RU2527457C2 - Inhibitors of human poly(adp-riboso)polymerase-1 based on uracyl derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel inhibitors of human poly(ADP-riboso)polymerase-1 based on uracyl derivatives of general formula (I), (II), (III) and (IV). Inhibitors of poly(ADP-riboso)polymerase-enzymes take part in DNA reparation. In general formula

and

R¹=H, Cl, Br, I, methyl, ethyl, propyl or isopropyl; R², R³, R⁴, R⁵=H, methyl, ethyl, propyl or phenyl; R⁶=(CH₂)_n, where n=1-4; X=OR² or NR²R³, R², R³=H, methyl, ethyl, propyl, phenyl, 3-hydroxypropyl.

EFFECT: obtaining novel inhibitors of human poly(ADP-riboso)polymerase-1.

1 tbl, 19 ex

Description

The invention relates to molecular biology, biomedical chemistry and biochemistry, specifically to inhibitors of the key enzyme of the DNA repair system of poly (ADP-ribose) polymerase-1 person (PARP) based on uracil derivatives of the general formula (I), (II), (III) and (Iv)

where R ¹ = H, Cl, Br, I, methyl, ethyl, propyl or isopropyl; R ² , R ³ , R ⁴ , R ⁵ = H, methyl, ethyl, propyl or phenyl; R ⁶ = (CH ₂ ) _n , where n = 1-4; X = OR ² or NR ² R ³ , R ² , R ³ = H, methyl, ethyl, propyl, phenyl, 2-hydroxyethyl, 3-hydroxypropyl.

The preferred field of use of the present invention is biomedical chemistry, since PARP inhibitors can be used in the treatment of diseases such as stroke, myocardial ischemia, diabetes and its complications, arthritis, colitis, traumatic damage to the central nervous system and several others.

Inhibition of PARP is also one of the ways to increase the activity of antitumor agents (complex therapy) by inhibiting necrosis and activating apoptosis. PARP inhibitors can also be used as independent agents for the treatment of certain types of tumors, initially characterized by a deficiency of certain DNA repair mechanisms. Alkylating drugs that cause DNA damage and ionizing radiation are used in treatment regimens for many forms of cancer. DNA repair systems resist the effects of DNA damaging agents; therefore, the therapeutic effect depends on the effectiveness of DNA repair systems. Selective action aimed at inhibiting DNA repair creates the prerequisites for effective accompanying therapy.

To date, a number of PARP inhibitors have been proposed based on various classes of chemical compounds [Ferraris D.V. Evolution of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors. From concept to clinic. J. Med. Chem., 53, 4561-4584 (2010)]: nicotinamide, 3-aminobenzamide and their analogues [Banasik M., Ueda K. Inhibitors and activators of ADP-ribosylation reactions. Mol. Cell Biochem., 138, 185-197 (1994)]; isoquinolinones and dihydroisoquinolinones (lactams) [US Patent 5391554; WO 9911645 A1]; benzimidazoles, indoles and related compounds [EP 0879820; US Patent 6310082]; isoindolinones [Southan G.J., Szabo C. Poly (ADP-ribose) polymeraze inhibitors. Curr. Med. Chem., 10 (4), 321-340, (2003)]; phthalazinones and quinazolinones [Banasik et al. J. Biol. Chem., 267 (3), 1569-75 (1992)]; phenanthridinones [WO 9307868 A1].

The disadvantages of the existing analogues of the present invention include their low solubility in water, a short life time in the body, as well as low specificity and toxicity for a number of analogues.

In addition to the above groups of inhibitors, PARP inhibitors based on derivatives of nucleic bases are known, characterized by high solubility in water and specificity for PARP. In particular, nucleosides have been proposed [Pivazyan AD et al. Inhibition of poly (ADP-ribose) polymerase activity by nucleoside analogs of thymidine. Biochemical Pharmacology, 44, 947-953 (1992)] and disaccharide nucleosides [CH Khodyreva et al. Means for inhibiting the enzyme poly (ADP-riboso) polymerase-1 person. RF patent 2411948 (2009)] with a relatively high inhibitory activity against PARP (IC ₅₀ ~ 50 μM). This group of compounds is the closest in structure to the inhibitors proposed in the present invention. It should be noted, however, that disaccharide nucleosides are obtained during multistage synthesis using relatively inaccessible compounds, which complicates and increases the cost of obtaining inhibitors of this type. DNA repair occurs in the cell nucleus, the active compound must not only penetrate the cell membrane, but also enter the nucleus. Nucleosides and disaccharide nucleosides can easily be converted in the cell, for example phosphorylated, which leads to a decrease in the concentration of the inhibitor.

