeatedly added every 24 hours) and the ΜΊΤ assay.

[00108] It was observed that DNC treatment significantly increased the antiproliferative potency of (-)-EGCG and (-)-EGCG octaacetate, but had a lesser enhancement of the antiproliferative activity of compounds (-)-2a and (-)-4a (Figure 8). These data support the hypothesis, and suggest that UtLM-ht cells express active COMT, and that COMT is involved in regulating the sensitivity of cells to (-)-EGCG-based therapy. Zhang et al. (Zhang, D. et al., Gynecol Obstet Invest (2014) 78, 109) also demonstrated the presence of active COMT in a human uterine leiomyoma cell line. Consistent with the observations reported herein, it was previously shown that DNC treatment increases the biological activity of (-)-EGCG and (-)-EGCG octaacetate in human breast cancer MDA-MB-231 cells - a cell line with a high level of COMT activity (Huo, C. et al., Bioorg. Med. Chem. (2010) 18, 1252; Landis-Pivvowar, K. R. et al, Oncol. Rep. (2010) 24, 563). Other COMT inhibitors have also been reported to synergistically enhance (-)-EGCG's gowth inhibition of cancer cells (Forester, S. C. and Lambert, J. D. Carcinogenesis (2014) 35, 365).

[00109 J Overall, the present data illustrates that the metabolites of prodrugs (-)-2a and (-)-4a have reduced susceptibility to COM 1-mediated methylation as compared to (- )-EGCG (Figures 1, 8), whereby such methylation could be responsible for decreased biological activity. This could account for an increased activity of compounds (-)-2a and (-)-4a over (-)-EGCG octaacetate and (-)-EGCG in all of the assays reported herein. Therefore, intracellular COMT activity is one aspect of cellular environment that dictates the biological activity of (-)-EGCG and (-)-EGCG octaacetate within the specific cellular context. However, there may be other contributing factors leading to several of the observations indicate that the biological activity of (-)-EGCG, (-)-EGCG-octaacetate as well as compound (-)-2a. varies depending on the cellular environment.

[00110] In human leiomyoma cells (with active COMT), compounds (-)-2a and (-)- 4a demonstrated greater antiproliferative activity, and potential for proapoptotic, antiangiogenic. and antifibrotic activity (as evidenced by biomarkers) than (-)-EGCG octaacetate and (-)-EGCG (Figures 4, 6, 7).

[001 1 1 J The results indicated that compounds (-)-2a and (-)-4a can inhibit Akt phosphorylation in leiomyoma cells (Figure 6). This mechanism can (at least partially) account for the activity of these compounds in assays and affecting biomarkers associated with cell proliferation, apoptosis, angiogenesis and fibrosis. For example, it has been shown that transcription of CoLI and aSMA is regulated by the Akt signaling pathway in leiomyoma cells (Salama, S. A. et al., Fertil Steril. (2012) 98, 178). It has been shown that Akt signaling is required for proliferation of human uterine leiomyoma cells (Yin, X. J. et al.. Am. J. Obstet. Gynecol. (2007) 196, 176). Also, Akt signaling has been shown to play a role in regulating VEGF expression (Jiang, B. H. and Liu, L. Z. Biochim. Biophys. Acta (2008) 1784, 150).

[00112] The antiproliferative and proapoptotic effects of Akt pathway inhibition may be (at least partially) affected by downstream cascades involving elevation of p27 and ΙκΒα levels (Lee, M. et al., Mol. Cancer Ther. (2011) 10, 1450; Hussain, A. R. et al., PLoS One (2012) 7, e39945). Both p27 and ΙκΒα protein levels are also enhanced by proteasome inhibition (Landis-Piwowar. K. R. et al., Drug Resist. Update (2006) 9, 263). Therefore, inhibition of proteasomal chymotrypsin-like activity and Akt signaling are likely to result in synergistic proapoptotic and antiproliferative effects. (-)-EGCG- octaacetate, and the racemic trans-substituted isomers of 2a and 4a have all been shown to elevate p27 and IKBU protein levels in human T cell leukemia Jurkat cells (Kuhn, D. et al.. Front. Biosci. (2005) 10, 1010). In the present study, (-)-EGCG octaacetate, (-)-2a and (-)-4a inhibited proteasomal chymotrypsin-like activity in human uterine leiomyoma cells (Figure 5).

