Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
  • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
  • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
  • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
  • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

• This invention relates to derivates of (-)-epigallocatechin gallate, particularly for use 5 as proteasome inhibitors and/or for inhibition of cancer cell growth.
• the polyphenols found in green tea extracts are (-)-epicatechin (EC), (-)- epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate
• L 5 responsible for the degradation of not only obsolete and misfolded proteins, but also regulatory proteins involved in cell cycle and apoptosis.
• ubiquitin is first conjugated to the substrate, followed by degradation of the substrate and recycling of the amino acids and ubiquitin.
• the ubiquitin/proteasome-dependent degradation pathway plays an essential role in up-regulation of cell proliferation, down-regulation of cell
• proteasome inhibitors show great potential as novel anticancer drugs (Dou, Q. P.; Li, B. Drug Resist
• the P13K/Akt signaling is a widely known tumor cell survival pathway
• Phosphorylated Akt (p- Akt) is the activated form of Akt.
• Akt Once Akt is activated, it can mediate cell cycle progression by phosphorylation and consequent inhibition of the cyclindependent kinase inhibitor p27.24 Recently, (-)-EGCG has been found to inhibit the Akt kinase activity via reducing the phosphatidylinositol 3-kinase signals in MMTV-Her-2/neu mouse mammary tumor NF639 cells, leading to reduced tumor cell growth (Pianetti, S.; Guo, S.; Kavanagh, K. T.; Sonenshein, G. E. Cancer Res 2002, 62, 652-5).
• (-)-EGCG has at least one limitation: it gives poor bioavailability.
• a study by Nakagawa et al. showed that only 0.012% of (-)-EGCG could be absorbed in rats given 56 mg of (-)-EGCG orally (Nakagawa, K.; Miyazawa, T. Anal Biochem. 1997, 248, 41-9). This low absorption was thought to be due to the poor stability of (-)-EGCG in neutral or alkaline solutions.
• pH value of the intestine and body fluid is neutral or slightly alkaline, GTCs will be unstable inside the human body, thus leading to reduced bioavailability.
• this invention provides a compound for inhibiting proteasome having the formula:
• R 11 , R 12 , R 13 , R 2 i, R 22 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H, and C 1 to C 10 acyloxyl group;
• R 5 is selected from the group consisting of -H, C 1 -C 10 -alkyl, C 2 -C 1O -alkenyl, C 2 -C 10 -alkynyl, C 3 -C 7 -cycloalkyl, phenyl, benzyl and C 3 -C 7 -cycloalkenyl, whereas each of the last mentioned 7 groups can be substituted with any combination of one to six halogen atoms;
• R 11 , R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4 is C 1 to C 10 acyloxyl group
• each Of R 11 R 2 , and R 4 is -H, and each of R 12 , R 13 , R 21 , R 22 , and R 3 is an acetate or benzoate group.
• R 11 is -H
• each Of R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4 is an acetate or benzoate group.
• each Of R 11 , R 13 , R 2 , and R 4 is -H, and each of R 12 , R 21 , R 22 , and R 3 is an acetate or benzoate group.
• R 5 is -H
• each Of R 11 , Ri 2 , Ri3 > R-21, and R 22 is an acetate group.
• This particular embodiment also provides the following three variations:
• R 2 is an acetate group, and each of R 3 and R 4 is -H;
• each ofR 2 and R 4 is an acetate group, and R 3 is -H. It is another aspect of this invention to provide a method of reducing tumor cell growth including the step of administering an effective amount of a compound having the formula:
• R 11 , R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H, and C 1 to C 10 acyloxyl group;
• R 5 is selected from the group consisting of -H, C 1 -C 10 -alkyl, C 2 -C 1O -alkenyl, C 2 -C 10 -alkynyl, C 3 -C 7 -cycloalkyl, phenyl, benzyl and C 3 -C 7 -cycloalkenyl, whereas each of the last mentioned 7 groups can be substituted with any combination of one to six halogen atoms; and
• R 11 , R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H, and C 1 to C 10 acyloxyl group;
• R 5 is selected from the group consisting of -H, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 10 -alkynyl, C 3 -C 7 -cycloalkyl, phenyl, benzyl and C 3 -C 7 -cycloalkenyl, whereas each of the last mentioned 7 groups can be substituted with any combination of one to six halogen atoms; and
• This invention also provides a compound for inhibiting proteasome having the formula:
• R 1 is -H
• R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H and -OH;
• R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H and -OH;
• R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H and -OH;
• Figure 1 shows the structures of the (-)-EGCG, and examples of the (-)-EGCG derivatives of this invention
• Figure 2 shows the degradation curve of (-)-EGCG and 1;
• Figure 3 shows the time-course results of peracetate EGCG (1) in culture medium with the presence of vitamin C (area vs time).
