Usnic acid derivatives as anti-tumour agents

FIELD: chemistry.

SUBSTANCE: invention provides benzylidene furanone derivatives of (+)-usnic acid of formula 6-13 as anti-tumour agents. The compounds exhibit cytotoxic activity with respect to tumour cell lines CEM-13, U-937, MT-4.

EFFECT: high activity.

2 dwg, 3 tbl, 8 ex

The technical field to which the invention relates.

The invention relates to medicinal chemistry and relates to derivatives of usninovoy acid containing benzylidenemalonate fragment. These compounds can be used as drugs with antitumor activity.

The level of technology

Modern regimens of various types of malignant tumors using surgical methods in the complex in aggressive high-dose therapy, a serious disadvantage is the high toxicity of modern anticancer drugs against vital organs and body systems. Related side effects reduce the efficiency, and in some cases restrict the use of anticancer agents. Another problem in cancer treatment is the problem of residual tumor clone. Tumor cells that survived chemotherapy, usually show drug resistance to a wide range of drugs and cause a relapse of the disease in a more severe form.

In this regard, it is important to search for new anticancer drugs, providing high selectivity and efficacy of treatment.

An important area of medical chemistry, allowing us to obtain new effective anticancer ven the rata is the use of synthetic transformations of plant metabolites. The most acceptable is the study of plant metabolites on the biological activity of which there is reliable information and which are currently available or will become available in the near future as the formation of the resource base. To this class of compounds is usnic acid, a broad spectrum of biological activity (anti-inflammatory, antiviral, antitumor, and so on) chains to her keen interest of researchers.

Antiproliferative activity of natural usninovoy acid (1) (Scheme 1) is described in the literature, both enantiomers exhibit moderate cytotoxic activity against a wide range of tumor cell cultures.

The first mention of the cytotoxic activity of usninovoy acids belong to 1975, in [Kupchan S., H. Kopperman 1-Usnic Acid: Tumor Inhibitor Isolated from Lichens Experientia, V. 31, Fasc.6, 625] examined the survival rate of mice with inoculated carcinoma Lewis, survival increased to 135-152% in the processing of (-)-usninovoy acid at doses of 20-200 mg/kg

Data on the cytotoxicity of (-)-usninovoy acid and some of its derivatives on the lung carcinoma cells (L1210) and leukemia (P388) published in [M. Takai, Y. Uehara, J. Beisler Usnic acid derivatives as potential antineoplastic agents Journal of Medicnal chemistry, 1979, V. 22, N 11, 1380-1384]. A high degree of inhibition (>90%) at a concentration of 140 µm showed itself (-)-usnic acid and some of its most lipophilic derivatives, modified on ring A. the Reduction of the cytotoxic activity was observed during the transition to a derivative, modification of which included the destruction of the triketone system of ring C.

(+)-Usnic acid (1) (Scheme 1) also shows a noticeable cytotoxic effect that was observed by the authors [B. Burlando, E. Ranzato, A. Volante, G. Appendino, F. Pollastro, L. Verotta Antiproliferative Effects on Tumour Cells and Promotion of Keratinocyte Wound Healing by Different Lichen Compounds Planta Med 2009; 75: 607-613] against several tumor cell mesothelioma MM, epidermoid carcinoma A431 and human keratinocytes Nasat, inhibitory concentration (+)-usninovoy acid IC₅₀ranged from 23 to 76 µM, which was significantly lower than that of several other lichen acids.

Cytotoxic activity of (+)-usninovoy acid against HeLa cells in vitro was investigated by the authors [M. Natić, Z. Tesbrevelić, K. Andbarelković, I. Brcbreveleski, S. Radulović, S. Manić, D. Sladić Synthesis and Biological Activity of Pd(II) and Cu(II) Complexes with Acylhydrazones of Usnic Acid Synthesis and Reactivity in Inorganic and Metal-Organic Chemistry Vol.34, No.1, pp.101-113, 2004], IC₅₀reached 14.9 μm, the increase of the cytotoxic activity against the cell line (cervical carcinoma) were observed with the use of its derivatives, and especially thiosemicarbazone usninovoy acid and its complex with copper (2) (Scheme 1), IC₅₀these compounds amounted to 2.4 and 1.8 µM, respectively.