[001 13] In summary, it was found that compounds (-)-2a and (-)-4a may have reduced susceptibility to COMT-mediated methylation and are proteasome and Akt pathway inhibitors. These mechanisms can create synergies and result in enhanced antiproliferative, antiangiogenic, and antifibrotic properties as compared to (-)-EGCG octaacetate, in cells with active COMT. Therefore, compounds (-)-2a and (-)-4a may have efficacy in a broader patient population than (-)-EGCG (or green tea extracts) including both high-activity and low-activity COMT phenotypes.

1001 14] The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.

[00115] While specific embodiments of the present invention have been described in the examples, it is apparent that modifications and adaptations of the present invention will occur to those skilled in the art. The embodiments of the present invention are not intended to be restricted by the examples. It is to be expressly understood that such modifications and adaptations which will occur to those skilled in the art are within the scope of the present invention, as set forth in the following claims. For instance, features illustrated or described as part of one embodiment can be used in another embodiment, to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the claims and their equivalents.

REFERENCES

Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence. Am. J. Obstet. Gynecol. 188: 100-107, 2003.

Carney SA, Tahara H, Swartz CD, Risinger Jl, He H, Moore AB, Haseman JK,

Barrett JC, Dixon D. 2002. Immortalization of human uterine leiomyoma and

myometrial cell lines after induction of telomerase activity: Molecular and

phenotypic characteristics. Lab Invest 82(6):719-728.

Chen D, Milacic V, Chen MS, Wan SB, Lam Wll, Huo C, Landis-Piwowar KR, Cui QC, Wali A, Chan TH, Dou QP. Tea polyphenols, their biological effects and potential molecular targets. Histol. Histopathol. 23: 487-496, 2008.

Chiou YS, Ma NJ, Sang S, Ho CT. Wang YJ, Pan MH. Peracetylated (-)- epigallocatechin-3-gallate (AcEGCG) potently suppresses dextran sulfate sodium-induced colitis and colon tumorigenesis in mice. J. Agric. Food Chem. 60: 3441-3451, 2012. Chiou YS, Sang S, Cheng KH, Ho CT, Wang YJ, Pan MH. Peracetylated (-)- epigallocatechin-3-gallate (AcEGCG) potently prevents skin carcinogenesis by- suppressing the PKD1 -dependent signaling pathway in CD34+ skin stem cells and skin tumors. Carcinogenesis 34: 1315-1322, 2013. Dou QP, Pardee AB. Transcriptional activation of thymidine kinase, a marker for cell cycle control. Prog. Nucleic Acid Res. Mol. Biol. 53: 197-217, 1996.

Du GJ, Zhang Z, Wen XD, Yu C, Calway T, Yuan CS, Wang CZ. Epigalloeateehin gal late (EGCG) is the most effective cancer chemopreventive polyphenol in green tea. Nutrients 4: 1679-1691, 2012.

Ebos J ML, Kerbel RS. Antiangiogenic therapy: impact on invasion, disease progression, and metastasis. Nat. Rev. Clin. Oncol. 8: 210-221 , 201 1. Forester SC. Lambert JD. Synergistic inhibition of lung cancer cell lines by (-)- epigallocatechin-3-gallate in combination with clinically used nitrocatechol inhibitors of catechol-O-methyltransferase. Carcinogenesis 35: 365-372, 2014.

Han DH, Kim JH. Difference in growth suppression and apoptosis induction of EGCG and EGC on human promyelocytic leukemia HL-60 cells. Arch. Pharm. Res. 32: 543-547, 2009. Hao X, Sun Y, Yang CS, Bose M, Lambert JD, Ju J, Lu G, Lee MJ, Park S, Husain A, Wang S. Inhibition of intestinal tumorigenesis in Apc(min/+) mice by green tea polyphenols (polyphenon E) and individual eatechins. Nutr. Cancer 59: 62-69, 2007.

Huo C, Shi G, Lam WH, Chen D, Cui QC, Dou QP, Chan TH. Semi-synthesis and proteasome inhibition of D-ring deoxy analogs of (-)-epigallocatechin gallate (EGCG), the active ingredient of green tea extract. Can. J. Chem. 86: 495-502, 2008.

