CZ309449B6 - Quinazoline derivatives as selective cyclooxygenase-1 inhibitors - Google Patents

Abstract

The solution provides quinazoline derivatives of general formula I, where- R1 is selected from the group comprising benzylamine substituted by at least one substituent R4 , aniline substituted by at least one substituent R4 , and phenol substituted by at least one substituent R4 ;- R 2 is selected from the group consisting of chlorine; bromine; a five- or six-member heteroaromatic ring containing at least one heteroatom selected from N, S, O;- R 3 is selected from the group consisting of chlorine; bromine; styryl; and a five- or six-member heteroalicyclic ring containing at least one heteroatom selected from N, S, O;- R4 is selected from the group consisting of fluorine, chlorine, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkylamino and C 1 -C 4 alkoxy.The substances of general formula I are selective COX-1 inhibitors and are therefore suitable for use as drugs.

Description

Quinazoline derivatives as selective cyclooxygenase-1 inhibitors

Field of technology

The present invention relates to quinazoline derivatives and their use.

Current state of the art

The body's inflammatory reaction occurs when the body is exposed to pathogens or an injury occurs. During this inflammatory reaction, the immune system is mobilized and a whole series of enzymes (e.g. cyclooxygenase, lipoxygenase, etc.) producing specific mediators of the inflammatory reaction, such as cytokinins, leukotrienes and prostaglandins, which mobilize other immune cells to the affected area, which is manifested by redness, swelling and pain at the site of damage. In addition, a large amount of reactive oxygen radicals are also produced, which damage DNA or proteins. Long-term exposure of the organism to an inflammatory reaction leads to aging and the development of age-related diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, etc.

The inflammatory reaction is traditionally treated with non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs (eg ibuprofen) are effective cyclooxygenase inhibitors. Cyclooxygenase (COX) is a membrane-bound enzyme that catalyzes two functions - peroxidase and fatty acid oxidation. The function of fatty acid oxidation is essential for the body's inflammatory reaction, it oxidizes arachidonic acid to prostaglandin H2, which subsequently produces other prostaglandins, mediators of the inflammatory reaction. Cyclooxygenase has two catalytically functional isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). The mentioned NSAIDs are non-specific cyclooxygenase inhibitors that inhibit both COX-1 and COX-2. With the development of next generations of NSAIDs, selective COX-2 inhibitors, so-called Coxibs, were developed. Behind this development was the finding that COX-1 genes are expressed in small amounts in most cells and tissues and are present in greater concentration only in the gastrointestinal tract and blood platelets, whereas COX-2 is not detectable under normal conditions, but its concentration increases rapidly during the inflammatory response. COX-1 was therefore considered to be a so-called constitutional enzyme, mainly ensuring the protection of the gastrointestinal tract and the proper function of blood platelets. For this reason, the development of selective COX-1 inhibitors has not been given attention.

Later, however, this idea was confronted by a number of studies, and currently COX-1 is considered to be the enzyme responsible for the initial response of the body to an inflammatory reaction, while the concentration of COX-2 only increases during it (Perrone et al., 2010, Curr. Med. Chem. 17, 3769-3805; Vitale et al., 2016, Med. Res. Rev. 36, 641-671). For example, COX-1 upregulation has been found to occur in some types of cancer (lung: Bauer, A.K. et al., 2000, Carcinogenesis. 21, 543-550, ovarian: Daikoku, T. et al., 2006, Cancer Res. , 66, 2527-2531; Gupta, R.A. et al., 2003, Cancer Res., 63, 906-911; prostate: Kirschenbaum, A. et al., 2000, Urology, 56, 671-676; cervix: Sales , K.J. et al., 2002, Cancer Res. 62, 424-432; head and neck: Erovic, B.M. et al., 2008, Eur. J. Clin. Investig. 38, 61-66; kidney: Yu, Z.H. etal. ., 2013, Asian Pac. J. Cancer Prev. 14, 3729-3734; endometrium: Sugimoto, T. et al., 2007, J. Med. Sci. 53, 177187; leukemia: Truffinet, V. et al., 2007, Int. J. Cancer. 121, 924-927; Rocca, B. et al., 2004, Leukemia. 18, 1373-1379; all reviewed in Pannunzio, A. and Coluccia, M., 2018, Pharmaceuticals. 11 , 101). In some types of cancer, the effectiveness of COX-1 inhibition on the growth and development of the disease has been directly demonstrated (breast cancer: Jeong, H.S. et al., 2010, Oncol. Rep. 24, 351-356; Kundu, N. and Fulton, A.M., 2002 , Cancer Res. 62, 2343-2346; McFadden, D.W. et al., 2006, Int. J. Oncol. 29, 10191023; ovarian: Cho M. et al., 2013, J. Cancer. 4, 6Ί\-6Ί %', Li, W. et al., 2009, Med. Oncol., 26, 170-177; Li, W. et al., 2010, Med. Oncol. 2010, 27, 98-104; Li, W. et al., 2012, Oncol. Lett. 2012, 4, 168-174; Zeng, X. and Yi, S., 2018, Int. J. Gynecol. Cancer. 28, 1085-1089; gut: Kitamura, T. et al., 2002, Carcinogenesis. 23, 1463-1466; Niho, N. et al., 2006, Cancer Sci. 97, 1011-1014; Wu,

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W. K. et al., 2009, Biochem. Biophys. Res. Commun. 382, 79-84; esophagus: Esquivias, P. et al., 2012, World J. Gastroenterol. 18, 4866-4874; Zhang, S. et al., 2014, Br. J. Cancer. 110, 2378-2388; leukemia: Habib, A. et al., 2008, Leukemia. 22, 1125-1130). Based on the results of these and others (Déry, M.C. et al. 2011, Biol. Reprod. 85, 1133-1142; Osman, W.M. et al., 2015, Int. J. Clin. Exp. Pathol. 8, 8165-8177; (Piazuelo, E. et al., 2012, Curr. Cancer Drug Targets. 12, 132-143; Tiano, H.F. et al., 2002, Cancer Res. 62, 3395-3401) research suggests that COX-1 inhibitors are potential drugs for a wide range of cancer types, such as breast, prostate, ovarian, cervical, lung, head and neck, bowel, kidney, skin, esophageal, endometrial, and leukemia.