A wide range of tumor cell cultures were studied in [M. Bazin, A. Le Lamer, J. Delcros, I. Rouaud, Uriac, P., J. Boustie, J. Corbel, S. Tomasi Synthesis and cytotoxic activities of usnic acid derivatives Bioorganic and Medicinal Chemistry, 2008, V. 16, 6860-6866 and S. Bezivin, S. Tomasi, I. Rouaud, J. Delcros, J. Boustie Cytotoxic activity of compounds from Lichen: Cladonia convolute Planta Med 2004; 70: 874-877], devoted (+)- and (-)-uninova acids and polyaminopropyl (+)-usninovoy acid. Both enantiomers of usninovoy acid showed moderate cytotoxic activity against cell lines L1210 (leukemia), CHO (ovarian cancer), 3LL (carcinoma of lung), DU145 (prostate cancer), MCF7 (breast adenocarcinoma), K-562 (leukemia), U251 (glioblastoma) with a concentration IC₅₀from 17 to 105 μm. Significant cytotoxic activity greater than native usnic acid, showed the compound (3) (Scheme 1) obtained by the reaction of usninovoy acid and diaminooctane, IC₅₀compound 3 against L1210 cells was 2.7 μm.

(+)- And (-)-uninove acid was investigated [Einarsdottir E, Groeneweg J, Bjornsdottir GG, Harethardottir G, Omarsdottir S, Ingolfsdottir K, Ogmundsdottir HM (2010) Cellular mechanisms of the anticancer effects of the lichen compound usnic acid. Planta Med 76(10): 969-974] on cytotoxic activity against breast cancer cells T-47D and liver cancer Capan-2. Both enantiomers showed similar antiproliferative activity against both cell l the deposits. IC₅₀amounted to 4.2 µg/ml and 4.0 mg/ml for (+) and (-)-usanovich acids against T-47D, 5.3 μg/ml and 5.0 μg/ml against Capan-2, respectively. The authors did not observe manifestations of classical apoptotic cells with fragmented DNA, however, the observed loss of mitochondrial membrane potential indicates that these connections use the mechanism of mitochondrial apoptosis in the corresponding signaling pathway cell death.

Usnic acid, the authors did not specify which of the enantiomers, exhibits cytotoxic activity against breast cancer cells (wild-type p53 MCF7 and non - functional p53 MDA-MB-231) and lung cancer (H1299) in a concentration of 29 µm [Mayer M, O'neill MA, Murray KE, Santos-Magalhaes NS, Carneiro-Leao AM, Thompson AM, Appleyard VC (2005) Usnic acid: a non-genotoxic compound with anti-cancer properties. Anticancer Drugs 16(8):805-809].

The disadvantages of natural usanovich acids are relatively low efficiency compared to existing drugs and poor bioavailability. Good results were obtained on some semi-synthetic derivatives, modified by various functional groups of usninovoy acid, which may indicate the prospects of synthetic versions of plant metabolites.

Disclosure of inventions

The objective of the invention is the creation of new, effective, non-lethal drugs with antitumor de is due and received from available plant materials.

The problem is solved by the use of compounds which are semisynthetic derivatives of (S)-usninovoy acid containing benzylidenemalonate fragment exhibiting cytotoxic activity against tumor cell lines CEM-13, U-937, MT-4.

To achieve this goal, we conducted a series of chemical modifications presented in scheme 2, the synthesis of the target compounds described in the article [Sokolov, A. N., Luzina O. A., Chernikov A. C., Salakhutdinov N. F. Synthesis of euronav based on usninovoy acid. Chemistry of natural compounds. 2012. No. 3. C. 350-355]. As the original connection was taken (S)-usnic acid, obtained by extraction of lichen Cladonia stellaris according to the method [N. F. Salakhutdinov, M. P. Spouse, M. Y. Panchenko, Pat. Of the Russian Federation No. S; bull. Fig. 2008, No. 5]. Bromination of usninovoy acid with bromine in the presence of bromoiodide gives the derivative 4, the treatment of which with potassium acetate leads to intramolecular cyclization with the formation of compounds with furninova fragment 5. The next step was to obtain the target compounds with antitumor activity by reaction of compound 5 with a number of aromatic aldehydes.