Huo C, Yang H, Cui QC. Dou QP, Chan TH. Proteasome inhibition in human breast cancer cells with high catechol-O-methyltransferase activity by green tea polyphenol EGCG analogs. Bioorg. Med. Chem. 18: 1252-1258, 2010.

Hussain AR, Ahmed SO, Ahmed M, Khan OS, Al Abdulmohsen S, Platanias LC, Uddin S. Cross-talk between NFkB and the PI3-kinase/AKT pathway can be targeted in primary effusion lymphoma (PEL) cell lines for efficient apoptosis. PLoS One 7: e39945, 2012.

Islam MS, Akhtar MM, Ciavattini A, Giannubilo SR, Protic O, Janjusevic M, Ciarmela P. Use of dietary phytochemica!s to target inflammation, fibrosis, proliferation, and angiogenesis in uterine tissues: promising options for prevention and treatment of uterine fibroids? Mol. Nutr. Food Res. 58: 1667-1684, 2014.

Jiang BH, Liu LZ. PI3K/PTLN signaling in tumorigenesis and angiogenesis. Biochim. Biophys. Acta 1784: 150- 158, 2008. Kohri T, Matsuinoto N, Yamakawa M, Suzuki M, Nanjo F, Hara Y, Oku N. Metabolic fate of (-)-[4-³H] epigallocatechin gallate in rats after oral administration. J. Agric. Food Chem. 49: 4102-41 12, 2001 .

Kohri T, Suzuki M, Nanjo F. Identification of metabolites of (-)-epicatechin gallate and their metabolic fate in the rat. J. Agric. Food Chem. 51 : 5561-5566, 2003.

Kuhn D, Lam WH, Kazi A, Daniel KG, Song S, Chow LM, Chan TH, Dou QP. Synthetic peracetate tea polyphenols as potent proteasome inhibitors and apoptosis inducers in human cancer cells. Front. Biosci. 10: 1010- 1023, 2005.

Lachman HM, Papolos DF, Saito T, Yu YM, Szumlanski CL, Weinshilbouni RM. Human catechol-O-methyltransfcrase pharmacogenetics: description of a functional polymorphism and its potential application to neuropsychiatric disorders. Pharmacogenetics 6: 243-250, 1996.

Lam WH, Kazi A, Kuhn DJ, Chow LMC, Chan ASC, Dou QP, Chan TH. A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallate [(-)-EGCG]. Bioorg. Med. Chem. 12: 5587-5593, 2004.

Lambert JD, Lee MJ, Lu H, Meng X, Hong JJ, Seril DN, Sturgill MG, Yang CS. Epi gal 1 ocatech in-3 -gal late is absorbed but extensively glucuronidated following oral administration to mice. J. Nutr. 133: 4172-4177, 2003.

Lambert JD, Sang S, Hong J, Kwon SJ, Lee MJ, Ho CT, Yang CS. Peracetylation as a means of enhancing in vitro bioactivity and bioavailability of epigallocatechin-3-gallate. Drug Metab. Dispos. 34: 21 1 1 -21 16, 2006. Landis-Piwowar K, Chen D, Chan TH, Dou QP. Inhibition of catechol-0 methyltransfera.se activity in human breast cancer cells enhances the biological effect of the green tea polyphenol (-)-EGCG. Oncol. Rep. 24: 563-569, 2010. Landis-Piwowar KR, Huo C, Chen D, Milacic V, Shi G, Chan TH, Dou QP. A novel prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate as a potential anticancer agent. Cancer Research 67: 4303-4310, 2007.

Landis-Piwowar KR, Milacic V, Chen D. Yang H, Zhao Y, Chan TH, Dou QP. The proteasome as a potential target for novel anticancer drugs and chemosensitizers. Drug Resist. Update 9: 263-273, 2006.

Landis-Piwowar KR, Wan SB, Wiegand RA, Kuhn DJ, Chan TH, Dou QP. Methylation suppresses the proteasome-inhibitory function of green tea polyphenols. J. Cell. Physiol. 213 : 252-260, 2007.

Lee M, Theodoropoulou M, Graw J, Roncaroli F, Zatelli MC, Pellegata NS. Levels of p27 sensitize to dual PBK/mTQR inhibition. Mol. Cancer Ther. 10: 1450-1459, 2011. Lee SK, Chan WK, Lee 1 W, Lam WH, Wang X, Chan TH, Wong YC. Effect of a prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate on the growth of androgen- independent prostate cancer in vivo. Nutr. Cancer 60: 483-491 , 2008.

