CZ309356B6 - Substituted purine compounds as protein kinase inhibitors, their use as medicaments and pharmaceutical preparations containing these derivatives - Google Patents

Abstract

The solution provides substituted formula I 2-piperazinylarylamino-8-oxopurines for use as pharmaceuticals. These substances are inhibitors of protein kinases CDK4, CDK6, FLT3 and PDGFR, and are thus suitable for treating oncological and hemato-oncological diseases.

Description

Substituted purine compounds as protein kinase inhibitors, their use as drugs and pharmaceutical preparations containing these derivatives

Field of technology

This invention relates to novel substituted 2-piperazinylarylamino-8-oxopurines which are potent inhibitors of cyclin dependent kinases 4 (CDK4) and 6 (CDK6) and receptor kinases FLT3 and PDGFR. The invention also relates to the use of said compounds as drugs, in particular for the treatment of cell proliferative diseases such as cancer.

Current state of the art

Protein kinases are enzymes responsible for regulating a number of cellular processes. The essence of their function is the transfer of a phosphate group from ATP to a protein, the properties of which change significantly. Qualitative or quantitative disorders of kinase activity and protein phosphorylation have been shown to be associated with many disorders and diseases, including autoimmune disorders, inflammatory, metabolic and neurodegenerative diseases, and cancer. For this reason, protein kinases are considered suitable targets for modern pharmaceuticals and their inhibitors are being developed for therapeutic use in both human and veterinary medicine.

Protein kinases from the cyclin-dependent kinase (CDK) family are used, among other things, in the regulation of the cell cycle. These enzymes are heterodimers formed by a catalytic (CDK) and a regulatory (cyclin) subunit (Genome Biol 2014; 15:122). Passage through the G1 phase is controlled by CDK4 and CDK6 in complex with D-type cyclins, induction of S phase by CDK2 activated by cyclin E. DNA replication during S phase depends on the activity of CDK2 in complex with cyclin A. CDK1 and cyclins A and B then regulate passage through G2 phases and M. Another CDK is involved in the regulation of processes unrelated to the cell cycle; eg CDK7, CDK8, CDK9 and CDK11 are involved in the regulation of mRNA transcription.

The activity of CDKs controlling the cell cycle is critically dependent on the association with cyclins, whose levels change significantly during the cell cycle and thus represent the most significant regulatory element. CDK activity is further regulated by activation loop phosphorylation by CDK7/CAK kinase and dephosphorylation by CDC25 phosphatases. The next level of CDK control is based on protein inhibitors from the INK4 and Cip/Kip family, which are mainly used to stop the cell cycle.

Deregulation of CDK activity is considered a typical property of tumor cells, and a number of mechanisms that cause it have already been described (Nat Rev Drug Discov 2009;8:547-66). Most frequently detected in tumors are inactivating mutations (deletions and point mutations) or silencing of genes encoding INK4 and Cip/Kip inhibitors and increased expression of cyclins and CDK. For example, increased expression of cyclin D1 has been demonstrated in breast, bladder, esophageal, and squamous cell carcinomas (Adv Cancer Res 1996;68:67-108.). In some cases, deregulation of CDK activity is caused by amplification or increased expression of the catalytic subunit; these are typically CDK4 and CDK6 (Leukemia 2008;22:387-392; Clin Cancer Res 2012;18:46124620). In rare cases, deregulation of CDK activity can be caused by a point mutation of CDK4, which reduces sensitivity to the inhibitory protein INK4a (Nat Genet 1996;12:97-99). The loss of the RB protein, which is one of the main subunits of CDK4 and CDK6, is also considered to be an important mechanism damaging the regulation of the cell cycle. The absence of a functional RB protein leads to the elimination of the cell cycle checkpoint in the G1 phase and subsequent deregulation of proliferation. This change is typical of retinoblastomas, osteosarcomas and some other types of cancer (Genome Biol 2014;15:122). CDKs are therefore currently considered important targets for antitumor therapy (Nat Rev Drug Discov 2009;8:547-66; Curr Drug Targets 2010;1 1:291302.).

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In accordance with the above facts, significantly more specific CDK4 and CDK6 inhibitors palbociclib, ribociclib and abemaciclib were approved as drugs for metastatic hormone-dependent and HER2-negative breast cancers (Pharmacol Res. 2019;139:471-488; Pharmacol Res. 2019;144: 19-50). However, preclinical experimental work indicates that specific inhibitors of CDK4 and CDK6 can also have a significant therapeutic effect in a number of other types of cancer.

The FLT3 gene encodes the membrane-bound receptor tyrosine kinase FLT3, which is activated by an extracellular signaling ligand (Biochim Biophys Acta Rev Cancer. 2019; 1872(1):80-88). The receptor then sizes, autophosphorylates, and activates downstream RAS/MAPK, JAK/STAT5, and PI3K/AKT signaling cascades that promote myeloid cell growth, proliferation, survival, and differentiation. Activating FLT3 mutations are typical of 30% of patients with acute myeloid leukemia and closely associated with poor prognosis (Cancer Sci. 2020; 111(2):312-322). The most common oncogenic mutation is an internal tandem duplication (ITD) domain that stabilizes the kinase in the active conformation and promotes its constitutive activation. Therefore, oncogenic FLT3 is also considered an important therapeutic target and its inhibitors are being studied in clinical experiments (Cancer. 2011, 117, 3293-304; Ther. Adv. Hematol. 2014, 5, 65-77). Small-molecule FLT3 inhibitors (eg, midostaurin, quizartinib, crenolanib, gilteritinib) show promising effects not only in preclinical models, but lead to significant partial and complete response in patients in clinical trials (Cancer Sci. 2020 Feb; 111(2):312-322 ). On the other hand, the developed inhibitors have undesirable side effects and prolonged treatment leads to the development of specific resistance complicating further therapy.

Compounds inhibiting CDK4 and CDK6 in combination with other kinases such as FLT3, JAK2 and AXL are therefore being developed as a very advantageous treatment concept, whose effect on selected leukemias, lymphomas and some other cancers is significantly stronger (Blood. 2017, 129, 257 -260; J. Comput. Aided Mol. Des. 2012, 26, 437-450; Leukemia. 2012, 26, 236-243; Mol. Cancer Ther. 2014, 13, 880-889).

The essence of this invention is therefore new compounds with significant antitumor activity that can be used to treat cancer.

The essence of the invention

The present invention therefore describes a group of new substituted 2-piperazinylarylamino-8oxopurines which are effective inhibitors of cyclin dependent kinases 4 and 6 and receptor kinases FLT3 and PDGFR. This group of new purine derivatives is notable for its high selectivity towards CDK4 and CDK6 and at the same time inhibition of FLT3 and PDGFR kinases, and the associated antitumor activity, especially against some hematological malignancies. The new compounds can therefore be used as antitumor drugs in oncology and hemato-oncology.

The subject of this invention are substituted 2-piperazinylarylamino-8-oxopurines of general formula I,

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(I) in which

X is CH, RI is methyl, ethyl, or isopropyl,

R 2 is selected from the group consisting of cyclohexyl, cycloheptyl, cyclooctyl, 3-methylcyclohexyl; and

R 3 is H or tert-butyloxycarbonyl, and pharmaceutically acceptable salts thereof, such as salts with alkali metals, ammonium or amines, or addition salts with acids, and pharmaceutically acceptable solvates thereof, including hydrates.

