RU2411229C2 - Therapeutic quinones - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention refers to new polyprenylated 1,4-benzoquinones of formula 1 or formula 3 or polyprenylated 1,4-hydroquinones of formula 2 or formula 4, or to their pharmaceutically acceptable salts exhibiting anticarcinogenic activity 1, 2, 3, 4. In formulae 1-4 each of R1 and R2, irrespectively of others, can be a hydrogen radical or (C1-C6)-alkoxy-group; each of R3 and R4 irrespectively of others, can be a hydrogen radical or (C1-C6)-alkyl group; n can be equal to 2 or 3. ^ EFFECT: provided application of new compounds and some common 1,4-benzoqunones and 1,4-hydroqunones for preparing a drug, a based pharmaceutical composition, development of a method of oncotherapy, a method of inducing AP-1-dependent or NF-kB-independent transcription activity or both activities simultaneously in a cell and a method of inducing apoptosis in a cell, as well as separating glabruquinones. ^ 21 cl, 3 tbl, 28 dwg, 26 ex

Description

Cross-references to relevant patent applications

This patent document claims the priority of US patent application number 60 / 612,472, filed September 23, 2004, which is presented here at this link.

Background

Polyprenylated 1,4-benzoquinones and hydroquinones, such as ubiquinones, plastoquinones and tocopherols, are widespread in plants and animals in which they play an important role in electron transport, photosynthesis and as antioxidants (see Thomson, RH Naturally Occurring Quinones, Academic Press London, 1971, pp. 1-197; and Pennock, JF B: Terpenoids in plants, JB Pridham, Ed. Academic Press, London, 1967, pp. 129-146).

Natural prenylated benzoquinones and hydroquinones having a terpenoid part of one to nine isoprene units in length were previously isolated from marine organisms, such as Fucales brown algae, sponges, and ascidia. Many algae contain tetraprenyl, triprenyl, and diprenyl quinones and hydroquinones (see Ochi, M .; Kotsuki, H .; Inooue, M .; Taniguchi, M .; Tokoroyama, T. Chem. Lett., 1979, 831-832 ; and Capon, RJ; Ghisalberti, EL; Jeffereis, PR Phytochemistry, 1981, 20, 2598-2600). In addition, sponges contain linear unsubstituted polyprenylated hydroquinones and benzoquinones with longer side chains and moderate antimicrobial activity, as well as ATPase inhibitors, sulfated polyprenylquinones (see Cimino, G .; De Stefano, S .; Minale, L. Tetrahedron, 1972, 28, 1315; Cimino, G .; De Stefano, S .; Minale, L. Experientia, 1972, 28, 1401; Pouches YF; Verbist, JF; Biard, JF; Boukef, KJ Nat. Prod., 1988, 51, 188; Lumsdon, D .; Capon, RJ; Thomas, SG; Beveridge, AA Aust. J. Chem. 1992, 45, 1321-1325; De Rosa, S .; De Giulio, Iodice, CJ 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, VA; Makarieva, TN; Dmitrenok, ASJ Nat. Prod., 1992, 55, 1256-1260; and Bifulco, G .; Bruno; I .; Minale; L., Riccio, R .; Debitus, S .; Bourdy, G .; Vassa, A .; Lavayre, JJ Nat. Prod. 1995 58, 1444-1449). About a dozen prenylated quinones were previously isolated from ascidia of the genus Aplidium, including the simplest of them, monoprenyl benzoquinones (see Guella, G; Mancini, I; Pietra, F. Helv. Chim. Acta. 1987, 70, 621-626; Howard, BM; Clarkson, K .; Bernstein, RL Tetrahedron. Lett. 1979, 4449 - argett, NM; Keeran, WSJ Nat. Prod., 1984, 47, 556-557; and Faulkner, DJ Nat. Prod. Rep., 1993 93, 1771-1791).

Cancer is one of the leading deaths in the United States. However, there is still a need for compounds having antitumor properties. There is also a need for pharmacological tools for further research on the physiological processes associated with cancer.

Summary of the invention

The present invention claims polyprenylated 1,4-benzoquinones (formula 1 or formula 3) or polyprenylated 1,4-hydroquinones (formula 2 or formula 4):

Where

each of R1 and R2, independently of the others, may be hydrogen

a radical or a (C ₁ -C ₆ ) alkoxy group;

each of R3 and R4, independently of the others, may be a hydrogen radical or a (C ₁ -C ₆ ) -alkyl group;

n may be 2 or 3;

or their pharmaceutically acceptable salts.

The present invention also claims the use of a compound of formula 1, or formula 2, or formula 3, or formula 4, as well as the known polyprenylated 1,4-benzoquinones of formula 5, or formula 6, or formula 7, or formula 8, or formula 9, or formula 10, or formula 11, or formula 12, or formula 13, or formula 14, or the polyprenylated 1,4-hydroquinones of formula 15 or their pharmaceutically acceptable salts for the preparation of a medicament that can be used to treat cancer in mammals.

The invention also claims the use of the above new and known substances or their pharmaceutically acceptable salts for the preparation of a medicament used for the treatment of cancerous tumors in mammals, i.e. to reduce the size and / or inhibition of cancer growth in mammals.

The invention also claims the use of a substance of the invention for the preparation of a medicament used to induce AP-1-dependent transcriptional activity in mammals.

The invention also claims the use of a substance of the invention for the preparation of a medicament used to induce NF-κB-dependent transcriptional activity in mammals.

The invention also claims the use of a substance of the invention for the preparation of a medicament used to induce simultaneously AP-1 and NF-κB-dependent transcriptional activity in mammals.

The invention also claims the use of a substance of the invention for the preparation of a medicament used to reduce the transformation of normal cells into tumor cells in mammals.

The invention also claims the use of a substance of the invention for the preparation of a medicament used to reduce the proliferation of tumor cells in mammals.

The invention also claims the use of a substance of the invention for the preparation of a medicament used to induce apoptosis in mammals.

The invention also claims a pharmaceutical composition having anti-cancer activity, comprising an effective amount of a compound of formula 1, or formula 2, or formula 3, or formula 4, or formula 5, or formula 6, or formula 7, or formula 8, or formula 9, or formula 10, or formula 11, or formula 12, or formula 13, or formula 15, or a pharmaceutically acceptable salt of this compound in combination with a pharmaceutically acceptable solvent or carrier.

The invention also claims a method of treating cancer, comprising administering to the mammal, if necessary in such treatment, an effective amount of a compound of formula 1, or formula 2, or formula 3, or formula 4, or formula 5, or formula 6, or formula 7, or formula 8 or formula 9 or formula 10 or formula 11 or formula 12 or formula 13 or formula 14 or formula 15 or an effective amount of a pharmaceutically acceptable salt of this compound or an effective amount of a pharmaceutical composition comprising these connections.

The invention also claims a method of treating cancer, where the administration of a compound or pharmaceutical composition reduces the size or inhibits the growth of a cancer tumor in a mammal, prevents the transformation of normal cells into tumor cells, reduces the proliferation of tumor cells, and causes apoptosis of tumor cells in a mammal.

The invention also claims a method for inducing in the cell an AP-1-dependent transcriptional activity in cells, comprising treating the cells with an effective amount of a substance of the invention.

The invention also claims a method for inducing NF-κB-dependent transcriptional activity in cells in a cell, comprising treating the cells with an effective amount of a substance of the invention.

The invention also claims a method for simultaneously inducing AP-1 and NF-κB-dependent transcriptional activity in a cell, comprising treating the cells with an effective amount of a substance of the invention.

The invention also claims a method for inducing apoptosis or cell death in cells, comprising treating the cells with an effective amount of a substance of the invention.

The invention also claims a method of inducing apoptosis or cell death in mammals, including, if necessary, the treatment, the introduction of an effective amount of a substance of the invention.

The invention also claims a method for the isolation of glabruquinone A (formula 5) and glabruquinone B (formula 6), including extraction of ascidium ascidium Aplidium glabrum with ethanol, evaporation of ethanol, extraction of the aqueous residue with chloroform, evaporation of the chloroform layer to obtain a brown oily residue, chromatography of the residue by column chromatography silica gel, in a hexane-ethyl acetate system, 10: 1 and separation of the mixture of the obtained glabruquinones A and B by HPLC on a column of silica gel in a hexane-ethyl acetate system, 7: 1 to obtain individual g labrukhinonov A and B.

A brief description of the figures.

Figure 1. Structural formulas of the compounds of the invention claimed as new.

Figure 2. Structural formulas of substances 5-15.

Figure 3. General scheme for the synthesis of glabruquinone A (5) and its analogues.

Figure 4. Synthesis of target quinones and hydroquinones from a phenol derivative 35.

Figure 5. Synthesis of target quinones and hydroquinones from a phenol derivative 36.

6. Synthesis of target quinones and hydroquinones from a phenol derivative 37.

7. Structures of compounds 5 and 6 and the establishment of structure 5 using NMVS and ¹ H- ¹ H-COZY correlations.

Figa. The cytotoxic effect of glabruquinone A (5) on JB6 P ⁺ Cl 41 cells, established using the MTS method. Cells were cultured in 96-well plates in 5% FBS / MEM. Then the medium was changed to 0.1% FBS / MEM and the cells were treated with the indicated concentrations of glabruquinone A (1). Cells were incubated with the substance for 22 hours and then 20 μl of MTS reagent was added to each well and incubated for another 2 hours. Data are presented as percentage of living cells compared to control, untreated glabruquinone cells. Each point on the graph reflects the obtained value ± SD (standard deviation from the mean) and is the result of two independent experiments, five samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Figv. Calculation of IC ₅₀ for glabruquinone A (5) when exposed to JB6 P ⁺ Cl 41 cells, performed by linear regression.

Figa. The cytotoxic effect of glabruquinone A (5) on NT-460 cells, established using the MTS method. Cells were cultured in 96-well plates in 10% FBS / RPMI. Then the medium was changed to 0.5% FBS / RPMI, and then the cells were treated as indicated for Figa. Data are presented as the percentage of living cells compared to control, untreated glabruquinone A cells. Each point on the graph reflects the obtained value ± SD (standard deviation from the mean value) and is the result of two independent experiments, three samples for each concentration in each experiment (* p <0.05; Mann-Whitney U-test).

Figv. Calculation of IC ₅₀ for glabruquinone A (5) when exposed to NT-460 cells, performed by linear regression.

Figa. Inhibition by glabruquinone A (5) of colony formation of HCT-116 cells in soft agar. HCT-116 cells were treated with the indicated concentrations of glabruquinone A (5) in soft agar. Cell colonies were counted after incubation with substance 5 for a week. Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Figv. Calculation of INCC ₅₀ for glabruquinone A (5) when exposed to HCT-116 cells in soft agar, performed by linear regression.

Figa. Inhibition of glabruquinone A (5) by EGF-induced JB6 P ⁺ Cl 41 cell transformation in soft agar. JB6 P ⁺ Cl 41 cells were treated with EGF (10 ng / ml) and indicated concentrations of glabruquinone A (5) in soft agar. Cell colonies were counted after incubation with substance 5 for a week. Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

11B. Calculation of INCC ₅₀ for glabruquinone A (5) upon action on soft agar cells transformed with EGF JB6 P ⁺ Cl 41 by linear regression.

Figa. Inhibition of glabruquinone A (5) TPA-induced JB6 P ⁺ Cl 41 cell transformation in soft agar. JB6 P ⁺ Cl 41 cells were treated with TPA (10 ng / ml) and indicated concentrations of glabruquinone A (5) in soft agar. Cell colonies were counted after incubation with substance 5 for two weeks. Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Figv. Calculation of INCC ₅₀ for glabruquinone A (5) on the action of linear regression using soft regar transformed with TPA JB6 P ⁺ Cl 41 cells in soft agar.

