RU2612875C1 - Means for human tyrosyl-dna-phosphodiesterase 1 enzyme inhibition - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: means which is a chromenone derivative:

I, where n = 1 or 2 or

II, developing the ability to inhibit the human tyrosyl-DNA phosphodiesterase 1 enzyme is proposed.

EFFECT: it expands the range of means for human tyrosyl-DNA-phosphodiesterase 1 enzyme inhibitors and can be used for development of anticancer drugs.

1 tbl, 7 ex

Description

The invention relates to molecular biology, biochemistry and biotechnology, specifically to compounds representing chromenone derivatives of the general formula I:

where, n = 1 or 2,

or II:

in which biological activity has been identified, consisting in the ability to inhibit the action of the enzyme tyrosyl DNA phosphodiesterase 1 person (Tdp1).

In recent years, active searches have been made for inhibitors of the enzyme tyrosyl DNA phosphodiesterase 1 (Tdp1), which is considered as a promising target enzyme in the development of drugs for the treatment of cancer and neurodegenerative diseases [Cortes Ledesma, 2009].

Tdp1 belongs to the class of phosphodiesterases - enzymes that break down phosphodiester bonds [Interthal, 2001]. Tdp1 plays an important role in removing DNA damage caused by topoisomerase 1 (Top1) in the presence of its camptothecin inhibitor. Thus, Tdp1 resists Top1 inhibitors, which are quite effective antitumor drugs [see Pommier 2010 reviews Pommier, 2006]. Tdp1 is believed to be responsible for the drug resistance of certain types of cancer [Dexheimer, 2008; Beretta, 2010]. This hypothesis is supported by a number of studies: Tdp1 knockout mice and human cell lines having the SCAN1 mutation are hypersensitive to camptothecin [El-Khamisy, 2009; Das, 2009; Katyal, 2007; Hirano, 2007]. Conversely, in cells with increased Tdp1 expression, camptothecin and etoposide cause less DNA damage [Barthelmes, 2004; Nivens, 2004]. Thus, a combination of drugs acting on Top1 and Tdp1 can significantly increase the effectiveness of chemotherapy. The therapeutic effect of Tdp1 inhibitors can be a selective increase in the activity of Top1 inhibitors in tumors with impaired DNA repair and cell cycle control.

In addition, it was shown that suppression of Tdp1 activity makes tumor cells hypersensitive to anticancer drugs with other mechanisms of action: temozolomide (purine methylation) [Alagoz, 2013], methyl methanesulfonate (formation of apurine / apyrimidine sites), bleomycin (single-chain / double-chain breaks from 3 ' -phosphoglycolates), hydrogen peroxide and ionizing radiation (tears and other types of damage) [Murai, 2012].

A few Tdp1 inhibitors have been described in the literature, and as a rule, they have a moderate inhibitory effect (in the range of 100-1 μM) [Antony, 2007; Nguyen, 2012; Dexheimer, 2008; Zakharenko, 2015].

Closest to the claimed agent - the prototype - is furamidine, which is a heterocyclic diamidine [Antony, 2007] of the general formula III:

A disadvantage of the known means is unsatisfactory inhibitory characteristics (IC ₅₀ for single-stranded DNA of the order of 100 μm).

An object of the invention is to provide a more effective Tdp1 inhibitor.

The stated technical problem is solved by the use of compounds representing chromenone derivatives of the general formula I or II, in which the biological activity of suppressing the activity of Tdp1 is revealed.

Compounds I and II can be synthesized by the reaction of bromide IV with substituted coumarins V and VI in accordance with the following scheme:

Compound IV can be synthesized, for example, from monoterpenoid Myrtenal VII in accordance with the following scheme:

To obtain both enantiomers of compound IV, (+) - and (-) - myrtenal (VII) were used as starting compounds.

Compounds Va, b and VI can be synthesized, for example, by reacting resorcinol VIII with esters of the corresponding β-keto acids according to the following scheme:

The structure of the obtained compounds I and II was confirmed by NMR spectroscopy and mass spectrometry.