If there are chiral centers in the molecule, then this invention also includes optically active isomers, their mixtures and racemates.

In another preferred embodiment, R 2 is selected from the stereoisomers in the 1 R,3R,\R,3S,\S,3R,1S,3S configuration for the 3-methylcyclohexyl group.

The following derivatives of general formula I are particularly preferred: 9-cyclohexyl-7-methyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-cycloheptyl-7-methyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-cyclooctyl-7-methyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-cyclohexyl-7-isopropyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-Cycloheptyl-7-isopropyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one.

The subject of this invention is also compounds of the general formula I for use as drugs, in particular for the treatment of proliferative diseases involving dysregulation (especially upregulation) of CDK4, CDK6, FLT3 and/or PDGFR kinases.

Another object of the present invention are compounds of general formula I for use in the treatment of diseases that involve hyperproliferation, in particular selected from the group comprising the following diseases and disorders: cancer, restenosis, rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, Alzheimer's disease, parasitic growth (animals, fungi, protists), graft rejection (host versus graft), graft versus host, and gout.

CDK4/6 regulates certain parts of the cell cycle, especially the G1 phase. Based on the activity of the compounds of the general formula as inhibitors of this kinase, it is expected that the compounds of the formula I and their pharmaceutically acceptable salts can be used as antimitotic compounds and for the treatment of proliferative diseases such as cancer and restenosis. In very low concentration (micromolar and

-3 CZ 309356 B6 lower) are therefore capable of inhibiting cell cycle transitions (Gl/S) in various animal tissues and embryos. In addition, these compounds are useful in the treatment of autoimmune diseases, eg, rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis; in the treatment of Alzheimer's disease, cardiovascular diseases such as restenosis, graft rejection (host vs. graft), graft vs. host, gout; and in the treatment of cancer, polycystic kidney disease and other proliferative diseases whose pathogenesis involves abnormal cell proliferation.

The subject of the invention are also compounds of the general formula I for the treatment of leukemias, in particular selected from the group including the following leukemias: acute myeloid leukemia, acute lymphocytemic leukemia, acute promyelocytemic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic neutrophilic leukemia, acute undifferentiated leukemia, anaplastic large cell lymphoma, prolymphocytemic leukemia, juvenile myelomonocytic leukemia, adult T-cell leukemia, myelodysplastic syndrome and myeloproliferative disorder.

Another object of the invention are compounds of general formula I for use as antileukemic drugs in the therapy of acute myeloid leukemia (AML) characterized by a standard or mutant form of the FLT3 kinase. For example, FLT3-ITD and FLT3 with point mutations FLT3-D835Y, FLT3-D835H, FLT3-D835V, FLT3-K663Q and FLT3-N841I can be considered mutant forms of FLT3. Mutant forms of FLT3 can be detected by conventional methods such as PCR or sequencing.

The effectiveness of compounds of general formula I for use as drugs against AML can also be derived from the mechanism of action based on the inhibition of protein kinases from the group of FLT3, CDK 4, CDK6, PDGFRA and PDGFRB, or their various combinations. Quantitative changes in the expression of FLT3, CDK and cyclins can be analyzed by RT-PCR. Genomic rearrangements (eg FIP1L1-PDGFRA translocation or other PDGFR rearrangements) are detected by cytogenetic examination, FISH, sequencing or comparative genomic hybridization. All the mentioned methods are commonly used in laboratory diagnostics.

Another subject of the invention are compounds of general formula I for use in the treatment of hematological disorders associated with eosinophilia and characterized by deregulation of PDGFRA, PDGFRB or FGFR1 kinases, but especially for chronic eosinophilic leukemia in which PDGFRA is oncogenically activated by translocation and fusion to form FIP1L1-PDGFRA. The compounds of general formula I can further be used for the treatment of other hematological disorders associated with eosinophilia, such as hypereosinophilic syndrome, lymphocytic variant of hypereosinophilia, idiopathic hypereosinophilic syndrome, acute myeloid leukemia, B-cell or T-cell acute lymphoblastic leukemia, lymphoblastic lymphoma or myeloid sarcoma expressing FIP1L1PDGFRA.

The invention also includes compounds of general formula I for the treatment of solid tumors with amplified, activated, deregulated or overexpressed CDK4, CDK6, cyclin D or PDGFRA genes, such as breast carcinomas, head and neck cancer, lung carcinomas, recurrent brain metastases, squamous cell carcinoma cancer, tumors of the central nervous system, gliomas, sarcomas, tumors of the colorectal and entire gastric system, tumors of the pancreas, liver, testicles, uterus, prostate, ovaries, bladder.

In addition to therapeutic applications (e.g. for human and veterinary use), it is clear that the subject compounds can be used as cell culture additives to regulate the proliferation and/or differentiation states of cells in vitro, for example by regulating the level of CDK4/CDK6/FLT3 activation.

The subject of this invention is also compounds of general formula I for use as inhibitors of cyclin dependent kinases (CDK), preferably CDK4 or CDK6, and/or FLT3 and PDGFR kinases.

Another subject of this invention are compounds of general formula I for use as inhibitors of cell proliferation or inducers of apoptosis in hyperproliferating cells in vivo or in vitro.

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Another subject of this invention are compounds of general formula I for use as inducers of senescence in hyperproliferating cells in vivo or in vitro.

Another object of the invention is also a pharmaceutical preparation containing at least one compound of general formula I, and at least one pharmaceutically acceptable carrier or excipient.

The newly described compounds can be used alone or as intermediates in the preparation of other compounds with therapeutic, diagnostic or technological use.

Pharmaceutical preparations

The therapeutic preparation contains from 1 to 95% of the active substance, whereby single doses preferably contain from 20 to 90% of the active substance, and in the case of methods of application that are not single-use, they preferably contain from 5 to 20% of the active substance. Unit dosage forms are, for example, coated tablets, tablets, ampoules, vials, suppositories or capsules. Other forms of application are e.g. ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions, etc. Examples are capsules containing from 0.05 g to 1.0 g of active substance.

The pharmaceutical preparations according to the present invention are prepared in a known manner, e.g. by conventional mixing, granulation, coating, dissolving or lyophilization processes.

Solutions of active substances are preferably used, as well as suspensions or dispersions, especially isotonic aqueous solutions, suspensions or dispersions, which can be prepared before use, e.g. in the case of lyophilized preparations containing the active substance alone or with a carrier such as mannitol. Pharmaceutical preparations may be sterilized and/or contain excipients such as preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizing agents, salts for regulating osmotic pressure and/or buffers. They are prepared in a known manner, e.g. by ordinary dissolution or lyophilization. Said solutions or suspensions may contain viscosity-increasing substances, such as sodium carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

Oil suspensions contain as an oil component vegetable, synthetic or semi-synthetic oils usual for injection purposes. The oils which may be mentioned here are especially liquid fatty acid esters which contain as an acid component a long-chain fatty acid having 8-22, preferably 12-22 carbon atoms, e.g. lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic, margaric, stearic, arachidonic and behenic acids, or corresponding unsaturated acids, e.g. oleic, alaidic, euric, brasidic and linoleic acids, possibly with the addition of antioxidants, e.g. vitamin E, β-carotene or 3,5-di-tert- butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters has no more than 6 carbon atoms and is mono- or polyhydric, e.g. mono-, di- or trihydric alcohols such as methanol, ethanol, propanol, butanol or pentanol and their isomers, but mainly glycol and glycerol. Fatty acid esters are preferably e.g. ethyl oleate, isopropyl myristate, isopropyl palmitate, "Labrafil M 2375" (polyoxyethylene glycerol trioleate, Gattefoseé, Paris), "Labrafil M 1944 CS" (unsaturated polyglycol glycerides prepared by alcoholysis of apricot kernel oil and composed of glycerides and polyethylene glycol esters; Gattefoseé, Paris), "Labrasol" (saturated polyglycol glycerides prepared by alcoholysis of TCM and composed of glycerides and polyethylene glycol esters; Gattefoseé, Paris) and/or "Miglyol 812" (a triglyceride of saturated fatty acids with Cx to C12 from Hůls AG, Germany) and especially vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and especially groundnut oil.