Figa. Induction of glabruquinone A apoptosis in JY cells (In human lymphoblasts). JY cells were grown and treated with the indicated concentrations of glabruquinone A (5). The percentage of apoptosis was calculated after 3 hours of incubation with the substance (5). Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Figv. Induction of apoptosis by glabruquinone A in JB6 P ⁺ Cl 41 mouse cells. JB6 P ⁺ Cl 41 cells were grown and treated with the indicated concentrations of glabruquinone A (5). The percentage of apoptosis was calculated after 3 hours of incubation with the substance (5). Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, two samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Figs. Induction of apoptosis by glabruquinone A in SK-MEL-28 cells. SK-MEL-28 human melanoma cells were grown and treated with the indicated concentrations of glabruquinone A (5). The percentage of apoptosis was calculated after 3 hours of incubation with the substance (5). Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Fig.13D. Induction of apoptosis by glabruquinone A in HT-460 cells. NT-460 human lung cancer cells were grown and treated with the indicated concentrations of glabruquinone A (5). The percentage of apoptosis was calculated after 3 hours of incubation with the substance (5). Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Fig.13E. Induction of glabruquinone A apoptosis in HCT-116 cells. Human intestinal cancer cells HCT-116 were grown and treated with the indicated concentrations of glabruquinone A (5). The percentage of apoptosis was calculated after 20 hours of incubation with the substance (5). Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, three samples for each concentration in each of the experiments (* p <0.05; Mann-Whitney U-test).

Fig.14. Induction of apoptosis by glabruquinone A (5) in COX2 ^{+ / +} or COX2 ^{- / -} MEF, determined using the DNA ladder method. COX2 ^{+ / +} or COX2 ^{- / -} MEF cells (mouse embryonic fibroblasts) were grown, treated with the indicated concentrations of glabruquinone A (5), and incubated for 24 hours. Then the cells were harvested and put to the test for apoptosis using the DNA ladder method.

Figa. Glabruquinone A (5) induces NF-κB-dependent transcriptional activity in JB6 Cl 41 cells. JB6 Cl 41 cells with a stably expressed luciferase reporter gene controlled by the NF-κB DNA-linked sequence were treated with the indicated concentrations of glabruquinone A (5) and incubated for 24 hours. Then the cells were extracted with lysis buffer and luciferase activity was measured.

Figv. Glabruquinone A (5) induces AP-1-dependent transcriptional activity in JB6 Cl 41 cells. JB6 Cl 41 cells with a stably expressed luciferase reporter gene controlled by the AP-1 DNA-linked sequence were treated with the indicated concentrations of glabruquinone A (5) and incubated for 24 hours. Then the cells were extracted with lysis buffer and luciferase activity was measured.

Fig.16. Inhibition of glabruquinone A (aka demethylubiquinone Q2, 5) of Ehrlich carcinoma growth in white non-linear mice. Mice were treated once by intraperitoneal administration, glabruquinone A dissolved in 50% DMSO, one day before tumor inoculation. Tumor sizes were measured on the 6th, 9th, 12th, 15th and 18th day. Data are presented as percent inhibition of tumor growth in mice treated with glabruquinone A compared with tumor sizes in untreated mice. Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, four mice in each of the experiments (* p <0.05; Mann-Whitney U-test).

Fig.17. Inhibition of glabruquinone A (aka demethylubiquinone Q2, 5) of Ehrlich carcinoma growth in white non-linear mice. Mice were treated once by intraperitoneal administration, glabruquinone A dissolved in 50% ethanol, one day before tumor inoculation. Tumor sizes were measured on the 6th, 9th, 12th, 15th and 18th day. Data are presented as percent inhibition of tumor growth in mice treated with glabruquinone A compared with tumor sizes in untreated mice. Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, four mice in each of the experiments (* p <0.05; Mann-Whitney U-test).

Fig. 18. Inhibition of glabruquinone A (aka demethylubiquinone Q2, 5) of Ehrlich carcinoma growth in white non-linear mice. Mice were treated once by intraperitoneal administration, glabruquinone A, dissolved in 50% DMSO, one day after tumor inoculation. Tumor sizes were measured on the 9th, 12th, 15th and 18th day. Data are presented as percent inhibition of tumor growth in mice treated with glabruquinone A compared with tumor sizes in untreated mice. Each column in the graph reflects the obtained value ± SD and is the result of two independent experiments, four mice in each of the experiments (* p <0.05; Mann-Whitney U-test).

DETAILED DESCRIPTION OF THE INVENTION

Substances of the invention showed anticancer preventive and therapeutic activity, which was shown using methods for determining tumor transformation of cells in soft agar, determining apoptosis by flow cytometry and DNA ladder methods, determining cytotoxic activity by the MTS method, as well as in vivo experiments on white non-linear mice with vaccinated Ehrlich carcinoma. For experiments, mouse epidermal JB6 P ⁺ Cl 41 cells, COX2 ^{+ / +} and COX2 ^{- / -} mouse embryonic fibroblasts (MEF), JY cells (human lymphoblasts), human tumor cells NT-460 (lung cancer), and HCT-116 were used (bowel cancer), SK-MEL-28 (skin cancer, melanoma). The studied polyprenylquinone and hydroquinone induced AP-1 and NF-κB-dependent transcriptional activity in JB6 Cl 41 cells. The substances of the invention also showed cancer-preventive activity in experiments on soft agar, in non-cytotoxic concentrations, which indicates that the antitumor effect of the substances of the invention is mediated through a non-toxic mechanism.

Relationships structure-activity on the example of a representative sample of substances of the invention

The structure – activity dependence of the presented compounds was studied using statistical analysis (computer program Statistica 6, Stat Soft Inc., USA). The studied compounds were divided into three groups depending on the number of isoprene units in the terpenoid part (Table 2). Group 1 contained quinones with two isoprene units (10 carbon atoms) in the side chain. Group 2 - quinones with three isoprene units (15 carbon atoms) in the terpenoid part and group 3 - quinones containing 4 to 6 isoprene units (20-30 carbon atoms) in the side chain. Significant differences and correlations between biological activity data obtained for various structural groups of quinones were determined using the Spearman and Mann-Whitney U-test non-parametric correlation using the data from Table. 2.

The results showed that the biological activity of the presented compounds depends on the side chain length of the terpenoid part of the molecule. Statistical analysis of the data from Table 2 showed that the cytotoxicity of quinones from group 1 has an average value of IC ₅₀ 20.0 ± 15.2 Mµ for JB6 Cl 41 cells. Quinones from group 2 have an average value of IC ₅₀ 9.7 ± 9.0 Mµ. Finally, quinones from group 3 showed an average IC ₅₀ value of 84.9 ± 63.8 Mµ. These results mean that quinones with three isoprene units in the terpenoid part are more toxic to JB6 Cl 41 cells than quinones from group 1 having 2 isoprene units. The opposite conclusion can be drawn by comparing the IC ₅₀ values for quinones from groups 1 and 3 or 2 and 3. Quinones from groups 1 or 2 are significantly more toxic to JB6 Cl 41 cells than compounds from group 3. Therefore, in compounds of groups 1, 2 with an increase in the length of the terpenoid part, the activity of the compounds increases, but the activity decreases significantly with a further increase in the length of the terpenoid part from group 2 to group 3.

Statistical analysis of the effect of the compounds on the EGF-induced transformation of JB6 Cl 41 cells showed that group 1 quinones have an average value of INCC ₅₀ 9.4 ± 5.3 Mµ, and group 2 quinones have an INCC _{50 of} 24.0 ± 16.7 Mµ. Group 3 again showed minimal activity among the three groups of compounds with INCC ₅₀ 59.7 ± 33.3 Mµ (Table 2). Thus, taking into account these data and the Mann-Whitney U-test data, it can be concluded that group 1 quinones have the strongest effect on the inhibition of cell transformation (p = 0.0283 when compared with group 2; p = 0.0253 when compared with group 3; p = 0.0084 when compared with the combined data for groups 2 and 3), and group 3 is the least effective. Significant correlations were observed between the length of the terpenoid part and INCC ₅₀ (p = 0.0002, R = 0.8556). This means that with an increase in the length of the terpenoid part, the INCC ₅₀ values for cell transformation also increase.

The toxicity and cell transformation of JB6 Cl 41 cells were compared, and it was found that the quinones from group 1 had an average INCC ₅₀ value of 1.4–4 times less than the IC ₅₀ for the corresponding cells (Table 2). On the other hand, most quinones from group 2 showed INCC ₅₀ values 4-10 times higher than IC ₅₀ . Based on these data, we can say that group 1 quinones are significantly more toxic for transformed JB6 cells than normal ones. Conversely, group 2 quinones are more toxic to normal JB6 cells than transformed ones. Thus, the quinones of group 1 have a stronger cancer-preventive activity than the quinones of group 2.

We also studied the effect of a representative sample of compounds 5, 7-18 on transcriptional activity, depending on some nuclear factors, in particular, p53, AP-1, and NF-κB. It has been shown that the activity of patentable compounds depends to some extent on the position of the methoxy group with respect to the terpenoid part. Several pairs of structural analogues with methoxy groups in the same position were selected: 1) ortho analogues, quinones 9 and 13; 2) meta-analogues, quinones 10 and 8; 3) para-analogues, quinones 11 and 12. From the data of tables 2 and 3 it follows that the carcinopreventive activity and the effect of quinones on AP-1 dependent transcriptional activity increases in the order of ortho-meta-pair. INCC ₅₀ has the following meanings: for quinones 9, 13: 15.1 and 24.6 Mµ, respectively; quinones 10, 8: 6.6 and 16.7 Mµ, respectively, for quinones 11, 12: 3.1 and 7.4 Mµ, respectively.

Induction of AP-1 dependent transcriptional activity by ortho compounds 9, 13 is 133.8% with respect to the control, and by meta compounds 10, 8 it is 188.7%. Para-compounds 11 and 12 showed the highest value of AP-1 dependent transcriptional activity - 486.9% compared with the control. Ortho-disubstituted quinones 9 and 13 are the least active compounds in comparison with meta- and para-analogues, not only with respect to the induction of AP-1 dependent transcriptional activity, but also with inhibition of cell transformation. Among the para-disubstituted derivatives, quinone 11 having two isoprene units in the side chain showed better activity compared to quinone 12 having three isoprene units. In addition, para-disubstituted quinones 11 and 12 have INCC _{50 of} 3.1 and 7.4 Mµ, respectively, against the EGF-induced cell transformation of JB6 P ⁺ Cl 41 cells. Quinone 9 also showed a higher induction of AP-1 dependent transcriptional activity (721.7%) compared with quinone 13 (252.2%).

The cancer-preventive properties of the patented compounds were studied using murine JB6 Cl 41 epithelial cells. Quinones with two isoprene units in the side chain showed a specific effect against JB6 Cl 41 transformed into tumor cells compared to normal cells. Effective concentrations could vary 4 times.

The structure-activity dependence of patentable compounds has been studied in relation to cytotoxicity or carcinopreventive activity. This study showed that the cytotoxicity of quinones increases with increasing number of carbon atoms, from quinones with two prenyl units in the side chain to quinones with three units and then decreases for compounds having 4 to 6 isoprene units. Carcinopreventive activity decreased with increasing length of the polyprenyl chain. The most active carcinopreventive polyprenylquinones have a side chain of two isoprene units.