The test results of compounds Ia (n = 1), Ib (n = 2) and II are shown in the table.

Technical result: a class of effective Tdp1 inhibitors with good inhibitory characteristics was obtained (IC ₅₀ for single-stranded DNA from 0.13 to 1.6 μM).

In the case of compounds Ia and II, derivatives of (+) - myrtenal were more active than the corresponding (-) - enantiomers, at the same time, both enantiomers of compound Ib showed the same activity.

Compounds of the general formula I and II, after conducting in-depth pharmacological studies, can be used to further develop new highly effective anti-cancer agents.

The following are specific examples of the implementation of the proposed technical solution.

Example 1. Synthesis of 7 - (((1R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methoxy) -2,3-dihydrocyclopenta [c] chromene-4 (1H ) -one ((-) - Ia)

To 0.101 g (0.5 mmol) of compound Va in 5 ml of ethanol was added 0.104 g (0.75 mmol) of K ₂ CO ₃ and 0.161 g (0.75 mmol) of bromide (-) - IV at 25 ° С with stirring. Stirred at room temperature for 15 minutes, then heated at 80 ° C for 5 hours. The hot solution was filtered, the filtrate was kept at -10 ° C for 48 hours. The product (-) - Ia was isolated by recrystallization from ethanol, the yield was 0.067 g (40%).

=-20.5 (EtOH, с=0.5). Спектр ЯМР ¹Н (CDCl₃, δ, м. д, J, Hz): 0.80 (с, 3Н, С²²Н₃); 1.16 (д, 1H, ²J=8.6, Н^20а); 1,28 (с, 3Н, С²¹Н₃); 2.10 (ддддд, 1Н, J_17,19=J_17,20s=5.6, J_17,16a=J_17,16s=2.9, J_17,15=1.3, Н¹⁷); 2.13-2.20 (м, 2Н, 2Н¹¹); 2.20 (ддд, 1Н, J_19,17=J_19,20s=5.6, J_19,15=1.4, Н¹⁹); 2.25 (д. м, 1Н, ²J=18.0, H^16s); 2.32 (д. м, 1Н, ²J=18.0, Н^16а); 2.40 (ддд, 1Н, ²J=8.6, J_20s,17=J_20s,19=5.6, H^20s); 2.84-2.88 (м, 2Н, 2Н¹⁰); 2.99-3.03 (м, 2Н, 2Н¹²); 4.42 (д. м, 1Н, ²J=12.4, другие J≤2.0, Н¹³); 4.44 (д. м, 1Н, ²J=12.4, другие J≤2.0, Н^13'); 5.60-5.63 (м, 1Н, Н¹⁵), 6.82 (дд, 1Н, J_7,6=8.6, J_7,9=2.4, Н⁷); 6.85 (д, 1Н, J_9,7=2.4, Н⁹); 7.29 (д, 1Н, J_6,7=8.6, Н⁶). Спектр ЯМР ¹³С (CDCl₃), δ, м. д.: 155.60 (с, С¹); 160.48 (с, С²); 124.28 (с, С³); 156.23 (с, С⁴); 112.21 (с, С⁵); 125.28 (д, С⁶); 112.85 (д, С⁷); 161.29 (с, С⁸); 101.67 (д, С⁹); 30.24 (т, С¹⁰); 22.47 (т, С¹¹); 31.93 (т, С¹²); 71.00 (т, С¹³); 143.07 (с, С¹⁴); 121.22 (д, С¹⁵); 31.17 (т, С¹⁶); 40.69 (д, С¹⁷); 37.99 (с, С¹⁸); 43.07 (д, С¹⁹); 31,39 (т, С²⁰); 26.01 (к, С²¹); 20.97 (к, С²²). Найдено: m/z=336.1722 [М]⁺ (С₂₂Н₂₄O³)⁺ Вычислено: m/z=336.1720.T _PL = 145 ° C,