Li C, Meng X, Winnik B, Lee MJ, Lu H, Sheng S, Buckley B, Yang CS. Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry. Chem. Res. Toxicol. 14: 702-707, 2001.

Liao S, Umekita Y, Guo J, Kokontis JM, Hiipakka RA. Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate. Cancer Lett. 96: 239-243, 1995. Lu I I, Meng X, Li C, Sang S, Patten C, Sheng S, Hong J, Bai N. Winnik B, Ho CT, Yang CS. Glucuronides of tea catechins: enzymology of biosynthesis and biological activities. Drug Metab. Disp. 31 : 452-461, 2003. Lu 11, Meng X, Yang CS. Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigalloeatechin gallate. Drug Metab. Dispos. 31 : 572-579, 2003.

Makker A, Goel MM, Das V, Agarwal A. PI3K-Akt-mTOR and MAPK signaling pathways in polycystic ovarian syndrome, uterine leiomyomas and endometriosis: an update. Gynecol. Endocrinol. 28: 175- 181 , 2012.

Meng X, Sang S, Zhu N, Lu II. Sheng S, Lee MJ, Ho CT, Yang CS. Identification and characterization of methylated and ring-fission metabolites of tea catechins formed in humans, mice, and rats. Chem. Res. Toxicol. 15 : 1042-1050. 2002.

Nakagawa K, Okuda S, Miyazawa T. Dose dependent incorporation of tea catechins, (-)- epigallocatechin-3 -gallate and (-)-epigallocatechin, into human plasma. Biosci. Biotechnol. Biochem. 61 : 1981 - 1985, 1997.

Nam S, Smith DM, Don QP. Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J. Biol. Chem. 276: 13322- 13330, 2001.

Naumann RW. The role of the phosphatidylinositol 3-kinase (PI3K) pathway in the development and treatment of uterine cancer. Gynecol. Oncol. 123 : 41 1 -420, 2011.

Okushio K, Suzuki M, Matsumoto N, Nanjo F, Hara Y. Methylation of tea catechins by rat liver homogenates. Biosci. Biotechnol. Biochem. 63: 430—432, 1999. Ozercan IH, Sahin N, Akdemir F, Onderci M, Seren S, Sahin K, Kucuk O. Chemoprevention of fibroid tumors by [-] -epigallocatechin-3 -gallate in quail. Nutr. Res. 28: 92-97, 2008. Paschka AG, Butler R, Young CY. Induction of apoptosis in prostate cancer cell lines by the green tea component, (-)-epigallocatechin-3-gallate. Cancer Lett. 130: 1-7, 1998.

Perez RA, Fernandez-Alvarez H, Nieto O, Piedrafita FJ. Inhibition of catechol-O- methyltransferase by 1 -vinyl derivatives of nitrocatechols and nitroguaiacols. Kinetics of the irreversible inhibition by 3 -(3 -hydroxy -4-methoxy-5 -n itro benzylidene)-2,4- pentanedione. Biochem. Pharmacol. 45: 1973- 1981 , 1993.

Roshdy E, Rajaratnam V, Maitra S, Sabry M, Ait Allah AS, Al-Hendy A. Treatment of symptomatic uterine fibroids with green tea extract: a pilot randomized controlled clinical study. Int. J. Women's Health 5: 477-486, 2013.

Salama SA, Diaz-Arrastia CR, Kilic GS, Kamel MW. 2-Methoxyestradiol causes functional repression of transforming growth factor β3 signaling by ameliorating Smad and non-Smad signaling pathways in immortalized uterine fibroid cells. Fertil S.teril. 98: 178- 1 84, 2012.

Tanaka H, Yamanouchi M, Miyoshi H, Hirotsu K, Tachibana H, Takahashi T. Solid- phase synthesis of a combinatorial methylated (±)-epigallocatechin gallate library and the growth-inhibitory effects of these compounds on melanoma B16 cells. Chem. Asian J. 5: 2231 -2248, 2010.

Tipoe GL, Leung TM, Liong EC, Lau ΊΎ, Fung ML, Nanji AA. Epigallocatechin-3- gallate (EGCG) reduces liver inflammation, oxidative stress and fibrosis in carbon tetrachloride (CCU)-induced liver injury in mice. Toxicology 273: 45-52, 2010.

