Field of Art

The invention relates to novel derivatives of nitrogen heterocyclic compounds, use thereof as medicaments, and to pharmaceutical preparations containing these compounds.

Background Art

Despite significant progress in targeted therapy and other procedures, conventional chemotherapy and radiotherapy are still indispensable tools in the fight against cancer. These techniques, known since the early 20th century, target the basic functions of dividing cells and are very effective against rapidly proliferating tumor cells. Unfortunately, these interventions also affect the vital processes of healthy cells and thus cause a number of side effects. The most commonly affected healthy cells include intestinal mucosa cells, bone marrow, reproductive organs, and hair follicles. As a result of anticancer therapy, a number of mutations occur that may lead to the subsequent development of secondary cancers . The second drawback of the conventional cytostatic therapy or radiation therapy is their insufficient effectiveness in some patients. Furthermore, these tumor cells often develop resistance to therapy due to rapid proliferation.

Tumor resistance to treatment is a serious problem. There are a number of mechanisms by which cells defend themselves. One of the key causes of resistance is the natural ability of all cells to repair their DNA. On the one hand, DNA repairs are indispensable for cellular life and development, but they reduce the effect of treatment during radiation and chemotherapy because they repair DNA lesions caused by the treatment. As a result, cellular DNA damage response has become a field of study in cancer research. DNA damage response contains complex, and not yet completely described, signaling cascades with a hundred different proteins. Minor interventions in these signaling pathways by means of drugs may contribute to increasing the efficacy of cytotoxic therapy. This is because tumor cells are often associated with many mutations in these pathways, as a result of which they can respond more sensitively to DNA repair processes than healthy cells, which have functional alternative pathways for DNA repair. Thus, by a specific intervention in the DNA damage response, it is possible to target the treatment to tumor cells and thus increase the efficacy of the conventional treatment, reduce the side effects of drugs, and kill tumor cells in monotherapy [JACKSON, Stephen P. and Jiri BARTEK. The DNA-damage response in human biology and disease. Nature [online]. 2009, 461(7267), 1071-1078. ISSN 1476-4687. Available from: doi: 10.1038/nature08467]. To date, a number of kinases have been identified that could be used for this purpose. The phosphatidylinositol 3 -kinase-related kinase (PIKK) family belongs amongst very interesting kinases which are widely studied in the literature. PIKK family plays a pivotal role in the control of DNA damage response. The most researched representatives are ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 -related (ATR) and DNA-dependent protein kinase (DNA-PK) [BLACKFORD, Andrew N. and Stephen P. JACKSON. ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Molecular Cell [online]. 2017, 66(6), 801-817. ISSN 1097-2765. Available from: doi: 10.1016/j.molcel.2017.05.015]. The ATR kinase is activated in response to replication stress which can be caused by a variety of endogenous and exogenous factors. In the event of replication stress, the replication fork stops and stabilizes. Inhibition of the ATR kinase results in a replication fork collapse leading to irreversible and potentially lethal DNA damage. If this damage is not resolved quickly, apoptosis can be triggered [[SALDIVAR, Joshua C., David CORTEZ and Karlene A. CIMPRICH. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nature reviews. Molecular cell biology [online] 2017, 18(10), 622-636. ISSN 1471-0072. Available from: doi: 10.1038/nrm.2017.67; LECONA, Emilio and Oscar FERNANDEZ-CAPETILLO. Targeting ATR in cancer. Nature Reviews Cancer [online]. 2018, 18(9), 586-595. ISSN 1474-1768. Available from: doi: 10.1038/s41568-018-0034-3] Due to the attractiveness of this kinase caused by its importance in cancer cells, several hundred inhibitors with varying effectiveness and selectivity have been developed [e g. GORECKI, Lukas, Martin ANDRS, Martina REZACOVA and Jan KORABECNY. Discovery of ATR kinase inhibitor berzosertib (VX-970, M6620): Clinical candidate for cancer therapy. Pharmacology & Therapeutics [online]. 2020, 107518. ISSN 1879-016X. Available from: doi: 10.1016/j.pharmthera.2020.107518; GORECKI, Lukas, Martin ANDRS and Jan KORABECNY. Clinical Candidates Targeting the ATR-CHK1-WEE1 Axis in Cancer. Cancers [online]. 2021, 13(4), 795. Available from: doi: 10.3390/cancersl3040795] These compounds can be used in the treatment of cancer both in monotherapy and in combination with standard chemo- or radiotherapeutics. Currently, the four most successful compounds are in clinical trials as monotherapeutics or as chemotherapy or radiotherapy sensitizers. In total, over 60 clinical trials have been opened. Although these known compounds have already confirmed their efficacy in the second phase of the clinical trial, progression to the third phase is still pending. This also leaves room for the development of new, more potent or more selective inhibitors. Disclosure of the Invention

The present invention provides nitrogen heterocyclic compounds of general formula I wherein X is C or N;

R ¹ is selected from C1-C6 alkyls;

Z- is -CH ₂ - or -C(O)-;

R ² is a primary or secondary amine bound via Z to the heterocyclic core, and selected from the group comprising anilino; anilino substituted by di(Cl-C6 alkyl)amino group in position 2, 3, or 4; benzylamino; benzylamino substituted by di(Cl-C6 alkyl)amino group in position 2, 3, or 4; 4- phenylpiperidin-l-yl, 1-(C1-C6 alkyl)piperazin-4-yl, 1,2,3,4-tetrahydroisoquinolinyl; 1-(C1-C6 alkanesulfonyl)piperazin-4-yl; 4-[4-(Cl-C6 alkyl)-piperazin-l-yl]piperidin-l-yl; and pharmaceutically acceptable salts thereof with alkali metals, or addition salts thereof with acids.

Addition salts with acids include in particular hydrochlorides, hydrobromides, hydrofluorides, hydroiodides, nitrates, nitrites, sulphates, bisulphates, borates, citrates, fumarates, lactates, malates, maleates, mesylates, tosylates, oxalates, tartrates, acetates, formates, salicylates, aspartates, adipates, benzoates, palmitates, stearates, besylates, phosphates, carbonates, hydrogencarbonates.

In some preferred embodiments, X is N and Z is -C(O)-.

C1-C6 alkyl is a saturated hydrocarbon residue, which may be linear, branched or cyclic. A branch or cyclic alkyl can exist for the C3-C6 alkyls. In particular, alkyl may be selected from the group comprising methyl, ethyl, propyl, butyl, isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In case of di(Cl-C6 alkyl)amino groups, the alkyls may be the same or different.

Thus, the present invention provides novel derivatives of nitrogen heterocyclic compounds, in particular 1 H-pyrrolo| 2.3-b Ipyridincs substituted in position 2 or 3 and in position 5, and lH-pyrazolo|3.4- b]pyridines substituted in the positions 3 and 5, and salts thereof. These compounds consist of three fundamental fragments that are necessary for proper ligand orientation in the ATP -binding cavity of the ATR kinase. The first fragment is a nitrogen heterocycle with a putative p-p interaction in the kinase cavity. In the present invention, the nitrogen heterocycle is a disubstituted lH-pyrrolo| 2.3-b Ipyridine or 1 H-pyrazolo| 3.4-b Ipyridine. From this central heterocycle, a bulky alkane sulfonylphenyl substituent is attached via a C-C bond at position 5. This substituent is necessary to maintain the selectivity for ATR kinase. This substituent also serves as a hydrogen bond donor to ensure the affinity for the enzyme. The other side of the molecule is preferably represented by basic substituents. The basic substituents provide for a higher binding to the enzyme cavity, especially through interactions with the anionic region of the protein. These basic „heads“ are attached to the nitrogen heterocycle at position 2 or 3 for 1 H- pyrrolo[2,3-b]pyridine and at position 3 for 1 H-pyrazolo| 3.4-b Ipyridine. via a methylene or carbonyl linker. Advantage of the methylene linker lies in the flexibility of the substituent with the presumed suitable orientation of the ligand into the anionic pocket. The carbonyl linker, on the other hand, can provide additional hydrogen bonds for the fixation of the molecule (rigid carbonyl character), whereby the kinase selectivity can be achieved in the overall proper construction of the compounds. Both of these approaches have proven to be effective.

The compounds of the present invention are preferably selected from the group:

The present invention further provides the nitrogen heterocyclic compounds of general formula I for use as medicaments, more specifically for use in the treatment of tumour diseases.

The compounds of general formula I are suitable for medical use in monotherapy or in combination therapy together with further antitumour medicaments (e.g., anthracyclin orplatinum cytostatics) and/or in combination with radiotherapy. Use in monotherapy involves the administration of the medicament of general formula I to stop the progression of the tumour growth and development, or to reduce the number of tumour cells. Use in combination therapy involves the administration of the medicament of general formula I in combination with standard radiotherapy or with standard chemotherapy, to potentiate the effect of the standard therapy against the tumour cells.