The aim of the present invention is the creation of effective PARP inhibitors based on derivatives of uracil, which can be obtained by single-stage synthesis.

The objective of the invention is solved by the fact that, as PARP inhibitors, compounds of the general formula (I-III) are proposed which are prepared by direct alkylation of substituted or unsubstituted nucleic bases in dimethylformamide in the presence of 1,8-diazobicyclo [5.4.0] undec-7-ene (DBU ) During the alkylation process, a mixture of N ¹ monosubstituted and N ¹ , N ³ disubstituted derivatives is formed, which are separated by silica gel column chromatography. The selected reaction conditions allow the synthesis of PARP inhibitors in one stage in a homogeneous environment. Esters of carboxymethyl derivatives of uracil (IV) are also obtained according to the same scheme [Mikhailov S.N. and others. Acyclic analogs of nucleosides with an amide bond. Bioorganic chemistry, 21 (2), 130-132 (1995)]. These compounds are converted to amides by reaction with the corresponding amines. The activity of inhibitors based on derivatives of uracil corresponds to the relatively high activity of the prototype (IC ₅₀ ~ 50 mM). IC ₅₀ values for some inhibitors based on uracil derivatives are shown in Table 1.

The uracil derivatives (I-IV) of this invention are first proposed as PARP-1 inhibitors. Some of the compounds obtained were previously described in the literature. The compounds of formula (I), where R ₁ = H, F, Cl, Br, I, methyl, and R ₂ = H are intermediates in the synthesis of antiviral agents based on triazole [Elayadi N. et al. Preparation of 1,4-disubstituted-1,2,3-triazoloribonucleosides by Na ₂ CuP ₂ O ₇ catalyzed azide-alkyne 1,3-dipolar cycloaddition. ARKIVOC, 8, 76-89 (2012); Parmenopoulou V. et al. Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis, biochemical and structural evaluation. Bioorganic & Medicinal Chemistry, 20 (24), 7184-7193 (2012)]. N ¹ -allyl substituted derivatives of 5-fluorouracil and thymine (formula (II), R ₁ = F, methyl, R ₃ , R ₄ , R ₅ = H) are used in the synthesis of a number of modified uracils with antiviral and antitumor activity [Chiacchio U. et al. Enantioselective synthesis of homo-carbocyclic-2′-oxo-3′-aza-nucleosides. Tetrahedron, 62 (6), 1171-1181 (2006)]. A known method of producing uracilcarboxylic acids, their esters and amides [Baker BR et al. Non-classical antimetabolites. Xviii. Simulation of 5′-phosphoribosyl binding. 2. ω-Uracilalkanoic acids related to 2′-deoxyuridylate. Journal of Pharmaceutical Sciences, 54 (1), 25-30 (1965)]. Derivatives of N ¹ -benzyluracil (III, R ¹ = H, R ⁶ = CH ₂ ) [Javaid ZZ et al. Pyrimidine nucleobase ligands of orotate phosphribosyltransferase from Toxoplasma gondii. Biochemical Pharmacology, 58 (9), 1457-1466 (1999)] are used to inhibit phosphoribosyltransferase, and N ¹ is (carboxymethyl) uracil (IV, R ¹ = OH, methyl) [EP 829542 A1], to obtain DNA analogues.

The structure of the claimed compounds is confirmed by UV and NMR spectroscopy. NMR spectra were recorded on a Bruker AMX400 instrument (Germany). Chemical shifts (δ) were measured relative to the internal standard tetramethylsilane (TMS, δ 0 ppm) and are given in parts per million (ppm). The values of the spin-spin interaction constants (J) are measured in hertz (Hz). When describing ¹ H-NMR spectra, the following abbreviations were adopted: c — singlet, broadened singlet, d — doublet, t — triplet, m — multiplet. UV spectra were recorded in water with a Cary 300 UV / VIS instrument (Varian, Australia). Melting points were determined on an Electrotermals device (Great Britain). Thin layer chromatography was performed on Kieselgel 260 F plates (Merck) with UV detection (λ = 254 nm). Column chromatography was performed on Kieselgel 60 silica gel (0.063-0.200 mm, Merck). Purification of solvents was carried out according to standard methods.