It was investigated the influence of usninovoy acid (1) and benzylideneaniline derivatives (6-13) on cell viability carcinoma lines man. CCID usninovoy acid (1) and its derivatives (6-13) for different carcinoma of human cell lines listed in table 1. As Comparators used (+)-uninova acid (1). In the result, it was shown that (+)usnic acid (1) shows a moderate, but the claimed compounds (6-13) - high antitumor activity against all used tumor cell cultures, namely CEM-13, U-937, MT-4. It is shown that the values CCID50 for connections (6-13) are similar in magnitude for all tumor cells are in the range 0.6-9.0 µg/ml For the most active compounds (6, 9, 10) was additionally investigated their ability to induce apoptosis in tumor cells. It is established that these compounds are able to induce apoptosis in tumor cells of monocytic origin U-937 from 28 to 69% and in tumor cells of lymphoid origin MT-4 from 6 to 19%. The obtained data on the antitumor activity of compounds (6-13) allow to consider them as promising medicinal agents. The invention is illustrated by the following examples.

Example 1.

The interaction of (+)-usninovoy acid with a twofold excess of bromine

To 1 mmol of usninovoy acid (344 mg) was added to the complex bromooctane (2 mmol bromide (0.10 ml) was dissolved in 14 ml of dioxane), a few drops of HBr and left for 7 days at room temperature. After concentration of the reaction mixture on a rotary evaporator was chromatographically receive the config residue on silica gel (60-200µ), eluent - CH₂Cl₂.

(S)-2-acetyl-6-(2-bromoacetyl)-3,7,9-trihydroxy-8,9 b-dimethylbenzo[b,d]furan-1(9bH)-4 he. Exit 283 mg (67%). So pl. 97-100°C. [α]_D+349 (c 0.5; CHCl₃). NMR¹H (CDCl₃, δ, M. D., J Hz): 1.75 (3H, s, H-15), 2.08 (3H, s, H-10), 2.64 (3H, s, H-12), 4.52 (2H, DD, J=12.4, J=14.0, H-14), 6.00(1H, s, H-4), 11.17 (1H, s, OH-9), 12.68 (1H, s, OH-7), 18.81 (1H, c, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 7.5 (C-10), 27.7 (C-12), 31.9 (C-15), 34.5 (C-14), 61.6 (C-9b), 98.7 (C-4), 99.0 (C-6), 104.3 (C-9a), 105.1 (C-2), 109.6 (C-8), 154.3 (C-5a), 158.4 (C-9), 164.1 (C-7), 178.5 (C-4aa), 191.5 (C-3), 192.7 (C-13), 197.7 (C-1), 201.7 (C-11). IR spectrum, ν, cm^-1): 842, 1140, 1292, 1458, 1628, 3013, 3497. Found: m/z 421.9976 [M]⁺C₁₈H₁₅O₇Br. Calculated: M=421.9996.

Example 2

The interaction of compounds 4 with potassium acetate

To a solution of 1 mmol of compound 4 (423 mg) in 25 ml of acetone was added 150 mg (1.5 mmol) of potassium acetate and heated the reaction mixture for 2 hours. Then diluted with water (up to 50-60 ml), acidified with HCl (1:4) to pH=3-4. Was extracted with CH₂Cl₂(3×10 ml), dried over calcined MgSO₄, solvent was removed and chromatographically the residue on a column of silica gel, eluent - CH₂Cl₂.

(10S)-8,13-dihydroxy-7,10-dimethyl-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12-pentaen-3,11-dione 5. The output 308 mg (90%). So pl. 202-203°C.[α]_D+397 (c 0.5; CHCl₃). NMR¹H (CDCl₃, δ, M. D., J Hz): 1.73 (3H, s, H-15), 2.13 (3H, s, H-10), 2.64 (3H, s, H-12), 4.66 (2H, s, H-14), 6.02 (1H, s, H-4), 11.27 (1H, s, OH-9), at 18.82 (1H, s, OH-3). NMR¹³C (CDCl_{3 , δ, M. D.): 6.91 (C-10), 27.80 (C-12), 31.91 (C-15), 58.71 (C-9b), 75.70 (C-14), 99.01 (C-4), 100.61 (C-9a), 105.10 (C-6), 105.7 (C-2), 107.10 (C-8), 149.13 (C-5a), 159.80 (C-9), 173.81 (C-7), 179.61 (C-4a), 191.61 (C-3), 194.10 (C-13), 197.90 (C-1), 201.71 (C-11). Found: m/z 342.0736 [M]⁺C18H14O7. Calculated: M=342.0734.}