COX-1 has also been identified as one of two key enzymes influencing the body's response to cancer treatment, and its inhibition could thus reverse resistance to chemotherapy (Roodhart, J.M., et al., 2011, Cancer Cell. 20, 370-383).

Inhibition of COX-1 in the spinal cord before surgery reduced postoperative pain (Zhu, X. et al., 2003, Pain, 104, 1523; Zhu, X. et al., 2005, Anesth. Analg., 100, 1390-1393). COX-1 inhibitors could also be used as analgesics.

The important role of COX-1 for the correct aggregation of platelets was also found, which can be used in the treatment of cardiovascular diseases such as atherosclerosis, high blood pressure, ischemic heart disease, or endothelial dysfunction (Belton, O. and Fitzgerald, D.J., 2003, Expert. Rev. Mol. Med. 5, 1-18; Francois, H. et al., 2004, Hypertension. 43, 364-369; Meek, I. L. et al., 2013, Eur. J. Clin. Pharmacol. 69, 365 -371; Mitchell, J.A. et al., 2019, Circ. Res. 125, 847-854; Toomey, S. et al., 2003, Biochem. Soc. Trans., 2003, 31, 1075-1079).

COX-1 also plays an important role in inflammatory reactions in the brain, which occur for example in Parkinson's or Alzheimer's disease, after stroke or heart attacks or during epileptic seizures (Aid, S. and Bosetti, F., 2011, Biochimie. 93 , 46-51; Calvello, R., et al., 2017, Front. Neurol. 8, 00251; Candelario-Jalil, E. et al., 2003, J. Neurochem. 86, 545-555; Szekely, C. A. et al. al., 2008, Neurology. 70, 2291-2298; Tanaka, S. et al., 2009, Neurosci. Res. 65, 79-87). In epileptic seizures, inhibition of COX-1 delayed the onset of epileptic seizures (Tanaka, S. et al., 2009, Neurosci. Res. 65, 79-87). The COX-1 inhibitors P6 and Mofezolac, in turn, effectively prevented the increase in prostaglandin production in brain inflammatory response models by regulating the NFkB signaling pathway (Calvello, R., et al., 2017, Front. Neurol. 8, 00251).

Selective inhibition of cyclooxygenase-1 prolonged pregnancy in mice without adverse effects on the ductus arteriosus, which may be useful in the treatment of tocolysis (Loftin, C.D. et al., 2002, J. Clin. Invest. 110, 549-557. doi : 10.1172/JCI14924.)

Since the long-term use of commercially available NSAIDs is associated with a variety of serious side effects, the demand for new, safer NSAIDs is still high. Selective COX-1 inhibitors can thus be a new direction in the development of anti-inflammatory substances with potential activity in diseases other than those treated with traditional NSAIDs.

Quinazolines are heterocyclic compounds active as anti-inflammatory substances, anticonvulsants, analgesics, etc. There are a few quinazoline substances used as anti-inflammatory substances on the market, e.g. tryptanthrin, proquazone, or fluoroquazone. Proquazone and fluproquazone belong to the third generation of NSAIDs, which excel in their effectiveness and safety. Nevertheless, the potential of quinazolines to inhibit COX is not well studied. Only a few quinazolines have been identified as selective COX-2 inhibitors. (Abdel-Aziz et al., 2016, Eur. J. Med. Chem. 121, 410-421; El-Feky, S.A. et al., 2017, Orient J. Chem. 33, 707-716; Hara et al. , 2017, J. Adv. Chem. 13, 5923-5931; Kavitha et al., 2019, Indo Am. J. Pharm. Sci. 06, 4032-4037; Kumar et al., 2003, Bioorg. Med. Chem. 11, 5293-5299; Laddha, S.S. et al., 2006, ARKIVOC. xi, 1-20; Rajput, C.S. and Singhal, S., 2013, J. Pharm. 2013, 907525; Sakr et al., 2019, Bioorg. .Chem. 84, 76-86).

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The essence of the invention

The subject of the invention are quinazoline derivatives of the general formula I (I) where

R 1 is selected from the group consisting of benzylamine substituted with at least one R 4 substituent, aniline substituted with at least one R 4 substituent, and phenol substituted with at least one R 4 substituent;

R 2 is selected from the group consisting of chlorine; bromine; a five- or six-membered heteroaromatic ring containing at least one heteroatom selected from N, S, O;

R 3 is selected from the group consisting of chlorine; bromine; styryl; and a five- or six-membered heteroalicyclic ring containing at least one heteroatom selected from N, S, O;

R 4 is selected from the group consisting of fluoro, chloro, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkylamino and C 1 -C 4 alkoxy.

Preferably, the five-membered or six-membered heteroaromatic ring containing at least one heteroatom selected from N, S, O is selected from thiophenyl, furanyl, pyrrolyl, thiazolyl, imidazolyl, oxazolyl, pyridinyl, pyrimidinyl, oxazinyl.

In a preferred embodiment, R 2 is selected from bromo athiophenyl substituents.

Preferably, the five-membered or six-membered heteroalicyclic ring containing at least one heteroatom selected from N, S, O is selected from the group consisting of morpholinyl, piperidinyl and tetrahydropyranyl.

In a preferred embodiment, R3 is selected from the substituents styryl, chlorine, morpholinyl.

Advantageously, the R 1 substituent is substituted by one R 4 substituent in the para position, and optionally also by another R 4 substituent.

Advantageously, the substituent R4 is selected from the group comprising fluorine, methyl, ethyl, isopropyl, propyl, methoxy, ethoxy, isopropoxy, propoxy.