Preparation of the injectable preparation is carried out under sterile conditions in the usual way, e.g. by filling into ampoules or vials and sealing the packages.

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E.g. pharmaceutical preparations for oral use can be obtained by mixing the active substance with one or more solid carriers, possibly granulating the resulting mixture and, if desired, processing the mixture or granules into tablets or coated tablets by adding other neutral substances.

Suitable carriers are especially fillers such as sugars, e.g. lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, preferably calcium phosphate or calcium hydrogen phosphate, further binders such as starches, preferably corn, wheat, rice or potato starch, methylcellulose . Other neutral substances are flow regulators and lubricants, preferably salicylic acid, talc, stearic acid and its salts such as magnesium and/or calcium stearate, polyethylene glycol or its derivatives. The cores of the coated tablets can be coated with suitable coatings which can be resistant to gastric juice, the coatings used being among others concentrated solutions of sugars which may contain gum arabic, talc, polyvinylpyrrolidine, polyethylene glycol and/or titanium dioxide, further coating solutions in suitable organic solvents or solvent mixtures, or for the preparation of coatings resistant to gastric juice, solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxypropyl methyl cellulose phthalate. Dyes or pigments are mixed into tablets or coated tablets, for example to identify or characterize different doses of the active ingredient.

Pharmaceutical preparations that can be taken orally are also hard capsules made of gelatin or soft closed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Hard capsules may contain the active substance in the form of granules, mixed for example with fillers such as corn starch, binders or lubricants such as talc or magnesium stearate, and with stabilizers. In soft capsules, the active substance is preferably dissolved or suspended in suitable liquid substances of a neutral nature, such as lubricating grease, paraffin oil or liquid polyethylene glycol or esters of fatty acids and ethylene or propylene glycol, while it is also possible to add stabilizers and detergents, e.g. of the type of polyethylene sorbitan fatty esters acids.

Other forms of oral administration are e.g. syrups prepared in a conventional manner, which contain the active ingredient e.g. in a suspended form and in a concentration of about 5 to 20%, preferably about 10% or a similar concentration that allows for a suitable individual dose, e.g. when 5 or 10 ml. Other forms are e.g. powder or liquid concentrates for preparing cocktails, e.g. in milk. Such concentrates may also be packaged in unit dose quantities.

Pharmaceutical preparations that can be used rectally are, for example, suppositories that contain a combination of an active substance and a base. Suitable bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alcohols.

Preparations suitable for parenteral administration are aqueous solutions of the active ingredient in a water-soluble form, e.g. a water-soluble salt or an aqueous injectable suspension that contains viscosity-increasing substances, e.g. sodium carboxymethylcellulose, sorbitol and/or dextran, and stabilizers where that appropriate. The active substance may also be present in the form of a lyophilizate together with excipients where appropriate and may be dissolved prior to parenteral administration by adding suitable solvents. Solutions that are used for parenteral application can also be used, for example, for infusion solutions. Antioxidants such as ascorbic acid or microbicides such as sorbic or benzoic acid are preferred canned foods.

Tinctures and solutions usually contain an aqueous-ethanol base to which humectants are mixed to reduce evaporation, such as polyalcohols, e.g. glycerol, glycols and/or polyethylene glycol, and lubricants such as esters of fatty acids and lower polyethylene glycols, i.e. lipophilic substances

-6CZ 309356 B6 soluble in an aqueous mixture replacing fatty substances removed from the skin with ethanol and, if necessary, also other excipients and additives.

The invention also relates to processes or methods for treating the diseases mentioned above. Substances can be administered prophylactically or therapeutically as such or in the form of pharmaceutical preparations, preferably in an amount that is effective against the mentioned diseases, while in warm-blooded animals, e.g. humans, requiring such treatment, the substance is used mainly in the form of a pharmaceutical preparation. A daily dose of about 0.01 to 1 g, preferably 0.05 to 1 g, is applied to a body weight of around 70 kg.

Clarification of drawings

Giant. 1 shows the effect of compound 9 on the cell cycle in MV4-11 cells after 24 hours.

Giant. 2 shows the effect of compound 9 on the cell cycle in EOL-1 cells after 24 hours.

Examples of implementation of the invention

The following examples serve to illustrate the invention without limiting its scope. Starting materials and auxiliary material for the preparation of compound I can be obtained from commercial sources (Merck, Fluorochem, etc.). Solvents were evaporated under reduced pressure using a rotary vacuum evaporator at temperatures below 60 °C. MS spectra (m/z) were measured on an AQUILTY UPLC system (Waters, USA) with a diode array detector and a mass spectrometer with a HESI and a quadrupole analyzer with a QDA detector. Merck silica gel Kieselgel 60 (grain size 230 to 400) was used for column chromatography. All compounds showed satisfactory elemental analyzes (± 0.4%).

Method A

Example 1

9-Cyclobutyl-7-methyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one (the compound does not fall within the scope of the invention)

A solution of 2,4-dichloropyrimidin-5-amine (5 g; 30.5 mmol) in MeOH (90 mL) was cooled to 0 °C. Subsequently, glacial acetic acid (10 mL; 183 mmol) and 37% aqueous formaldehyde solution (2.7 mL; 36.6 mmol) were added to the solution. The mixture was stirred for 1 hour, then taken out of the ice bath, when an additional amount of glacial acetic acid (20 mL) was added. After 3 h, NaBFLCN (4.6 g; 73.2 mmol) was added portionwise to the mixture. After 20 h, when the mixture was stirred at 5 °C, the solvent was evaporated under reduced pressure, the residue was diluted with distilled water (100 mL) and cooled in an ice bath. Acetic acid was neutralized with solid NaHCCL.

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Subsequently, the aqueous phase was extracted with DCM (2x 80 mL) and the combined organic phases were washed with brine, dried over anhydrous MgSO4 and evaporated under reduced pressure. The residue was diluted with MeOH (10 mL), cooled in an ice bath, and 80 mL of distilled water was added. After thorough mixing, the solid was filtered and washed with water. 2,4-Dichloro-A-mcthylpyrimidin-5-amine was obtained as a white solid in a yield of 4.4 g (81%).

To a solution of 2,4-dichloro-N-methylpyrimidin-5-amine (178 mg, 1.00 mmol) in butanol (10 mL) was added cyclobutylamine (71 mg; 82 μΐ; 1.00 mmol), Λ'.Λ ′-diisopropylethylamine (259 mg; 348 μΐ; 2.00 mmol) and the mixture was stirred at 80 °C. After 48 hours, the butanol was evaporated under reduced pressure and the crude product was purified on a hexane/EtOAc (1:1) mobile phase chromatography column. The product 2-chloro-A ⁴ -cyclobutyl-A ⁵ -methylpyrimidine-4,5-diamine was obtained in a yield of 179 mg (84%).