As shown by flow cytometry and DNA ladder methods, a representative sample of patented compounds induced apoptosis in JB6 P ⁺ Cl 41 cells and MEF. The p53 tumor suppressor protein, which is part of the cell’s “safety system” and functions according to the principle of negative regulation of cell growth in cells with damaged DNA, is often involved in apoptosis induced by some external stimuli, including chemopreventive agents and antitumor drugs. However, patentable compounds not only do not activate p53, but even vice versa, many of the studied patentable compounds demonstrate significant inhibition of p53-dependent transcriptional activity. In addition, patentable compounds induce significant activation of AP-1 or NF-κB-dependent transcriptional activity. The AP-1 transcription factor regulates many cellular processes, such as proliferation, differentiation, apoptosis, and was previously considered primarily as an oncogen. Recently, it has been shown that some of the constituent AP-1 proteins, such as Jun-B and c-Fos, have cancer-preventive activity, both in vitro and in vivo. Activation of another AP-1 protein, c-Jun, is required for the induction of Fas L-mediated apoptosis in PC 12 cells and human leukemia cells HL-60. The activation of both AP-1 and NF-κB nuclear factors is important for the action of DNA-damaging agents and apoptosis induced by ceramides in T-lymphocytes and Jurkat T-cells. The balance between the member proteins of the AP-1 family, c-Jun and ATF-2 regulates the choice between directions for differentiation or apoptosis in PC 12 cells. Anticancer drugs, such as vinblastine, which inhibit microtubules, activate the AP-1 transcription factor in human carcinoma cells, KB-3. This activation is necessary for the effective apoptosis induced by this drug.

NF-κB, a member of the highly regulated family of dimeric nuclear transcription factors, is involved in the activation of a large number of genes that respond to infection, inflammation and other stressful situations. NF-κB has been reported to be involved in both induction and inhibition of apoptosis.

The results presented in the present invention suggest that apoptosis induced by a representative sample of patentable compounds occurs independently of the p53 nuclear transcription factor, but can instead be mediated via AP-1 and NF-κ B nuclear factors.

Thus, the results obtained show that methoxylated polyprenylquinones and their synthetic analogues are a new promising group of anticancer drugs of marine origin. Patented compounds show cytotoxic activity and induce apoptosis in JB6 P ⁺ Cl 41 and MEF cells. The most active of these compounds are potent inducers of AP-1 and NF-κB-dependent transcriptional activity and, at the same time, inhibitors of p53-dependent transcriptional activity. The cancer-preventive effect of these compounds can be explained by the induction of p53-independent apoptosis.

It was found that quinones having a side chain of 10 carbon atoms have a specific effect of inhibiting transformed JB6 P ⁺ Cl 41 cells, in contrast to quinones having a side chain of 15 or 20 to 30 carbon atoms. Since the present compounds are active against the transformation of epidermal JB6 cells, they can be used as anti-skin cancer agents, i.e. skin cancer prevention agents. Quinone 11, having a side chain in the para position relative to the methoxy group, has the strongest cancer-preventive effect of the studied compounds.

Anticancer and therapeutic properties of the studied compounds were manifested in relatively non-toxic concentrations. This was confirmed by a cytotoxicity test using the MTS method, a test for tumor cell transformation in soft agar, and apoptosis was determined by flow cytometry and DNA ladder methods. The study was performed on mouse epidermal JB6 Cl 41 cells, human JY lymphoblasts, COX2-deficient and normal mouse embryonic fibroblasts (MEF), as well as some human tumor cell lines including lung cancer (HT460), colon cancer (HCT) and melanoma (SK- MEL-28). The carcinopromotor effect of epidermal growth factor (EGF) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was significantly reduced with the use of non-toxic doses of glabruquinone A or other patented compounds.

In addition to those already mentioned, the following definitions were used, if others are not described: alkyl, alkoxy, etc., denoting both branched and unbranched groups, but a reference to such a radical as “propyl” means only an unbranched radical, and “isopropyl” refers only to a branched radical.

Basically, the term “isolated and purified” means that the compound is substantially purified from biological material (eg, blood, tissues, cells, plant material, etc.). In the framework of the patent, in one case, this means that the substance is purified by 75%, in the other by at least 90%, in the third by 98 or, finally, by 99% of the biological material. In other cases, the term refers to synthesized compounds.

The term "treatment" refers to both therapeutic and prophylactic treatment, when the goal is to prevent or reduce unwanted physiological changes or disorders, such as the development and spread of cancer. Desirable clinical outcomes include, but are not limited to, alleviating symptoms, limiting the spread of the disease, stabilizing the condition of the disease, limiting or slowing the progress of the disease, improving or temporarily alleviating the disease state, and remission (partial or complete), whether or not definable. “Treatment” may also mean prolonging survival when compared to expected survival if untreated. Clinical results, if necessary, treatment include the results of an existing condition or disorder, as well as propensity for this condition or disorder, or the results in which this condition or disorder is prevented.

The terms “cancer” or “cancerous” refer to the physiological state of a mammal in which uncontrolled cell growth is characteristic. "Cancer" may include one or more tumor lines. A list of cancers, such as, for example, skin cancer, is included in US Pat. No. 6,833,373.

The specific and preferred meanings listed below for radicals, substituents and ranks are used for illustration only, they do not exclude other specific meanings or other meanings within certain ranks for radicals and substituents.

Specifically, (C ₁ -C ₆ ) alkyl may be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C ₁ -C ₆ ) alkoxy may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 3-pentoxy or hexyloxy.

The process for preparing patentable compounds is given in the following examples of the invention and is illustrated by procedures in which radicals are designated as described above, unless otherwise qualified.

In those cases where the patented compounds are strong enough bases or acids to form stable non-toxic salts, the introduction of substances in the form of salts may be preferred. Examples of pharmaceutically acceptable salts are organic acid salts which are formed with acids forming a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. inorganic salts can also be prepared, including hydrochloride, sulfate, nitrate, bicarbonate and carbonate.

Pharmaceutically acceptable salts can be prepared using standard, well-known procedures, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to give a physiologically acceptable anion. Salts of alkali metals (e.g. sodium, potassium or lithium) or alkaline earth metals (e.g. calcium) and organic acids can also be obtained.

Patentable compounds can also be formulated as pharmaceutical compositions and administered to a mammal, for example a human, in various forms adapted to the chosen route of administration, i.e. orally or parenterally, intravenously, intramuscularly, topically or subcutaneously.

Thus, the compounds provided can be systemically administered, for example orally, in combination with a pharmaceutically acceptable excipient, such as an inert solvent or an edible carrier. They can be in the form of hard or soft gelatin capsules, can be formed into tablets, or can be incorporated directly into the patient’s food as a dietary supplement. For oral therapeutic use, the active substance may be combined with one or more inert excipients and used in the form of swallowable tablets, cheek tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the active substance. The percentage of active substance in the compositions and preparations can, of course, vary and usually can be from 2 to 60% of the weight of a unit dosage form. The amount of active substance must be such that an effective dose level is achieved.

Tablets, pills, capsules, etc. may also contain the following: binders such as gum, acacia, gelatin; fillers such as calcium phosphate; disintegrants, for example starch, alginic acid; lubricants such as magnesium stearate; sweeteners, for example sucrose, fructose, lactose or aspartame; or perfumes, such as mint, wintergreen, cherries. When the dosage form is in the form of a capsule, it may contain, in addition to the above, a liquid carrier, such as vegetable oil or polyethylene glycol. Various materials may be used as shells or other modifications to the solid dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar and the like. The syrup or elixir may contain the active substance, sucrose or fructose as a sweetener, methyl or propyl parabens as antioxidants, colorants and perfumes, such as cherry or orange aromas. Of course, any material used for the preparation of dosage forms must be pharmaceutically acceptable and non-toxic in the quantities used. In addition, the active substance can be placed in a preparation or device, from where it will enter the body of a mammal by continuous long-term release.

The active substance may also be administered intravenously or intraperitoneally by injection or injection. Solutions of the active substance or its salts can be prepared in water, to which a surfactant can be added. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations may contain antioxidants to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection or injection may include sterile aqueous solutions or dispersions or sterile powders, including the active ingredient, adapted for sterile injection or injection, and optionally encapsulated in liposomes. In any case, the final dosage form must be sterile, liquid, and stable under the conditions of preparation and storage. The liquid carrier may be a solvent, or a liquid dispersion medium containing, for example, water, ethanol, a polyol (e.g. glycerin, propylene glycol, liquid polyethylene glycols, etc.), vegetable oils, non-toxic glycerol esters, and their suitable mixtures. Suitable viscosity of the liquid can be achieved, for example, by forming liposomes or by obtaining particles of the desired size in the case of dispersions or by using surfactants. Prevention of exposure to microorganisms can be achieved by the use of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosol, etc. In many cases, it is preferable to include isotonic agents, for example sugars, buffers or sodium chloride. Prolonged absorption of the injected compounds can be achieved by the use of compositions of agents delaying absorption, for example aluminum monostearate or gelatin.

Sterile injectable solutions are prepared by incorporating the active substance in the required amount of a suitable solvent with the various other ingredients listed above, if necessary, followed by sterilization by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum and lyophilic drying, which give the powder of the active ingredient plus any desired additional ingredient present in pre-sterilized solutions, are preferred methods of preparation.

For topical use, the compounds can be used in pure form, if it is a liquid. However, it may be desirable to apply them to the skin in the form of compositions, in combination with a dermatologically acceptable carrier, which may be solid or liquid.

Used solid carriers include finely ground solids, for example talc, clay, microcrystalline cellulose, silica gel, aluminum, etc. Used liquid carriers include water, alcohols, glycols, or mixtures of water with glycols in which the active substances can be dissolved or dispersed, possibly with the use of non-toxic surfactants. Aids such as perfumes and additional antimicrobial agents can be added to optimize the properties of the dosage form. The resulting liquid compositions can be applied with absorbent aids, such as bandages and other dressings or in the form of sprays on the affected area. Thickeners, for example synthetic polymers, fatty acids, salts and esters of fatty acids, aliphatic alcohols, modified celluloses or modified mineral materials can also be used together with liquid carriers to form pastes, gels, ointments, soaps, etc., for use directly on the skin the patient.

Examples of dermatological compositions used to apply patentable compounds directly to the skin are well known, for example, see Jacquet et al. (US Pat. No. 4,608,392), Geria (US Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Used doses of patentable compounds can be determined by comparing their in vitro and in vivo activities in animal models. Methods of extrapolating effective doses obtained for mice and other animals to humans are also well known; see, for example, US Pat. US No. 4,938,949.

The amount of the compound or active salt or derivative thereof required for use in the treatment will vary not only depending on the type of salt selected, but also depending on the chosen route of administration, the conditions of administration, the age and condition of the patient, and will ultimately Depend on the discretion of the doctor.

The desired dose may be presented in a single dose, or may be administered in multiple doses at predetermined intervals, for example two, three, four or more sub-doses per day. Subdoses themselves can also be divided into a number of discrete introductions, with free gaps between them; it can be, for example, numerous inhalations or eye drops.

Thus, it is an object of the present invention to provide industrially applicable and effective use of mono- or non-methoxylated di- or tri-prenylbenzoquinones (substances of formulas 1, 3, 5-14) or the corresponding hydroquinones (substances of formulas 2, 4, 15) as anticancer preventive and therapeutic components of medical or cosmetic preparations for the treatment of any mammals, and as scientific tools for the study of AP-1 or NF-κB proteins.

Patentable substances can be obtained by isolating them from natural sources (marine invertebrates) or by chemical synthesis. Many of the patented compounds are unique, since methoxyl groups are present in their structure, and the polyprenyl side chains are shorter when comparing the patented compounds with ubiquinones also having polyprenyl side chains. For example, glabruquinone A (formula 5) (or, in other words, demethylubiquinone Q2) differs from ubiquinones in the absence of a methyl group in the aromatic nucleus and the presence of a shorter side chain.

The invention is further illustrated by the following examples.

