WO2006034392A2 - Therapeutic quinones - Google Patents

Abstract

The present invention provides quinone and hydroquinone compounds that have, for example, anti-cancer activity.

Description

THERAPEUTIC QUINONES

Cross-Reference to Related Application

This patent document claims priority to U.S. Application Serial No. 60/612,472, filed on September 23, 2004, which application is incorporated by reference herein.

Background Polyprenylated 1,4-benzoquinones and hydroquinones such as ubiquinones, plastoquinones, and tocopherols are widespread in plants and animals, in which they play important roles in electron transport, photosynthesis and as antioxidants {see Thomson, R.H. Naturally Occurring Quinones, Academic Press, London, 1971, p. 1-197; and Pennock, J.F. hi Terpenoids in plants; J.B. Pridham, Ed. Academic Press, London, 1967, pp. 129-146).

Naturally occurring marine prenyl benzoquinones and hydroquinones having a terpenoid portion ranging from one to nine isoprene units and differing structurally from the above-mentioned groups have been described from marine organisms, and especially from brown algae of the order Fucales, sponges and ascidians. Many algae contain tetraprenyl, triprenyl and diprenylquinones and dihydroquinones {see Ochi, M.; Kotsuki, H.; Inooue,M.; Taniguchi, M.; Tokoroyama, T. Chem. Lett., 1979, 831-832; and Capon, R.J.; Ghisalberti, EX.; Jeffereis, P.R. Photochemistry, 1981, 20, 2598-2600). Additionally, sponges contain linear unsubstituted polyprenylated hydroquinones and benzoquinones with longer side chains and moderate antimicrobial activity as well as ATPase inhibiting sulfated prenylhydroquinones {see Cimino, G.; De Stefano, S.; Minale, L. Tetrahedron, 1972, 28, 1315; Cimino, G.; De Stefano, S.; Minale, L. Experientia, 1972, 28, 1401; Pouches Y.F.; Verbist, J.F.; Biard, J.F.; Boukef, K. J. Nat. Prod, 1988, 51, 188; Lumsdon, D.; Capon, R. J.; Thomas, S.G.; Beveridge, A.A. Aust. J. Chem. 1992, ¥5,1321-1325; De Rosa, S.; De Giulio, Iodice, C. J. Nat. Prod. 1994, 57, 1711-1716; Fusetani, N.; Sugano, M.; Matsunaga, S.; Hashimoto, K.; Shikama, H.; Ohta, A., Nagano, H. Experientia, 1987, 43, 1233; Stonik, V.A.; Makarieva, T.N.; Dmitrenok, A.S. J. Nat. Prod., 1992, 55, 1256-1260; and Bifulco, G.; Bruno; L; Minale; L., Riccio, R.; Debitus, S.; Bourdy, G.; Vassa, A.; Lavayre, J. J. Nat. Prod. 1995, 58, 1444-1449). Ascidians of the genus Aplydium have previously yielded about a dozen prenylated quinones including the most simple of them, monoprenylbenzo- quinone {see Guella, G; Mancini, I; Pietra, F. HeIv. Chim. Acta. 1987, 70, 621- 626; Howard, B.M.; Clarkson, K.; Bernstein, R.L. Tetrahedron. Lett. 1979, 4449-4452; Targett, N.M.; Keeran, W.S. J. Nat. Prod., 1984, 47, 556-557; and Faulkner, DJ. Nat. Prod. Rep., 1993, 93, 1771-1791).

Cancer is one of the leading causes of death in the United States. However, there is still a need for compounds with anti-cancer properties. There is also a need for pharmacological tools for the further study of the physiological processes associated with cancer.

Summary of Certain Embodiments of the Invention

The present invention provides quinone and hydroquinone compounds that have anti-cancer activity. Accordingly, there is provided a compound of the invention which is a compound of formula I or II:

wherein: each OfR₁, R₂, and R₃ is independently hydrogen, (CrC₆)alkoxy, or a group of formula III ;

III each OfR₄ and R₅ is independently hydrogen or (Q-C^alkyl; n is 2 or 3; and each m is 1, 2, or 3; or a pharmaceutically acceptable salt thereof. The invention also provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier.

The invention also provides a composition (e.g. a cosmetic composition, a sunscreen, a dietary supplement, or a lotion) comprising a compound of the invention and an acceptable diluent or carrier.

The invention also provides a method for treating cancer comprising administering to a mammal in need of such treatment, an effective amount of a compound of the invention. In certain embodiments of the invention, the compound reduces tumor size in the mammal and/or inhibits tumor growth in the mammal.

The invention also provides a method for inducing AP-I dependent transcriptional activity in a cell comprising contacting the cell with an effective amount of a compound of the invention. The invention also provides a method for inducing NF-κB dependent transcriptional activity in a cell comprising contacting the cell with an effective amount of a compound of the invention.

The invention also provides a method for inducing AP-I and NF-κB dependent transcriptional activity in a cell comprising contacting the cell with an effective amount of a compound of the invention.

The invention also provides a method for decreasing the transformation of normal cells into tumor cells in a mammal in need of such treatment, comprising administering an effective amount of a compound of the invention.

The invention also provides a method for decreasing tumor cell proliferation in a mammal in need of such treatment, comprising administering an effective amount of a compound of the invention.

The invention also provides a method for inducing apoptosis or cell death in a cell comprising contacting the cell with an effective amount of a compound of the invention. The invention also provides a method for inducing apoptosis or cell death in a mammal in need of such treatment, comprising administering an effective amount of a compound of the invention. The invention also provides a method for isolating a compound of formula rV or XXXVI wherein R₁ and R₂ are each methoxy and R₃ is hydrogen, comprising extracting one or more marine invertebrate animals (e.g., ascidians) comprising the compound of formula IV or XXXVI with a suitable solvent (e.g. an alcohol such as ethanol) followed by purification with column chromotography to provide the compound of formula IV or XXXVI.

The invention provides a compound of the invention for use in medical therapy (e.g. for use in treating cancer).

The invention also provides the use of a compound of the invention to prepare a medicament useful for treating cancer in a mammal, e.g. , for reducing tumor size and/or inhibiting tumor growth in the mammal.

The invention also provides the use of a compound of the invention to prepare a medicament useful for inducing AP-I dependent transcriptional activity in a mammal. The invention also provides the use of a compound of the invention to prepare a medicament useful for inducing NF -KB dependent transcriptional activity in a mammal.

The invention also provides the use of a compound of the invention to prepare a medicament useful for inducing AP-I and NF-κB dependent transcriptional activity in a mammal.

The invention also provides the use of a compound of the invention to prepare a medicament useful for decreasing the transformation of normal cells into tumor cells in a mammal.

The invention also provides the use of a compound of the invention to prepare a medicament useful for decreasing tumor cell proliferation in a mammal.

The invention also provides the use of a compound of the invention to prepare a medicament useful for inducing apoptosis in a mammal.

The invention also provides processes and intermediated disclosed herein, e.g., that are useful for preparing compounds of formula (I) or formula (II), or salts thereof. Brief Description of the Figures

Fig. 1. The general formulae of compounds 1-33 that include benzoquinone compounds (shown) and the corresponding hydroquinone compounds (not shown, e.g., compounds of formula I or II having hydroxy- groups in positions 1 and 4, instead, of the oxo-groups shown). Fig. 2. The detailed formulae of the compounds 1-33. Fig. 3. The general scheme of synthesis of glabruquinone A (1) and its analogues.

Fig. 4. Syntheses of target qiαinones and hydroquinones from phenol 34. Fig. 5. Syntheses of target quinones and hydroquinones from phenol 35.

Fig. 6. Syntheses of target qαώiones and hydroquinones from phenol 36. Fig. 7. Structures of 1 and 2 and key HMBC and ¹H-¹H-COSY correlations.

Fig. 8 A. The effect of glabruquinone A (1) on MTS reduction in JB 6 P⁺ Cl 41 cells. The cells were cultured in 96-well plates. Then the media were changed to 0.1% FBS/MEM and the cells were treated with the indicated concentrations of glabruquinone A- (1). The cells were then incubated for 22 h and the MTS reagent (20 μl/well) Λvas then added and the cells were incubated for two more hours. Data represent the percentage of viable cells compared to percentage of untreated control cells. Each data point indicates the mean + SD of two independent experiments (*p < 0.05, Mann- Whitney U test).

Fig. 8B. Calculation of the IC₅₀ for glabruquinone A (1) in JB6 P+ Cl 41 cells by the method of linear regression.

Fig. 9 A. The effect of glabruquinone A (1) on MTS reduction in HT-460 cells. The cells were cultured and treated in 96-well plates as described for Fig. IA. Data represent the percentage of viable cells compared to percentage of untreated control cells. Each data point indicates the mean ± SD of two independent experiments (*p < 0.O5, Mann- Whitney U test).

Fig. 9B. Calculation of the IC₅₀ for glabruquinone A (1) in HT-460 cells by the method of linear regression. Fig. 1OA. HCT-116 cells were treated with the indicated concentrations of glabruquinone A (1) in soft agar. The cell colonies were scored and counted after 1 wk of incubation with the compound (1). Eacli bar indicates the mean + SD of two independent experiments (*p < 0.05, Mann- Whitney U test). Fig. 1OB. Calculation of INCC₅₀ of glabruquinone A (1) for HCT-116 cells by linear regression.

Fig. HA. JB6 P⁺ Cl 41 cells were treated with the indicated concentrations of glabruquinone A (1) with EGF, in soft agar. The cell colonies were scored and counted after 1 wk of incubation with the compound (1). Each bar indicates the mean ± SD of two independent experiments (*p < 0.05, Mann- Whitney U test).

Fig. HB. Calculation of INCC₅₀ of Glabruquinone A (1) for JB6 P⁺ Cl 41 cells transformed by EGF, using linear regression.

Fig. 12 A. JB 6 P⁺ Cl 41 cells were treated with the indicated concentrations of glabruquinone A (1) with TPA, in soft agar. The cell colonies were scored and counted after 2 wk of incubation with the compound (1). Each bar indicates the mean ± SD of two independent experiments (*p < 0.05, Mann- Whitney U test).

Fig. 12B. Calculation of INCC₅₀ of Glabruquinone A (1) for JB6 P⁺ Cl 41 cells transformed by TPA, using linear regression.

Fig. 13 A. JY cells were grown and treated with the indicated concentrations of glabruquinone A (1). The percentage of apoptosis was calculated after 3 h incubation with compound (1). Each bar indicates the mean ± SD of two independent experiments. (*p < 0.05 Mann — Whitney U test). Fig 13B. JB6 P⁺ Cl 41 cells were grown and treated with the indicated concentrations of glabruquinone A (1). The percentage of apoptosis was calculated after 3 h incubation with compound (1). Each bar indicates the mean + SD of two independent experiments. (*p < 0.05 Mann - Whitney U test).

Fig. 13C. Human skin melanoma SK-MEL-28 cells were grown and treated with the indicated concentrations of glabruquinone A (1). The percentage of apoptosis was calculated after 3 h incubation with, compound (1). Each bar indicates the mean ± SD of two independent experiments. (*p < 0.O5 Mann — Whitney U test).

Fig. 13D. Human lung tumor HT-460 cells were grown and treated with the indicated concentrations of glabruquinone A (1). The percentage of apoptosis was calculated after 3 h incubation with compound (1). Each bar indicates the mean ± SD of two independent experiments. (*p < 0.05 Mann — Whitney U test).

Fig. 13E. Human colon cancer HCT-116 cells were grown and treated with the indicated concentrations of glabruquinone A (1). The percentage of apoptosis was calculated after 20 h incubation with compound (1). Each bar indicates the mean ± SD of two independent experiments. (*p < 0.05 Mann — Whitney U test).

Fig. 14. COX2^+/+ or COX2^"A MEFs were treated with different concentrations of the glabruquinone A (1) for 24 h. The cells were harvested and a DNA fragmentation assay was performed.

Fig. 15A. Glabruquinone A (1) induces NF-κB dependent transcriptional activity in JB6 Cl 41 cells. JB6 Cl 41 cells stably expressing a luciferase reporter gene controlled by an NF-κB DNA binding sequence were treated with different concentrations of glabruquinone A as indicated for 24 h. The cells were extracted with lysis buffer and luciferase activity was assessed.

Fig. 15B. Glabruquinone A (1) induces AP-I dependent transcriptional activity in JB6 Cl 41 cells. JB6 Cl 41 cells stably expressing a luciferase reporter gene controlled by an AP-I DNA binding sequence were treated with different concentrations of glabruquinone A as indicated for 24 h. The cells were extracted with lysis buffer and luciferase activity was assessed.