2-Chloro-A ⁴ -cyclobutyl-A ⁵ -methylpyrimidine-4,5-diamine (150 mg; 0.70 mmol) was dissolved in anhydrous THF (10 mL) under an inert nitrogen atmosphere and cooled to -10 °C. To the solution was slowly added 15 wt% COC12 in toluene (560 μΐ; 0.85 mmol) and 1M LiHMDS in hexane (1.4 mL; 1.40 mmol) was added dropwise. After 1 hour, the mixture was brought to room temperature and stirred for 20 min. The reaction mixture was evaporated under reduced pressure and purified by column chromatography with EtOAc/hexane (2:1). The product 2-chloro-9-cyclobutyl-7-methyl-7,9dihydro-8H-purin-8-one was obtained in a yield of 130 mg (77%).

A microwave reactor vial was filled with 2-chloro-9-cyclobutyl-7-methyl-7,9-dihydro-8Hpurin-8-one (65 mg; 0.27 mmol), a 4:1 dioxane/H2O solvent mixture (1 mL ), tert-butyl 4-(4aminophenyl)piperazine-1-carboxylate (60 mg; 0.22 mmol), K 2 CO 3 (150 mg; 1.08 mmol) and XPhos Pd G 2 (4 mg; 2 mol %). The reaction mixture was stirred at 100 °C under microwave radiation (150 W) for 90 min. The mixture was evaporated under reduced pressure and purified by column chromatography with hexane/EtOAc (1:1) mobile phase. The product tert-butyl 4-(4-((9-cyclobutyl-7methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)amino)phenyl)piperazine-1-carboxylate was obtained in a yield of 60 mg (57%).

To a solution of tert-butyl 4-(4-((9-cyclobutyl-7-methyl-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1-carboxylate (60 mg; 0 .13 mmol) in 1:1 DCM/MeOH (2.5 mL) was added with 36% HCl (200 μΐ). After 48 h, the solid product was filtered off, dissolved in distilled water (1.3 mL) and the mixture was neutralized with solid K 2 CO 3 . The crystalline product was filtered, washed with water and dried under reduced pressure. The product 9-cyclobutyl-7-methyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 29 mg (61%).

Example 2

7-ethyl-2-((4-(piperazin-1-yl)phenyl)amino)-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin8-one (the compound does not fall within the scope of the invention)

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2,4-Dichloro-N-cthylpyrimidin-5-amine was prepared according to Example 2 using 1,1-dimethoxyethane.

To a solution of 2,4-dichloro-A-ethylpyrimidin-5-amine (192 mg; 1.00 mmol) in butanol (10 mL) was added tetrahydro-2H-pyran-4-amine hydrochloride (137 mg; 1.00 mmol) ), Α,Α-diisopropylethylamine (389 mg; 522 µL; 3.00 mmol) and the mixture was stirred at 85 °C. After 48 h, the butanol was evaporated under reduced pressure and the crude product was purified by column chromatography with EtOAc/hexane (2:1). The product 2-chloro-A ⁵ -ethyl-A ⁴ -(tetrahydro-2H-pyran-4-yl)pyrimidin-4,5-diamine was obtained in a yield of 205 mg (80%).

2-Chloro-A ⁵ -ethyl-A ⁴ -(tetrahydro-2H-pyran-4-yl)pyrimidin-4,5-diamine (189 mg; 0.74 mmol) was dissolved in anhydrous THF (10 mL) under inert nitrogen atmosphere and cooled to -10 °C. To the solution was slowly added 15 wt% COCh in toluene (592 µL; 0.89 mmol) and 1 M LiHMDS in hexane (1.5 mL; 1.50 mmol) was added dropwise. After 1 hour, the mixture was brought to room temperature and stirred for 20 minutes. The reaction mixture was evaporated under reduced pressure and purified by column chromatography with EtOAc/hexane (2:1). The product 2-chloro-7-ethyl-9(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 86 mg (41%).

A microwave reactor vial was charged with 2-chloro-7-ethyl-9-(tetrahydro-277-pyran-4-yl)-7,9dihydro-8H-purin-8-one (79 mg; 0.28 mmol), a mixture solvents 4:1 dioxane/H2O (1.4 mL), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (62 mg; 0.22 mmol), K2CO3 (155 mg; 1.12 mmol) and XPhos Pd G2 (4.5 mg; 2 mol %). The reaction mixture was stirred at 100 °C under microwave radiation (150 W) for two hours. The mixture was evaporated under reduced pressure and purified by column chromatography with EtOAc/hexane (2:1). Product tert-butyl 4-(4-((7ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenyl)piperazine -lcarboxylate was obtained in a yield of 107 mg (93%).

To a solution of tert-butyl 4-(4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2yl)amino)phenyl) To piperazine-1-carboxylate (102 mg; 0.20 mmol) in 1:1 DCM/MeOH (4.9 mL) was added 36% HCl (384 μΐ). After 72 h, distilled water (1 mL) and solid K 2 CO 3 were added to achieve pH >12. The solution was evaporated and the product purified by mobile phase column chromatography (10:1 DCM/MeOH). The product 7-ethyl-2-((4-(piperazin-1-yl)phenyl)amino)-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 53 mg (64%).

Example 3

9-Cyclopentyl-7-isopropyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one (the compound does not fall within the scope of the invention)

A round bottom flask was charged with 2,4-dichloro-pyrimidin-5-amine (5 g; 30.5 mmol), 2,2-dimethoxypropane (61 mL; 50 mmol) and glacial acetic acid (7.3 mL; 134 mmol). The mixture was cooled in an ice bath, to which a solution of NaBH(OAc)3 (26 g; 122 mmol) in

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DCM (61 mL). After two hours, the reaction mixture was removed from the ice bath and stirred for an additional 20 minutes at room temperature. The mixture was concentrated under reduced pressure, diluted with DCM (150 mL), washed with 10% K 2 CO 3 (100 mL), distilled water (100 mL), brine (100 mL) and then dried over anhydrous MgSO 4 and evaporated under reduced pressure. The crude product 2,4-dichloro-Isopropylpyrimidin-5-amine was obtained by column chromatography with a mobile phase of hexane/EtOAc (1:1) in a yield of 6.18 g (98%).

To a solution of 2,4-dichloro-A-isopropylpyrimidin-5-amine (412 mg; 2.00 mmol) in butanol (20 mL) was added cyclopentylamine (170 mg; 197 µL; 2.00 mmol), Λζ/V- diisopropylethylamine (518 mg; 696 μΐ; 4.00 mmol) and the mixture was stirred at 90 °C. After 48 h, the butanol was evaporated under reduced pressure and the crude product was purified by column chromatography with a mobile phase of hexane/EtOAc (7:2). The product 2-chloro-A ⁴ -cyclopentyl-A ⁵ -isopropylpyrimidine-4,5-diamine was obtained in a yield of 377 mg (74%).

