Substituted pyridines as DNMT1 inhibitors

Abstract

This invention provides a novel compound that inhibits the activity of DNA methyltransferase 1 (DNMT1). [Solution] A compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof is provided. TIFF2026136150000051.tif43160

Description

[Technical Field]

[0001] The present invention relates to substituted pyridine derivatives that are inhibitors of DNA methyltransferase 1 (DNMT1) activity. The present invention also relates to pharmaceutical compositions comprising such compounds, as well as methods of using such compounds for the treatment of cancer, precancerous syndromes, beta-hemoglobin disorders, and other diseases associated with inappropriate DNMT1 activity. [Background technology]

[0002] Epigenetics is a means of turning genes on and off independently of the underlying DNA sequence. DNA methylation occurring at gene promoters is an example of repressive epigenetic marking, which causes chromatin condensation and gene silencing. DNA methylation is mediated by the DNA methyltransferase (DNMT) family, which consists of five family members. Three of the family members, DNMT1, DNMT3A, and DNMT3B, contain DNA methyltransferase activity. These three members respond to the establishment of de novo DNA methylation patterns, but DNMT1 also responds to the maintenance of methylation patterns in daughter strands after DNA replication.

[0003] In cancer, DNA methylation patterns become abnormal, leading to comprehensive hypomethylation and localized hypermethylation within promoter regions. This triggers downstream silencing of tumor suppressor genes (Ting et al., Genes Dev. 2006; 20: pp. 3215-3231). Furthermore, silencing of DNMT1 leads to DNA demethylation, and the re-expression of tumor suppressor genes inhibits tumor growth (Zhou et al., Oncol. Lett. 2014; 5: pp. 2130-2134).

[0004] DNA methylation inhibitors (also referred to as DNA hypomethylating agents) are clinically effective anticancer therapies utilized for the treatment of MDS, AML, and CMML. While these agents are available, there remains significant room for improvement with respect to toxicity, usefulness in solid tumors, and oral bioavailability. Accordingly, novel DNMT inhibitors for treating cancer and / or any disease or condition mediated by DNA methylation may be of interest. Of particular interest in the present invention is specifically targeting DNMT1 to prevent the proliferation of abnormal methylation patterns (such as those occurring in cancer) on daughter strands during replication.

[0005] Hemoglobin disorders, such as sickle cell anemia and beta-thalassemia, represent the most commonly seen hereditary blood disorders worldwide. Sickle cell anemia and beta-thalassemia are characterized by disorders of hemoglobin, a protein complex that transports oxygen in red blood cells. Structurally, hemoglobin typically consists of two pairs of proteins and four heme molecules. Adults and children over about four months express a form of hemoglobin called adult hemoglobin, which consists mainly of two alpha-globin proteins paired with two beta-globin proteins and four heme molecules. However, fetuses and infants typically express fetal hemoglobin, which consists of two alpha-globin proteins paired with two gamma-globin proteins and four heme molecules. It should be 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 highly functionally equivalent, and fetal hemoglobin refers to any combination of a pair of G-gamma and / or A-gamma and a pair of alpha-globin proteins, along with four heme molecules.

[0006] In sickle cell anemia, the gene encoding beta-globin contains mutations that cause abnormal hemoglobin structures, leading to red blood cells taking on a characteristic sickle shape under certain conditions. This sickle shape causes a decrease in the flexibility of red blood cells, an extended transit time through capillaries, and a frequent vascular occlusion process that damages tissues and can cause disease in patients. In contrast, beta-thalassemia is characterized by insufficient production of beta-globin that can combine with normally produced alpha-globin. The resulting accumulation of alpha-globin is toxic to red blood cell precursors, causing ineffective erythropoiesis and extensive red blood cell hemolysis.

[0007] Currently, there are no approved pharmacological treatments to cure sickle cell anemia or beta-thalassemia. However, an increase in the number of red blood cells producing fetal hemoglobin, in combination with an overall increase in fetal hemoglobin levels per red blood cell, has been shown to provide clinical benefit by reducing the frequency of acute vaso-occlusive events in patients with sickle cell anemia and sickle cell disease. Furthermore, although not clinically proven, the disease biology of beta-thalassemia suggests that an increase to high levels of fetal hemoglobin production may also be a viable strategy for the treatment of this disease.

[0008] The objective of this therapeutic approach, which desuppresses the silenced HBG1 and HBG2 genes, is to target erythropoiesis through intervention in epigenetic processes. Changes in DNA methylation are crucial determinants in hematopoiesis, representing differentiation milestones that involve various cell lineages. During erythropoiesis, a rapid decrease in comprehensive DNA methylation defines points involved in the expression of erythroid-specific regulators GATA1 and KLF1, as well as the suppression of hematopoietic precursor regulators GATA2 and PU.1 (1, 2). In erythroid progenitor cells in adult bone marrow, DNA in the promoter region of the beta-globin (HBB) gene becomes unmethylated in response to high levels of beta-globin protein expression. In contrast, the promoters of the HBG1 and HBG2 loci are highly methylated, leading to a significant decrease in gamma-globin protein expression (3). DNA methyltransferases DNMT1, DNMT3A, and DNMT3B are expressed in erythroid precursors, and the relatively high expression of DNMT1, particularly in the final stage of erythroid differentiation, suggests that it plays a major role in regulating globin genes (2). 5-azacitidine and 5-aza-2'-deoxycytidine (decitabine) are known whole-DNMT inhibitors that induce fetal hemoglobin in erythroid progenitor cells. In erythroid cell cultures and in vivo models of fetal hemoglobin induction (4, 5), treatment with these agonists corresponds to increased gammaglobin protein expression and decreased methylation of the CpG site in the HBG promoter. Furthermore, in a limited set of clinical studies, both agonists induced increased fetal hemoglobin in patients with sickle cell anemia, sickle cell disease, and beta-thalassemia (6-9). While these agents are effective in inducing fetal hemoglobin, they are not widely used to treat sickle cell anemia, sickle cell disease, or beta-thalassemia due to concerns about long-term safety, dose-limiting toxicity, and unsuitable routes of administration. [Prior art documents] [Non-patent literature]

[0009]

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[0010] The object of the present invention is to provide a novel compound that is an inhibitor of DNMT1. [Means for solving the problem]

[0011] This invention focuses on novel compounds.

[0012] Specifically, the present invention relates to formula (I)

[0013] [ka] The subject is the compound and its prodrug, as well as its salts.

[0014] The present invention further relates to pharmaceutical compositions comprising a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0015] The present invention further relates to a method for treating a disease associated with inadequate DNMT1 activity, comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0016] The present invention further relates to compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, for use in drug therapy.

[0017] The present invention further relates to compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, for use in treating diseases associated with inappropriate DNMT1 activity.

[0018] The present invention further relates to the use of a compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, in the manufacture of a pharmaceutical for use in treating diseases associated with inadequate DNMT1 activity.

[0019] The present invention further covers combinations comprising a compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents. [Brief explanation of the drawing]

[0020] [Figure 1] This is an X-ray powder diffraction analysis of the glycinate salt of the compound from Example 1. [Figure 2] This is a differential scanning calorimetry measurement of the glycinate salt of the compound from Example 1. [Figure 3] This is a differential scanning calorimetry measurement of the glycinate salt of the compound from Example 1. [Figure 4] This is an X-ray powder diffraction pattern of the glycinate salt monohydrate of the compound of Example 1. [Figure 5] This is a differential scanning calorimetry measurement of the glycinate monohydrate of the compound from Example 1. [Figure 6] This is a thermogravimetric analysis of the glycinate monohydrate of the compound from Example 1. [Figure 7] This is an X-ray powder diffraction pattern of compound example 1. [Figure 8] This is a differential scanning calorimetry measurement of the compound in Example 1. [Figure 9] This is the thermogravimetric analysis of the compound in Example 1. [Modes for carrying out the invention]

[0021] In one embodiment, the present invention is based on formula (I)

[0022] [ka]

[0023] The present invention applies to compounds and their prodrugs, and their salts (hereinafter referred to as "compounds of the present invention").