Example 1. Synthesis of 5-iodo-1- (propin-2-yl) uracil

To a solution of 0.308 g (2.026 mmol) of 1,8-diazobicyclo [5.4.0] undec-7-ene in 6 ml of dry DMF was added 0.482 g (2.025 mmol) of 5-iodouracil and stirred for 15 minutes. To the resulting mixture at 20 ° C, 0.241 g (2.023 mmol) of propargyl bromide was added dropwise over 1 min. The reaction was carried out with stirring for 4.5 hours at a temperature of 20 ° C. The mixture was left overnight at room temperature. After completion of the reaction, the solvent was distilled off in vacuo, the residue was diluted with methylene chloride (70 ml), transferred to a separatory funnel and washed with water (40 ml). The aqueous layer was extracted with methylene chloride (4 × 15 ml). The organic layers after washing and extraction were combined, washed with water (4 × 40 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue was applied to a silica gel column (10 g) for further chromatographic purification. The column was washed with methylene chloride, the products were eluted with methylene chloride - ethanol systems 100: 1 and 100: 2 (v / v). Fractions containing the same products were combined, evaporated to dryness in vacuo. The yield of 5-iodo-1- (propin-2-yl) uracil 0.244 g (44% based on 5-iodouracil), R _f 0.44 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, DMSO-D ₆ ): 11.73 ss (1H, NH), 8.23 s (1H, 6-H), 4.50 d (2H, ³ J _{CH, CH3} = 2.6, CH ₂ ), 3.41 t (1H, ³ J _{CH, CH 2} = 2.6, CH).

Example 2. Synthesis of 5-iodo-1- (3-methylbut-2-en-1-yl) uracil

To a solution of 0.451 g (2.963 mmol) of 1,8-diazobicyclo [5.4.0] undec-7-ene in 8 ml of dry DMF was added 0.690 g (2.890 mmol) of 5-iodouracil and stirred for 15 minutes. 0.444 g (2.976 mmol) of isoprenyl bromide was added dropwise to the resulting mixture at 20 ° C over 1 min. The reaction was carried out with stirring for 4.5 hours at a temperature of 20 ° C. The mixture was left overnight at room temperature. After completion of the reaction, the solvent was distilled off in vacuo, the residue was diluted with methylene chloride (70 ml), transferred to a separatory funnel and washed with water (35 ml). The aqueous layer was extracted with methylene chloride (3 × 30 ml). The organic layers after washing and extraction were combined, washed with water (4 × 30 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue was applied to a silica gel column (25 g) for chromatographic separation. The column was washed with methylene chloride, the products were eluted with methylene chloride – ethanol systems, 100: 1 and 100: 2 (v / v). The fractions containing the product were combined, evaporated to dryness in vacuo.

The yield of 5-iodo-1- (3-methylbut-2-en-1-yl) uracil 0.466 g (53% based on 5-iodouracil), R _f 0.45 (methylene chloride - ethanol, 20: 1, v / v) . T _pl = 146-148 ° C. UV (pH 1-7): λ _max 291 nm (ε 7849); (pH 13): λ _max 281 nm (ε 6046). ¹ H NMR (400 MHz, DMSO-D ₆ ): 11.57 ss (1H, NH), 8.09 s (1H, 6-H), 5.21 m (1H, ³ J _{CH, CH2} = 6.9, ⁴ J _{CH3, CH} = 1.2, CH), 4.26 d (2H, ³ J _{CH2, CH} = 6.9, CH ₂ ), 1.71 d (3H, ⁴ J _{CH3, CH} = 1.2, CH ₃ ), 1.70 d (3H, ⁴ J _{CH3, CH} = 1.2, CH ₃ ).