2-Chloro-A ⁴ -cyclopentyl-A ⁵ -isopropylpyrimidine-4,5-diamine (203 mg; 0.80 mmol) was dissolved in anhydrous THF (12 mL) under an inert nitrogen atmosphere and cooled to -10 °C. To the solution was slowly added 15 wt% COC12 in toluene (640 μΐ; 0.96 mmol) and 1M LiHMDS in hexane (1.6 mL; 1.60 mmol) was added dropwise. After 1 hour, the mixture was brought to room temperature and stirred for 20 minutes. The reaction mixture was evaporated under reduced pressure and purified by column chromatography with hexane/EtOAc (3:1) mobile phase. The product 2-chloro-9-cyclopentyl-7-isopropyl-7,9dihydro-8H-purin-8-one was obtained in a yield of 207 mg (92%).

A microwave reactor vial was charged with 2-chloro-9-cyclopentyl-7-isopropyl-7,9-dihydro8H-purin-8-one (94 mg; 0.33 mmol), a 4:1 dioxane/H2O solvent mixture (1, 65 mL), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (73 mg; 0.26 mmol), K2CO3 (182 mg; 1.32 mmol), and XPhos Pd G2 (5 mg; 2 mol . %). The reaction mixture was stirred at 100 °C under microwave radiation (150 W) for 90 min. The mixture was evaporated under reduced pressure and purified by column chromatography with hexane/EtOAc (2:1 to 1:1) mobile phase. The product tert-butyl 4-(4-((9cyclopentyl-7-isopropyl-8-oxo-8,9-dihydro-7H-purin-2-yl)amino)phenyl)piperazine-1-carboxylate was obtained in a yield of 85 mg (70%).

To a solution of tert-butyl 4-(4-((9-cyclopentyl-7-isopropyl-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1-carboxylate (80 mg; 0 .15 mmol) in 1:1 DCM/MeOH (3 mL) was added 36% HCl (240 μΐ). After 72 hours, distilled water (0.75 mL) and solid K 2 CO 3 were added to achieve pH >12. The solution was evaporated and purified by column chromatography with mobile phase DCM/MeOH (10:1). The product 9-cyclopentyl-7-isopropyl-2-((4-(piperazin-lyl)phenyl)amino)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 16 mg (25%).

Example 4

7-methyl-9-(1-methylpiperidin-4-yl)-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8one (the compound does not fall within the scope of the invention)

-10 CZ 309356 B6

A solution of 2,4-dichloropyrimidin-5-amine (5 g; 30.5 mmol) in MeOH (90 mL) was cooled to 0 °C. Subsequently, glacial acetic acid (10 mL; 183 mmol) and 37% aqueous formaldehyde solution (2.7 mL; 36.6 mmol) were added to the solution. The mixture was stirred for 1 hour, then taken out of the ice bath, when an additional amount of glacial acetic acid (20 mL) was added. After 3 h, NaBFLCN (4.6 g; 73.2 mmol) was added portionwise to the mixture. After 20 h, when the mixture was stirred at 5 °C, the solvent was evaporated under reduced pressure, the residue was diluted with distilled water (100 mL) and cooled in an ice bath. The acetic acid was neutralized with solid NaHCOv. Subsequently, the aqueous phase was extracted with DCM (2x 80 mL) and the combined organic phases were washed with brine, dried over anhydrous MgSO4 and evaporated under reduced pressure. The residue was diluted with MeOH (10 mL), cooled in an ice bath, and 80 mL of distilled water was added. After thorough mixing, the solid was filtered and washed with water. 2,4-Dichloro-N-methylpyrimidin-5-amine was obtained as a white solid in a yield of 4.4 g (81%).

To a solution of 2,4-dichloro-N-methylpyrimidin-5-amine (356 mg; 2.00 mmol) in butanol (10 mL) was added 1-methylpiperidin-4-amine (228 mg; 2.00 mmol) and Λζ N-diisopropylethylamine (518 mg; 696 µl; 4.00 mmol) and the mixture was stirred at 60 °C. After 48 h, 1-methylpiperidin-4-amine (228 mg; 2.00 mmol) and Λζ/V-diisopropylethylamine (518 mg; 696 μΐ; 4.00 mmol) were added and the mixture was stirred at 60 °C for 5 days. The butanol was evaporated under reduced pressure and the crude product was purified by column chromatography with EtOAc/hexane (2:1). The product 2-chloro-A ⁵ -methylΛ' ⁴ -( 1 -mcthylpipecridin-4-yl)pyrimidin-4,5-diamine was obtained in a yield of 480 mg (94%).

2-Chloro-A ⁵ -methyl-A ⁴ -(1-methylpiperidin-4-yl)pyrimidin-4,5-diamine (224 mg; 0.88 mmol) was dissolved in anhydrous THF (12 mL) under an inert nitrogen atmosphere and cooled to -10 °C. 15 wt% COCE in toluene (725 μΐ; 1.10 mmol) was slowly added to the solution and 1 M LiHMDS in hexane (1.76 mL; 1.76 mmol) was added dropwise. After 1 hour, the mixture was brought to room temperature and stirred for 20 minutes. The reaction mixture was evaporated under reduced pressure and purified by column chromatography with EtOAc/hexane (2:1). The product 2-chloro-7-methyl-9-(1-methylpiperidin-4-yl)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 101 mg (41%).

A microwave reactor vial was charged with 2-chloro-7-methyl-9-(1-methylpiperidin-4-yl)-7,9dihydro-8H-purin-8-one (76 mg; 0.27 mmol), solvent mixture 4 :1 dioxane/H2O (1 mL), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (60 mg; 0.22 mmol), K2CO3 (150 mg; 1.08 mmol) and XPhos Pd G2 (4 mg ; 2 mol.%). The reaction mixture was stirred at 100 °C under microwave radiation (150 W) for 60 min. The mixture was evaporated under reduced pressure and purified by column chromatography with mobile phase DCM/MeOH (30:1 to 10:1). Product tert-butyl 4-(4-((7-methyl-9-(1-methylpiperidin-4-yl)-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1 -carboxylate was obtained in a yield of 103 mg (95%).

To a solution of tert-butyl 4-(4-((7-methyl-9-(1-methylpiperidin-4-yl)-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine- 1-carboxylate (103 mg; 0.20 mmol) in 1:1 DCM/MeOH (3.85 mL) was added 36% HCl (308 μΐ). After 24 hours, 36% HCl (308 μΐ) was added and the mixture was stirred for another 24 hours. Subsequently, the solid product was filtered, dissolved in distilled water (1.3 mL) and the mixture was neutralized with solid K 2 CO 3 . The crystalline product was filtered, washed with water and dried under reduced pressure. The product 7-methyl-9-(1-methylpiperidin-4-yl)-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one was obtained in yield 26 mg (31%).

Example 5

7-isopropyl-9-(4-methylcyclohexyl)-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8one (the compound does not fall within the scope of the invention)

-11 CZ 309356 B6

A round bottom flask was charged with 2,4-dichloropyrimidin-5-amine (5 g; 30.5 mmol), 2,2-dimethoxypropane (61 mL; 50 mmol) and glacial acetic acid (7.3 mL, 134 mmol) . The mixture was cooled in an ice bath, to which was subsequently added a solution of NaBH(OAc) 3 (26 g; 122 mmol) in DCM (61 mL). After two hours, the reaction mixture was taken out of the ice bath and stirred for another 20 min at room temperature. The mixture was concentrated under reduced pressure, diluted with DCM (150 mL) and washed with 10% K 2 CO 3 (100 mL), distilled water (100 mL), brine (100 mL) and then dried over anhydrous MgSO 4 and evaporated under reduced pressure. The crude product 2,4-dichloro-isopropylpyrimidin-5-amine was obtained by mobile phase column chromatography (1:1 hexane/EtOAc) in a yield of 6.18 g (98%).