= -20.5 (EtOH, s = 0.5). ¹ H NMR spectrum (CDCl ₃ , δ, ppm, J, Hz): 0.80 (s, 3H, C ²² H ₃ ); 1.16 (d, 1H, ² J = 8.6, H ^20a ); 1.28 (s, 3H, C ²¹ H ₃ ); 2.10 (ddddd, 1H, J _17.19 = J _17.20s = 5.6, J _17.16a = J _17.16s = 2.9, J _17.15 = 1.3, H ¹⁷ ); 2.13-2.20 (m, 2H, 2H ¹¹ ); 2.20 (ddd, 1H, J _19.17 = J _{19.20 s} = 5.6, J _19.15 = 1.4, H ¹⁹ ); 2.25 (dm, 1H, ² J = 18.0, H ^16s ); 2.32 (dm, 1H, ² J = 18.0, H ^16a ); 2.40 (ddd, 1H, ² J = 8.6, J _{20s, 17} = J _{20s, 19} = 5.6, H ^20s ); 2.84-2.88 (m, 2H, 2H ¹⁰ ); 2.99-3.03 (m, 2H, 2H ¹² ); 4.42 (d. M, 1H, ² J = 12.4, other J≤2.0, H ¹³ ); 4.44 (d. M, 1H, ² J = 12.4, others J≤2.0, H ^{13 '} ); 5.60-5.63 (m, 1H, H ¹⁵ ), 6.82 (dd, 1H, J _7.6 = 8.6, J _7.9 = 2.4, H ⁷ ); 6.85 (d, 1H, J _9.7 = 2.4, H ⁹ ); 7.29 (d, 1H, J _6.7 = 8.6, H ⁶ ). ¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 155.60 (s, C ¹ ); 160.48 (s, C ² ); 124.28 (s, C ³ ); 156.23 (s, C ⁴ ); 112.21 (s, C ⁵ ); 125.28 (d, C ⁶ ); 112.85 (d, C ⁷ ); 161.29 (s, C ⁸ ); 101.67 (d, C ⁹ ); 30.24 (t, C ¹⁰ ); 22.47 (t, C ¹¹ ); 31.93 (t, C ¹² ); 71.00 (t, C ¹³ ); 143.07 (s, C ¹⁴ ); 121.22 (d, C ¹⁵ ); 31.17 (t, C ¹⁶ ); 40.69 (d, C ¹⁷ ); 37.99 (s, C ¹⁸ ); 43.07 (d, C ¹⁹ ); 31.39 (t, C ²⁰ ); 26.01 (q, C ²¹ ); 20.97 (q, C ²² ). Found: m / z = 336.1722 [M] ⁺ (C ₂₂ H ₂₄ O ³ ) ⁺ Calculated: m / z = 336.1720.

Example 2. Synthesis of 7 - (((1S, 5R) -6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methoxy) -2,3-dihydrocyclopenta [c] chromene-4 (1H ) -one ((+) - Ia)

To 0.101 g (0.5 mmol) of compound Va in 5 ml of ethanol were added 0.104 g (0.75 mmol) of K ₂ CO ₃ and 0.161 g (0.75 mmol) of bromide (+) - IV at 25 ° С with stirring. Stirred at room temperature for 15 minutes, then heated at 80 ° C for 5 hours. The hot solution was filtered, the filtrate was kept at -10 ° C for 48 hours. The product (+) - Ia was isolated by recrystallization from ethanol, the yield was 0.093 g (55%).

=+27.6 (EtOH, с=0.65). Спектры ЯМР ¹Н и ¹³С соединения (+)-Ia соответствуют спектрам энантиомера (-)-Ia. Найдено: m/z=336.1718 [М]⁺ (С₂₂Н₂₄O₃)⁺ Вычислено: m/z=336.1720. _Mp = 140 ° C.

= + 27.6 (EtOH, s = 0.65). ¹ H and ¹³ C NMR spectra of the compound (+) - Ia correspond to the spectra of the enantiomer (-) - Ia. Found: m / z = 336.1718 [M] ⁺ (C ₂₂ H ₂₄ O ₃ ) ⁺ Calculated: m / z = 336.1720.