Example 3. Synthesis of 5-iodine-1-benzyluracil

To a solution of 0.152 g (1.0 mmol) of 1,8-diazobicyclo [5.4.0] undec-7-ene (DBU) in 3.0 ml of dry DMF was added 238 mg (1.0 mmol) of 5-iodouracil and stirred at room temperature until a homogeneous solution was formed . 0.12 ml (1.0 mmol) of benzyl bromide (BnBr) was added to the reaction mixture. The reaction was carried out at 20 ° C with stirring. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was distilled off in vacuo, the residue was diluted with methylene chloride (30 ml), transferred to a separatory funnel and washed with water (35 ml). The aqueous layer was extracted with methylene chloride (3 × 30 ml). The organic layers after washing and extraction were combined, washed with water (4 × 30 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue was applied to a silica gel column (10 g) for chromatographic separation. The column was washed with methylene chloride, the products were eluted with methylene chloride – ethanol systems, 100: 1, 100: 2 and 100: 3 (v / v). The fractions containing the product were combined, evaporated to dryness in vacuo. The yield is 233 mg (71%). R _f 0.42 (methylene chloride - ethanol, 20: 1, v / v). ¹ H-NMR (400 MHz, DMSO-D ₆ ): 11.67 ss (1H, NH), 8.32 s (1H, 6-H), 7.39-7.28 m (5H, Ph), 4.88 s (2H, CH ₂ ) . ¹³ C-NMR (100 MHz, DMSO-D ₆ ): 160.90 (C4), 150.66 (C2), 149.66 (C6), 136.59 (C1 in Ph), 128.58 (C2 and C6 in Ph), 127.67 (C4 in Ph ), 127.44 (C3 and C5 in Ph), 68.53 (C5), 50.44 (CH ₂ ). T _pl = 216-217 ° C. UV (pH 1-7): λ _max 292 nm (ε 7546); (pH 13): λ _max 282 nm (ε 5670).

Example 4. Synthesis of 5-iodo-1- (3-phenylpropyl) uracil

To a solution of 0.506 g (3.326 mmol) of 1,8-diazobicyclo [5.4.0] undec-7-ene in 6 ml of dry DMF was added 0.791 g (3.324 mmol) of 5-iodouracil and stirred for 15 minutes. A solution of 0.972 g (3.347 mmol) of 3-phenylpropanol tosylate in 3.5 ml of dry DMF was added dropwise to the resulting mixture at 20 ° C over 1 min. The reaction was carried out with stirring for 4.5 hours at a temperature of 20 ° C. The mixture was left overnight at room temperature. After completion of the reaction, the solvent was distilled off in vacuo, the residue was diluted with methylene chloride (70 ml), transferred to a separatory funnel and washed with water (35 ml). The aqueous layer was extracted with methylene chloride (4 × 15 ml). The organic layers after washing and extraction were combined, washed with water (4 × 40 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The product was chromatographed on silica gel (30 g). The column was washed with methylene chloride, the products were eluted with methylene chloride – ethanol systems, 100: 1 and 100: 2. (v / v). The fractions containing the product were combined, evaporated to dryness in vacuo. The yield of 5-iodo-1- (3-phenylpropyl) uracil 0.760 g (64% based on 5-iodouracil), R _f 0.46 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, DMSO-d ₆ ): 11.53 ss (1H, NH), 8.16 s (1H, 6-H), 7.30-7.14 m (5H, Ph), 3.71 t (2H, ³ J _{CH2, CH2} = 7.2, CH ₂ -N), 2.58 t (2H, ³ J _{CH2, CH2} = 7.8, CH ₂ -Ph), 1.92 m (1H, ² J _{CHA, CHB} = 14.8, ³ J _{CHA, CH2} = 7.8, ³ J _{CHA, CH2} = 7.2 Hz, CH _A ), 1.88 m (1H, ² J _{CHB, CHА} = 14.8, ³ J _{CHB, CHА} = 7.8, ³ J _{CHB, CH2} = 7.2, CH _B ).

Example 5. Synthesis of 5-iodo-1- (3-phenylethyl) uracil

The substance was obtained analogously to example 4 with a yield of 40%. R _f 0.46 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, DMSO-D ₆ ): 11.58 ss (1H, NH), 8.03 s (1H, 6-H), 7.34-7.19 m (5H, Ph), 3.89 t (2H, ³ J _{CH2, CH2} = 7.6, CH ₂ -N), 2.89 t (2H, ³ J _{CH2, CH2} = 7.6, CH ₂ -Ph).

Example 6. Synthesis of 5-bromo-1- (3-methyl-but-2-en-1-yl) uracil

The substance was obtained analogously to example 2 with a yield of 68%. R _f 0.48 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 11.70 ss (1H, NH), 8.10 s (1H, 6-H), 5.22 m (1H, ³ J _{CH, CH2} = 6.9, ⁴ J _{CH3, CH} = 1.2, CH), 4.27 d (2H, ³ J _{CH2, CH} = 6.9, CH ₂ ), 1.71 d (6H, ⁴ J _{CH3, CH} = 1.2, CH ₃ ), 1.70 d (6H, ⁴ J _{CH3, CH} = 1.2, CH ₃ ). T _pl = 146-148 ° C. UV (pH 1-7): λ _max 285 nm (ε 8487); (ph 13): λ _max 280 nm (ε 6361).