Fig. 16. Inhibition of Ehrlich carcinoma growth by glabruquinone A (demethylubiquinone Q2, 1) in white nonlinear mice. The mice were treated once with glabruquinone A dissolved in 50% DMSO, one day before the inoculation of the tumor. The size of the tumor was measured on Day 6, 9, 12, 15, and 18. Data represent the percent inhibition of tumor growth in mice treated with glabruquinone A compared to tumor growth in untreated control mice. Each bar indicates the mean + S.D. from 8 mice in 2 independent experiments. Asterisk (*) indicates p < 0.05. Fig. 17. Inhibition of Ehrlich carcinoma growth by glabruquinone A (demethylubiquinone Q2, 1) in white nonlinear mice. The mice were treated once with glabruquinone A dissolved in 50% DMSO, one day before the inoculation of the tumor. The size of the tumor was measured on Day 6, 9, 12, 15, and 18. Data represent the percent inhibition of tumor growth in mice treated with glabruquinone A compared to tumor growth in untreated control mice. Each bar indicates the mean + S. D. from 8 mice in 2 independent experiments. Asterisk (*) indicates p < 0.05.

Fig. 18. Inhibition of Ehrlich carcinoma growth by glabruquinone A (demethylubiquinone Q2, 1) in white nonlinear mice. The mice were treated once with glabruquinone A dissolved in 50% DMSO, one day after the inoculation of the tumor. The size of the tumor was measured on Day 6, 9, 12, 15, and 18. Data represent the percent inhibition of tumor growth in mice treated with glabruquinone A compared to tumor growth in untreated control mice. Each bar indicates the mean + S. D. from 8 mice in 2 independent experiments. Asterisk (*) indicates p < 0.05.

Fig. 19. The inhibition of EGF-induced JB6 P⁺ C141 cell transformation by demethylubiquinone Q2 (1) in soft agar (anchorage-independent assay). JB6 P+ C141 cells (8χl0³/ml in 6-well plates), were activated with EGF (10 ng/ml), treated with the indicated concentrations of quinone (1), maintained for 1 week, and cell colonies were then scored. Data represent the percentage of EGF- activated, quinone (l)-treated cell colonies compared to percentage of EGF- activated, untreated cells. Each bar represents the mean ± SD from six samples of two independent experiments. *, indicates a significant inhibition by quinone (1) (p < 0.05) compared to EGF-activated untreated control.

Fig. 20. The effect of demethylubiquinone Q2 (1) on JB6 P⁺ C141 cell viability. The cells were cultured in 96-well plates. Then, the medium was replaced with 0.1% FBS-MEM containing the indicated concentrations of quinone (1). The cells were incubated with the quinone (1) for 22 h. The MTS reagent was then added and its reduction was measured spectrophotometrically 2 h later. Data represent the percentage of quinone (l)-treated viable cells compared to percentage of untreated control cells. Each data point represents the mean ± SD from ten samples of two independent experiments. *, indicates a significant decrease in viability induced by quinone (1) (p < 0.05) compared to untreated control cells. Fig. 21. The induction of apoptosis by demethylubiquinone Q2 (1) in JB6

P⁺ C141 cells measured by flow cytometry. The cells (3xlO⁵/dish) were grown in 6-cm dishes and treated with the indicated concentrations of quinone (1). Cells were harvested and processed for detection of apoptosis using Annexin V- FITC and propidium iodide staining according to the manufacturer's protocol. Each bar represents the mean ± SD from four samples of two independent experiments. *, indicates a significant increase in apoptosis by quinone (1) (p < 0.05) compared to untreated control cells.

Fig. 22. The induction of apoptosis by demethylubiquinone Q2 (1) in murine embryonic fibroblasts (MEFs) determined by the method of DNA- laddering. MEFs were grown in 10-cm dishes, treated with the indicated concentrations of quinone (1) for 24 h, and harvested. The isolated DNA fragments were separated by 1.8% agarose gel electrophoresis. DNA laddering in the gel was stained with ethidium bromide and photographed under UV light. A representative experiment is shown. Detailed Description

Compounds of the invention showed anticancer preventive and therapeutic activities as determined by the anchorage-independent neoplastic transformation assay, flow cytometery and DNA laddering assays for apoptosis, and MTS assay for determination of cell viability. Mouse epidermal JB6 C141 cells, COX2- deficient and wildtype mouse embryonic fibroblasts (MEFs), lymphocytes, and human lung cancer (HT-460), human colon cancer (HCT-116), and human skin melanoma (SK-MEL28) cell lines were used in these assays. Representative quinones induced activator protein-1 (AP-I) and/or nuclear factor-kappaB (NF- κB)-dependent transcriptional activity in JB6 Cl 41 cells. Several representative compounds of the invention demonstrated cancer preventive activity in the anchorage-independent transformation assay in doses significantly less than used in the cell viability assay, suggesting activity occurs through non-toxic mechanisms.

Structure-Activity Relationships for Representative Compounds

The structure-activity relationships (SARs) of representative compounds of the invention were studied using statistical analysis (Statistica 6.0). The quinone compounds were divided into three groups according to the number of isoprene units included in their terpenoid parts {see Table 2). Group 1 consisted of quinones which have two isoprene units (10 carbon atoms) in their side chains. Group 2 quinones contained three isoprene units (15 carbon atoms) in their terpenoid parts and group 3 quinones had four to six isoprene units (20 to 30 carbon atoms) in their side chains. Significant differences and correlations between the data regarding the biological activities obtained for different structural groups of the quinones were determined using the nonparametric Spearman correlation method and the Mann- Whitney U Test and the data in Table 2.

The results indicated that the biological activity of representative compounds of the invention depends on the length of the terpenoid side chains in the molecule. Statistical analysis of data from Table 2 indicates that quinones in group 1 averaged an IC₅₀ of 20.0 ± 15.2 μM for toxicity in JB6 C141 cells. Quinones in group 2 had an average IC₅₀ of 9.7 ± 9.0 μM. Finally, the quinones in group 3 showed an average IC_5O of 84.9 ± 63.8 μM. These results suggest that quinones with three isoprene units in their terpenoid portion are on the whole more toxic against JB6 cells than the quinones in group 1, which have only two isoprene units in their side chains. The opposite conclusion can be drawn when comparing the ICs₀ of quinones in groups 1 and 3 or for those in groups 2 and 3. The quinones of groups 1 or 2 are significantly more toxic for JB6 C141 cells than those of group 3. Therefore, as the length of the terpenoid portion increases as from group 1 to group 2 the toxicity of the quinones of this series also increases; but it decreases dramatically by further increasing of the length of the terpenoid portion as from group 2 to group 3.

In the statistical analysis of the effect of representative compounds of the invention on EGF-induced cell transformation in JB6 C141 cells, it was found that the quinones in group 1 displayed an average INCC₅O of 9.4 ± 5.3 μM and the quinones in group 2 averaged an INCCs₀ of 24.0 + 16.7 μM. Group 3 again possessed minimal activity among all three groups of compounds with an average INCC_5O of 59.7 ± 33.3 μM (Table T). Therefore, based on this analysis and the results of the Mann- Whitney U Test, group 1 quinones had the most potent effect on inhibition of cell transformation (p = 0.0283 vs. group 2; p = 0.0253 vs. group 3; p = 0.0084 vs. groups 2 and 3) and group 3 was the least effective. A significant correlation was observed between the length of the terpenoid portion and INCC₅₀ (p = 0.0002, R = 0.8556). These results indicate that when the length of the terpenoid portion increases, the INCC₅₀ values for cell transformation also increase.

The toxicity with cell transformation in JB6 P⁺ C141 cells was compared, and it was found that the quinones in group 1 displayed an average INCC₅₀ 1.4 to 4 times less than the IC₅₀ for the corresponding cells (Table T). On the other hand, the majority of quinones in group 2 showed an INCC₅₀ at doses 4 to 10 times higher than the IC₅₀ values (Table T). Based on these data, quinones of group 1 are distinctly more toxic to transformed JB 6 cells than to normal JB 6 cells. In contrast, the quinones in group 2 are more toxic to normal JB6 cells than to those that have been transformed. Therefore, the quinones in group 1 have more potential in respect to cancer preventive activity than the quinones from group 2.

The activity of representative compounds of the invention was shown to depend at least in part on the position of the methoxy group relative to the terpenoid part. Several pairs of structurally similar quinones were selected which have the methoxy groups in the same position. These pairs are as follows: 1) o/tø-analogues, quinones 5 and 9; T) /netα-analogues, quinones 6 and 4; and 3) jrarø-analogues, quinones 7 and 8. Based on the data from Tables 2 and 3, the cancer preventive activity and the effect of quinones on AP-I transcriptional activity, increased in the line of orto -» meta -> para. The INCC₅₀ had following values: for quinones 5, 9: 15.1 and 24.6 μM, respectively; quinones 6, 4: 6.6 and 16.7 μM, respectively; and quinones 7, 8: 3.1 and 7.4 μM, respectively.

Induction of AP-I transcriptional activity by the orto compounds 5, 9 averaged 133.8% of control and by the meta derivatives 6, 4, the average was 187.7% of control. The para derivatives 7 and 8 had the highest induction of AP- 1 activation at 486.9% of control. The ørto-disubstituted quinones 5 and 9 are the least active compounds not only in the induction of AP-I transcriptional activity, but also in the inhibition of cell transformation compared to the meta- anάpara- analogues. Among the /wra-disubstituted derivatives, quinone 7 having two isoprene units in the side chain showed better activities compared with quinone 8 having three isoprene units in the side chain. Indeed, the para- disubstituted quinones 7 and 8 showed an INCC₅₀ of 3.1 and 7.4 μM, respectively, against EGF -induced JB6 P⁺ C141 cell transformation. Quinone 5 also demonstrated a higher induction of AP-I transcriptional activity (721.7%) compared to quinone 9 (252.2%). The cancer preventive properties for representative compounds of the invention were studied using mouse epithelial JB6 P⁺ C141 cells and MEFs. The quinones having two isoprene units in the side chain showed specific effects against the malignantly transformed JB6 C141 cells compared to normal cells. The active doses differed up to 4-fold. SARs for representative compounds of the invention were studied in respect to cytotoxic or cancer preventive properties were examined. The present study indicated that cytoxicity of quinones increased with the number of carbon atoms from quinones having two prenyl units in their side chain to their analogues having three prenyl units and then decreased for compounds with 4 to 6 isoprene units.. Cancer preventive activity decreased when the polyprenyl side chain became longer. The most active cancer preventive polyprenylquinones, among those studied herein, have a side chain containing two isoprene units.

Using flow cytometry and the DNA laddering method, it was shown that representative compounds of the invention induced apoptosis in JB6 C141 cells and MEFs. The tumor suppressor protein, p53, which is a part of the cell's emergency team and functions to negatively regulate cell growth following DNA damage, is often involved in apoptosis induced by various stimuli including chemopreventive agents and drugs. However, representative compounds of the invention did not activate p53, but instead, most of the quinones studied demonstrated significant inhibition of p53 dependent transcriptional activity. In addition, these compounds induced a substantial activation of AP-I or NF-κB- dependent transcriptional activities. The AP-I transcription factor regulates a variety of cellular processes, including proliferation, differentiation, apoptosis and has been considered primarily to be an oncogene. Recently, some of the AP- 1 proteins, such as Jun-B and c-Fos, were shown to have tumor-suppressor activity both in vitro and in vivo. Activation of another AP-I protein, c-Jun, is required for induction of Fas L-mediated apoptosis in PC 12 and human leukemia HL-60 cells. Activation of both AP-I and NF-κB nuclear factors is important for DNA damaging agents and ceramide-induced apoptosis in T lymphocytes and Jurkat T cells. The balance between AP-I family members c-Jun and ATF-2 governs the choice between differentiation and apoptosis in PC 12 cells. Anticancer drugs, such as vinblastine, which inhibit microtubules, activate AP-I in human KB-3 carcinoma cells. This activation is required for efficient apoptosis induced by these drugs. NF-κB, a member of a family of highly regulated dimeric transcription factors, is involved in the activation of a large number of genes that respond to infections, inflammation, and other stressful situations. NF-κB is reported to be involved in both induction and inhibition of apoptosis. The results presented herein suggest that apoptosis induced by representative compounds of the invention occurs independently of p53 activation but instead may be related to the induction of AP-I and NF-κB transcriptional activity. Thus the results show that methoxylated polyprenylquinones and their synthetic analogues represent a new prospective group of marine secondary metabolites as anti-cancer compounds. Representative compounds of the invention show cytotoxic properties and induce apoptosis of JB6 P⁺ C141 cells and MEFs. The most active of these compounds are potent inducers of AP-I- and NF-κB -activation and, at the same time, inhibitors of p53 transcriptional activities. The cancer preventive effects of representative compounds may be explained by the induction of p53-independent apoptosis. It was also found that quinones having a side chain of 10-carbon atom length showed specificity in the inhibitory effect for transformed JB6 P+ C141 cells in contrast to quinones with 15 or 20 to 30 carbon atoms in the side chain. As representative compounds were active against transformation of the epithelial JB6 cells, representative compounds may be used as anti-skin cancer agents, e.g., as skin cancer preventive agents. Quinone 7, which has a diprenylated side chain in the para position relative to the methoxy-group, appears to be the most potent among the representative compounds of the invention studied with respect to a cancer preventive effect. The anticancer and therapeutic properties of the compounds are developed at doses that are relatively non-toxic. This was confirmed by using the MTS cell viability assay, the anchorage-independent transformation assay, and detection of apoptosis by flow cytometry and DNA laddering. These parameters were tested in mouse epidermal JB6 C141 cells, COX2 -deficient and normal mouse embryonic fibroblasts (MEFs), and several human tumor cell lines, including lung (HT-460), colon (HCT-116), and skin melanoma (SK-MEL-28). The tumor promoting effects of epidermal growth factor (EGF) or 12-O-tetradecanoyl- phorbol- 13 -acetate (TPA) were significantly diminished by the application of non-toxic doses of glabruquinone A or other compounds of the invention. The following definitions are used, unless otherwise described: Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to.