Example 3. Synthesis of 3 - (((1R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methoxy) -7,8,9,10-tetrahydro-6H-benzo [s] chromen-6-one ((-) - Ib)

To 0.108 g (0.5 mmol) of compound Vb in 5 ml of ethanol were added 0.104 g (0.75 mmol) of K ₂ CO ₃ and 0.161 g (0.75 mmol) of bromide (-) - IV at 25 ° С with stirring. Stirred at room temperature for 15 minutes, then heated at 80 ° C for 5 hours. The hot solution was filtered, the filtrate was kept at -10 ° C for 48 hours. The product (-) - Ib was isolated by recrystallization from ethanol, the yield was 0.067 g (38%).

=-25.0 (EtOH, с=0.75). Спектр ЯМР ¹Н (CDCl₃, δ, м. д, J, Hz): 0.80 (с, 3Н, С²³Н₃); 1.16 (д, 1Н, ²J=8.7, Н^21а); 1,27 (с, 3Н, С²²Н₃); 1.74-1.85 (м, 4Н, 2Н¹¹, 2Н¹²); 2.09 (ддддд, 1Н, J_18,20=J_18,21s=5.6, J_18,17a=J_18,17s=2.8, J_18,16=1.3, Н¹⁸); 2.20 (ддд, 1Н, J_20,18=J_20,21s=5.6, J_20,16=1.4 Н²⁰); 2.24 (д. м, 1Н, ²J=18.0, H^17s); 2.32 (д. м, 1Н, ²J=18.0, Н^17а); 2.39 (ддд, 1Н, ²J=8.7, J_21s,18=J_21s,20=5.6, H^21s); 2.53 (т. м, 2Н, J_10,11=6.3, 2Н¹⁰); 2.72 (т. м, 2Н, J_13,12=6.3, 2Н¹³); 4.41 (д. м, 1Н, ²J=12.4, другие J≤2.0, Н¹⁴); 4.43 (д. м, 1Н, ²J=12.4, другие J≤2.0, Н¹⁴); 5.59-5.62 (м, 1H, Н¹⁶), 6.79 (д, 1Н, J_9,7=2.4, Н⁹); 6.81 (дд, 1H, J_7,6=8.7, J_7,9=2.4, Н⁷); 7.40 (д, 1H, J_6,7=8.7, Н⁶). Спектр ЯМР ¹³С (CDCl₃), δ, м. д.: 153.30 (с, С¹); 162.12 (с, С²); 120.29 (с, С³); 147.17 (с, С⁴); 113.54 (с, С⁵); 123.79 (д, С⁶); 112.62 (д, С⁷); 160.69 (с, С⁸); 101.49 (д, С⁹); 23.72 (т, С¹⁰); 21.60 (т, С¹¹); 21.29 (т, С¹²); 25.10 (т, С¹³); 70.93 (т, С¹⁴); 143.12 (с, С¹⁵); 121.12 (д, С¹⁶); 31,16 (т, С¹⁷); 40.69 (д, С¹⁸); 37.98 (с, С¹⁹); 43.06 (д, С²⁰); 31.38 (т, С²¹); 26.01 (к, С²²), 20.96 (к, С²³). Найдено: m/z=350.1875 [М]⁺ (С₂₃Н₂₆O₃)⁺ Вычислено: m/z=350.1877. _Mp = 110 ° C.