Example 7. Synthesis of 5-bromo-1- (2-phenylethyl) uracil

The substance was obtained analogously to example 4 with a yield of 46%. R _f 0.47 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, DMSO-D ₆ ): 11.71 ss (1H, NH), 8.06 s (1H, 6H), 7.34-7.19 m (5H, Ph), 3.90 t (2H, ³ J _{CH2, CH2} = 7.6, CH ₂ -N), 2.90 t (2H, ³ J _{CH2, CH2} = 7.6, CH ₂ -Ph).

Example 8. Synthesis of 5-bromo-1- (3-phenylpropyl) uracil

The substance was obtained analogously to example 4 with a yield of 51%. R _f 0.47 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, DMSO-D ₆ ): 11.66 ss (1H, NH), 8.18 s (1H, 6H), 7.31-7.13 m (5H, Ph), 3.72 t (2H, ³ J _{CH2, CH2} = 7.3, CH ₂ -N), 2.60 t (2H, ³ J _{CH2, CH2} = 7.8, CH ₂ -Ph), 1.94 m (1H, ² J _{CHA, CHB} = 14.8, ³ J _{CHA, CH2} = 7.8, ³ J _{CHA, CH2} = _7.3, CH _A), 1.90 m (1H, ² J _{CHB, CHA} = 14.8, J ³ _{CHB, CH2} = _7.8, ³ J _{CHB, CH2} = _7.3, CH _B).

Example 9. Synthesis of 1- (propin-2-yl) -5-ethyluracil

To a solution of 0.308 g (2.026 mmol) of 1,8-diazobicyclo [5.4.0] undec-7-ene in 6 ml of dry DMF was added 0.278 g (1.984 mmol) of 5-ethyluracil and stirred for 15 minutes. To the resulting mixture at 20 ° C, 0.238 g (2.005 mmol) of propargyl bromide was added dropwise over 1 min and left overnight at room temperature. After completion of the reaction, the solvent was distilled off in vacuo, the residue was diluted with methylene chloride (60 ml), transferred to a separatory funnel and washed with water (30 ml). The aqueous layer was extracted with methylene chloride (4 × 15 ml). The organic layers after washing and extraction were combined, washed with water (4 × 30 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue was applied to a silica gel column (15 g) for separation. The column was washed with methylene chloride, the products were eluted with methylene chloride – ethanol systems, 100: 1 and 100: 2. (v / v). The fractions containing the product were combined, evaporated to dryness in vacuo. The yield of 1- (propin-2-yl) -5-ethyluracil 0.118 g (33% based on 5-ethyluracil), R _f 0.44 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 9.00 ct (1H, NH), 7.19 ct (1H, 6-H), 4.55 d (2H, ³ J _{CH2, CH} = 2.5, CH ₂ ), 2.46 t (1H , ³ J _{CH, CH2} = 2.5, CH), 2.39 q (2H, ³ J _{CH2, CH3} = 7.5, CH ₂ ), 1.15 t (3H, ³ J _{CH3, CH2} = 7.5, CH ₃ ). ¹³ C NMR (100 MHz, CDCl ₃ ): 163.60 (C4), 150.44 (C2), 137.70 (C6), 117.54 (C5), 76.53 (CH≡), 75.24 (CH≡), 36.85 (CH ₂ ), 20.21 (CH ₂ ), 12.89 (CH ₃ ).