Main procedures

¹ H and ¹³ C NMR spectra were recorded on Bruker WM-250 spectrometers at 250 and 62.9 MHz respectively and Broker DPX 300 at 300 and 75 MHz, respectively. High resolution mass spectra (HREIMS) were obtained on an AMD-604S mass spectrometer. The substances were purified by high performance liquid chromatography (HPLC) using a DuPont 8800 chromatograph equipped with a refractometer detector and an Ultrasphere Si silica gel column. IR spectra were recorded on a Bruker FT-IR "Vector 22" spectrometer. UV spectra were recorded in CCLl ₄ on a Cecil CE 7200 spectrophotometer. The onset of apoptosis was analyzed by flow cytometry using a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The MTS cytotoxicity test was performed using a Multiscan MS plate reader spectrophotometer (Labsystems, Finland). Soft agar cell colonies were counted using an Leica DM IRB inverted microscope (Leica Mikroskopie und Systeme GmbH, Germany) and Image-Pro Plus computer software, version 3.0 for Windows (Media Cybernetics, Silver Spring, MD). Chemiluminescence in the method for determining p53, AP-1, or NF-kB-dependent transcriptional activity was measured using a Luminoscan Ascent Type 392 plate reader (Labsystems, Finland).

Reagents

Nutrient media MEM and DMEM were from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Bovine embryo serum (FBS) was from Gemini Bio-Products (Calabasas, USA). Penicillin / streptomycin and gentamicin were from Bio-Whittaker (Walkersville, MD, USA), L-glutamine was from Mediatech, Inc. (Herndon, Virginia, USA). Epidermal Growth Factor (EGF) was from Collaborative Research (Bedford, MA, USA). The substrate for the luciferase method and the MTS reagent for the cytotoxicity assay were from Promega (Madison, WA, USA). Annexin V-FITC Apoptosis Detection Kit was an apoptosis flow cytometry kit from Medical & Biological Laboratories (Watertown, MA). Silica gel L (40/100 μm) for low pressure column chromatography was from Chemapol (Praha, Czech Republic). Silica gel plates for thin layer chromatography (4.5 × 6.0 cm, 5-17 μm) were from Sorbfil (Russian Federation).

Cell culture

The mouse epidermal cell lines JB6 P ⁺ Cl 41 and its stable transfectants JB6 Cl 41-NF-κB, JB6 Cl 41-AP-1, JB6 Cl 41-p53 (PG-13) were cultured in a monolayer at 37 ° C and 5% CO ₂ in a MEM containing 5% FBS, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin. Mouse embryonic fibroblasts (MEFs) were grown at 37 ° C and 5% CO ₂ in DMEM containing 10% FBS, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin. Human lymphoblastoid JY cells were grown at 37 ° C and 5% CO ₂ in RPMI medium containing 10% FBS, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin. Human tumor cells, HCT-116, NT-460 and SK-MEL-28 were cultured at 37 ° C and 5% CO ₂ in RPMI medium (for NT-460), McCoy's (for HCT-116) or MEM (for SK- MEL-28) containing 10% FBS, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin.

Example 1. Preparation and isolation of patentable compounds.

Isolation of glabruquinones A (5) and B (6) from ascidium Aplidium glabrum.

Frozen ascidia were extracted with ethanol (1: 1 by weight), ethanol was evaporated and the aqueous residue was extracted with chloroform (1: 1 by volume). The chloroform layer was evaporated in vacuo to obtain a brown oily residue, which was chromatographed on a silica gel column using a 10: 1 hexane-ethyl acetate system (system A). Isolation was controlled by thin layer chromatography (TLC) in system A. R _{f of the} mixture of glabruquinones A (5) and B (6) under these conditions was approximately 0.35. After evaporation of the solvents from the appropriate fractions, a mixture of glabruquinones A (5) and B (6) was obtained, which was separated by HPLC on an Altex Ultrasphere Si silica gel column (4.6 mm × 25 cm), in a hexane-ethyl acetate system, 7: 1 (system B).

Synthesis procedure

The main description.

The synthesis of polyprenylbenzoquinones 5-14, 16-21 and hydroquinones 15, 22-34 was carried out according to the scheme depicted in Figure 3. It involves the alkylation of commercially available phenols with geraniol or farnesole under acid catalysis conditions, followed by oxidative demethylation of the obtained phenylated phenols and reduction of the desired benzoquinones to the corresponding hydroquinones. Glabruquinones A (5), B (6) and a number of analogues (7-34) were obtained. Analogs 7-34 differ from glabruquinones A and B in the number and / or position of methoxy groups and / or the structure and number of polyprenyl side chains or the presence of a hydroquinone fragment instead of benzoquinone.

Synthesis of benzoquinones 5-14, 16-21.

The best results were obtained by alkylation of 35-37 phenols with trans-geraniol (38) or trans-farnesole (39) in the presence of boron trifluoride etherate as an acid catalyst (Figs. 4-6). Under these conditions, the total yield of alkylation products was about 60%. Oxidative demethylation of the obtained fractions of cerium ammonium nitrate (CAN) gave a mixture of the corresponding benzoquinones (5-13, 16-21), after the division of which individual compounds were obtained. Prenylbenzoquinone 14 was synthesized from unsubstituted hydroquinone monomethyl ether (60) via intermediate 2-geranyl or 2-farnesyl-4-methoxyphenols (61, 62, not shown in the diagram).

Stage 1

Boron trifluoride etherate (0.5 ml) was added with stirring to a mixture of 1 mmol of the corresponding phenol (35, 36 or 37) and 4 mmol of geraniol (38) or farnesol (39) in 10 ml of absolute ether. The mixture was kept at room temperature for 12 hours, after which 30 ml of water was added and extracted with ether (3 × 15 ml). The extract was washed with 10% NaCl and dried over Na ₂ SO ₄ . The solvent was removed and the residue was partitioned on a silica gel column. A mixture of prenylated quinones 40, 42, 44 or 41, 43, 45 (Figure 4), 46, 48, 50 or 47, 49, 51 (Figure 5), or 52, 54, 56, 58 or 53, 55, 57, 59 (FIG. 6), 61 or 62 (not shown in the diagram) were eluted in a hexane: acetone gradient system, 50: 1 → 20: 1. These mixtures were used in stage 2 of the reaction. The average yield was about 60%. In some cases, individual pre-phenylated phenols were isolated by HPLC on an Altex Ultrasphere Si column (4.6 mm × 25 cm) in system B. In this way, individual prenylated phenols 40, 41, 46, 51 and 61 were obtained, which were used for ¹ H NMR analysis.

Stage 2

A solution of CAN (0.9 mmol) in 3 ml of a mixture of CH ₃ CH: H ₂ O (1: 1) was added with stirring to a cooled (0 ° C) solution of phenolic fractions 40, 42, 44 or 41, 43, 45 (Figure 4 ), 46, 48, 50 or 47, 49, 51 (Fig. 5), 52, 54, 56, 58 or 53, 55, 57, 59 (Fig. 6), 61 or 62 (0.3 mmol) in 7 ml CH ₃ CN. After stirring at 0 ° C for 1-2 hours, the mixture was poured into 25 ml of 10% NaCl and extracted with ether (3 × 15 ml). The extract was dried over Na ₂ SO ₄ and evaporated. The corresponding benzoquinones 5, 17 and 7, 20 (FIG. 4), 11, 9, 21 or 12, 13, 18 (FIG. 5), 9, 10, 11, 19 or 8, 12, 13, 16 (FIG. 6), or 14 (not shown in the diagram) were obtained by thin-layer chromatography on silica gel in the hexane: acetone system (8: 1). Each benzoquinone (5, 7-14, 16-21) contained as an impurity (less than 6%) the corresponding 2'-3'- or 2``-3 '' - cis isomer. The formation of these isomers can be explained by the isomerization of geraniol and / or farnesol under the action of boron trifluoride etherate at the phenyl phenylation stage. As a rule, the cis isomer impurity was not separated from the main product (trams isomer), with the exception of mixtures 5 and 6, which were separated by HPLC. A comparison of actions 5 and 6 showed that they have the same level of activity. This means that for practical use it is not necessary to separate these products. The yield of target products at this stage was about 70%. The total yield after two stages was approximately 45%.

Synthesis of hydroquinones 15, 22-34 (The main method)

A solution of Na ₂ S ₂ O ₄ (3 mmol) in 3 ml of water was added to a solution of 1 mmol of the corresponding benzoquinone (5, 7-14, 16-21) in 7 ml of acetone. The mixture was stirred for one hour, diluted with water and extracted with ether (3 × 15 ml). The extract was dried over Na ₂ SO ₄ and evaporated. As a result, hydroquinones 15, 22-34 containing a small amount of impurities (less than 6%) of 2'-3 'cis or 2''-3''cis isomers were obtained.

Structures of glabruquinones A (5) and B (6).

The electron impact mass spectra (EIMS) of glabruquinones A (5) and B (6) contained, in addition to the molecular ion peak at m / z 304, a peak characteristic at benzoquinones at m / z 306 (M + 2). UV spectra showed an absorption maximum at 264 nm (ε = 15000), and IR spectra showed an absorption at 1675, 1657 and 1603 cm ^-1 , which confirms the presence of the p-benzoquinone part of the molecule.

^{The 1} H NMR spectrum 5 was very similar to the spectrum of verapliquinone A from Aplidium sp., But was distinguished by the presence of an additional singlet at 4.02, characteristic of the OMe group. Quartets at 61.2 and 61.3 ppm and singlets at 145.2 and 144.9 ppm the ¹³ C NMR spectrum indicated the presence of two methoxy groups in 5. This meant 5,6- or 3,5-dimethoxy substitution in the 1,4-benzoquinone part of the molecule. The attachment of the terpenoid part at C-2 of the benzoquinone part and the 5,6-position of methoxy groups was confirmed by analysis of NMR data, including ¹ H- ¹ H COSY, NOESY and NMVS (Figure 7). The multiplicity of H-3 (narrow triplet at 6.34 ppm) and the cross-peaks of H-3 / H-1 'in ¹ H- ¹ H COZY and H-3 / H-1' interactions in the NOESY spectrum were very indicative . The signals of two trisubstituted double bonds and three methyl groups attached to these double bonds in the ¹³ C NMR spectrum confirmed the presence of a diprenyl side chain.

Comparison of NMR spectra 5 and 6 showed that glabruquinones A and B contain geranyl and non-aryl types of side chains, respectively (Table 1; also see Shubina et.al., Tetrahedron Letters, 46, 559-562 (2005)). Indeed, these compounds differ from each other in the configuration of the C2 ′, C3′-double bond, which follows from the chemical shift values of C10 ′. In more sterically hindered. The E-isomer shows this signal in a stronger field than the Z-isomer. The chemical shift values of С-10 'in the spectra of compounds 5 and 6 differ significantly and are equal to 16.2 and 22.8 ppm. respectively. The structures of glabruquinones A and B differ from the structures of verapliquinones A and B by the presence of an additional methoxy group at position 5. Structures 5 and 6 were confirmed by synthesis, as described above.

Trimethoxyphenol 35 was obtained from commercially available 2,3,4-trimethoxybenzaldehyde as previously described (Matsumoto, et. Al., 1984.) Acid-catalyzed alkylation of phenol 35 with trans-geranyl bromide gave trans-geranyl phenol 40 with an admixture of cis isomer ( total yield 18%, trans: cis ratio = 95: 5). Phenylated phenol 40 was purified by HPLC and its structure was determined by analyzing ¹ H NMR spectra and comparing them with the spectra of glabruquinones 5 and 6. Oxidative demethylation 40 led to a mixture of benzoquinones 5 and 6 (14%). A mixture of synthetic 5 and 6 was separated by HPLC, and synthetic 5 and 6 were identified with glabruquinones A and B by comparing their NMR spectra.

