EA038355B1 - Substituted pyridines as inhibitors of dnmt1 - Google Patents

Abstract

The invention relates to substituted pyridine derivatives. Specifically, the invention relates to compounds according to Formulaeor pharmaceutically acceptable salts thereof. The compounds of the invention are selective inhibitors of DNMT1 and can be useful in the treatment of cancer, pre-cancerous syndromes, beta hemoglobinopathy disorders, sickle cell disease, sickle cell anemia and beta thalassemia, and diseases associated with DNMT1 inhibition. The invention further relates to pharmaceutical compositions/combinations comprising a compound of the invention as an active agent. The invention still further relates to methods of inhibiting DNMT1 activity.

Description

Scope of invention

The present invention relates to substituted pyridine derivatives that are selective inhibitors of DNA methyltransferase 1 (DNMT1) activity. The present invention also relates to pharmaceutical compositions containing such compounds and methods of using such compounds in the treatment of malignant neoplasm, precancerous symptoms, beta hemoglobinopathies, sickle cell disease, sickle cell anemia and betathalassemia, and other diseases associated with inhibition of DNMT1.

Background of the invention

Epigenetics is a way of turning genes on and off regardless of the DNA sequence on which they are located. DNA methylation that occurs in gene promoters is an example of a repressive epigenetic tag leading to chromatin compaction and gene silencing. DNA methylation is mediated by the DNA methyltransferase (DNMT) family of proteins, which consists of four family members. Three family members, DNMT1, DNMT3A and DNMT3B, contain DNA methyltransferase activity. These three members are responsible for establishing the de novo DNA methylation pattern, while DNMT1 is primarily responsible for maintaining the daughter strand methylation pattern after DNA replication.

In malignant neoplasms, the DNA methylation scheme becomes aberrant, which leads to global hypomethylation and local hypermethylation in the promoter zones. This can lead to a decrease in the silencing of tumor suppressor genes (Ting et al., Genes Dev. 2006; 20: 3215-3231). In addition, DNMT1 silencing leads to DNA demethylation and re-expression of tumor suppressor genes, which leads to inhibition of tumor growth (Zhou et al., Oncol. Lett. 2014; 5: 2130-2134).

DNA methylation inhibitors (called DNA hypomethylating agents) are clinically proven anti-cancer drugs used to treat MDS, AML, and CMML. Although these agents are available, there is still significant room for improvement in toxicity, utility in solid tumors, and oral bioavailability. Therefore, the new DNMT inhibitor will be of interest for the treatment of cancer and / or any disease or condition mediated by DNA methylation. Of particular interest to the present invention is direct targeting of DNMT1 to prevent abnormal methylation patterns (such as those occurring in malignant neoplasm) from spreading to daughter filaments during replication.

In the documents US 2008/0132525 and WO 2006/078752 inhibitors of DNA methyltransferase are described. CA 2030875 describes methods and probes for detecting a nucleoside transporter and a method for producing probes.

Hemoglobinopathy.

Hemoglobin disorders such as sickle cell disease and beta thalassemia are the most common inherited blood disorders in the world. Heart anemia and beta thalassemia are characterized by abnormalities in hemoglobin, which is an oxygen-carrying protein complex in red blood cells. Structurally, hemoglobin usually consists of two pairs of proteins plus four topic molecules. Adults and children over four months of age express a form of hemoglobin called adult hemoglobin, which is predominantly composed of two alpha globin proteins paired with two beta globin proteins and four tema molecules. However, fetuses and infants usually express mainly embryonic hemoglobin, which consists of two alpha globin proteins paired with two gamma globin proteins plus four tema molecules. It is noted that there are two forms of gamma globin, called G-gamma and A-gamma, which are encoded by two different genes (HBG1 and HBG2), but are functionally equivalent to a large extent; fetal hemoglobin refers to the combination of a G-gamma and / or A-gamma pair plus a pair of alpha globin proteins plus four heme molecules.

In sickle cell anemia, the gene encoding beta-globin contains a mutation that results in an abnormal hemoglobin structure and causes red blood cells to take on the characteristic sickle shape under certain conditions. This sickle shape leads to a decrease in the plasticity of red blood cells, an increase in capillary transit time and frequent vaso-occlusive processes that can damage tissues and lead to morbidity in patients. In contrast, beta thalassemia is characterized by inadequate beta globin production in combination with normally produced alpha globin. As a result, the accumulation of alpha-globin is toxic to erythrocyte precursors and leads to ineffective erythropoiesis and extensive hemolysis of erythrocytes.

There is currently no approved pharmacological treatment for sickle cell disease or beta thalassemia. However, it has been shown that an increase in the number of red blood cells that produce fetal hemoglobin, combined with an overall increase in the level of fetal hemoglobin per red blood cell, has a clinical effect in patients with sickle cell disease and sickle cell disease, reducing the incidence of acute vaso-occlusive crises.

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Generally, although not clinically proven, the biology of beta thalassemia disease suggests that increasing fetal hemoglobin production to high levels may also be a viable strategy for treating this disease.

The object of this therapeutic approach, the derepression of the silent HBG1 and HBG2 genes, can be aimed at interfering with the epigenetic process in erythropoiesis. Changes in DNA methylation are key defining events during hematopoiesis, marking the stages of differentiation that lead to the commitments of different cell lines. During erythropoiesis, a rapid decrease in global DNA methylation demarcates the committed v point for the expression of erythroid specific regulators GATA1 and KLF1 and suppression of the hematopoietic progenitor regulators GATA2 and PU.1 (1, 2). For erythroid progenitor cells in adult bone marrow, the DNA in the promoter region of the HBB beta-globin gene becomes unadapted, which corresponds to high-level beta-globin protein expression. In contrast, the promoters of the HBG1 and HBG2 loci are methylated, which leads to a significant decrease in the expression of gamma globin proteins (3). Although DNA methyltransferase DNMT1, DNMT3A, and DNMT3B are each expressed in erythroid progenitors, the relatively increased expression of DNMT1, especially at the final stages of erythroid differentiation, suggests that it plays a dominant role in the regulation of the globin gene (2). 5-Azacytidine and 5-aza-2'-deoxycytidine (decitabine) are inhibitors of rap-DNMT, which are known inducers of fetal hemoglobin in erythroid progenitor cells. In erythroid cell culture and in an in vivo model of fetal hemoglobin induction (4,5) with these agents, treatment leads to a decrease in methylation of CpG regions in HBG promoters with a corresponding increase in the expression of the gamma globin protein. Moreover, in a limited set of clinical studies, both agents caused an increase in fetal hemoglobin in patients with sickle cell disease, sickle cell disease, and beta thalassemia (6-9). While effective in inducing fetal hemoglobin, these agents have not been widely used to treat sickle cell disease, sickle cell disease, or beta thalassemia due to concerns about long-term safety, dose-limiting toxicity, and inappropriate dosing routes.

Links:

(1) Pop R, Shearstone JR, Shen Q, Liu Y, Hallstrom K, Koulnis M, et al. A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.l and S-phase progression. 2010; eight.

- 2 038355 (2) Shearstone JR, Pop R, Bock C, Boyle P, Meissner A, Socolovsky M. Global DNA demethylation during mouse erythropoiesis in vivo. 2011; 334: 799-802.

(3) Mabaera R, Richardson CA, Johnson K, Hsu M, Fiering S, Lowrey CH. Developmental- and differentiation-specific patterns of human + | - and + | -globin promoter DNA methylation. 2007; 110: 1343-52.

(4) Chin J, Singh M, Banzon V, Vaitkus K, Ibanez V, Kouznetsova T, et al. Transcriptional activation of the + | globin gene in baboons treated with decitabine and in cultured erythroid progenitor cells involves different mechanisms. 2009; 37: 1131-42.

(5) Akpan I, Banzon V, Ibanez V, Vaitkus K, DeSimone J, Lavelle D. Decitabine increases fetal hemoglobin in Papio anubis by increasing + | -globin gene transcription. 2010; 38: 98993.

(6) Dover GJ, Charache SH, Boyer SH, Talbot J, Smith KD. 5 Azacytidine increases fetal hemoglobin production in a patient with sickle cell disease. 1983; 134: 475-88.

(7) Saunthararajah Y, Hillery CA, Lavelle D, Molokie R, Dorn L, Bressler L, et al. Effects of 5-aza-2GGI-deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease. 2003; 102: 3865-70.

(8) Ley TJ, DeSimone J, Noguchi CT, Turner PH, Schechter AN, Heller P, et al. 5-Azacytidine increases 3 + | -globin synthesis and reduces the proportion of dense cells in patients with sickle cell anemia. 1983; 62: 370-80.

(9) Lowrey CH, Nienhuis AW. Brief report: Treatment with azacitidine of patients with end-stage + | - thalassemia. 1993; 329: 845-8.

The object of the present invention is to provide new compounds that are selective DNMT1 inhibitors.

It is also an object of the present invention to provide compounds that increase the production of gamma globin, and thus also increase the production of fetal hemoglobin in human erythroid cells. The compounds of this invention are thus useful in the treatment of sickle cell anemia and sickle cell disease. Beta thalassemia can also be alleviated by treatment with these compounds.

The subject of the present invention is also the preparation of a pharmaceutical composition which contains a pharmaceutical excipient and compounds of the formula:

In addition, it is an object of the present invention to provide a method for treating malignant neoplasms, precancerous symptoms, beta hemoglobinopathies such as sickle cell disease, sickle cell anemia and beta thalassemia, which includes the administration of novel selective inhibitors of DNMT1 activity.

The essence of the invention

The invention relates to substituted pyridine derivatives. In particular, the invention relates to compounds corresponding to the formulas:

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The present invention also relates to the discovery that the compounds

are active as DNMT1 inhibitors and are selective for DNMT3A and DNMT3B.

In a preferred embodiment, the invention relates to a compound represented by the following formula:

or a pharmaceutically acceptable salt thereof.

This invention also relates to a pharmaceutical composition containing compounds

or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient thereof.

This invention also relates to a method of treating a disease selected from malignant neoplasm, precancerous conditions, beta hemoglobinopathies, sickle cell disease in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount of the compounds

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, this invention relates to a method for treating sickle cell 4038355 anemia.

In a more preferred embodiment, this invention relates to a method for treating betathalassemia.

In an even more preferred embodiment, this invention relates to a method of treatment in which the mammal is a human.

In the most preferred embodiment, this invention relates to a method in which said malignant neoplasm is selected from the following: brain tumor (glioma), glioblastoma, astrocytoma, glioblastoma multiforme, Bunyan-Zonan syndrome, Cowden's disease, Lermitte-Duclos disease, breast cancer, cancer colon, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, adenocarcinoma, ductal adenocarcinoma, adenosquamous carcinoma, acinar cell carcinoma, glucagonoma, insulinoma, prostate cancer, sarcoma and thyroid gland.

This invention also relates to the use of compounds

or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or amelioration of cancer.

This invention also relates to a method for inhibiting DNMT1 (DNA methyltransferase) activity in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount of the compounds

or a pharmaceutically acceptable salt thereof.

In a more preferred embodiment, this invention relates to an inhibition method wherein the mammal is a human.

This invention also relates to a method for treating cancer in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount:

a) connections

or a pharmaceutically acceptable salt thereof; and

b) at least one anticancer agent.

In a preferred embodiment, the present invention also relates to a method of treatment in which at least one anticancer agent is selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs , signaling pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, cell cycle signaling inhibitors, proteasome inhibitors, cancer metabolism inhibitors, anti-PD-L1 agents, PD-1 antagonist, immunomodulators 5 038355 STING, CD39 inhibitors, A2a and A2a adenosine antagonists, TLR4 antagonists, anti-ICOS antibodies and anti-OX40 antibodies.

This invention also relates to a pharmaceutical combination comprising:

a) connections

or a pharmaceutically acceptable salt thereof; and

b) at least one anticancer agent.

In a preferred embodiment, the invention relates to a pharmaceutical combination for the treatment of cancer.

In a more preferred embodiment, the invention relates to a method for malignant neoplasm in a mammal in need thereof, wherein said malignant neoplasm is selected from the following: breast cancer, inflammatory breast cancer, ductal carcinoma, lobular carcinoma, colon cancer, pancreatic cancer, insulinoma, adenocarcinoma, ductal adenocarcinoma, adenosquamous carcinoma, acinar cell carcinoma, glucagonoma, skin cancer, melanoma, metastatic melanoma, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma, large cell carcinoma, gliocystroblastoma, a brain tumor (gliocystroblastoma) , Banyan-Zonan syndrome, Cowden's disease, Lermitte-Duclos disease, Wilms tumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, head and neck cancer, kidney cancer, liver cancer, melanoma, ovarian cancer, prostate cancer, sarcoma, osteosarcoma, giant cell bones, cancer of the shield prominent gland, lymphoblastic T cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T cell leukemia, large plasmacytoma cell leukemia, immunoblast cell leukemia myeloma, megakaryoblocal leukemia, acute megakaryocytic leukemia, promyelocytic leukemia, erythroleukemia, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphomas, T-cell lymphoblastic lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, cervical cancer , mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular carcinoma, stomach cancer, nasopharyngeal cancer, cheek cancer, oral cancer, GIST (gastrointestinal stromal tumor), neuroendocrine cancers, and testicular cancer.

In a preferred embodiment, the invention relates to a method for malignant neoplasm in a mammal in need thereof, in which the mammal is a human.

In a more preferred embodiment, the invention relates to a method for malignant neoplasm in a mammal in need thereof, in which said precancerous condition is selected from: intraepithelial cervical neoplasia, monoclonal gammopathy of undetermined etiology (MGUS), myelodysplastic syndrome, aplastic anemia, cervical disease of the skin (premelanomas, nevus ), prostatic intraepithelial (intraductal) neoplasia (PIN), ductal carcinoma in situ (DCIS), colon polyps, and severe hepatitis or cirrhosis.

This invention also relates to a pharmaceutical combination comprising:

a) connections

or a pharmaceutically acceptable salt thereof; and

b) at least one agent used to treat beta hemoglobinopathies.

In a preferred embodiment, the present invention relates to a pharmaceutical combination for the treatment of sickle cell disease.

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In a more preferred embodiment, the present invention relates to a pharmaceutical combination for the treatment of beta-thalassemia.

This invention also relates to a method of treating a disease selected from diabetic nephropathy, diabetes, podocyte injury, atherosclerosis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, liver cirrhosis, rheumatoid arthritis and Alzheimer's disease in a mammal in need thereof, which comprises administering to such a mammal therapeutically effective number of compounds:

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the present invention relates to a method for treating a disease in which the mammal is a human.

This invention also relates to a pharmaceutical composition containing from 0.5 to 1000 mg of compounds:

or a pharmaceutically acceptable salt thereof, and from 0.5 to 1000 mg of a pharmaceutically acceptable excipient.