The compounds of general formula I are selective COX-1 inhibitors, which predisposes them to use in medicine.

The subject of the present invention is therefore quinazoline derivatives of general formula I for use as medicine.

More specifically, the subject of the present invention are quinazoline derivatives of general formula I for use in the treatment of diseases selected from cancer, tocolysis, neurodegenerative diseases, cardiovascular diseases, and postoperative pain. The effectiveness of substances selectively inhibiting COX-1 in the treatment of these diseases and conditions is generally known from the literature.

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Cancers suitable for treatment with selective COX-1 inhibitors include, in particular, breast cancer; prostate cancer; epithelial ovarian cancer; cervical cancer, lung cancer; neck and head cancer; bowel cancer; kidney cancer; skin cancer; esophageal cancer; endometrial cancer; leukemia.

Cardiovascular diseases for which treatment with selective COX-1 inhibitors is suitable include mainly atherosclerosis, high blood pressure, ischemic heart disease, and endothelial dysfunction.

Furthermore, the subject of the present invention of the present invention are quinazoline derivatives of the general formula I for use as drugs reducing the resistance of cancer cells to chemotherapy.

Substances of the general formula I can be prepared by analogs of the procedures known from the literature or the procedures listed below.

Derivatives of general formula I with R3 = styryl can be prepared from anthranilic or 2-amino-4-bromobenzoic acid by successive reactions, first with acetic anhydride, then with aqueous ammonia, then with benzaldehyde, and finally with POCl3 (Scheme 1). This synthesis is simple and cheap, and can be used to obtain gram quantities of the product. The yield of the reaction ranges from 80 to 90%.

Further derivatives of general formula I can be obtained by subsequent substitution of chlorine (Ri) in position 4 with substituted aniline, benzylamine or phenol. By substituting bromine in position 7 with five-membered or six-membered heteroaromatics by reaction with appropriate boronic acids or their esters, other derivatives of general formula I are obtained.

Scheme 1: Reagents and examples of suitable conditions: a) Acetic anhydride, 120 °C, 3 h; b) aqueous ammonia, reflux, 3 h; c) benzaldehyde, acetic acid, reflux, 12 h; d) POCl 3 , dimethylaminopyridine, toluene, reflux, 5 h.

Derivatives of general formula I with R 3 = Cl can be prepared from 2-amino-4-bromobenzoic acid by successive reactions, first with urea and then with POCl 3 (Scheme 2). The yield of the reaction is around 75%. Subsequently, further substitutions can be made in positions 4 and possibly 7.

Scheme 2: Reagents and examples of suitable conditions: a) urea, 200 °C, 3 h; b) POCl 3 , dimethylamine, 120 °C, 4 h.

Other derivatives of general formula I can be prepared from the starting material 7-bromo-2,4-dichloroquinazoline by substituting chlorine in position 4 with substituted aniline, benzylamine, or phenol. And then possibly by reaction of bromine in position 7 with organic boronic acids or their esters.

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Derivatives of general formula I with R3 = morpholinyl can be prepared from the above-mentioned derivatives prepared from 7-bromo-2,4-dichloroquinazoline by reacting them with morpholine in a basic medium. The yield of the reaction is around 90%.

Examples of implementation of the invention

General preparation of 4-chloro-2-styrylquinazoline and 7-bromo-4-chloro-2-styrylquinazoline:

To anthranilic or 2-amino-4-bromobenzoic acid (36.5 mmol) was added acetic anhydride (AC 2 O, 20 mL, 36.5 mmol) and the mixture was stirred for 3 h at 120 °C. The AC2O was then evaporated and aqueous ammonia (NH4OH, 28% vol, 40 mL) was added to the residue. The reaction mixture was stirred for 3 h at 120 °C, then cooled to room temperature, filtered through a Buchner funnel, washed with water and methanol, and the solid portion was evaporated. To this solid (31.2 mmol) was then added acetic acid (AcOH, 30 mL) and benzaldehyde (32.5 mmol) and the reaction mixture was stirred at 120 °C overnight. The reaction mixture was then cooled to room temperature, the resulting solid was filtered through a Buchner funnel, washed with methanol and dried on the evaporator. This solid (18.7 mmol) was dissolved in toluene and POCl 3 (37.4 mmol) and A/A-dimethylaniline (37.4 mmol) were added and the resulting reaction mixture was stirred for 5 h at 120 °C. Then the toluene was evaporated on the evaporator, the evaporator was dissolved in chloroform and washed with water. The chloroform was evaporated and the residue was purified by column chromatography on silica gel. Chloroform was used as eluent.

General preparation of 7-bromo-2,4-dichloroquinazoline:

Urea (370 mmol) was added to 2-amino-4-bromobenzoic acid (37.0 mmol) and the substances were rubbed together in a mortar and pestle. The mixture was then placed in a drying oven heated to 200 °C for 3 h. Subsequently, the mixture was cooled, resuspended in water and filtered through a Bůchner funnel, and the solid portion was dried on the evaporator. POCl3 (30 mL, 328 mmol) and ΛξΑ-dimethylaniline (49.8 mmol) were added to the obtained solid (24.9 mmol) and the mixture was stirred for 4 h at 120 °C. The POCl3 was then evaporated, the residue dissolved in dichloromethane and washed with water. Dichloromethane was evaporated on the evaporator and the residue was purified by column chromatography on silica gel, where a petroleum ether/ethyl acetate gradient from 10/1 (v/v) to 5/1 (v/v) was used as eluent. Pure 7-bromo-2,4-dichloroquinazoline was obtained as a yellowish solid in 74% yield.

General procedure for the preparation of quinazoline derivatives of general formula I with R3 = styryl:

The synthesis is based on 4-chloro-2-styrylquinazoline or 7-bromo-4-chloro-2-styrylquinazoline, which reacts with the appropriate benzylamine, aniline or phenol in a basic environment. The first series of quinazoline derivatives of general formula I with R3 = styryl was obtained by the reaction. The reaction yield ranges from 60 to 80%.