• Compound 1 ⁇ ; compound A (di-acetate): x; compound B (mono-acetate): A; EGCG: ⁇ ;
• Figure 4 shows the time-course results of peracetate EGCG (1) in culture medium with the presence of vitamin C with the addition of lysate (area vs time).
• Compound 1 ⁇ ; compound A (di-acetate): x; compound B (mono-acetate): ⁇ ; EGCG: ⁇ ;
• Figure 5 shows the inhibition of the chymotrypsin-like activity of the purified 2OS proteasome by 1 and (-)-EGCG;
• Figure 6 shows (a) the inhibition of Proteasome Actvitiy by 1 and (-)-EGCG in vivo; (b) Western blot assay of ubiquitin after treatment with 1 and (-)-EGCG; Figure 7 shows the amount of p-Akt levels with 1 and (-)-EGCG treatment;
• Figure 8 shows the cell viability in Jurkat cells treated with 1 and (-)-EGCG
• Figure 9 shows the results of treating Jurkat cells with 25 ⁇ M of each indicated polyphenol for 4 h (A), up to 8 (C), or 24 h (B), or of LNCaP cells treated with 25 ⁇ M of indicated compound for 24 h (D), followed by Western blot analysis using specific antibodies to Ubiquitin, Bax, I ⁇ B ⁇ , p27 and Actin.
• the bands indicated by an arrow are possible ubiquitinated forms of Bax and I ⁇ B ⁇ .
• A, Lane 4, Ub- I ⁇ B ⁇ band may be result of spillage from Lane 5.
• Data shown are representative from three independent experiments;
• Figure 10 shows the results of treating Jurkat T cells (A and B) or VA- 13 (C and D) cells with 25 ⁇ M of indicated polyphenols for 24 h.
• A Trypan blue incorporation assay. The data represented are as the mean number of dead cells over total cell population ⁇ SD.
• B Western blot for PARP cleavage.
• C Fluorescent microscopy studies of late-stage apoptosis using a specific antibody to the p85 cleaved PARP fragment conjugated to FITC. Counterstaining with DAPI is used as a control for non-apoptotic cells. Images were obtained with Axio Vision software utilizing an inverted fluorescent microscope (Zeiss, Germany).
• D Quantification of apoptotic cells in C by counting the number of apoptotic cells over the total number of cells in the same field. Data are mean of duplicate experiments ⁇ SD;
• Figure 11 shows the effects of synthetic acetylated polyphenols on breast and prostate cancer cells.
• A MTT assay. Breast cancer MCF-7 cells were treated with each indicated compound at 5 or 25 ⁇ M for 24 h.
• B Morphological changes. Prostate cancer LNCaP cells were treated with 25 ⁇ M of (-)-EGCG or a protected analog for 24 h, followed by morphological assessment. Images were obtained using a phase-contrast microscope at 4OX magnification (Leica, Germany).
• C Soft agar assay. LNCaP cells were plated in soft agar with the solvent DMSO or 25 pM of (-)-EGCG or protected analogs. Cells were cultured for 21 days without further addition of drug. Data shown are representative scanned wells from triplicate experiments.
• D Colonies in C were quantified with an automated counter and presented as mean values ⁇ SD;
• Figure 12 shows the results of treating normal WI-38 and SV-40-transformed VA- 13 cells with 25 ⁇ M of indicated compounds for 24 h (A and B) or 36 h (C), or leukemic Jurkat T and non-transformed YT cells were treated with each compound at 25 ⁇ M for 24 h (D).
• A Chymotrypsin-like activity of the proteasome in intact cells.
• B and C Nuclear staining for apoptotic morphology of both detached and attached cells at 1OX (B) or 4OX (C) magnification. Missing panels indicate that no detachment of cells occurred.
• Figure 15 shows the accumulation of proteasome target and ubiquitinated proteins when Jurkat T cells were treated with the solvent (DMSO), and 25 ⁇ M of the compounds in Figure 13;
• Figure 17 shows the activation of caspase-3 when Jurkat T cells were treated with the solvent (DMSO), and 25 ⁇ M of the compounds in Figure 13;
• prodrugs form of (-)-EGCG is synthesized that improves its bioavailability.
• the prodrugs exhibit: [i] improved stability in physiological conditions at a neutral pH; [ii] remain biologically inactive until enzymatic hydrolysis in vivo, leading to the release of the parent drug; [iii] and lastly, the promoiety groups possess low systemic toxicity Further, three derivatives of (-)-EGCG and their prodrug forms are synthesized, and surprisingly, are found to have higher potency than the natural form of (-)-EGCG itself.