To a solution of 2,4-dichloro-A-isopropylpyrimidin-5-amine (615 mg; 3.00 mmol) in butanol (30 mL) was added 4-methylcyclohexaneamine (340 mg; 400 µL; 3.00 mmol), ΛζΑ-diisopropylethylamine (777 mg; 1.05 mL; 6.00 mmol) and the mixture was stirred at 90 °C. After 48 h, the butanol was evaporated under reduced pressure and the crude product was purified by column chromatography with a mobile phase of hexane/EtOAc (7:2). The product 2-chloro-A ⁵ -isopropyl-A ⁴ -(4-methylcyclohexyl)pyrimidine-4,5-diamine was obtained in a yield of 509 mg (60%).

2-Chloro-A ⁵ -isopropyl-A ⁴ -(4-methylcyclohexyl)pyrimidine-4,5-diamine (226 mg; 0.80 mmol) was dissolved in anhydrous THF (12 mL) under an inert nitrogen atmosphere and cooled to - 10 °C. 15 wt% COCh in toluene (640 µl; 0.96 mmol) was slowly added to the solution and IMLiHMDS in hexane (1.6 mL; 1.60 mmol) was added dropwise. After 1 hour, the mixture was brought to room temperature and stirred for 20 minutes. The reaction mixture was evaporated under reduced pressure and purified by column chromatography with hexane/EtOAc (4:1) mobile phase. The product 2-chloro-7-isopropyl-9-(4methylcyclohexyl)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 159 mg (65%).

A microwave reactor vial was filled with 2-chloro-7-isopropyl-9-(4-methylcyclohexyl)-7,9dihydro-8H-purin-8-one (103 mg; 0.33 mmol), a solvent mixture of 4:1 dioxane / H 2 O (1.65 mL), tert -butyl 4-(4-aminophenyl)piperazine-1-carboxylate (73 mg; 0.26 mmol), K 2 CO 3 (182 mg; 1.32 mmol) and XPhos Pd G 2 (5 mg ; 2 mol.%). The reaction mixture was stirred at 100 °C under microwave radiation (150 W) for 3 hours. The mixture was evaporated under reduced pressure and purified by column chromatography with a mobile phase of hexane/EtOAc (2:1 to 3:2). The product tert-butyl 4(4-((7-isopropyl-9-(4-methylcyclohexyl)-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1-carboxylate was obtained in yield 78 mg (55%).

To a solution of tert-butyl 4-(4-((7-isopropyl-9-(4-methylcyclohexyl)-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1-carboxylate ( 36 mg; 0.065 mmol) in 1:1 DCM/MeOH (1.25 mL) was added 36% HCl (104 μΐ). After 72 hours, distilled water (0.75 mL) and solid K 2 CO 3 were added to achieve pH >12. The solution was evaporated and purified by column chromatography with mobile phase DCM/MeOH (10:1). The product 7-isopropyl-9-(4-methylcyclohexyl)-2-((4-(piperazin1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 26 mg (90 %).

Method B

-12 CZ 309356 B6

Example 6

7-Methyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one (the compound does not fall within the scope of the invention)

Me ji J · ^Ώ : H .N. ·· N

H

(2,4-dimethoxyphenyl)methanamine (356 mg; 320 μΐ; 2, 13 mmol), Λζ/V-diisopropylethylamine (552 mg; 741 μΐ; 4.26 mmol) and the mixture was stirred at 90 °C. After 48 hours, the butanol was evaporated under reduced pressure and the crude product was purified by column chromatography with a mobile phase of hexane/EtOAc (1:1). The product 2-chloro-A' ⁴ -(2,4-dimcthoxybenzyl)-A' ⁵ -mcthylpyrimidine-4,5-diamine was obtained in a yield of 518 mg (79%).

2-Chloro-A ⁴ -(2,4-dimethoxybenzyl)-A ⁵ -methylpyrimidine-4,5-diamine (309 mg, 1.00 mmol) was dissolved in anhydrous THF (14 mL) under an inert nitrogen atmosphere and cooled to -10 °C. 15 wt % COCh in toluene (960 μΐ; 1.20 mmol) was slowly added to the solution and 1 M LiHMDS in hexane (2 mL; 2.00 mmol) was added dropwise. After 1 hour, the mixture was brought to room temperature and stirred for 20 minutes. The reaction mixture was evaporated under reduced pressure and purified by column chromatography with hexane/EtOAc (3:2 to 2:1) mobile phase. The product 2-chloro-9-(2,4dimethoxybenzyl)-7-methyl-7,9-dihydro-8H-purin-8-one was obtained in a yield of 314 mg (94%).

A microwave reactor vial was charged with 2-chloro-9-(2,4-dimethoxybenzyl)-7-methyl-7,9dihydro-8H-purin-8-one (188 mg; 0.56 mmol), a 4:1 solvent mixture dioxane/H2O (2 mL), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (124 mg; 0.5 mmol), K2CO3 (309 mg; 2.24 mmol) and XPhos Pd G2 (9 mg; 2 mol.%). The reaction mixture was stirred at 100 °C under microwave radiation (150 W) for 90 min. The mixture was evaporated under reduced pressure and purified by column chromatography eluting with hexane/EtOAc (2:1) to EtOAc/hexane (2:1). Product tert-butyl 4-(4-((9-(2,4-dimethoxybenzyl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1-carboxylate was obtained in a yield of 252 mg (88%).

To a solution of tert-butyl 4-(4-((9-(2,4-dimethoxybenzyl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2yl)amino)phenyl)piperazine-1- carboxylate (219 mg; 0.38 mmol) in 1:1 DCM/MeOH (7.6 mL) was added 36% HCl (608 μΐ). After 48 h, the solid product was filtered, dissolved in distilled water (4 mL) and the mixture was neutralized with solid K 2 CO 3 . The crystalline product was filtered, washed with water and dried under reduced pressure. The product 9-(2,4-dimethoxybenzyl)-7methyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 110 mg (61%).

9-(2,4-Dimethoxybenzyl)-7-methyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8one (109 mg; 0.23 mmol) was dissolved in anisole (1.2 mL), then trifluoroacetic acid (1 mL) was added and the reaction mixture was heated in a reflux round flask at 90 °C for 5 days. The TFA was blown off with a stream of nitrogen and the anisole was evaporated under reduced pressure and purified by DCM/MeOH (5:1) column chromatography. After evaporation, the product was

-13 CZ 309356 B6 diluted with 1:1 H2O/MeOH (2 mL) and the solid was filtered. The product 7-methyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one was obtained in a yield of 26 mg (35%).