By substituting bromine in position 7 with five-membered or six-membered heteroaromatics in the first series of quinazoline derivatives of the general formula I with R3 = styryl, by reaction with the appropriate boronic acids or esters, a second series of quinazoline derivatives of the general formula I with R3 = styryl can be prepared. The yield of the reaction ranged from 60 to 90%.

General procedure for the preparation of quinazoline derivatives of general formula I with R3 = Cl:

The synthesis is based on 7-bromo-2,4-dichloroquinazoline, which reacts with the appropriate benzylamine/aniline/phenol in a basic environment. The reaction yield ranged from 75 to 90%. The products thus obtained were used in the next reaction with the respective boronic acids or esters. A third series of quinazoline derivatives was thus obtained, this time of general formula I with R3 = Cl. The yield of the reaction ranged from 60 to 90%.

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The subsequent reaction of derivatives of the third series with a secondary amine (morpholine) produces derivatives of the fourth series. The yield of the reaction is around 90%.

Example 1: Preparation of (E)-2-styryl-N-(p-tolyl)quinazolin-4-amine (comparative example) (£)-4-Chloro-2-styrylquinazoline (0.50 g, 1.87 mmol) was dissolved in tetrahydrofuran (THF) and triethylamine (0.53 mL, 3.75 mmol), dimethylaminopyridine (DMAP, 46.0 mg, 0.38 mmol) and 4-methylaniline (p-toluidine, 402 mg, 3 .75 mmol). The reaction mixture was stirred for 20 h at 80 °C. Then the solvent was evaporated on the evaporator, the evaporator was dissolved in chloroform and washed with water. The chloroform was then evaporated and the resulting derivative was purified by column chromatography. As eluent, a mixture of hexane/ethyl acetate was used in a gradient from 2/1 (v/v) to 1/1 (v/v). (£)-2-styryl-A-(p-tolyl)quinazolin-4-amine was obtained as a yellowish solid in 58% yield.

Ή NMR (CDCl 2 ): δ 8.00 (1H, d, J = 15.8 Hz, CH); 7.91-7.88 (1H, m, Ar); 7.85 - 7.82 (1H, m, Ar); 7.80-7.73 (3H, m, Ar); 7.65 - 7.61 (2H, m, Ar); 7.49 (1H, ddd, J = 1.2, 7.0, 8.2 Hz, Ar); 7.42 - 7.37 (3H, m, Ar); 7.35 - 7.30 (1H, m, Ar); 7.29-7.23 (2H, m, Ar, CH); 2.41 (3H, s, CH ₃ ). ¹³ C NMR (CDCl 2 ): δ 160.7; 156.8; 150.9; 137.9; 136.5; 135.9; 133.8; 132.9; 129.5; 129.4; 128.8; 128.7; 127.6; 125.9; 121.4; 120.3; 113.9; 21.0. HRMS(EI) m/z: calcd for C23H19N3: 337.1579. Found: 337.1580.

Example 2: Preparation of (E)-N-(4-Methoxyphenyl)-2-styrylquinazolin-4-amine (Comparative Example) (£)-4-Chloro-2-styrylquinazoline (0.50 g, 1.87 mmol) was dissolved in THF and triethylamine (0.53 mL, 3.75 mmol), DMAP (46.0 mg, 0.38 mmol) and 4-methoxyaniline (p-anisidine, 462 mg, 3.75 mmol) were added. The reaction mixture was stirred for 20 h at 80 °C. Then the solvent was evaporated on the evaporator, the evaporator was dissolved in chloroform and washed with water. The chloroform was then evaporated and the resulting derivative was purified by column chromatography. As eluent, a mixture of hexane/ethyl acetate was used in a gradient from 2/1 (v/v) to 1/1 (v/v). (£)-A-(4-Methoxyphenyl)-2-styrylquinazolin-4-amine was obtained as a yellow solid in 54% yield.

Ή NMR (CDCl 2 ): δ 7.96 (1H, d, J = 15.8 Hz, CH); 7.89 (1H, dq, J = 0.6; 8.4 Hz, Ar); 7.84-7.81 (1H, m, Ar); 7.80-7.73 (3H, m, Ar); 7.64-7.60 (2H, m, Ar); 7.49 (1H, ddd, J = 1.2; 7.0; 8.2 Hz, Ar); 7.41 - 7.29 (3H, m, Ar); 7.24 (1H, d, J = 15.8 Hz, CH); 7.03 - 6.98 (2H, m, Ar); 6.77-6.63 (1H, m, NH); 3.88 (3H, s, CH ₃ ). ¹³ C NMR (CDCl 2 ): δ 160.7; 157.0; 156.5; 150.8; 137.8; 136.5; 132.9; 131.5; 128.7; 127.6; 125.9; 123.3; 120.3; 114.1; 113.9; 55.5. HRMS(EI) m/z: calcd for C23H19N3O: 353.1528. Found: 353.1531.

Example 3: Preparation of (E)-4-((2-Styrylquinazolin-4-yl)oxy)aniline (Comparative Example) (£)-4-Chloro-2-styrylquinazoline (0.50 g, 1.87 mmol) was dissolved in THF and triethylamine (0.53 mL, 3.75 mmol), DMAP (46.0 mg, 0.38 mmol) and 4-aminophenol (409 mg, 3.75 mmol) were added. The reaction mixture was stirred for 20 h at 80 °C. Then the solvent was evaporated on the evaporator, the evaporator was dissolved in chloroform and washed with water. The chloroform was then evaporated and the resulting derivative was purified by column chromatography. As eluent, a mixture of hexane/ethyl acetate was used in a gradient from 2/1 (v/v) to 1/1 (v/v). (£)-4-((2-styrylquinazolin-4-yl)oxy)aniline was obtained as a yellowish solid in 71% yield.