Brief description of the figures

FIG. 1A shows the effect of Compound A on erythroid progenitor cells (EPC). Representative results (n = 3 studies each) of 5 days Compound A treatment on fetal hemoglobin (HbF) ELISA (open circles) and cell growth assay (closed circles).

FIG. 1B shows the effect of Compound A on HBG1 and HBG2 DNA methylation. Erythroid progenitor cells (EPC) were treated for 3 days with carrier medium (gray bars) or 5 μM compound A (black bars), genomic DNA was extracted and sequenced with bisulfite for nine loci in the HBG1 and HBG2 promoter regions, which were previously described as sites DNMT1 cytosine methylation. Methylation sites are marked as positions relative to the respective starting sites.

FIG. 2A shows the effect of Compound A on fetal hemoglobin in a transgenic mouse model. Compound A, orally administered to transgenic mice of sickle cell disease (SCD) model at 10 or 50 mg / kg, twice a day, caused a daily dose-dependent increase in% HbF protein as determined by HPLC.

FIG. 2B shows the effect of Compound A on fetal hemoglobin in a transgenic mouse model. Compound A, administered orally to SCD transgenic mice at 10 or 50 mg / kg, twice daily, caused a dose-dependent increase in% F reticulocytes and% F erythrocytes as determined by flow cytometry daily.

Detailed description of the invention

This invention relates to compounds of the formula:

- 7,038355 and their pharmaceutically acceptable salts and their use in the methods of the invention.

The person skilled in the art will understand that pharmaceutically acceptable salts of the compounds can be obtained

Indeed, in some embodiments, pharmaceutically acceptable salts of the aforementioned compounds may be preferred over the corresponding free or non-salt compound. Accordingly, the invention further relates to pharmaceutically acceptable salts of the compounds:

Pharmaceutically acceptable salts of the compounds of the invention are readily obtainable by those skilled in the art.

Typical pharmaceutically acceptable acid additions include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (capratzylate), (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecyl sulfate (estolate), edetate (ethylenediamine tetraacetate), estolate (lauryl sulfate), 1,2 -disulfonate (edizylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, gluosulfatex, glycerolpurate hydrabamine (N, N'-di (dehydroabietil) ethyldene diamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, methanesulfonate (mesylate), methylsulfate, mucosa -disulfonate (napa disilate), naphthalene-2-sulfonate (napsilate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate (embonate) pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacite, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, theoclate (8-chloroteophyllinate), thiocyanate, triethiodide, undecylenate, and y

Typical pharmaceutically acceptable base addition salts include, but are not limited to, aluminum salts, 2-amino-2- (hydroxymethyl) -1,3-propanediol (TRIS, tromethamine), arginine, benetamine (N-benzylphenethylamine), benzathine (N, N'-dibenzylethylenediamine), bis- (2hydroxyethyl) amine, bismuth, calcium, chloroprocaine, choline, clemisole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine , ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylgluamine), pi-8 038355 perazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, tert- butylamine and zinc.

The compounds of the invention can exist as a solid. As a solid, a compound of the invention can exist in a range of solid states ranging from fully amorphous to fully crystalline. The term amorphous refers to a state in which a substance lacks long-range order at the molecular level and, depending on temperature, may exhibit the physical properties of a solid or liquid. Typically, such materials do not give distinctive X-ray diffraction parameters and, exhibiting the properties of a chemical substance, are more formally described as a liquid. When heated, there is a transition from the properties of a solid to the properties of a liquid, which is characterized by a change in state, usually with a large order (glass shift). The term crystalline refers to the phase of a solid in which the substance has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with distinct peaks. Such substances, sufficiently heated, will also exhibit liquid properties, but the transition from solid to liquid is characterized by a phase change, usually of the first order (melting point).

The compounds of the invention may have the ability to crystallize in more than one form, a characteristic that is known as polymorphism (s). Polymorphism can usually occur as a reaction to changes in temperature or pressure, or both, it can also be the result of changes in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art, such as X-ray diffraction patterns, solubility and melting point.

Definitions

As used herein, the symbols and conventions used in these processes, diagrams and examples correspond to those used in modern scientific literature, for example, in the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard one-letter or three-letter abbreviations are usually used to indicate amino acid residues that are assumed to be in the L-configuration, unless otherwise indicated. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. In particular, the following abbreviations may be used in the examples and throughout the description:

Ac - acetyl;

Ac2O - acetic anhydride;

A-CN is acetonitrile;

AIBN - azo-bis-isobutyronitrile;

BINAP - 2,2'-bis- (diphenylphosphino-1,1'-binaphthyl);

BMS - borane-dimethyl sulfide complex;

Bn is benzyl;

Boc is tert-butoxycarbonyl;

Boc ₂ O - di-tert-butyl dicarbonate;

BOP - benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate;

CAN - cerium ammonium nitrate;

Cbz is benzyloxycarbonyl;

CSI is chlorosulfonyl isocyanate;

CSF - cesium fluoride;

DABCO - 1,4-diazabicyclo [2.2.2] octane;

DAST, diethylaminosulfur trifluoride;

DBU - 1,8-diazabicyclo [5.4.0] undec-7-ene;

DCC, dicyclohexyl carbodiimide;

DCE - 1,2-dichloroethane;

DCM for dichloromethane;

DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone;

ATP, adenosine triphosphate;

bis-pinacolato diborone - 4,4,4 ', 4', 5.5,5 ', 5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane;

BSA - bovine serum albumin;

C18 - refers to an 18-carbon alkyl group on silica in an HPLC stationary phase;

CH ₃ -CN - acetonitrile;

Cy is cyclohexyl;

DCM - dichlomethane;

DIEA for diisopropylethylamine;

DIPEA, Hunig's base, N-ethyl-N- (1-methylethyl) -2-propanamine;

dioxane - 1,4-dioxane;

DMAP, 4-dimethylaminopyridine;

DME - 1,2-dimethoxyethane;

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DMEDA - Ν, Ν'-dimethylethylenediamine;

DMF - Ν, Ν-dimethylformamide;

DMSO - dimethyl sulfoxide;

DPPA, diphenylphosphorylazide;

EDC - N- (3-dimethylaminopropyl) -N'ethylcarbodiimide hydrochloride salt

EDTA - ethylenediaminetetraacetic acid;

EtOAc - ethyl acetate;

EtOH is ethanol;

Et2O - diethyl ether;

HEPES - 4- (2-hydroxyethyl) -1-piperazinyl-ethanesulfonic acid;

HATU - O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate;

HOAt = 1-hydroxy-7-azabenzotriazole;

HOBt = 1-hydroxybenzotriazole;

HOAc - acetic acid;

HPLC = high pressure liquid chromatography;

HMDS, hexamethyldisilazide;

Junig's base is N, N-diisopropylethylamine;

IPA, isopropyl alcohol;

indoline - 2,3-dihydro-1H-indole;

KHMDS - potassium hexamethyldisilazide;

LAH - lithium aluminum hydride;

LDA - lithium diisopropylamide;

LHMDS, lithium hexamethyldisilazide;

MeOH - methanol;

MTBE - methyl tert-butyl ether;

μM - micromolar;

mCPBA, m-chloroperbenzoic acid;

NaHMDS, sodium hexamethyldisilazide;

NCS - N-chlorosuccinimide;

NBS - N-bromosuccinimide;

PE - petroleum ether;

Pd ₂ (dba) ₃ - tris- (dibenzylideneacetone) dipalladium (0;

complex Pd (dppf) Cl ₂ -DCM - complex [1,1'-bis- (diphenylphosphino) ferrocene] dichloropalladium (P) · dichloromethane;

PyBOP - benzotriazol-1-yl-hydroxytripyrrolidinophosphonium hexafluorophosphate;

PyBrOP - bromotripyrrolidinophosphonium hexafluorophosphate;

RPZhKhVD - reverse phase high pressure liquid chromatography;

RT - room temperature;

saturate - saturated;

SFC - supercritical fluid chromatography;

SGC - silica gel chromatography;

SM is the starting material;

TLC - thin layer chromatography;

TEA - triethylamine;

TEMPO - 2,2,6,6-tetramethylpiperidinyl-1-oxyl, free radical;

TFA - trifluoroacetic acid;

THF - tetrahydrofuran;

Ts-Cl - p-toluenesulfonyl chloride).

All references to ether refer to diethyl ether and to brine refer to saturated aqueous NaCl.

Getting connections.

The compounds of the present invention are prepared using conventional organic synthesis techniques. Suitable synthetic routes are further illustrated by the following general reaction schemes. All starting materials are commercially available or can be readily obtained by those skilled in the art from commercially available starting materials.

One skilled in the art will understand that if a substituent described herein is incompatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is resistant to the reaction conditions. The protecting group can be removed at a suitable point in the reaction sequence to provide the desired intermediate or target compound. Suitable protecting groups and methods of protecting and deprotecting various substituents are well known to those skilled in the art.

- 10 038355 using such suitable protecting groups; examples of which can be found in the review T. Greene and P. Wuts, Protecting Groups in Organic Synthesis ( 4 ^{th ed.), John Wiley &} Sons, NY ( 2006). In some cases, a substituent may be selected as reactive under the reaction conditions. Under these reaction conditions, the selected substituent is converted to another substituent, which can either be used as an intermediate or is a desired substituent in the target compound.

Scheme 1

In some cases, the intermediate of Formula 13 was used to prepare the intermediate of Formula 4 as shown in Scheme 2 and used in subsequent steps as shown in Schemes 1 and 2.

Scheme 2

In other cases, the intermediate compound of formula was used to obtain the compound HCI, 50C

Ivte ^ GO of formula 16, which was used in subsequent steps to obtain the compound of formula 1, 8, as described in Scheme 3.

Scheme 3

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In some cases, compounds of Formulas 19 and 20 have been used to prepare compounds of Formula 21 as described in Scheme 4.

Scheme 4

Compounds of Formula 24 were prepared synthetically as shown in Scheme 5. Intermediates of Formula 22 are commercially available compounds that may or may not be separate enantiomers. When the compounds of Formula 22 are separate enantiomers, then the corresponding compounds of Formula 23 and Formula 24 are also.

Scheme 5

To prepare compounds of Formula 26, an intermediate of Formula 14 and an intermediate of Formula 25 were used as shown in Scheme 6. Intermediates of Formula 25 are commercially available or have been synthesized using conventional organic synthetic methods that can be reproduced by any skilled artisan.

Scheme 6

Ι ^ΝΟ γ ^ Α ^Ν Cl KSAC _r ^NC rJv ^CN ψΑ * <dm *, kt '> A

GB ^5g

25

Alternatively, to prepare a compound of Formula 26, an intermediate of Formula 14 and an intermediate of Formula 27 were used as shown in Scheme 7. The intermediate of Formula 27 is commercially available or has been synthesized using conventional organic synthetic methods that can be reproduced by any skilled artisan. ...

Scheme 7

Compounds of formulas 33 and 34 were prepared by the synthetic routes outlined in Scheme 8. Intermediates 28 and 29 are commercially available compounds. The intermediate of formula 31 is commercial or has been synthesized using conventional organic synthetic methods that can be reproduced by any skilled artisan.

Scheme 8

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Compounds of Formula 40 were prepared by the synthetic routes outlined in Scheme 9. Intermediates 35 and 36 are commercially available compounds. The intermediate of formula 31 is commercially available or has been synthesized using conventional organic synthetic methods that can be reproduced by any skilled artisan.

Scheme 9

The compound of Formula 43 was prepared by the synthetic routes outlined in Scheme 10. Intermediate 41 is commercially available.

Scheme 10

The compound of Formula 48 was prepared by the synthetic routes outlined in Scheme 11. Intermediates 1 and 44 are commercially available.

Scheme 11

The compound of Formula 52 was prepared by the synthetic routes outlined in Scheme 12. Intermediate 49 is commercially available and the preparation of intermediate 46 is shown in Scheme 11 above.

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Scheme 12

Compounds of Formula 58 were prepared by the synthetic routes outlined in Scheme 13. Intermediates 1 and 53 are commercially available.

Scheme 13

Application methods.

Compounds of the formulas and their pharmaceutically acceptable salts are selective DNMT1 inhibitors. These compounds are potentially useful in the treatment of conditions that respond to selective inhibition of DNMT1.

The term treatment and its derivatives, as used herein, in relation to a condition means (1) an improvement in a condition or one or more biological manifestations of a condition; (2) obstructing (a) one or more points in the biological cascade that lead to the condition or are responsible for the condition; or (b) one or more biological manifestations of the condition; (3) relief of one or more symptoms or effects associated with the condition; or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

The term treatment and its derivatives refers to curative therapy. Curative therapy is suitable for relieving symptoms or treating early signs of illness or progression.

A person skilled in the art will understand that warning is not an absolute term. In medicine, prevention is understood as referring to the prophylactic administration of a drug to significantly reduce the likelihood or severity of a condition or its biological manifestation, or to delay the onset of such a condition or its biological manifestation.

Prophylactic therapy is appropriate when the subject has, for example, a well-defined family history of malignant neoplasm or, on the other hand, is at high risk of developing a malignant neoplasm, or when the subject is exposed to a carcinogen, or when the subject has a well-defined family history of beta-14 038355 hemoglobinopathies such as sickle cell disease, sickle cell disease, or beta thalassemia.

As used herein, the term effective amount and its derivative means that the amount of a medicinal or pharmaceutical agent that will elicit such a biological or medical response of a tissue, system, animal or human that is sought by, for example, a researcher or physician. In addition, the term therapeutically effective amount and its derivative means any amount that, compared to a corresponding subject not receiving such an amount, results in improved treatment, healing, prevention, or amelioration of a disease or side effect, or a decrease in the rate of progression of a disease or disorder. The term also encompasses an amount effective to enhance normal physiological function.

As used herein, the term patient or subject refers to a human or other mammal. A suitable patient or subject is a human.

Compounds of formulas

or a pharmaceutically acceptable salt thereof can be administered by any suitable route of administration, including systemic administration.

Systemic administration includes oral administration and parenteral administration. Parenteral administration refers to routes of administration other than enteral, transdermal, or inhalation, and is usually by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion.

Compounds of formulas

or their pharmaceutically acceptable salts may be administered once or in accordance with a dosing regimen in which a number of doses are administered at different intervals over a period of time.

For example, doses can be administered one, two, three, or four times a day. Doses can be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosage regimens for a compound of the invention depend on the pharmacokinetic properties of the compound, such as absorption, distribution and half-life, which can be determined by one of ordinary skill in the art. In addition, appropriate dosage regimens are prescribed, including the duration of such regimens for a compound of the invention, depending on the condition to be treated, the severity of the condition to be treated, the age and physical condition of the patient being treated, the medical history of the patient being treated, the nature simultaneous therapy, the desired therapeutic effect and similar factors in the knowledge and experience of a qualified specialist. Those of skill in the art will understand that suitable dosing regimens may need to be adjusted based on the response of an individual patient to the dosing regimen or over time if an adjustment is needed for an individual patient.