The term "treatment" refers to the administration of a medicament with the aim of reducing the symptoms of a disease. This effect may be related to slowing the progression of the disease, or improving the patient's health in any way. This includes reducing or stopping tumour cell proliferation, preventing or suppressing invasion and metastasis, promoting genomic instability and mutagenicity, preventing resistance to cell death, avoiding replication immortality, or promoting tumour suppressors.

The invention further provides a pharmaceutical composition comprising at least one nitrogen heterocyclic compound of formula I according to the invention and at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients may include carriers, fillers, binders, solvents, and the like, as known to those skilled in the art of pharmaceutical formulation.

The compounds of the invention may in particular be administered orally or parenterally. The pharmaceutical composition can be formulated into various pharmaceutical formulations, such as granules, powders, tablets, gels, capsules, syrups, emulsions, suspensions and forms for parenteral administration such as injectable formulations, infusion formulations, sprays or suppositories. These formulations can be prepared by generally known methods. A pharmaceutical formulation for injection can be obtained, for example, by the following procedure. The active ingredient is dissolved, suspended or emulsified in an aqueous medium, for example water, saline or Ringer's solution, or in an oily medium (e.g. olive oil, sesame oil, linseed oil, com oil or propylene glycol), with a dispersing ingredient (e.g. Tween ^® 80, HCO ^® 60, polyethylene glycol, carboxymethylcellulose or sodium alginate), with a preservative (e.g. methyl p-hydroxybenzoate. propyl p-hydroxybenzoate. benzyl alcohol, chlorobutanol or phenol), an isotonic agent (e.g. sodium chloride, glycerol, sorbitol or glucose), and other additives such as solubilizers (e.g. sodium salicylate, sodium acetate), or stabilizers (e.g. human serum albumin).

Nitrogen heterocyclic compounds of formula I derived from 1 H-pyrrolo[2,3-b]pyridinc. wherein the secondary amine in position 3 of the ring is attached by a methylene linker, can be prepared according to Scheme 1. In the first step, reductive amination is performed using sodium triacetoxyborohydride. In this step, an aromatic aldehyde 21 is reacted with a secondary amine to give an iminium intermediate, which is immediately reduced to a tertiary amine (compounds 22-24). In the second step, a modified Suzuki-Miyaura Cross-Coupling is performed, using a microwave reactor. This reaction of aromatic bromide (22-24) with arylboronic acid is catalyzed by Pd(dppf)Cl ₂ .DCM ([1,1'- bis(diphenylphosphino)ferrocene]dichloropalladium (II), complex with dichloromethane) and gives products 1 and 2.

²¹ 22 R = 4-phenylpiperidin-1-yl 1 R = 4-phenylpiperidin-1-yl

23 R = 4-(4-ethylpiperazin-1-yl)piperidin-1-yl 2 R = 4-(4-ethylpiperazin-1-y)piperidin-1-yl

24 R = 4-ethylpiperazin-1-yl

Scheme 1 Preparation of compounds of formula I with a structural pattern of 1 H-pyrrolo| 2.3-b Ipyridinc with a methylene linker in position 3 to the secondary amine. Reaction conditions: a) secondary amine, NaBH(OAc) ₃ (sodium triacetoxyborohydride), AcOH (acetic acid), 90 °C, THF (tetrahydrofuran)/ methanol (MeOH) (1:1), Ar (argon), 24 hours; b) arylboronic acid, Pd(dppf)Cl ₂ .DCM ([1,T- bis(diphenylphosphino)ferrocene]dichloropalladium (II), dichloromethane complex), Na ₂ CO ₃ (sodium carbonate), dioxane/water (4: 1), Ar, reaction in a microwave reactor (100 W power) at 110 °C for one hour.

Nitrogen heterocyclic compounds of formula I derived from 1 H-pyrrolo| 2.3-b Ipyridinc or 1 H- pyrazolo[3,4-b]pyridine, wherein a primary or secondary amine is attached via a carbonyl linker in position 2 or 3 of the ring, can be prepared according to the procedure shown in Scheme 2 (25-27). In the first step, a modified Suzuki-Miyaura Cross-Coupling catalyzed by Pd(dppf)Cl ₂ .DCM is performed. For products 30-35, this reaction is performed under standard conditions with heating at 110 °C for three days. In the case of products 32-35, the ester is directly hydrolyzed and the resulting products are isolated as hydrochloride salts of carboxylic acids (32 and 33), or as neutral carboxylic acid compounds (34 and 35). On the other hand, products 30 and 31 are initially isolated in the form of esters (36 and 37) which are subsequently hydrolyzed with boiling sodium hydroxide. Acids 30 and 31 are then isolated as hydrochloride salts. All carboxylic acids 30-35 are finally reacted with a primary or secondary amine to form the corresponding secondary or tertiary amide. This reaction is catalyzed by EDC.HC1 (N-(3- dimcthylaminopropyl)-N'-cthylcarbodiimidc hydrochloride), HOBt.H ₂ O (1-hydroxybenzotriazole hydrate) and DIPEA (AN-diisopropylethylamine) to give final products 3-20.

Scheme 2 Preparation of compounds of formula I derived from 1 H-pyrrolo| 2.3-b Ipyridine or 1 H- pyrazolo[3,4-b]pyridine, wherein a primary or secondary amine is attached by a carbonyl linker in position 2 or 3 of the cycle. Reaction conditions: a) arylboronic acid 28 or 29, Pd(dppf)Cl ₂ .DCM, Na ₂ CO ₃ , dioxane/water (4: 1), Ar, 110 °C, 72 hours; b) 2M NaOH (sodium hydroxide), MeOH, 110 °C, 24 hours; c) primary or secondary amine, EDC.HC1 (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), HOBt.H ₂ O (1-hydroxybenzotriazole hydrate), DIPEA (AN-diisopropylethylamine). THF, room temperature, Ar, 24 hours b) arylboronic acid 28 or 29, Pd(dppf)Cl ₂ .DCM, Na ₂ CO ₃ , dioxane/water (4: 1), Ar, reaction in a microwave reactor (100 W power) at 110 °C for one hour.

Examples of carrying out the Invention

The invention is further described in more detail by way of examples which should however not be construed as limiting the scope of the invention.

General chemical methods

Thin layer chromatography was performed on aluminum plates coated with silica gel 60 F254 (Merck, Prague, Czech Republic). Column chromatography was performed at atmospheric pressure on silica gel 100 (particle size 0.063-0.200 mm, 70-230 mesh ASTM, Fluka, Prague, Czech Republic). The chemicals necessary for the synthesis were purchased from Sigma Aldrich Co. LLC (Prague, Czech Republic) and were used without further purification. The CEM Explorer SP 12 S Class apparatus was used for reactions performed under microwave activation conditions. The analytical system consisting of Dionex Ultimate 3000 LC-MS combined with an Orbitrap Q Exactive Plus spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used to determine the mass spectrometry. The LC-MS system consists of a binary pump HHG-3400RS which is connected to a vacuum degasser, of a heated column compartment TCC-3000, an autosampler WTS-3000 and an ultraviolet detector VWD-3000. The quadrupole mass spectrometer was equipped with an electron-spray ionization source and the data were recorded in positive mode with the following parameters: spray voltage was 3.2 kV, capillary temperature was 350 °C, gas temperature was 300 °C. 'H-NMR and ¹³ C-NMR spectra were recorded on a Varian S500 spectrometer (500 and 126 MHz, respectively) in CDCl ₃ -dl (CHCl ₃ -dl : 7.26 (D), 77.16 (C) ppm), in hexadeuteriodimethylsulfoxide (DMSO-d6: 2.50 (D), 39.7 (C) ppm) or CD.OD-d4 (CH3OH -d4; 3.35, 4.78 (D), 49.3 (C) ppm). The characteristics of the individual signals were as follows: s (singlet), d (doublet), (dd) (doublet of doublets), t (triplet), p (pentet) or m (multiplet). Chemical shifts are reported in ppm (parts per million, d) relative to TMS (tetramethylsilane). The chemical shift assignment is based on standard NMR experiments ('H. ¹³ C, ¹ H- ¹ H COSY, 'H- ¹³ C HSQC, HMBC, DEPT).