= -25.0 (EtOH, s = 0.75). ¹ H NMR spectrum (CDCl ₃ , δ, ppm, J, Hz): 0.80 (s, 3H, C ²³ H ₃ ); 1.16 (d, 1H, ² J = 8.7, H ^21a ); 1.27 (s, 3H, C ²² H ₃ ); 1.74-1.85 (m, 4H, 2H ¹¹ , 2H ¹² ); 2.09 (ddddd, 1H, J _18.20 = J _{18.21 s} = 5.6, J _{18.17 a} = J _{18.17 s} = 2.8, J _18.16 = 1.3, H ¹⁸ ); 2.20 (ddd, 1H, J _20.18 = J _{20.21 s} = 5.6, J _20.16 = 1.4 H ²⁰ ); 2.24 ( ^dm , 1H, ² J = 18.0, H ^17s ); 2.32 (dm, 1H, ² J = 18.0, H ^17a ); 2.39 (ddd, 1H, ² J = 8.7, J _{21s, 18} = J _{21s, 20} = 5.6, H ^21s ); 2.53 (t, m, 2H, J _10.11 = 6.3, 2H ¹⁰ ); 2.72 (t, m, 2H, J _13.12 = 6.3, 2H ¹³ ); 4.41 (d. M, 1H, ² J = 12.4, other J≤2.0, H ¹⁴ ); 4.43 (d. M, 1H, ² J = 12.4, others J≤2.0, H ¹⁴ ); 5.59-5.62 (m, 1H, H ¹⁶ ), 6.79 (d, 1H, J _9.7 = 2.4, H ⁹ ); 6.81 (dd, 1H, J _7.6 = 8.7, J _7.9 = 2.4, H ⁷ ); 7.40 (d, 1H, J _6.7 = 8.7, H ⁶ ). ¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 153.30 (s, C ¹ ); 162.12 (s, C ² ); 120.29 (s, C ³ ); 147.17 (s, C ⁴ ); 113.54 (s, C ⁵ ); 123.79 (d, C ⁶ ); 112.62 (d, C ⁷ ); 160.69 (s, C ⁸ ); 101.49 (d, C ⁹ ); 23.72 (t, C ¹⁰ ); 21.60 (t, C ¹¹ ); 21.29 (t, C ¹² ); 25.10 (t, C ¹³ ); 70.93 (t, C ¹⁴ ); 143.12 (s, C ¹⁵ ); 121.12 (d, C ¹⁶ ); 31.16 (t, C ¹⁷ ); 40.69 (d, C ¹⁸ ); 37.98 (s, C ¹⁹ ); 43.06 (d, C ²⁰ ); 31.38 (t, C ²¹ ); 26.01 (q, C ²² ), 20.96 (q, C ²³ ). Found: m / z = 350.1875 [M] ⁺ (C ₂₃ H ₂₆ O ₃ ) ⁺ Calculated: m / z = 350.1877.

Example 4. Synthesis of 3 - (((1S, 5R) -6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methoxy) -7,8,9,10-tetrahydro-6H-benzo [s] chromen-6-one ((+) - Ib)

To 0.108 g (0.5 mmol) of compound Vb in 5 ml of ethanol were added 0.104 g (0.75 mmol) of K ₂ CO ₃ and 0.161 g (0.75 mmol) of bromide (+) - IV at 25 ° С with stirring. Stirred at room temperature for 15 minutes, then heated at 80 ° C for 5 hours. The hot solution was filtered, the filtrate was kept at -10 ° C for 48 hours. The product (+) - Ib was isolated by recrystallization from ethanol, the yield was 0.067 g (38%).

=+32.6 (EtOH, с=0.65). Спектры ЯМР ¹Н и ¹³С соединения (+)-Ib соответствуют спектрам энантиомера (-)-Ib. Найдено: m/z=350.1872 [М]⁺ (С₂₃Н₂₆О₃)⁺ Вычислено: m/z=350.1876.

= + 32.6 (EtOH, s = 0.65). ¹ H and ¹³ C NMR spectra of the compound (+) - Ib correspond to the spectra of the enantiomer (-) - Ib. Found: m / z = 350.1872 [M] ⁺ (C ₂₃ H ₂₆ O ₃ ) ⁺ Calculated: m / z = 350.1876.

Example 5. Synthesis of 7 - (((1R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methoxy) -4-methyl-2H-chromen-2-one (( -) - II)

To 0.108 g (0.5 mmol) of compound VI in 5 ml of ethanol was added 0.104 g (0.75 mmol) of K ₂ CO ₃ and 0.132 g (0.75 mmol) of bromide (-) - IV at 25 ° С with stirring. Stirred at room temperature for 15 minutes, then heated at 80 ° C for 5 hours. The hot solution was filtered, the filtrate was kept at -10 ° C for 48 hours, and evaporated. The product (-) - II was isolated by silica gel column chromatography, and the eluent was a solution containing 25 to 100% chloroform in hexane. The yield was 0.084 g (54%).