Vyas S, Sharma M, Sharma PD, Singh TV. Design, semisynthesis, and evaluation of o- acyl derivatives of (-)-epigallocatechin-3-ga11ate as antitumor agents. J. Agric. Food Chem. 55 : 6319-6324, 2007.

Wang CC, Xu H, Man GC, Zhang T, Chu KO, Chu CY, Cheng JT, Li G, He YX, Qin L, Lau TS, Kwong J, Chan I^'l l. Prodrug of green tea epigallocatechin-3 -gallate (Pro-EGCG) as a potent anti-angiogenesis agent for endometriosis in mice. Angiogenesis 16: 59-69, 2013.

Wu AH, Tseng CC, Van Den Berg D, Yu MC. Tea intake, COMT genotype, and breast cancer in Asian-American women. Cancer Res. 63 : 7526-7529, 2003.

Xu H, Becker CM, Lui WT, Chu CY, Davis TN, Kung AL, Wang CC. Green tea epigailocatechin-3-gallate inhibits angiogenesis and suppresses vascular endothelial growth factor C/vascular endothelial growth factor receptor 2 expression and signaling in experimental endometriosis in vivo. Ferti!. Steril. 96: 1021 - 1028, 201 1.

Yang H, Dou QP. Targeting apoptosis pathway with natural terpenoids: implications for treatment of breast and prostate cancer. Curr. Drug Targets 1 1 : 733-744, 2010. Yin XJ, Wang G, Khan-Dawood FS. Requirements of phosphatidylinositol-3-kinase and mammalian target of rapamycin for estrogen-induced proliferation in uterine leiomyoma- and myometrium-derived cell lines. Am. J. Obstet. Gynecol. 196: e l -5, 2007.

Yoshino K, Suzuki M, Sasaki K, Miyase T, Sano M. Formation of antioxidants from (-)- epigallocatechin gallate in mild alkaline fluids, such as authentic intestinal juice and mouse plasma. J. Nutr. Biochem. 10: 223-229, 1999.

Zhang D, Rajaratnam V, Al-Hendy O, Haider S, Al-Hendy A. Green tea extract inhibition of human leiomyoma cell proliferation is mediated via catechol-o methyhransferase. Gynecol. Obstet. Invest. 78: 109-118, 2014.

Zhu BT, Patel UK, Cia MX, Conney AH. O-Methylation of tea polyphenols catalyzed by- human placental cytosolic eatechol-O-methyltransferase. Drug Metab. Dispos. 28: 1024- 1030, 2000.

Claims

A compound having the formula:

OR

wherein R is either H or Ac, and R' is either OH or OAc, with the proviso that when R is H, then R' is OH, and when R is Ac, then R' is OAc,

2. A method for the synthesis of a compound of claim 1 , wherein R is H and R' is OH said method comprising the steps of:

a. reacting (-)-EGCG with benzyl bromide under conditions to produce perbenzyl- (-)-EGCG;

b. reacting perbenzyl-(-)-EGCG under conditions suitable to produce 5, 7, 3', 4',

5 ' -pentabenzyl-(-)-EGC ;

c. reacting 5, 7, 3 ', 4', 5'-pentabenzyl-(-)-EGC with 3, 5 dibenzyloxybenzoic acid under conditions to produce 5, 7, 3', 4', 5 ' -pentabenzy l-(-)-EGC 3,5- dibenzyloxybenzoate; and

d. reacting 5, 7, 3', 4', 5' -pentabenzy l-(-)-EGC 3,5- dibenzyloxybenzoate with palladium on carbon in a mixture of tetrahydrofuran and methanol under conditions to produce (-)-EGC 3.5-dihydroxybenzoate.

3. A method for the synthesis of a compound of claim 1 , wherein R is Ac and R' is OAc, comprising the method of claim 2 and further comprising:

e. reacting EGC 3.5-d ihydroxy benzoate with pyridine and acetic anhydride under conditions to produce (-)-EGC 3 , 5 -dihydroxybenzoate heptaacetate.