Example 10. Synthesis of 1- (3-methylbut-2-en-1-yl) -5-ethyluracil

To a solution of 0.270 g (1.771 mmol) of 1,8-diazobicyclo [5.4.0] undec-7-ene in 5.2 ml of dry DMF was added 0.243 g (1.734 mmol) of 5-ethyluracil and stirred for 15 minutes. 0.258 g (1.734 mmol) of isoprenyl bromide was added dropwise to the reaction mixture at 20 ° C over 1 min and left overnight at room temperature. After completion of the reaction, the solvent was distilled off in vacuo, the residue was diluted with methylene chloride (60 ml), transferred to a separatory funnel and washed with water (30 ml). The aqueous layer was extracted with methylene chloride (4 × 15 ml). The organic layers after washing and extraction were combined, washed with water (4 × 30 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue was applied to a silica gel column (15 g) for separation. The column was washed with methylene chloride, the products were eluted with methylene chloride – ethanol systems, 100: 1 and 100: 2. (v / v). The fractions containing the product were combined, evaporated to dryness in vacuo. The yield of the product is 0.133 g (31% based on 5-ethyluracil), R _f 0.46 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 8.93 wh (1H, NH), 6.89 d (1H, ⁴ J _{6-H, CH2} = 1.1, 6-H), 5.22 m (1H, ³ J _{CH, CH2} = 7.3, ³ J _{CH, CH3} = 1.4, CH), 4.31 d (2H, ³ J _{CH2, CH} = 7.3, CH ₂ ), 2.35 dc (1H, ³ J _{CH2, CH3} = 7.4, ⁴ J _CH2.6H = 1.1 , CH ₂ ), 1.78 d (3H, ⁴ J _{CH3, CH} = 1.4, CH ₃ ), 1.76 g (3H, CH ₃ ), 1.12 t (6H, ⁴ J _{CH3, CH} = 7.4, CH ₃ ). ¹³ C-NMR (100 MHz, CDCl ₃ ): 163.89 (C4), 151.03 (C2), 139.27 (C =), 138.96 (C6), 116.64 (C5), 118.10 (CH =), 45.40 (CH ₂ ), 25.86 (CH ₃ ), 20.19 (CH ₂ ), 18.20 (CH ₃ ), 13.02 (CH ₃ ).

Example 11. Synthesis of 5-isopropyl-1- (propin-2-yl) -uracil.

The substance was obtained analogously to example 9 with a yield of 25%. R _f 0.45 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 9.04 ct (1H, NH), 7.15 ct (1H, 6-H), 4.55 d (2H, ³ J _{CH2, CH} = 2.6, CH ₂ ), 2.92 sept (1H , ³ J _{CH, CH3} = 6.9, CH), 2.46 t (1H, ³ J _{CH2, CH} = 2.6, CH), 1.17 d (6H, ³ J _{CH3, CH2} = 6.9, CH ₃ ). ¹³ C-NMR (100 MHz, CDCl ₃ ): 163.27 (C4), 150.32 (C2), 136.82 (C6), 121.94 (C5), 76.54 (C≡), 75.22 (CH≡), 36.94 (CH ₂ ), 21.64 (2XCH ₃ ), 26.07 (CH).

Example 12. Synthesis of 5-isopropyl-1- (3-methylbut-2-en-1-yl) uracil.

The substance was obtained analogously to example 10 with a yield of 31%. R _f 0.47 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 8.80 wh (1H, NH), 6.86 d (1H, ³ J _{6-H, CH} = 0.9, 6-H), 5.23 m (1H, ³ J _{CH, CH2} = 7.3, ⁴ J _{CH, CH3} = 1.4, CH), 4.31 d (2H, ³ J _{CH2, CH} = 7.3, CH ₂ ), 2.89 m (1H, ³ J _{CH, CH2} = 7.3, ⁴ J _{CH, CH3} = 0.9 , CH), 1.77 d (3H, ⁴ J _{CH3, CH} = 1.4, CH ₃ ), 1.76 us (3H, CH ₃ ), 1.14 d (6H, ⁴ J _{CH3, CH} = 7.3, CH ₃ ). ¹³ C NMR (100 MHz, CDCl ₃ ): 163.43 (C4), 150.80 (C2), 139.25 (C =), 138.01 (C6), 121.03 (C5), 118.11 (CH =), 45.55 (CH ₂ ), 25.96 (CH ₃ ), 25.85 (CH ₃ ), 21.68 (2XCH ₃ ), 18.21 (CH =).