Example 3

Interaction of compound 5 with aldehydes (General method)

To a solution of 1 mmol of compound 5 (342 mg) in 24 ml of MeOH was added 1.1 mmol of the corresponding aldehyde, 1 ml of 50% aqueous KOH and heated (in the case of obtaining compounds 6-12) or stirred at room temperature for 1.5 hours (in the case of receiving a connection 13). Then the reaction mixture was diluted with water (up to 50-60 ml), acidified with HCl (1:4) to pH=3-4. Was extracted with CH₂Cl₂(3×10 ml), dried over calcined MgSO₄, solvent was removed and chromatographically the residue on a column of silica gel, eluent - CH₂Cl₂.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(phenylmethylene)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 6. The output 262 mg (61%). So pl. 203°C. [α]_D+267 (c 0.45; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.78 (3H, s, H-15), 2.32 (3H, s, H-10), 2.67 (3H, s, H-12), 6.07 (1H, s, H-4), 6.81 (1H, s, H-16), 7.39 (1H, m, H-20), 7.46 (2H, m, H-19, H-21), 7.87 (2H, m, H-18, H-22), 11.42 (1H, s, OH-9), 18.86 (1H, s, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 7.50 (C-10), 27.86 (C-12), 31.96 (C-15), 58.70 (C-9b), 99.13 (C-4), 100.85 (C-6), 105.12 (C-2), 105.77 (C-8), 108.23 (C-9a), 112.12 (C-16), 128.81 (C-18, C-22), 129.69 (C-20), 131.23 (C-19, C-21), 132.13 (C-17), 147.44 (C-14), 149.81 (C-5a),159.36 (C-9), 165.72 (C-7), 179.57 (C-13, C-4a), 191.62 (C-3), 197.86 (C-1), 201.79 (C-11). Found:m/z 430.1039 [M]⁺C₂₅H₁₈O₇. Calculated: M=430.1047.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(n-brompheniramine)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 7. Yield 270 mg (53%). So pl. 218-219°C.[α]_D+406 (c 0.3; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.76 (3H, s, H-15), 2.28 (3H, s, H-10), 2.65 (3H, s, H-12), 6.06 (1H, H-4), 6.72 (1H, s, H-16), 7.56 (2H, m, H-19, H-21), 7.70 (2H, m, H-18, H-22), 11.44 (1H, c, OH-9), 18.84 (1H, s, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 7.19 (C-10), 27.53 (C-12), 31.63 (C-15), at 58.35 (C-9b), 98.88 (C-4), 100.41 (C-6), 104.81 (C-2), 105.49 (C-8), 108.05 (C-9a), 110.23 (C-16), 123.72 (C-20), 130.76 (C-17), is 131.75 (C-19, C-21), 132.10 (C-18, C-22), 147.36 (C-14), 149.54 (C-5a), 159.18 (C-9), 165.28 (C-7), 178.97 (C-4a), 179.14 (C-13), 191.30 (C-3), 197.51 (C-1), 201.48 (C-11). Found: m/z 508.0161 [M]⁺C₂₅H₁₇O₇Br. Calculated: M=508.0152.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(o-methoxyphenylethylamine)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 8. Yield 340 mg (74%). So pl. 206-208 °C. [α]_D+362 (c 0.4; CHCl₃). NMR¹H (CDCl₃, 8, M. D., J/Hz): 1.76 (3H, s, H-15), 2.29 (3H, s, H-10), 2.65 (3H, s, H-14), 3.89 (3H, s, H-23), 6.06 (1H, s, H-4), 6.91 (1H, DD, J₁₉,₂₀=8.3, J₁₉,₂₁=0.7, H-19), 7.05 (1H, DDD, J₂₁,₂₂=7.8, J₂₁,₂₀=7.4, J₂₁,₁₉=0.7, H-21), 7.35 (1H, DDD, J₂₀,₁₉=8.3, J₂₀,₂₁=7.4, J₂₀,₂₂=1.7, H-20), 7.39 (1H, s, H-16), 8.23 (1H, DD, J₂₂,₂₁=7.8, J₂₂,₂₀=1.7, H-22), 11.37 (1H, s, OH-9), 18.86 (1H, s, OH-3). NMR 13C (CDCl₃, δ, M. D.): 7.54 (C-10), at 27.94 (C-12), 31.99 (C-15), 55.46 (C-23), 58.77 (C-9b), 99.08 (C-4), 101.14 (C-6), 105.11 (C-2), 105.69 (C-8), 106.26 (C-16), 108.02 (C-9a), 110.63 (C-19), 120.75 (C-21), 121.15 (C-17), 131.29 (C-20), 131.56 (C-22), 147.49 (C-14), 149.74 (C-5a), 158.65 (C-18), 159.06 (C-9), 165.62 (C-7), 179.60 (C-13), 179.76 (C-4a), 191.65 (C-3), 197.91 (C-1), 201.82 (C-11). Found: m/z 460.1154 [M]⁺C₂₆H₂₀O₈. Calculated: M=460.1153.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(n-performation)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 9. Exit 273 mg (61%). So pl. 198°C.[α]_D+281 (c 0.4; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.75 (3H, s, H-15), 2.26 (3H, s, H-10), 2.66 (3H, s, H-12), 6.04 (1H, s, H-4), 6.69 (1H, s, H-16), 7.10 (2H, m, H-19, H-21), 7.81 (2H, m, H-18, H-22), 11.39 (1H, c, OH-9), 18.84 (1H, s, OH-3). NMR¹³C (CDCl₃, 5, M. D., J/Hz): 7.48 (C-10), 27.82 (C-12), 31.95 (C-15), 58.67 (C-9b), 99.10 (C-4), 100.76 (C-6), 105.10 (C-2), 105.69 (C-8), 108.27 (C-9a), 110.67 (C-16), 116.00 (l, J_C-F=22 Hz, C-19, C-21), 128.43 (l, J_C-F=3.18 Hz, C-17), 133.10 (l, J_C-F=8.41 Hz, C-18, C-22), 147.01 (l, J_C-F=2.64 Hz, C-14), 149.76 (C-5a), 159.29 (C-9), 163.00 (d, J_C-F=252.39 Hz, C-20), 165.55 (C-7), 179.22 (C-4a), 179.48 (C-13), 191.60 (C-3), 197.82 (C-1), 201.77 (C-11). Found: m/z 448.0957 [M]⁺C₂₅H₁₇O₇F₁. Calculated: M=448.0953.