[0024] The compounds of the present invention contain at least one chiral center and therefore may exist as individual enantiomers, diastereomers, or other stereoisomers, or as mixtures thereof. A chiral center, such as a chiral carbon atom, may also be present in substituents, such as alkyl groups. If the stereochemistry of a chiral center present in a compound of the present invention or in any of the chemical structures illustrated herein is not specified, the structure is intended to encompass all stereoisomers and mixtures thereof. Accordingly, the compounds of the present invention may be used as racemic mixtures, enantiomerically enriched mixtures, or enantiomerically pure individual stereoisomers.

[0025] Therefore, in one embodiment, the compound of the present invention is of formula (II)

[0026] [ka] It is a compound or prodrug thereof, or a salt thereof.

[0027] In another embodiment, the compound of the present invention is of formula (II)

[0028] [ka] It is a compound or a prodrug thereof.

[0029] In further embodiments, the compound of the present invention is of formula (III)

[0030] [ka] It is a compound or prodrug thereof, or a salt thereof.

[0031] The individual stereoisomers of the compounds of the present invention can be separated by methods known to those skilled in the art. For example, such separation can be carried out (1) by the formation of diastereoisomer salts, complexes, or other derivatives; (2) by selective reaction with a stereoisomer-specific reagent, for example, by enzymatic oxidation or reduction; or (3) by gas-liquid or liquid chromatography in a chiral environment, for example, on a chiral support, for example, on silica having a bound chiral ligand, or in the presence of a chiral solvent. Those skilled in the art will recognize that if a desired stereoisomer is converted to another chemical entity by one of the separation procedures described above, further steps may be required to liberate the desired form. Alternatively, specific stereoisomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by asymmetric transformation, converting one enantiomer to another.

[0032] The compounds of the present invention may also contain geometrically asymmetric centers. Unless the stereochemistry of a geometrically asymmetric center present in a compound of the present invention or any of the chemical structures illustrated herein is specified, the structure is intended to encompass all trans-geometric isomers, cis-geometric isomers, and mixtures thereof. Similarly, all tautomers are also included, whether such tautomers exist in equilibrium or one form is predominant.

[0033] The compounds of the present invention may be administered as prodrugs. As used herein, a “prodrug” of the compound of formula (I) is a functional derivative of the compound that, when administered to a patient, ultimately releases the compound of formula (I) in vivo. Administration of the compound of formula (I) as a prodrug may enable those skilled in the art to perform one or more of the following: (a) alteration of the compound’s in vivo solubility; (b) alteration of the compound’s in vivo activity; (c) alteration of the compound’s in vivo duration of action; (d) alteration of the compound’s in vivo transport or distribution; and (e) overcoming side effects or other difficulties the compound faces. Typical functional derivatives used to prepare prodrugs include modifications of compounds that can be chemically or enzymatically cleaved in vivo. Such modifications, including the preparation of phosphates, amides, esters, thioesters, carbonates, and carbamates, are well known to those skilled in the art. In one embodiment, the prodrug portion is -P(O)(OH)2.

[0034] Therefore, in one embodiment, the compound of the present invention is of formula (IV)

[0035] [ka] It is a prodrug or a salt thereof.

[0036] In another embodiment, the compound of the present invention is of formula (V)

[0037] [ka] It is a prodrug or a salt thereof.

[0038] In another embodiment, the compound of the present invention is of formula (V)

[0039] [ka] It is a prodrug.

[0040] In further embodiments, the compound of the present invention is of formula (VI)

[0041] [ka] It is a prodrug or a salt thereof.

[0042] References herein to the compound of formula (I) and its prodrug, and its salts, should be understood to cover the compound of formula (I) and its prodrug as a free acid or free base, or as a salt thereof, for example, a pharmaceutically acceptable salt thereof. Accordingly, in one embodiment, the present invention covers the compound of formula (I) or its prodrug as a free acid or free base. In another embodiment, the present invention covers the compound of formula (I) or its prodrug, or a salt thereof. In a further embodiment, the present invention covers the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof.

[0043] Those skilled in the art will recognize that pharmaceutically acceptable salts of the compound according to formula (I), or prodrugs thereof, can be prepared. In fact, in some embodiments of the present invention, pharmaceutically acceptable salts of the compound according to formula (I), or prodrugs thereof, may be preferred over the respective free bases or free acids because such salts can impart greater stability or solubility to the molecules, thereby facilitating formulation into dosage forms.

[0044] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the compound in question and exhibits minimal undesirable toxicological effects. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound with its free acid or free base form, or with a pharmaceutically acceptable salt, with a suitable base or acid, respectively.

[0045] Salts and solvates having pharmaceutically unacceptable counterions or associated solvents are within the scope of the present invention when used, for example, as intermediates in the preparation of the compound of formula (I) or its prodrug, and its pharmaceutically acceptable salt. Accordingly, one embodiment of the present invention includes the compound of formula (I) and its prodrug, and its salt.

[0046] Pharmaceutically acceptable salts include those listed in particular Berge, J. Pharm. Sci., 1977, 66, pp. 1-19, or those listed in P.H. Stahl and C.G. Wermuth (eds.), Handbook of Pharmaceutical Salts; Properties, Selection and Use, 2nd edition, Stahl / Wermuth: Wiley-VCH / VHCA, 2011 (see http: / / www.wiley.com / WileyCDA / WileyTitle / productCd-3906390519.html).

[0047] In some embodiments, the compound according to formula (I) or its prodrug may contain an acidic functional group. Suitable pharmaceutically acceptable salts include salts of such acidic functional groups. Typical salts include aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris,tromethamine), arginine, benetamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, cremisole (1-p-chlorobenzyl-2-pyrrolildine-1'-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, and ethanolamine. This includes, but is not limited to, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, L-lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t-butylamine, betaine (tri-methylglycine), L-proline, L-phenylalanine, L-alanine, L-tyrosine, L-leucine, imidazole, glycine, L-valine, L-serine, morpholine, tricholine, diethylenetriamine (diethyenetriamine), 1-(2-hydroxyethyl)-2-pyrrolidine, and zinc.

[0048] Such base addition salts can be formed by the reaction of a compound of formula (I) or its prodrug (e.g., containing a carboxylic acid or other acidic functional group) with a suitable base in an optionally suitable solvent, such as an organic solvent, to obtain a salt, which can then be isolated by a variety of methods, including crystallization and filtration.

[0049] If the compound of formula (I) or its prodrug contains two or more base moieties, it is understood that the stoichiometry of salt formation may contain one or more equivalents of acid. Such salts may contain one or more acid counterions, such as dihydrochloride salts.

[0050] pharmaceutically acceptable salts of the compound of formula (I) or its prodrug in stoichiometric and non-stoichiometric forms are included within the scope of the present invention, including, for example, quasi-stoichiometric salts containing an acidic proton with more than one counterion.

[0051] In some embodiments, the compound according to formula (I) or its prodrug may contain a basic functional group, and therefore, a pharmaceutically acceptable acid addition salt can be formed by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic acids and pharmaceutically acceptable organic acids. Typical pharmaceutically acceptable acid addition salts include 4-acetamidebenzoate, acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate (besilate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphor sulfonate (cansilate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecyl sulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), Formate, fumarate, galactarate (mucinate), gentisinate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate, hydravamin (N,N'-di(dehydroabiethyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucinate, naphthalene-1,This includes, but is not limited to, 5-disulfonates (napadisylates), naphthalene-2-sulfonates (napsylates), nicotinates, nitrates, oleates, palmitates, p-aminobenzenesulfonates, p-aminosalicylates, pamoates (embonates), pantothenates, pectinates, persulfates, phenylacetates, phenylethylbarbiturates, phosphates, polygalacturonates, propionates, p-toluenesulfonates (tosylate), pyroglutamates, pyruvates, salicylates, sebacinates, stearates, basic acetates, succinates, sulfamates, sulfates, tannates, tartrates, theoclates (8-chlorotheophylline), thiocyanates, triethiodides, undecanoates, undecylenates, and valerates.

[0052] Such acid addition salts can be formed by the reaction of a compound of formula (I) or its prodrug (e.g., containing a basic amine or other basic functional group) with a suitable acid in an optionally suitable solvent, such as an organic solvent, to obtain a salt, which can then be isolated by a variety of methods, including crystallization and filtration.