Ή NMR (CDCl 2 ): δ 8.32 (1H, ddd, J= 0.7; 1.5; 8.2 Hz, Ar); 7.76 (1H, d, J = 15.8 Hz, CH); 7.59-7.53 (3H, m, Ar); 7.38 - 7.27 (3H, m, Ar); 7.19 (1H, d, J = 15.8 Hz, CH); 7.17 - 7.12 (2H, m, Ar); 6.83-6.77 (2H, m, Ar); 3.73 (2H, s, NH ₂ ). ¹³ C NMR (CDCl 2 ): δ 166.5; 160.5; 152.3; 144.7; 143.9; 138.5; 138.5; 136.2; 133.9; 128.7; 127.9; 127.7; 127.5; 126.6; 123.8; 123.7; 122.6; 122.5; 115.7; 115.6; 115.3. HRMS(EI) m/z: calcd for C22H17N3O: 339.1372. Found: 339.1374.

Example 4: Preparation of (E)-7-bromo-2-styryl-N-(p-tolyl)-4-aminoquinazoline

-6CZ 309449 B6 (£)-7-Bromo-4-chloro-2-styrylquinazoline (646 mg, 1.87 mmol) was dissolved in toluene and triethylamine (0.53 mL, 3.75 mmol) was added, DMAP ( 46.0 mg, 0.38 mmol) and 4-methylaniline (p-toluidine, 402 mg, 3.75 mmol). The reaction mixture was stirred for 20 h at 120 °C. Then the solvent was evaporated on the evaporator, the evaporator was dissolved in chloroform and washed with water. The chloroform was then evaporated and the resulting derivative was purified by column chromatography. A mixture of hexane/ethyl acetate 4/1 (v/v) was used as eluent. (£)-7-Bromo-2-styryl-N-(p-tolyl)-4-aminoquinazoline was obtained as a yellowish solid in 53% yield.

Ή NMR (CDCl 2 ): δ 9.83 (1H, s, Ar); 8.48 (1H, d, J = 8.9 Hz, Ar); 7.94 (1H, d, J = 2.0 Hz, Ar); 7.88-7.81 (3H, m, Ar, CH); 7.73 (1H, dd, J = 2.1; 8.8 Hz, Ar); 7.71-7.67 (2H, m, Ar); 7.46 7.40 (2H, m, Ar); 7.40-7.34 (1H, m, Ar); 7.28 (2H, d, J = 8.2 Hz, Ar); 7.17 (1H, d, J = 15.9 Hz, CH); 2.34 (3H, s, CH ₃ ). ¹³ C NMR (CDCl 2 ): δ 160.9; 157.2; 151.6; 137.5; 136.5; 135.7; 132.9; 129.5; 129.1; 128.9; 128.6; 127.6; 126.6; 125.3; 122.2; 113.0; 20.6. HRMS(ESI) m/z: calcd for C 23 Hi ₉ N ₃ Br (M + H) ⁺ : 416.07569. Found: 416.07529.

Example 5: Preparation of (E)-7-bromo-N-(3,4-difluorobenzyl)-2-styryl-4-aminoquinazoline (£)-7-Bromo-4-chloro-2-styrylquinazoline (646 mg, 1, 87 mmol) was dissolved in toluene and triethylamine (0.53 mL, 3.75 mmol), DMAP (46.0 mg, 0.38 mmol) and 3,4-difluorobenzylamine (537 mg, 3.75 mmol) were added. . The reaction mixture was stirred for 20 h at 120 °C. Then the solvent was evaporated on the evaporator, the evaporator was dissolved in chloroform and washed with water. The chloroform was then evaporated and the resulting derivative was purified by column chromatography. A mixture of hexane/ethyl acetate 4/1 (v/v) was used as eluent. (£)-7-Bromo-A-(3,4-difluorobenzyl)-2-styryl-4-aminoquinazoline was obtained as a yellowish solid in 93% yield.

Ή NMR (CDCl 3 ): δ 8.03 (1H, dd, J = 0.6; 1.8 Hz, Ar); 7.97 (1H, d, J = 15.8 Hz, CH); 7.65-7.59 (2H, m, Ar); 7.54 (1H, dd, J = 0.6; 8.8 Hz, Ar); 7.50 (1H, dd, J = 1.8; 8.7 Hz, Ar); 7.42 - 7.37 (2H, m, Ar); 7.36 - 7.31 (1H, m, Ar); 7.31 - 7.26 (1H, m, Ar); 7.22 - 7.12 (3H, m, Ar); 5.93 (1H, t, J = 5.7 Hz, HH); 4.93 (2H, d, J = 5.5 Hz, CH ₂ ); ¹³ C NMR (CDCl 2 ): δ 161.7; 158.6; 151.8 (d, J = 9.7 Hz); 151.6; 151.1 (d, J = 12.6 Hz); 138.2; 136.2; 135.5 (d, J= 6.6 Hz); 135.4 (d, J = 5.0 Hz); 131.0; 129.0; 128.93; 128.86; 128.8; 128.4; 128.2; 127.6; 127.2; 123.9 (dd, J= 3.6; 6.2 Hz); 121.8; 117.6 (d, J = 17.3 Hz); 117.0 (d, J = 17.4 Hz); 112.3; 105.0; 44.3. HRMS(ESI) m/z: calcd for C 23 Hi ₇ N ₃ BrF 2 (M + H) ⁺ : 452.05684. Found: 452.05619.

Example 6: Preparation of (E)-2-styryl-N-(p-tolyl)-7-(thiophen-2-yl)-4-aminoquinazoline (£)-7-Bromo-2-styryl-A-(p- tolyl)-4-aminoquinazone (300 mg, 0.72 mmol) was dissolved in toluene/dioxane/water (6 mL, 10/5/8, v/v/v) and K2CO3 (300 mg, 2, 16 mmol), thiophene-2-boronic acid pinacholester (212 mg, 1.01 mmol), and 5% PdCl 2 (PPh 3 ) 2 (340 mg). The reaction mixture was stirred at 90 °C overnight. The reaction mixture was then cooled to room temperature and extracted with ethyl acetate. The combined organic extracts were washed with water, dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography on silica gel. A mixture of hexane/ethyl acetate 4/1 (v/v) was used as eluent. (£)-2-Styryl-A-(p-tolyl)-7-(thiophen-2-yl)-4-aminoquinazoline was obtained as a yellowish solid in 77% yield.