• Rn, R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of -H, and C 1 to C 10 acyloxyl group;
• R 5 is selected from the group consisting of -H, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 1O -alkynyl, C 3 -C 7 -cycloalkyl, phenyl, benzyl and C 3 -C 7 -cycloalkenyl, whereas each of the last mentioned 7 groups can be substituted with any combination of one to six halogen atoms;
• R 11 , R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4 is C 1 to C 10 acyloxyl group
• C 1 -C 6 acyl having the structure -(CO)-R, wherein R is hydrogen or straight-chain or branched alkyl groups having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, 1- methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 2-methylbutyl.
• R is hydrogen or straight-chain or branched alkyl groups having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, 1- methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 2-methylbutyl.
• the alkyl group R can be partially or fully halogenated".
• partially or fully halogenated is meant to express that in the groups characterized in this manner the hydrogen atoms may be partially or fully replaced by identical or different halogen atoms, for example chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chlorofluoromethyl, dichlorofluoromethyl, chlorodifluoromethyl, 1-fiuoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-chloro-2-fluoroethyl, 2-chloro-2,2-difluoroethyl, 2,2- dichloro-2-fluoroethyl, 2,2,2-trichloroethyl and pentafluoroethyl;
• C 1 -Ci O acyloxyl having the structure -0-(CO)-R, wherein R can be any one of -H, Ci- C 9 - alkyl, C 2 -C 9 -alkenyl, C 2 -C 9 -alkynyl, C 3 -C 7 -cycloalkyl, phenyl, benzyl and C 3 -C 7 - cycloalkenyl, whereas each of the last mentioned 7 groups can be substituted with any straight chain or branched alkyl groups, such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 2-methylbutyl.
• peracetate (-)-EGCG, 1 was synthesized ( Figure 1). 1 was found to be more stable than (-)-EGCG.
• the prodrug was biologically inactive against a purified 2OS proteasome, but potently inhibited the proteasome in intact tumor cells. Furthermore, administration of the prodrug, but not its parent compound, to intact tumor cells resulted in the loss of phosphorlyated Akt (p-Akt), indicating inactivation of this cancer- associated kinase. Finally, treatment of leukemia Jurkat T cells with 1 induced cell death.
• Monoclonal antibody to p27 (554069) was purchased from BD Biosciences (San Diego, CA). Vectashield Mounting Medium with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA). The Polyclonal antibody, specific to the PARP cleavage site and FITC-conjugated, was acquired from Biosource (Camarillo, CA). CaspACE FITC-V AD- FMK marker was purchased fiom Promega (Madison, WI).
• the title compound was prepared in a similar manner as described for 2b, using 5 (0.08 g, 0.1 mrnol) and 4-(benzyloxy)benzoic acid (0.049 g, 0.22 mmol) giving 3b as a white solid (0.087 g, 90%).
• the title compound was prepared in a similar manner as described for 2b, using 5 (0.3 g, 0.4 mmol) and 3,5-bis(benzyloxy)benzoic acid (0.27 g, 0.81 mmol) giving 4b as a white solid (0.36 g, 85%).
• Pd(OH) 2 /C (20%, 100 mg) was added to a solution of 102
• Ethanetliiol (1Og, 216 mmol) was added dropwise to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 2.4 g, 100 mmol) in dry DMF (120 mL) at 0 0 C.
• the propene (#e) (3.4 g, 4.6 mmol) was dissolved in dry DMF (30 mL), and to this solution imidazole (1.03 g, 15.2 mmol) and TBSCl (1.2 g, 7.8 mmol) were added successively. The resulting mixture was stirred at rt for 3 days, and then saturated Na 2 CO 3 solution was added to quench the reaction. The mixture was extracted with EtOAc. The organic layers were combined, dried (MgSO 4 ), and evaporated.
• AD-mix- ⁇ (13.0 g) and methanesulfonamide (0.87 g) were dissolved in a solvent mixture of t-BuOH (50 mL) and H 2 O (50 mL). The resulting mixture was stirred at rt for 5 min, then the mixture was cooled to 0 0 C and a solution of [3,5-bis(benzyloxy)-6-benzyl-2-[3-[3,4- ⁇ z,s(benzyloxy)phenyl]-allyl]phenoxy]-tert-butyldimethylsilane in dichloromethane (50 mL) was added.
• a solution of 3,4,5-tris(benzyloxy)benzoic acid (170 mg, 0.39 mmol) was refluxed with oxally chloride (1 mL) in dry CH 2 Cl 2 (10 mL) and one drop of DMF for 3 h. The excess oxally chloride and solvent were removed by distillation and the residue was dried under vacuum for 3 h and dissolved in CH 2 Cl 2 (2 mL).