Typical daily doses will vary depending on the particular route of administration chosen. Typical oral doses range from 1 mg to 1000 mg per person per dose. Preferred dosages are 1-500 mg once daily or BID per person.

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The compounds of the formulas and their pharmaceutically acceptable salts can be co-administered together with at least one other active agent previously used in the treatment of cancer or precancerous symptoms.

By the term co-administration, as used herein, is meant either the simultaneous administration or any method of separate sequential administration of an inhibitor of DMNT1 activity described herein and another active agent or agents known to be useful in the treatment of cancer, including chemotherapy and radiation therapy.

The term additional active agent or agents, as used herein, includes any compound or therapeutic agent known or which exhibits beneficial properties when administered to a patient in need of cancer treatment. Preferably, when the administration is not simultaneous, the compounds are administered at a time interval relative to each other. In addition, it does not matter if the compounds are administered in the same dosage form, for example, one compound can be injected and the other compound can be administered orally.

Examples of additional active ingredient or ingredients (antineoplastic agent) for use in combination or co-administration with the combinations of the present invention are shown below. This list is not limiting. Additional antineoplastic agents are intended for use with the compounds of the present invention.

In general, any antineoplastic agent that has activity against the susceptible tumor to be treated can be co-administered in the present invention to treat cancer. Examples of such agents can be found in the review Cancer Principles and Practice of Oncology by VT Devita and S. Hellman (editors), 6 ^th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. Typical antineoplastic agents used in the present invention include, but are not limited to, anti-microtubule agents such as diterpenoid alkaloids and vinca alkaloids; coordination complexes of platinum; alkylating agents such as nitrogen mustard, oxazaphosphorines, alkyl sulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclines, actinomycins, and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogs and antifolates; topoisomerase I inhibitors such as camptothecins; hormones and hormone analogues; signal transduction inhibitors; inhibitors of angiogenesis of non-receptor tyrosine kinase; immunotherapeutic agents; proapoptotic agents; cell cycle signaling inhibitors; proteasome inhibitors; and inhibitors of cancer metabolism.

Examples of additional active ingredient or ingredients (antineoplastic agent) for use in combination or co-administration with the combinations of the present invention are chemotherapeutic agents.

Anti-microtubule or antimitotic agents are phase-specific agents active against the microtubules of tumor cells during the M-phase or mitotic phase of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids. Diterpenoids, which are derived from natural sources, are phase-specific anti-cancer agents that act during the G2 / M phases of the cell cycle. It is believed that diterpenoids stabilize the β-tubulin subunit of microtubules by binding to this protein. Apparently, then protein disassembly is inhibited with arrest of mitosis and subsequent cell death. Examples of diterpenoids include, but are not limited to, paclitaxel and its analogue docetaxel.

Paclitaxel, 5 c, 20-epoxy-1,2α, 4,7β, 10β, 13а-hexa-hydroxytax-11-en-9-one-4,10-diacetate-2benzoate 13-ester with (2R, 3S) - N-benzoyl-3-phenylisoserine, is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as TAXOL® injection. It is a member of the taxane terpene family. Paclitaxel has been approved in the United States for clinical use in the treatment of refractory ovarian cancer and breast cancer.

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Docetaxel, (2R, 3S) -N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5b-20-epoxy-1,2а, 4.7b, 10b, 13а-hexahydroxytax-11- en-9-one-4-acetate-2-benzoate, a trihydrate, is commercially available as an injection as TAXOTERE®. Docetaxel is indicated for the treatment of breast cancer. Docetaxel is a semi-synthetic derivative of paclitaxel q.v. prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from European yew needles. The dose-limiting toxicity of docetaxel is neuropenia.

Vinca alkaloids are phase anticancer agents derived from the vinca plant. Vinca alkaloids act on the M-phase (mitosis) of the cell cycle by specifically binding to tubulin. Consequently, the bound tubulin molecule cannot polymerize into microtubules. It is believed that mitosis stops at the metaphase stage with subsequent cell death. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine.

Vinblastine, vincaleucoblastine sulfate, is commercially available as VELBAN® as an injection. Although it may be indicated as a second line therapy for a variety of solid tumors, it is primarily indicated for the treatment of testicular cancer and various lymphomas, including Hodgkin's disease and lymphocytic and histiocytic lymphomas. A dose-limiting side effect of vinblastine is myelosuppression.

Vincristine, vincaleucoblastine, 22-oxo, sulfate, is commercially available as ONCOVIN® injectable solution. Vincristine is indicated for the treatment of acute leukemia and has also found use in the treatment of malignant Hodgkin's lymphoma and non-Hodgkin's lymphoma. Alopecia and neurologic effects are the most common side effects of vincristine and to a lesser extent the effects of myelosuppression and gastrointestinal mucositis.

Vinorelbine, 3 ', 4'-didehydro-4'-deoxy-8'-norvinsaleucoblastine [R- (R *, R *) - 2,3-dihydroxybutanedioate (1: 2) (salt)], commercially available as an injection solution of vinorelbine tartrate (NAVELBINE®), is a semi-synthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents such as cisplatin in the treatment of various solid tumors, in particular non-small cell lung cancer, advanced breast cancer and hormone-refractory prostate cancer. Myelosuppression is the most common dose-limiting side effect of vinorelbine.

Platinum coordination complexes are nonphase-specific anticancer agents that interact with DNA. Platinum complexes penetrate into tumor cells, hydrate and form intra- and inter-stranded cross-links with DNA, causing adverse biological effects on the tumor. Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatin, is commercially available as PLATINOL® as an injection. Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer. The main dose-limiting side effects are nephrotoxicity, which can be controlled by hydration and diuresis, and ototoxicity.

Carboplatin, platinum, diamino [1,1-cyclobutane dicarboxylate (2 -) - O, O '], is commercially available as PARAPLATIN® as an injection solution. Carboplatin is primarily indicated in the first and second line of treatment for advanced ovarian carcinoma. Bone marrow suppression is the dose-limiting toxicity of carboplatin.

Alkylating agents are nonphase-specific anticancer agents and are potent electrophiles. Typically, alkylating agents form covalent bonds with DNA via alkylation through the nucleophilic groups of the DNA molecule, such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. This alkylation disrupts the function of nucleic acids, leading to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine.

Cyclophosphamide, 2- [bis- (2-chloroethyl) amino] temrahydro-2H-1,3,2-oxazaphosphorin-2-oxide monohydrate, is commercially available as an injection solution or tablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of malignant lymphoma, multiple myeloma, and leukemia. Alopecia, nausea, vomiting, and leukopenia are the most common dose-limiting side effects of cyclophosphamide.

Melphalan, 4- [bis- (2-chloroethyl) amino] -L-phenylalanine, is commercially available as an injection or tablets as ALKERAN®. Melphalan is indicated for the palliative treatment of multiple myeloma and inoperable epithelial ovarian cancer. Bone marrow suppression is the most common dose-dependent side effect of melphalan.

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Chlorambucil, 4- [bis- (2-chloroethyl) amino] benzenebutanoic acid, is commercially available as LEUKERAN® tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphocytic leukemia and malignant lymphoma such as lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease. Bone marrow suppression is the most common dose-limiting side effect of chlorambucil.

Busulfan, 1,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® tablets. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Bone marrow suppression is the most common dose-limiting side effect of busulfan.

Carmustine, 1,3- [bis- (2-chloroethyl) -1-nitrosourea, is commercially available as single vials with lyophilized material as Bi-CNU®. Carmustine is indicated for palliative care as a single agent or in combination with other agents for the treatment of brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphoma. Delayed myelosuppression is the most common dose-limiting side effect of carmustine.

Dacarbazine, 5- (3,3-dimethyl-1-triazeno) imidazole-4-carboxamide, is commercially available as single vials of lyophilisate as DTIC-Dome®. Dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents in the second line of treatment for Hodgkin's disease. Nausea, vomiting, and anorexia are the most common dose-limiting side effects of dacarbazine.

Antibiotic chemotherapeutic agents are non-phase-specific agents that bind to or intercalate with DNA. This usually results in stable DNA complexes or strand damage that disrupts the normal functioning of nucleic acids, leading to cell death. Examples of antibiotic anticancer agents include, but are not limited to, actinomycins such as dactinomycin; anthrocyclines such as daunorubicin and doxorubicin; and bleomycins.

Dactinomycin, also known as actinomycin D, is commercially available in injectable form as COSMEGEN®. Dactinomycin is indicated for the treatment of Wilms tumor and rhabdomyosarcoma. Nausea, vomiting, and anorexia are the most common dose-limiting side effects of dactinomycin.

Daunorubicin, (8S-cis -) - 8-acetyl-10 - [(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl) oxy] 7,8,9,10-tetrahydro-6,8 , 11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride, is commercially available as a liposomal injection as DAUNOXOME® or as an injection as CERUBIDINE®. Daunorubicin is indicated for the induction of remission in the treatment of acute non-lymphocytic leukemia and progressive HIV associated with Kaposi's sarcoma. Myelosuppression is the most common dose-limiting side effect of daunorubicin.

Doxorubicin, (8S, 10S) -10 - [(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl) oxy] -8glycoloyl-7,8,9,10-tetrahydro-6,8,11 β-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride, is commercially available as an injectable form as RUBEX® or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloid leukemia, but may also be useful in the treatment of some solid tumors and lymphomas. Myelosuppression is the most common dose-limiting side effect of doxorubicin.

Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated from strains of Streptomyces verticillus, is commercially available as BLENOXANE®. Bleomycin is indicated for paleomycin therapy as a single agent or in combination with other agents of squamous cell carcinoma, lymphoma, and ovarian cancer. Pulmonary and cutaneous toxicity are the most common dose-limiting side effects of bleomycin.

Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.

Epipodophyllotoxins are phase-specific antineoplastic agents isolated from the mandrake plant. Epipodophyllotoxins usually act on cells in the S and G2 phases of the cell cycle by forming ternary complexes with topoisomerase II and DNA, which leads to DNA strand breaks. Chain breaks accumulate, resulting in cell death. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide.

Etoposide, 4'-demethylpipodophyllotoxin-9- [4,6-O- (R) -ethylidene-β-D-glycopyranoside], is commercially available as an injection solution or capsule as VePESID® and is commonly known as VP-16. Etoposide is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of testicular cancer and non-small cell lung cancer. Myelosuppression is the most common side effect of etoposide. Cases of leukopenia tend to be more severe than thrombocytopenia.

Teniposide, 4'-demethylepipodophyllotoxin-9- [4,6-O-R) -2-tenilidene] -e-D-glucopyranoside, is 18 038355 commercially available as an injection solution as VUMON® and is commonly known as

VM-26. Teniposide is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of acute leukemia in children. Myelosuppression is the most common dose-limiting side effect of teniposide. Teniposide can cause both leukopenia and thrombocytopenia.

Neoplastic antimetabolites are phase-specific anticancer agents that act in the S phase (DNA synthesis) of the cell cycle, inhibiting DNA synthesis or inhibiting the synthesis of purine or pyrimidine bases, thereby limiting DNA synthesis. As a result, the S phase does not proceed and cell death follows. Examples of neoplastic antimetabolites include, but are not limited to, fluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine, and gemcitabine.

5-Fluorouracil, 5-fluoro-2,4- (1H, 3H) -pyrimidinedione, is commercially available as fluorouracil. Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result is usually cell death. 5-fluorouracil is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of breast, colon, rectal, stomach and pancreatic cancers. Myelosuppression and mucositis are dose-limiting side effects of 5-fluorouracil. Other fluoropyrimidine analogs include 5-fluoro-deoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate.

Cytarabine, 4-amino-1-β-D-arabinofuranosyl-2 (1H) -pyrimidinone, is commercially available as CYTOSAR-U® and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase specificity in the S-phase by inhibiting DNA strand elongation by terminal incorporation of cytarabine into the growing DNA strand. Cytarabine is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of acute leukemia. Other analogs of cytidine include 5-azacytidine and 2 ', 2'-difluorodeoxycytidine (gemcitabine). Cytarabine induces leukopenia, thrombocytopenia, and mucositis.

Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PURINETHOL®. Mercaptopurine exhibits S-phase cell phase specificity by inhibiting DNA synthesis through an as yet unknown mechanism. Mercaptopurine is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of acute leukemia. Myelosuppression and gastrointestinal mucositis are expected side effects of high dose mercaptopurine. The used analogue of mercaptopurine is azathioprine.

Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID®. Thioguanine exhibits cell phase specificity in the S-phase by inhibiting DNA synthesis through an as yet unknown mechanism. Thioguanine is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of acute leukemia. Myelosuppression, including leukopenia, thrombocytopenia, and anemia, is the most common dose-limiting side effect of thioguanine administration. However, there are gastrointestinal side effects that can be dose-limiting. Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine.

Gemcitabine, 2'-deoxy-2 ', 2'-difluorocytidine monohydrochloride (β-isomer), is commercially available as GEMZAR®. Gemcitabine exhibits S-phase cell phase specificity by blocking cell progression across the G1 / S border. Gemcitabine is indicated in combination with cisplatin in the treatment of local progressive non-small cell lung cancer and separately in the treatment of local progressive pancreatic cancer. Myelosuppression, including leukopenia, thrombocytopenia, and anemia, is the most common dose-limiting side effect of gemcitabine administration.

Methotrexate, N- [4 [[(2,4-diamino-6-pteridinyl) methyl] methylamino] benzoyl] -L-glutamic acid, is commercially available as sodium methotrexate. Methotrexate exhibits cell phase specificity in the S-phase by inhibiting DNA synthesis, repair and / or replication through inhibition of dihydrofolic reductase, which is required for the synthesis of purine nucleotides and thymidylate. Methotrexate is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and breast, head, neck, ovary, and bladder cancers. Myelosuppression (leukopenia, thrombocytopenia, and anemia) and mucositis are expected side effects of methotrexate administration.

Camptothecins, including camptothecin and camptothecin derivatives, are available or under development as topoisomerase I inhibitors. It is believed that the cytotoxic activity of camptothecins should be related to its topoisomerase I inhibiting activity. Examples of camptothecins include, but are not limited to, irinotecan, topotecan, and various optical forms of 7- (4-methylpiperazino-methylene) -10,11-ethylenedioxy-20-camptothecin, described below.

Irinotecan HCl, (4S) -4,11-diethyl-4-hydroxy-9 - [(4-piperidinopiperidino) carbonyloxy] -1H-19 038355 pyrano [3 ', 4', 6,7] -indolizino [1,2 -B] quinoline-3.14 (4H, 12H) -dione hydrochloride, is commercially available as CAMPTOSAR® injection.

Irinotecan is a camptothecin derivative that binds, together with its active metabolite SN-38, to the topoisomerase I-DNA complex. This cytotoxicity is thought to result from irreparable breaks in two strands caused by the interaction of the ternary complex topoisomerase I: DNA: irinotecan or SN-38 with replication enzymes. Irinotecan is indicated for the treatment of metastatic colon or rectal cancer. The dose-limiting side effects of irinotecan HCl are myelosuppression, including neutropenia and GI effects, including diarrhea.