Generally, the term "isolated and purified" means that the compound is substantially free from biological materials {e.g. blood, tissue, cells, plant material, etc.). In one specific embodiment of the invention, the term means that the compound of the invention is at least about 75 wt.% free from biological materials; in another specific embodiment, the term means that the compound of the invention is at least about 90 wt.% free from biological materials; in another specific embodiment, the term means that the compound of the invention is at least about 98 wt.% free from biological materials; and in another embodiment, the term means that the compound of the invention is at least about 99 wt.% free from biological materials. In another specific embodiment, the invention provides a compound of the invention that has been synthetically prepared (e.g., ex vivo).

The terms "treat" or "treatment" as used herein refer to both therapeutic treatment and prophylactic or preventative treatment, wherein the goal is to prevent or decrease an undesired physiological change or disorder, such as the development or spread of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

The terms "cancer" and "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells. A list of cancers, such as skin cancer, is included in U.S. Pat No. 6,833,373. Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; and (CrC₆)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy.

Processes for preparing compounds of the invention are provided, as further embodiments of the invention and are illustrated by the procedures herein in which the meanings of the generic radicals are as given above unless otherwise qualified. hi cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The compounds of the irrvention can be formulated as pharmaceutical compositions and administered, to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, /. e. , orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes. Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet, e.g., as a dietary supplement. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wimtergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed- In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity caαi be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. For example, the compounds may be administered as a cosmetic, a sunscreen, and/or as a lotion.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the . affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the compounds of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.

4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g. _s into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

Thus, an objective of the present invention is to provide industrially relevant and effective use of mono- or dimethoxylated or nonmethoxylated di- or triprenylquinones (compounds 1-17) or corresponding hydroquinones

(compounds 18-33) as anticancer preventive and therapeutic components of medicinal or cosmetic preparations to treat any mammal including humans and as a scientific tool for study of AP-I and/or NF-κB proteins.

The compounds can be obtained through their isolation from natural sources (ascidians) and/or by chemical syntheses. Many of the compounds are unique because of the presence of methoxyl groups and shorter polyprenyl side chains when compared with ubiquinones having polyprenyl side chains. For example, glabruquinone A differs from ubiquinones because of the absence of a methyl group in the nucleus and the presence of shorter side chain.

The invention will now be illustrated by the following non-limiting Examples.

General Procedures

¹H and ¹³C NMR spectra were recorded on a Bruker WM-250 spectrometer at 250 and 62.9 MHz₅ respectively, Bruker DPX 300 at 300 and 75 MHz, respectively. HREIMS were obtained on an AMD-604S mass spectrometer. HPLC separations were conducted on a DuP ont 8800 chromatograph equipped with differential refractometer using an Ultrasphere Si column. The IR spectra were measured on a Bruker FT-IR "Vector 22" spectrophotometer. UV spectra were determined in CCl₄ on a Cecil CE 7200 spectrophotometer. The onset of apoptosis was analyzed by flow cytometry using the Becton Dickinson FACSCalibur (BD Biosciences, San Jose, CA). The MTS reduction assay to determine cell viability was measured using the Multiskan MS microplate reader (Labsystems, Finland). Cell colonies in the anchorage independent transformation assay were scored using the LEICA DM IRB inverted research microscope (Leica Mikroskopie und Systeme GmbH, Germany) and Image-Pro Plus software, version 3.0 for Windows (Media Cybernetics, Silver Spring, MD). The luminescence assay for p53, AP-I and NF-κB nuclear factor-dependent transcriptional activity was measured using the Luminoscan Ascent Type 392 microplate reader (Labsystems, Finland). Reagents Minimum essential medium (MEM) and DMEM were from Gibco

Invitrogen Corporation (Carlsbad, CA). Fetal bovine serum (FBS) was from Gemini Bio-Products (Calabasas, CA). Penicillin/streptomycin and gentamycin were from Bio-Whittaker (Walkersville, MD), L-glutamine was from Mediatech, Inc. (Herndon, Virginia). Epidermal growth factor (EGF) was from Collaborative Research (Bedford, MA). Luciferase assay substrate and Cell Titer 96 Aqueous One Solution Reagent (MTS) for the cell proliferation assay were from Promega (Madison, WI). The Annexin V-FITC Apoptosis Detection Kit was from Medical & Biological Laboratories (Watertown, MA). Silica gel L (40/100 μm) for low-pressure column liquid chromatography was from Chemapol (Praha, Czech Republic). Silica gel plates for thin-layer chromatography (4.5 x 6.0 cm, 5-17 μ,) were from Sorbfil (Russia). Cell Culture

The JB6 P⁺ C141 mouse epidermal cell line and its stable transfectants C141-NF-κB, C141-AP-1, C141-p53 (PG-13) were cultured in monolayers at 37°C and 5% CO₂ in MEM containing 5% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. The mouse embryonic fibroblasts (MEFs) were grown at 37°C and 5% CO₂ in DMEM containing 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin.

Example 1. Preparation and Isolation of Compounds of the Invention Isolation of glabruquinones A (1) and B (2) from the ascidian Λplidium glahrum.

Frozen ascidians were extracted with ethanol (1:1, w/w) and, after evaporation of the ethanol in vacuo, the aqueous residue was extracted with chloroform (1:1, v/v). The chloroform was eliminated in vacuo to obtain a brown oil, which was purified by chromatography on a silica gel column using a hexane-ethyl acetate (10:1) system (System A). In System A, the isolation of glabruquinones can be controlled by using TLC (thin layer chromatography). The R_f of the mixture of glabruquinones A (1) and B (2) under these conditions is equal to 0.35. After evaporation of the solvents from the corresponding fractions, a mixture of glabruquinones A (1) and B (2) was obtained and they were then separated by repeated HPLC on a silica gel Altex Ultrasphere Si column (4.6 mm x 25 cm) using a hexane-ethyl acetate (7:1) system (System B). Synthetic procedures

GENERAL DESCRIPTION. The syntheses ofpolyprenylquinones (1-17) and corresponding hydroquinones (18-33) were carried out in accordance with a general scheme as shown in Fig. 3. This involved the alkylation of corresponding available phenols by geraniol or farnesol under acid catalysis conditions, which was followed by oxidative demethylation of the obtained prenylated phenols and the reduction of target benzoquinones for 18-33. Glabruquinone A (1), B (2) and a series of analogues (3-33) were obtained. The analogues 3-33 differed from glabruquinones A (1) and B (2) in the number and/or position of methoxyls and/or in structures of polyprenyl side chains. SYNTHESES OF QUINONES 1-17.

The best results were obtained after the alkylation of phenols 34-36 by trøTM-geraniol (37) or trarø-farnesol (38) in the presence of boric trifluoride etherate as the acid catalyst (Figs. 4-6). Under these conditions, total yields of the alkylation products were about 60%. The oxidative demethylation of the fractions obtained with cerium-ammonium nitrate (CAN) gave mixtures of corresponding benzoquinones (1-9, 12-17), which were separated to obtain individual compounds. The synthesis of prenyl-benzoquinones 10, 11 has been carried out in nonsubstituted monomethyl ether of hydroquinone (59) through intermediate 2-geranyl- or 2-pharnesyl-4-methoxyphenols (60, 61; scheme not shown).

STAGE 1. Boric trifluoride etherate (0.5 ml) was added with agitation to a mixture of 1 mmol of the corresponding phenol (34, 35 or 36) and 4 mmol of geraniol (37) or farnesόl (38) in 10 ml of absolute ether. The mixture was kept for 12 h at room temperature, after which time, 30 ml of water was added to the mixture and products were extracted with ether (3x15 ml). The extract was washed out with 10% NaCl and dried under Na₂SO₄. The solvent was removed and the residue was separated using a column of silica gel. The mixture of the prenylated phenols 39, 41, 43 or 40, 42, 44 (Fig. 4), 45, 47, 49 or 46, 48, 50 (Fig. 5), or 51, 53, 55, 57 or 52, 54, 56, 58 (Fig. 6), 60 or 61 (scheme not shown) were eluted under gradient conditions with the solvent system hexane: acetone, 50:1 — » 20:1. These purified mixtures were used for the Stage 2 reaction. The mean yield was about 60%. In some cases, individual prenylated phenols were isolated by HPLC on an Altex Ultrasphere Si column (4.6 mm x 25 cm) using solvent system B. Thus, purified samples of the individual prenylated phenols 39, 40, 45, 51 and 60 were obtained for ¹H NMR analysis. STAGE 2. A solution of CAN (0.9 mmol) in 3 ml of the mixture CH₃CN:H₂O (1 :2) was added to the cooled (0 degrees centigrade) and agitating solution of phenolic fractions 39, 41, 43 or 40, 42, 44 (Fig.4), 45, 47, 49 or 46, 48, 50, (Fig. 5), 51, 53, 55, 57 or 52, 54, 56, 58, (Fig. 6), 60 or 61 (0.3 mmol) in 7 ml Of CH₃CN. After agitation at 0 degrees centigrade for 1-2 h, the mixture was poured into 25 ml of 10% NaCl and extracted with ether (3x15 ml). The extract was dried by Na₂SO₄ and evaporated. The corresponding benzoquinones 1, 14 and 3, 15, (Fig. 4), 7, 5, 16 and 8, 9, 17, (Fig. 5), 6, 5, 7, 13 and 2, 8, 9, 12 (Fig. 6), 10 and 11 (scheme not shown) were obtained by preparative thin-layer chromatography on silica gel in a hexane: acetone (8:1) system. Every benzoquinone [(1), (3-17)] contained less than 6% corresponding 2'-3'-C/,?- or 2 "-3" czs-isomer as an impurity. The formation of these isomers may be explained by the isomerization of geraniol and/or farnesol at the action of boric trifmoride etherate at the stage of prenylation of phenols. As a rule, impurities of cw-isomers were not separated from major products (trans-isomtrs) except for the mixture of 1 and 2, which was separated by HPLC. A comparison of the effects of 1 and 2 showed that the major products and impurities possess the same level of activity. This means that for practical application, separating the major products from the czs-isomers is not needed. Yields of target products were about 70% at this stage. Mean total yield calculated for the two stages was about 45%.