Table 1: Compounds prepared by method A and B (X = CH)

C. PURINE SUBSTITUTE CHN ANALYSIS MS (ZMD) ANALYSIS R1 R2 R3 [%] [M-H] a) [M+H] ⁺ b) 8 Methyl Cyclohexyl -Shit C 63.95; H 7.55; N 19,16 506.3 508.3 9 Methyl Cyclohexyl -H C 64.74; H 7.35; N 24.20 406.2 408.3 10 Methyl Cycloheptyl -Shit C 64.60; H 7.74; N 18.67 520.3 522.3 11 Methyl Cycloheptyl -H C 65.45; H 7.50; N 23.33 420.3 422.3 12 Methyl Cyclooctyl -Shit C 65.20; H 7.61; N 18.45 534.3 536.3 13 Methyl Cyclooctyl -H C 66.40; H 7.75; N 22.55 434.3 436.3 26 Methyl (1R,3R)-3Methylcyclohexyl -Shit C 64.60; H 7.42; N 18.98 520.3 522.3 27 Methyl (1R,3R)-3Methylcyclohexyl -H C 65.72; H 7.33; N 23.35 420.3 422.3 28 Methyl (1R,3S)-3- Methylcyclohexyl -Shit C 64.71; H 7.65; N 18.72 520.3 522.3 29 Methyl (1R,3S)-3Methylcyclohexyl -H C 65.62; H 7.54; N 23.33 420.3 422.3 30 Methyl (1S,3R)-3- Methylcyclohexyl -Shit C 64.61; H 7.47; N 18.96 520.3 522.3 31 Methyl (1S,3R)-3Methylcyclohexyl -H C 65.45; H 7.49; N 23.35 420.3 422.3 32 Methyl (15,35)-3Methylcyclohexyl -Shit C 64.52; H 7.43; N 18.99 520.3 522.3 33 Methyl (15,35)-3Methylcyclohexyl -H C 65.70; H 7.51; N 23.05 420.3 422.3 66 Ethyl Cyclohexyl -Shit C 64.54; H 7.42; N 18.57 520.3 522.3 67 Ethyl Cyclohexyl -H C 65.42; H 7.51; N 23.01 420.3 422.3 72 Isopropyl Cyclohexyl -Shit C 65.11; H 7.66; N 18.03 534.3 536.3 73 Isopropyl Cyclohexyl -H C 66.22; H 7.71; N 22.28 434.3 436.3 74 Isopropyl Cycloheptyl -Shit C 65.72; H 7.93; N 17.65 548.3 550.4 75 Isopropyl Cycloheptyl -H C 66.85; H 7.74; N 21.52 448.3 450.3

-14 CZ 309356 B6

Example 7 Inhibition of kinases

The ability of the newly prepared compounds to inhibit the given protein kinases was determined and quantified by enzyme measurement with recombinant protein kinases. Protein kinases were produced in the insect cell line Sf9 after infection with a baculovirus vector and purified by separation on a NiNTA column. Kinase activity was measured in the following arrangement: 1 mg/ml RB peptide (for CDK4 and CDK6) or AGLT peptide (for FLT3 and PDGFRα), 1 μΜ ATP, 0.05 pCi [γ ³³ P]ATP, test compound, all in of buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCF. 3 mM MnCE, 3 μΜ Na-orthovanadate, 1.2 mM DTT, 2.5 pg / 50 μl PEG20,000), in a total volume of 10 μl. The reaction was stopped by adding 5 µl of 3% aq H 3 PO 4 . Phosphopeptides were separated using P-81 phosphocellulose (Whatman) which was washed with 3* 0.5% aq H 3 PO 4 and dried. Kinase activity was quantified with a Fujifilm FLA7000 digital laser analyzer and expressed as IC50, i.e. the concentration of substance required to reduce enzyme activity to 50%. Examples of results are shown in Table 2 and demonstrate that the compounds are potent inhibitors of CDK4, CDK6, FLT3-ITD or PDGFR kinases. At the same time, the selectivity of most compounds was verified by measuring the inhibitory effect on other protein kinases, while all tested compounds showed a significantly weaker effect on CDK1, CDK2 and CDK9. The IC50 values for these enzymes were at least 20x (but typically up to 100x) higher than the IC50s for CDK4 or FLT3-ITD.

Table 2: Examples of kinase inhibition by novel purine derivatives.

compound IC50 (nmol/1) % inhibition @ 1 μΜ CDK4 FLT3-ETC CDK6 PDGFRa 9 50 25 >80 >80 10 100 100 >80 >80 11 10 30 >80 >80 12 50 50 >80 >80 13 20 10 >80 >80 29 10 30 >80 >80 30 50 100 >80 >80 31 10 30 >80 >80 32 50 50 >80 >80 33 10 30 >80 >80 66 200 200 73 >1000 80 75 1000 30

Example 8 Antitumor activity of new compounds

The antitumor activity of the compounds was verified on tumor cell lines in vitro. One of the standard ways to determine cell proliferation and the number of live cells in a culture is to measure the metabolic activity of the cells, which is proportional to the number of live cells. Resazurin, for example, is commonly used to determine the number of living cells in the search and characterization of cytotoxic compounds. Metabolically active living cells reduce resazurin to resorufin and the amount of reosufin formed is proportional to the number of living cells in the culture. The effect of the new purine derivatives was verified on the following cell lines: MV4-11 (acute myeloid leukemia, FLT3-ITD positive), MOLM-13 (acute myeloid leukemia, FLT3-ITD positive), EOL-1 (eosinophilic leukemia, FIP1L1-PDGFRA fusion ), H1703 (non-small cell lung cancer, PDGFRA amplification), MCF7 (breast adenocarcinoma), BT549 (ductal breast carcinoma), U2932 (diffuse large B-cell lymphoma), JEKO1 (mantle cell lymphoma).

-15 CZ 309356 B6

For the measurements, the cells were planted in a 96-well panel and affected by the new derivatives in different concentrations. Four days after the addition of derivatives, a resazurin solution (final concentration 100 μΜ) was added to the culture, and after one hour of incubation, the fluorescence of resorufm produced by living cells was measured (570 nm/610 nm, ex/em) with a Fluoroskan Ascent fluorimeter (Labsystems). The effectiveness of the derivatives was expressed by the EC50 value, which expresses the concentration reducing the number of living cells to 50%.

Very significant activities were detected especially against the cell lines MV4-11 and MOLM-13 expressing the FLT3-ITD oncogene and EOL-1 expressing the FIP1L1-PDGFRA oncogene, for which nanomolar EC50 values were achieved. Some compounds were also active in other cell lines derived from lymphomas and lung carcinomas. Tumor cell lines without these typical oncogenic alterations (eg, MCF7 or K562) were significantly less sensitive, with EC50 values in the units to tens of micromoles/1.

Table 3: Example of antitumor activity of some 2,7,9-substituted 8-oxopurines.

compound EC50 (nmol/1) MV4-11 MOLM13 EOL1 9 200 100 100 10 100 500 800 11 50 50 50 12 80 200 200 13 60 80 30 29 100 300 100 30 200 500 400 31 100 300 100 32 400 500 400 33 80 200 80 66 100 200 100 73 50 100 80 75 50 100 200

Example 9 Effect of derivatives on the cell cycle

Inhibition of the CDK4 and FLT3 kinases is manifested at the cellular level by stopping proliferation in the G1 phase of the cell cycle. To verify the presumed mechanism of action, MV4-11 and EOL-1 cell cultures were affected by the new derivatives and their effect on the cell cycle was analyzed by flow cytometry. After 24 h exposure, cells were harvested by centrifugation and fixed with 70% ethanol. After staining with propidium iodide (final concentration 10 μg/ml), the relative DNA content in individual cells was quantified with a BD FacsVerse flow cytometer (Beckman Coulter, USA).