[0053] Salts can be prepared in situ during the final isolation and purification of the compound of formula (I) or its prodrug. When a basic compound of formula (I) or its prodrug is isolated as a salt, the corresponding free base form of the compound can be prepared by any suitable method known in the art, including treatment of the salt with an inorganic or organic base. Similarly, when a compound of formula (I) or its prodrug containing a carboxylic acid or other acidic functional group is isolated as a salt, the corresponding free acid form of the compound can be prepared by any suitable method known in the art, including treatment of the salt with an inorganic or organic acid.

[0054] The compounds of formula (I) and their prodrugs, as well as all optical isomers, stereoisomers, polymorphs, and radiolabeled derivatives of their salts, are included within the scope of "compounds of the present invention."

[0055] The compounds of the present invention may exist in solid or liquid form. In the solid state, the compounds of the present invention may exist in crystalline or amorphous form, or as a mixture thereof. In the crystalline form of the compounds of the present invention, those skilled in the art will recognize that pharmaceutically acceptable solvates may be formed in which solvent molecules are incorporated into the crystalline lattice during crystallization. The compounds of the present invention may exist in solvated and non-solvated forms. Solvates may be accompanied by a non-aqueous solvent, such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ¼, or these may be accompanied by water as the solvent incorporated into the crystalline lattice. Solvates in which water is the solvent incorporated into the crystalline lattice are typically called “hydrates.” Hydrates include stoichiometric hydrates and compositions containing a variable amount of water.

[0056] Those skilled in the art will further recognize that certain compounds of the present invention, existing in crystalline form, including various solvates thereof, may exhibit polymorphism (i.e., the ability to arise as different crystalline structures). These different crystalline forms are typically known as “polymorphs.” The present invention encompasses all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometric arrangement, and other descriptive properties of the crystalline solid state. Polymorphs may therefore have different physical properties, such as shape, density, hardness, deformability, stability, and solubility. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which can be used for identification. Those skilled in the art will recognize that different polymorphs can be produced, for example, by changing or adjusting the reaction conditions or reagents used to make the compound. For example, changes in temperature, pressure, or solvent can produce polymorphs. Furthermore, one polymorph may spontaneously transform into another under certain conditions.

[0057] The present invention also includes isotope-labeled compounds that are identical to the compounds of the present invention, except that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that may be incorporated into the compounds of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, for example.2 H, 3 H, 11 C, 14 C and 18 Includes F.

[0058] "Enantiomerically enriched" refers to products where the enantiomer excess is greater than zero. For example, enantiomerically enriched products are those with enantiomer excesses greater than 50%ee, 75%ee, and 90%ee.

[0059] "Enantiomer excess" or "ee" is the excess of one enantiomer to the other, expressed as a percentage. As a result, both enantiomers are present in equal amounts in the racemic mixture, so the enantiomer excess is zero (0%ee). However, if one enantiomer is concentrated and consequently constitutes 95% of the product, the enantiomer excess can be 90%ee (95% of the concentrated enantiomer minus 5% of the other enantiomer). In some embodiments, the compounds of the present invention may have at least 50%ee, at least 60%ee, at least 65%ee, at least 70%ee, at least 75%ee, at least 80%ee, at least 85%ee, at least 90%ee, at least 95%ee, at least 96%ee, at least 97%ee, at least 98%ee, or at least 99%ee with respect to the R or S enantiomer.

[0060] "Enantiomerically pure" refers to a product with an enantiomer excess of 99%ee or higher.

[0061] "Pharmacologically acceptable" means a compound, salt, material, composition, or dosage form that is within the bounds of medical common sense, free from excessive toxicity, irritation, or other problems or complications, and that is suitable for use in contact with human and animal tissues, with a reasonable benefit-to-risk ratio.

[0062] The compounds of the present invention are useful as selective DNMT1 inhibitors in mammals that require them, particularly humans. Compounds that are DNMT1 inhibitors may be useful in treating diseases in which the underlying pathology is (at least partially) caused by inappropriate DNMT1 activity, such as cancer. "Inappropriate DNMT1 activity" refers to any DNMT1 activity that deviates from the normal DNMT1 activity expected in a particular patient. Inappropriate DNMT1 can take the form of, for example, an abnormal increase in activity, or abnormalities in the timing and / or control of DNMT1 activity. Therefore, in another embodiment, the present invention relates to methods for treating such diseases.

[0063] In some aspects of the present invention, compounds of formula (I) and their prodrugs, as well as salts thereof, are potent inhibitors of DNMT1 and are more selective to DNMT1 than DNMT3A and DNMT3B.

[0064] Such conditions include cancer, precancerous syndromes (sometimes called precancerous conditions), or beta-hemoglobin disorders. A precancerous condition is a condition or lesion involving abnormal cells that have a high risk of developing into cancer. Clinically, precancerous conditions encompass a wide range of conditions or lesions that have a high risk of developing into cancer.