Ή NMR (CDCl 2 ): δ 8.09 (1H, d, J = 1.8 Hz, Ar); 7.98 (1H, d, J = 15.8 Hz, CH); 7.80 (1H, d, J = 8.6 Hz, Ar); 7.78-7.73 (2H, m, Ar); 7.70 (1H, dd, J = 1.9; 8.5 Hz, Ar); 7.65-7.60 (2H, m, Ar); 7.50 (1H, dd, J = 1.2; 3.6 Hz, Ar); 7.43 - 7.37 (4H, m, Ar, HH); 7.36 - 7.31 (1H, m, Ar); 7.28 7.22 (3H, m, Ar, CH); 7.13 (1H, dd, J = 3.6; 5.1 Hz, Ar); 2.41 (3H, s, CH ₃ ). ¹³ C NMR (CDCl 2 ): δ 161.2; 156.6; 142.7; 138.7; 138.2; 136.4; 135.9; 133.9; 129.4; 128.8; 128.7; 128.4; 127.7; 126.6; 124.8; 124.2; 123.8; 121.4; 121.2; 112.7; 21.0. HRMS(ESI) m/z: calcd for C 27 H 22 N 3 S (M + H) ⁺ : 420.15289. Found: 420.15221.

Example 7: Preparation of (E)-N-(3,4-difluorobenzyl)-2-styryl-7-(thiophen-2-yl)-4-aminoquinazoline

-7CZ 309449 B6

7-Bromo- N -(3,4-difluorobenzyl)-2-styryl-4-aminoquinazoline (326 mg, 0.72 mmol) was dissolved in toluene/dioxane/water (6 mL, 10/5/8, v/v/v) and K 2 CO 3 (300 mg, 2.16 mmol), thiophene-2-boronic acid pinacholester (212 mg, 1.01 mmol), and 5% PdChiPPlbE (350 mg) were added. The reaction mixture was stirred at 90 °C overnight. The reaction mixture was then cooled to room temperature and extracted with ethyl acetate. The combined organic extracts were washed with water, dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography on silica gel. A mixture of hexane/ethyl acetate 3/1 (v/v) was used as eluent. (£)-A-(3,4difluorobenzyl)-2-styryl-7-(thiophen-2-yl)-4-aminoquinazoline was obtained as a yellowish solid in 85% yield.

Ή NMR (CDCl 2 ): δ 8.08 (1H, dd, J = 0.7; 1.8 Hz, Ar); 7.97 (1H, d, J = 15.8 Hz, CH); 7.72 - 7.59 (4H, m, Ar); 7.50 (1H, dd, J = 1.2; 3.6 Hz, Ar); 7.42 - 7.36 (3H, m, Ar); 7.35 - 7.27 (2H, m, Ar); 7.23 (1H, d, J = 15.8 Hz, CH); 7.20 - 7.10 (3H, m, Ar); 6.02 (1H, t, J = 5.6 Hz, NH): 4.93 (2H, d, 7 = 5.6 Hz, CH ₂ ); ¹³ C NMR (CDCl 3 ): δ 161.3; 158.6; 151.6 (d, J = 12.7 Hz); 151.0 (m, 2C); 149.2 (d, J = 12.7 Hz); 148.5 (d, J = 12.6 Hz); 142.8; 138.6; 137.6; 136.4; 135.8 (dd, J = 4.0; 5.1 Hz); 128.8; 128.7; 128.4; 127.6; 126.5; 124.7; 124.2; 123.8 (dd, 7=3.6; 6.4 Hz); 123.6; 121.2; 117.5 (d, 7= 17.3 Hz); 116.9 (d, 7= 17.4 Hz); 112.5; 105.0; 44.3. HRMS(ESI) m/z: calcd for C 27 H 20 N 3 F 2 S (M + H) ⁺ : 456.13405. Found: 456.13374.

Example 8: Preparation of 2-chloro-N-(4-methoxybenzyl)-7-(thiophen-2-yl)-4-aminoquinazoline

7-Bromo-2,4-dichloroquinazoline (500 mg, 1.80 mmol) was dissolved in THF and 4-methoxybenzylamine (271 mg, 2.00 mmol) and sodium acetate (164 mg, 2.00 mmol) were added to the solution ). The reaction mixture was stirred at 65 °C overnight. Then the reaction mixture was cooled to room temperature, washed with water and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography on silica gel. As eluent, a mixture of hexane/ethyl acetate was used in a gradient from 6/1 (v/v) to 4/1 (v/v). 7-Bromo-2-chloro- N -(4-methoxybenzyl)-4-aminoquinazoline was obtained as a white solid in 88% yield.

7-Bromo-2-chloro- N -(4-methoxybenzyl)-4-aminoquinazoline (400 mg, 1.06 mmol) was dissolved in toluene/dioxane/water (9 mL, 10/5/8, v/ v/v) and K2CO3 (438 mg, 3.17 mmol), thiophene-2-boronic acid pinacholester (312 mg, 1.48 mmol), and 5% PdCh1PPlbE (508 mg) were added. The reaction mixture was stirred at 90 °C overnight. The reaction mixture was then cooled to room temperature and extracted with ethyl acetate. The combined organic extracts were washed with water, dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography on silica gel. A mixture of ethyl acetate/methanol in a gradient from 10/0 (v/v) to 9/1 (v/v) was used as eluent. 2-Chloro-N-(4-methoxybenzyl)-7-(thiophen-2-yl)-4-aminoquinazoline was obtained as a white solid in 86% yield.