Example 13. Synthesis of 1- (propin-2-yl) thymine

To a suspension of 1.26 g (10 mmol) of thymine in 16 ml (78 mmol) of hexamethyldisilazane (HMDS) was added a catalytic amount of ammonium sulfate and the reaction mixture was refluxed without moisture in the air until the heterocyclic base was completely dissolved. The excess GMDS was removed by evaporation in vacuo, the residue was evaporated with toluene (2 × 15 ml) and dissolved in dichloroethane (DCE). 1.1 ml (10 mmol) of an 80% solution of propargyl bromide in toluene were added dropwise to the resulting solution at 20 ° C. The reaction mixture was heated under reflux to 65 ° C for 8-9 hours without access to moisture, after which the mixture was neutralized with a 10% aqueous sodium bicarbonate solution (25 ml). The mixture was transferred to a separatory funnel, the substance was extracted from the aqueous phase with methylene chloride (4 × 30 ml). The extracts were combined, washed with saturated aqueous NaCl (25 ml), dried over anhydrous sodium sulfate, filtered, evaporated to dryness in vacuo. The material was purified by recrystallization from ethyl alcohol (15 ml). The yield of 1- (propin-2-yl) thymine is 808 mg (49%) as white fluffy crystals. R _f 0.45 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 8.84 wh (1H, NH ³ ), 7.18 q (1H, ⁴ J _{6H, 5-CH3} = 1.2, 6H), 4.46 d (2H, ⁴ J _{CH2, CH} = 2.6 , CH ₂ ), 2.39 t (1H, CH), 1.89 d (3H, ⁴ J _5-CH3,6H = 1.2, 5-CH ₃ ).

Example 14. Synthesis of 1-benzylthymine

The substance was obtained analogously to example 3 with a yield of 53%. R _f 0.53 (methylene chloride-ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 8.59 wh (1H, NH ³ ), 7.35-7.17 m (5H, Bn), 6.90 k (1H, ⁴ J _{6H, 5-CH3} = 1.2, 6H), 4.82 s (2H, CH ₂ in Bn), 1.81 d (3H, ⁴ J _5-CH3.6H = 1.2, 5-CH ₃ ).

Example 15. Synthesis of 1-benzyluracil

The substance was obtained analogously to example 3 with a yield of 54%. R _f 0.50 (methylene chloride - ethanol, 20: 1, v / v). ¹ H NMR (400 MHz, CDCl ₃ ): 8.73 us (1H, NH ³ ), 7.34-7.19 m (5H, Bn), 7.08 d (1H, ³ J _{6H, 5H} = 7.9, 6H), 5.62 dd (1H , ³ J _5H.6H = 7.9, ⁴ J _{5H, N (3) H} = 1.8, 5H), 4.84 s (2H, CH ₂ , Bn).

Example 16. The synthesis of ethyl ester of 1-carboxymethylthymine

A mixture of 3.15 g (25 mmol) of thymine, 20 ml of hexamethyldisilazane (HMDS) and 10 mg of ammonium sulfate was boiled until completely dissolved without moisture, evaporated to dryness in vacuo, then evaporated with dry 1,2-dichloroethane (10 ml). To the residue were added 50 ml of dichloroethane (DCE) and 3.0 ml (27 mmol) of bromoethyl acetate. The reaction mixture was heated for 2.5 hours at 80 ° C without access to air moisture. After cooling to 20 ° C, the mixture was evaporated to dryness in vacuo, 100 ml of ethanol was added to the residue, and the precipitate formed was filtered off and washed with chloroform (100 ml). The combined filtrates containing the product were evaporated to dryness; the residue was recrystallized from ethanol. Yield 3.9 g (74%). R _ƒ 0.41 (dichloromethane: ethanol -9: 1, v / v). T _pl, 170-171 ° C. ¹ H-NMR (400 MHz, CDCl ₃ ): 7.19 s (1H, J _6.5 = 1.2, H-6 Thy), 4.24 s (2H, CH ₂ ), 3.98 k (2H, CH ₂ CH ₃ ), 1.74 d (3H, CH ₃ Thy), 1.02 t (3H, CH ₂ CH ₃ ).

Example 17. Synthesis of 1- (carboxamidomethyl) thymine

100 mg (0.472 mmol) of 1-carboxymethylthymine ethyl ester was treated with a 5M solution of ammonia in methanol (47.2 mmol, 9.4 ml). The reaction mixture was kept at room temperature for 20 hours. During the course of ammonolysis, the reaction product precipitated as white crystals. The reaction mixture was evaporated in vacuo, evaporated with methanol (2 × 5 ml) and dried over P ₂ O ₅ for 2 hours. Yield 70 mg (81%). R _ƒ 0.08 (dichloromethane: ethanol - 9: 1 v / v). ¹ H-NMR (400 MHz, DMSO-D ₆ ): 10.67 us (NH ³ Thy), 7.55 br. from. (1H, C (O) NH ₂ ), 7.40 q (1H, J _6.5 = 1.1, H-6 Thy), 7.16 br. from. (1H, C (O) NH ₂ ), 4.23 s (2H, CH ₂ ), 1.74 d (3H, CH ₃ -Thy).