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(n-chlorpheniramine)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 10. The output of 255 mg (55%). So pl. 217°C. [α]_D+262 (c 0.3; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.75 (H, c, H-15), 2.26 (3H, s, H-10), 2.66 (3H, s, H-12), 6.04 (1H, s, H-4), 6.67 (1H, s, H-16), 7.36 (2H, m, H-19, H-21), 7.7 (2H, m, H-18, H-22), 11.41 (1H, s, OH-9), 18.84 (1H, s, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 7.49 (C-10), at 27.84 (C-12), 31.96 (C-15), 58.66 (C-9b), 99.14 (C-4), 100.68 (C-6), 105.12 (C-2), at 105.75 (C-8), 108.35 (C-9a), 110.42 (C-16), 129.06 (C-19, C-21), 130.64 (C-17), 132.21 (C-18, C-22), 135.54 (C-14), 147.52 (C-20), 149.80 (C-5a), 159.41 (C-9), 165.53 (C-7), 179.15 (C-4a), 179.43 (C-13), 191.59 (C-3), 197.81 (C-1), 201.77 (C-11). Found: m/z 464.0660 [M]⁺C₂₅H₁₇O₇C1₁. Calculated: M=464.0657.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(n-methoxyphenylethylamine)-5,16-doxytetracycline[7.7,0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 11. Yield 300 mg (65%). So pl. 258-260°C. [α]_D+300 (c 0.45; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.75 (3H, s, H-15), 2.29 (3H, s, H-10), 2.65 (3H, s, H-12), 3.84 (3H, s, H-23), 6.04 (1H, s, H-4), 6.78 (1H, s, H-16), 6.95 (2H, m, H-19, H-21), 7.81 (2H, m, H-18, H-22), 11.34 (1H, s, OH-9), 18.83 (1H, s, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 7.54 (C-10), 27.88 (C-12), 32.00 (C-15), 55.28 (C-23), 58.80 (C-9b), 99.06 (C-4), 102.93 (C-6), 105.14 (C-2), 105.65 (C-8), 108.06 (C-9a), 112.38 (C-16), 114.43 (C-19, C-21), 124.92 (C-17), 133.10 (C-18, C-22), 146.42 (C-14), 149.81 (C-5a), 158.99 (C-9), 160.85 (C-20), 165.56 (C-7), 179.42 (C-4a), 179.77 (C-13), 191.65 (C-3), 197.94 (C-1), 201.79 (C-11). Found: m/z 460.1148 [M]⁺C₂₆H₂₀O₈. Calculated: M=460.1153.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(3,4,5-trimethoxyphenethylamine)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 12. The output of 385 mg (72%). So pl. 174-176°C. [α]_D+224 (c 0.4; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.72 (3H, s, H-15), 2.23 (3H, s, H-10), 2.63 (3H, s, H-12), 3.87 (3H, s, H-24), 3.89 (6H, s, H-23, H-25), 6.02 (1H, s, H-4), 6.65 (1H, s, H-16), 7.09 (2H, c, H-18, H-22), 11.39 (1H, s, OH-9), at 18.82 (1, s, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 7.28 (C-10), 27.80 (C-12), at 31.98 (C-15), 55.80 (C-24, c-25), 58.67 (C-9b), 60.83 (C-23), 99.11 (C-4), 100.87 (C-6), 105.09 (C-2), 105.32 (C-8), 108.24 (C-9a), 108.30 (C-18, C-22), 112.23 (C-16), 127.43 (C-17), 139.56 (C-20), 146.95 (C-14), 149.79 (C-5a), 153.04 (C-19, C-21), 159.15 (C-9), 165.41 (C-7), 179.11 (C-4a), 179.47 (C-13), 191.61 (C-3), 197.80 (C-1), 201.79 (C-11). Found: m/z 520.1374 [M]⁺C₂₈H₂₄O₁₀. Calculated: M=520.1364.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(m-nitrophenylamino)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 13. Exit 167 mg (35%). So pl. 230°C decomp. [α]_D+364 (c 0.2; CH₂Cl₂). NMR¹H (CD₂Cl₂δ, M. D.): 1.80 (3H, s, H-15), 2.34 (3H, s, H-10), 2.66 (3H, s, H-12), 6.09 (1H, s, H-4), 6.77 (1H, c H-16), 7.64, 8.12, 8.21 (3H, 3M, H-20, H-21, H-22), 8.83 (4H, c, H-18), 11.53 (1H, c, OH-9), 18.88 (1H, s, OH-3). NMR¹³C (CD₂Cl₂, δ, M. D.): 6.34 (C-10), 27.26 (C-12). 31.34 (C-15), 58.39 (C-9b), 98.81 (C-4), 100.05 (C-6), 104.89 (C-2), 105.63 (C-8), 107.73 (C-16), 108.49 (C-9a), 123.21, 124.81, 129.43, 136.06 (C-18, C-19, C-20, C-21), 133.60 (C-17), 148.17 (C-14), 148.46 (C-5a), 159.57 (C-9), 165.33 (C-7), 178.45 (C-4a), 179.09 (C-13), 191.38 (C-3), 197.70 (C-1), 201.62 (C-11). Found: m/z 475.0895 [M]⁺C₂₅H₁₇O₉N₁. Calculated: M=475.0898.