SYNTHESIS OF HYDROQUINONES 18-33 (GENERAL METHOD). A solution of Na₂S₂O₄ (3 mmol) in 3 ml of water was added to 1 mmol of a corresponding benzoquinone (1, 3-17) in 7 ml of acetone. The mixture was agitated for 1 h, diluted with water and extracted with ether (3x15 ml). The extract was dried by Na₂SO₄ and evaporated. As a result, hydroquinones (18-33) were obtained. They contained small impurities (less than 6 %) of I'-T-cis- or 2"-3"-cκ'-isomers. Structures of the glabruquinones A (1) and B (2). Besides molecular ion peaks at m/z 304, in their EIMS, both glabruquinones A (1) and B (2) had ion peaks characteristic of benzoquinones (M⁺+ 2) at m/z 306. The UV and IR spectra of 1 and 2 showed an absorption maxima at 264 nm (ε=15000) and at 1675, 1657, 1603 cm^'1 , confirming the presence of a p-benzoquinone moiety. The ¹H NMR spectrum of 1 was similar to that of verapliquinone A from the Aplidium sp. but differed in an additional singlet signal at 4.02 ppm typical for MeO-group. Quartet signals at 61.2 and 61.3 ppm and singlets at 145.2 and 144.9 ppm in ¹³C NMR spectrum indicated the presence of two methoxyls in 1. This suggested a 5,6- or 3,5-dimethoxy-substitution in 1,4-benzoquinone moiety. The attachment of a terpenoid fragment to C-2 of the benzoquinone moiety and 5,6-positions of methoxyls were established by a detailed inspection of the NMR spectra, including ¹H-¹H-COSY, NOESY and HMBC (Fig. 7). The multiplicity of H- 3 (the narrow triplet at 6.34 ppm) and cross peaks corresponding to H-3/1'- H allylic coupling in ¹H-¹H-COSY and H-3/1 '-H interaction in NOESY were particularly important. The signals of two tri-substituted double bonds and three methyl groups attached to these bonds in the ¹³C NMR spectrum clearly showed the presence of a diprenyl side chain.

The comparison of NMR spectra of 1 and 2 showed that glabruquinones A and B contain geranyl and neryl types of side chains, respectively (Table 1; also, see Shubina et al, Tetrahedron Letters, 46, 559-562 (2005)). In fact, these compounds differ from each other by configurations of the C2', C3'-double bond as was established on the basis of chemical shifts of ClO¹. hi the sterically more congested E-isomers, this signal is observed in a higher field when compared with the Z-isomer. The C-IO¹ chemical shifts in the spectra of 1 and 2 differ from each other significantly, 16.2 and 22.8 ppm, respectively. The structures of glabruquinones A (1) and B (2) differ from those of the verapliquinone A and verapliquinone B by the presence of an additional methoxyl group in position 5. Structures 1 and 2 have been confirmed by synthesis as was described above. Trimethoxyphenol (34) was obtained from commercial 2,3,4— tri- methoxybenzaldehyde by a previously described method (Matsumoto, et al, 1984). The acid-catalyzed alkylation of phenol 34 with trαrø-geranyl bromide led to traws-geranyl-phenol (39) with an admixture of its c/s-isomer (a total yield of 18%, the ratio of trans :cis=95:5). Prenylated phenol 39 was purified by HPLC and its structure was established using ¹H NMR spectrum and by comparison with the ¹H NMR spectra of glabruquinones 1 and 2. The oxidative demethylation of the 39 resulted in the mixture of benzoquinones 1 and 2 (14%). The mixture of synthetic 1 and 2 was separated by HPLC and synthetic 1 and 2 were identified with glabruquinones A and B by comparison of their NMR spectra.

Table 1. ¹³C- and ¹H NMR data of 1 and 2.

*,** - values can be interchanged Structures of the synthetic polyprenylated compounds. The structures of the target prenyl-benzoquinones and -hydroquinones as well as intermediate prenylphenols were established using ¹H-NMR spectra in comparison with the corresponding data for the structures of the glabruquinones A (1) and B (2). The spectral characteristics of some synthetic analogues of glabruquinones 1 and 2 as well as some intermediate prenylphenols are provided below.

Example 2 3-demethyIut>iquinone Q2 or 2,3-dimethoxy-5-(3',7'- dimethyl- octa-2'(E),6'- dienyl)-[l,4]t>enzoquinone (1): yellow oil, HREIMS m/z 304.1655 [M]⁺, calcd for C₁₈H₂₄O₄ 304.1675, IR (CHCl₃): 1675, 1657, 1603. ¹H NMR (CDCl₃, 250MHz) δ: 6.34 (t, J=I.7, IH, H-6); 5.13 (m, IH, H-2'); 5.08 (m, IH, H-6¹); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.10 (dd, J=7.3, 1.7, 2H₅ H-I¹); 2.09 (m, 2H, H-5¹); 2.O8 (m, 2H, H-4'); 1.70 (d, J=I.2, 3H, H-8¹); 1.62 (d, J=I.2, 3H, H- 10'); 1.60 (br.s, 3H, H-9¹). ¹³C NMR (CDCl₃, 62.9 MHz) δ: 16.20 (q, C-IO¹), 17.79 (q, C-9'), 25.77 (q, C-8¹), 26.52 (t, C-5¹), 27.17 (t, C-I'), 39.72 (t, C-4¹), 61.20 (q, OMe)₅ 61.30 (q, OMe), 117.78 (d, C-2'), 123.98 (d, C-6'), 130.45 (d, C- 6), 131.95 (s, C-7')₅ 140.17 (s, C-3'), 144.91 (s, C-2 or C-3), 145.16 (s, C-3 or C- 2), 146.92 (S₃, C-5), 184.38 (s, C-4 or C-I), 184.54 (s, C-I or C-4).

Example 3

2,3-Dimethoxy-5-(3',7'-dimethyl-octa-2'(Z),6'-dienyl)-[l,4]benzoquinone (2): yellow oil, HREIMS m/z 304.1662 [M]⁺, calcd for C₁₈H₂₄O₄ 304.1675, ¹H NMR (CDCl₃, 250MHz) δ: 6.37 (t, J=I.7, IH, H-6); 5.13 (m, IH, H-2¹); 5.07 (m, IH, H-6¹); 4.02 (s, 3H₅ OMe); 4.00 (s, 3H₅ OMe); 3.11 (br. d, J=7.1, 1.2, 2H₅ H-I¹); 2.04 (m, 2H, H-5¹); 2.04 (m, 2H, H-4¹); 1.75 (q, J=I.2, 3H, H-10¹); 1.66 (br. d, J=I.2, 3H₅ H-8¹); 1.59 (d, J=I .2, 3H, H-9¹). ¹³C NMR (CDCl₃, 62.9 MHz) δ: 17.79 (q, C-9¹), 22.76 (q, C-IO¹), 25.77 (q, C-8¹), 26.52 (t₅ C-5¹), 27.27 (t, C-I¹), 32.01 (t, C-4'), 61.20 (q, OMe), 61.30 (q, OMe)₅ 117.78 (d, C-2¹), 123.98 (d, C- 6¹), 130.45 (d, C-6), 131.95 (s, C-7¹), 140.17 (s, C-3¹), 144.91 (s, C-2 or C-3), 145.16 (s, C-3 or C-2), 146.92 (s, C-5), 184.38 (s, C-4 or C-I)₅ 184.54 (s, C-I or C-4). Example 4

2-Methoxy-3-(3',7'-dimethyl-octa-2',6'-dienyl)-[l,4]benzoquinone (5): yellow oil,HREIMS m/z 274.1558 [M]⁺, calcd for C₁₇H₂₂O₃ 274.1569, ¹H-NMR (250 MHz, CDCl₃) δ 6.68 (d, J=I O.O, IH, H-5); 6.59 (d, J=I 0.0, IH, H-6); 5.05 (m, 2H, H-2', H-6'); 4.02 (s, 3H, OMe); 3.15 (br. d, J=7.3, 2H, H-I'); 2.01 (m, 4H, H- 4', H-5¹); 1.73 (br. s, 3H, Me); 1.65 (br. s, 3H, Me); 1.58 (br. s, 3H, Me).

Example 5

2-Methoxy~6-(3 ' ,7' -Dimethyl-octa-2 ' ,6 ' -dienyl)- [1 ,4] benzoquinone (6) : yellow oil, HREIMS m/z 274.1576 [M]⁺, calcd for C₁₇H₂₂O₃ 274.1569, ¹H-NMR (250 MHz, CDCl₃) δ 6.45 (q, J=2.1, IH, H-5); 5.87 (d, J=2.4, IH, H-6); 5.15 (m, IH, H-2'); 5.08 (m, IH, H-6¹); 3.82 (s, 3H, OMe); 3.14 (br. d, J=7.3, 2H, H-I'); 2.07 (m, 4H, H-4', H-5'); 1.70 (br. s, 3H, Me); 1.63 (br. s, 3H, Me); 1.60 (br. s, 3H, Me).

Example 6

2-Methosy-5-(3',7^!-dimethyI-octa-2',6'-dienyl)-[l,4]benzoquinone (7): yellow crystals, HREIMS m/z 274.1582 [M]⁺, calcd for C₁₇H₂₂O₃ 274.1569, ¹H-NMR (250 MHz, CDCl₃) δ 6.46 (t, J=1.7, IH, H-6), 5.92 (s, IH, H-3), 5.15 (m, IH, H- T), 5.08 (m, IH, H-6¹), 3.82 (s, 3H, OMe), 3.14 (br. d, J=7.3, 2H₅ H-I¹), 2.08 (m, 4H, H-4', H-5'), 1.70 (br. s, 3H, Me), 1.62 (br. s, 3H, Me), 1.60 (br. s, 3H, Me).

Example 7

2-(3',7'-Dimethyl-octa-2',6'-dienyl)-[l,4]beuzoquinone (11): yellow oil, HREIMS m/z 244.1454 [M]⁺, calcd for C₁₆H₂₀O₂ 244.1463, ¹H-NMR (250 MHz, CDCl₃) δ 6.77 (d, J=I 0.2, IH, H-6); 6.69 (dd, J=I 0.2, 2.3, IH, H-5); 6.53 (q, J=I .9, IH, H-3); 5.15 (m, IH, H-2¹); 5.07 (m, IH₅ H-6'); 3.13 (br. d, J=7.5, 2H, H-I'); 2.08 (m, 4H, H-4', H-5'); 1.69 (br. s, 3H, Me); 1.62 (br. s, 3H, Me); 1.60 (br. s, 3H₅ Me).

Example 8

2,3-Dimethoxy-5-(3',7',ll'-trimethyl-dodeca-2',6,'10'-trienyl)- [l,4]benzoquinone (3): yellow oil, HREIMS m/z 372.2316 [M]⁺, calcd for C₂₃H₃₂O₄ 372.2300, ¹H-NMR (250 MHz, CDCl₃) δ 6.34 (t, J=I.8, IH, H-6); 5.10 (m, 3H, H-2', H-6¹, H-IO'); 4.02 (s, 3H, OMe); 4.00 (s, 3H₅ OMe); 3.11 (br. d, J=7.3, 2H₅ H-I¹); 2.05 (m, 8H₅ H-4¹, H-5', H-8¹, H-9¹); 1.68 (br. s, 3H₅ Me); 1.62 (br. S₅ 3H₅ Me); 1.60 (br. s, 6H₅ 2Me). Example 9

2-Methoxy-6-(3',7',ll'-trimethyl-dodeca-2',6',10'-trienyI)-[l,4]benzoquinone

(4): yellow oil, HREIMS m/z 342.2182 [M]⁺, calcd for C₂₂H₃₀O₃ 342.2195, ¹H- NMR (250 MHz, CDCl₃) δ 6.45 (q, J=2.0, IH, H-5); 5.87 (d, J=2.4, IH, H-3); 5.15 (m, IH, H-2¹); 5.10 (m, 2H, H-6¹, H-IO¹); 3.81 (s, 3H₅ OMe); 3.14(br. d, J=7.3, 2H, H-I¹); 2.06 (m, 8H, H-4', H-5', H-8', H-9'); 1.67 (br. s, 3H, Me); 1.63 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me).

Example 10

2-Methoxy-5-(3',7',ll'-trimethyl-dodeca-2',6',10'-trienyl)-[l,4]benzoquinone

(8): yellow crystals, HREMS m/z 342.2172 [M]⁺, calcd for C₂₂H₃₀O₃ 342.2195, ¹H-NMR (250 MHz, CDCl₃) δ 6.47 (t, J=I.7, IH, H-6); 5.93 (s₉ IH, H-3); 5.16 (m, IH, H-2¹); 5.10 (m, 2H, H-6', H-IO'); 3.82 (s, 3H, OMe); 3.14(br. d, J=7.3, 2H, H-I'); 2.05 (m, 8H, H-4', H-5', H-8', H-9'); 1.68 (br. s, 3H, Me); 1.62 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me).