New derivatives demonstrably and very strongly stop proliferation in the G1 phase of the cell cycle in both used cell lines already in nanomolar concentrations. Higher concentrations also caused an increase in apoptotic cells, observed as a subdiploid population. Examples of representative cell cycle phases of MV4-11 and EOL-1 cell lines treated with different concentrations of the new derivative are shown in Figures 1 and 2. The vertical axis shows cell numbers.

Example 10 Induction of senescence

In addition to stopping cell proliferation, CDK4/6 inhibitors are also able to induce cell senescence. To verify the ability to induce senescence, MCF-7 cells were cultured with the new derivatives (1 μΜ concentration) for 3 to 10 days. β-galactosidase was used as a senescence marker,

-16 CZ 309356 B6 which was detected by an established method (Nature Protocols 2009:4:1798-1806). After said cultivation, the cells were rinsed with PBS and fixed with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in PBS (10 min). β-Galactosidase activity was detected by color reaction by incubating in a solution of 5-bromo-4-chloro-3-indolyl β-D-galactoside (1 mg/ml), K4[Fe(CN)e] (5 mM), K3[Fe (CN)e] (5 mM), NaCl (105 mM), MgCl (2 mM), all in 40 mM sodium citrate buffer (pH 6.0). Microscopic observation (after rinsing with PBS) revealed a significant number of enlarged and flattened cells with a pronounced blue color, indicating their senescence. Palbociclib, which induced a similar phenotype, was used as a positive control.

Table 4: Example of induction of MCF7 cell secession by novel 2,7,9-substituted 8-oxopurines.

compound % of galactosidase positive cells* palbociclib ++ 9 ++ 11 ++ 13 + 29 ++ The (+) symbol indicates the positivity of 40-79% of cells; (++) expresses the positivity of 80-100% of cells.

Example 11 Dry capsules

5000 capsules, each containing as active ingredient 0.25 g of one of the compounds of formula I are prepared by the following procedure:

Ingredients

Active folder 1250 g Talc 180 g Wheat starch 120 g Magnesium stearate 80 g Lactose 20 g

Preparation procedure: The rubbed substances are pushed through a sieve with a mesh size of 0.6 mm. A dose of 0.33 g of the mixture is transferred into a gelatin capsule using a capsule filling machine.

Example 12 Soft capsules

5000 soft gelatin capsules, each of them containing as an active ingredient 0.05 g of one of the substances of the general formula I as an active ingredient, are prepared by the following procedure:

Ingredients

Active ingredient Lauroglycol

250 g per liter

Preparation procedure: The powdered active substance is suspended in Lauroglycol® (propylene glycol laurate, Gattefoseé S.A., Saint Priest, France) and ground in a wet pulverizer to a particle size of about 1 to 3 pm. A 0.419 g dose of the mixture is then transferred into soft gelatin capsules using a capsule filling machine.

- 17 CZ 309356 B6

Example 13 Soft capsules

5000 soft gelatin capsules, each of them containing as an active ingredient 0.05 g of one of the 5 compounds of the general formula I as an active ingredient are prepared by the following procedure:

Ingredients

The active ingredient is also PEG 400

Tween 80's

250 g liter liter

Preparation procedure: The powdered active ingredient is suspended in PEG 400 (polyethylene glycol with Mr between 380 and 420, Sigma, Fluka, Aldrich, USA) and Tween® 80 (polyoxyethylene sorbitan monolaurate, 15 Atlas Chem. Ind., Inc., USA, supplied Sigma, Fluka, Aldrich, USA) and crushed in a wet pulverizer to particles of 1 to 3 µm in size. A 0.43 g dose of the mixture is then transferred into soft gelatin capsules using a capsule filling machine.

Claims (11)

PATENT CLAIMS 1. Substituted 2-piperazinylarylamino-8-oxopurine of general formula I, R ¹ yO (I) in which X is CH, R1 is methyl, ethyl, or isopropyl, R 2 is selected from the group consisting of cyclohexyl, cycloheptyl, cyclooctyl, 3-methylcyclohexyl; and R 3 is H or tert-butyloxycarbonyl, and pharmaceutically acceptable salts thereof, which are salts with alkali metals, ammonium or amines, or addition salts with acids, and pharmaceutically acceptable solvates thereof, including hydrates. 2. A compound of general formula I according to claim 1, wherein R2 is selected from the group comprising stereoisomers in the 1Λ,3Λ, 1Λ,3Ν, 15,3Λ, IS,3S configuration of the 3-methylcyclohexyl group. 3. The compound of general formula I according to claim 1, selected from the group comprising 9-cyclohexyl-7methyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purine-8- he; 9-cycloheptyl-7-methyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-cyclooctyl-7-methyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-cyclohexyl-7-isopropyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one; 9-Cycloheptyl-7-isopropyl-2-((4(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one. 4. A compound of general formula I according to any one of claims 1 to 3, for use as a medicine. 5. A compound of general formula I according to any one of claims 1 to 3, for use in the treatment of proliferative diseases involving dysregulation, in particular upregulation, of CDK4, CDK6, FLT3 and/or PDGFR kinases. 6. A compound of general formula I according to any one of claims 1 to 3, for use in the treatment of diseases selected from the group comprising the following diseases and disorders: cancer, restenosis, rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, Alzheimer's disease, parasitic growth, which are animals, fungi, protists, graft rejection - host versus graft, graft versus host, polycystic kidney disease and gout. - 19CZ 309356 B6 7. A compound of the general formula I according to any one of claims 1 to 3, for use in the treatment of leukemias, in particular selected from the group comprising the following leukemias: acute myeloid leukemia, acute lymphocytemic leukemia, acute promyelocytemic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic neutrophilic leukemia, acute undifferentiated leukemia, anaplastic large cell lymphoma, prolymphocytemic leukemia, juvenile myelomonocytic leukemia, adult T-cell leukemia, myelodysplastic syndrome and myeloproliferative disorder. 8. Compound of general formula I according to any one of claims 1 to 3, for use for the treatment of hematological disorders associated with eosinophilia, in particular for the treatment of chronic eosinophilic leukemia, hypereosinophilic syndrome, lymphocytoma variant of hypereosinophilia, idiopathic hypereosinophilic syndrome, acute myeloid leukemia, B-cell or T-cell acute lymphoblastic leukemia, lymphoblastic lymphoma, myeloid sarcoma expressing FIP1L1PDGFRA. 9. A compound of general formula I according to any one of claims 1 to 3, for use for the treatment of solid tumors with an amplified, activated, deregulated or overexpressed CDK4, CDK6, cyclin D or PDGFRA gene, in particular for the treatment of breast cancer, head and neck cancer, lung cancer, recurrent brain metastases, squamous cell carcinoma, tumors of the central nervous system, gliomas, sarcomas, colorectal tumors, tumors of the gastric system, pancreatic tumors, liver tumors, testicular tumors, uterine tumors, prostate tumors, ovarian tumors, bladder tumors. 10. Use of the compound of general formula I according to any one of claims 1 to 3, in cell cultures as an inhibitor of cell proliferation or inducers of apoptosis in hyperproliferating cells in vitro, or as an inducer of senescence in hyperproliferating cells in vitro. 11. A pharmaceutical preparation, characterized in that it contains at least one compound of general formula I according to any one of claims 1 to 3, and at least one pharmaceutically acceptable carrier or excipient. 2 drawings