[0065] Cancers that can be treated include adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, carcinosarcoma, melanoma, adrenal carcinoma, adrenocortical carcinoma, pheochromocytoma, breast cancer, ductal carcinoma in situ, lobular carcinoma, inflammatory breast cancer, invasive ductal carcinoma, papillary Paget's disease, papillary breast cancer, medullary carcinoma, breast cancer (mammary carcinoma), anal cancer, cloacal carcinoma, anorectal melanoma, appendiceal cancer, appendiceal neuroendocrine neoplasia, appendiceal mucocystadenocarcinoma, colonic appendiceal adenocarcinoma, signet ring cell adenocarcinoma, goblet cell carcinoma / adenoneuroendocrine carcinoma (adenoneuroendocrine carcinomas), cholangiocarcinoma, intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma, distal extrahepatic cholangiocarcinoma cholangiocarcinoma), colorectal cancer (CRC), mucinous carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, esophageal cancer, small cell carcinoma, leiomyoma, gallbladder cancer, non-papillary adenocarcinoma, breast Capitate adenocarcinoma, gastric cancer, gastric adenocarcinoma, liver cancer, hepatocellular carcinoma, fibrolamellar carcinoma, angiosarcoma, lymphangiosarcoma, angiosarcoma, hepatoblastoma, pancreatic cancer, ductal carcinoma, acinar adenocarcinoma, acinar cell carcinoma, colloid carcinoma, giant cell tumor, hepatoid adenocarcinoma, mucinous cystic neoplasm (mucinous) cystic neoplasms), pancreatoblastoma, serous cystadenoma, intraductal papillary mucinous neoplasms neoplasm, pancreatic neuroendocrine tumors, gastrinoma, insulinoma, glucagonoma, VIPoma, somatostatinoma, PPoma, small intestine cancer, eye cancer, intraocular melanoma, intraocular lymphoma, intraocular retinoblastoma, conjunctival melanoma, eyelid cancer, sebaceous carcinoma, lacrimal gland tumor, malignant mixed epithelial tumor, adenoid cystic carcinoma, bladder cancer, urothelial carcinoma, kidney cancer, renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobic RCC, collecting duct RCC, multilocular cystic RCC, renal mucinous tubular and spindle cell Cancer, tubular cystic RCC, thyroid-like follicular RCC, acquired cystic kidney disease-associated RCC, t(6;11) translocation RCC (TFEB), hybrid oncocytoma / chromophobic RCC, Wilms' tumor, penile cancer, prostate cancer, castration-resistant prostate cancer, transitional cell carcinoma, testicular cancer, seminomas, classical seminoma, spermatogenic seminoma, non-seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, Leydig cell tumor, Sertoli cell tumor, reticular carcinoma, urethral cancer, extracranial germ cell tumor, germ cell tumor,Gonadal buds, mixed germ cell tumors, gonadal ectodermal tumors, endoderm cavitary tumors, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, epithelial cancer, undifferentiated germ cell tumors, sex cord-stromal tumors, gestational villonoma, primary peritoneal cancer, uterine sarcoma, papillary serous uterine carcinoma, vaginal cancer, leukocyte adenocarcinoma, vulvar cancer, verrucous cancer, head and neck cancer, head and neck squamous cell carcinoma, pharyngeal cancer, hypopharyngeal cancer, hyperpharyngeal cancer, middle pharyngeal cancer, non-keratinizing squamous cell carcinoma, undifferentiated carcinoma, laryngeal cancer, oral cavity cancer, mouth cancer Cancer), mucoepidermal carcinoma, paranasal cavity and nasal cavity carcinoma, sensory nerve lesion, salivary gland carcinoma, epithelial fascia carcinoma, parathyroid carcinoma, thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, hydatid cell carcinoma, pyloric thyroid carcinoma, undifferentiated thyroid carcinoma, paraneuropathy, carotid paraneuropathy, tympanic paraneuropathy (jugulotympanic).Paraganglioma, paraganglioma of the vagus nerve, leukemia, acute lymphoblastic leukemia, T-lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia, acute myeloid leukemia (AML), acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, erythroleukemia, chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell prelymphoblastic leukemia, T-cell prelymphoblastic leukemia, macrogranulocyte lymphocytic leukemia T-cell macrogranular lymphocytic leukemia, NK-cell granular lymphocytic leukemia, hairy cell leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, plasma cell leukemia, lymphoma, Hodgkin lymphoma, classical Hodgkin lymphoma, nodular sclerosis classical Hodgkin lymphoma, mixed cell classical Hodgkin lymphoma, lymphocyte-rich classical Hodgkin lymphoma, lymphopenic classical Hodgkin lymphoma, nodular lymphocyte-dominant Hodgkin lymphoma Lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, primary exudative lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, lymphoplasmacytic lymphoma, lymphoblastic lymphoma, small lymphocytic lymphoma, double-hit / triple-hit lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, small non-incisional nuclear cell lymphoma, follicular lymphoma, follicular large cell lymphoma, immunoblastic lymphoma, Intravascular large cell lymphoma, primary splenic lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, marginal zone lymphoma (MZL), extranodal MZL, nodal MZL, splenic MZL, splenic MZL with hairy lymphocytes, peripheral T-cell lymphoma, vascular immunity Blastic T-cell lymphoma, adult T-cell lymphoma / leukemia, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, T-cell non-Hodgkin's lymphoma NOS (T-cell) non-Hodgkin's lymphoma not otherwise(specified), gamma / delta T-cell lymphoma, mucosal-associated lymphoid tissue lymphoma, post-transplant lymphoproliferative disorder, HIV-associated lymphoma, Langerhans cell histiocytosis, multiple myeloma, smoldering multiple myeloma, active multiple myeloma, plasmacytoma, solitary plasmacytoma of bone, extramedullary plasmacytoma, primary amyloidosis, myelodysplastic syndrome (MDS), refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with blast plaque, refractory anemia with blast plaque during transformation, myeloproliferative neoplasm, polycythemia vera, Essential thrombocythemia, myelofibrosis, systemic mastocytosis, bone cancer, Ewing's sarcoma, osteosarcoma, intramedullary osteosarcoma, paraosteal osteosarcoma, extraosseous osteosarcoma, malignant fibrous histiocytoma of bone, chordoma, classic chordoma, chondrosarcoma, dedifferentiated chordoma, chondrosarcoma, classic chondrosarcoma, clear cell chondrosarcoma, myxoid chondrosarcoma, mesenchymal chondrosarcoma, dedifferentiated chondrosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, staphylosid rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, soft tissue sarcoma, extraosseus sarcoma Sarcoma, dermatofibrosarcoma protuberans, epithelioid sarcoma, Kaposi's sarcoma, liposarcoma, malignant peripheral nerve schwannoma, fibrosarcoma, myxosarcoma, synoviomas, brain cancer, undifferentiated astrocytoma, glioblastoma, glioblastoma multiforme, meningioma, pituitary cancer, Schwann cell tumor, oligodendroglioma, ependymoma, medulloblastoma, astrocytoma, brainstem glioma, atypical teratomatoid / rhabdoid tumor, pineal gland tumor, neuroblastoma, This includes CNS lymphoma, primitive neuroectodermal tumors, diffuse endogenous pontine glioma, lung cancer, non-small cell lung cancer (NSCLC), undifferentiated NSCLC, small cell lung cancer, pleuroblastoma, bronchogenic carcinoma, malignant mesothelioma, malignant pleural mesothelioma, malignant peritoneal mesothelioma, thymoma, thymic carcinoma, skin cancer, keratoacanthoma, sebaceous gland carcinoma, sweat gland carcinoma, apocrine carcinoma, eccrine gland carcinoma, clear cell eccrine carcinoma, Merkel cell carcinoma, cutaneous T-cell lymphoma, mycosis fungoides, Sézary syndrome, chondrosyringoma, HPV-related cancer, tumors containing transformed cells, tumors containing cells in a precancerous state, precancerous hyperplasia, precancerous metaplasia, precancerous dysplasia, carcinoma in situ, mixed tumors, malignant mixed tumors, and complex cancers.

[0066] Cancers that can be treated include cancers with high tumor mutational load (TMB), cancers exhibiting DNA mismatch repair deficiency (dMMR), cancers exhibiting high-frequency microsatellite instability (MSI-H), cancers exhibiting low-frequency microsatellite instability (MSI-L), cancers exhibiting increased microsatellite alteration (EMAST) at selected tetranucleotide repeats, microsatellite stability (MSS), cancers containing mutations in polymerase delta (POLD), cancers containing mutations in polymerase epsilon (POLE), or cancers with homologous recombination repair deficiency (HRD).

[0067] Cancers that can be treated further include breast cancers defined by expression profiling (triple-negative breast cancer, HER2-positive breast cancer, luminal type A breast cancer, luminal type B breast cancer, normal-like breast cancer) or breast cancers with BRCA1 or BRCA2 mutations.

[0068] In one embodiment of the present invention, the cancer treated is myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), colorectal cancer (CRC), non-Hodgkin lymphoma (NHL), melanoma, or breast cancer. In another embodiment of the present invention, the cancer is acute myeloid leukemia (AML). In a further embodiment of the present invention, the cancer is colorectal cancer (CRC).

[0069] The treatment method of the present invention comprises the step of administering a safe and effective amount of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, to a patient in need. Individual embodiments of the present invention include a method of treating any one of the disorders mentioned above by administering a safe and effective amount of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0070] As used herein, “to treat” means (1) to relieve or prevent one or more of the disorder or one or more of its biological signs; (2) to (a) inhibit one or more of the biological cascade points that cause or respond to the disorder, or (b) inhibit one or more of the biological signs; (3) to alleviate one or more of the symptoms or effects associated with the disorder; or (4) to slow the progression of the disorder or one or more of the biological signs of the disorder.

[0071] As used herein, “safe and effective amount” means an amount of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, or other pharmaceutically active agent, that is sufficient to treat the patient’s condition, but within the bounds of medical common sense, in a small enough (reasonable benefit / risk ratio) to avoid serious side effects. The safe and effective amount of the compound will vary depending on the specific compound selected (e.g., considering potency, efficacy and half-life of the compound); the route of administration selected; the disorder being treated; the severity of the disorder being treated; the age, size, weight, and physical condition of the patient being treated; the patient’s medical history; the duration of treatment; the nature of concurrent treatment; the desired therapeutic effect; and similar factors, but can still be routinely determined by those skilled in the art.

[0072] As used herein, “patient” refers to a human being (including adults and children) or another animal. In one embodiment, “patient” refers to a human being.

[0073] The present invention therefore relates to a method for treating a disease associated with inadequate DNMT1 activity, comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0074] In one embodiment, the present invention provides a method for treating cancer, a precancerous syndrome, or a beta-hemoglobin disorder, comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0075] In another embodiment, the present invention provides a method for treating cancer, a precancerous syndrome, or a beta-hemoglobin disorder, comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0076] In another embodiment, the present invention provides a method for treating myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), colorectal cancer (CRC), non-Hodgkin lymphoma (NHL), melanoma, or breast cancer, comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0077] In another embodiment, the present invention provides a method for treating acute myeloid leukemia (AML), comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0078] In another embodiment, the present invention provides a method for treating colorectal cancer (CRC), comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need.