Topotecan HCl, (S) -10 - [(dimethylamino) methyl] -4-ethyl-4,9-dihydroxy-1H-pyrano [3 ', 4', 6,7] indolizino [1,2-b] quinoline- 3.14- (4H, 12H) -dione monohydrochloride, is commercially available as HYCAMTIN® injection solution. Topotecan is a camptothecin derivative that binds to the topoisomerase I-DNA complex and prevents the repetition of single strand breaks caused by topoisomerase I in response to the twisting deformation of the DNA molecule. Topotecan is indicated for the second line treatment of metastatic ovarian carcinoma and small cell lung cancer. The dose-limiting side effect of topotecan HCl is myelosuppression, primarily neutropenia.

Also of interest is a camptothecin derivative of formula A, including the racemic mixture form (R, S), as well as the R- and S-enantiomers:

known under the chemical name 7- (4-methylpiperazino-methylene) -10,11-ethylenedioxy20 (R, S) -camptothecin (racemic mixture), or 7- (4-methylpiperazino-methylene) -10,11-ethylenedioxy20 (R) -camptothecin (R enantiomer), or 7- (4-methylpiperazinomethylene) -10,11-ethylenedioxy-20 (S) camptothecin (S enantiomer). Such compound, as well as related compounds, are described, including methods of preparation, in US patents No. 6,063,923, 5342947, 5559235 and 5491237.

Hormones and hormone analogs are useful compounds for treating malignant neoplasms where there is a relationship between the hormone (s) and the growth and / or lack of growth of the malignant neoplasm. Examples of hormones and hormone analogs that can be used in the treatment of malignant neoplasms include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone, which can be used in the treatment of malignant lymphoma and acute leukemia in children; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole and exemestane, which can be used in the treatment of adrenocortical carcinoma and hormone-dependent breast carcinoma containing estrogen receptors; progestins such as megestrol acetate, which can be used in the treatment of hormone-dependent breast cancer and endometrial carcinoma; estrogens, androgens and antiandrogens such as flutamide, nilutamide, bicalutamide, cyproterone acetate and 5a-reductase such as finasteride and dutasteride, which can be used in the treatment of prostate carcinoma and benign prostatic hyperplasia; antiestrogens such as tamoxifen, toremifene, raloxifene, droloxifene and iodoxifene, as well as selective estrogen receptor modulators (SERMs), such as those described in US Pat. and gonadotropin-releasing hormone (GnRH) and analogs thereof, which stimulate the release of luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH), for the treatment of prostate carcinoma, for example, LHRH agonists and antagonists such as goserelin acetate and luprolide acetate and luprolide.

Inhibitors of signal transduction pathways are those inhibitors that block or inhibit a chemical process that causes intracellular change. As used herein, this change represents cell proliferation or differentiation. Inhibitors of signal transduction that can be used in accordance with the present invention include inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, blockers of SH2 / SH3 domains, serine / threonine kinases, phosphatidyl-inositol-3-kinases, myoinositol-oncogenes.

Several protein tyrosine kinases catalyze the phosphorylation of specific tyrosine residues in various proteins involved in the regulation of cell growth. Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.

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Receptor tyrosine kinases are transmembrane proteins having an extracellular ligand-binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in the regulation of cell growth and are commonly referred to as growth factor receptors. It has been shown that inappropriate or uncontrolled activation of many of these kinases, i. E. aberrant kinase receptor activity against growth factors, for example, through overexpression or mutation, leads to uncontrolled cell growth. Accordingly, the aberrant activity of such kinases has been associated with the growth of malignant tissues. Therefore, inhibitors of such kinases could provide methods for treating malignant neoplasms. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr, ErbB2 and ErbB4), platelet growth factor receptor (PDGFr), vascular endothelial growth factor receptor (VEGFR), tyrosine kinase with domains homologous to immunoglobulin-like and epidermal growth factor (TIE) 2), insulin growth factor-I (IGF-I) receptor, macrophage-colony-stimulating factor (cfms), BTK, ckit, cmet, fibroblast growth factor (FGF) receptors, Trk receptors (TrkA, TrkB and TrkC), ephrin- receptors (eph) and RET-protooncogenes. Several inhibitors of growth factor receptors are under development and include ligand antagonists, antibodies, tyrosine kinase inhibitors, and antisense oligonucleotides. Growth factor receptors and agents that inhibit the function of growth factor receptors are described, for example, in the works of Kath, John C., Exp. Opin. Ther. Patents (2000), 10 (6): 803-818; Shawver et al., DDT Vol. 2, No. 2, February 1997; and Lofts, F.J. et al., Growth factor receptors as targets, New Molecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr, David, CRC press 1994, London.

Accordingly, the pharmaceutically active compounds of the invention can be used in combination with a VEGFR inhibitor, for example 5 - [[4 - [(2,3-dimethyl-2H-indazol-6-yl) methylamino] -2pyrimidinyl] amino] -2-methylbenzenesulfonamide or a pharmaceutically acceptable salt thereof, for example a monohydrochloride salt, which are disclosed and claimed in WO PCT / US01 / 49367 with an international filing date of December 19, 2001, WO 02/059110 and international publication of August 1, 2002. , the full disclosure of which is incorporated herein by reference and which is the compound of Example 69. 5 - [[4 - [(2,3-Dimethyl-2H-indazol-6-yl) methylamino] -2-pyrimidinyl] amino] - 2-methylbenzenesulfonamide can be prepared as described in WO PCT / US01 / 49367.

Suitably 5 - [[4 - [(2,3-dimethyl-2H-indazol-6-yl) methylamino] -2-pyrimidinyl] amino] -2methylbenzenesulfonamide is presented as the monohydrochloride salt. This salt form can be obtained by a person skilled in the art in accordance with the description of International Application No. PCT / US01 / 49367, with an international filing date of December 19, 2001.

5 - [[4 - [(2,3-Dimethyl-2H-indazol-6-yl) methylamino] -2-pyrimidinyl] amino] -2-methylbenzenesulfonamide is sold commercially as a monohydrochloride salt and is known collectively as pazopanib and the trade name Votrient®.

Pazopanib has been implicated in the treatment of malignant neoplasms and ocular diseases / angiogenesis.

Accordingly, the present invention relates to the treatment of malignant neoplasms and ocular diseases / angiogenesis, for example age-related macular degeneration, which comprises administering a compound of formula (I) alone or in combination with pazopanib.

Tyrosine kinases that are not growth factor receptor kinases are called non-receptor tyrosine kinases. Non-receptor tyrosine kinases that can be used in the present invention that are targets or potential targets of anticancer drugs include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Such non-receptor kinases and agents that inhibit the functioning of non-receptor tyrosine kinases are described in Sinn, S. and Corey, SJ, (1999), Journal of Hematotherapy and Stem Cell Research, 8 (5): 465-80 and Bolen, JB, Brugge, JS, (1997), Annual review of Immunology. 15: 371-404.

SH2 / SH3 domain blockers are agents that disrupt binding to SH2 or SH3 domains in a number of enzymes or adapter proteins, including the PI3-K p85 subunit, Src kinase family, adapter molecules (Shc, Crk, Nek, Grb2) and Ras -GAP. SH2 / SH3 domains as targets for anticancer drugs have been discussed by Smithgall, T.E. (1995) Journal of Pharmacological and Toxicological Methods 34 (3): 125-32.

Serine / threonine kinase inhibitors include blockers of the MAP kinase cascade, including blockers of Raf kinases (rafk), mitogen or extracellular regulated kinases (MEK), and extracellular regulated kinases (ERK); and blockers of the protein kinase C family, including blockers of PKC subtypes (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta), IkB kinase family (IKKa, IKKb), kinases of the PKB family, members of the Akt kinase family and TGF-beta receptor kinases. Such serine / threonine kinases and their inhibitors are described in Yamamoto, T., Taya, S., Kaibuchi, K., (1999), Journal of Biochemistry. 126 (5): 799-803; Brodt, P., Samani, A., and Navab, R. (2000), Biochemical Pharmacology, 60. 1101-1107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys. 27: 41-64; Philip, P.A., and Harris, A.L.

21 038355 (1995) Cancer Treatment and Research. 78: 3-27, Lackey, K. et al., Bioorganic and Medicinal Chemistry Letters, (10), 2000, 223-226; U.S. Patent No. 6,268,391; Pearce, L. R et al., Nature Reviews Molecular Cell Biology (2010) 11, 9-22; and Martinez-Iacaci, L., et al., Int. J. Cancer (2000), 88 (1), 44-52.

Accordingly, the pharmaceutically active compounds of the invention can be used in combination with a MEK inhibitor, for example N- {3- [3-cyclopropyl-5- (2-fluoro-4-iodo-phenylamino) -6.8 dimethyl-2,4,7 -trioxo-3,4,6,7-tetrahydro-2H-pyrido [4,3-d] pyrimidin-1-yl] phenyl} acetamide or its pharmaceutically acceptable salts or solvate, for example dimethyl sulfoxide solvate, which are described and claimed in the international Application No. PCT / JP2OO5 / O11O82 with an international filing date of June 10, 2005; international publication number WO 2005/121142 and international publication dated December 22, 2005, the full disclosure of which is incorporated herein by reference. N- {3- [3-Cyclopropyl-5- (2-fluoro-4-iodo-phenylamino) -6,8-dimethyl-2,4,7-trioxo-3,4,6,7tetrahydro-2H-pyrido [ 4,3-d] pyrimidin-1-yl] phenyl} acetamide can be prepared as described in United States Patent Publication No. US 2006/0014768, published January 19, 2006, the entire disclosure of which is incorporated herein by reference.

Accordingly, the pharmaceutically active compounds of the invention can be used in combination with a B-Raf inhibitor, for example N- {3- [5- (2-amino-4-pyrimidinyl) -2- (1,1-dimethylethyl) 1,3- thiazol-4-yl] -2-fluorophenyl} -2,6-difluorobenzenesulfonamide or a pharmaceutically acceptable salt thereof, which are described and claimed in International Application No. PCT / US2009 / 042682 with an international filing date of May 4, 2009, the full disclosure of which incorporated herein by reference. N- {3- [5- (2-Amino-4-pyrimidinyl) -2- (1,1-dimethylethyl) -1,3-thiazol-4-yl] -2-fluorophenyl} -2,6 difluorobenzenesulfonamide can be obtained as described in International Application No. PCT / US2009 / 042682.

Accordingly, the pharmaceutically active compounds of the invention can be used in combination with an Akt inhibitor, for example N - {(1S) -2-amino-1 - [(3,4-difluorophenyl) methyl] ethyl} -5-chloro4- (4- chloro-1-methyl-1H-pyrazol-5-yl) -2-furancarboxamide or a pharmaceutically acceptable salt thereof as described and claimed in WO PCT / US2008 / 053269 with an international filing date of February 7, 2008; international publication number WO 2008/098104 and international publication dated August 14, 2008, the full disclosure of which is incorporated herein by reference. N - {(1S) -2-Amino-1 - [(3,4-difluorophenyl) methyl] ethyl} -5-chloro-4- (4-chloro-1-methyl-1Hpyrazol-5-yl) -2- furancarboxamide is the compound of Example 224 and can be prepared as described in International Application No. PCT / US2008 / 053269.

Accordingly, the pharmaceutically active compounds of the invention can be used in combination with an Akt inhibitor, for example N - {(1S) -2-amino-1 - [(3-fluorophenyl) methyl] ethyl} -5-chloro-4- (4chloro -1-methyl-1H-pyrazol-5-yl) -2-thiophenecarboxamide or a pharmaceutically acceptable salt thereof as described and claimed in WO PCT / US2008 / 053269 with an international filing date of February 7, 2008; international publication number WO 2008/098104 and international publication dated August 14, 2008, the full disclosure of which is incorporated herein by reference. N - {(1 S) -2-Amuho-1 - [(3-fluorophenyl) methyl] ethyl} -5-chloro-4- (4-chloro-1-methyl-1Hpyrazol-5-yl) -2-thiophenecarboxamide is the compound of Example 96 and can be prepared as described in International Application No. PCT / US2008 / 053269. For example, N - {(1S) -2-amino-1 - [(3fluorophenyl) methyl] ethyl} -5-chloro-4- (4-chloro-1-methyl-1H-pyrazol-5-yl) -2- thiophenecarboxamide is presented as the hydrochloride salt. The salt form can be obtained by a person skilled in the art in accordance with the description of International Application No. PCT / US2010 / 022323, with an international filing date of January 28, 2010.

Inhibitors of members of the phosphotidylinositol-3 kinase family, including blockers of PI3 kinase, aTM, DNA-PK and Ku, can also be used in the present invention. Such kinases are discussed in the works of Abraham, R.T. (1996), Current Opinion in Immunology. 8 (3): 412-8; Canman, C.E., Lim, D.S. (1998) Oncogene 17 (25): 3301-3308; Jackson, S.P. (1997), International Journal of Biochemistry and Cell Biology. 29 (7): 935-8; and Zhong, H. et al., Cancer res, (2000), 60 (6), 1541-1545.

Also of interest in accordance with the present invention are inhibitors of the action of myoinositol on signaling pathways, such as phospholipase C blockers and myoinzitol analogs. Such signaling pathway inhibitors are described by Powis, G., and Kozikowski A. (1994), New Molecular Targets for Cancer Chemotherapy ed., Paul Workman and David Kerr, CRC press 1994, London.

Another group of inhibitors of signal transduction pathways are inhibitors of Ras oncogenes. Such inhibitors include farnesyl transferase, geranyl geranyl transferase and CAAX protease inhibitors, as well as antisense oligonucleotides, ribozymes, and immunotherapy. Such inhibitors have been shown to block Ras activation in cells containing wild-type mutant Ras, thus acting as antiproliferative agents. Inhibition of Ras oncogenes was discussed in the works of Scharovsky, O.G., Rozados, V.R., Gervasoni, S.I. Matar, P. (2000), Journal of Biomedical Science. 7 (4): 292-8; Ashby, M.N. (1998), Current Opinion in Lipidology. 9 (2): 99-102; and BioChim. Biophys. Acta, (1999, 1423 (3): 19-30.

As mentioned above, antibodies against ligand-binding receptor kinase can also serve as inhibitors of signal transduction. This group of inhibitors of signal transduction pathways includes the use of humanized antibodies against the extracellular ligand-binding domain of receptor tyrosine kinase. For example, Imclone C225 EGFR-specific antibody (see Green, M.C. et al., Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev. (2000), 26 (4), 269286); Herceptin® erbB2 antibody (see Tyrosine Kinase Signaling in Breast Cancer: ErbB Family Receptor Tyrosine Kinase, Breast Cancer Res., 2O00, 2 (3), 176-183); and a 2CB VECFR2-specific antibody (see Brekken, R.A. et al., Selective Inhibition of VEGFR2 Activity by a Monoclonal Anti-VEGF Antibody Blocks Tumor Growth in Mice, Cancer Res. (2000), 60, 5117-5124).