Example 11

2-Methoxy-3-(3',7',ll'-trimethyl-dodeca-2',6^l,10'-trienyl)-[L,4]benzoquinone (9): yellow oil, HREMS m/z 342.2212 [M]⁺, calcd for C₂₂H₃₀O₃ 342.2195, ¹H- NMR (250 MHz, CDCl₃) δ 6.68 (d, J=I 0.0, IH, H-6); 6.57 (d, J=I 0.0, IH, H-5); 5.07 (m, 3H, H-2', H-6¹, H-IO'); 4.02 (s, 3H, OMe); 3.16(br. d, J=7.3, 2H, H-I¹); 2.01 (m, 8H, H-4¹, H-5¹, H-8¹, H-9¹); 1.73 (br. s, 3H, Me); 1.67 (br. s, 3H, Me); 1.60 (br. s, 3H, Me); 1.57 (br. s, 3H, Me).

Example 12

l^-Dimethoxy-S-CS'JSll'-trimethyl-dodeca-lSόSlO'-trienyO-benzene-l^- diol (19): yellow oil, HREIMS m/z 31A2A12 [M]⁺, calcd for C₂₃H₃₄O₄ 374.2457, ¹H-NMR (250 MHz, CDCl₃) δ 6.49 (s, IH, H-6); 5.31 (s, IH, OH); 5.30 (m, IH, H-2¹); 5.17 (s, IH, OH); 5.12 (m, 2H, H-6¹, H-IO¹); 3.91 (s, 3H, OMe); 3.88 (s, 3H, OMe); 3.28(br. d, J=7.6, 2H, H-I'); 2.05 (m, 8H, H-4', H-5¹, H-8¹, H-9¹); 1.70 (br. s, 3H, Me); 1.68 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me).

Example 13

2,3-Dimethoxy-5,6-bis-(3',7'-dimethyl-octa-2',6'-dienyI)-[l,4]benzoquin(>iie (14): yellow oil, HREMS m/z 440.2944 [M]⁺, calcd for C₂₈H₄₀O₄, 440.2927, IH-NMR (250 MHz, CDCl₃) δ 5.04 (m, 2H, H-2¹, H-2¹¹); 4.94 (m, 2H, H-6', H- 6"); 3.99 (s, 6H, 2OMe); 3.19(br. d, J=6.8, 2H, H-I', H-I"); 2.00 (m, 8H, H-4-', H- 5', H-4", H-5"); 1.73 (br. s, 6H, 2Me); 1.66 (br. s, 6H, 2Me); 1.58 (br. s, 6H, 2Me). Example 14

12

2-Methoxy-5,6-bis-(3',7',ll'-trimethyl-dodeca-2',6',10'-trienyl)- [l,4]benzoquinone (12): yellow oil, HREIMS m/z 546.4048 [M]⁺, calcd for C₃₇H₅₄O₃, 546.4073, ¹H-NMR (250 MHz, CDCl₃) δ 5.87 (s, IH, H-3); 5.00 (m, 6H, H-2', H-6', H-IO', H-2", H-6", H-IO"); 3.79 (s, 3H, OMe); 3.22(br. d, J=6.8, 4H, H-r, H-I"); 2.01 (m, 16H, H-4', H-5¹, H-8', H-9¹, H-4", H-5", H-8", H-9"); 1.73 (m, 3H, Me); 1.67 (m, 9H, 3Me); 1.60 (m, 12H, 4Me).

Example 15

2-Methoxy-3,5-bis-(3',7^t,ll'-trimethyl-dodeca-2^t,6',10'-trienyl)- [l,4]benzoquinone (17): yellow oil, HREIMS m/z 546.4052 [M]⁺, calcd for C₃₇H₅₄O₃, 546.4073, ¹H-NMR (250 MHz, CDCl₃) δ 6.33 (t, J=I .2, IH, H-6); 5.09 (m, 6H, H-2¹, H-6¹, H-IO¹, H-2", H-6", H-IO"); 4.00 (s, 3H, OMe); 3.14(m, 4H, H-r, H-I"); 2.04 (m, 16H, H-4¹, H-5¹, H-8¹, H-9¹, H-4", H-5", H-8", H-9"); 1.74 (m, 3H, Me); 1.68 (m, 9H, 3Me); 1.60 (m, 12H, 4Me).

Example 16

2,3,4-Trimethoxy-6-(3',7'-dimethyl-octa-2',6^f-dienyl)-phenol (39): pale yellow oil, HREMS m/z 320.1974 [M]⁺, calcd for C₁₉H₂₈O₄ 320.1987, IR (CCl₄): 3541, 2935, 1498, 1464 cm-¹. ¹H NMR (CDCl₃, 250MHz) δ: 6.44 (s, IH, H-5), 5.45 (s, IH, OH), 5.31 (m, IH, H-2^!), 5.11 (m, IH, H-6') 3.95 (s, 3H, OMe), 3.86 (s, 6H, OMe), 3.79 (s, 3H, OMe), 3.31 (br.d, J=7.1, 2H, H-I'), 2.07 (m, 4H, H-4', H-5¹), 1.72 (d, J=I.2, 3H, Me), 1.67 (d, J=I.2, 3H, Me), 1.60 (d, J=0.7, 3H, Me). ¹³C NMR (CDCl₃, 62.9 MHz) δ: 16.12 (q, C-IO'), 17.66 (q, C- 9'), 25.66 (q, C-8% 26.73 (t, C-5'), 27.90 (t, C-I¹), 39.75 (t, C-4'), 56.62 (q, OMe), 60.89 (q, OMe), 61.16 (q, OMe), 108.30 (d, C-5), 121.61 (s, C-6), 121.98 (d, C-2' or C-6'), 124.20 (d, C-6' or C-2'), 128.89 (s, C-I), 131.41 (s, C-7'),

136.59 (s, C-4), 140.04 (s, C-3'), 140.81 (s, C-3 or C-2), 146.14 (s, C-2 or C-3).

Example 17

2,3,4-Trimethoxy-6-(3',7',ll'-trimethyl-dodeca-2',6',10'-trienyl)-phenol (40): pale yellow oil, HREIMS m/z 388.2648 [M]⁺, calcd for C₂₄H₃₆O₄ 388.2635, ¹H- NMR (250 MHz, CDCl₃) δ 6.44 (s, IH, H-5); 5.45 (s, IH, OH); 5.32 (br. t, J=7.3, IH, H-2'); 5.12 (m, 2H, H-6', H-IO'); 3.95 (s, 3H, OMe); 3.87 (s, 3H, OMe); 3.79 (s, 3H, OMe); 3.31(br. d, J=7.3, 2H, H-I'); 2.06 (m, 8H, H-4', H-5', H-8', H-9'); 1.72 (br. s, 3H, Me); 1.67 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me).

Example 18

3,4-Dimethoxy-6-(3',7'-dimethyl-octa-2',6'-dienyl)-phenol (45): pale yellow oil, HREMS m/z 290.1876 [M]⁺, calcd for C₁₈H₂₆O₃ 290.1882, ¹H-NMR (250 MHz, CDCl₃) δ 6.73 (s, IH, H-2); 6.49 (s, IH, H-5); 5.27 (br t, J=7.3, IH, H-2'); 5.15 (s, IH, OH); 5.10 (m, IH, H-6'); 3.87 (s, 3H, OMe); 3.79 (s, 3H, OMe); 3.24(br. d, J=7.3, 2H, H-I'); 2.05 (m, 4H, H-4', H-5¹); 1.68 (br. s, 6H, 2Me); 1.60 (br. s, 3H, Me).

Example 19

2,4 -Dimethoxy-6-(3',7'-dimethyI-octa-2',6'-dienyl)-phenol (51): pale yellow oil, HREIMS m/∑ 290.1898 [M]⁺, calcd for Ci₈H₂₆O₃ 290.1882, ¹H-NMR (250 MHz, CDCl₃) δ 6.36 (d, J=2.7, IH, H-3 or H-5); 6.31 (d, J=2.7, IH, H-3 or H-5); 5.33 (br. t, J=7.1, IH, H-2¹); 5.26 (s, IH, OH); 5.11 (br. t, J= 6.6, IH, H-6¹); 3.86 (s, 3H, OMe); 3.75 (s, 3H, OMe); 3.35 (br. d, J=7.3, 2H₅ H-I'); 2.08 (m, 4H, H- 4', H-5'); 1.72 (br. s, 3H, Me); 1.67 (br. s, 3H₅ Me); 1.60 (br. s, 3H, Me).

Example 20

¹H-NMR (250 MHz, CDCl₃): 6.69 (m, 3H, H-3, H-5, H-6); 5.31 (br. t, J=7.32, IH, H-2'); 5.07 (m, IH, H-6'); 3.75 (s, 3H, OMe); 3.33(br. d, J=7.08₅ 2H, H-I'); 2.09 (m, 4H, H-4', H-5'); 1.76 (br. s, 3H₅ Me); 1.68 (br. s, 3H₅ Me); 1.60 (br. s₅ 3H₅ Me)

Example 21. Cytotoxicity

The MTS-method was used to evaluate the cytotoxic effect of the representative compounds of the invention by assessing cell viability. The effect of representative compounds of the invention on the viability of JB 6 P+ C141 cells, JB6 cells transfected with NF-kappaB luciferase promoter, JB6 cells transfected with AP-I luciferase promoter and human lung cancer HT-460 cells was determined. Quinones 1, 5-7, 10, which possess a terpenoid portion consisting of two isoprene units (or 10 carbon atoms in length), showed an IC₅₀ for JB6 Cl 41 cells in a range of concentrations from 8.3 to 45.1 μM and for HT- 460 cells from 25.8 to 81.8 μM. The quinones with the terpenoid portion consisting of a 15 carbon atom length (three isoprene units) showed an IC₅₀ for JB6 Cl 41 cells in a range of concentrations from 3.6 to 29.0 μM and for HT-460 cells from 12.1 to 72.0 μM. The average IC₅₀ of the quinones 1, 5-7, 10 for JB6 P⁺ Cl 41 cells was 18.7 μM and for HT-460 cells it was 44.2 μM. The average IC₅₀ of the quinones 3, 4, 8, 9, 19 for JB6 Cl 41 cells was 10.4 μM and for HT- 460 cells, 30.5 μM. Therefore quinones 3, 4, 8, 9, 19 characterized by a longer terpenoid part are almost twice as toxic for JB6 cells and 1.5 times more toxic for HT-460 cells than quinones 1, 5-7, 10, which possess a shorter terpenoid portion. Both groups of compounds are about two times more toxic for JB6 cells (15.9 μM average) than for HT-460 cells (37.8 μM average). The third group of the compounds, quinones 12, 14, 17, which possess two terpenoid side chains in the molecule, showed the least cytotoxicity for both JB6 C141 and HT-460 cells among the three groups of quinones. The cytotoxicity effects of quinones 1-33 against human lung tumor (HT-460) cells and mouse JB6 Cl 41 cells has never before been reported.

The effect of glabruquinone A (1) on cell viability was evaluated using MTS reduction into its formazan product (CellTiter 96^R AQ_ueOus One Solution Cell Proliferation Assay, Promega, Madison, WI). JB6 P⁺ Cl 41 or HT-460 cells were cultured for 12 h in 96-well plates (4,000 cells per well) using 5% FBS/MEM (for JB6 P⁺ Cl 41 cells) or 10% FBS/RPMI (for HT-460 cells). Then the media were replaced with 0.1% FBS/MEM (for JB6 P⁺ Cl 41 cells) or 0.1% FBS/RPMI (for HT-460 cells) containing glabruquinone A (1) at various concentrations in a total volume of 0.1 ml and the cells were incubated with this quinone for 22 h. Then 20 μl of the MTS reagent was added into each well and MTS reduction was measured 2 h later spectrophotometrically at 492 nm and 690 nm as background using the Multiskan MS microplate reader (Labsystems, Finland). Data shown in Fig. 8A and 8B represent the percentage of viable JBb P+ Cl 41 and HT-460 cells, respectively, compared to untreated control cells. Fig. 9 A and Fig. 9B shows the IC₅₀ calculation for JB6 P+ Cl 41 and HT-460 cells, respectively, using linear regression built from data in Fig. 8A and 8B respectively.

Example 22. Anchorage-Independent Transformation Assay Representative compounds of the invention inhibit phenotype expression (colony formation) of human^" lung (HT-460) cancer, human colon (HCT-116) cancer, and human melanoma (SK-MEL-28) cancer cell lines. They also inhibit EGF- or TPA-induced JB6 Cl 41 malignant cell transformation in soft agar or anchorage-independent transformation.