[0079] In further embodiments, the present invention provides a method for treating sickle cell disease, sickle cell anemia, or beta-thalassemia, comprising the step of administering a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need. Sickle cell anemia is a single specific disease characterized by a homozygous E6V mutation in both alleles of the betaglobin gene. In contrast, sickle cell disease is a collection of several related diseases, all of which present similar symptoms of varying severity. Patients with sickle cell disease have one allele of the betaglobin gene with an E6V mutation (as in sickle cell anemia), and the allele of the second betaglobin gene may have any number of mutations, among others, that cause beta-thalassemia. The most common signs of sickle cell disease are called "sickle beta-zero" and "sickle beta-plus," but others exist as well. While mutations in the second betaglobin allele are not entirely absent in patients with sickle cell disease (the presence of an E6V betaglobin allele in addition to one normal betaglobin allele is a known characteristic of sickle cell anemia, and although not harmless overall, it is generally not treated), it should be noted that the mutation in the second allele is simply not an E6V mutation. It should also be noted that the characteristics of sickle cell anemia are not considered indicative of sickle cell disease.

[0080] The present invention further relates to compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, for use in drug therapy.

[0081] The present invention further relates to compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, for use in treating diseases associated with inappropriate DNMT1 activity.

[0082] The present invention further relates to the use of a compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, in the manufacture of a pharmaceutical for use in treating diseases associated with inadequate DNMT1 activity.

[0083] Compounds of formula (I) and their prodrugs, as well as pharmaceutically acceptable salts thereof, are not essential but are usually incorporated into pharmaceutical compositions before administration to the patient.

[0084] Accordingly, in one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0085] In another embodiment, the present invention relates to a pharmaceutical composition comprising 0.5 to 3500 mg of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, and 0.1 to 2 g of one or more pharmaceutically acceptable excipients.

[0086] In a further embodiment, the present invention relates to a pharmaceutical composition for treating diseases mediated by inadequate DNMT1 activity, comprising a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof.

[0087] Compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, may be administered by any preferred route of administration, including both systemic and topical administration. Systemic administration includes oral, parenteral, transdermal, and rectal administration. Parenteral administration refers to routes of administration other than enteral or transdermal, typically by infusion or injection. Parenteral administration includes intravenous, intramuscular, and subcutaneous infusion or injection. Topical administration includes application to the skin, as well as intraocular, ear, and vaginal, inhalation, and intranasal administration. Inhalation refers to administration into the patient's lungs, whether through the oral cavity or via a nasal route. In one embodiment, compound (I) or its prodrug, or pharmaceutically acceptable salts thereof, may be administered orally.

[0088] In some embodiments, a prodrug of a compound of formula (I) may be particularly suitable for oral administration due to improved solubility, which leads to increased oral bioavailability.

[0089] The compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, may be administered in single or multiple doses at various time intervals over a predetermined period of time. For example, the dose may be administered once, twice, three times, four times, five times, or six times per day. The dose may be administered until the desired therapeutic effect is achieved or to maintain the desired therapeutic effect indefinitely. The preferred dosage of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, depends on the pharmacokinetic properties of the compound, such as absorption, distribution, and half-life, which can be determined by those skilled in the art. Furthermore, the preferred dosage of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, including the duration for which such a regimen is performed, depends on the disorder being treated, the severity of the disease being treated, the age and physical condition of the patient being treated, the patient's medical history, the nature of concurrent treatment, the desired therapeutic effect, and similar factors within the knowledge and expertise of those skilled in the art. It will be further understood by those skilled in the art that the preferred method of use may need to be adjusted in consideration of the individual patient's response to the method of use, or over time as the individual patient requires change.

[0090] The dose of the invented pharmaceutically active compound in the drug dosage units described above is an effective non-toxic dose, preferably selected from the range of the active compound from 0.001 to 500 mg / kg, preferably from 0.01 to 100 mg / kg. When treating human patients requiring a DNMT1 inhibitor, the selected dose is preferably administered orally or parenterally, 1 to 6 times per day. Preferred forms of parenteral administration include topical administration, rectal administration, transdermal administration, administration by injection, and continuous administration by infusion. Oral dosage units for human administration preferably contain 0.5 to 3500 mg of the active compound. Preferably, oral dosage units for human administration preferably contain 0.5 to 1000 mg of the active compound. Oral administration using lower doses is preferred. However, parenteral administration at higher doses may also be used if it is safe and convenient for the patient.

[0091] Compounds of formula (I) and pharmaceutically acceptable salts thereof are usually incorporated into pharmaceutical compositions before administration to the patient, although this is not essential.

[0092] Accordingly, in one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0093] In another embodiment, the present invention relates to a pharmaceutical composition comprising 0.05 to 1000 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and 0.1 to 2 g of one or more pharmaceutically acceptable excipients.

[0094] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, for treating or preventing disorders mediated by inadequate DNMT1 activity.

[0095] The pharmaceutical compositions of the present invention may be prepared and packaged in bulk form, for example, as a powder or syrup, after a safe and effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof has been extracted. Alternatively, the pharmaceutical compositions of the present invention may be prepared and packaged in unit dosage forms, each of which is physically separate and contains the compound of formula (I) or a pharmaceutically acceptable salt thereof. When prepared in unit dosage forms, the pharmaceutical compositions of the present invention may typically contain, for example, 0.5 to 1,000 mg, or 1 mg to 700 mg, or 5 mg to 100 mg of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof.

[0096] The pharmaceutical compositions of the present invention typically contain a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof.

[0097] As used herein, “pharmaceutically acceptable excipients” means pharmaceutically acceptable materials, compositions, or vehicles that contribute to imparting form or consistency to a pharmaceutical composition. Each excipient must be compatible with the other components of the pharmaceutical composition when mixed, so as to avoid interactions that, when administered to a patient, could substantially reduce the efficacy of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, or interactions that could result in a pharmaceutically unacceptable pharmaceutical composition. Furthermore, each excipient must, of course, be pharmaceutically acceptable, for example, of sufficiently high purity.

[0098] Compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients(s) are typically formulated into dosage forms suitable for administration to a patient via a preferred route of administration. For example, dosage forms include (1) those suitable for oral administration, such as tablets, capsules, caplets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets and cachets; (2) those suitable for parenteral administration, such as sterile solutions for reconstitution, suspensions and powders; (3) those suitable for transdermal administration, such as transdermal patches; (4) those suitable for rectal administration, such as suppositories; (5) those suitable for inhalation, such as aerosols, solutions and dry powders; and (6) those suitable for topical administration, such as creams, ointments, lotions, solutions, pastes, sprays, foams and gels.

[0099] Suitable pharmaceutically acceptable excipients vary depending on the specific dosage form selected. Furthermore, suitable pharmaceutically acceptable excipients may be selected for the specific function they can perform in the composition. For example, a pharmaceutically acceptable excipient may be selected for its ability to promote the formation of a uniform dosage form. A pharmaceutically acceptable excipient may be selected for its ability to promote the formation of a stable dosage form. A pharmaceutically acceptable excipient may be selected for its ability, once administered to a patient, to promote the retention or transport of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, from one organ or part of the body to another. A pharmaceutically acceptable excipient may be selected for its ability to improve medication adherence.

[0100] Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, flow enhancers, granulators, coatings, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anticaking agents, hygroscopic agents (hemectants), chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Those skilled in the art will recognize that a pharmaceutically acceptable excipient may perform more than one function, and that depending on the amount of excipient present in the formulation and which other excipients are present, it may perform alternative functions.

[0101] Those skilled in the art will possess the knowledge and skills of the art to select suitable, pharmaceutically acceptable excipients in appropriate amounts for use in the present invention. Furthermore, there are several resources available to those skilled in the art that describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (American Pharmaceutical Association and the Pharmaceutical Press).

[0102] The pharmaceutical compositions of the present invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

[0103] Accordingly, in another embodiment, the present invention relates to a method for preparing a pharmaceutical composition comprising a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, the method comprising the step of mixing the raw materials. A pharmaceutical composition comprising a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, can be prepared, for example, by mixing at ambient temperature and atmospheric pressure.

[0104] In one embodiment, the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, is formulated for oral administration. In a further embodiment, the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, is formulated for parenteral administration.

[0105] In one embodiment, the present invention relates to a solid oral dosage form, such as a tablet or capsule, comprising a safe and effective amount of a compound of formula (I) or a prodrug thereof, or a pharmaceutically acceptable salt thereof, and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g., corn starch, potato starch, and pregelatinized starch), cellulose and its derivatives (e.g., microcrystalline cellulose), calcium sulfate, and calcium hydrogen phosphate. The oral solid dosage form may further include a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pregelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose, and their derivatives (e.g., microcrystalline cellulose). The oral solid dosage form may further include a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethylcellulose. The oral solid dosage form may further contain a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

[0106] Where appropriate, formulations of oral dosage units may be microencapsulated. Compositions may also be prepared to extend or prolong release by, for example, coating or embedding particulate materials with polymers, waxes, etc.