Example 1 - Preparation of compounds of formula I wherein a secondary amine in position 3 is attached to 1H-pyrrolo [2, 3-b] pyridine via a methylene bridge iperidin-1-yl

23 R = 4-(4-ethylpiperazin-1 -yl)piperidin-1 -yl

24 R = 4-ethylpiperazin-1-yl

To compound 21 (1.0 eq) suspended in anhydrous THF and anhydrous MeOH (1: 1), secondary amine (2.0 eq) and acetic acid (2.0 eq) were added under an inert argon atmosphere. The mixture was then stirred for one hour at room temperature. Subsequently, sodium triacetoxyborohydride (3.0 eq) was added portionwise and the reaction was heated at 90 °C for 24 hours. After cooling to room temperature, the solvents were distilled off in vacuo and the reaction mixture was extracted between ethyl acetate (EA; 30 mL) and sodium bicarbonate (NaHCO ₃ ; 30 mL). The aqueous phase was then shaken twice more with additional EA (2 x 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate (Na ₂ SO ₄ ), the mixture was filtered and the filtrate was evaporated to dryness. The crude product was purified by column chromatography to give compounds 22-24. l-({5-bromo-lH-pyrrolo[2,3-b]pyridin-3-yl}methyl)-4-phenylpi peridine (22): Aldehyde 21 (64 mg;

0.28 mmol); 4-phenylpiperidine (90 mg; 0.56 mmol); AcOH (32 μL; 0.56 mmol) and NaBH(OAc)3 (178 mg; 0.84 mmol) in anhydrous THF (3 mL) and anhydrous MeOH (3 mL). After extraction, the resulting mixture was purified by column chromatography using mobile phase (dichloromethane (DCM) / MeOH / ammonium hydroxide (NH4OH; aqueous 25% solution)) (20: 1 :0.1) to give a pure product 22 as a pale orange solid. Yield 56%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 11.73 (s, 1H), 8.29 - 8.20 (m, 2H), 7.44 (d, J= 2.4 Hz, 1H), 7.29

7.12 (m, 5H), 3.63 (s, 2H), 3.01 - 2.93 (m, 2H), 2.48 - 2.39 (m, 1H), 2.05 - 1.98 (m, 2H), 1.75 - 1.68

(m, 2H), 1.66 - 1.55 (m, 2H). ¹³ C NMR (126 MHz, DMSO) δ 147.21, 146.46, 142.53, 129.37, 128.44, 127.09, 126.84, 126.09, 121.90, 110.52, 110.22, 53.58, 53.24, 42.06, 33.32. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₉ H ₂₁ BrN ₃ ⁺ (m/z): 370.09134; found: 370.09195.

1- [l-({5-bromo-l H-pyrrolo [2,3-b]pyridin-3-yl}methyl)piperidin-4-yl] -4-ethylpiperazine (23) :

Aldehyde 21 (174 mg; 0.773 mmol); l-ethyl-4-(piperidin-4-yl)piperazine (310 mg; 1.546 mmol); AcOH (90 μL; 1.55 mmol) and NaBH/OAcf (491 mg; 2.32 mmol) in anhydrous THF (7 mL) and anhydrous MeOH (7 mL). After extraction, the resulting mixture was purified by column chromatography using mobile phase (DCM/MeOH/NH ₄ OH (aqueous 25% solution) (9: 1:0.1) to give a pure product 23 as a pale orange solid. Yield 49%. 1HNMR(500 MHz, CD ₃ OD-d ₄ ) δ 8.26 (d, J= 2.1 Hz, 1H), 8.23 (d, J= 2.1 Hz, 1H), 7.39 (s, 1H), 3.68 (s, 2H), 3.06 - 2.97 (m, 2H), 2.75 - 2.39 (m, 10H), 2.21 (tt, J= 11.5, 3.8 Hz, 1H), 2.07 (td, J= 12.0, 2.3 Hz, 2H), 1.92 - 1.85 (m, 2H), 1.59 - 1.46 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H). ¹³ C NMR (126 MHz, CD3OD) δ 148.07, 143.94, 131.16, 128.71, 123.65, 112.09, 110.55, 62.90, 53.61, 53.56, 53.49, 53.18, 49.78, 28.89, 11.68. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for Ci ₉ H ₂ 9BrN ₅ ⁺ (m/z): 406.16008; found: 406.15994. l-({5-bromo-lH-pyrrolo[2,3-b]pyridin-3-yl}methyl)-4-ethylpip erazine (24): Aldehyde 21 (151 mg; 0.67 mmol); 1-ethylpiperazine (170 μL; 1.34 mmol); AcOH (80 μL; 1.30 mmol) and NaBH(OAc) ₃ (426 mg; 2.01 mmol) in anhydrous THF (7 mL) and anhydrous MeOH (7 mL). After extraction, the resulting mixture was purified by column chromatography using mobile phase (DCM/MeOH/NH ₄ OH (aqueous 25% solution) (15: 1:0.1) to give a pure product 24 as a pale orange solid. Yield 75%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 11.82 - 11.62 (m, 1H), 8.23 (d, J= 2.3 Hz, 1H), 8.20 (d, J= 2.2 Hz, 1H), 7.41 (d, J= 2.4 Hz, 1H), 3.57 (s, 2H), 2.48 - 2.30 (m, 8H), 2.27 (q, J= 7.2 Hz, 2H), 0.95 (t, J = 7.2 Hz, 3H). ¹³ C NMR (126 MHz, DMSO) δ 147.23, 142.55, 129.41, 126.98, 121.75, 110.46, 110.11, 53.10, 52.65, 51.74, 12.22. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₄ H ₂₀ BrN ₄ ⁺ (m/z): 323.08659; found: 323.08694.

22 R = 4-phenylpiperidin-1-yl 1 R = 4-phenylpiperidin-1-yl

23 R = 4-(4-ethylpiperazin-1-yl)piperidin-1-yl 2 R = 4-(4-ethylpiperazin-1-yl)piperidin-1-yl

24 R = 4-ethylpiperazin-1-yl To compound 22-24 (1.0 eq) suspended in anhydrous 1,4-dioxane, arylboronic acid (1.3 eq) was added under an inert argon atmosphere. Subsequently, Na ₂ CO ₃ (10.0 eq), Pd(dppf)Cl ₂ .DCM catalyst (0.01 eq) and argon purged water were added sequentially. The reaction mixture was then subjected to 100 W microwave irradiation at 110 °C for one hour. After cooling, the mixture was diluted with water (30 mL) and shaken three times with EA (3 x 30 mL). The organic phases were collected, dried over anhydrous Na ₂ SO ₄ , the mixture was filtered and the filtrate was evaporated. The result was purified by column chromatography to obtain pure products 1 and 2.

4-phenyl-l-({5-[4-(propane-2-sulfonyl)phenyl]-l H-pyrrolo[2,3-b]pyridin-3-yl}methyl)piperidine (1): Compound 22 (47 mg; 0.127 mmol); arylboronic acid 29 (38 mg; 0.165 mmol); Na ₂ CO ₃ (135 mg; 1.27 mmol) in 0.3 mL H2O; Pd(dppf)Cl ₂ .DCM (1 mg); and 3 mL anhydrous 1,4-dioxane. After extraction, the resulting mixture was purified by column chromatography using mobile phase (DCM/MeOH/NHziOH (aqueous 25% solution) (15:1:0,1)) to give a pure product 1 as a pale orange solid. Yield 52%. ¹ HNMR (500 MHz, CD3OD-J4) δ 8.55 (d, J= 2.1 Hz, 1H), 8.45 (d, J= 2.1 Hz, 1H), 7.98 - 7.91 (m, 4H), 7.45 (s, 1H), 7.24 - 7.09 (m, 5H), 3.80 (s, 2H), 3.37 - 3.34 (m, 1H), 3.11 (dt, J= 11.6, 3.4 Hz, 2H), 2.51 - 2.42 (m, 1H), 2.24 - 2.13 (m, 2H), 1.83 - 1.69 (m, 4H), 1.28 (d, J= 6.9 Hz, 6H). ¹³ C NMR (126 MHz, CD3OD) δ 149.59, 147.32, 146.41, 142.71, 136.52, 130.80, 129.39, 128.85, 128.54, 128.45, 127.94, 127.75, 127.16, 122.36, 111.39, 56.63, 54.95, 54.00, 43.51, 34.16, 15.91. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₈ H ₃₂ N ₃ 0 ₂ S ⁺ (m/z): 474.22097; found: 474.22144. LC-MS purity 99% l-ethyl-4-[l-({5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2 ,3-b]pyridin-3-yl}methyl)piperidin- 4-yl] piperazine (2): Compound 23 (105 mg; 0.258 mmol); arylboronic acid 29 (76 mg; 0.335 mmol); Na ₂ CO ₃ (273 mg; 2.58 mmol) in 0.5 mL H2O; Pd(dppf)Cl ₂ .DCM (2 mg; 0.0026 mmol); and 3 mL anhydrous 1,4-dioxane. After extraction, the resulting mixture was purified by column chromatography using mobile phase (DCM/MeOH/NHziOH (aqueous 25% solution) (9: 1 :0, 1) to give a pure product 2 as a pale orange solid. Yield 69%. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 11.63 (s, 1H), 8.56 (s, 1H), 8.33 (s, 1H), 8.00 - 7.95 (m, 2H), 7.89 (s, 2H), 7.38 (s, 1H), 3.61 (s, 2H), 3.41 (h, J= 6.8 Hz, 1H), 2.88 (d, J= 11.0 Hz, 2H), 2.44 - 2.23 (m, 8H), 2.20 (q, J= 7.2 Hz, 2H), 2.05 - 1.96 (m, 1H), 1.91 - 1.83 (m, 2H), 1.67 - 1.59 (m, 2H), 1.30 (d,J= 12.1 Hz, 2H), 1.19 - 1.12 (m, 6H), 0.90 (t, J = 7.1 Hz, 3H). ¹³ C NMR (151 MHz, DMSO-A,) δ 148.75,

144.44, 141.69, 134.82, 129.31, 127.43, 126.23, 125.85, 125.79, 119.92, 110.87, 61.27, 54.21, 52.85, 52.80, 52.36, 51.62, 48.69, 28.07, 15.25, 11.99. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₈ H ₄₀ N ₅ O ₂ S ⁺ (m/z): 510.28972; found: 510.28970. LC-MS purity 98%

Example 2 - Preparation of compounds of formula I derived from 1H-pyrrolo [2, 3-b] pyridine or from 1H-pyrazolo [3 ,4-b] pyridine, wherein the primary or secondary amine in position 2 or 3 of the basic ring is attached by a carbonyl bridge.