• fetal bovine serum 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
• the non- transformed natural killer cells (YT line) were grown in RPMI medium containing with 10% (vlv) fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, ImM MEM sodium pyruvate, and 0.1 mM MEM nonessential amino acids solution.
• Human breast cancer MCF-7 cells normal (WI-38) and simian virus-transformed (VA- 13) human fibroblast cells were grown in Dulbecco's modified Eagle's mediums supplemented with 10% (vlv) fetal bovine serum, 100 U/ml penicillin, 100 pg/ml streptomycin. All cell cultures were maintained in a 5% CO 2 atmosphere at 37' C.
• Measurement of the chyrnotrypsin-like activity of the 20s proteasome was performed by incubating 0.5 ⁇ g of purified rabbit 20s proteasome with 40 ⁇ M fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC, with or without various concentrations of natural and synthetic tea polyphenols as described previously (Nam S et. al.).
• VA-13 or WI-38 cells were grown in 24 well plates (2 ml/well) to 70-80% confluency, followed by 24 h treatment with 25 pM (-)-EGCG, 2, or 2a.
• 40 pM Suc-Leu-Leu-Val-Tyr- AMC substrate was then added for 2.5 h at 37 0 C and the chyrnotrypsin-like activity was measured as described above.
• cells were treated with each compound at 25 ⁇ M for 4 or 24 h, and harvested and lysed.
• Suc-Leu-Leu-Val-Tyr-AMC (40 ⁇ M) was then incubated with the prepared cell lysates for 2.5 h and the chymotrypsin-like activity was measured as described above.
• Immunostaining of apoptotic cells was performed by addition of a FITC-conjugated polyclonal antibody that recognizes cleaved poly(ADP-ribose) polymerase (PARP) and visualized on an Axiovert 2 5 (Z m 1 " "3TY) microscope.
• PARP cleaved poly(ADP-ribose) polymerase
• Cells were grown to about 80% confluency in 60 mm dishes.
• VA- 13 cells were then treated with VP- 16, 2, or 2a (25 ⁇ M) for 24 h. Following treatment, both suspension and adhering cells were collected and washed twice in PBS pH 7.4. The cells were washed between all steps listed below and all washes are 1 min duration with PBS.
• MTT Assay MTT was used to determine effects of polyphenols on overall proliferation of tumor cells.
• Human breast MCF-7 cells were plated in a 96 well plate and grown to 70-80% confluency, followed by addition of analogs for 24 h.
• MTT (1 mg/ml) in PBS was then added to wells and incubated at 37 0 C for 4 hours to allow for complete cleavage of the tetrazolium salt by metabolically active cells.
• the MTT was removed and 100 ⁇ l of DMSO was added and colorimetric analysis performed using a multilabel plate reader at 560 nm (Victor 3 ; Perkin Elmer). Absorbance values plotted are the mean from triplicate experiments.
• Soft agar assay LNCaP cells (2 x 10 4 ) were plated in soft agar on 6 well plates in the presence of (-)- EGCG or protected tea analogs (25 pM) or in DMSO (control) to determine cellular transformation activity as described previously (Kazi A et al).
• both detached and attached VA- 13 and WI-38 cells were stained Hoechst 33342 to assess for apoptosis. Briefly, cells were washed 2X in PBS, fixed for 1 h with 70% ethanol at 4 0 C, washed 3X in PBS, and stained with 50 pM Hoechst for 30 min in the dark at room temperature. Detached cells were plated on a slide and attached cells were visualized on the plate with a fluorescent microscope at 1OX or 4OX resolution (Zeiss, Germany). Digital Scientific obtained images with Axio Vision 4.1 and adjusted using Adobe Photoshop 6.0.
• Acetylated Synthetic Tea Polyphenols Do Not Inhibit the Purified 20s Proteasome Activity
• a purified 20s proteasome and a fluorogenic substrate for chyrnotrypsin activity for 30 rnin.
• the half-maximal inhibitory concentration or IC 50 was then determined.
• (-)-EGCG showed to be the most potent with an IC 50 of 0.2 ⁇ M, followed by 2 (IC 50 about 9.9 ⁇ M).
• IC 50 values of compounds 3 and 4 were found to be 14-15 ⁇ M.
• the protected analogs were much less active: ⁇ 35% inhibition at 25 ⁇ M. This is consistent with the results above.