Inhibitors of angiogenesis of non-receptor kinases may also find use in the present invention. Angiogenesis inhibitors associated with VEGFR and TIE2 are discussed above for signal transduction inhibitors (both receptors are receptor tyrosine kinases). It has been shown that angiogenesis is mainly associated with erbB2 / EGFR signaling, since inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, primarily VEGF expression. Accordingly, non-receptor tyrosine kinase inhibitors can be used in combination with the compounds of the present invention. For example, anti-VEGF antibodies that do not recognize VEGFR (receptor tyrosine kinase) but bind to a ligand; low molecular weight integrin inhibitors (alpha _to beta ₃ ), which will inhibit angiogenesis; endostatin and angiostatin (non-RTK) may also show utility in combination with the compounds described. (See, Bruns CJ et al. (2000) Cancer Res. 60: 2926-2935; Schreiber AB, Winkler ME, and Derynck R. (1986) Science 232: 1250-1253; Yen L et al. (2000) Oncogene 19: 3460-3469).

Agents used in immunotherapeutic regimens can also be used in combination with compounds of formula (I). There are a number of immunological strategies for generating an immune response. These strategies generally relate to the field of tumor vaccination. The effectiveness of immunological approaches can be significantly enhanced by combined inhibition of signaling pathways using a small molecule inhibitor. For a discussion of the erbB2 / EGFR immunological / tumor vaccine approach, see Reilly R.T. et al. (2000) Cancer Res. 60: 3569-3576.

Agents used in pro-apoptotic treatment regimens (eg, bcl-2 antisense oligonucleotides) can also be used in combination of the present invention. Members of the Bcl-2 family of proteins block apoptosis. Therefore, upregulation of bcl-2 has been associated with chemoresistance. Studies have shown that epidermal growth factor (EGF) stimulates anti-apoptotic members of the bcl-2 family (i.e. mcl-1). Therefore, strategies developed to down-regulate bcl-2 expression in tumors have shown beneficial effects in the clinic and are currently in phase II / III trials, namely the G3139 bcl-2 antisense oligonucleotide. Such pro-apoptotic strategies using the bcl-2 antisense oligonucleotide methodology have been reviewed by Waters J.S. et al. (2000) J. Clin. Oncol. 18: 18121823.

Cell cycle signaling inhibitors inhibit molecules involved in cell cycle control. A family of protein kinases called cyclin-dependent kinases (CDKs) and their interaction with a family of proteins called cyclins control the entire cell cycle in eukaryotes. Coordination between activation and inactivation of various cyclin / CDK complexes is necessary for the normal course of the entire cell cycle. Several inhibitors of cell cycle signaling are under development. For example, examples of cyclin-dependent kinases, including CDK2, CDK4 and CDK6, and their inhibitors are described, for example, Rosania et al., Exp. Opin. Ther. Patents (2000), 10 (2): 215-230. In addition, p21WAF1 / CIP1 has been described as a possible and versatile inhibitor of cyclin-dependent kinase (Cdks) (Ball et al., Progress in Cell Cycle Res., 3: 125 (1997)). Compounds known to induce p21WAF1 / CIP1 expression have been implicated in suppressing cell proliferation and exhibiting tumor suppressive activity (Richon et al., Proc. Nat Acad. Sci. USA 97 (18): 10014-10019 (2000)) , and are included as inhibitors of cell cycle signaling. Histone deacetylase (HDAC) inhibitors are involved in the transcriptional activation of p21WAF1 / CIP1 (Vigushin et al., Anticancer Drugs, 13 (1): 1-13 (Jan 2002)) and are suitable cell cycle signaling inhibitors for use in combination herein.

Examples of such HDAC inhibitors include the following.

1. Vorinostat, including its pharmaceutically acceptable salts. Marks et al., Nature Biotechnology, 25, 84-90 (2007); Stenger, Community Oncology, 4, 384-386 (2007).

Vorinostat has the following chemical structure and name:

N-hydroxy-N'-phenyl-octanediamide

2. Romidepsin, including its pharmaceutically acceptable salts. Vinodhkumar et al., Biomedicine &

- 23 038355

Pharmacotherapy, 62 (2008) 85-93.

Romidepsin has the following chemical structure and name:

(1S, 4S, 7Z, 10S, 16E, 21R) -7-ethylidene-4,21-di (propan-2-yl) -2-oxa-12,13-dithia-5,8,20,23 tetrazabicyclo [8 , 7,6] tricos-16-en-3,6,9,19,22-pentone

3. Panobinostat, including its pharmaceutically acceptable salts. Drugs of the Future, 32 (4): 315-322 (2007).

Panobinostat has the following chemical structure and name:

(2E) -N-hydroxy-3- [4 - ({[2- (2-methyl-1H-indol-3-yl) ethyl] amino} methyl) phenyl] acrylamide

4. Valproic acid, including its pharmaceutically acceptable salts. Gottlicher, et al., EMBO J. 20 (24): 6969-6978 (2001).

Valproic acid has the following chemical structure and name:

sn ₃ - sn _g - sn ₂ \ no. he - s sn _y - sn ₂ - sn / An

2-propylpentanoic acid

5. Mocetinostat (MGCD0103), including its pharmaceutically acceptable salts. Balasubramanian et al. Cancer Letters 280: 211-221 (2009).

Mocetinostat has the following chemical structure and name:

N- (2-aminophenyl) -4 - [[(4-pyridin-3-ylpyrimidin-2-yl) amino] methyl] benzamide

The following examples of such HDAC inhibitors are presented in Bertrand European Journal of Medicinal Chemistry, 45, (2010), 2095-2116, in particular the compounds of table. 3 below.

- 24 038355

Proteasome inhibitors are drugs that block the action of proteasomes, cell complexes that degrade proteins such as the p53 protein. Several proteasome inhibitors are commercially available or are being studied in the treatment of cancer. Suitable proteasome inhibitors for use in combination herein include the following.

1. Bortezomib (Velcade®) including its pharmaceutically acceptable salts. Adams J., Kauffman M. (2004) Cancer Invest. 22 (2): 304-11.

Bortezomib has the following chemical structure and name:

[(1 R) -3 -methyl-1 - ({(2S) -3-phenyl-2- [(pyrazin-2-ylcarbonyl) amino] propanoyl} amino) butyl] boric acid

2. Disulfiram, including its pharmaceutically acceptable salts. Bouma et al. (1998). J. Antimicrob. Chemother. 42 (6): 817-20.

Disulfiram has the following chemical structure and name:

1, G, G ', G- [disulfanediyl-bis- (carbonothioylnitrilo)] tetraethane

3. Epigallocatechin gallate (EGCG), including its pharmaceutically acceptable salts. Williamson et al. (December 2006), The Journal of Allergy and Clinical Immunology, 118 (6): 1369-74.

Epigallocatechin gallate has the following chemical structure and name:

- 25 038355

[(2R, 3R) -5,7-dihydroxy-2- (3,4,5-trihydroxyphenyl) chroman-3-yl] -3,4,5-trihydroxybenzoate

4. Salinosporamide A, including its pharmaceutically acceptable salts. Feling et al. (2003) Angew.

Chem. Int. Ed. Engl. 42 (3): 355-7.

Salinosporamide A has the following chemical structure and name:

(4R, 5S) -4- (2-chloroethyl) -1 - ((1S) -cyclohex-2-enyl (hydroxy) methyl) -5-methyl-6-oxa-2azabicyclo [3,2,0] heptane- 3,7-dione

5. Carfilzomib, including its pharmaceutically acceptable salts. Kuhn D. J., et al., Blood 2007,110: 3281-3290.

Carfilzomib has the following chemical structure and name:

(S) -4-methyl-N - ((S) -1 - (((S) -4-methyl-1 - ((R) -2-methyloxiran-2-yl) -1-oxopentan-2-yl ) amino) -1-oxo-3 phenylpropan-2-yl) -2 - ((S) -2- (2-morpholinoacetamido) -4-phenylbutanamido) pentanamide

Heat shock proteins of 70 kDa (Hsp70) and heat shock proteins of 90 kDa (Hsp90) are a family of ubiquitous heat shock proteins. Hsp70 and Hsp90 are overexpressed in some malignant tumors. The use of some Hsp70 and Hsp90 inhibitors in cancer treatment is being studied. Suitable inhibitors of Hsp70 and Hsp90 for use in combination herein include the following.

1. 17-AAG (Geldanamycin), including its pharmaceutically acceptable salts. Jia W et al., Blood. 2003 Sep 1; 102 (5): 1824-32.

17-AAG (Geldanamycin) has the following chemical structure and name:

17- (allylamino) -17-demethoxygeldanamycin

2. Radicicol, including its pharmaceutically acceptable salts. (Lee et al., Mol. Cell Endocrinol. 2002, 188, 47-54)

Radicicol has the following chemical structure and name:

(1 aR, 2Z, 4E, 14R, 15 aR) -8-chloro-9,11-dihydroxy-14-methyl-15,15а-dihydro-1 aH-benzo [c] oxireno [2,3k] [1] oxacyclotetradecin-6,12 (7H, 14H) -dione

Cancer metabolism inhibitors.

Many tumor cells exhibit a metabolism that is markedly different from that of normal tissues. For example, the rate of glycolysis, a metabolic process that converts

- 26 038355 glucose into pyruvate increases, and the resulting pyruvate is reduced to lactate, and does not undergo additional oxidation in mitochondria through the tricarboxylic acid (TCA) cycle. This effect is often observed even under aerobic conditions and is known as the Warburg effect.

Lactate dehydrogenase A (LDH-A), an isoform of lactate dehydrogenase expressed in muscle cells, plays a key role in the metabolism of tumor cells, reducing pyruvate to lactate, which can then be cleared from the cell. The enzyme has been shown to be activated in many types of tumors. The alteration of glucose metabolism, described as the Warburg effect, is critical for the growth and proliferation of cancer cells, and lowering LDH-A levels using RNA-i has been shown to result in decreased cell proliferation and tumor growth in xenograft models.

D.A. Tennant et al., Nature Reviews, 2010, 267.

P. Leder, et al., Cancer Cell, 2006, 9, 425.

High levels of fatty acid synthase (FAS) have been found in precancerous lesions. Pharmacological inhibition of FAS affects the expression of key oncogenes involved in the development and maintenance of malignant neoplasm. Alli et al., Oncogene, (2005), 24, 39-46. doi: 10.1038.

Inhibitors of cancer metabolism, including inhibitors of LDH-A and inhibitors of fatty acid biosynthesis (or FAS inhibitors), are suitable for use in combination with the compounds of the present invention.

Further examples of the additive active ingredient or ingredients (antineoplastic agent) for use in combination or for coadministration with the CD73 inhibiting compounds of the present invention are α anti-PD-L1 agents.

Anti-PD-L1 antibodies and methods for their preparation are known in the art.

Such anti-PD-L1 antibodies can be polyclonal or monoclonal and / or recombinant and / or humanized.

Examples of PD-L1 antibodies are described:

U.S. Patent No. 8217149; 12/633339;

US patent No. 8383796; 13/091936;

US patent No. 8552154; 13/120406;

U.S. Patent Publication No. 20110280877; 13/068337;

U.S. Patent Publication No. 20130309250; 13/892671;

WO 2013/019906;

WO 2013079174;

US Application No. 13/511538 (filed August 7, 2012 which is the US national phase of international application No. PCT / US10 / 58007 (filed 2010); and US Application No. 13/478511 (filed May 23, 2012).

Additional examples of anti-PD-L1 antibodies (also referred to as CD274 or B7-H1) and methods of use are described in US Pat. No. 7,943,743; uS20130034559, WO2014055897, US Pat. No. 8,168,179 and US Pat. No. 7,595,048. PD-L1 antibodies are under development as immunomodulatory agents for the treatment of cancer.

In one embodiment, the anti-PD-L1 antibody is as described in US Pat. No. 8,217,149. In another embodiment, the anti-PD-L1 antibody comprises the CDRs of the antibodies described in US Pat. No. 8,217,149.

In another embodiment, the anti-PD-L1 antibody is as described in US Application No. 13/511538. In another embodiment, the anti-PD-L1 antibody comprises the CDRs of the antibodies described in US Application No. 13/511538.

In another embodiment, the anti-PD-L1 antibody is the antibody described in Application No. 13/478511. In another embodiment, the anti-PD-L1 antibody comprises the CDRs of the antibodies described in US Application No. 13/478511.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (MDX1105). In another embodiment, the anti-PD-L1 antibody is MPDL3280A (RG7446). In another embodiment, the anti-PD-L1 antibody is MEDI4736.

A further example of an additional active ingredient or ingredients (antineoplastic agent) for use in combination or for coadministration with the CD73 inhibiting compounds of the present invention is a PD-1 antagonist.

PD-1 antagonist means any chemical compound or biological molecule that blocks the binding of PD-L1 expressed in a cancer cell to PD-1 expressed in an immune cell (T cell, B cell or NKT cell), and preferably also blocks the binding of PD-L2 expressed in a cancer cell to PD-1 expressed in an immune cell. Alternative names or synonyms for PD-1 and its ligands include PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1 and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In embodiments of aspects or embodiments of the present invention,

- 27 038355 for which human treatment is required, a PD-1 antagonist blocks the binding of human PD-L1 to

Human PD-1 and preferably blocks the binding of both human PD-L1 and human PD-L2 to

Human PD-1. A description of the amino acid sequences of human PD-1 can be found in NCBI Locus #: NP 005009. A description of the amino acid sequences of human PD-L1 and PD-L2 can be found in NCBI Locus #: NP_054862 and NP_079515, respectively.

PD-1 antagonists that can be used in any aspect of the present invention include a monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 and preferably specifically binds to human PD-1 or PD -L1 person. The MAb can be a human antibody, a humanized antibody, or a chimeric antibody, and can include a human constant region. In some embodiments, the human antibody constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human antibody constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F (ab ') ₂ , scFv, and Fv fragments.

Examples of mAbs that bind to human PD-1 and can be used in various aspects and embodiments of the present invention are described in US 7488802, US 7521051, US 8008449, US 8354509, US 8168757, WO 2004/004771, WO 2004/072286, WO 2004/056875 and US 2011/0271358.

Specific anti-human PD-1 mAbs useful as a PD-1 antagonist in any aspect and embodiment of the present invention include MK-3475, a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 2, 161-162 (2013), and which contains the amino acid sequences of the heavy and light chains shown in FIG. 6; nivolumab, a human IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 1, 68-69 (2013), and which contains the amino acid sequences of the heavy and light chains shown in FIG. 7; the humanized antibodies h409A11, h409A16 and h409A17, as described in WO 2008/156712, and AMP-514, developed by Medimmune.