[0107] Compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, may also be combined with soluble polymers as targetable drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacrylamide-phenol, polyhydroxyethyl aspartamidophenol, or polyethylene oxide polylysine substituted with palmitoyl residues. Furthermore, compounds of formula (I) or their prodrugs, or pharmaceutically acceptable salts thereof, may be combined with certain classes of biodegradable polymers useful for achieving controlled drug release, such as crosslinked or amphiphilic block copolymers of polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogels.

[0108] In another embodiment, the present invention relates to liquid oral dosage forms. Oral liquids, such as liquids, syrups, and elixirs, can be prepared in dosage unit form such that a predetermined amount contains a predetermined amount of the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof. Syrups can be prepared by dissolving the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, in a suitably flavored aqueous solution, while elixirs can be prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, in a non-toxic vehicle. Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservatives, flavoring additives, such as peppermint oil, or natural sweeteners, saccharin, or other artificial sweeteners may also be added.

[0109] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient organism, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Compositions may be presented in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0110] A compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, may be administered concurrently with one or more other activators. Therefore, in one embodiment, the present invention provides a combination comprising a compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, and one or more other activators. In further embodiments, the other activators are known to be useful in the treatment of cancer or precancerous syndromes.

[0111] As used herein, the term “simultaneous administration” means the simultaneous administration of the DMNT1 activity inhibitors described herein, as well as any further activators or agents known to be useful in the treatment of cancer, including chemotherapy and radiotherapy, or the individual sequential administration of any of these means. As used herein, any further terms “active ingredient,” “active ingredients,” “active agent,” or “active agents” include any compound or therapeutic agent known to be, or demonstrably, beneficial when administered to a patient. Preferably, if administration is not simultaneous, the compounds are administered at close intervals. Furthermore, this is not problematic even if the compounds are administered in the same dosage form; for example, one compound may be administered by injection and another by oral administration.

[0112] Typically, any antineoplastic agent active against tumors sensitive to treatment can be administered concurrently with the treatment of cancer in the present invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology, VT Devita, TS Lawrence and SA Rosenberg (eds.), 10th edition (December 5, 2014), Lippincott Williams & Wilkins Publishers. Those skilled in the art will be able to identify combinations of agents that may be useful based on the specific properties of the drugs and the cancers involved. Typical antineoplastic agents useful in the present invention include, but are not limited to, microtubule inhibitors or antimitotic agents, platinum coordination complexes, alkylating agents, antibiotic formulations, topoisomerase I inhibitors, topoisomerase II inhibitors, antimetabolites, hormones and hormone analogs, signaling pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, cell cycle signaling inhibitors, proteasome inhibitors, heat shock protein inhibitors, cancer metabolism inhibitors, and oncogenetic agents.

[0113] Examples of further active ingredients or raw materials for use in combination with or concurrently administered with the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof, are antineoplastic agents. Examples of antineoplastic agents include, but are not limited to, chemotherapeutic agents, immunomodulators, immunomodulatory factors, and immunostimulatory adjuvants.

[0114] Microtubule inhibitors or antimitotic agents are cell cycle-specific agonists that are active against microtubules in tumor cells during the M phase of the cell cycle, i.e., mitosis. Examples of microtubule inhibitors include, but are not limited to, diterpenoids and vinca alkaloids.

[0115] Platinum-coordinated complexes are non-cell cycle-specific anticancer agents that interact with DNA. Platinum complexes enter tumor cells, undergo aqualation, and form intra- and inter-strand crosslinks with DNA, leading to harmful biological effects on the tumor. Examples of platinum-coordinated complexes include, but are not limited to, cisplatin and carboplatin.

[0116] Alkylating agents are non-cell cycle-specific anticancer agents and are strong electrophiles. Typically, alkylating agents form covalent bonds to the nucleophilic portion of the DNA molecule, such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups, through alkylation. Such alkylation interferes with the function of nucleic acids and causes cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards, e.g., cyclophosphamide, melphalan, and chlorambucyl; alkyl sulfonates, e.g., busulfan; nitrosoureas, e.g., carmustine; and triazenes, e.g., dacarbazine.

[0117] Antibiotic antineoplastic agents are non-cell cycle-specific agonists that bind to or intercalate DNA. This action interferes with the normal function of nucleic acids and causes cell death. Examples of antibiotic antineoplastic agents include, but are not limited to, actinomycin, e.g., dactinomycin; anthracyclines, e.g., daunorubicin and doxorubicin; and bleomycin.

[0118] Topoisomerase I inhibitors include, but are not limited to, camptothecin. The cytotoxic activity of camptothecin is thought to be related to its topoisomerase I inhibitory activity.

[0119] Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins. Epipodophyllotoxins are cell cycle-specific antineoplastic agents derived from the mandrake plant. Typically, epipodophyllotoxins affect cells during the S and G2 phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA, causing DNA strand disruption. Strand disruption accumulates, leading to cell death. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide.

[0120] Antimetabolites are cell cycle-specific antineoplastic agents that act during the S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or purine or pyrimidine base synthesis, thereby limiting DNA synthesis. As a result, the S phase does not progress, and cell death continues. Examples of antimetabolites include, but are not limited to, fluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine, and gemcitabine.

[0121] Hormones and hormone analogs are compounds useful in treating cancer that are related to hormones (plural) and growth, and / or the reduction of cancer growth. Examples of hormones and hormone analogs useful in treating cancer include corticosteroids, e.g., prednisone and prednisolone; aminoglutethimides and other aromatase inhibitors, e.g., anastrozole, letrazole, vorazole and exemestane; progestins, e.g., megestrol acetate; estrogens, androgens and antiandrogens, e.g., flutamide, nilutamide, bicalutamide, cyproterone acetate and 5α-reductase, e.g., The following are included, but are not limited to, finasteride and dutasteride; anti-estrogens, such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene, and selective estrogen receptor modulators (SERMS); and gonadotropin-releasing hormone (GnRH) and its analogues, LHRH agonists and antagonists, such as goserelin acetate and leuprolide, which stimulate the release of luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH).

[0122] Signaling pathway inhibitors are inhibitors that block or inhibit chemical processes that induce intracellular changes. As used herein, these changes are cell proliferation or differentiation. Signaling inhibitors useful to the present invention include, but are not limited to, inhibitors of receptor tyrosine kinases, inhibitors of non-receptor tyrosine kinases, SH2 / SH3 domain blockers, inhibitors of serine / threonine kinases, inhibitors of phosphatidylinositol-3 kinases, inhibitors of myo-inositol signaling, and inhibitors of Ras oncogenes.

[0123] Some protein tyrosine kinases catalyze the phosphorylation of specific tyrosyl residues in various proteins involved in regulating cell growth. Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.

[0124] Receptor tyrosine kinases are transmembrane proteins that possess an extracellular ligand-binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in regulating cell growth and are commonly referred to as growth factor receptors. For example, inappropriate or uncontrolled activation of many of these kinases, i.e., abnormal kinase growth factor receptor activity, due to overexpression or mutation, has been shown to result in uncontrolled cell growth. Therefore, abnormal activity of such kinases leads to malignant tissue growth. Consequently, inhibitors of such kinases provide methods for treating cancer. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), erbB2, erbB4, vascular endothelial growth factor receptor (VEGFR), tyrosine kinase with immunoglobulin-like and epidermal growth factor homologous domains (TIE-2), insulin growth factor-I (IGFI) receptor, macrophage colony-stimulating factor (Cfms), BTK, ckit, cmet, fibroblast growth factor (FGF) receptor, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph) receptor, and the RET oncogene. Several inhibitors of growth receptors are under development and include ligand antagonists, antibodies, tyrosine kinase inhibitors, and antisense oligonucleotides. Aggressors that inhibit growth factor receptors and growth factor receptor function are described, for example, in Kath JC, Exp. Opin. Ther. Patents, 10(6):803-818 (2000); Shawver LK et al., Drug Discov. Today, 2(2):50-63 (1997); and in New Molecular Targets for Cancer Chemotherapy, Kerr DJ and Workman P (eds.), Lofts, FJ and Gullick WJ, "Growth factor receptors as targets." (June 27, 1994), CRC Press. Non-exclusive examples of growth factor receptor inhibitors include pazopanib and sorafenib.