To ester 25-27 (1.0 eq) δissolved in anhydrous 1,4-dioxane, arylboronic acid 28 or 29 (1.3 eq) was added under an inert argon atmosphere. Subsequently, Na ₂ CO ₃ (10.0 eq), Pd(dppf)Cl ₂ .DCM (0.01 eq) and argon purged water were added sequentially. The reaction mixture was heated to 110 °C for three days. After cooling, dioxane was evaporated using a rotary evaporator. The mixture was diluted with water and the aqueous phase of the resulting acids 30-35 was washed twice with EA (2 ^c 100 mL) and then twice with DCM (2 ^c 100 mL). The resulting aqueous phase was carefully acidified with 10% HCI to pH ~ 1. The resulting precipitate was filtered, carefully rinsed with water (3 ^c 15 mL) followed by hexane (3 ^c 20 mL) to give pure carboxylic acids 32 and 33 as hydrochloride salts, and carboxylic acids 34 and 35 as neutral compounds.

The aqueous phase of the resulting ester compounds 30 and 31 was washed three times with EA (3 ^c 100 mL). The organic phases were collected, dried over anhydrous NajSCL. the mixture was filtered and the filtrate was evaporated. The result was immediately used in the following reaction: hydrolysis of the ester to the carboxylic acid. methyl 5-(4-methanesulfonylphenyl)-lH-pyrrolo[2,3-b]pyridine-3-carb oxylate (30): Compound 25 (470 mg; 1.84 mmol); arylboronic acid 28 (480 mg; 2.4 mmol); Na ₂ CO ₃ (1.95 g; 18.4 mmol) in 6.1 mL H2O; Pd(dppf)Cl ₂ .DCM (15 mg; 0.018 mmol); and 30 mL anhydrous 1,4-dioxane. Dark brown solid 30 was directly used into the following reaction (hydrolysis) without NMR characterization. Quantitative yield was calculated. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₆ H ₁₅ N ₂ O ₄ S ⁺ (m/z): 331.07470; found: 331.07428. methyl 5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridine-3 -carboxylate (31):

Compound 25 (551 mg; 2.16 mmol); arylboronic acid 29 (640 mg; 2.8 mmol); Na ₂ CO ₃ (2.29 g; 21.6 mmol) in 7.2 mL H2O; Pd(dppf)Cl ₂ .DCM (18 mg; 0.022 mmol); and 40 mL anhydrous

1,4-dioxane. Dark brown solid 31 was directly used into the following reaction (hydrolysis). Quantitative yield was calculated. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 8.71 (d, J= 2.3 Hz, 1H), 8.58 (d, J= 23 Hz, 1H), 8.32 (s, 1H), 8.05 - 8.00 (m, 2H), 7.97 - 7.92 (m, 2H), 3.85 (s, 3H), 3.51 - 3.40 (m, 1H), 1.29 - 1.14 (m, 6H). ¹³ C NMR (126 MHz, DMSO) δ 164.31, 148.94, 143.92, 143.37, 135.60, 134.48, 129.58, 128.68, 128.02, 127.32,

118.23, 105.86, 54.40, 51.18, 15.42. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₈ H ₁₉ N ₂ O ₄ S ⁺ (m/z): 359.10600; found: 359.10562.

5-(4-methanesulfonylphenyl)-lH-pyrrolo[2,3-b]pyridine-2-c arboxylic acid hydrochloride (32):

Compound 26 (407 mg; 1.6 mmol); arylboronic acid 28 (416 mg; 2.1 mmol); Na ₂ CO ₃ (1.70 g;

16.0 mmol) in 5.3 mL H ₂ 0; Pd(dppf)Cl ₂ .DCM (13 mg; 0.016 mmol); and 30 mL anhydrous

1,4-dioxane. Brownish powder 32 in the yield 56%. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 12.55 (s, 1H), 8.79 (s, 1H), 8.49 (s, 1H), 8.02 (s, 4H), 7.19 (s, 1H), 3.27 (s, 3H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 162.84, 149.19, 145.99, 144.03, 139.85, 130.77, 129.70,

128.23, 128.20, 127.96, 119.80, 107.14, 44.16. HRMS (ESI ⁺ ): [M+Hf: calculated for C ₁₅ H ₁₃ N ₂ O ₄ S ⁺ (m/z): 317.05905; found: 317.05948.

5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin e-2-carboxylic acid hydrochloride (33): Compound 26 (431 mg; 1.7 mmol); arylboronic acid 29 (504 mg; 2.21 mmol); Na ₂ CO ₃ (1.79 g; 16.9 mmol) in 5.6 mL H ₂ 0; Pd(dppf)Cl ₂ .DCM (14 mg; 0.017 mmol); and 30 mL anhydrous

1,4-dioxane. Brownish powder 33 in the yield 96%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.58 - 12.54 (m, 1H), 8.82 (s, 1H), 8.51 (d, J= 2.3 Hz, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.95 (d, J= 8.3 Hz, 2H), 7.21 (d, J= 2.0 Hz, 1H), 3.47 (p, J= 6.8 Hz, 1H), 1.21 (d, J= 6.7 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 162.81, 149.19, 145.99, 144.16, 135.70, 130.67, 129.81, 129.63, 128.04, 127.80, 119.71, 107.10, 54.67, 15.71. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₇ H ₁₇ N ₂ O ₄ S ⁺ (m/z): 345.09035; found: 345.09070.

5-(4-methanesulfonylphenyl)-lH-pyrazolo[3,4-b] pyridine-3- carboxylic acid (34): Compound 27 (437 mg; 1.7 mmol); arylboronic acid 28 (442 mg; 2.21 mmol); Na ₂ CO ₃ (1.80 g; 17.0 mmol) in 5.6 mL H ₂ 0; Pd(dppf)Cl ₂ .DCM (14 mg; 0.017 mmol); and 30 mL anhydrous 1,4-dioxane. Dark brown powder

34 in the yield 86%. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 9.01 (d, J= 2.1 Hz, 1H), 8.68 (d, J= 2.2 Hz, 1H), 8.11 - 8.04 (m, 4H), 3.28 (s, 3H). HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₄ H ₁₂ N ₃ O ₄ S ⁺ (m/z): 318.05430; found: 318.05396.

5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrazolo[3,4-b]pyridi ne-3-carboxylic acid (35): Compound 27 (880 mg; 3.44 mmol); boronic acid 29 (1.02 g; 4.5 mmol); Na ₂ CO ₃ (3.65 g; 34.4 mmol) in 11.5 mL H ₂ 0; Pd(dppf)Cl ₂ .DCM (28 mg; 0.034 mmol); and 50 mL anhydrous 1,4-dioxane. Dark brown powder

35 in the yield 70%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 13.38 (s, 1H), 9.01 (d, J= 2.2 Hz, 1H), 8.56 (d, J= 2.2 Hz, 1H), 8.12 - 8.06 (m, 2H), 8.00 - 7.94 (m, 2H), 3.51 - 3.43 (m, 1H), 1.19 (d, J = 6.8 Hz, 6H). HRMS (ESf): [M+H] ⁺ : calculated for C ₁₆ H ₁₆ N ₃ O ₄ S ⁺ (m/z): 346.08560; found: 346.08578.

To ester 30 or 31 dissolved in MeOH, 10% NaOH solution was added and the reaction mixture was heated to 110 °C for 24 h. After cooling, the MeOH was evaporated using a rotary evaporator and the residue was diluted with water (100 mL). The aqueous phase was washed twice with EA (2 x 100 mL) and then twice with DCM (2 x 100 mL). The resulting aqueous phase was carefully acidified with 10% HC1 to pH ~ 1. The resulting precipitate was filtered, carefully rinsed with water (3 x 15 mL) followed by hexane (3 x 20 mL) to give pure carboxylic acids 36 and 37 as hydrochloride salts.