Other PD-1 antagonists useful in any aspect and embodiment of the present invention include immunoadhesion that specifically binds to PD-1 and preferably specifically binds to human PD-1, for example, a fusion protein containing an extracellular or PD-1 binding portion of PD- L1 or PD-L2 fused to a constant region such as the Fc region of an immunoglobulin molecule. Examples of immunoadhesive molecules that specifically bind to PD-1 are described in WO 02010/027827 and WO 2011/066342. Specific fusion proteins useful as a PD-1 antagonist in the treatment, medicaments and uses of the present invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to PD -1 person.

Other examples of mAbs that bind to human PD-1 and can be used in the method of treatment, medicaments and uses of the present invention are described in WO 2013/019906, WO 2010/077634 A1 and US 8383796. Specific anti-human PD-L1 mAbs, which can be used as a PD-1 antagonist in the method of treatment, medicaments and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C.

Keytruda / pembrolizumab is an anti-PD-1 antibody marketed by Merck for the treatment of lung cancer. The amino acid sequence of pembrolizumab and methods of use are disclosed in US Pat. No. 8,168,757.

Opdivo / nivolumab is a fully human monoclonal antibody marketed by Bristol Myers Squibb, directed against the negative immune regulation PD-1 receptor on the surface of human cells (programmed death-1 or programmed cell death 1 / PCD-1) with immunopotentiating activity. Nivolumab binds and blocks the activation of PD-1, a transmembrane protein of the Ig superfamily, by its ligands PD-L1 and PD-L2, resulting in the activation of T cells and cell-mediated immune responses against tumor cells or pathogens. Activated PD-1 negatively regulates T cell activation and effector function by suppressing activation of the P13k / Akt pathway. Other names for nivolumab include BMS-936558, MDX-1106, and ONO-4538. The amino acid sequence of nivolumab and methods of use and preparation are disclosed in US Pat. No. 8,008,449.

Further examples of the additive active ingredient or ingredients (antineoplastic agent) for use in combination or for co-administration with the CD73 inhibiting compounds of the present invention are immunomodulators.

As used herein, the term immunomodulators refers to a substance, including monoclonal antibodies, that attacks the immune system. The ICOS binding proteins of the present invention can be regarded as immunomodulators. Immunomodulators can be used as antineoplastic agents for the treatment of malignant neoplasms. For example, immunomodulators include, but are not limited to, anti-CTLA-4 antibodies,

- 28 038355 such as ipilimumab (ERVOY) and anti-PD-1 antibodies (opdivo / nivolumab and keytruda / pembrolizumab).

Other immunomodulators include, but are not limited to, OX-40 antibodies, PD-L1 antibodies,

LAG3 antibodies, TIM-3 antibodies, 41BB antibodies, and GITR antibodies.

Ervoy (ipilimumab) is a fully human CTLA-4 antibody marketed by Bristol Myers Squibb. The protein structure of ipilimumab and methods of use are described in US Pat. Nos. 6,984,720 and 7,605,238.

CD134, also known as anti-OX40 antibody, is a member of the TNFR receptor superfamily that is not expressed constitutively on dormant naive T cells, unlike CD28. anti-OX40 antibody is a secondary costimulatory molecule expressed 24-72 hours after activation; its ligand, an anti-OX40 antibody, is also not expressed on dormant antigen-presenting cells, but is accompanied by their activation. Anti-OX40 antibody expression is dependent on complete T cell activation; without CD28, expression of anti-OX40 antibody is delayed and four times lower. OX-40 antibodies, OX-40 fusion proteins and methods of their use are disclosed in US Pat. Nos. US Pat. No. 750501; US 7758852; US 7858765; US 7550140; US 7960515; WO 2012/027328; WO 2013/028231.

Additional examples of additional active ingredient or ingredients (antineoplastic agent) for use in combination or for coadministration with the CD73 inhibiting compounds of the present invention are toll-like receptor 4 (TLR4) antagonists.

It is known that aminoalkylglucosaminide phosphates (AGPs) can be used as vaccine adjuvants and immunostimulating agents to stimulate the production of cytokines, activate macrophages, stimulate an innate immune response, and enhance antibody production in immunized animals. Aminoalkylglucosaminide phosphates (AGPs) are synthetic ligands of the toll-like receptor 4 (TLR4). AGPs and their immunomodulatory effects through TLR4 are disclosed in patent publications such as WO 2006/016997, WO 2001/090129 and / or US Pat. No. 61131918 and have been described in the literature. Other AGP derivatives are described in US Pat. No. 7,122,919, US Pat. No. 6,525,028 and US Pat. No. 6,911,434. Some AGPs act as TLR4 agonists, while others are recognized as TLR4 antagonists.

Further examples of the additive active ingredient or ingredients (antineoplastic agent) for use in combination or for coadministration with the CD73 inhibiting compounds of the present invention are anti-ICOS antibodies.

CDRs for murine anti-human ICOS antibodies with antagonistic activity are shown in PCT / EP2012 / 055735 (WO 2012/131004). Antibodies to ICOS are also described in WO 2008/137915, WO 2010/056804, EP1374902, EP 1374901 and EP 1125585.

Further examples of additional active ingredient or ingredients (antineoplastic agent) for use in combination or for co-administration with the compound of formula (I) of the present invention are STING modulating compounds, CD39 inhibitors and adenosine antagonists A2a and A2a.

In one embodiment, a method for treating malignant neoplasms in accordance with the present invention comprises co-administering a compound of formula (I) and / or a pharmaceutically acceptable salt thereof and at least one anti-tumor agent such as selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signaling pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, cell cycle signaling inhibitors; proteasome inhibitors; and inhibitors of cancer metabolism.

The compounds of formula (I) and their pharmaceutically acceptable salts can be administered together with at least one other active agent known to be used in the treatment of bethemoglobinopathy such as sickle cell disease, sickle cell disease, and betathalassemia.

An example of an additional active ingredient or ingredients for use in combination or for coadministration with the combinations of the present invention is hydroxyurea.

Compositions.

Pharmaceutically active compounds within the scope of the present invention can be used as selective inhibitors of DNMT1 in mammals in need thereof, in particular in humans.

The present invention relates to a pharmaceutical composition containing a pharmaceutically acceptable excipient and an effective amount of compounds of the formulas:

- 29 038355 or a pharmaceutically acceptable salt thereof.

Thus, the present invention relates to a method for the treatment of malignant neoplasm

treatment, precancerous symptoms and other conditions requiring inhibition of DNMT1, which includes the administration of an effective amount of compounds of the formulas:

or a pharmaceutically acceptable salt thereof.

The aforementioned compounds also provide a method for treating the aforementioned disease states in view of their demonstrated ability to act as DNMT1 inhibitors. The drug can be administered to a patient in need thereof by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, topical, subcutaneous, intradermal, intraocular, and parenteral.

The pharmaceutically active compounds of the present invention are formulated into convenient dosage forms such as capsules, tablets, or injections. Solid or liquid pharmaceutical carriers are used. Solid carriers include starch, lactose, calcium sulfate dihydrate, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, and water. Likewise, the carrier or diluent can include any sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies widely, but is preferably from about 25 mg to about 1 g per dosage unit. When a liquid carrier is used, the formulation will be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injection liquid such as an ampoule, or an aqueous or non-aqueous liquid suspension.

Pharmaceutical compositions are prepared by conventional pharmaceutical chemistry techniques including mixing, granulating and compressing as needed to form tablets, or mixing, filling and dissolving ingredients as needed to produce the desired oral or parenteral products.

Doses of the inventive pharmaceutically active compounds in a pharmaceutical dosage unit as described above are an effective, non-toxic amount, preferably in the range of 0.001-500 mg / kg of active compound, preferably 0.01100 mg / kg. When treating a person in need of a DNMT1 inhibitor, the selected dose is preferably administered 1-6 times a day, orally or parenterally. Preferred forms of parenteral administration include topical, rectal, transdermal, injection, and continuous infusion. Oral dosage units for administration to humans preferably contain from 0.5 to 3500 mg of active compound. Acceptable oral dosage units for human administration preferably contain from 0.5 to 1000 mg of active compound. Oral administration is preferred, using lower doses. Parenteral administration in high doses, however, can also be used when it is safe and convenient for the patient.

The optimal dosages can be readily determined by those skilled in the art and can vary depending on the particular DMNT1 inhibitor used, the efficacy of the formulation, the route of administration, and the improvement in the disease state. Adaptive factors depending on the particular patient being treated, including patient age, weight, diet and time of administration, may necessitate dosage adjustments.

The method of the present invention for inducing DNMT1 inhibitory activity in mammals, including humans, comprises administering to a subject in need of such activity an effective amount of a DNMT1 inhibiting pharmaceutically active compound of the present invention.

The invention also relates to the use of compounds of the formulas:

- 30 038355

or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use as a DNMT1 inhibitor.

In addition, the pharmaceutically active compounds of the present invention can be co-administered with additional active ingredients such as other compounds known to be useful in the treatment of cancer or compounds known to be successful when used in combination with a DNMT1 inhibitor.

The invention also relates to a pharmaceutical composition containing from 0.5 to 1000 mg of compounds of the formulas:

or a pharmaceutically acceptable salt thereof and from 0.5 to 1000 mg of a pharmaceutically acceptable excipient.

Without further elaboration, it is believed that one skilled in the art can, using the foregoing description, make full use of the present invention. Therefore, the following examples should be construed simply as illustrative and not limiting in any way the scope of the present invention.

Examples of

The following examples illustrate the invention. These examples are not intended to limit the scope of the present invention, but rather to provide guidance to those skilled in the art on the preparation and use of the compounds, compositions and methods of the present invention. While descriptions of specific embodiments of the present invention have been provided, one skilled in the art will appreciate that various changes and modifications can be made without departing from the spirit and scope of the invention.

Example 168.

(R) -2 - ((3,5-Dicyano-4-ethyl-6 - ((S) -3-hydroxypyrrolidin-1-yl) pyridin-2-yl) thio) -2-phenylacetamide

Stage 1. (S) -2-Chloro-4-ethyl-6- (3-hydroxypyrrolidin-1-yl) pyridine-3,5-dicarbonitrile

To a solution of 2,6-dichloro-4-ethylpyridine-3,5-dicarbonitrile (described in example 3, stage 2, 678 mg, 3.00 mmol) and (S) -pyrrolidin-3-ol (261 mg, 3, 00 mmol) in N, N-dimethylformamide (20 ml) triethylamine (0.418 ml, 3.00 mmol) was added at room temperature and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into water (100 ml) and extracted EtOAc (100 mlx2). The combined organic layers were dried and concentrated to give (S) -2-chloro-4-ethyl-6- (3-hydroxypyrrolidin-1-yl) pyridine-3,5-dicarbonitrile (620 mg, 75% yield) as a pale solid.

LCMS m / z = 276.9 [M + H] +.

Stage 2.2 - ((3,5-Dicyano-4-ethyl-6 - ((S) -3-hydroxypyrrolidin-1-yl) pyridin-2-yl) thio) -2phenylacetamide

- 31 038355

A solution of (S) -2-chloro-4-ethyl-6- (3-hydroxypyrrolidin-1-yl) pyridine-3,5-dicarbonitrile (500 mg,

1.81 mmol), potassium thioacetate (248 mg, 2.17 mmol) in Ν, Ν-dimethylformamide (DMF) (15 ml) was stirred at room temperature for 30 min, then 2-amino-2-oxo -1phenylethyl methanesulfonate (synthesis described in example 3, step 5, 497 mg, 2.17 mmol) and triethylamine (0.50 ml, 3.6 mmol). The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was poured into water (100 ml) and extracted with EtOAc (100 ml x 2). The combined organic layers were dried, concentrated and purified on a silica gel column (eluting with MeOH / DCM 0-2%) to give 2 - ((3,5-dicyano-4-ethyl-6 - ((S) -3-hydroxypyrrolidine- 1-yl) pyridin-2yl) thio) -2-phenylacetamide (380 mg, 51% yield) as a white solid.

LCMS m / z = 407.9 [M + H] +.

Step 3. (R) -2 - ((3,5-Dicyano-4-ethyl-6 - ((S) -3-hydroxypyrrolidin-1-yl) pyridin-2-yl) thio) -2phenylacetamide

2 - ((3,5-Dicyano-4-ethyl-6 - ((S) -3-hydroxypyrrolidin-1-yl) pyridin-2-yl) thio) -2-phenylacetamide (250 mg, 0.62 mmol) separated by chiral HPLC (chiralpak-IC column, HEX-EtOH (FA) to give (R) -2 - ((3,5-dicyano-4-ethyl-6 - ((S) -3-hydroxypyrrolidine-1- yl) pyridin-2-yl) thio) -2phenylacetamide The absolute configuration of the chiral center near the sulfur atom was confirmed by VCD analysis (30 mg).

LCMS m / z = 408.1 [M + H] +.

1H NMR (400 MHz, DMSO-d ₆ ) δ ppm 7.91 (br s, 1H), 7.52 (br s, 1H), 7.52-7.49 (m, 1H) , 7.44-7.22 (m, 4H), 5.61 (s, 1H), 5.14 (d, J = 3.4 Hz, 1H), 4.41 (s, 1H), 4, 05-3.65 (m, 4H), 2.74 (q, J = 7.4 Hz, 2H), 2.07-1.85 (m, 2H), 1.21 (q, J = 7, 6 Hz, 3H).

Example 418.

(S) -1- (6 - (((R) -2-Amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4-ethylpyridin-2-yl) pyrrolidin-3-yl dihydrogen phosphate ...

Stage 1. (S) -2-Chloro-4-ethyl-6- (3-hydroxypyrrolidin-1-yl) pyridine-3,5-dicarbonitrile

TEA (154 ml, 1106 mmol) and 2.6 -dichloro-4-ethylpyridine-3,5-dicarbonitrile (synthesis is described in example 3, step 2, 100 g, 442 mmol). The resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (2000 ml) and extracted with DCM (2x2000 ml). The combined organic layers were washed with water (2x2000 ml), brine (1000 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the crude material. The crude material was triturated in diethyl ether (1000 ml), filtered and dried to give (S) -2-chloro4-ethyl-6- (3-hydroxypyrrolidin-1-yl) pyridine-3,5-dicarbonitrile (110 g, 89% ) as a pale brown solid.

LCMS m / z = 277.1 [M + H] +.