Example 4.

The influence of usninovoy acid (1), it purananooru derivative (5) and compounds (6-13) on the viability of tumor cells T-cell human leukemia MT-4.

Cell line MT-4 cells T-cell leukemia, human) were cultured in medium RPMI 1640 containing 10% fetal calf serum, antibiotics (100 units/ml pen is cellina and 0.1 mg/ml streptomycin), in an atmosphere of 5% Co₂at 37°C. cell Viability after incubation with compounds (1, 6-13) was determined using the MTT test, which is based on the ability of living cells to make connections on the basis of tetrazole (MTT) in brightly colored crystals formazan that allows spectrophotometric estimate the number of living cells in the preparation. For this, cells were planted in 96-well plates (100 μl of cells with a concentration of 500 thousand cells/ml). Then the cells were added to the solution of compounds (1,5-13) in DMSO to a final concentration in the medium from 0.1 to 100 µg/ml Cells were incubated in the presence of compounds for 3 days under the same conditions. At the end of incubation, without changing the environment to the cells was added a solution of MTT (5 mg/ml) in phosphate-buffered saline to a concentration of 0.5 mg/ml and incubated for 3 h in the same conditions. The medium was removed, cells were added to 100 ál of DMSO, in which the dissolution occurs resulting in the cells of the crystal formazan, and measured the optical density for multi-channel spectrophotometer at wavelengths of 570 and 630 nm, where a₅₇₀- absorption formosana, and A₅₇₀- background cells. Data were represented as the number of live cells relative to control. 100% took the number of cells in the control, where cells were incubated in the absence of a connection, but in the presence of the solvent DMSO.