The effect of quinones 1, 3-10, 12, 14, 17, 19 on colony formation in human tumor cells HT-460, HCT-116, MEL-28, and mouse JB6 P+ Cl 41 cells was evaluated by the method of anchorage-independent transformation assay. The results confirm that the representative compounds of the invention possess anticancer preventive and therapeutic properties. In the anchorage-independent transformation assay, the quinones with side chains of at least ten carbon atoms in length demonstrated anticancer activities on EGF-induced transformation of JB6 Cl 41 cells, at an INCC₅₀ (inhibition of number of the colonies) that was 1.4- 4 times less than IC_5O observed in the cell viability assay. On the other hand, quinones with a longer side chain of 15 -carbon atom length displayed anticancer activity mostly at doses equal to or 4 to 10 times higher than IC₅₀ obtained in the cell viability assay. Therefore, the quinones having a side chain of 10-carbon atom length appear to be more potent anticancer agents compared to quinones with a 15 -carbon atom length side chain. The same conclusion is true for anticancer activity of the quinones investigated for their effects against the HT- 460 cell line. Data for IC₅₀ and INCC₅₀ for quinones 1, 3-10, 12, 14, 17, 19 were obtained from cell viability and anchorage -independent transformation assays using the regressions, which were built using the computer program

STATISTICA 6.0 (StatSoft, Inc., USA). The IC₅₀ refers to the concentration of quinone at which 50% of cells are still viable compared to untreated control. The INCC₅O refers to the concentration of quinone at which colony formation is inhibited by 50% compared to EGF-stimulated control. The anticancer properties of representative compounds of the invention against expression of phenotype of human lung cancer (HT-460), human colon cancer (HCT-116) and human skin cancer (SK-MEL-28) cell lines as well as EGF- and TPA- induced JB6 Cl 41 cell transformation have not been reported.

The anticancer preventive and/or therapeutic effects of glabruquinone A (1) were evaluated using an anchorage-independent neoplastic transformation assay. The assay was carried out in 6-well tissue culture plates. For evaluation of the anticancer effects of glabruquinone A (1) we used the human colon cancer cell line, HCT-116, to assess whether this quinone could prevent phenotype expression (colony formation) in soft agar — no additional stimulus is required. JB6 P⁺ Cl 41 cells, activated EGF (10 ng/ml) or TPA (20 ng/ml) were also used, to assess whether this glabruquinone A (1) can prevent tumor promoter-induced neoplastic transformation as indicated by colony formation. This assay is a well- accepted tool to determine whether a compound can be a potentially effective anticancer agent in either humans or animals. HCT-116 cells or JB6 P⁺ Cl 41 cells (8 x 10³ per ml) were treated with the indicated concentrations of glabruquinone A (1) in 1 ml of 0.33% BME (basal medium Eagle) agar containing 10% FBS over 3.5 ml of 0.5% BME agar containing 10% FBS and the indicated concentrations of glabruquinone A (1). The cultures were maintained in a 37°C, 5% CO₂ incubator for 1 wk (JB6 cells, activated with EGF or HCT-116 cells) or 2 wk (JB6 cells, activated with TPA). Cell colonies were then scored using an LEICA DM IRB inverted research microscope (Leica Mikroskopie und Systeme GmbH, Germany) and Image-Pro Plus software, version 3.0 for Windows (Media Cybernetics, Silver Spring, MD). The effects of glabruquinone A (1) on HCT-116 cells colony growth (Fig. 10A), or EGF- induced transformation of JB6 P⁺ Cl 41 cells (Fig. HA), or TPA-induced transformation of JB6 P⁺ Cl 41 cells (Fig. 12A) are presented as a percentage of cell colony growth compared with untreated control cells. Fig. 1OB, HB₅ or 12B represent the calculation OfINCC₅₀ for HCT-116 cells. EGF- induced JB6 P+ Cl 41 cells transformation, or for TPA- induced JB6 P+ Cl 41 cells transformation, respectively using linear regression built from the data in Fig. 1OA, HA, 12A respectively.

Example 23. Apoptosis Assay Using Flow Cytometry and DNA Laddering Representative compounds of the invention induce apoptosis in the JB 6 P+

Cl 41 cells, human lymphoblast JY cells, COX2^"A and COX2^+/+ MEFs and human tumor melanoma (SK-MEL-28), lung (HT-460), and colon (HCT-116) cells. For example, the induction of apoptosis by quinones 3-10, 14, 17, 19 was evaluated by flow cytometry in mouse JB6 P+ Cl 41cells. The induction of apoptosis by glabruquinone A (1) was also evaluated by flow cytometry in the JB6 P+ Cl 41 cells, human lymphoblast JY cells, and human tumor melanoma (SK-MEL- 28), lung (HT-460), and colon (HCT-116) cells. Induction of apoptosis for glabruquinone A (1) was also evaluated by DNA laddering in COX2^"/" and COX2^+/"1" MEFs (Fig. 14). The ability of representative compounds of the invention to induce apoptosis in human tumor HT-460, HCT-116, and MEL-28 cells, human lymphoblast JY cells, mouse JB6 P+ Cl 41 cells, or in COX2^"A and COX2^+A+ MEFs has not been reported before.

The induction of early and late apoptosis by the glabruquinone A (1) was analyzed by flow cytometry using the Becton Dickinson FACs Calibur Flow Cytometer (BD Biosciences, San Jose, CA). JY, JB6 P⁺ Cl 41, SK-MEL-28, HT- 460, or HCT-116 cells (3x10⁵ cells per dish), were grown in 6 cm dishes for 24 h in 10% FBS/MEM for JY and SK-MEL-28 cells, 5% FBS/MEM for JB6 P⁺ Cl 41, 10%> FBS/RPMI for HT-460, or 10% FBS/McCoy's for HCT-116 cells. Cells were then treated with glabruquinone A (1) in 0.1% FBS/medium for three hours for JY, JB6 P⁺ Cl 41 , SK-MEL-28, and HT-460 cells or for 24 hours for HCT- 116 cells. After treatment with glabruquinone A-(I), the medium was collected and attached cells were harvested with 0.025% trypsin in 0.1% EDTA in PBS. Trypsinization was stopped by adding 2 ml of 5% FBS in PBS. For floating JY cells trypsinization was unnecessary. Cells were washed by centrifugation at 1 ,000 rpm (170 rcf) for 5 min and processed for detection of early and late apoptosis using Annexin V- FITC and propidium iodide staining according to the manufacturer's protocol, hi brief, 1-5x10⁵ cells were collected after centrifugation, and resuspended in 500 μl of Ix binding buffer (Annexin V-FITC Apoptosis Detection Kit, Medical & Biological Laboratories (Watertown, MA)). Then, 5 μl of Annexin V-FITC, and 5 μl of propidium iodide were added, and the cells were incubated at room temperature for 5 min in the dark and analyzed by flow cytometry. The Glabruquinone A (1) - induced apoptosis in the indicated above cells is shown in Figures 13A, B, C, D, E.

COX2 ^+/+ and ^"Λ MEFs were grown in 10-cm dishes and treated with glabruquinone A (1) when, cells were 80% confluent. Both detached and attached cells were harvested by scraping followed by centrifugation. Then the cells were disrupted with lysis buffer (5 mM Tris-HCl, pH 8.0, 20 mM EDTA, and 0.5% Triton X-100) and left on ice for 45 min. After centrifugation at 14,000 rpm (45 min, 4 C), the supernatant fraction containing fragmented DNA was extracted twice with phenol/chlorofbrm/isopropyl alcohol (25:24:1, v/v) and once with chloroform. Then the fragmented DNA was precipitated overnight at -20⁰C after addition of two volumes of 100% ethanol and 1/10 volume of 5 M NaCl. The DNA pellet was saved by centrifugation at 14,000 rpm for 45 min, washed once with 70% ethanol, dried, and resuspended in TE buffer (10 mM Tris-HCl, ImM EDTA, pH 8.0). After addition of 100 μg/ml RNAse A (Sigma), the mixture was incubated at 37°C for 2 h. The DNA fragments were separated by 1.8% agarose gel electrophoresis. DNA laddering in the gel was stained with ethidium bromide and photographed under UV light. The results are shown in Fig. 14.

Example 24. AP-I and NF-κB Nuclear Factors Dependent Transcriptional

Activity Representative compounds of the invention induce AP-I and/or NF-κB- dependent transcriptional activity in JB6 Cl 41 cells. For example, the induction of AP-I- or NF-κB-dependent transcriptional activity by the quinones 1, 3-10, 14, 17, 19 was assessed with a JB6 Cl 41 cell line stably expressing a luciferase reporter gene controlled by an AP-I or NF-κB DNA binding sequence. Viable JB6 Cl 41 AP-I or NF-κB cells (6xlO³) suspended in 100 μl 5% FBS/MEM were added into each well of a 96-well plate. Plates were incubated for 24 h and then were or were not treated with various concentrations of individual quinones in 100 μl of 0.1% FBS/MEM. After incubation with quinones for 24 h, the cells were extracted for 1 h at room temperature with 100 μl/well of lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA). Then 30 μl of lysate from each well were transferred into a plate for luminescent analysis and the luciferase activity was measured using 100 μl/well of the luciferase assay buffer (1 mM D-luciferase, pH = 6.1-6.5; 40 mM Tricin, 2.14 mM magnesium carbonate (MgCO₃)₄ x Mg(OH)₂ x 5H₂O, 5.34 mM MgSO₄ x 7H₂O, 66.6 mM DTT, 1.06 mM ATP, 0.54 mM COA, 0.2 mM EDTA₅ pH = 7.8) and the Luminoscan Ascent Type 392 microplate reader (Labsystems, Finland). The ability of representative compounds of the invention to induce AP- 1- or NF-κB-dependent transcriptional activity in mouse JB6 Cl 41 cell line has not been reported before.

The ability of glabruquinone A (1) to induce AP-I- or NF-κB-dependent transcriptional activity in mouse JB6 Cl 4- 1 cells was evaluated using the luciferase method. Viable JB6 Cl 41 AP- 1 or NF-κB cells (6x10³) suspended in 100 μl 5% FBS/MEM were added into each well of a 96-well plate. Plates were incubated for 24 h and then treated with various concentrations of (e.g. ,) glabruquinone A (1) in 100 μl of 0.1% FBS-MEM. After incubation with glabruquinone A (1) for 24 h, the cells were extracted for Ih at the room temperature with 100 μl/well of lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT₅ 2 mM EDTA). Then 30 μl of lysate from each well were transferred in the plate for luminescent analysis and the luciferase activity was measured using 1OO μl/well of the luciferase assay buffer (1 mM D-luciferase, pH = 6.1 - 6.5; 40 mM Tricin, 2.14 mM magnesium carbonate (MgCO₃)₄ x Mg(OH)₂ x 5H₂O, 5.34 mM MgSO₄ x 7H₂O, 66.6 mM DTT, 1.06 mM ATP, 0.54 mM COA, 0.2 mM EDTA, pH = 7.8) and the Luminoscan Ascent Type 392 microplate reader (Labsystems, Finland). Results for glabruquinone A (1) are shown in Fig. 15A, B and are expressed as percentage of NF-κB- or AP-I -dependent transcriptional activation relative to untreated control cells. Example 25. Anticancer Preventive and Therapeutic Activities The study of anti-neoplastic and cancer preventive activities of 3- demethylubiquinone Q₂ (1) has been carried out using modem non-invasive technical equipment, Magnetic Resonance Tomograph, and white non-linear mice inoculated with Ehrlich carcinoma. After only one therapeutic or preventive injection of 3-demethyl-ubiquinone Q₂, the size of solid Ehrlich carcinomas in the mice was significantly (25% -50%), diminished. The anticancer preventive and therapeutic effects of 3-demethylυbiquinone Q₂ against Ehrlich carcinoma were developed at doses that are relatively non-toxic. This finding was confirmed by the study of the toxicity of 3 -demethylubiquinone Q₂ for nonlinear mice as previously described (G. Karber, Arch. Exp. Pathol. Pharm.,1931, v. 162, p.48O). Biologic activities. The 3 -demethylubiquinone Q₂ showed anticancer preventive and therapeutic activities in relatively non-toxic doses as determined by the methods of Magnetic Resonance Tomography and a previously described method of determination of toxicity in mice.

Toxicity in mice. The toxicity of 3 -demethylubiquinone Q₂ was determined using Karber's method (G. Karber, Arch. Exp. Pathol. Pharm.,1931, v. 162, p.480). The LD₁₀₀ of 3 -demethylubiquinone Q₂ for nonlinear mice was 60 mg/kg and the LD₅₀ was 35 mg/kg. Medical doses were 30 mg/kg dissolved in 50% DMSO or 50% EtOH.