Table 1 ¹³ C and ¹ H NMR spectra of glabruquinones A (5) and B (6) 5 6 Atom ¹³ C ¹ H ^{13 s 1} H one 184.38 * s 184.38 * s 2 146.92 s 146.92 s 3 130.45 d 6.34t, 1H, J = 1.70 130.45 d 6.37t, 1H, J = 1.71 four 184.54 * s 184.54 * s 5 145.16 ** s 145.16 ** s 6 144.91 ** s 144.91 ** s one' 27.17 t 3.10 dd, 2H, J = 7.32, 1.70 27.27 t 3.11 br.d, 2H, J = 7.08, 1.22 2 ' 117.78 d 5.13 t sextet, 1H, J = 7.32, 1.22, 1.22 117.78 d 5.13 m, 1H 3 ' 140.17 s 140.17 s four' 39.72 t 2.08 m, 2H 32.01 t 2.04 m, 2H 5' 26.52 t 2.09 m, 2H 26.52 t 2.04 m, 2H 6 ' 123.98 d 5.08 t septet, 1H, J = 6.83, 1.22, 1.22 123.98 d 5.07 m, 1H 7 ' 131.95 s 131.95 s 8' 25.77 q 1.70 d, 3H, J = 1.22 25.77 q 1.66 br.d, 3H, J = 1.22 9' 17.79 q 1.60 br.s, 3H 17.79 q 1.59 d, 3H, J = 1.22 10' 16.2 q 1.62 d, 3H, J = 1.22 22.76 q 1.75 q 3H, J = 1.22 OCH ₃ 61.3 q; 61.2 q 4.00 s; 4.02 s 4.00 s; 4.02 s *, ** - values can be swapped.

Structures of synthetic polypropylene compounds.

The structures of the target prenyl benzoquinones and β-hydroquinones, as well as intermediate prenyl phenols, were determined using ¹ H-NMR spectra in comparison with the corresponding data for the structures of glabruquinones A (5) and B (6). The spectral characteristics of some synthetic analogues of glabruquinones 5 and 6, as well as some intermediate prenylphenols, are given below.

Example 2

3-Demethylubiquinone Q2 or 2,3-dimethoxy-5- (3 ', 7'-dimethyl-octa-2' (E), 6'-dienyl) - [1,4] benzoquinone (5): yellow oil, HREIMS m / z 304.1655 [M] ⁺ , calculated for C ₁₈ H ₂₄ O ₄ 304.1675, IR (CHCl ₃ ): 1675, 1657, 1603. ¹ H-NMR (CDCl ₃ , 250 MHz) δ: 6.34 (t, J = 1.7, 1H, H-6); 5.13 (m, 1H, H-2 '); 5.08 (m, 1H, H-6 '); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.10 (dd, J = 7.3, 1.7, 2H, H-1 '); 2.09 (m, 2H, H-5 '); 2.08 (m, 2H, H-4 '); 1.70 (d, J = 1.2, 3H, H-8 '); 1.62 (d, J = 1.2, 3H, H-10 '); 1.60 (br. S, 3H, H-9 '). ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ: 16.20 (q, C-10 '), 17.79 (q, C-9'), 25.77 (q, C-8 '), 26.52 (t, C-5 '), 27.17 (t, C-1'), 39.72 (t, C-4 '), 61.20 (q, OMe), 61.30 (q, OMe), 117.78 (d, C-2'). 123.98 (d, C-6 '), 130.45 (d, C-6), 131.95 (s, C-7'), 140.17 (s, C-3 '), 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 Cl), 184.54 (s, C-1 or C-4).

Example 3

2,3-dimethoxy-5- (3 ', 7'-dimethyl-octa-2' (Z), 6'-dienyl) - [1,4] benzoquinone (6): yellow oil, HREIMS m / z 304.1662 [ M] ⁺ , calculated for C ₁₈ H ₂₄ O ₄ 304.1675, ¹ H-NMR (CDCl ₃ , 250 MHz) δ: 6.37 (t, J = 1.7, 1H, H-6); 5.13 (m, 1H, H-2 '); 5.07 (m, 1H, H-6 '); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.11 (br. D, J = 7.1, 1.2, 2H, H-1 '); 2.04 (m, 2H, H-5 '); 2.04 (m, 2H, H-4 '); 1.75 (q, J = 1.2, 3H, H-10 '); 1.66 (br. D, J = 1.2, 3H, H-8 '); 1.59 (d, J = 1.2, 3H, H-9 '). ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ: 17.79 (q, C-9 '), 22.76 (q, C-10'), 25.77 (q, C-8 '), 26.52 (t, C-5 '), 27.27 (t, C-1'), 32.01 (t, C-4 '), 61.20 (q, OMe), 61.30 (q, OMe), 117.78 (d, C-2'), 123.98 (d , C-6 '), 130.45 (d, C-6), 131.95 (s, C-7'), 140.17 (s, C-3 '), 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-1), 184.54 (s, C-1 or C-4).

Example 4

2-Methoxy-3- (3 ', 7'-dimethyl-octa-2', 6'-dienyl) - [1,4] benzoquinone (9): yellow oil, HREIMS m / z 274.1558 [M] ⁺ , calculated for C ₁₇ H ₂₂ O ₃ 274.1569, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.68 (d, J = 10.0, 1H, H-5); 6.59 (d, J = 10.0, 1H, H-6); 5.05 (m, 2H, H-2 ', H-6'); 4.02 (s, 3H, OMe); 3.15 (br. D, J = 7.3, 2H, H-1 '); 2.01 (m, 4H, H-4 ', H-5'); 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 (10): yellow oil, HREIMS m / z 274.1576 [M] ⁺ , calculated for C ₁₇ H ₂₂ O ₃ 274.1569, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.45 (q, J = 2.1, 1H, H-5); 5.87 (d, J = 2.4, 1H, H-6); 5.15 (m, 1H, H-2 '); 5.08 (m, 1H, H-6 '); 3.82 (s, 3H, OMe); 3.14 (br. D, J = 7.3, 2H, H-1 '); 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-Methoxy-5- (3 ', 7'-dimethyl-octa-2', 6'-dienyl) - [1,4] benzoquinone (11): yellow crystals, HREIMS m / z 274.1582 [M] ⁺ , calculated for C ₁₇ H ₂₂ O ₃ 274.1569, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.46 (t, J = 1.7, 1H, H-6), 5.92 (s, 1H, H-3), 5.15 ( m, 1H, H-2 '), 5.08 (m, 1H, H-6'), 3.82 (s, 3H, OMe), 3.14 (br. d, J = 7.3, 2H, H-1 '), 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) - [1,4] benzoquinone (14): yellow oil, HREIMS m / z 244.1454 [M] ⁺ , calculated for C ₁₆ H ₂₀ O ₂ 244.1463, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.77 (d, J = 10.2, 1H, H-6); 6.69 (dd, J = 10.2, 2.3, 1H, H-5); 6.53 (q, J = 1.9, 1H, H-3); 5.15 (m, 1H, H-2 '); 5.07 (m, 1H, H-6 '); 3.13 (br. D, J = 7.5, 2H, H-1 '); 2.08 (m, 4H, H-4 ', H-5'); 1.69 (br. S, 3H, Me); 1.62 (br. S, 3H, Me); 1.60 (br. S, 3H, Me).

Example 8

2,3-Dimethoxy-5- (3 ', 7', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) - [1,4] benzoquinone (7): yellow oil, HREIMS m / z 372.2316 [M] ⁺ , calculated for C ₂₃ H ₃₂ O ₄ 372.2300, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.34 (t, J = 1.8, 1H, H-6); 5.10 (m, 3H, H-2 ', H-6', H-10 '); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.11 (br. D, J = 7.3, 2H, H-1 '); 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 9

2-Methoxy-6- (3 ', 7', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) - [1,4] benzoquinone (8): yellow oil, HREIMS m / z 342.2182 [M] ⁺ , calculated for C ₂₂ H ₃₀ O ₃ 342.2195, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.45 (q, J = 2.0, 1H, H-5); 5.87 (d, J = 2.4, 1H, H-3); 5.15 (m, 1H, H-2 '); 5.10 (m, 2H, H-6 ', H-10'); 3.81 (s, 3H, OMe); 3.14 (br. D, J = 7.3, 2H, H-1 '); 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', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) - [1,4] benzoquinone (12): yellow crystals, HREIMS m / z 342.2172 [M] ⁺ , calculated for C ₂₂ H ₃₀ O ₃ 342.2195, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.47 (t, J = 1.7, 1H, H-6); 5.93 (s. 1H, H-3); 5.16 (m, 1H, H-2 '); 5.10 (m, 2H, H-6 ', H-10'); 3.82 (s, 3H, OMe); 3.14 (br. D, J = 7.3, 2H, H-1 '); 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', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) - [1,4] benzoquinone (13): yellow oil, HREIMS m / z 342.2212 [M] ⁺ , calculated for C ₂₂ H ₃₀ O ₃ 342.2195, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.68 (d, J = 10.0, 1H, H-6); 6.57 (d, J = 10.0, 1H, H-5); 5.07 (m, 3H, H-2 ', H-6', H-10 '); 4.02 (s, 3H, OMe); 3.16 (br. D, J = 7.3, 2H, H-1 '); 2.01 (m, 8H, H-4 ', H-5', H-8 ', H-9'); 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

2,3-Dimethoxy-5- (3 ', 7', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) benzene-1,4-diol (15): yellow oil, HREIMS m / z 374.2472 [M] ⁺ , calculated for C ₂₃ H ₃₄ O ₄ 374.2457, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.49 (s, 1H, H-6); 5.31 (s, 1H, OH); 5.30 (m, 1H, H-2 '); 5.17 (s, 1H, OH); 5.12 (m, 2H, H-6 ', H-10'); 3.91 (s, 3H, OMe); 3.88 (s, 3H, OMe); 3.28 (br. D, J = 7.6, 2H, H-1 '); 2.05 (m, 8H, H-4 ', H-5', H-8 ', H-9'); 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'-dienyl) - [1,4] benzoquinone (17): yellow oil, HREIMS m / z 440.2944 [M] ⁺ , calculated for C ₂₈ H ₄₀ O ₄ , 440.2927, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 5.04 (m, 2H, H-2 ', H-2``); 4.94 (m, 2H, H-6 ', H-6``); 3.99 (s, 6H, 20Me); 3.19 (br. D, J = 6.8, 2H, H-1 ', H-1``); 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