=-36 (EtOH, с=0.8). Спектр ЯМР ¹Н (CDCl₃, δ, м. д, J, Hz): 0.80 (с, 3Н, С²⁰Н₃); 1.17 (д, 1Н, ²J=8.7, Н^18а); 1,28 (с, 3Н, С¹⁹Н₃); 2.08-2.13 (м, 1Н, Н¹⁵); 2.20 (ддд, 1Н, J_17,15=J_17,18s=5.6, J_17,13=1.4, Н¹⁷); 2.25 (д. м, 1Н, ²J=18.0, H^14s); 2.33 (д. м, 1Н, ²J=18.0, Н^14а); 2.35 (д, 3Н, J_10,3=1.2, С¹⁰Н₃); 2.40 (ддд, 1H, ²J=8.7, J_18s,17=J_18s,17=5.6, H^18s); 4.43 (д. м, 1H, ²J=12.4, другие J≤2.0, Н¹¹); 4.45 (д. м, 1Н, ²J=12.4, другие J≤2.0, Н^11'); 5.60-5.63 (м, 1Н, Н¹³), 6.10 (к 1H, J_3,10=1.2, Н³); 6.80 (д, 1H, J_9,7=2.4, Н⁹); 6.83 (дд, 1Н, J_7,6=8.7, J_7,9=2.4, Н⁷); 7.44 (д, 1Н, J_6,7=8.7, Н⁶). Спектр ЯМР ¹³С (CDCl₃), δ, м. д.: 155.10 (с, С¹); 161.28 (с, С²); 111.74 (с, С³); 152.44 (с, С⁴); 113.37 (с, С⁵); 125.21 (д, С⁶); 112.91 (д, С⁷); 161.96 (с, С⁸); 101.75 (д, С⁹); 18.51 (к, С¹⁰); 71.06 (т, С¹¹); 142.96 (с, С¹²); 121.30 (д, С¹³); 31.18 (т, С¹⁴); 40.70 (д, С¹⁵); 37.99 (с, С¹⁶); 43.10 (д, С¹⁷); 31.39 (т, С¹⁸); 26.01 (к, С¹⁹); 20.96 (к, С²⁰). Найдено: m/z=310.1564 [М]⁺ (С₂₀Н₂₂О₃)⁺ Вычислено: m/z=310.1563. _Mp = 70 ° C.

= -36 (EtOH, c = 0.8). ¹ H NMR Spectrum (CDCl ₃ , δ, ppm, J, Hz): 0.80 (s, 3H, C ²⁰ H ₃ ); 1.17 (d, 1H, ² J = 8.7, H ^18a ); 1.28 (s, 3H, C ¹⁹ H ₃ ); 2.08-2.13 (m, 1H, H ¹⁵ ); 2.20 (ddd, 1H, J _17.15 = J _{17.18 s} = 5.6, J _17.13 = 1.4, H ¹⁷ ); 2.25 (dm, 1H, ² J = 18.0, H ^14s ); 2.33 (d. M, 1H, ² J = 18.0, H ^14a ); 2.35 (d, 3H, J _10.3 = 1.2, C ¹⁰ H ₃ ); 2.40 (ddd, 1H, ² J = 8.7, J _{18s, 17} = J _{18s, 17} = 5.6, H ^18s ); 4.43 (d. M, 1H, ² J = 12.4, other J≤2.0, H ¹¹ ); 4.45 (d. M, 1H, ² J = 12.4, other J≤2.0, H ^{11 '} ); 5.60-5.63 (m, 1H, H ¹³ ), 6.10 (q 1H, J _3.10 = 1.2, H ³ ); 6.80 (d, 1H, J _9.7 = 2.4, H ⁹ ); 6.83 (dd, 1H, J _7.6 = 8.7, J _7.9 = 2.4, H ⁷ ); 7.44 (d, 1H, J _6.7 = 8.7, H ⁶ ). ¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 155.10 (s, C ¹ ); 161.28 (s, C ² ); 111.74 (s, C ³ ); 152.44 (s, C ⁴ ); 113.37 (s, C ⁵ ); 125.21 (d, C ⁶ ); 112.91 (d, C ⁷ ); 161.96 (s, C ⁸ ); 101.75 (d, C ⁹ ); 18.51 (q, C ¹⁰ ); 71.06 (t, C ¹¹ ); 142.96 (s, C ¹² ); 121.30 (d, C ¹³ ); 31.18 (t, C ¹⁴ ); 40.70 (d, C ¹⁵ ); 37.99 (s, C ¹⁶ ); 43.10 (d, C ¹⁷ ); 31.39 (t, C ¹⁸ ); 01/26 (q, C ¹⁹ ); 20.96 (q, C ²⁰ ). Found: m / z = 310.1564 [M] ⁺ (C ₂₀ H ₂₂ O ₃ ) ⁺ Calculated: m / z = 310.1563.