4. A method for the synthesis of (-)-EGC 3-hydroxybenzoate, said method comprising the steps of:

a. reacting (-)-EGCG with benzyl bromide under conditions to produce perbenzyl-(-)-EGCG

b. reacting perbenzyl-(-)-EGCG under conditions suitable to produce 5, 7, 3', 4', 5 ' -pentabenzyl-(-)-EGC;

c. reacting 5, 7, 3 ', 4', 5'-pentabenzyl-(-)-EGC with 3-benzyloxybenzoic acid under conditions to produce 5, 7, 3 ', 4', 5'-pentabenzyl-(-)-EGC 3- benzyloxybenzoate; and

d. reacting 5, 7, 3 ', 4'. 5'-pentabenzyl-(-)-EGC 3 -benzy loxybenzoate with palladium on carbon in a mixture of tetrahydrofuran and methanol under conditions to produce EGC 3-hydroxybenzoate.

5. A method for the synthesis of (-)-EGC 3-hydroxybenzoate hexaacetate comprising the method of claim 4 and further comprising:

e. reacting EGC 3 -hydroxybenzoate with pyridine and acetic anhydride under conditions to produce (-)-EGC 3 -hydroxybenzoate hexaacetate.

6. A method for treating a condition requiring an induction of cell apoptosis, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1.

7. A method for treating a condition requiring a reduction in proteasomal chymotrypsin-like activity, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1.

8 . A method for treating a condition requiring a reduction in cell proliferation, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, (-)-EGC 3-hydroxybenzoate, or (-)-EGC 3-hydroxybenzoate hexaacetate.

9. A method for treating a condition requiring a reduction in angiogenesis, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3-hydroxybenzoate. or (-)-EGC 3-hydroxybenzoate hexaacetate.

10. A method for treating a condition requiring a reduction in fibrosis, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, (-)-EGC 3-hydroxybenzoate, or (-)-EGC 3-hydroxybenzoate hexaacetate.

1 1. A method for treating a condition requiring a reduction in Akt pathway signaling, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, (-)-EGC 3 -hydro xybenzoate, or (-)-EGC 3 -hydroxybenzoate hexaacetate.

12. A method for treating a condition requiring a reduction in Akt pathway signaling and a reduction in proteasomal chymotrypsin-like activity the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3-hydroxybenzoate hexaacetate.

13. A method for treating leiomyoma, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, (-)-EGC 3-hydroxybenzoate, or (-)-

EGC 3-hydroxybenzoate hexaacetate.

14. The method of claim 13 wherein the leiomyoma is a uterine leiomyoma.

15. A method for treating endometriosis, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or (-)-EGC 3-hydroxybenzoate, or (- )-EGC 3-hydroxybenzoate hexaacetate.

16. A method of any one of claims 8 to 15 wherein the compound is a compound of claim 1.

1 7. A method of claim 6, 7 or 16 wherein the compound is (-)-EGC 3,5- dihydroxybenzoate heptaacetatc.

18. A method of any one of claims 6 - 17, wherein the pharmaceutical compositions comprises one or more pharmaceutically acceptable carriers.

19. A method of any one of claims 6 - 17, wherein the pharmaceutical compositions comprise pharmaceutically acceptable salts of said compounds.

20. A method for treating leiomyoma in patients with the low activity COMT genotype, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of (-)- EGCG octaacetate.

21. A method for treating leiomyoma, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a combination of (-)-EGCG octaacetate and a COMT inhibitor.

22. The method of claim 21 wherein the patient has high or intermediate activity COMT genotype.

23. The method of claim 21 or 22 where the COMT inhibitor is selected from the group consisting of: dinitrocatechol, entacapone, tolcapone, and nitecapone.

24. A method for treating leiomyoma in patients with the high and intermediate activity COMT genotypes, the method comprising administering to a subject in need, a pharmaceutical composition comprising up to a four-fold higher dose of (- )-EGCG octaacetate than used in patients with the low activity COMT genotype, and measuring surrogate markers of efficacy.

25. The method of any one of claims 20 - 24, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

26. A method for treating leiomyoma, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of proteasomal chymotrypsin-like activity.

27. A method for treating leiomyoma, the method comprising administering to a subject in need, a pharmaceutical composition comprising a therapeutically effective amount of a dual inhibitor of proteasomal chymotrypsin-like activity and

Akt signaling.

28. A pharmaceutical composition comprising compounds of claim 1, (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3 -hydroxybenzoate hexaacetate and one or more pharmaceutically acceptable carriers.