2-Methoxy-5,6-bis- (3 ', 7', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) - [1,4] benzoquinone (16): yellow oil, HREIMS m / z 546.4048 [M] ⁺ , calculated for C ₃₇ H ₅₄ O ₃ , 546.4073, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 5.87 (s, 1H, H-3); 5.00 (m, 6H, H-2 '. H-6', H-10 ', H-2``, H-6'',H-10``); 3.79 (s, 3H, OMe); 3.22 (br. D, J = 6.8, 4H, H-1 ', H-1``); 2.01 (m, 16H, H-4 ', H-5', H-8 ', H-9', 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', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) - [1,4] benzoquinone (18): yellow oil, HREIMS m / z 546.4052 [M] ⁺ , calculated for C ₃₇ H ₅₄ O ₃ , 546.4073, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.33 (t, J = 1.2, 1H, H-6); 5.09 (m, 6H, H-2 ', H-6', H-10 ', H-2``, H-6'',H-10``); 4.00 (s, 3H, OMe); 3.14 (m, 4H, H-1 ', H-1``); 2.04 (m, 16H, H-4 ', H-5', H-8 ', H-9', 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'-dienyl) phenol (40): yellow opalescent oil, HREIMS m / z 320.1974 [M] ⁺ , calculated for C ₁₉ H ₂₈ O ₄ 320.1987, IR (CCl ₄ ): 3541, 2935, 1498, 1464 cm ^-1 . ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.44 (s, 1H, H-5), 5.45 (s, 1H, OH), 5.31 (m, 1H, H-2 '), 5.11 (m, 1H , 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-1' ), 2.07 (m, 4H, Н-4 ', Н-5'), 1.72 (d, J = 1.2, 3Н, Me), 1.67 (d, J = 1.2, 3Н, Me), 1.60 (d, J = 0.7. 3H, Me). ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ: 16.12 (q, C-10 '), 17.66 (q, C-9'), 25.66 (q, C-8 '), 26.73 (t, C-5 '), 27.90 (t, C-1'), 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-1), 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', 11'-trimethyl-dodeca-2 ', 6', 10'-trienyl) phenol (41): yellow opalescent oil, HREIMS m / z 388.2648 [M] ⁺ , calculated for C ₂₄ H ₃₆ O ₄ 388.2635, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.44 (s, 1H, H-5); 5.45 (s, 1H, OH); 5.32 (br. T, J = 7.3, 1H, H-2 '); 5.12 (m, 2H, H-6 ', H-1``); 3.95 (s, 3H, OMe); 3.87 (s, 3H, OMe); 3.79 (s, 3H, OMe); 3.31 (br.d, J = 7.3, 2H, H-1 '); 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 (46): yellow opalescent oil, HREIMS m / z 290.1876 [M] ⁺ , calculated for C ₁₈ H ₂₆ O ₃ 290.1882, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.73 (s, 1H, H-2); 6.49 (s, 1H, H-5); 5.27 (br t, J = 7.3, 1H, H-2 '); 5.15 (s, 1H, OH); 5.10 (m, 1H, H-6 '); 3.87 (s, 3H, OMe); 3.79 (s, 3H, OMe); 3.24 (br. D, J = 7.3, 2H, H-1 '); 2.05 (m, 4H, H-4 ', H-5'); 1.68 (br. S, 6H, 2Me); 1.60 (br. S, 3H, Me).

Example 19

2,4-Dimethoxy-6- (3 ', 7'-dimethyl-octa-2', 6'-dienyl) phenol (52): yellow opalescent oil, HREIMS m / z 290.1898 [M] ⁺ , calculated for C ₁₈ H ₂₆ O ₃ 290.1882, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.36 (d, J = 2.7, 1H, H-3 or H-5); 6.31 (d, J = 2.7, 1H, H-3 or H-5); 5.33 (br. T, J = 7.1, 1H, H-2 '); 5.26 (s, 1H, OH); 5.11 (br. T, J = 6.6, 1H, H-6 '); 3.86 (s, 3H, OMe); 3.75 (s, 3H, OMe); 3.35 (br. D, J = 7.3, 2H, H-1 '); 2.08 (m, 4H, H-4 ', H-5'); 1.72 (br. S, 3H, Me); 1.67 (br. S, 3H, Me); 1.60 (br. S, 3H, Me).

Example 20

4-Methoxy-6- (3 ', 7'-dimethyl-octa-2', 6'-dienyl) phenol (61): yellow opalescent oil, ¹ H-NMR (250 MHz, CDCl ₃ ) δ: 6.69 ( m, 3H, H-3, H-5, H-6); 5.31 (br. T, J = 7.32, 1H, H-2 '); 5.07 (m, 1H, H-6 '); 3.75 (s, 3H, OMe); 3.33 (br. D, J = 7.08, 2H, H-1 '); 2.09 (m, 4H, H-4 ', H-5'); 1.76 (br. S, 3H, Me); 1.68 (br. S, 3H, Me); 1.60 (br. S, 3H, Me).

Example 21. Cytotoxic activity

To evaluate the cytotoxic effect of a representative sample of patented compounds, the MTS method was used. The effect of a representative sample of patentable compounds on the cell survival of JB6 P ⁺ Cl 41 cells, transfect JB6 cells with NF-κB luciferase promoter, transfect JB6 cells with AP-1 luciferase promoter and human lung cancer cells NT-460 was investigated. Quinones 5, 9-11, 14, having a terpenoid part consisting of two isoprene units (or 10 carbon atoms), showed IC ₅₀ for JB6 Cl 41 cells in the concentration range from 8.3 to 45.1 μM, and for NT- 460 cells - from 25.8 to 81.8 μM. Quinones 7, 8, 12, 13, 15, having a terpenoid part containing 15 carbon atoms (or three isoprene units), showed IC ₅₀ for JB6 Cl 41 cells in the concentration range from 3.6 to 29.0 μM, and for NT -460 cells - from 12.1 to 72.0 μM. The average IC _{50 of} quinones 5, 9-11, 14 for JB6 Cl 41 cells was 18.7 μM, and for NT-460 cells 44.2 μM. The average IC ₅₀ value of quinones 7, 8, 12, 13, 15 JB6 Cl 41 cells was 10.4 uM and for NT-460 cells - 30.5 .mu.M. Therefore, quinones 7, 8, 12, 13, 15, characterized by a longer terpenoid part, are almost two times more toxic for JB6 Cl 41 cells and one and a half times more toxic for NT-460 cells than quinones 5, 9-11, 14 having a short terpenoid part. Both of these groups of compounds are approximately two times more toxic for JB6 Cl 41 cells (15.9 μM on average) than for HT-460 cells (37.8 μM on average). The third group of compounds, quinones 16-18, having two terpenoid side chains in the molecule, showed the lowest cytotoxic activity for both JB6 Cl 41 and HT-460 cells, among all three groups of compounds. The cytotoxic effect of quinones 5, 7-18 in relation to human lung cancer cells (NT-460) and mouse epidermal JB6 Cl 41 cells has not been previously published by anyone.

The effect of glabruquinone A (5) on cell survival was evaluated using the reduction of the MTS reagent to formazan (CellTiter 96 ^R AQ _ueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA). JB6 P ⁺ Cl 41 or HT-460 cells were cultured for 12 hours in 96-well plates (4000 cells per well) using 5% FBS / MEM (for JB6 P ⁺ Cl 41 cells) or 10% FBS / RPMI (for NT-460 cells). Then the medium was replaced with 0.1% FBS / MEM (for JB6 P ⁺ Cl 41 cells) or 0.1% FBS / RPMI (for NT-460 cells) containing glabruquinone A (5) in various concentrations, in a volume of 0 , 1 ml of medium per well, and cells were incubated with the substance for 22 hours. Then, 20 μl of the MTS reagent was added to each well and the absorption density of the recovery product of the MTS reagent, formazan, was measured two hours later, spectrophotometrically, at 492 nm, and also at 690 nm (as background), using a Multiscan plate reader spectrophotometer MS (Labsystems, Finland). The data are presented in FIGS. 8A and 8B as a percentage of live JB6 P ⁺ Cl 41 or HT-460 cells, respectively, compared to control, untreated cells. FIGS. 9A and 9B show IC ₅₀ calculated for JB6 P ⁺ Cl 41 or NT-460 cells, respectively, using linear regressions based on the data presented in FIGS. 8A and 8B.

Example 22. Test for tumor transformation of normal cells or for the phenotypic manifestation of cancer cells in soft agar.

A representative sample of patentable compounds inhibits phenotypic expression (colony formation) in soft agar of human lung cancer cells (NT-460), intestinal cancer (HCT-116), melanoma (SK-MEL-28). They also inhibit EGF or TPA-induced tumor transformation of JB6 P ⁺ Cl 41 cells in soft agar. The results of the corresponding experiments are presented in table 2 as the concentrations of the corresponding quinones (in μM), in which the tumor transformation of JB6 P ⁺ Cl 41 cells or the colony formation of human tumor cells is inhibited by 50% compared to untreated, control cells. Moreover, most of the tested substances 5, 7-18 significantly inhibit the tumor transformation of JB6 P ⁺ Cl 41 cells in concentrations lower than cytotoxic (see table 2).

The effect of quinones 5, 7-18 on the colony formation of human cancer cells NT-460, HCT-116, SK-MEL-28, as well as on the transformation of murine JB6 P ⁺ Cl 41 cells in soft agar was evaluated using the soft agar method. Inhibition of cell transformation is usually a good indicator that a substance will have cancer-preventive activity. The results obtained confirmed that a representative sample of patentable compounds has anti-cancer prophylactic and therapeutic activities (see table 2). In experiments on soft agar, quinones with side chains having a length of 10 carbon atoms showed anti-cancer activity against the EGF-induced transformation of JB6 P ⁺ Cl 41 cells with INCC ₅₀ (the concentration at which the inhibition of the number of colonies is 50% of the number of control cell substances) is 1.4-4 times less than the IC ₅₀ obtained for the same substances on the same cells in experiments on cytotoxic activity. On the other hand, quinones with a longer side chain of 15 carbon atoms showed anticancer preventive activity for the most part in doses that were equal to or even d 4-10 times higher than the IC ₅₀ values obtained in experiments on cytotoxic activity. Therefore, quinones having side chains 10 carbon atoms long are more powerful agents for preventing the transformation of normal cells into cancer cells than quinones with side chains 15 carbon atoms long (see Table 2). The same conclusion is true for the anti-cancer therapeutic activity of quinones, which were investigated on their effect against colony formation of NT-460 cells in soft agar and on cytotoxic activity against the same cells. Data on IC ₅₀ and INCC ₅₀ for quinones 5, 7-18 in these experiments were obtained on the basis of regressions constructed using the statistical computer program STATISTICA 6.0 (StatSoft, Inc., USA). IC ₅₀ means the concentration of quinone at which 50% of the treated cells die compared to untreated, control cells. INCC ₅₀ means the concentration of quinone at which the colony formation of the treated cells is inhibited by 50% compared with untreated, control cells.

Anticancer properties of a representative sample of patentable compounds for inhibition of colony formation of human cancer cells NT-460 (lung cancer), HCT-116 (intestinal cancer), SK-MEL-28 (melanoma, skin cancer), as well as inhibition of EGF- or TPA-induced the pro-cancer transformation of murine JB6 P ⁺ Cl 41 cells has not been previously published.