5-(4-methanesulfonylphenyl)-lH-pyrrolo[2,3-b]pyridine-3-c arboxylic acid hydrochloride (36):

Compound 30 (607 mg; 1.84 mmol) was dissolved in 20 mL MeOH and 20 mL 10% NaOH was added. 88% yield of dark solid 36 after two reaction steps. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 12.60 (d, J= 3.0 Hz, 1H), 8.70 (d, J= 2.3 Hz, 1H), 8.58 (d, J= 2.3 Hz, 1H), 8.23 (d, J= 2.9 Hz, 1H), 8.06 - 8.01 (m, 4H), 3.26 (s, 3H). ¹³ C NMR (151 MHz, DMSO-D ₆ ) d 165.81, 149.28, 144.17, 143.51, 139.96, 134.47, 128.94, 128.40, 128.33, 127.73, 118.97, 107.34, 44.18. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₅ H ₁₃ N ₂ O ₄ S ⁺ (m/z): 317.05905; found: 317.05865. 5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridine-3 -carboxylic acid hydrochloride (37): Compound 31 (774 mg; 2.16 mmol) was dissolved in 20 mL MeOH and 20 mL 10% NaOH was added. 78% yield of pale brown compound 37 after two reaction steps. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.66 (s, 1H), 8.70 (s, 1H), 8.58 (s, 1H), 8.22 (d, J= 2.5 Hz, 1H), 8.02 (d, J= 8.0 Hz, 2H), 7.94 (d, J= 8.1 Hz, 2H), 3.52 (m, 1H), 1.18 (d, J= 6.8 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 165.75, 149.17, 144.28, 143.43, 135.76, 134.41, 129.91, 128.77, 128.26, 127.71, 118.88, 107.24, 54.75, 15.71. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₁₇ H ₁₇ N ₂ O ₄ S ⁺ (m/z): 345.09035; found: 345.08936. 30 3-subst; X = C; R ¹ = Me; .HCI 3-9 3-subst; X = C; R ¹ = iPr

31 3-subst; X = C; R ¹ = iPr; .HCI 10-11 3-subst; X = C; R ¹ = Me

32 2-subst; X = C; R ¹ = Me; .HCI 12-13 2-subst; X = C; R ¹ = Me

33 2-subst; X = C; R ¹ = iPr; .HCI 14-16 2-subst; X = C; R ¹ = iPr

34 3-subst; X = N; R ¹ = Me; 17-19 3-subst; X = N; R ¹ = iPr

35 3-subst; X = N; R ¹ = iPr; 20 3-subst; X = N; R ¹ = Me

EDC.HC1 (1.0 eq), HOBt.H ₂ O (1.1 eq) and DIPEA (3.0 eq) were added sequentially to carboxylic acid 30-35 (1.0 eq) δissolved in anhydrous THF, under an inert argon atmosphere. Finally, the corresponding primary or secondary amine (1.0 eq) was added and the reaction was stirred at room temperature for 24 hours. The resulting mixture was diluted with water (30 mL) and the organic phase was collected. The aqueous phase was then shaken twice more with new EA (2 x 30 mL). The organic phases were collected, dried over anhydrous sodium sulfate (Na ₂ SO ₄ ), the mixture was fdtered and the filtrate was evaporated. The crude product was purified by column chromatography to give pure amide products, or MeOH was added directly to precipitate the pure product. l-ethyl-4-(l-{5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2, 3-b]pyridine-3-carbonyl}piperidin- 4-yl)piperazine (3): Carboxylic acid 31 (97 mg; 0,.255 mmol); EDC.HC1 (49 mg; 0.255 mmol); HOBt.H ₂ O (43 mg; 0.280 mmol); DIPEA (133 μL; 0.765 mmol) and l-ethyl-4-(piperidin-4- yl)piperazine (51 mg; 0.255 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/MeOH/NHziOH (aqueous 25% solution) (15: 1:0.1), to yield pure product 3 as an orange solid. Yield 28%. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 12.34 (s, 1H), 8.69 (d, J= 2.2 Hz, 1H), 8.38 (d, J= 2.2 Hz, 1H), 8.05 - 7.99 (m, 2H), 7.95 - 7.92 (m, 2H), 7.91 (s, 1H), 4.32 (s, 2H), 3.51 - 3.41 (m, 1H), 3.31 - 3.26 (m, 1H), 3.01 (s, 2H), 2.50 - 2.29 (m, 8H), 2.27 (q, J= 7.2 Hz, 2H), 1.85 - 1.76 (m, 2H), 1.45 - 1.33 (m, 2H), 1.20 (d, J = 6.8 Hz, 6H), 0.96 (t, J = 7.2 Hz, 3H). ¹³ C NMR (151 MHz, DMSO- D ₆ ) δ 164.59, 148.63, 144.47, 143.25, 135.73, 129.89, 129.82, 128.22, 127.96, 127.66, 119.28, 109.80, 61.50, 54.77, 53.39, 52.19, 49.17, 40.62, 28.89, 15.79, 12.57. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for (m/z): 524.26899; found: 524.26855. LC-MS purity >99%. l-ethyl-4-{5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2,3-b ]pyridin-3-carbonyl}piperazine (4):

Carboxylic acid 31 (85 mg; 0.223 mmol); EDC.HC1 (43 mg; 0.223 mmol); HOBt.H ₂ 0 (38 mg; 0.245 mmol); DIPEA (117 μL; 0.669 mmol) and 1-ethylpiperazine (29 μL; 0.223 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/MeOH/NHziOH (aqueous 25% solution) (15: 1:0.1), to yield pure product 4 as pale brown solid. Yield 37%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 12.36 (s, 1H), 8.69 (d, J= 2.2 Hz, 1H), 8.39 (d, J= 2.2 Hz, 1H), 8.06

— 8.00 (m, 2H), 7.93 (d, J= 8.5 Hz, 3H), 3.72 - 3.64 (m, 4H), 3.46 (p , J = 6.8 Hz, 1H), 2.44 - 2.38 (m, 4H), 2.35 (q, J = 7.2 Hz, 2H), 1.19 (d, J= 6.8 Hz, 6H), 1.01 (t, J= 7.1 Hz, 3H). ¹³ C NMR (126 MHz,

DMSO) δ 164.29, 148.27, 144.05, 142.93, 135.37, 129.69, 129.52, 127.84, 127.64, 127.31, 118.90, 109.16, 62.96, 54.39, 52.74, 51.70, 15.42, 12.08. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₃ H ₂₉ N ₄ O ₃ S ⁺ (m/z): 441.19549; found: 441.19580. LC-MS purity >99%.

4-phenyl-l-{5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2 ,3-b]pyridine-3-carbonyl}piperidine (5): Carboxylic acid 31 (100 mg; 0.263 mmol); EDC.HC1 (50 mg; 0.263 mmol); HOBt.H ₂ 0 (44 mg; 0.290 mmol); DIPEA (137 μL; 0.790 mmol) and 1-phenylpiperidine (42 mg; 0.263 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1: 1), to yield pure product 5 as brownish solid. Yield 32%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 12.36 (s, 1H), 8.70 (d, J= 2.2 Hz, 1H), 8.44 (d, J= 2.3 Hz, 1H), 8.08 - 8.01 (m, 2H), 7.98 - 7.94 (m, 2H), 7.93 (d, J= 1.9 Hz, 1H), 7.33 - 7.25 (m, 4H), 7.23 - 7.16 (m, 1H), 4.51 - 4.45 (m, 2H), 3.52 - 3.42 (m, 1H), 3.16 - 3.04 (m, 2H), 2.87 - 2.79 (m, 1H), 1.86 - 1.80 (m, 2H), 1.71 - 1.59 (m, 2H), 1.20 (d, J= 6.8 Hz, 5H). ¹³ C NMR (126 MHz, DMSO) δ 164.62, 148.59, 146.19, 144.41, 143.19, 135.66, 129.83, 129.78, 128.87, 128.15, 127.90, 127.66, 127.24, 126.66, 119.27,

109.80, 54.70, 42.48, 33.71, 15.73. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₈ H ₃₀ N ₃ O ₃ S ⁺ (m/z): 488.20024; found: 488.19879. LC-MS purity 99%.