Example 6. Synthesis of 7 - (((1S, 5R) -6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methoxy) -4-methyl-2H-chromen-2-one (( +) - II)

To 0.108 g (0.5 mmol) of compound VI in 5 ml of ethanol was added 0.104 g (0.75 mmol) of K ₂ CO ₃ and 0.132 g (0.75 mmol) of bromide (+) - IV at 25 ° С with stirring. Stirred at room temperature for 15 minutes, then heated at 80 ° C for 5 hours. The hot solution was filtered, the filtrate was kept at -10 ° C for 48 hours, and evaporated. Product (+) - II was isolated by silica gel column chromatography, and the eluent was a solution containing from 25 to 100% chloroform in hexane. The yield was 0.047 g (30%).

=+36 (EtOH, с=0.8). Т_пл=85°С. Спектры ЯМР ¹Н и ¹³С соединения (+)-II соответствует спектрам энантиомера (-)-П. Найдено: m/z=310.1559 [М]⁺ (С₂₀Н₂₂О₃)⁺ Вычислено: m/z=310.1563,

= + 36 (EtOH, c = 0.8). _Mp = 85 ° C. ¹ H and ¹³ C NMR spectra of the compound (+) - II correspond to the spectra of the enantiomer (-) - P. Found: m / z = 310.1559 [M] ⁺ (C ₂₀ H ₂₂ O ₃ ) ⁺ Calculated: m / z = 310.1563,

Example 7. The study of the effect of the proposed compounds on the activity of Tdp1

Recombinant human tyrosyl DNA phosphodiesterase 1 (EC 3.1.4.) Was expressed in the Escherichia coli system (plasmid pET 16B-Tdp1 kindly provided by Dr. Caldecott K.U., University of Sussex, UK) and isolated as described [Interthal, 2001 ].

As a test system for determining the inhibitory properties of the proposed compounds, the reaction of removing the Black Hole Quencher 1 fluorescence quencher (BHQ1) from the 3'-end of the oligonucleotide catalyzed by Tdp1 was used. At the 5'-end of the oligonucleotide is (5,6) -FAM - a fluorophore, the fluorescence intensity of which increases with the removal of the quencher. To measure fluorescence, a POLARstar OPTIMA fluorometer manufactured by BMG LABTECH was used.

200 μl reaction mixtures contained a buffer (50 mM Tris-HCl, pH 8.0; 50 mM NaCl; 7 mM mercaptoethanol), 50 nM oligonucleotide, and various inhibitor concentrations. The reaction was started by adding Tdp1 to a final concentration of 1.3 nM. The measurements were carried out in a linear range of the reaction rate versus time (up to 8 minutes) every 55 seconds. The effect of the proposed compounds was evaluated by the value of IC ₅₀ (the concentration of the inhibitor at which the enzyme activity is reduced by half). The calculation of IC ₅₀ values was carried out using the MARS Data Analisys 2.0 program (BMG LABTECH).

IC ₅₀ values for the compounds studied are shown in the table.

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Claims (6)

An agent representing a chromenone derivative of the general formula I:

where n = 1 or 2, or II:

exhibiting an inhibitory effect on the enzyme tyrosyl DNA phosphodiesterase 1 person.