From the data presented in table 1, it is seen that the compounds (6-13) have more pronounced antitumor effect in relation to the investigated tumor cells MT-4 in comparison with (+)-usninovoy acid (1).

Example 5.

The influence of usninovoy acid (1) and compounds (6-13) on the viability of tumor cells T-cell human leukemia CEM-13.

Cell line CEM-13 cell line T-cell leukemia, human) were cultured in medium RPMI 1640 containing 10% fetal calf serum, antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin), in an atmosphere of 5% CO₂at 37°C.

Cell viability after incubation with compounds (1, 6-13) was determined using the MTT test, which is based on the ability of living cells to make connections on the basis of tetrazole (MTT) in brightly colored crystals formazan that allows spectrophotometric estimate the number of living cells in the preparation. For this, cells were planted in 96-well plates (100 μl of cells with a concentration of 500 thousand cells/ml). Then the cells were added to the solution of compounds (1, 5-13) in DMSO to a final concentration in the medium from .1 to 100 µg/ml As the comparison drug used (+)-uninova acid (1). Cells were incubated in the presence of investigated compounds for 3 days under the same conditions. At the end of incubation, without changing the environment to the cells was added a solution of MTT (5 mg/ml) in phosphate-buffered saline to a concentration of 0.5 mg/ml and incubated for 3 h in the same conditions. The medium was removed, cells were added to 100 ál of DMSO, in which the dissolution occurs resulting in the cells of the crystal formazan, and measured the optical density for multi-channel spectrophotometer at wavelengths of 570 and 630 nm, where A₅₇₀- absorption formosana, and A₆₃₀- background cells. The calculated values of CD were performed as described in example 4. Value CD₅₀the concentration of compound at which there is a loss of 50% of the cells, as well as CD₈₀and CD₉₀(concentration at which there is death of 80 and 90% of cells, respectively) are shown in table 1. All studied compounds (6-13) have more pronounced antitumor effect in relation to the investigated tumor cells SEM-13 in comparison with (+)-usninovoy acid (1).

Example 6.

The influence of usninovoy acid (1) and compounds (6-13) on the viability of human tumor cells, U-937

Cell line U-937 (tumor line human monocytes) were cultured in medium RPMI 1640 containing 10% of embryonal the Yu calf serum, antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin), in an atmosphere of 5% CO₂at 37°C. cell Viability after incubation with compounds (1, 6-13) was determined using the MTT test, which is based on the ability of living cells to make connections on the basis of tetrazole (MTT) in brightly colored crystals formazan that allows spectrophotometric estimate the number of living cells in the preparation. For this, cells were planted in 96-well plates (100 μl of cells with a concentration of 400 thousand cells/ml). Then the cells were added to a solution of the compounds in DMSO to a final concentration in the medium from 0.1 to 100 µg/ml as the reference product used (+)-uninova acid (1). Cells were incubated in the presence of compounds for 3 days under the same conditions. At the end of incubation, without changing the environment to the cells was added a solution of MTT (5 mg/ml) in phosphate-buffered saline to a concentration of 0.5 mg/ml and incubated for 3 h in the same conditions. The medium was removed, cells were added to 100 ál of DMSO, in which the dissolution occurs resulting in the cells of the crystal formazan, and measured the optical density for multi-channel spectrophotometer at wavelengths of 570 and 630 nm, where A₅₇₀- absorption formosana, and A₆₃₀- background cells. The calculated values of CD were performed as described in example 4.

From the data, set the built in table 1, it is seen that the compounds (6-13) have more pronounced antitumor effect in relation to the investigated tumor cells U-937, compared with (+)-usninovoy acid (1), except for the connection (7), the antitumor effect of which on the cells U-937 less pronounced in comparison with (+)-usninovoy acid (1).