[0125] Tyrosine kinases that are not growth factor receptor kinases are called non-receptor tyrosine kinases. Non-receptor tyrosine kinases useful in the present invention that are targets or potential targets of anticancer drugs include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (focal adhesion kinase), Bruton's tyrosine kinase, and Bcr-Abl. Such non-receptor kinases and agonists that inhibit the function of non-receptor tyrosine kinases are described in Sinha S. and Corey SJ, J. Hematother. Stem Cell Res., 8(5):465-480 (2004) and Bolen, JB, Brugge, JS, Annu. Rev. Immunol., 15:371-404 (1997).

[0126] SH2 / SH3 domain blockers are agents that interfere with SH2 or SH3 domain binding in a variety of enzymes or adapter proteins, including the PI3-K p85 subunit, Src family kinases, adapter molecules (Shc, Crk, Nck, Grb2), and Ras-GAP. The SH2 / SH3 domain as a target for anticancer drugs is discussed in Smithgall TE, J. Pharmacol. Toxicol. Methods, 34(3):125~32 (1995).

[0127] Serine / threonine kinase inhibitors include, but are not limited to, mammalian targets of rapamycin (mTOR) inhibitors, including MAP kinase cascade blockers, including Raf kinase (rafk), mitogen or extracellular regulatory kinases (MEKs), and extracellular regulatory kinases (ERKs); protein kinase C family member blockers, including PKC (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta) blockers; IκB kinases (IKKa, IKKb); PKB family kinases; AKT kinase family members; TGF beta receptor kinases; and rapamycin (FK506) and rapalog, RAD001 or everolimus (AFINITOR®), CCI-779 or temsirolimus, AP23573, AZD8055, WYE-354, WYE-600, WYE-687 and Pp121. Examples of serine / threonine kinase inhibitors include, but are not limited to, trametinib, dabrafenib, and the Akt inhibitor afrecertib, as well as N-{(1S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-1-methyl-1H-pyrazole-5-yl)-2-francarboxamide.

[0128] Inhibitors of phosphatidylinositol 3-kinase family members, including PI3-kinase, ATM, DNA-PK, and Ku blockers, are also useful in the present invention. Such kinases are discussed in Abraham RT, Curr. Opin. Immunol., 8(3):412-418 (1996); Canman CE and Lim DS, Oncogene, 17(25):3301-3308 (1998); Jackson SP, Int. J. Biochem. Cell Biol., 29(7):935-938 (1997); and Zhong H. et al., Cancer Res., 60(6):1541-1545 (2000).

[0129] Myo-inositol signaling inhibitors, such as phospholipase C blockers and myo-inositol analogs, are also useful in the present invention. Such signaling inhibitors are described in "Inhibitors of Myo-Inositol Signaling" by Powis G. and Kozikowski A., in *New Molecular Targets for Cancer Chemotherapy*, edited by Kerr DJ and Workman P., (June 27, 1994), CRC Press.

[0130] Another group of signaling pathway inhibitors are Ras oncogene inhibitors. Such inhibitors include farnesyltransferase, geranyl-geranyltransferase, and CAAX protease inhibitors, as well as antisense oligonucleotides, ribozymes, and other immunotherapies. Such inhibitors have been shown to block ras activation in cells containing wild-type mutant ras, thereby acting as antiproliferative agents. Ras oncogene inhibition has been discussed in Scharovsky OG et al., J. Biomed. Sci., 7(4):292-298 (2000); Ashby MN, Curr. Opin. Lipidol., 9(2):99-102 (1998); and Bennett CF and Cowsert LM, Biochim. Biophys. Acta., 1489(1):19-30 (1999).

[0131] Antagonists of receptor kinase ligand binding can also act as signaling inhibitors. This group of signaling pathway inhibitors includes the use of humanized antibodies or other antagonists against the extracellular ligand-binding domain of receptor tyrosine kinases. Examples of antibodies or other antagonists against receptor kinase ligand binding include, but are not limited to, cetuximab (ERBITUX®), trastuzumab (HERCEPTIN®); trastuzumab emtansine (KADCYLA®); pertuzumab (PERJETA®); ErbB inhibitors including lapatinib, erlotinib, and gefitinib; and 2C3 VEGFR2-specific antibodies (see Brekken RA et al., Cancer Res., 60(18):5117-5124 (2000)).

[0132] Non-receptor kinase angiogenesis inhibitors may also find use in the present invention. Inhibitors of angiogenesis-related VEGFR and TIE2 have been discussed above in relation to signaling inhibitors (both receptors are receptor tyrosine kinases). Since inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, mainly VEGF expression, angiogenesis generally leads to erbB2 / EGFR signaling. Therefore, non-receptor tyrosine kinase inhibitors may be used in combination with the EGFR / erbB2 inhibitors of the present invention. For example, anti-VEGF antibodies that do not recognize VEGFR (receptor tyrosine kinase) but bind to its ligand; integrins (alpha) that inhibit angiogenesis. v Small molecule inhibitors of beta-3), such as endostatins and angiostatins (non-RTKs), have also been shown to be useful in combination with the disclosed compounds (see Bruns CJ et al., Cancer Res., 60(11):2926-2935 (2000); Schreiber AB et al., Science, 232(4755):1250-1253 (1986); Yen L. et al., Oncogene, 19(31):3460-3469 (2000)).

[0133] The agonists used in immunotherapy regimens may also be useful in combination with the present invention. Several immunological strategies exist to generate an immune response against erbB2 or EGFR. These strategies generally fall within the realm of tumor vaccine administration. The efficacy of immunological approaches can be significantly enhanced through combined inhibition of the erbB2 / EGFR signaling pathway using small molecule inhibitors. Discussions of immunological / tumor vaccine approaches against erbB2 / EGFR can be found in Reilly RT et al., Cancer Res., 60(13):3569-3576 (2000); and Chen Y. et al., Cancer Res., 58(9):1965-1971 (1998).

[0134] The agonists used in pro-apoptotic regimens (e.g., Bcl-2 antisense oligonucleotides) can also be used in the combination of the present invention. Members of the Bcl-2 family of proteins block apoptosis. Upregulation of Bcl-2 has therefore led to chemical resistance. Studies have shown that epidermal growth factor (EGF) stimulates the anti-apoptotic member of the Bcl-2 family (i.e., Mcl-1). Therefore, strategies designed to downregulate Bcl-2 expression in tumors have demonstrated clinical benefits. Such pro-apoptotic strategies using antisense oligonucleotide strategies for Bcl-2 are discussed in Waters JS et al., J. Clin. Oncol., 18(9): pp. 1812-1823 (2000); and Kitada S. et al., Antisense Res. Dev., 4(2): pp. 71-79 (1994).

[0135] Cell cycle signaling inhibitors inhibit molecules involved in the regulation of the cell cycle. The interaction between a family of protein kinases called cyclin-dependent kinases (CDKs) and a family of proteins called cyclins controls the progression of the entire eukaryotic cell cycle. Cooperative activation and inactivation of different cyclin / CDK complexes are necessary for the normal progression 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, in Rosania GR and Chang YT, Exp. Opin. Ther. Patents, 10(2):215-230 (2000). Furthermore, p21WAF1 / CIP1 has been described as a potent and universal inhibitor of cyclin-dependent kinases (Cdk) (Ball KL, Prog. Cell Cycle Res., 3:125-134 (1997)). Compounds known to induce p21WAF1 / CIP1 expression are associated with the suppression of cell proliferation and are considered to possess tumor suppressor activity (Richon VM et al., Proc. Natl. Acad. Sci. USA, 97(18):10014-10019 (2000)), and are included as cell cycle signaling inhibitors. Histone deacetylase (HDAC) inhibitors are associated with the transcriptional activation of p21WAF1 / CIP1 (Vigushin DM, and Coombes RC, Anticancer Drugs, 13(1):1-13 (2002)), and are suitable cell cycle signaling inhibitors for combined use as described herein. Examples of such HDAC inhibitors include, but are not limited to, vorinostat, romidepsin, panobinostat, valproic acid, and mosetinostat.