Ή NMR (CDCl 3 ): δ 7.99 (1H, d, 7 = 1.7 Hz, Ar); 7.66 (1H, dd, 7=1.8; 8.6 Hz, Ar); 7.61 (1H, d, 7 = 8.6 Hz, Ar); 7.48 (1H, dd, 7 = 1.1; 3.7 Hz, Ar); 7.39 (1H, dd, 7 = 1.1; 5.1 Hz, Ar); 7.34 (2H, ddd,7 = 2.1; 3.0; 8.6 Hz, Ar); 7.13 (1H, dd, 7 = 3.7; 5.1 Hz, Ar); 6.91 (2H, ddd,7 = 2.1; 3.0; 8.7 Hz, Ar); 6.00 (1H, t, 7 = 5.3 Hz, NH): 4.78 (2H, d, 7 = 5.1 Hz, CH ₂ ); 3.82 (3H, s, OCH ₃ ). ¹³ C NMR (CDCl 2 ): δ 160.3; 159.5; 158.3; 151.4; 142.3; 139.3; 129.8; 129.2; 128.5; 127.0; 125.1; 124.0; 123.5; 121.4; 114.3; 111.9; 55.3; 45.3. HRMS(ESI) m/z: calcd for C20H17N3OCIS (M + H) ⁺ : 382.07754. Found: 382.07743.

Example 9: Preparation of 2-chloro-N-(3,4-difluorobenzyl)-7-(thiophen-2-yl)-4-aminoquinazoline

7-Bromo-2-chloro- N -(3,4-difluorobenzyl)-4-aminoquinazoline (408 mg, 1.06 mmol) was dissolved in toluene/dioxane/water (9 mL, 10/5/8, v/v/v) and K2CO3 (438 mg, 3.17 mmol), thiophene-2-boronic acid pinacholester (312 mg, 1.48 mmol), and 5% PdCh1PPlbE (510 mg) were added. The reaction mixture was stirred at 90 °C overnight. The reaction mixture was then cooled to room temperature

-8CZ 309449 B6 rooms and extracted with ethyl acetate. The combined organic extracts were washed with water, dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography on silica gel. A mixture of ethyl acetate/methanol in a gradient from 10/0 (v/v) to 9/1 (v/v) was used as eluent. 2-Chloro-N-(3,4-difluorobenzyl)-7-(thiophen-2-yl)-4-aminoquinazoline was obtained as a white solid in 68% yield.

Ή NMR (CDCl 2 ): δ 8.01 (1H, d, J = 1.8 Hz, Ar); 7.71 (1H, dd, J = 1.8; 8.6 Hz, Ar); 7.66 (1H, d, J = 8.6 Hz, Ar); 7.49 (1H, dd, J = 1.2; 3.7 Hz, Ar); 7.41 (1H, dd, J = 1.1; 5.1 Hz, Ar); 7.26 - 7.20 (1H, m, Ar); 7.19 - 7.09 (3H, m, Ar); 6.12 (1H, t, J = 5.9 Hz, NH): 4.84 (2H, d, J = 5.6 Hz, CH ₂ ). ¹³ C NMR (CDCl 2 ): δ 160.4; 158.2; 151.7 (d, J = 12.8 Hz); 151.6; 151.3 (d, J= 12.4 Hz); 149.2 (d, J = 12.7 Hz); 148.8 (d, J = 12.5 Hz); 142.1; 139.6; 134.4 (dd, J= 3.9; 5.3 Hz); 128.5; 127.1; 125.2; 124.3; 124.2 (dd, J = 3.6; 6.2 Hz); 121.3; 117.7 (d, J = 17.1 Hz); 117.3 (d, J = 17.4 Hz); 111.7; 44.6. HRMS(ESI) m/z: calcd for C 19 H 13 N 3 CIF 2 S (M + H) ⁺ : 388.04813. Found: 388.04802.

Example 10: Preparation of N-(3,4-difluorobenzyl)-2-morpholino-7-(thiophen-2-yl)-4-aminoquinazoline

2-Chloro-A-(3,4-difluorobenzyl)-7-(thiophen-2-yl)-4-aminoquinazoline (290 mg, 0.75 mmol) was dissolved in DMF and morpholine (0.20 mL , 2.25 mmol), KI (137 mg, 0.83 mmol), K 2 CO 3 (311 mg, 2.25 mmol) and diisopropylethylamine (0.40 mL, 2.25 mmol). The reaction mixture was stirred at 110 °C overnight. The reaction mixture was then cooled to room temperature and extracted with THF and ethyl acetate. The combined organic extracts were washed with water, dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography on silica gel. A mixture of hexane/ethyl acetate in a gradient from 3/1 (v/v) to 2/1 (v/v) was used as eluent. A-(3,4-difluorobenzyl)-2-morpholino-7-(thiophen-2-yl)-4-aminoquinazoline was obtained as a yellowish solid in 91% yield.

Ή NMR (CDCl 2 ): δ 7.73 (1H, d, J = 1.9 Hz, Ar); 7.54-7.42 (2H, m, Ar); 7.40-7.31 (2H, m, Ar); 7.20 (1H, ddd, J = 1.9; 7.5; 10.9 Hz, Ar); 7.16 - 7.06 (3H, m, Ar); 5.81 (1H, t, J= 5.6 Hz, NH): 4.76 (2H, d, J= 5.5 Hz, CH ₂ )· 3.87 (4H, dd, J= 3.7 ; 5.8 Hz, 2xCH ₂ ); 3.76 (4H, dd, J = 3.9; 5.6 Hz, 2xCH ₂ ). ¹³ C NMR (CDCl 2 ): δ 159.4; 159.2; 152.8; 151.6 (d, J = 13.0 Hz); 150.7 (d, J = 12.6 Hz); 149.1 (d, J = 12.5 Hz); 148.4 (d, J = 12.7 Hz); 143.5; 138.5; 135.8 (dd, J = 3.9; 5.0 Hz); 128.2; 125.9; 124.3; 123.5 (dd,J=3.7; 6.2 Hz); 122.1; 121.3; 119.5; 117.4 (d, J = 17.2 Hz); 116.6 (d, J = 17.6 Hz); 109.3; 67.0; 44.5; 44.2. HRMS(ESI) m/z: calcd for C 23 H 21 N 4 OF 2 S (M + H) ⁺ : 439.13986. Found: 439.13920.