Example 7.

The influence of compounds 6, 9, 10 on the induction of apoptosis in tumor cells of human MT-4

For compounds 6, 9, 10 with the highest cytotoxicity was determined by their ability to induce apoptosis in tumor cells.

In the activation of apoptosis is DNA fragmentation due to the activation of endonucleases. To determine the fragmented DNA of cells stained with dye propidium iodide and then determine the percentage fragmentirovannoj DNA, using the flowing cytofluorimetry. Fixed cells in suspension in 70% ethanol by adding 1 ml of cells suspended in phosphate-buffered saline (FSB) (1-5×10⁶cells) to 9 ml of 70% ethanol in a test tube on ice. Next, the cells are centrifuged at 200 g for 3 min, the ethanol is removed, cells are suspended in 10 ml PBS and centrifuged at 300 g for 5 minutes the cells are Then suspended in 0.5 ml PBS and incubated at room temperature for 5 minutes After centrifugation at 300 g for 5 min the precipitated cells are suspended in 1 ml of the solution is La staining DNA. The solution for staining DNA is prepared as follows: dissolve 200 mg of propidium iodide in 10 ml of the FSB, and then add 10 ál of Triton X-100 and 2 mg RNase. The resulting solution is incubated for 15 min at 70°C. For staining of cells incubated for 30 min at room temperature.

Analysis of the cells is carried out by flow cytometry for flow cytofluorimetry FACS Canto. For excitation of fluorescence using laser (wavelength 488 nm), measuring the fluorescence at a length of 600 nm and the light diffusing. Calculate 10000 cells. The number of cells in which the observed apoptosis corresponds to the area SubGl. The results are shown in Fig.1 and table 2.

From these data it is seen that the processing cell line MT-4 leads to induction of apoptosis after 24 hours in 6-12% of the cells, and after 72 hours in 13-19% of the cells. The greatest activity against induction of apoptosis in cells MT-4 showed connections 9, 10. The table shows the data of two independent experiments, each was calculated 10 thousand cells.

Example 8.

The influence of compounds 6,9,10 on the induction of apoptosis in tumor cells of human U-937

For compounds 6, 9, 10 with the highest cytotoxicity was determined by their ability to induce apoptosis in tumor cells.

In the activation of apoptosis is DNA fragmentation due to the activation of endonucleases. Fordetermination fragmented DNA of cells stained with dye propidium iodide and then determine the percentage fragmentirovannoj DNA using the flowing cytofluorimetry. Fixed cells in suspension in 70% ethanol by adding 1 ml of cells suspended in phosphate-buffered saline (FSB) (1-5×10⁶cells) to 9 ml of 70% ethanol in a test tube on ice. Next, the cells are centrifuged at 200 g for 3 min, the ethanol is removed, cells are suspended in 10 ml PBS and centrifuged at 300 g for 5 minutes the cells are Then suspended in 0.5 ml PBS and incubated at room temperature for 5 minutes After centrifugation at 300 g for 5 min the precipitated cells are suspended in 1 ml solution for staining DNA. The solution for staining DNA is prepared as follows: dissolve 200 mg of propidium iodide in 10 ml of the FSB, and then add 10 ál of Triton X-100 and 2 mg RNase. The resulting solution is incubated for 15 min at 70°C. For staining of cells incubated for 30 min at room temperature.

Analysis of the cells is carried out by flow cytometry for flow cytofluorimetry FACS Canto. For excitation of fluorescence using laser (wavelength 488 nm), measuring the fluorescence at a length of 600 nm and the light diffusing. Calculate 10000 cells. The number of cells in which the observed apoptosis corresponds to the area of SubG1. The results are shown in Fig.2 and table 3. From these data it is seen that the processing cell line U-937 leads to the induction of apoptosis after 24 hours in 28-63% tile is to, and after 48 hours in 28-69% of the cells. The greatest activity was shown by the connection 6. The table shows the data of two independent experiments, each was calculated 10 thousand cells.

Thus, compounds (6-13) have more pronounced antitumor effect in relation to the investigated tumor cells MT-4, CEM-13 and U-937, compared with (+)-usninovoy acid (1).

Table 1

Application benzylideneaniline derivatives of (+)-usninovoy acid of the formula:



as anticancer agents.