Example 18. Synthesis of 1- (hydroxypropylaminocarbonylmethyl) thymine

A mixture of 412 mg (2 mmol) of 1-carboxymethylthymine ethyl ester and 0.76 ml (10 mmol) of 3-aminopropanol in 5 ml of ethyl alcohol was boiled for 5 hours. The precipitate was filtered off, washed with ethanol and dried over P ₂ O ₅ . Yield 450 mg (93%). R _ƒ 0.66 (chloroform: ethanol - 8: 2, v / v). _Mp 244-246 ° C. ¹ H-NMR (400 MHz, DMSO-D ₆ ): 10.29 cs (1H, NH ³ Thy), 7.92 t (1H, J _CH-NH = 5.0 Hz, C (O) NH), 7.39 k (1H, J _6.5 = 1.2 Hz, H-6 Thy), 4.28 s (2H, CH ₂ ), 3.35-2.99 m (4H, C (O) NHCH ₂ CH ₂ CH ₂ OH), 1.76 d (3H, CH ₃ - Thy), 1.63 m (2H, C (O) NHCH ₂ CH ₂ CH ₂ OH). UV (pH 1-7): λ _max 272 nm (ε 9580); (pH 13): λ _max 272 nm (ε 7960).

Example 19. Determining the inhibitory ability of derivatives of nucleic bases in relation to PARP using spectrofluorimetric detection of residual NAD ⁺ .

Pre-prepared: 2M solution of KOH in distilled water; 20% solution of acetophenone in ethyl alcohol; 88% solution of formic acid in distilled water; Enzymatic reaction buffer (PARP buffer): 50 mM Tris (pH 8.0), 2 mM MgCl ₂ in distilled water (milli-Q); 1.25 μM solution of NAD + in the PARP buffer; solutions of PARP inhibitors in dimethyl sulfoxide at a concentration of 5 mM and diluted them 10 times with PARP buffer.

Next, the activity of the commercial preparation PARP-1 was determined and IC ₅₀ values were measured. 20 μl of activated DNA, 20 μl of inhibitor solution, 1 μl of PARP-1 in 19 μl of PARP buffer were added to 40 μl of a NAD + solution in PARP buffer and incubated at 20 ° C for 30 min. After incubation, 40 μl of KOH and acetophenone solutions were added, shaken and incubated for 10 min at 4 ° C. Then, 180 μl of formic acid solution was added, vortexed and held for 10 min at 110 ° C. The resulting mixture was diluted to 1500 μl with PARP buffer and the fluorescence of the solution was measured at 440 nm.

Fluorescence of the solution in the absence of PARP inhibitors corresponds to the minimum content of residual NAD + and maximum enzyme activity.

Fluorescence of a solution containing no enzyme, inhibitor, and activated DNA corresponds to the maximum NAD + content and zero enzyme activity.

For calibration purposes, the fluorescence of solutions with NAD + content was measured to be 2, 10, and 100 times lower than indicated above.

The determination of the residual enzyme activity and IC ₅₀ values was carried out according to three independent experiments.

Inhibitor solutions with varying degrees of dilution were used to determine IC ₅₀ values.

Table 1 IC ₅₀ values for some uracil derivatives Compound IC ₅₀ , µM nicotinamide 230 3-aminobenzamide fifty 5-iodo-1- (propin-2-yl) uracil 80 5-iodo-1- (3-methylbut-2-en-1-yl) uracil 45 5-iodo-1- (3-phenylpropyl) uracil 90 1- (propin-2-yl) -5-ethyluracil 90 1- (3-methylbut-2-en-1-yl) -5-ethyluracil 85 1- (carboxamidomethyl) thymine 85

Claims (1)

Inhibitors of poly (ADP-ribose) polymerase-1 person based on uracil derivatives of the general formula (I), (II), (III) and (IV)

where R ¹ = H, Cl, Br, I, methyl, ethyl, propyl or isopropyl; R ² , R ³ , R ⁴ , R ⁵ = H, methyl, ethyl, propyl or phenyl; R ⁶ = (CH ₂ ) _n , where n = 1-4; X = OR ² or NR ² R ³ , R ² , R ³ = H, methyl, ethyl, propyl, phenyl, 3-hydroxypropyl.