Stage 2. (S) -Di-tert-butyl (1- (6-chloro-3,5-dicyano-4-ethylpyridin-2-yl) pyrrolidin-3-yl) phosphate

To a stirred solution of (S) -2-chloro-4-ethyl-6- (3-hydroxypyrrolidin-1-yl) pyridine-3,5 dicarbonitrile (160 g, 578 mmol) in tetrahydrofuran (THF) (1500 ml) at room temperature 1H-tetrazole (81 g, 1156 mmol) and di-tert-butyl diethylphosphoramidite (202 g, 809 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The resulting reaction

- 32 038355 the mixture was cooled to a temperature of 0 ° C, then 2-hydroperoxy2-methylpropane (145 ml, 867 mmol) was added to the reaction mixture dropwise and stirred at room temperature for 1 h. The reaction mixture was quenched with a saturated solution of sodium bisulfite 1.5 L) and extracted with ethyl acetate (3x1.5 L). The combined organic layers were washed with water (1.0 L), brine (1.0 L), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a crude material. The crude material was purified by neutral alumina column chromatography (eluent: 20% EtOAc in hexanes) to give (S) -di-tert-butyl (1- (6-chloro-3,5-dicyano-4ethylpyridin-2-yl ) pyrrolidin-3-yl) phosphate (125 g, 46%) as an off-white solid.

LCMS m / z = 469.2 [M + H] +.

Step 3. (S) -2-amino-2-oxo-1-phenylethyl 4-methylbenzenesulfonate

OTs

DIPEA (104 ml, 595 mmol) and DMAP (2.425 g,

19.85 mmol). The reaction mixture was cooled to 0 ° С, then Ts-Cl (56.8 g, 298 mmol) was added to the reaction mixture in portions and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into ice water (2000 ml) was stirred for 10 min and a solid precipitated. The precipitated solid was collected by filtration and dried to give the crude compound. The crude material was triturated in diethyl ether (2x500 ml) to give (S) -2-amino-2-oxo-1-phenylethyl 4-methylbenzenesulfonate (38 g, 58%) as an off-white solid.

LCMS m / z = 306.1 [M + H] +.

Step 4. (S) -1- (6 - (((R) -2-amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4-ethylpyrudin-2yl) pyrrolidin-3-yl di-tert-butyl phosphate

In a stirred solution of (S) -di-tert-butyl (1- (6-chloro-3,5-dicyano-4-ethylpyridin-2yl) pyrrolidin-3-yl) phosphate (40.0 g, 84 mmol) in N , N-dimethylformamide (DMF) (400 ml), potassium thioacetate (14.46 g, 127 mmol) was added at room temperature and stirred for 2 h at room temperature. TEA (17.65 ml, 127 mmol) and (S) -2-amino-2-oxo-1-phenylethyl 4-methylbenzenesulfonate (28.1 g, 84 mmol) were added to the reaction mixture at room temperature and stirred for 18 h. The reaction mixture was diluted with ice water (2000 ml) and was extracted with EtOAc (3x1500 ml). The combined organic layers were washed with ice water (3x2000 ml), brine (1000 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the crude material. The crude material was triturated in diethyl ether (200 ml) and n-pentane (1000 ml), filtered and dried to give a pale brown solid. The solid product was dissolved in EtOAc (500 ml) and filtered through Celite®. The filtrate was concentrated under reduced pressure to give (S) -1- (6 - (((R) -2-amino-2-oxo-1phenylethyl) thio) -3,5-dicyano-4-ethylpyridin-2-yl) pyrrolidine -3-yl di-tert-butyl phosphate (38.0 g, 59%) as a pale brown solid.

LCMS m / z = 600.3 [M + H] +.

Step 5. (S) -1- (6 - (((R) -2-Amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4-ethylpyridin-2yl) pyrrolidin-3-yl dihydrogen phosphate

^but 'bn

Into a stirred solution of (S) -1- (6 - (((R) -2-amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4ethylpyridin-2-yl) pyrrolidin-3-yl di-tert-butyl phosphate (38.0 g, 50.2 mmol) in ethanol (400 ml) and diethyl ether (800 ml) in diethyl ether at 0 ° C was added 2.0 M hydrochloric acid

- 33 038355 (400 ml, 800 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give a crude material. The crude material was co-distilled with EtOH (400 ml) and diethyl ether (800 ml) (3 times) and dried to give a pale brown solid. The solid was triturated in EtOH (400 ml) and diethyl ether (800 ml), filtered and dried to give (S) -1- (6 - (((R) -2-amino-2-oxo-1phenylethyl) thio) - 3,5-dicyano-4-ethylpyridin-2-yl) pyrrolidin-3-yl dihydrogen phosphate (18.5 g, 75%) as an off-white solid.

LCMS (m / z): 488.0 [M + H] ⁺ .

Step 6. (S) -1- (6 - (((R) -2-amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4-ethylpyridin-2yl) pyrrolidin-3-yl dihydrogen phosphate

Two lots of (S) -1- (6 - (((R) -2-amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4-ethylpyridin-2yl) pyrrolidin-3-yl dihydrogen phosphate (4.7 g and 18.5 g) were combined and dissolved in ethanol (4.0 L). The solution was evaporated under reduced pressure to give an off-white solid. The solid was triturated in EtOH and diethyl ether (200 ml: 1000 ml), filtered and dried to give an off-white solid. The solid product was thoroughly ground with a pestle and mortar to obtain (S) -1- (6 - (((R) -2-amino-2-oxo-1-phenylethyl) thio) -3,5-dicyano-4-ethylpyridin-2yl ) pyrrolidin-3-yl dihydrogen phosphate (22.5 g, 98%) as an off-white solid.

LCMS (m / z) = 488.0 [M + H] ⁺ .

Ή NMR (400 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.52-7.48 (m, 2H), 7.40-7.23 (m, 4H), 5.59 ( s, 1H), 4.92 (s, 1H), 4.05-3.96 (m, 2H), 3.95-3.82 (m, 2H), 2.73 (q, J = 7, 5 Hz, 2H), 2.24-2.06 (m, 2H), 1.19 (t, J = 7.6 Hz, 3H).

Two phosphate protons are not observed.

Example 700 In vitro DNMT1 assay.

Compounds of the invention have been assayed for DNMT1 selective activity in one or more of the following DNMT1 assays, and may also be assayed in one or more of the following DNMT3 assays.

Breakliqht DNMT1 analysis.

In vitro breaklight DNMT1 assays were performed in a total volume of 50 μl in a standard 384 well format using black non-binding surface microplates (Corning 3575). 5 μl of full-length human DNMT1 (internally produced) used at a final concentration of 40 nM was added to 5 μl of a substrate mixture containing a final concentration of 125 nM hemimethylated oligonucleotide (synthesized by ATD Bio;

5'-fam-atctagcg5atcagttttctgatg5G5tagat-3 ', where 5 = methyldeoxycytidine) and 2 μM ultrapure SAM (Cisbio # 62SAHZLD). For negative control wells, 5 μl DNMT1 and 5 μl oligonucleotide were used, but 0 μM SAM. All reagents were mixed in 1x assay buffer (20 mM Tris pH 6.8, 25 mM NaCl, 0.5 mM MgCl ₂ , 0.01% Triton X100, and 1 mM DTT). The reaction was incubated at 26 ° C for 45 min and then stopped by adding 40 μl of developing reagent. The developing reagent in 1x Gla assay buffer (20 mM Tris pH 8.0, 80 mM NaCl, 0.75 mM MgCl ₂ , 0.01% Triton X100 and 1 mM DTT) has a final concentration of 100 μM SAH (Sigma # A9384) and 0.0008 units / μl of Gla1 restriction endonuclease (Sibenzyme # E494). After adding the developing reagent, the plate was incubated in the dark at room temperature for 5 hours. The fluorescence intensity was then measured by PHERAstar FS (BMG Labtech) at Ex 485 nm and Em 520 nm with a gain of 400 and an integration time of 100 ms.

Breaklight Gla1 counting screening assay

In vitro breaklight Gla1 counting screening assay was performed in a total volume of 50 μl in a standard 384 well format using black non-binding surface microplates (Corning 3575). Ten μl of substrate mixture containing final concentrations of 100 nM hemimethylated oligonucleotide (synthesized in ATD Bio; 5'-FAM-ATCTAGCG5ATCAGTTTTTTTGATG5G5TAGAT-3 'Bio;5'-FAM-ATCTAG5G5ATCAGTTTTCTGATG5G5TAGAT-3', where 5 = methyldeoxycytidine). The negative control wells used 10 μl of 125 nM hemimethylated oligonucleotide. All reagents were mixed in 1x assay buffer (20 mM Tris pH 6.8, 25 mM NaCl, 0.5 mM MgCl ₂ , 0.01% Triton X100, and 1 mM DTT). 40 μl of the Gla1 developing reagent in 1x Gla analyte was added immediately.

- 34 038355 buffer (20 mM Tris pH 8.0, 80 mM NaCl, 0.75 mM, MgCl ₂ , 0.01% Triton X100 and 1 mM DTT) with a final concentration of 100 μM SAH (Sigma # A9384) and 0 , 0008 units / μl of restriction endonuclease Gla1 (Sibenzyme # E494). After the addition of the developing reagent, the plate was incubated in the dark at room temperature for 5 hours. The fluorescence intensity was then measured on a PHERAstar FS (BMG Labtech) at Ex 485 nm and Em 520 nm with a gain of 400 and an integration time of 100 ms.

Breakliqht DNMT3a / 3l analysis.

In vitro breaklight DNMT3a / 3l assays were performed in a total volume of 50 μl in a standard 384 well format using black non-binding surface microplates (Corning # 3575). 5 μl of full-length human DNMT3a / 3l (internally produced) used at a final concentration of 600 nM was added to 5 μl of a substrate mixture containing a final concentration of 125 nM hemimethylated oligonucleotide (synthesized in ATD Bio; 5'-FAM-ATCTAGCG5ATCATGT5TTCTAG, where 5 = methyl deoxycytidine) and 2 μM ultrapure SAM (Cisbio # 62SAHZLD). The negative control wells used 5 μl DNMT3a / 3l and 5 μl oligonucleotide, but also included 200 μM SAH. All reagents were mixed in 1x assay buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 1.5 mM EDTA, 0.1 mM MgCl ₂ , 1 mM CHAPS, and 1 mM DTT). The reaction was incubated at 37 ° C for 90 min and then stopped by adding 40 μl of developing reagent. The developing reagent in 1x Gla assay buffer (20 mM Tris pH 8.0, 80 mM NaCl, 0.75 mM MgCl ₂ , 0.01% Triton X100 and 1 mM DTT) had a final concentration of 200 μM SAH (Sigma # A9384) and 0.0008 units / μl of Gla1 restriction endonuclease (Sibenzyme # E494). After adding the developing reagent, the plate was incubated in the dark at room temperature for 5 hours. The fluorescence intensity was then measured on a PHERAstar FS (BMG Labtech) at Ex 485 nm and Em 520 nm with a gain of 400 and an integration time of 100 ms.

Analysis methods DNMT1 SPA.

The biochemical activity of DNMT1 was analyzed using SPA technology. Plates (Griener 784075) were pre-coated with 100 nL / well compound (11-point, 3-fold serial dilution). The reaction was initiated by adding 5 μl of 2x enzyme mix to wells containing 5 μl of 2x substrate mix. The wells of the lower control contained 5 μl of buffer instead of the enzyme. After a 40 minute incubation, the reaction was quenched by the addition of 10 μl of a stop mixture containing 1 mM SAM (Sigma A7007) and 2 mg / ml PEI beads (Perkin Elmer RPNQ0098). Plates were sealed, centrifuged for 1 min and then read on Viewlux (Perkin Elmer) using a 613 nm emission filter / 300 sec read time after> 30 min dark adaptation. The assay conditions before quenching consisted of 40 nM DNMT1 (601-1600 internally produced), 100 nM 3H-SAM (American Radiolabeled Chemicals ART 0288), 900 nM SAM (New England BioLabs B9003S), and 200 nM hemimethylated DNA oligonucleotide (synthesized according to integrated DNA technologies, 5'-CCTCTTCTAACTGCCAT5GATCCTGATAGCAGGTGCATGC-3 ') in 50 mM HEPES (pH 8.0), 2 mM MgCl ₂ , 1 mM DTT, 0.01% NP-40 and 0.01% BSA.

Example 701. Effect on erythroid progenitor cells (EPC) - in vitro.

7-day-old EPCs were cultured in serial dilution plates of compound A (2 - {[3,5-dicyano-6- (dimethylamino) -4-ethylpyridin-2-yl] sulfanyl} -2-phenylacetamide) for 5 days at 37 ° C. The wells were then analyzed for fetal hemoglobin induction (HbF, HbF ELISA) or cell growth (Cell Titer-Glo). In both cases, the signal was normalized relative to the cells treated with the control carrier medium. Compound A induced an increase in HbF with an average pEC ₅₀ = 6.5 and inhibited cell growth by 50% with an average pGI ₅₀ = 5.9. Representative curves for each assay are shown in FIG. 1A.

To analyze the effect of Compound A on DNA methylation, EPCs were collected, treated for 3 days with Compound A, and genomic DNA was sequenced with bisulfite. Methylation of nine CpG sites at or near the promoter regions of the HBG1 and HBG2 gene loci was selected and analyzed based on their previous characterization as DNMT1 methylation sites during erythropoiesis (Mabaera et al., Blood: 110 (4). 2007). As shown in FIG. 1B, all nine sites showed a decrease in methylation by an average of 65 ± 5% compared to cells treated with carrier medium.

Ways.

Culture, distribution and characterization of erythroid progenitor cells (EPC) on day 7.

All donors gave written informed voluntary consent for the use of their samples, as well as the collection and use of samples that received official approval from the expert council of the organization. All cryopreserved CD34 + human bone marrow cells used herein were obtained from AllCells (Emeryville, Calif.) And are generally from different donors. CD34 + cells were cultured to generate EPC on day 7. Briefly, 1 million cells were cultured in 5% CO ₂ , 5% O ₂ at 37 ° C in Stemspan H3000 medium (StemCell Technologies Vancouver, BC,

- 35 038355

Canada) supplemented with 2 mM L-glutamine, 40 μg / ml human low-density lipoproteins (StemCell Technologies), 10 ng / ml recombinant human (rh) interleukin IL-3 (R&D Systems, Minneapolis, MN), 100 ng / ml factor rh stem cells (R&D Systems) and 0.5 U / ml rh erythropoietin (Invitrogen, Grand Island, NY). Cells were separated and maintained on day 4 with the complete culture medium described above and harvested on day 7 to assess erythroid marker expression and to assess γ-globin induction. 7 day old EPCs were then frozen in liquid nitrogen at 5 to 10 million cells / ml in 95% fetal bovine serum (FBS, Invitrogen) with 5% DMSO for later use.

During compound treatment, frozen 7 day old EPCs obtained as described above were thawed, washed once and resuspended in complete culture medium as described above, except for increasing rhEPO to 3 U / ml. Cells were counted and diluted to 3.3x103 cells / ml for loading onto assay plates. The cells were then dispensed at 30 µl / well using a Multidrop ™ Combi Reagent (Thermo Scientific) into 384-well cell culture plates pre-dosed with test compounds at 100 nl / well. Black transparent bottom (Greiner Bio-One, 781090) and white (Greiner Bio-One; 781080) plates were used for ELISA and Cell Titer-Glo assays, respectively. The final cell density in the assays was 1000 cells per well with final compound concentrations between 33 nM (highest) and 6.6 nM (lowest) for a 22-point serial dilution.