2-{5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2, 3-b]pyridine-3-carbonyl}-l, 2,3,4- tetrahydroisoquinoline (6): Carboxylic acid 31 (95 mg; 0.250 mmol); EDC.HC1 (48 mg; 0.250 mmol); HOBt.H ₂ 0 (42 mg; 0.275 mmol); DIPEA (131 μL; 0.750 mmol) and 1,2,3,4-tetrahydroisoquinoline (31 μL: 0.250 mmol) in 5 mL anhydrous THF. After the extraction, the resulting mixture was directly precipitated in MeOH, to yield pure product 6 as white solid. Yield 53%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 13.73 (s, 1H), 10.01 (d, J = 2.3 Hz, 1H), 9.75 (d, J = 2.3 Hz, 1H), 9.35 (s, 1H), 9.30 (d, J= 8.2 Hz, 2H), 9.24 - 9.18 (m, 2H), 8.55 - 8.41 (m, 4H), 6.17 (s, 2H), 5.22 (t, J = 5.9 Hz, 2H), 4.75 (hept, J= 6.8 Hz, 1H), 4.23 (t, J= 5.9 Hz, 2H), 2.49 (d, J= 6.9 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 148.64, 144.38, 143.31, 135.65, 135.34, 134.19, 129.81, 129.01, 128.13, 127.99, 127.84, 126.93, 126.62, 119.49, 109.62, 60.22, 54.69, 29.05, 21.23, 15.72. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₆ H ₂₆ N ₃ O ₃ S ⁺ (m/z): 460.16894; found: 460.16757. LC-MS purity 98%. N-benzyl-5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2,3-b]p yridine-3-carboxamide (7):

Carboxylic acid 31 (95 mg; 0.250 mmol); EDC.HC1 (48 mg; 0.250 mmol); HOBt.H ₂ O (42 mg; 0.275 mmol); DIPEA (131 μL; 0.750 mmol) and benzylamine (27 μL; 0.250 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1:1), to yield pure product 7 as a brownish solid. Yield 44%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.33 (s, 1H), 8.81 (d, J= 2.3 Hz, 1H), 8.72 - 8.64 (m, 2H), 8.30 (d, J= 1.7 Hz, 1H), 8.07 - 8.00 (m, 2H), 7.99 - 7.92 (m, 2H), 7.40 - 7.31 (m, 4H), 7.27 - 7.22 (m, 1H), 4.53 (d, J = 5.9 Hz, 2H), 3.47 (hept, J = 6.8 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 164.20, 148.97, 144.49, 143.20, 140.62, 135.67, 129.96, 129.87, 128.75, 128.20, 128.13, 127.72, 127.15, 118.99, 110.28, 54.70, 42.41, 15.72. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₄ H ₂₄ N ₃ O ₃ S ⁺ (m/z): 434.15329; found: 434.15210. LC-MS purity 98%. N-{[4-(diethylamino)phenyl]methyl}-5-[4-(propane-2-sulfonyl) phenyl]-lH-pyrrolo[2,3- b]pyridine-3-carboxamide (8): Carboxylic acid 31 (95 mg; 0.250 mmol); EDC.HC1 (48 mg; 0.250 mmol); HOBt.H ₂ 0 (42 mg; 0.275 mmol); DIPEA (131 μL; 0.750 mmol) and (4- aminomethylphenyl)diethylamine (45 μL; 0.250 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1:1), to yield pure product 8 as brownish solid. Yield 44%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.27 (s, 1H), 8.81 (d, J= 2.3 Hz, 1H), 8.68 (d, J= 2.3 Hz, 1H), 8.47 (t, J= 5.9 Hz, 1H), 8.26 (d, J= 1.8 Hz, 1H), 8.05 - 8.00 (m, 2H), 7.97 - 7.92 (m, 2H), 7.18 - 7.12 (m, 2H), 6.65 - 6.58 (m, 2H), 4.36 (d, J= 5.8 Hz, 2H), 3.46 (hept, J= 6.9 Hz, 1H), 3.29 (q, J= 7.0 Hz, 4H), 1.20 (d, J= 6.8 Hz, 6H), 1.04 (t, J= 7.0 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 163.97, 148.95, 146.89, 144.53, 143.12, 135.65, 129.88, 129.76, 129.15, 128.25, 128.11, 126.78, 119.05, 112.06, 110.49, 54.71, 44.17, 42.01, 15.73, 12.84. HRMS (ESI ⁺ ) [M+H] ⁺ : calculated for C ₂₈ H ₃₃ N ₄ O ₃ S ⁺ (m/z): 505.22679; found: 505.22733. LC-MS purity 98%.

N- [4-(dimethylamino)phenyl] -5- [4-(pr opane-2-sulf onyl)phenyl]-l H-pyrrolo [2,3-b] pyridine-3- carboxamide (9): Carboxylic acid 31 (105 mg; 0.276 mmol); EDC.HC1 (53 mg; 0.276 mmol); HOBt.lTO (47 mg; 0.304 mmol); DIPEA (144 μL; 0.828 mmol) and N,N-dimethyl-/;- phenylenediamine (38 mg; 0.276 mmol) in 5 mL anhydrous THF. After the extraction, the resulting mixture was directly precipitated in MeOH, to yield pure product 9 as pale green solid. Yield 51%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 12.39 (s, OH), 9.65 (s, 1H), 8.82 (d, J= 2.3 Hz, 1H), 8.71 (d, J= 2.3 Hz, 1H), 8.44 (d, J= 2.8 Hz, 1H), 8.04 (d, J= 8.6 Hz, 1H), 7.96 (d, J= 8.5 Hz, 2H), 7.57 (d, J= 9.1 Hz, 1H), 6.74 (d, J= 9.1 Hz, 2H), 3.46 (p, J = 6.8 Hz, 1H), 2.87 (s, 6H), 1.20 (d, J= 6.8 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 162.40, 149.01, 147.47, 144.48, 143.31, 135.69, 130.10, 129.91, 129.60, 128.35, 128.26, 128.14, 121.96, 119.29, 113.12, 110.66, 54.72, 41.03, 15.73. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₅ H ₂₇ N ₄ O ₃ S ⁺ (m/z): 463.17984; found: 463.17841. LC-MS purity 95% l-[5-(4-methanesulfonylphenyl)-lH-pyrrolo[2,3-b]pyridine-3-c arbonyl]-4-phenylpiperidine (10):

Carboxylic acid 30 (85 mg; 0.242 mmol); EDC.HC1 (46 mg; 0.242 mmol); HOBt.H ₂ 0 (41 mg; 0.266 mmol); DIPEA (126 μL; 0.725 mmol) and 1-phenylpiperidine (39 mg; 0.242 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1: 1). The purified mixture was then precipitated from MeOH to yield pure product 10 as white solid. Yield 32%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 12.35 (s, 1H), 8.69 (d, J= 2.2 Hz, 1H), 8.42 (d, J= 2.3 Hz, 1H), 8.03 (s, 4H), 7.96 (d, J= 1.7 Hz, 1H), 7.34 - 7.27 (m, 4H), 7.23 - 7.16 (m, 1H), 4.51 - 4.46 (m, 2H), 3.27 (s, 3H), 3.19 - 3.04 (m, 1H), 2.88 - 2.79 (m, 1H), 1.88 - 1.81 (m, 2H), 1.72 - 1.60 (m, 2H). ¹³ C NMR (126 MHz, DMSO) δ 164.64, 148.57, 146.19, 144.21, 143.19, 139.74, 129.79, 128.88, 128.22, 128.18, 127.99, 127.57, 127.25, 126.67, 119.25, 109.79, 44.10, 42.48, 33.71. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₆ H ₂₆ N ₃ O ₃ S ⁺ (m/z): 460.16894; found: 460.16849. LC-MS purity 98% N-{[4-(diethylamino)phenyl]methyl}-5-(4-methanesulfonylpheny l)-lH-pyrrolo[2,3-b] pyridine-3- carboxamide (11): Carboxylic acid 30 (107 mg; 0.303 mmol); EDC.HC1 (58 mg; 0.303 mmol); HOBt.H ₂ 0 (51 mg; 0.334 mmol); DIPEA (158 μL; 0.910 mmol) and (4- aminomethylphenyl)diethylamine (55 μL; 0.303 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1:1) to yield pure product 11 as white solid. Yield 21%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.27 (s, 1H), 8.79 (d, J= 2.3 Hz, 1H), 8.67 (d, J= 2.3 Hz, 1H), 8.47 (t, J= 5.9 Hz, 1H), 8.26 (s, 1H), 8.08 - 7.96 (m, 4H), 7.19 - 7.08 (m, 2H), 6.65 - 6.56 (m, 2H), 4.36 (d, J= 5.9 Hz, 2H), 3.30 (q, J= 7.0 Hz, 4H), 3.27 (s, 3H), 1.05 (t, J= 7.0 Hz, 6H). ¹³ C NMR (126 MHz,

DMSO) δ 163.98, 148.93, 146.89, 144.33, 143.12, 139.73, 129.74, 129.14, 128.23, 128.21, 128.18,

126.77, 119.06, 112.07, 110.46, 44.17, 44.11, 42.00, 12.85. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₆ H ₂₉ N ₄ O ₃ S ⁺ (m/z): 477.19549; found: 477.19504. LC-MS purity 98%. l-ethyl-4-[5-(4-methanesulfonylphenyl)-lH-pyrrolo[2,3-b]pyri dine-2-carbonyl]piperazine (12):

Carboxylic acid 32 (110 mg; 0.312 mmol); EDC.HC1 (60 mg; 0.312 mmol); HOBt.H ₂ 0 (53 mg; 0.343 mmol); DIPEA (163 μL; 0.936 mmol) and 1-ethylpiperazine (40 μL; 0.312 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase DCM/MeOH/NH ₄ OH (25% aqueous solution) (15: 1:0.1) to yield pure product 12 as brownish solid. Yield 60%. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 12.32 (s, 1H), 8.71 (s, 1H), 8.40 (s, 1H), 8.04 - 7.97 (m, 4H), 6.81 (s, 1H), 3.75 - 3.64 (m, 4H), 3.27 (s, 3H), 2.47 - 2.40 (m, 4H), 2.37 (q, J= 7.2 Hz, 2H), 1.03 (t, J= 7.2 Hz, 3H). ¹³ C NMR (151 MHz, DMSO-D ₆ ) δ 161.47, 148.00, 143.99, 143.74, 139.18, 132.15, 128.09, 127.67, 127.60, 127.18, 119.00, 102.47, 52.36, 51.46, 43.61, 11.86. HRMS (ESI ⁺ ) [M+H] ⁺ : calculated for C _2i H ₂₅ N ₄ O ₃ S ⁺ (m/z): 413.16419; found: 413.16434. LC-MS purity >99% l-[5-(4-methanesulfonylphenyl)-lH-pyrrolo[2,3-b]pyridine-2-c arbonyl]-4-phenylpiperidine (13):