The anti-cancer prophylactic and therapeutic effect of glabruquinone A (5) was evaluated using the soft agar method. The experiments were carried out in 6-well plates. To assess the anti-cancer therapeutic effect, human intestinal cancer cells of the HCT-116 line were used. EGF (10 ng / ml) - or TPA (20 ng / ml) -activated JB6 P ⁺ Cl 41 cells were used to evaluate the anti-cancer prophylactic effect. The soft agar method is a widespread tool that is used to determine whether a given substance is a potentially effective anti-cancer agent for use in animals or humans. HCT-116 or JB6 P ⁺ Cl 41 cells (8 × 10 ³ per ml) were treated with the indicated concentrations of glabruquinone A in 1 ml BME (Needle medium) containing 0.33% agar and 10% FBS on top of 3.5 ml BME, containing 0.5% agar and 10% FBS, as well as the indicated concentrations of glabruquinone A. The experimental plates were incubated at 37 ° C and 5% CO ₂ in an incubator for 1 week (for HCT-116 or JB6 P ⁺ Cl 41 cells activated EGF) or 2 weeks (JB6 P ⁺ Cl 41 TPA-activated cells). Cell colonies were then counted using a LEICA DM IRB inverted microscope (Leica Mikroskopie und Systeme GmbH, Germany) and Image Pro Plus 3.0 software for Windows (Media Cybernetics, Silver Spring, MD, USA). The effects of glabruquinone A on colony formation of HCT-116 cells (Figure 10A) or on the EGF-induced transformation of JB6 P ⁺ Cl 41 cells (Figure 11A) or on the TPA-induced transformation of JB6 P ⁺ Cl 41 cells (Figure 12A) are presented in as a percentage of colony formation compared to untreated glabruquinone, control cells. FIGS. 10B, 11B, or 12B show INCC ₅₀ subtractions of glabruquinone A for colony formation of HCT-116 cells, EGF-induced transformation of JB6 P ⁺ Cl 41 cells, or TPA-induced transformation of JB6 P ⁺ Cl 41 cells, respectively, using data-based regressions Figa, 11A, 12A.

table 2 IC ₅₀ and INCC _{50 of the} presented quinones in relation to JB6 P ⁺ Cl 41 cells Connections IC ₅₀ , µM INCC ₅₀ , μM Structural Group 1 1. Quinone 5 11.4 7.3 2. Quinone 9 45.1 15.1 3. Quinone 10 8.3 6.6 4. Quinone 11 11.8 3.1 5. Quinone 14 23.4 14.7 Structural group 2 6. Quinone 7 5.1 19.4 7. Quinone 8 4.7 16.7 8. Quinone 12 8.6 7.4 9. Quinone 13 25.5 24.6 10. Quinone 15 4.6 51.7 Structural Group Z 11. Quinone 17 > 140 * 29.2 12. Quinone 16 > 15 * 54.7 13. Quinone 18 99.6 95.3 * all numbers used indicated numbers

Methods

The carcinative effect of the present compounds was evaluated in six-well plates using EGF (10 ng / ml) - or TPA (20 ng / ml) -activated JB6 P ⁺ Cl 41 cells. The soft agar method is a widespread tool that is used to determine whether a given substance is a potentially effective anti-cancer agent for use in animals or humans. JB6 P ⁺ Cl 41 cells (8 × 10 ³ per ml) were treated with the indicated concentrations of quinones in 1 ml of BME (Needle medium) containing 0.33% agar and 10% FBS over 3.5 ml of BME containing 0.5% agar and 10% FBS, as well as the indicated concentrations of quinones. The experimental plates were incubated at 37 ° C and 5% CO ₂ in an incubator for 1 week (for JB6 P ⁺ Cl 41 cells activated by EGF) or 2 weeks (for JB6 P ⁺ Cl 41 cells activated by TPA). Cell colonies were then counted using a LEICA DM IRB inverted microscope (Leica Mikroskopie und Systeme GmbH, Germany) and Image Pro Plus 3.0 software for Windows (Media Cybernetics, Silver Spring, MD, USA). Two independent experiments were performed for each compound, in triplicate for each concentration.

Example 23. Tests for apoptosis using flow cytometry and DNA ladder methods.

A representative sample of patentable compounds induces apoptosis in JB6 P ⁺ Cl 41 cells, human lymphoblasts, JY cells, COX2 ^{- / -} and COX2 ^{+ / +} MEF, as well as human cancer cells NT-460 (lung cancer), HCT-116 (intestinal cancer ) and SK-MEL-28 (melanoma, skin cancer). For example, the induction of apoptosis by quinones 5, 7-18 was evaluated by flow cytometry in murine JB6 P ⁺ Cl 41 cells. The induction of apoptosis by glabruquinone A was also investigated by flow cytometry in JY cells, as well as human cancer cells NT-460 (lung cancer), HCT-116 (intestinal cancer) and SK-MEL-28 (melanoma, skin cancer). The induction of apoptosis by glabruquinone A was also investigated by DNA ladder in COX2 ^{- / -} and COX2 ^{+ / +} MEPs (Fig. 14).

The ability of a representative sample of patentable compounds to induce apoptosis in JB6 P ⁺ Cl 41 cells, human lymphoblasts, JY cells, COX2 ^{- / -} and COEF2 ^{+ / +} MEF, as well as human cancer cells NT-460 (lung cancer), HCT-116 (cancer intestines) and SK-MEL-28 (melanoma, skin cancer) have not been previously published.

Induction of early and late apoptosis with glabruquinone A was analyzed on a Becton Dickinson FACs Calibur Flow Cytometer flow cytometer (BD Biosciences, San Jose, CA, USA). JB6 P ⁺ Cl 41, JY, COX2 ^{- / -} or COX2 ^{+ / +} . HT-460, HCT-116 or SK-MEL-28 cells (3 × 10 ⁵ cells per dish) were cultured in 6 cm plates for 24 hours in 10% FBS / MEM for JY and SK-MEL-28 cells, 5% FBS / MEM for JB6 P ⁺ Cl 41 cells, 10% FBS / RPMI for HT-460 or 10% FBS / McCoy's for HCT-116 cells. The cells were then treated with glabruquinone A in 0.1% FBS / medium for 3 hours for JB6 P ⁺ Cl 41, JY, HT-460, and SK-MEL-28 cells, or for 24 hours for HCT- 116 cells. Then, the cells (except for unattached JY) were trypsinized with 0.025% trypsin in 0.1% EDTA in PBS buffer. Trypsinization was stopped by the addition of 2 ml of 5% FBS in PBS buffer. Cells were washed with PBS buffer by centrifugation at 1000 rpm (170 rcf) for 5 min and processed to detect early and late apoptosis using Annexin V-FITC and PI (propidium iodide) fluorescent dyes according to protocol from manufacturer. Briefly: 1-5 × 10 ⁵ cells were collected after centrifugation and resuspended in 500 μl of binding buffer solution (Annexin V-FITC Apoptosis Detection Kit, Medical & Biological Laboratories (Waterton, MA, USA)). Then 5 μl Annexin V-FITC and 5 μl PI were added and the cells were incubated for 5 min in the dark, at room temperature, and then analyzed in a flow cytometer. Glopruquinone A-induced apoptosis in the above cells is shown in Figs. 13A, B, C, D, E.

COX2 ^{- / -} and COX2 ^{+ / +} MEF were grown in 10 cm Petri dishes and treated with glabruquinone A to achieve 80% confluency within 24 hours. Then, both attached and non-attached cells were collected by scraping, followed by centrifugation. Then, the cells were lysed with lysis buffer solution (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 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 reprecipitated overnight at -20 ° C, after adding two volumes of 100% ethanol and 1/10 volume of 5 M NaCl. The precipitated DNA was centrifuged at 14,000 rpm for 45 minutes, washed with 70% ethanol, dried and resuspended in TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). After adding 100 μg / ml RNAse A (Sigma), the mixture was incubated at 37 ° C for 2 hours. DNA fragments were separated by electrophoresis on a 1.8% agarose gel. The DNA ladder in the gel was stained with ethidium bromide and photographed in UV light. The result is shown in FIG.

Example 24. Test for AP-1 - and NF-κ-dependent transcriptional activity

Several key transcriptional nuclear factors, including p53, AP-1, or NF-κB, are usually involved in the induction or inhibition of apoptosis in the cell, in response to various external stimuli, including exposure to chemopreventive substances or anti-tumor drugs. Therefore, the effect of a representative sample of patentable compounds 5, 7-18 on these three key nuclear transcription factors was investigated. For this, JB6 Cl 41 cell lines with a stably expressed luciferase reporter gene controlled by p53, AP-1 and / or NF-κB-linked sequence were used. A representative sample of patentable compounds showed significant (up to 8 times) induction of AP-1 and / or NF-κB-dependent transcriptional activity and also significant (up to 4 times) inhibition of p53-dependent transcriptional activity (see table 3).

Table 3 The effect of the presented compounds 5, 7-18 on AP-1-, NF-κB-, and p53-dependent transcriptional activity in JB6 Cl41 cells Connections AP-1-dependent transcriptional activity,% in relation to the control NF-κB-dependent transcriptional activity, in% relative to the control p53-dependent transcriptional activity,% relative to control Structural Group 1 Quinone 5 190.3 216.9 71.7 Quinone 9 159.8 194.8 55.8 Quinone 10 293.3 197.9 36.3 Quinone 11 721.7 201.2 31.8 Quinone 14 880.7 421.7 47.0 Structural group 2 Quinone 7 125.0 99.2 24.6 Quinone 8 97.2 138.8 57.9 Quinone 12 252.2 223.0 26.9 Quinone 13 107.7 179.3 31.5 Quinone 15 84.0 403.5 22.8 Structural group 3 Quinone 17 139.5 225.7 * Quinone 16 * * * Quinone 18 198.5 549.7 36.5 *, significant differences were not observed.

The ability of a representative sample of patentable compounds to induce AP-1 and / or NF-κB-dependent transcriptional activity in murine JB6 Cl 41 cells has not been previously published. The results for glabruquinone A (5) are shown in FIGS. 15A, B, which depict AP-1 and / or NF-κB-dependent transcriptional activity, expressed as a percentage in relation to untreated control cells.

Methods The ability of the present compounds to influence AP-1, NF-κB, and p53-dependent transcriptional activity in murine JB6 Cl 41 cells was evaluated by the luciferase method. JB6-LucPG-13, JB6-LucAP-l or JB6-LucNF-kB cells (6 × 10 ³ ) suspended in 100 μl of 5% FBS-MEM were added to each well of a 96-well plate. The plates were incubated for 24 hours and then treated with various concentrations of quinones in 100 μl of 0.1% FBS / MEM. After incubation with quinones for 24 hours, the cells were extracted for 1 hour at room temperature with 100 μl per well of lysis buffer solution (0.1 M Na-phosphate buffer solution, pH 7.8, 1% Triton X-100, 1 mm DTT, 2 mm EDTA). Then 30 μl of the lysate from each well was transferred to a luminescent assay plate and luciferase activity was measured using 100 μl per well of luciferase test buffer solution (1 mM D-luciferin, pH 6.1-6.5; 40 mM tricine; 2.14 mM magnesium carbonate ( MgCO ₃ ) ₄ × Mg (OH) ₂ × 5H ₂ O; 5.34 mM MgSO ₄ × 7H ₂ O, 66.6 mM DTT, 1.06 mM ATP; 0.54 mM coenzyme A, 0.2 mM EDTA, pH 7.8) and a plate reader Luminoscan Ascent Type 392 (Labsystems, Finland). Two independent experiments were performed for each compound, five samples for each concentration.

Example 25. Anti-cancer preventive and therapeutic activity

The anti-cancer preventive and therapeutic activity of 3-demethylubiquinone Q2 (5) was studied using the modern method of magnetic resonance imaging on white nonlinear mice inoculated with Ehrlich carcinoma. After just one preventive or therapeutic injection of 3-demethylubiquinone Q2 (5), the size of the tumor was significantly (25-50%) reduced compared to a similar tumor in control, untreated mice. The anti-cancer preventive and therapeutic activity of 3-demethylubiquinone Q2 (5) against Ehrlich carcinoma was manifested at relatively non-toxic doses. This conclusion was confirmed by studying the toxicity of 3-demethylubiquinone Q2 for nonlinear white mice by the Karber method (G. Karber, Arch. Exp. Pathol. Pharm., 1931, v. 162, p. 480).

Toxicity of 3-demethylubiquinone Q2 (5) in mice.

Using the Karber method, it was shown that the LD _{100 of} glabruquinone A for mice was 60 mg / kg, while the LD ₅₀ was 35 mg / kg. The medical dose of glabruquinone A for mice was chosen equal to 30 mg / kg, a solution in 50% DMSO or 50% ethanol in water.

Magnetic resonance imaging (MRI).

MRI is a powerful and reliable method for research on animal models. Monitoring tumor growth or inhibition in response to in vivo chemotherapy is one of the most commonly used uses for MRI. The advantages of this method are the possibility of obtaining holistic images of the tumor in any projection on animal models and in the absence of radiation.