• Soft agar assay is used to determine the transforming activity of tumor cells. Abrogation of colony formation is linked to GI arrest and/or apoptosis. LNCaP cells were added to soft agar in 6-well plates, and were then treated one time at initial plating with 25 ⁇ M of (-)EGCG or a protected analog (Figure HC). After 21 days, colony formation was evaluated. Cells treated with (-)-EGCG showed a significant decrease in colony formation compared to the control cells treated with DMSO ( Figures HC and HD). Protected polyphenols also inhibited tumor cell transforming activity, with 2a and 4a being the most potent inhibitors of colony formation ( Figure HD).
• the green tea polyphenol (-)-EGCG contains a B-ring with three -OH groups ( Figure 1) and has an IC 50 value of 0.3 ⁇ M to a purified 2OS proteasome (Table 1).
• the inhibitory activities of the protected polyphenol analogs to purified proteasome are also determined.
• the peracetate-protected analogs (indicated by *) were not potent inhibitors of the chymotrypsin-like activity of the purified 2OS proteasome, compared to their unprotected analogs, perhaps due to the lack of cellular esterases required for conversion. All the protected analogs at 25 ⁇ M inhibited ⁇ 20% of the purified 2OS proteasome activity (Table 1).
• the pro-apoptotic protein Bax and the cyclin-dependent kinase inhibitor p27 are natural proteasome targets. If the peracetate-protected polyphenols inhibit the intact proteasome activity, we would expect accumulation of Bax and p27 in these cells. To test this idea, Jurkat T cells were treated with both protected and unprotected compounds and lysates were analyzed by Western blot using specific antibodies to Bax and p27 ( Figure 15). After 4 h of treatment, the peracetate-protected analogs 24* and 19*, but not their unprotected analogs, were able to increase accumulation of both Bax (by 3- and 2.5-fold, respectively) and p27 (6- and 11 -fold, respectively) (Figure 15A).
• Natural (-)-EGCG from green tea has been converted to its peracetate compound 1.
• synthetic analogs to (-)-EGCG that possess deletions of the hydroxyl groups on the gallate ring are synthesized.
• the hydroxyl groups were converted to acetate groups to create a prodrug, which could be cleaved via esterases inside the cell and converted to the parent drug.
• the protected analogs were much more potent proteasome inhibitors in intact tumor cells than their unprotected counterparts.
• the peracyloxyl, particularly peracetate-protected analogs are also found to be more potent proteasome inhibitors than their unprotected, hydroxylated counterparts. It was suggested that the iV-terminal Ttu " ' ⁇ n ' in-like subunit (/35) of the proteasome executes a nucleophilic attack on the ester-bonded carbon of (-)-EGCG, which initiates irreversible acylation to the /35-subunit and inhibits its protease activity (Nam S, Smith DM and Dou QP: J Biol Chem 276: 13322-13330, 2001.).
• the peracetate-protected polyphenols were much more potent inhibitors of the proteasomal chymotrypsin-like activity than the unprotected polyphenols.
• Peracetate-protected analogs appear to be stable for extended time points evidenced by the decrease in cellular proteasomal chymotrypsin-like activity at 24 h.
• the unprotected analogs appear to lose stability after 24 h treatment.
• Cellular proteasomal chymotrypsin-like activity was inhibited 97% by analog 19* after 24 h while its unprotected counterpart, 6, inhibited only 30% of the activity. Compared to other pairs of protected vs.
• proteasome target proteins were also evaluated after proteasome inhibition with synthetic tea polyphenols (Figure 15).
• the data provided another piece of evidence that peracetate-protected analogs require the milieu of the cell and/or cell extract to be converted into effective proteasome inhibitors, represented by the accumulation of Bax, p27, and ubiquitinatied proteins.
• the prote * " ible to accumulate target proteins with greater efficacy than their unprotected counterparts in a cellular model compared to the in vitro model.
• the unprotected analogs did not appear to accumulate target proteins to the extent of the protected analogs, which may be a result of proton donation from the -OH groups and subsequent degradation.
• the data further indicates that the peracetate-protected analogs undergo conversion to a new compound and act as prodrugs in vivo.
• This invention provides a variety of derivatives of (-)-EGCG that is at least as potent as (-)-EGCG.
• the carboxylate protected forms of (-)-EGCG and its derivatives are found to be more stable than the unprotected forms, which can be used as proteasome inhibitors to reduce tumor cell growth. Further, from the structures of 1, 2, 3, 4, 19*, 21*, 22* and 23*it can be seen that some of the hydroxyl groups of the gallate ring of (-)-EGCG may not be important to the potency. While the preferred embodiment of the present invention has been described in detail by the examples, it is apparent that modifications and adaptations of the present invention will occur to those skilled in the art.

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