Carboxylic acid 32 (97 mg; 0.275 mmol); EDC.HC1 (53 mg; 0.275 mmol); HOBt.H ₂ 0 (46 mg; 0.302 mmol); DIPEA (144 μL; 0.825 mmol) and 1-phenylpiperidine (44 mg; 0.275 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1: 1) to yield pure product 13 as white solid. Yield 28%. ¹ HNMR (600 MHz, DMSO-d ₆ ) δ 12.33 (s, 1H), 8.71 (d, J= 2.2 Hz, 1H), 8.40 (d, J= 2.2 Hz, 1H), 8.02 (s, 4H), 7.39 - 7.24 (m, 4H), 7.22 - 7.16 (m, 1H), 6.85 (s, 1H), 4.83 - 4.16 (m, 2H), 3.27 (s, 3H), 3.21 - 2.93 (m, 2H), 2.93 - 2.83 (m, 1H), 1.94 - 1.81 (m, 2H), 1.74 - 1.60 (m, 2H). ¹³ C NMR (151 MHz, DMSO-A,) δ 162.16, 148.58, 146.06, 144.41, 144.34, 139.73, 133.14, 128.98, 128.58, 128.23, 128.16, 127.73, 127.30, 126.81, 119.63, 102.60, 44.16, 42.33, 33.62. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₆ H ₂₆ N ₃ O ₃ S ⁺ (m/z): 460.16894; found: 460.16803. LC-MS purity 98% N-benzyl-5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2,3-b]p yridine-2-carboxamide (14):

Carboxylic acid 33 (99 mg; 0.260 mmol); EDC.HC1 (50 mg; 0.260 mmol); HOBt.H ₂ 0 (44 mg; 0.286 mmol); DIPEA (136 μL; 0.780 mmol) and benzylamine (28 μL; 0.260 mmol) in 5 mL anhydrous THF. After the extraction, the resulting mixture was directly precipitated in MeOH, to yield pure product 14 as white solid. Yield 73%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.35 (s, 1H), 9.16 (t, J= 6.0 Hz, 1H), 8.75 (d, J= 2.2 Hz, 1H), 8.50 (d, J= 2.3 Hz, 1H), 8.08 - 8.02 (m, 2H), 7.96 - 7.91 (m, 2H), 7.40 - 7.32 (m, 4H), 7.30 - 7.23 (m, 2H), 4.54 (d, J = 5.9 Hz, 2H), 3.46 (hept, J = 6.9 Hz, 1H), 1.19 (d, J = 6.8 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 160.93, 148.88, 145.00, 144.35, 139.84, 135.58, 133.82, 129.77, 129.13, 128.84, 128.02, 127.82, 127.56, 127.37, 119.88, 102.72, 54.68, 42.80, 15.72. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for 434.15329; found: 434.15277. LC-MS purity 96%

2-{5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrrolo[2, 3-b]pyridine-2-carbonyl}-l, 2,3,4- tetrahydroisoquinoline (15): Carboxylic acid 33 (105 mg; 0.276 mmol); EDC.HC1 (53 mg; 0.276 mmol); HOBt.H ₂ 0 (46 mg; 0.303 mmol); DIPEA (144 μL; 0.827 mmol) and 1,2,3,4-tetrahydroisoquinoline (35 μL; 0.276 mmol) in 5 mL anhydrous THF. After the extraction, the resulting mixture was directly precipitated in MeOH, to yield pure product 15 as white solid. Yield 58%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.39 (s, 1H), 8.75 (d, J= 2.3 Hz, 1H), 8.45 (d, J= 2.3 Hz, 1H), 8.04 (d, J= 8.4 Hz, 2H), 7.94 (d, J= 8.2 Hz, 2H), 7.23 - 7.17 (m, 4H), 6.96 (s, 1H), 4.86 (s, 2H), 3.93 (s, 2H), 3.47 (hept, J= 6.8 Hz, 1H), 2.98 - 2.92 (m, 2H), 1.20 (d, J= 6.8 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 148.56, 144.70, 144.41, 135.62, 135.07, 133.59, 132.75, 129.82, 129.00, 128.79, 128.02, 127.59, 127.10, 126.76, 119.58, 54.67, 15.73. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₆ H ₂₆ N ₃ O ₃ S ⁺ (m/z): 460.16894; found: 460.16739. LC-MS purity 98% N-{[4-(diethylamino)phenyl]methyl}-5-[4-(propane-2-sulfonyl) phenyl]-lH-pyrrolo[2,3- b]pyridine-2-carboxamide (16): Carboxylic acid 33 (92 mg; 0.242 mmol); EDC.HC1 (46 mg; 0.242 mmol); HOBt.H ₂ 0 (41 mg; 0.266 mmol); DIPEA (126 μL; 0.725 mmol) and (4- aminomethylphenyl)diethylamine (44 μL; 0.242 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1:1) to yield pure product 16 as pale orange solid. Yield 25%. ¹ HNMR (500 MHz, DMSO-d ₆ ) δ 12.29 (s, 1H), 8.95 (t, J= 5.9 Hz, 1H), 8.73 (d, .7= 2.2 Hz, 1H), 8.47 (d, J= 2.3 Hz, 1H), 8.07 - 8.01 (m, 2H), 7.95 - 7.89 (m, 2H), 7.24 (s, 1H), 7.18 - 7.10 (m, 2H), 6.65 - 6.59 (m, 2H), 4.37 (d, J= 5.8 Hz, 2H), 3.45 (hept, J= 6.8 Hz, 1H), 3.29 (q, J= 7.0 Hz, 4H), 1.19 (d, J = 6.8 Hz, 6H), 1.05 (t, .7= 7.0 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 160.59, 148.84, 147.03, 144.88,

144.37, 135.56, 134.06, 129.76, 129.31, 129.05, 128.01, 127.51, 125.91, 119.89, 112.03, 102.61, 54.68,

44.16, 42.47, 15.72, 12.85. HRMS (ESf): [M+H] ⁺ : calculated for C ₂₈ H ₃₃ N ₄ O ₃ S ⁺ (m/z): 505.22679; found: 505.22714. LC-MS purity 98% N-benzyl-5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrazolo[3,4-b] pyndine-3-carboxamide (17):

Carboxylic acid 35 (128 mg; 0.370 mmol); EDC.HC1 (71 mg; 0.370 mmol); HOBt.H ₂ 0 (62 mg; 0.410 mmol); DIPEA (193 μL; 1.11 mmol) and benzylamine (41 μL; 0.370 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1: 1) to yield pure product 17 as white solid. Yield 25%. ¹ HNMR (500 MHz, DMSO-tT,) δ 14.32 (s, 1H), 9.14 (t, J= 6.3 Hz, 1H), 8.95 (d, J= 2.3 Hz, 1H), 8.74 (d, J= 2.3 Hz, 1H), 8.02 (d, J= 8.6 Hz, 2H), 7.90 (d, J= 8.5 Hz, 2H), 7.35 - 7.20 (m, 4H), 7.17 (t, J = 7.2 Hz, 1H), 4.46 (d, J= 6.3 Hz, 2H), 3.41 (p, J= 6.8 Hz, 1H), 1.13 (d, .7= 6.8 Hz, 6H). ¹³ C NMR (126

MHz, DMSO) δ 162.02, 152.81, 149.46, 143.30, 140.20, 138.91, 136.28, 129.90, 129.65, 128.72,

128.49, 127.85, 127.20, 114.01, 54.68, 42.46, 15.69. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂ 3H ₂ 3N ₄ 03S ⁺ (m/z): 435.14854; found: 435.14798. LC-MS purity 98% N-{[4-(diethylamino)phenyl]methyl}-5-[4-(propane-2-sulfonyl) phenyl]-lH-pyrazolo[3,4- b]pyridine-3-carboxamide (18): Carboxylic acid 35 (200 mg; 0.580 mmol); EDC.HC1 (111 mg; 0.580 mmol); H0Bt.H ₂ 0 (98 mg; 0.640 mmol); DIPEA (303 μL; 1.74 mmol) and (4- aminomethylphenyl)diethylamine (105 μL; 0.580 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1:1) to yield pure product 18 as pale orange solid. Yield 17%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 14.33 (s, 1H), 9.00 (d, J= 2.3 Hz, 1H), 8.97 (t, J= 6.3 Hz, 1H), 8.81

(d, J= 2.3 Hz, 1H), 8.08 (d, J= 8.5 Hz, 1H), 7.97 (d, J= 8.5 Hz, 2H), 7.17 (d, J= 8.8 Hz, 1H), 6.60 (d, J= 8.8 Hz, 1H), 4.37 (d, J= 6.2 Hz, 2H), 3.48 (p, J= 6.8 Hz, 1H), 3.28 (q, J= 7.0 Hz, 4H), 1.20 (d, J= 6.7 Hz, 6H), 1.04 (t, J= 7.0 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 161.71, 152.79, 146.90, 143.32,

139.11, 136.27, 129.91, 129.69, 129.57, 129.31, 128.47, 126.47, 113.99, 111.96, 54.69, 44.14, 41.99, 15.70, 12.84. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₃₂ H ₂₇ N ₅ O ₃ S ⁺ (m/z): 506.22204; found: 506.22067.