The study of the inhibition of a solid variant of Ehrlich carcinoma after treatment with 3-demethylubiquinone Q2 was carried out in vivo in white non-linear mice. 3-demethylubiquinone Q2 was injected into mice as a solution in a mixture of DMSO with water (1: 1) or ethanol with water (1: 1). After treatment with 3-demethylubiquinone Q2 in DMSO or ethanol with water, the mice lost about 0.5-1% of body weight. The size of Ehrlich solid carcinoma in mice treated with 3-demethylubiquinone Q2 prophylactically (one day before tumor inoculation) was significantly (≈ 50%) smaller compared to control untreated mice (Figs. 16 and 17). When 3-demethylubiquinone Q2 was used as a therapeutic drug and injected into mice one day after tumor inoculation, inhibition of tumor size was about 25% (Fig. 18). Thus, quinone (5) and other patentable compounds can be used as carcinogenic or therapeutic agents for the treatment of carcinomas.

In addition to measuring the size of the tumor, experimental mice also monitored the intravital state of the liver, spleen and thymus. No significant changes were found in the size, type or general condition of these immunocompetent organs in mice that were injected with 3-demethylubiquinone Q2, compared to control mice that were not treated. The same results were obtained after euthanasia, dissection of mice and measurement of these same organs.

Methods

Part 1. The study of acute toxicity in mice. The toxicity of 3-demethylubi-quinone Q2 for mice was determined by the method of Karber (G. Karber, Arch. Exp. Pathol. Pharm., 1931, v. 162, p. 480). Briefly: white non-linear mice weighing 20 g each were used for experiments. 3-Demethylubiquinone Q2 was dissolved in a mixture of DMSO and water (1: 1) or ethanol and water (1: 1) and injected into the mouse intraperitoneally at the indicated concentrations and in a volume of 0.1 ml. Toxicity was evaluated by the Karber formula:

LD ₅₀ = LD ₁₀₀ −Σ (z × d) / m. LD ₅₀ or LD ₁₀₀ means the death of 50% or 100% of experimental mice, respectively; z is equal to half the number of animals killed by the last two adjacent doses; d is equal to the interval between every two adjacent doses; m is equal to the number of animals used to study each concentration of the substance.

Part 2. Magnetic resonance imaging. The anti-cancer or cancer-preventive activity of 3-demethylubiquinone Q2 was studied using a modern, non-aggressive technical instrument, a PharmaScan US 70/16 magnetic resonance imager (Brucker, Germany), which is equipped with a 7 Tesla superconducting magnet and a frequency of 300 MHz. For experiments used white nonlinear mice weighing 20 g each, an Ehrlich carcinoma ascites tumor was injected into the animals under the right scapula at a concentration of 5 million / ml Treatment with 3-demethylubiquinone Q2 in one embodiment was started a day before the inocul and tumors (in the case of experiments on cancer-preventive activity), and in the other, a day after tumor inoculation (if the anti-cancer therapeutic activity was investigated) 3-Demethylubiquinone Q2 was dissolved in a mixture of DMSO and water (1: 1) or ethanol and water ( 1: 1) and injected into the mouse intraperitoneally, once or twice, in a volume of 0.1 ml, at the indicated time and at a final concentration of 30 mg / kg The antitumor preventive and therapeutic effects of 3-demethylubiquinone Q2 were evaluated after measuring the size of Ehrlich solid carcinoma n and the 6th, 9th, 12th, 15th and 18th day after tumor transplantation.

Mice were anesthetized with xylazine solution, 1 mg / ml (SPORA, Praha), at a final concentration of 0.3 mg / kg, and placed in the instrument in the dorsal position so that the tumor was concentrated in the center of the coil surface. The radio frequency has been adapted for experiments with mice. T2-weighted spin-echo sequencing was used with the following parameters: repeat time / to echo time, 2579.8 / 44.5 ms; FOV 3.2 × 3.2 cm; acquisition time 3.46 min; matrix size 128 × 128; layer thickness 1 mm; the distance between the layers is 1.5 mm. The gain and signal-to-noise ratio were determined separately for each scan session. TARE-weighted RARE_8 images gave the best contrast between the tumor and the surrounding normal tissues. Animals, including untreated control individuals, were studied by MRI every third day during the experiments. Tumor volume was determined from two orthogonal sets of RARE_8 images, including the entire tumor. Images were analyzed using the ROI (Region of Interest Tool), Para Vision computer tomography software, version 3.0.1. Statistical analysis of the data was performed using the non-parametric Mann-Whitney U-test method. The cancer-preventive or therapeutic effects of 3-demethylubiquinone Q2 for mice are shown in FIGS. 16-18 as a percentage of inhibition of tumor growth in experimental mice compared to control untreated mice.

Example 26

The following illustrations represent the composition of pharmaceutical forms containing patentable substances ("Substance X") for therapeutic or prophylactic use by humans. These formulations can be obtained using conventional procedures widely known in the pharmaceutical community.

(i) Tablet 1 mg / tablet Substance X = 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 Total 300,0 (ii) Tablet 2 mg / tablet Substance X = 20,0 Microcrystalline cellulose 410.0 Starch 50,0 Starch glycolate sodium 15.0 Magnesium stearate 5,0 Total 500,0 (iii) Capsule mg / capsule Substance X = 10.0 Colloidal silicon dioxide 1,5 Lactose 465.5 Gelatinized starch 120.0 Magnesium stearate 3.0 Total 600,0 (iv) Injection 1 (1 mg / ml) mg / ml Substance X = (as free acid) 1,0 Disubstituted Sodium Phosphate 12.0 Monosubstituted Sodium Phosphate 0.7 Sodium chloride 4,5 1 N caustic soda solution (up to pH 7-7.5) how much is required Water for injections up to 1 ml (v) Injection 2 (10 mg / ml) mg / ml Substance X = (as free acid) 10.0 Monosubstituted Sodium Phosphate 0.3 Disubstituted Sodium Phosphate 1,1 Polyethylene glycol 400 200,0 0.1 N sodium hydroxide solution (up to pH 7-7.5) how much is required Water for injections up to 1 ml (vi) Aerosol mg / dose Substance X = 20,0 Oleic acid 10.0 3-Chloro-monofluoro-methane 10000,0 Dichlorotetrafluoroethane 5000.0

All publications, patents, and patent documents referenced herein are incorporated individually by reference. The invention has been described with reference to various specific and preferred descriptions and methods. However, it must be understood that numerous other variations and modifications may be made that will be made in the spirit and letter of this patent.

Claims (21)

1. Polyprenylated 1,4-benzoquinones of formula 1 or formula 3 or polyprenylated 1,4-hydroquinones of formula 2 or formula 4

where each of R1 and R2, independently of the others, may be a hydrogen radical or a (C ₁ -C ₆ ) alkoxy group;
each of R3 and R4, independently of the others, may be a hydrogen radical or a (C ₁ -C ₆ ) -alkyl group;
n may be 2 or 3;
or their pharmaceutically acceptable salts. 2. The use of polyprenylated 1,4-benzoquinones of formula 1 or formula 3 or polyprenylated 1,4-hydroquinones of formula 2 or formula 4

where each of R1 and R2, independently of the others, may be a hydrogen radical or a (C ₁ -C ₆ ) alkoxy group;
each of R3 and R4, independently of the others, may be a hydrogen radical or a (C ₁ -C ₆ ) -alkyl group;
n may be 2 or 3;
or polyprenylated 1,4-benzoquinones of formula 5 or formula 6 or formula 7 or formula 8 or formula 9 or formula 10 or formula 11 or formula 12 or formula 13 or formula 14 or polyprenylated 1,4 β-hydroquinones of formula 15 or their pharmaceutically acceptable salts for the preparation of a medicament that can be used to treat cancer in mammals

. 3. The use according to claim 2, where the drug is used to reduce the size of the cancer in mammals. 4. The use according to claim 2, where the drug is used to inhibit the growth of cancer in mammals. 5. The use according to claim 2 for the preparation of a drug used to induce AP-1-dependent transcriptional activity in mammals. 6. The use according to claim 2 for the preparation of a drug used to induce NF-κB-dependent transcriptional activity in mammals. 7. The use according to claim 2 for the preparation of a medicament used for the simultaneous induction of AP-1 and NF-κB-dependent transcriptional activity in mammals. 8. The use according to claim 2 for the preparation of a medicament used to inhibit the transformation of normal cells into cancer cells in mammals. 9. The use according to claim 2 for the preparation of a medicinal product used to reduce the proliferation of cancer cells in mammals. 10. The use according to claim 2 for the preparation of a drug used to induce apoptosis in mammals. 11. A pharmaceutical composition having anti-cancer activity, comprising an effective amount of a compound of formula 1, or formula 2, or formula 3, or formula 4, or formula 5, or formula 6, or formula 7, or formula 8, or formula 9, or formula 10, or formula 11, or formula 12, or formula 13, or formula 15, as described in claim 2, or a pharmaceutically acceptable salt of this compound in combination with a pharmaceutically acceptable solvent or carrier. 12. A method of treating cancer, comprising administering to the mammal, if necessary, such treatment, an effective amount of a compound of formula 1, or formula 2, or formula 3, or formula 4, or formula 5, or formula 6, or formula 7, or formula 8, or formula 9, or formula 10, or formula 11, or formula 12, or formula 13, or formula 14, or formula 15, as described in claim 2, or an effective amount of a pharmaceutically acceptable salt of this compound or an effective amount of a pharmaceutical composition according to claim 11, including these compounds eniya. 13. The method of claim 12, wherein administering the compound or pharmaceutical composition reduces the size or inhibits the growth of a cancerous tumor in a mammal. 14. The method according to item 12, where the introduction of a compound or pharmaceutical composition prevents the transformation of normal cells into tumor cells in a mammal. 15. The method of claim 12, wherein administering the compound or pharmaceutical composition reduces the proliferation of tumor cells in a mammal. 16. The method of claim 12, wherein administration of the compound or pharmaceutical composition causes apoptosis of tumor cells in a mammal. 17. A method of inducing in a cell an AP-1-dependent or NF-κB-dependent transcriptional activity, or both, simultaneously, comprising treating the cell with an effective amount of a compound of formula 1, or formula 2, or formula 3, or formula 4, or formula 5, or formula 6, or formula 7, or formula 8, or formula 9, or formula 10, or formula 11, or formula 12, or formula 13, or formula 14, or formula 15, as indicated in claim 2, or an effective amount of a pharmaceutically an acceptable salt of this compound or an effective amount of a pharmaceutical The composition of claim 11, comprising these compounds. 18. A method of inducing apoptosis in a cell, comprising treating the cell with an effective amount of a compound of formula 1, or formula 2, or formula 3, or formula 4, or formula 5, or formula 6, or formula 7, or formula 8, or formula 9, or formula 10, or formula 11, or formula 12, or formula 13, or formula 14, or formula 15, as described in claim 2, or an effective amount of a pharmaceutically acceptable salt of this compound or an effective amount of a pharmaceutical composition according to claim 11, comprising these connections. 19. The method according to any one of paragraphs.17 and 18, where the cell is a mammalian cell. 20. The method according to any one of paragraphs.17 and 18, where the processing is carried out in vitro or in vivo. 21. The method of separation of glabruquinones A (formula 5) and B (formula 6), including extraction of ascidium ascidium glabrum with ethanol, evaporation of ethanol, extraction of the aqueous residue with chloroform, evaporation of the chloroform layer to obtain a brown oily residue, chromatography of the residue on a silica gel column, in the system hexane-ethyl acetate, 10: 1 and separation of the mixture of the obtained glabruquinones A and B by HPLC on a column of silica gel in a hexane-ethyl acetate system, 7: 1 to obtain individual glabruquinones A and B

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