Example 11

Testing the ability of prepared derivatives to inhibit COX-1 and COX-2 isoenzymes

The inhibitory activity of the substances was tested using an in vitro enzymatic assay. The inhibitory activity of the compounds was tested using an in vitro enzymatic assay. Sheep COX-1 (1 unit/reaction) or human recombinant COX-2 (0.5 units/reaction; both purchased from Sigma-Aldrich, USA) was added to 180 µl of 100 mM Tris buffer (pH 8.0), containing 5 μΜ porcine hematin, 18 mM Z-epinephrine, and 50 μΜ Na2EDTA. Ten μΐ of the test substance dissolved in DMSO or pure DMSO (as a control) was added to the reaction. (S)-(+)-Ibuprofen or SC-560 (5-(4-chlorophenyl)1-(4-methoxyphenyl)-3-trifluoromethylpyrazole) (both Sigma-Aldrich, USA) were used as reference inhibitors. After 5 min of incubation at room temperature, the reaction was started by adding 5 μΐ of arachidonic acid (10 μΜ) and the reaction was incubated for 20 min at 37 °C. The reaction was then terminated by adding 20 μΐ formic acid (10% v/v). The concentration of prostaglandin E2 (PGE2) was measured using the Prostaglandin E2 ELISA kit (Enzo Life Sciences, USA). The reaction mixture was diluted 1:15 in the assay buffer and incubated according to the instructions in the assay manual. The concentration of PGE2 formed during the reaction was calculated based on the measured absorbance at 405 nm, measured on a Tecan Infinite M200 microplate reader (Tecan Group, Switzerland). Inhibitory activity was calculated as percent inhibition of PGE2 production relative to the blank. First

-9CZ 309449 B6, the inhibitory activity was tested at 20 μΜ concentration of the test substance to determine whether the substance exhibits any inhibitory activity. IC50 values were then determined for substances that showed at least 90% COX-1 inhibition or 50% COX-2 inhibition at this concentration (Table 1). The level of percent inhibition was determined by the percent inhibition exhibited by the reference inhibitor, ibuprofen. All experiments were performed at least twice in duplicate.

Table 1. IC50 values of individual substances on COX-1.

Substance IC50 COX-1 (pM) IC50 COX-2 (pM) Selectivity index (COX-2/COX-1) Substance according to example 1 l.57±0.54 43.0±10.3 27.4 Substance according to example 2 1.89±0.63 37.3±9.36 19.7 Substance according to example 3 3.I4±O.99 >50 > 15.9 Substance according to ex. 4 0.78±0.64 > 50 > 64.1 Substance according to example 5 1.58±0.89 >50 >31.6 Substance according to ex. 6 0.141±0.045 > 50 > 355 Substance according to ex. 7 0.064±0.044 >50 >781 Substance according to ex. 8 0.376±0.189 > 50 > 133 Substance according to ex. 9 0.142±0.014 >50 > 355 Substance according to ex. 10 L39±0.72 > 50 > 36.0 Ibuprofen 2.19±0.78 3.30±0.96 1.51 SC-560 ().()()6:().()03 1.03:0.40 172

-10 CZ 309449 B6

Claims (10)

1. Quinazoline derivatives of general formula I where R 1 is selected from the group consisting of benzylamine substituted with at least one R 4 substituent, aniline substituted with at least one R 4 substituent, and phenol substituted with at least one R 4 substituent; R 2 is selected from the group consisting of chlorine; bromine; a five- or six-membered heteroaromatic ring containing at least one heteroatom selected from N, S, O; R 3 is selected from the group consisting of chlorine; bromine; styryl; and a five- or six-membered heteroalicyclic ring containing at least one heteroatom selected from N, S, O; R 4 is selected from the group consisting of fluoro, chloro, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkylamino and C 1 -C 4 alkoxy. 2. Quinazoline derivatives according to claim 1, general formula I, where a five-membered or six-membered heteroaromatic ring containing at least one heteroatom selected from N, S, O is selected from thiophenyl, furanyl, pyrrolyl, thiazolyl, imidazolyl, oxazolyl, pyridinyl, pyrimidinyl, oxazinyl. 3. Quinazoline derivatives according to claim 1, general formula I, where R 2 is selected from the substituents bromine and thiophenyl. 4. Quinazoline derivatives according to any one of claims 1 to 3, general formula I, where the five-membered or six-membered heteroalicyclic ring containing at least one heteroatom selected from N, S, O is selected from the group comprising morpholinyl, piperidinyl and tetrahydropyranyl. 5. Quinazoline derivatives according to any one of claims 1 to 3, general formula I, where R3 is selected from the substituents styryl, chlorine, morpholinyl. 6. Quinazoline derivatives according to any one of claims 1 to 5, general formula I, where the substituent R 1 is substituted by one substituent R 4 in the para position, and optionally also by another substituent R 4 . 7. Quinazoline derivatives according to any one of claims 1 to 6, general formula I, where the substituent R4 is selected from the group comprising amino, fluorine, methyl, ethyl, isopropyl, propyl, methoxy, ethoxy, isopropoxy, propoxy. 8. Quinazoline derivatives of general formula I according to any one of claims 1 to 7 for use as medicine. 9. Quinazoline derivatives of general formula I according to any one of claims 1 to 7 for use in the treatment of diseases selected from cancer, tocolysis, neurodegenerative diseases, cardiovascular diseases and postoperative pain. - 11 CZ 309449 B6 10. Quinazoline derivatives of general formula I according to any one of claims 1 to 7 for use as drugs for reducing the resistance of cancer cells to chemotherapy.