29. A pharmaceutical composition comprising a compound of claim 1 and one or more pharmaceutically acceptable carriers.

30. A pharmaceutical composition of claim 1 wherein the compound is (-)-EGC 3,5- dihydroxybenzoate or a pharmaceutically acceptable salt thereof.

3 1. A pharmaceutical composition comprising (-)-EGC 3,5 -dihydroxy benzoate heptaacetate and one or more pharmaceutically acceptable carriers.

32. A pharmaceutical composition useful in treating or inhibiting leiomyoma in a subject in need thereof, such composition comprising (-)-EGCG octaacetate and one or more pharmaceutically acceptable carriers.

33. The use of claims 20 - 27 or the pharmaceutical composition of claim 32, wherein the leiomyoma is a uterine leiomyoma.

34. The method of any one of claims 6 - 27 wherein the subject is a human.

35. The pharmaceutical composition of claim 32 or 33 wherein the subject is a human.

36. A use of a therapeutically effective amount of a compound of claim 1 for treating a condition requiring an induction of cell apoptosis

37. A use of a therapeutically effective amount of a compound of claim 1 for treating a condition requiring a reduction in proteasomal chymotrypsin-like activity.

38. A use of a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3-hydroxybenzoate hexaacetate for treating a condition requiring a reduction in cell proliferation.

39. A use of a of a therapeutically effective amount of a compound of claim 1 , (-)- EGC 3-hydroxybenzoate, or (-)-EGC 3 -hydroxybenzoate hexaacetate for treating a condition requiring a reduction in angiogenesis.

40. A use of a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3- hydroxybenzoate. or (-)-EGC 3-hydroxybenzoate hexaacetate for treating a condition requiring a reduction in fibrosis.

41. A use of a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3 -hydroxybenzoate hexaacetate for treating a condition requiring a reduction in Akt pathway signaling.

42. A use of a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3- hydroxybenzoate. or (-)-EGC 3 -hydroxybenzoate hexaacetate for treating a condition requiring a reduction in Akt pathway signaling and a reduction in proteasomal ehymotrypsin-like activity.

43. A use of a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3 -hy droxybenzoate hexaacetate for treating leiomyoma.

44. The use of claim 43 wherein the leiomyoma is a uterine leiomyoma.

45. A use of a therapeutically effective amount of a compound of claim 1 , (-)-EGC 3- hydroxybenzoate, or (-)-EGC 3-hydroxybenzoate hexaacetate for treating endometriosis.

46. A use of any one of claims 38 to 45 wherein the compound is a compound of claim 1.

47. A use of claim 36, 37 or 46 wherein the compound is (-)-EGC 3,5- dihydroxybenzoate heptaacetate.

48. A use of any one of claims 36 - 47, wherein the use is of a pharmaceutical composition comprising any one of said compounds.

49. The use of claim 48 wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

50. The use of any one of claims 36 -- 49. wherein the pharmaceutical compositions comprise pharmaceutically acceptable salts of said compounds.

51. A use of a therapeutically effective amount of (-)-EGCG octaacetate for treating leiomyoma in a patient with the low activity COMT genotype.

52. A use a therapeutically effective amount of (-)-EGCG octaacetate and a COMT inhibitor for treating leiomyoma in a patient in need thereof.

53. The use of claim 52 wherein the patient has high or intermediate activity COMT genotype.

54. The use of claim 52 or 53 where the COMT inhibitor is selected from the group consisting of: dinitrocatechol, entacapone, tolcapone, and nitecapone.

55. A use of up to a four-fold higher dose of (-)-EGCG octaacetate than used in patients with the low activity COMT genotype for treating leiomyoma in patients with high and intermediate activity COMT genotypes.

56. The use of any one of claims 51 - 55, wherein a pharmaceutical composition comprising any one of said compounds and one or more pharmaceutically acceptable carriers is used.

57. A use of a therapeutically effective amount of an inhibitor of proteasomal chymotrypsin-like activity for treating leiomyoma.

58. A use of a therapeutically effective amount of a dual inhibitor of proteasomal chymotrypsin-like activity and Akt signaling for treating leiomyoma.

59. A use of any one of claims 51 - 58 wherein the leiomyoma is a uterine leiomyoma.

60. The use of any one of claims 36 - 59 in a human.