Magnetic Resonance Tomography CMRT). MRT is a powerful and versatile imaging method for animal studies. Monitoring of tumor growth or inhibition of the response to drug therapy in vivo is one the most useful applications of MRT. The advantages of the method include the possibility of obtaining a holistic image of a tumor in any projection on a. living model and in the absence of radiation. The inhibition of the solid Ehrlich carcinoma after treatment with 3- demethyl-ubiquinone Q₂ was studied in vivo using nonlinear mice. 3-demethyl- ubiquinone Q₂ was injected into the mouse as a solution in a mixture of DMSO and H₂O (1 :1) or EtOH and H₂O (1:1). After treatment with 3- demethylubiquinone Q₂ in DMSO or EtOH, the mice lost about 0.5-1% of ttαeir body weight. The size of the solid Ehrlich carcinoma in these mice was significantly (about 50%) diminished with injection of 3-demethylubiquinone Q₂ dissolved in 50% DMSO or 50% EtOH (Figs. 16 and 17). When 3- demethylubiquinone Q₂ was used as a therapeutic preparation and injected into mice one day after tumor inoculation, inhibition of tumor growth was about 25% (Fig. 18). Quinone 1 and other compounds of the invention can be used a cancer preventive agent and/or as a therapeutic agent for the treatment of established carcinomas.

Besides tumor measurements, the state of the liver, spleen or thymus over the lifetime of the mice was observed visually. No significant differences in the size, appearance or general condition of these immunocompetent organs in the 3- demethylubiquinone Q₂-treated mice were observed compared to untreated control mice. Similar results were obtained after removing and measuring these organs from the mice. Methods

PART 1. STUDY OF TOXICITY IN MICE. The toxicity of 3- demethylubiquinone Q₂ in mice was determined by Karber's method (G. Karber, Arch. Exp. Pathol. Pharm., 1931 , v. 162, p.480). hi brief, linear white mice weighing about 20 g each were used for the experiments. 3-Demethyl- ubiquinone Q₂ was dissolved in a mixture of DMSO and H₂O (1:1) or EtOH and H₂O (1 :1) and injected into each mouse intraperitoneally (i.p.)_> in a volume of 0.1 ml, at the indicated final concentrations. The toxicity was evaluated using Karber's formula: LD₅₀ = LD₁₀₀ - ∑(z x d)/m. LD₅₀ or LD₁₀₀ refers to 50% or 100% death of the mice, respectively; z indicates half of the number of anLmals that died from the two last adjacent doses; d indicates the interval between every two last adjacent doses; and m indicates the number of the animals used for the study of every dose of the compound. PART 2. MAGNETIC RESONANCE TOMOGRAPHY. The study of 3- demethylubiquinone Q₂ anti-neoplastic or cancer preventive activities was carried out using a modern non-invasive technical instrument, the Magnetic Resonance Tomograph "PharmaScan US 70/16" (Bruker, Germany), which has a superconductive magnet of 7 Tesla power and 300 MHz frequency. Nonlinear white mice weighing 20 g each were used for the experiments. Ascites tumors from Ehrlich carcinoma were inoculated into animals under the right shoulder- blade in a concentration of 5 mln/ml. Treatment was started one day before (for the study of the cancer preventive effect) or one day after the transplantation of the tumor. 3-Demethylubiquinone Q₂ was dissolved in the mixture of DMSO and H₂O (l:l-v/v) or in EtOH and H₂O (l :l-v/v) and injected into the mouse intraperitoneally (Lp.), once or twice, at the indicated time, in a volume of 0.1 ml, at a final 3 -demethylubiquinone Q₂ concentration of 30 mg/kg. Anti¬ neoplastic preventive and therapeutic effects of 3 -demethylubiquinone Q₂ were estimated by the measuring the size of the solid Ehrlich carcinoma on the 6^th, 9^th, 12^th, 15^th, and 18^th day after the tumor inoculation.

Mice were deeply anaesthetizised i.p. with a lmg/ml solution of xylazine (SPORA, Praha), 0.3 ml/kg final concentration, and placed supinely with the tumor located at the center of the coil surface. Radiofrequency was adapted for mouse experiments. A T2- weighted spin-echo sequence was used with the following parameters: repetition time/echo time, 2579.8/44.5 ms; FOV, 3.2x3.2 cm; acquisition time, 3.46 min; matrix size, 128x128; slice thickness, lmm; distance between slices, 1.5 mm. Gain and signal-to-noise ratios were determined at each individual scanning session. T2 -weighted images of RARE_8 gave the best contrast between the tumor and surrounding normal tissue. The animals, including untreated controls, were imaged every third day during the experiments. The tumor volume was defined from two orthogonal sets of RARE_8 images covering the entire tumor. Images were analyzed using ROI (Region of Interest Tool), the tomography software version Para Vision 3.0.1. Statistical analysis of the data was done using Mann- Whitney U-test non- parametric method. The cancer preventive or therapeutic effects of 3- demethylubiquinone Q₂ in mice (Figs. 16-18) are presented as a percentage of the inhibition of tumor growth in the experimental mice compared with untreated control mice. Example 26. Inhibition of Malignant JB6 P⁺ C141 Cell Transformation

Representative compounds were assayed for cancer preventive activity using the anchorage-independent JB6 P+ C141 cell transformation assay in a soft agar. Generally, inhibition of cell transformation is a good indication that a compound will have an effective cancer preventive activity. Toxicity of each compound for JB6 C141 cells was determined by the MTS cell viability assay. For one of the quinones, 3-demethylubiquinone Q2 A (1), the corresponding data are shown in Figs. 19 and 20. Using the obtained data and statistical computer program Statistica 6.0, the corresponding regressions were built and the IC₅₀ for decreased cell viability and the INCC_5O (Inhibition of the Number of the Colonies C₅₀) for inhibition of cell transformation were determined for each quinone studied. These data are summarized in Table 2. The obtained results indicated that all quinones studied inhibited cell transformation induced by EGF or TPA in dose-dependent manner in JB6 C141 cells. For many of the quinones, the dose that inhibited malignant transformation was well below that which was toxic (Table 2). To understand the possible inhibitory signalling pathway activated by the quinones studied, whether the JB6 cells were undergoing apoptosis induced by quinones was investigated.

Table 2. IC₅₀ and INCC₅₀ of representative quinones for JB6 C141 P⁺ cells.

*, in all calculations the designated numbers were used.

Methods

The cancer preventive effects of representative compounds were evaluated in 6-well plates using JB6 P⁺ C141 cells, activated with EGF (10 ng/ml) or TPA (20 ng/ml). JB6 P⁺ C141 cells (8 x 10³/ml) were treated with the indicated concentrations of the quinones in 1 ml of 0.33% BME (basal medium Eagle) agar containing 10% FBS over 3.5 ml of 0.5% BME agar containing 10% FBS and indicated concentrations of the quinones. The cultures were maintained in a 37°C, 5% CO₂ incubator for 1 wk (JB6 P⁺ C141 cells, activated with EGF) or 2 weeks (JB6 P⁺ C141 cells, activated with TPA). Cell colonies were then scored using the LEICA DM IRB inverted research microscope (Leica Mikroskopie und Systeme GmbH, Germany) and Image-Pro Plus software, version 3.0 for Windows (Media Cybernetics, Silver Spring, MD). For each compound, two independent experiments in triplicate for each concentration were performed.

Example 27. Induction of Apoptosis in JB6 C141 Cells and MEFs The ability of representative compounds to induce apoptosis was determined by flow cytometry. The results indicated that representative compounds induced apoptosis in JB6 C141 cells in a dose-dependent manner (Fig. 21). For 3-demethylubiquinone Q2 A (1), apoptosis was also demonstrated by DNA laddering in MEFs (Fig. 22).

Apoptosis Assay Using Flow Cytometry - Methods JB6 P⁺ C141 cells (3xlO⁵ cells/dish) were grown in 6-cm dishes for 24 hrs in 5% FBS-MEM. Then cells were treated with different concentrations of representative compounds dissolved in 0.1% FBS-medium for 3 hrs. Then the medium was removed and attached cells were harvested with 0.025% trypsin in 0.1% EDTA in PBS. Trypsinization was stopped by adding 2 ml of 5% FBS in PBS. Cells were then washed by centrifugation at 1,000 rpm (170 rcf) for 5 mm and processed for detection of apoptosis using Annexin V- FITC and propidium iodide staining according to the manufacturer's protocol. In brief, 1-5x10⁵ cells were collected after centrifugation, and resuspended in 500 μl of Ix binding buffer (Annexin V-FITC Apoptosis Detection Kit, Medical & Biological Laboratories (Watertown, MA)). Then, 5 μl of Annexin V-FITC and 5 μl of propidium iodide were added and the cells were incubated at room temperature for 5 min in the dark and analyzed by flow cytometry. For each compound, two independent experiments in duplicate for each concentration were performed. Apoptosis Assay Using DNA Ladder - Methods MEFs were grown in 10-cm dishes and treated with 3- demethylubiquinone Q2 (1) when cells were 80% confluent. The cells were incubated with quinone 1 for 24 h. Then, both detached and attached cells were harvested by scraping followed by centrifugation. The obtained cells were disrupted with lysis buffer (5 niM Tris-HCl, pH 8.0, 20 mM EDTA, and 0.5% Triton X-100) and left on ice for 45 min. After centrifugation at 14,000 rpm (45 min, 4^°C), the supernatant fraction containing fragmented DNA was extracted twice with phenol/chloroform/isopropyl alcohol (25:24:1, v/v) and once with chloroform. Then the fragmented DNA was precipitated overnight at -20°C after addition of two volumes of 100% ethanol and 1/10 volume of 5 M NaCl. The DNA pellet was saved by centrifugation at 14,000 rpm for 45 min, washed once with 70% ethanol, dried, and resuspended in TE buffer (10 mM Tris-HCl, ImM EDTA, pH 8.0). After addition of 100 μg/ml RNAse A (Sigma), the mixture was incubated at 37°C for 2 h. The DNA fragments were separated by 1.8% agarose gel electrophoresis. DNA laddering in the gel was stained with ethidium bromide and photographed under UV light. Two independent experiments were performed.

Example 28. Inhibition of p53 and Induction of AP-I or NF-κB Transcriptional

Activity

Several key transcription factors, including the p53 tumor suppressor protein, AP-I or NF -KB are often implicated in the induction or inhibition of apoptosis by various stimuli, including chemopreventive compounds or drugs. Therefore, the effect of representative compounds on these three transcription factors was investigated. JB6 C141 cell lines stably expressing a luciferase reporter gene controlled by an AP-I, NF -KB, or p53 DNA binding sequence were used. Representative compounds showed a significant (up to an 8-fold) induction of AP-I or NF-κB-dependent transcriptional activation, and a substantial (up to 4 fold) inhibition of p53-dependent transcriptional activity {see Table 3). Table 3. The effect of representative compounds on AP-I-, NF-κB-, and p53-dependent transcriptional activity in JB6 C141 cells.

*, significant differences were not determined. Methods

The ability of representative compounds to influence AP-I-, NF-κB- and p53-dependent transcriptional activities in the mouse JB6 C141 cell line was evaluated using the luciferase method. Viable JB6-LucPG-13, JB6-LucAP-l, or JB6-LucNF-κB cells (6x10³) suspended in 100 μl 5% FBS-MEM were added into each well of a 96-well plate. Plates were incubated for 24 h and then treated with various concentrations of quinones in 100 μl of 0.1% FBS-MEM. After incubation with representative compounds for 24 h, the cells were extracted for 1 h at room temperature with 100 μl/well of lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA). Then 30 μl of lysate from each well were transferred in a plate used for luminescent analysis and luciferase activity was measured using 100 μl/well of the luciferase assay buffer (1 mM D-luciferase, pH = 6.1 - 6.5; 40 mM Tricin, 2.14 mM magnesium carbonate (MgCO₃)₄ x Mg(OH)₂ x 5H₂O, 5.34 mM MgSO₄ x 7H₂O, 66.6 mM DTT, 1.06 mM ATP, 0.54 mM COA, 0.2 mM EDTA, pH = 7.8) and the Luminoscan Ascent Type 392 microplate reader (Labsystems, Finland). For each compound, two independent experiments with five samples for each concentration were performed.

Example 29

The following illustrate representative pharmaceutical dosage forms, containing a compound of the invention ('Compound X'), for therapeutic or prophylactic use in humans. The formulations may be obtained by conventional procedures Λvell known in the pharmaceutical art.