To monitor cell health and cell growth, cell growth assays were performed during cell seeding (day 0) using Cell Titer-Glo (detailed below). For compound treatment, cell culture plates were incubated for 5 days at 37 ° C. with 5% CO2.

Fetal Hemoglobin (HbF) ELISA.

Anti-HbF Ab coating (Bethyl Lab; cat. A80-136A) was diluted 100-fold in coating buffer (0.05 M carbonate bicarbonate, pH 9.6) and then added at 20 μl / well per 384 well MaxiSorp ELISA plate (Thermo Fisher, cat. 464718). After 1 h incubation at room temperature, plates were washed twice with ELISA wash buffer (50 mM Tris, 0.05% Tween 20, pH 8.0) using an EL406 plate washer (BioTek, Winooski, VT). Then, 40 μl / well of blocking buffer consisting of 50 mM Tris and 1% BSA (pH 8.0) was added to the plate and stored at 4 ° C with a capping layer overnight or until assay time. The coated ELISA plates were stable for 30 days at 4 ° C. On the day of assay, before adding cell lysate, plates were washed twice with ELISA wash buffer.

After 5 days incubation at 37 ° C with 5% CO _{2, the} cell culture plates for ELISA assay were placed in a freezer at -80 ° C for at least 2 hours. After thawing at room temperature, 30 μl of cell lysis buffer (Invitrogen; cat. FNN0011, supplemented with 1x protease inhibitor) was added to each well, and the resulting cell lysate was mixed eight times using a Cybi-Well pipette (Jena, Germany). After the mixing procedure, 20 μl / well of the lysate was transferred to the coated ELISA plates described above, and then incubated for 1 hour at room temperature. ELISA plates were washed three times with ELISA wash buffer. Then 20 μl / well of 1: 75,000 to 100,000 diluted horseradish peroxidase (HRP) anti-HbF Ab conjugate (Bethyl Labs; cat. A80-136P, diluted in 50 mM Tris [pH 8.0], 1% BSA, 0 , 05% Tween-20). After an additional 1 hour incubation at room temperature, the plates were washed four times and then 20 μl / well of tetramethylbenzidine substrate ELISA (Thermo Scientific; cat. 34028) was added. After incubation for 3-10 min at room temperature in the dark, 20 μl / well of stopping solution (0.2 M sulfuric acid) was added. Plates were then read at 450 nm absorbance (OD450) using an Envision plate reader (PerkinElmer, Waltham, MA). The average reading of the control wells (16 wells in column 6 of each assay plate) containing only DMSO was used as the basal level for normalization. The γ-globin level for each treated well was calculated as a percentage of the basal level (100%).

Normalized responses of 22 concentrations of each test compound were subjected to curve fitting using a specialized statistical computing tool based on R (R basis for statistical calculations). The EC ₅₀ value (compound concentration at V ₂ Max%) and the corresponding Max% were determined from the established curve of each active compound.

Analysis of cell growth of 7-day-old EPCs.

Cell growth assays were performed on cell culture plates after 5 days of incubation at 37 ° C. with 5% CO2. To assay plates, 15 μl / well of Cell Titer-Glo assay reagent (Promega, Madison, WI) was added. The plates were then incubated at room temperature for 10 min, then read on a ViewLux 1430 (Perkin Elmer) using a luminescence protocol. The average reading of the control wells (16 wells in column 6 of each assay plate) containing only DMSO was used as the basal level for normalization. The Cell Titer-Glo signal of each test well was calculated as a percentage of basal level (100%). Normalized responses of 22 concentrations of each test compound were subjected to curve fitting using a specialized statistical computing tool based on R (R basis for statistical calculations).

To monitor cell health and cell growth, the Cell Titer-Glo signal of the DMSO control wells was compared to the signal obtained on day 0 (see above). For healthy cell growth, approximately

A 20-fold increase was usually observed on day 5 relative to day 0.

Bisulfite sequence analysis.

Genomic DNA was extracted in duplicate from 7 day old EPCs, cultured at 37 ° C with 5% CO ₂ and 5% O ₂ for 3 days in the presence of the compound using the ZYMO gDNA kit (Zymo Research, Irvine, Calif.). The isolated DNA was subjected to bisulfite conversion using the Zymo EZ DNA methylation kit. Primer sets for bisulfite treated regions in HBG1 and HBG2 were developed using ABI Methyl Primer Express software. The regions analyzed included 9 previously described DNMT1-dependent β-dependent DNA methylation sites (Mabaera et al. Blood: 110 (4). 2007). DNA was further amplified with fluidigm and QC'd using an Agilent bioanalyzer. MiSeq was performed using the Illumina system and the result was analyzed using ArrayStudio v8 and CLC-GWB v8.1.

Example 702 Antiproliferative activity MV-4-11 (pEC ₅₀ category).

Cell proliferation assay. The optimum cell seeding density was determined empirically for AML MV-4-11 cells by examining the growth of a wide variety of seeding densities in a 384-well format to determine conditions that assumed maximum tolerable and sustained proliferation for 6 days. MV-4-11 cells were plated in duplicate on plates 24 hours prior to treatment with a 20-point two-fold compound dilution series or 0.15% DMSO. The plates were incubated for 6 days at 37 ° C in 5% CO ₂ . The cells were then lysed with CellTiter-Glo (CTG) (Promega) and the chemiluminescent signal was detected with an appropriate microplate reader. In addition, an untreated cell plate was harvested to quantify the initial cell density at the time of compound addition (T _0). The CTG values obtained after 6 days of treatment were expressed as a percentage of the T ₀ value and plotted against the concentration of the compound. The data were matched with a four-parameter equation to construct a concentration versus curve from which growth _{IC 50 values were calculated.}

Example 703 In vivo sickle cell assay.

A mouse model of sickle cell anemia was used to determine the in vivo efficacy of Compound A, previously shown to have potent in vitro activity against primary human erythroid progenitor cells derived from normal bone marrow or peripheral blood mononuclear cells of a sickle cell patient.

Ways.

Preparing the experiment.

The in vivo activity of Compound A was studied in a mouse model with sickle cells in accordance with US / UK animal care standards.

Male and female mice transgenic for human hemoglobin [B6; 129Hbatm1 (HBA) Tow / Hbbtm2 (HBG1, HBB *) Tow / J Mice (Jackson Laboratories, ME)], at the beginning of the study were approximately 6-8 weeks of age and weighed approximately 15-25 g. As far as possible, the groups were balanced by sex and consisted of 6 mice.

Description of the experience.

Mice were dosed with vehicle (10% DMA / 90% PEG400), 10 or 50 mg / kg Compound A twice a day (BID) by the oral route five days a week for two weeks.

At the end of the dosing period, mice were euthanized by CO2 asphyxiation, and blood from the vena cava was collected in EDTA tubes for fetal hemoglobin analysis. % HbF protein was determined using high performance liquid chromatography and% F cells (expressing HbF erythrocytes) were determined using flow cytometry. An anti-human HbF mouse monoclonal antibody conjugated to APC (Life Technologies, Grand Island, NY) was used to identify HbF-expressing erythroid cells. The nuclear dye syto16 (Life Technologies) was used to separate populations of reticulocytes and RBCs. Protein and cell data were collected on a BioRad D10 analyzer (Bio-Rad, Benicia, CA) and FACs Canto I (BD BioSciences, San Jose, CA), respectively. Flow cytometry data were analyzed using Flowjo v7 software (Treestar, Inc. Ashland. OR). The mean and standard deviation were determined for the control and treatment groups. Data were pooled and analyzed using one-way ANOVA with Tukey post-test (Graphpad Prism v5 [La Jolla, CA]).

Results.

Compound A, administered orally at a dose of 10 or 50 mg / kg, BID daily (5 days of dosing, 2 days of rest, 4 days of dosing) caused an increase in the% HbF protein, which was dose dependent and statistically significant (5.4 times, p <0.05; 10.3 times, p <0.001, respectively) [Fig. 2A].

Compound A treatment caused an increase in% F reticulocytes and% F RBC after oral administration

- 37,038355 10 or 50 mg / kg BID infusions over a 2 week dosing. There was a dose-dependent and statistically significant increase in% F reticulocytes (4-fold and 9-fold, respectively; p <0.001) and% F RBC (6-fold and 8-fold, respectively; p <0.001) [FIG. 2B].

Biological activity.

It will be understood by those skilled in the art that the above assays may undergo variability from experiment to experiment. Accordingly, it should be understood that the values below are given as examples only.

Compounds of the invention were tested for activity against DNMT1, typically according to the above breaklight assay and / or SPA assay.

DNMT1 datasheet.

Binning category (pXC ₅₀ ):

Category A: <4.5.

Category B: 4.5 <x <6.0.

Category C: 6.0 <x <6.5.

Category D:> 6.5 .________________________________________________________________

Example# DNMT1 BioChemical (pEC value ₅₀ ) EPC ELISA (pEC value ₅₀ ) MV4-11 Antiproliferate activity (EU value ₅₀ ) DNMT3A Breaklight 1C ₅₀ (micromolar) DNMT3B Breaklight 1C ₅₀ (micromolar) 168 D WITH 418 V A

Comments.

1. Compounds show the indicated level of activity in both the breaklight assay and the SPA assay described above.

2. If the field is blank, then the compound was not tested in this assay.

The compound of Example 168 was tested essentially in accordance with the above DNMT1 breaklight assay and / or SPA assay and showed an average pIC ₅₀ > 6.5 in repeated experimental runs.

While the preferred embodiments of the invention are illustrated above, it should be understood that the invention is not limited to the instructions prescribed herein and that all changes within the scope of the following claims are reserved.

Claims (24)

CLAIM 1. Compounds represented by the following formulas: or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, represented by the following formula: or a pharmaceutically acceptable salt thereof. 3. A pharmaceutical composition comprising a compound according to claim 1 or 2, an acceptable salt and a pharmaceutically acceptable excipient. or pharmaceutically - 38 038355 4. A method of treating a disease selected from malignant neoplasm, precancerous conditions, beta hemoglobinopathies, sickle cell disease in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount of a compound described in any one of claims 1, 2, or its pharmaceutically acceptable salt. 5. The method of claim 4, wherein the disease is sickle cell anemia. 6. The method of claim 4, wherein the disease is beta thalassemia. 7. A method according to claims 4-6, wherein the mammal is a human. 8. The method according to claim 4, wherein said malignant neoplasm is selected from the following: brain tumor (glioma), glioblastoma, astrocytoma, glioblastoma multiforme, Banyan-Zonan syndrome, Cowden's disease, Lermitte-Duclos disease, breast cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, adenocarcinoma, ductal adenocarcinoma, adenosquamous carcinoma, acinar cell carcinoma, glucagonoma, insulinoma, prostate cancer, sarcoma, and thyroid cancer. 9. The method according to claim 4, wherein said malignant neoplasm is selected from the following: breast cancer, inflammatory breast cancer, ductal carcinoma, lobular carcinoma, colon cancer, pancreatic cancer, insulinoma, adenocarcinoma, ductal adenocarcinoma, adenosquamous cell carcinoma, acini cancer, glucagonoma, skin cancer, melanoma, metastatic melanoma, lung cancer, small cell lung cancer, non small cell lung cancer, squamous cell carcinoma, large cell carcinoma, brain tumor (glioma), glioblastoma, astrocytoma, glioblastoma multiforme, Bunyan-Zonan disease Lermitte-Duclos disease, Wilms tumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, head and neck cancer, kidney cancer, liver cancer, melanoma, ovarian cancer, prostate cancer, sarcoma, osteosarcoma, giant cell bone tumor, thyroid cancer, lymphoblast T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairball summer leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma, megakaryoblocal leukemia, acute megakaryotic leukemia, leukemia Hodgkin's lymphoma, non-Hodgkin's lymphomas, T-cell lymphoblastic lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, vulvar cancer, cervical cancer, endometrial cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatic cell cancer, stomach cancer nasopharyngeal cancer, cheek cancer, oral cancer, GIST (gastrointestinal stromal tumor), neuroendocrine cancers, and testicular cancer. 10. The method of claim 9, wherein the mammal is a human. 11. The method according to claim 4, wherein said precancerous condition is selected from intraepithelial cervical neoplasia, monoclonal gammopathy of undetermined etiology (MGUS), myelodysplastic syndrome, aplastic anemia, cervical disease, skin nevi (premelanoma), prostatic intraepithelial non-PIN ), ductal carcinoma in situ (DCIS), colon polyps, and severe hepatitis or cirrhosis. 12. The use of a compound described in any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or reduction of the severity of cancer. 13. A method of inhibiting DNMT1 (DNA methyltransferase) activity in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount of a compound described in any one of claims 1, 2, or a pharmaceutically acceptable salt thereof. 14. The method of claim 13, wherein the mammal is a human. 15. A method of treating cancer in a mammal in need thereof, which comprises administering to such a mammal a therapeutically effective amount: a) a compound described in any one of claims 1, 2, or a pharmaceutically acceptable salt thereof; b) at least one anticancer agent. 16. The method according to claim 15, wherein at least one antitumor agent is selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, transmission inhibitors signals, inhibitors of angiogenesis of non-receptor tyrosine kinase, immunotherapeutic agents, proapoptotic agents, inhibitors of cell cycle signaling, proteasome inhibitors, inhibitors of cancer metabolism, αNu-PD-L1 agents, PD-1 antagonist, immunomodulators, STING-modulating compounds, CD39 inhibitors , TLR4 antagonists, anti-ICOS antibodies, and anti-OX40 antibodies. 17. Pharmaceutical combination containing: a) a compound described in any one of claims 1, 2, or a pharmaceutically acceptable salt thereof; b) at least one anticancer agent. 18. A pharmaceutical combination according to claim 17 for the treatment of malignant neoplasm. 19. Pharmaceutical combination containing: - 39 038355 a) a compound described in any one of claims 1, 2, or a pharmaceutically acceptable salt thereof; b) at least one agent used to treat beta hemoglobinopathies. 20. A pharmaceutical combination according to claim 19 for the treatment of sickle cell disease. 21. A pharmaceutical combination according to claim 19 for the treatment of beta thalassemia. 22. A method of treating a disease selected from diabetic nephropathy, diabetes, podocyte injury, atherosclerosis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, liver cirrhosis, rheumatoid arthritis and Alzheimer's disease in a mammal in need thereof, which comprises administering to such a mammal a therapeutically effective amount of a compound described in any of claims 1, 2, or a pharmaceutically acceptable salt thereof. 23. The method of claim 22, wherein the mammal is a human. 24. A pharmaceutical composition containing from 0.5 to 1000 mg of a compound or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 or 2, and from 0.5 to 1000 mg of a pharmaceutically acceptable excipient.