LC-MS purity 98%

4-phenyl-l-{5-[4-(propane-2-sulfonyl)phenyl]-lH-pyrazolo[ 3,4-b]pyridine-3-carbonyl}piperidine (19): Carboxylic acid 35 (200 mg; 0.580 mmol); EDC.HC1 (111 mg; 0.580 mmol); HOBt.H ₂ O (98 mg; 0.640 mmol); DIPEA (303 μL; 1.74 mmol) and 1-phenylpiperidine (94 mg; 0.580 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1: 1) to yield pure product 19 as a brownish solid. Yield 18%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 14.28 (s, 1H), 9.02 (d, J= 2.3 Hz, 1H), 8.72 (d, J= 2.3 Hz, 1H), 8.17 - 8.05 (m, 2H), 8.02 - 7.91 (m, 2H), 7.42 - 7.25 (m, 4H), 7.23 - 7.09 (m, 1H), 5.00 (d, J= 13.3 Hz, 1H), 4.78 (d, J= 13.0 Hz, 1H), 3.56 - 3.42 (m, 1H), 3.31 - 3.26 (m, 1H), 3.01 - 2.83 (m, 2H), 1.96 -

1.81 (m, 2H), 1.74 - 1.61 (m, 2H), 1.20 (d, J = 6.9 Hz, 6H). ¹³ C NMR (126 MHz, DMSO) δ 161.37, 152.14, 149.49, 146.05, 143.32, 139.85, 136.24, 129.91, 129.89, 129.27, 128.90, 128.47, 127.23, 126.70, 115.38, 54.67, 47.25, 43.06, 42.38, 34.22, 33.33, 15.70. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₇ H ₂₉ N ₄ O ₃ S ⁺ (m/z): 489.19549; found: 489.19391. LC-MS purity 99% l-[5-(4-methanesulfonylphenyl)-lH-pyrazolo[3,4-b]pyndine-3-c arbonyl]-4-phenylpipendine (20): Carboxylic acid 34 (109 mg; 0.308 mmol); EDC.HC1 (59 mg; 0.308 mmol); HOBt.H ₂ O (52 mg; 0.339 mmol); DIPEA (161 μL; 0.924 mmol) and 1-phenylpiperidine (50 mg; 0.308 mmol) in 5 mL anhydrous THF. After the extraction, the reaction mixture was purified by column chromatography using mobile phase (DCM/EA) (1 : 1) to yield pure product 20 as white solid. Yield 46%. 1HNMR (500 MHz, DMSO-d ₆ ) δ 14.25 (s, 1H), 9.00 (d, J= 2.3 Hz, 1H), 8.71 (d, J= 2.3 Hz, 1H), 8.07 (q, J= 8.3 Hz, 4H), 7.34 - 7.25 (m, 4H), 7.20 (t, J= 6.9 Hz, 1H), 5.03 - 4.96 (m, 1H), 4.81 - 4.75 (m, 1H), 3.29 (s, 3H), 3.01 - 2.85 (m, 2H), 1.99 - 1.81 (m, 2H), 1.75 - 1.64 (m, 2H). ¹³ C NMR (126 MHz, DMSO) δ 161.38, 152.12, 149.50, 146.05, 143.11, 140.28, 139.82, 129.81, 129.35, 128.91, 128.53, 128.24, 127.24, 126.70, 115.37, 47.25, 44.03, 43.07, 42.38, 34.22, 33.34. HRMS (ESI ⁺ ): [M+H] ⁺ : calculated for C ₂₅ H ₂₅ N ₄ O ₃ S ⁺ (m/z): 461.16419; found: 461.16458. LC-MS purity 98%

Example 3 - Determination of the decrease in proliferation of tumor cell lines by the action of the inhibitor alone, and potentiation of the effect of cisplatin by the inhibitor

Nine human stabilized cell lines of different histogenetic origins were selected for a comprehensive assessment of the cytotoxic effect of inhibitors. The cell lines were: Jurkat (acute T- leukemia), MOLT-4 (acute lymphoblastic leukemia), A549 (pulmonary adenocarcinoma), HT-29 (colorectal adenocarcinoma), PANC -1 (pancreatic cancer), A2780 (ovarian cancer), HeLa (cervical cancer), MCF-7 (breast adenocarcinoma) and SAOS-2 (osteosarcoma). The lines were purchased from ATCC (Manassas, USA) and Sigma- Aldrich (St. Louis, USA) and maintained according to the supplier's recommendations. All lines were cultured in an incubator at a constant temperature of 37 °C and a controlled air atmosphere with 5% carbon dioxide. Exponentially growing cells up to a maximum of the twentieth passage were used during the study.

The cells were resuspended and seeded at an optimal density of 1 ^c 10 ³ to 50* 10 ³ cells per well (determined from preliminary experiments) in 96-well plates (TPP, Trasadingen, Switzerland), to the bottoms of which they adhered by the next day. For the next 48 hours, the cells were cultured in test substance medium at a final concentration of 10 mM. Doxorubicin (Sigma-Aldrich, St. Louis, USA) at a final concentration of 1 mM was used as a positive control for the standard testing procedure.

After the incubation, the proliferation of the treated cells and control cells was determined and compared. The used WST-1 proliferation assay (Roche, Basel, Switzerland) was performed exactly according to the manufacturer's instructions, the absorbance was measured on a Tecan Spark instrument (Tecan, Mannedorf, Switzerland). Each value is an average of three independent experiments and is presented relative to the proliferation of control cells that were treated with solvent alone (0.1% DMSO) as 100%. In addition to the activity of the inhibitors themselves, their chemosensitizing effect was also tested, i.e. the ability to increase the efficacy of standard chemotherapeutics. The cell lines were again cultured for 48 hours with a test substance (10 pM) together with cisplatin, which was used according to the sensitivity of the respective cell line at a concentration of 2 pM (MOLT-4, Jurkat, A2780), 10 pM (MCF-7) and 15 pM (A549, HT-29, HeLa, SAOS-2, PANC-1). At the end of the culture, the decrease in proliferation relative to the control cells proliferation was determined again, and this time the chemosensitizing effect was evaluated, i.e. a decrease in proliferation in a given cell line after treatment with a combination of cisplatin and test substance versus cisplatin alone and test substance alone. The standard ATR kinase inhibitor VE-821 was used to compare efficacy.

Table 1 shows the decrease in proliferation of Jurkat, MOLT-4, A549, HT-29, PANC-1, A2780, HeLa, MCF-7, SAOS-2 tumor cell lines by the action of compounds of formula I compared to the VE 821 standard at 10 mM concentration. The decrease in proliferation of the cells treated with compounds of formula I is expressed as a percentage of the proliferation of control cells treated with solvent only (0.1% DMSO) (100%).

Table 2 shows the potentiation of the effect of cisplatin on Jurkat, MOLT 4, A549, HT-29, PANC-1, A2780, HeLa, MCF-7, SAOS-2 tumor cell lines. The table includes cisplatin alone, the compound of formula I alone and the combination of the compound of formula I with cisplatin. The decrease in proliferation of the cells treated with compounds of formula I and cisplatin is expressed as a percentage of the proliferation of control cells treated with solvent only (0.1% DMSO) (100%).

Cisplatin at the concentration used (2 to 15 pM) reduces cell proliferation to 40 to 80% of control cell proliferation. The novel inhibitors increased its cytotoxic properties, leading to a further decrease in proliferation in the tumor cell lines MOLT 4, A459, HT-29, PANC-1, A2780, HeLa, MCF-7, SAOS-2.

Table 1: Decrease of proliferation in tumor cell lines Jurkat, MOLT-4, A549, HT-29, PANC-1, A2780, HeLa, MCF-7, SAOS-2 by the action of the inibitors (10 mM) alone, in comparison with VE-821 (10 pM) standard, expressed as % of control cell proliferation (100%).

Table 2: Potentiation of the effect of cisplatin by the inhibitor (10 mM) compared to the action of the inhibitor alone and cisplatin alone, on tumor cell lines MOLT-4, A549, HT-29, HeLa, SAOS-2, PANC-1, A2780 and MCF-7.