(T) Tablet 1 mg/tablet

Compound X= 100.0

Lactose 77.5

Povidone 15.0 Croscarmellose sodium 12.0

Microcrystalline cellulose 92.5

Magnesium stearate 3.0

300.0

(if) Tablet 2 mg/tablet

Compound X= 20.0

Microcrystalline cellulose 410.0 Starch 50.0

Sodium starch glycolate 1 5.0

Magnesium stearate 5.0

500.0

J

(iii) Capsule rag/capsule

Compound X= 1 0.0

Colloidal silicon dioxide 1 .5

Lactose 465.5 0 Pregelatinized starch 120.0

Magnesium stearate 3.0

6O0.0

(iv) Injection 1 Cl mε/ml) mg/ml 5 Compound X= (free acid form) 1.0

Dibasic sodium phosphate 12.0

Monobasic sodium phosphate 0.7

Sodium chloride 4.5

1.0 N Sodium hydroxide solution 0 (pH adjustment to 7.0-7.5) q.s.

Water for injection q.s. ad 1 mL

Cv) Injection 2 (10 mε/ml) mg/ml

Compound X= (free acid form) 10.0 5 Monobasic sodium phosphate 0.3

Dibasic sodium phosphate 1.1

Polyethylene glycol 400 200.0

01 N Sodium hydroxide solution

(pH adjustment to 7.0-7.5) q.s. 0 Water for injection q.s. ad 1 mL (vi) Aerosol mg/can

Compound X= 20.0

Oleic acid 10.0

Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0

Dichlorotetrafluoroethane 5,000.0

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. The use of a compound of formula I or II:

¹ π or a pharmaceutically acceptable salt thereof, to prepare a medicament useful for treating cancer in a mammal, wherein: each OfR₁, R₂, and R₃ is independently hydrogen,

or a group of formula III;

III

each OfR₄ and R₅ is independently hydrogen or (Q-C^aLkyl; n is 2 or 3; and each m is 1, 2, or 3.

2. The use of claim 1 , wherein the medicament is useful for reducing tumor size in the mammal.

3. The use of claim 1 or 2, wherein the medicament is useful for inhibiting tumor growth in the mammal.

4. The use of a compound as described in claim 1 to prepare a medicament useful for inducing AP-I dependent transcriptional activity in a mammal.

5. The use of a compound as described in claim 1 to prepare a medicament useful for inducing NF-κB dependent transcriptional activity in a mammal.

6. The use of a compound as described in claim 1 to prepare a medicament useful for inducing AP-I and NF-κB dependent transcriptional activity in a mammal.

7. The use of a compound as described in claim 1 to prepare a medicament useful for decreasing the transformation of normal cells into tumor cells in a mammal.

8. The use of a compound as described in claim 1 to prepare a medicament useful for decreasing tumor cell proliferation in a mammal.

9. The use of a compound as described in claim 1 to prepare a medicament useful for inducing apoptosis in a mammal.

10. The use as described in any one of claims 1-9, wherein the compound is a compound of formula I.

11. The use as described in any one of claims 1 -9, wherein the compound is a compound of formula II.

12. The use as described in any one of claims 1-11, wherein at least one of R₁, R₂, and R₃ is hydrogen.

13. The use as described in any one of claims 1-11, wherein at least two of R₁, R₂, and R₃ are hydrogen.

14. The use as described in any one of claims 1-11, wherein R₁, R₂, and R₃ are each hydrogen.

15. The use as described in any one of claims 1-11, wherein at least one of R₁, R₂, and R₃ is (C_rC₆)alkoxy.

16. The use as described in any one of claims 1-11, wherein at least two of R₁, R₂, and R₃ are are each independently (Cj-C₆)alkoxy.

17. The use as described in any one of claims 1-11, wherein R₁ , R₂, and R₃ are each independently

18. The use as described in any one of claims 1-11, wherein at least one of R₁, R₂, and R₃ is a group of formula III.

19. The use as described in any one of claims 1-11, wherein at least two of R₁, R₂, and R₃ are each independently a group of formula III.

20. The use as described in any one of claims 1-11, wherein R₁, R₂, and R₃ are each independently a group of formula HI.

21. The use as described in any one of claims 1-11, wherein R₁ is hydrogen.

22. The use as described in any one of claims 1-11, wherein R₁ is (C₁- C₆)alkoxy.

23. The use as described in any one of claims 1-11, wherein R₁ is methoxy.

24. The use as described in any one of claims 1-11 and 21-23, wherein R₂ is hydrogen.

25. The use as described in any one of claims 1-11 and 21-23, wherein R₂ is (C]-C₆)alkoxy.

26. The use as described in any one of claims 1-11 and 21-23, wherein R₂ is methoxy.

27. The use as described in any one of claims 1-11 and 21-26, wherein R₃ is hydrogen.

28. The use as described in any one of claims 1-11 and 21-26, wherein R₃ is (C]-C₆)alkoxy.

29. The use as described in any one of claims 1-11 and 21-26, wherein R₃ is methoxy.

30. The use as described in any one of claims 1-9 and 11-29, wherein R₄ is hydrogen.

31. The use as described in any one of claims 1-9 and 11-29, wherein R₄ is (Ci-QOalkyl.

32. The use as described in any one of claims 1-9 and 11-29, wherein R₄ is methyl.

33. The use as described in any one of claims 1-9 and 11-32, wherein R₅ is hydrogen.

34. The use as described in any one of claims 1-9 and 11-32, wherein R₅ is (d-QOalkyl.

35. The use as described in any one of claims 1-9 and 11-32, wherein R₅ is methyl.

36. The use as described in any one of claims 1-35, wherein n is 2.

37. The use as described in any one of claims 1-35, wherein n is 3.

38. The use as described in any one of claims 1-9 and 12-29, wherein the compound is a compound of formula FV:

IV

39. The use as described in any one of claims 1-9 and 12-29, wherein the compound is a compound of formula V:

40. The use as described in any one of claims 1-9 and 21-26, wherein the compound is a compound of formula VI:

41. The use as described hi any one of claims 1-9 and 21-26, wherein the compound is a compound of formula VII:

42. The use as described in any one of claims 1-9 and 21-26, wherein the compound is a compound of formula VIII:

43. The use as described in any one of claims 1-9 and 21-26, wherein the compound is a compound of formula IX:

44. The use as described in any one of claims 1-9 and 21-26, wherein the compound is a compound of formula X:

45. The use as described in any one of claims 1-9 and 21-26, wherein the compound is a compound of formula XI:

46. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XII:

47. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XDI:

48. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XTV:

49. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XV:

50. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XVI:

51. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XVII:

52. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XVIII:

53. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XIX:

54. The use as described in any one of claims 1-9, 21-26, and 30-35, wherein the compound is a compound of formula XX:

55. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXI:

56. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXII:

57. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXIII:

58. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXTV:

59. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXV:

60. The use as described in any one of claims 1 -9 and 24-29, wherein the compound is a compound of formula XXVI:

61. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula IXXVII:

XXVII.

62. The use as described in any one of claims 1 -9 and 24-35, wherein the compound is a compound of formula XXVIII:

63. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula XXTX:

64. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula XXX:

65. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula XXXI:

66. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula XXXII:

67. The use as described in any one of claims 1-9 and 21-26, wherein the compound is a compound of formula XXXIII:

68. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXXTV:

69. The use as described in any one of claims 1 -9 and 24-35, wherein the compound is a compound of formula XXXV:

70. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXXVI:

71. The use as described in any one of claims 1-9 and 24-29, wherein the compound is a compound of formula XXXVII:

72. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula XXXVIII:

73. The use as described in any one of claims 1-9 and 24-35, wherein the compound is a compound of formula XXXIX:

74. The use as described in any one of claims 1-9, wherein the compound is not a compound of IV wherein R₁ is methoxy and R₂ and R₃ are each hydrogen.

75. The use as described in any one of claims 1-9 and 74, wherein the compound is not a compound of IV wherein R₁, R₂, and R₃ are each hydrogen.

76. The use as described in any one of claims 1-9, 74, and 75, wherein the compound is not a compound of V wherein R_{1 ,} R₂, and R₃ are each hydrogen.

77. The use as described in any one of claims 1-9 and 74-76, wherein the compound is not a compound of V wherein R₁ and R₂ are each methoxy and R₃ is hydrogen.

78. The use as described in any one of claims 1-9 and 74-77, wherein the compound is not a compound of XII wherein R₁- R₅ are each hydrogen.

79. The use as described in any one of claims 1-9 and 74-78, wherein the compound is not a compound of XIII wherein R₁- R₅ are each hydrogen.

80. The use as described in any one of claims 1-9 and 74-79, wherein the compound is not a compound of XII wherein R₁ is methoxy and R₂-R₅ are each hydrogen.

81. The use as described in any one of claims 1 -y , wnerem tne compounu is a compound of formula IV or XXXVI wherein R₁ and R₂ are each methoxy and R₃ is hydrogen.

82. The use as described in any one of claims 1-81, wherein the compounds is isolated and purified.

83. A compound as described in any one of claims 1 and 10-82, wherein: the compound is not a compound of IV wherein R₁ is methoxy and R₂ and R₃ are each hydrogen; the compound is not a compound of IV wherein Ri₅ R₂, and R₃ are each hydrogen; the compound is not a compound of V wherein R₁, R₂, and R₃ are each hydrogen; the compound is not a compound of V wherein R₁ and R₂ are each methoxy and R₃ is hydrogen; the compound is not a compound of XII wherein R₁- R₅ are each hydrogen; the compound is not a compound of XIII wherein R₁- R₅ are each hydrogen; and the compound is not a compound of XII wherein R₁ is methoxy and R₂- R₅ are each hydrogen.

84. A compound as described in claim 83 for use in medical treatment or diagnosis.

85. A composition comprising a compound as described as described in any one of claims 1 and 10-83 and an acceptable diluent or carrier.

86. The composition of claim 85 which is formulated as a cosmetic.

87. The composition of claim 85 or 86 which is formulated as a sunscreen.

88. The composition of any of claims claim 85-87 which is formulated as a lotion.

89. The composition of claim 85 which is formulated as a dietary supplement.

90. A method for treating cancer comprising administering to a mammal in need of such treatment, an effective amount of a compound as described in any one of claims 1 and 10-83.

91. The method of claim 90, wherein the administration of the compound reduces tumor size in the mammal.

92. The method of claim 90 or 91 , wherein the administration of the compound inhibits tumor growth in the mammal.

93. The method of any one of claims 90-92, wherein the mammal has been diagnosed with cancer prior to the administration of the compound.

94. A method for inducing AP-I dependent transcriptional activity in a cell comprising contacting the cell with an effective amount of a compound as described in any one of claims 1 and 10-83.

95. A method for inducing NF -KB dependent transcriptional activity in a cell comprising contacting the cell with an effective amount of a compound as described in any one of claims 1 and 10-83.

96. A method for inducing AP-I and NF-κB dependent transcriptional activity in a cell comprising contacting the cell with an effective amount of a compound as described in any one of claims 1 and 10-83.

97. A method for inducing apoptosis or cell death in a cell comprising contacting the cell with an effective amount of a compound as described in any one of claims 1 and 10-83.

98. The method of any one of claims 90-97, wherein the cell is a mammalian cell.

99. The method of any one of claims 90-98, wherein the cell is a human cell.

100. The method of any one of claims 90-99, wherein the contacting is in vitro.

101. The method of any one of claims 90-99, wherein the contacting is in vivo.

102. A method for decreasing the transformation of normal cells into tumor cells in a mammal in need of such treatment, comprising administering an effective amount of a compound as described in any one of claims 1 and 10-83 to the mammal.

103. A method for decreasing tumor cell proliferation in a mammal in need of such treatment, comprising administering an effective amount of a compound as described in any one of claims 1 and 10-83 to the mammal.

104. A method for inducing apoptosis or cell death in a mammal in need of such treatment, comprising administering an effective amount of a compound as described in any one of claims 1 and 10-83 to the mammal.

105. A method for isolating a compound of formula IV or XXXVI wherein R₁ and R₂ are each methoxy and R₃ is hydrogen, comprising extracting one or more marine invertebrate animals comprising the compound of formula IV or XXXVI with a suitable solvent followed by purification with column chromotography to provide the compound of formula FV or XXXVI.

106. The method of claim 105, wherein the animals are ascidian ApIi diwn glahrum.

107. The method of claim 105 or 106, wherein the chromatography is silica gel chromatography with hexane: ethyl acetate as the eluent.

108. The method of any one of claims 105-107, wherein the chromatography is repeated two or more times.

109. The method of any one of claims 105-108, wherein the solvent is an alcohol.

110. The method of claim 109, wherein the alcohol is ethanol.

111. The method of any one of claims 105-110, wherein the hexane:ethyl acetate is about 10:1.