[0136] Proteasome inhibitors are drugs that block the action of the proteasome, a cellular complex that degrades proteins such as the p53 protein. Several proteasome inhibitors are on the market or being studied for the treatment of cancer. Suitable proteasome inhibitors for combined use as described herein include, but are not limited to, bortezomib, disulfiram, epigallocatechin gallate, salinosporamide A, and carfilzomib.

[0137] 70-kilodalton heat shock proteins (Hsp70) and 90-kilodalton heat shock proteins (Hsp90) are families of ubiquitously expressed heat shock proteins. Hsp70 and Hsp90 are overexpressed in certain types of cancer. Several Hsp70 and Hsp90 inhibitors have been studied for cancer treatment. Examples of Hsp70 and Hsp90 inhibitors for use in combination as described herein include, but are not limited to, tanespimycin and radisicol.

[0138] Many tumor cells exhibit metabolism significantly different from that of normal tissues. For example, the rate of glycolysis, the metabolic process that converts glucose to pyruvate, is increased, and the resulting pyruvate is reduced to lactate rather than being further oxidized in the mitochondria via the tricarboxylic acid (TCA) cycle. This effect is often observed even under aerobic conditions and is known as the Warburg effect.

[0139] Lactate dehydrogenase A (LDH-A), an isoform of lactate dehydrogenase expressed in muscle cells, plays a crucial role in tumor cell metabolism by reducing pyruvate to lactate, which can then be transported extracellularly. The enzyme has been shown to be upregulated in many tumor types. The glucose metabolic changes described by the Warburg effect are important for cancer cell growth and proliferation, and RNA-i-mediated knockdown of LDH-A has been shown to reduce cell proliferation and tumor growth in xenograft models (Tennant DA et al., Nat. Rev. Cancer, 10(4):267-277 (2010); Fantin VR et al., Cancer Cell, 9(6):425-434 (2006)).

[0140] 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 both cancer development and maintenance. Alli PM et al., Oncogene, 24(1):39-46 (2005).

[0141] Inhibitors of cancer metabolism, including LDH-A inhibitors and fatty acid biosynthesis inhibitors (or FAS inhibitors), are suitable combinations for use as described herein.

[0142] Oncogene therapy involves the selective delivery of recombinant DNA / RNA using viral or nonviral gene delivery vectors that modify cancer cells for therapeutic purposes. Examples of oncogene therapy include, but are not limited to, suicide and oncolytic gene therapies, as well as adoptive T-cell therapies.

[0143] As used herein, “immunomodulatory agent” refers to any substance, including monoclonal antibodies, that affect the immune system. Compounds of formula (I) of the present invention, their prodrugs, or pharmaceutically acceptable salts thereof are considered immunomodulators. Immunomodulatory agents may be used as antineoplastic agents for treating cancer. For example, immunomodulatory agents include, but are not limited to, antibodies or other antagonists against CTLA-4, e.g., ipilimumab (YERVOY®) and tremelimumab; those against PD-1, e.g., dostallimab, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®) and semiprimab (LIBTAYO®); and those against TIM-3, e.g., covolimab. Other immunomodulatory agents include, but are not limited to, antibodies or other antagonists against PD-L1, OX-40, LAG3, TIM-3, 41BB, and GITR.

[0144] As used herein, “PD-1 antagonist” means any chemical compound or biological molecule that blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells, or NKT cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. 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. The human PD-1 amino acid sequence can be found at NCBI locus No.: NP_005009. The amino acid sequences of human PD-L1 and PD-L2 can be found at NCBI loci No.: NP_054862 and NP_079515, respectively.

[0145] A PD-1 antagonist useful in any aspect of the present invention comprises a monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to PD-1 or PD-L1, preferably to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is 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.

[0146] Examples of mAbs that bind to human PD-1 and are useful in various aspects and embodiments of the present invention are described in U.S. Patents 8,552,154; 8,354,509; 8,168,757; 8,008,449; 7,521,051; 7,488,802; WO2004072286; WO2004056875; and WO2004004771.

[0147] Other PD-1 antagonists useful in any aspect and embodiment of the present invention include immunoadhesins that specifically bind to PD-1, preferably specifically to human PD-1, such as fusion proteins containing an extracellular or PD-1 binding moiety of PD-L1 or PD-L2 that fuses to a constant region, such as the Fc region of an immunoglobulin molecule. Examples of immunoadhesin molecules that specifically bind to PD-1 are described in WO2010027827 and WO2011066342. In the treatment methods, pharmaceuticals and uses of the present invention, a specific fusion protein useful as a PD-1 antagonist includes AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein that binds to human PD-1.

[0148] Nivolumab is a humanized monoclonal anti-PD-1 antibody marketed as OPDIVO®. Nivolumab is indicated for the treatment of several unresectable or metastatic melanomas. Nivolumab binds to PD-1, an Ig superfamily transmembrane protein, and blocks its activation by its ligands PD-L1 and PD-L2, leading to T-cell activation and a cell-mediated immune response against tumor cells or pathogens. Activated PD-1 negatively modulates T-cell activation and effector function through the suppression of P13k / Akt pathway activation. Other names for nivolumab include BMS-936558, MDX-1106, and ONO-4538. The amino acid sequence of nivolumab, as well as methods for its use and preparation, are disclosed in U.S. Patent No. 8,008,449.

[0149] Pembrolizumab is a humanized monoclonal anti-PD-1 antibody marketed as KEYTRUDA®. Pembrolizumab is indicated for the treatment of several unresectable or metastatic melanomas. The amino acid sequence of pembrolizumab and the method of use are disclosed in U.S. Patent No. 8,168,757.

[0150] Anti-PD-L1 antibodies and methods for producing them are well known in the art. Such antibodies against PD-L1 may be polyclonal or monoclonal, and / or recombinant, and / or humanized. PD-L1 antibodies are being developed as immunomodulatory agents for treating cancer.

[0151] Exemplary PD-L1 antibodies are disclosed in U.S. Patent Nos. 9,212,224; 8,779,108; 8,552,154; 8,383,796; 8,217,149; U.S. Patent Publication No. 20110280877; WO2013079174; and WO2013019906. Further exemplary antibodies against PD-L1 (also known as CD274 or B7-H1) and methods for their use are disclosed in U.S. Patent Nos. 8,168,179; 7,943,743; 7,595,048; WO2014055897; WO2013019906; and WO2010077634. Specific anti-human PD-L1 monoclonal antibodies useful as PD-1 antagonists in the treatment methods, pharmaceuticals, and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, and MSB0010718C.

[0152] Atezolizumab, marketed as TECENTRIQ®, is a fully humanized monoclonal anti-PD-L1 antibody. Atezolizumab is indicated for the treatment of several locally advanced or metastatic urothelial carcinomas. Atezolizumab blocks the interaction between PD-L1 and PD-1 and CD80. Other exemplary PD-L1 antibodies include avelumab (BAVENCIO®) and durvalumab (IMFINZI®).

[0153] Bifunctional fusion proteins targeting PD-1, or PD-L1 in conjunction with another target, may also be useful in the present invention. A bifunctional fusion protein, vintrafusp alfa, designed to simultaneously block the PD-L1 and TGF-β pathways, is disclosed in U.S. Patent No. 9,676,863.

[0154] CD134, also known as OX40, is a member of the TNFR-superfamily of receptors that, unlike CD28, is not constitutively expressed in quiescent naive T cells. OX40 is a secondary costimulatory molecule expressed 24 to 72 hours after activation, and its ligand, OX40L, is also not expressed in quiescent antigen-presenting cells, but follows their activation. OX40 expression depends on the full activation of T cells, and in the absence of CD28, OX40 expression is delayed to a quarter of the normal level. OX-40 antibodies, specifically OX-40 fusion proteins, and methods for using them are disclosed in U.S. Patents: US7, 504, 101, US7, 758, 852, US7, 858, 765, US7, 550, 140, US7, 960, 515, WO2012027328, and WO2013028231.

[0155] Examples of additional active ingredients or raw materials (antineoplastic agents) for use in combination with or co-administered with the disclosed compounds include antibodies against CD20 or other antagonists, retinoids, or other kinase inhibitors. Examples of such antibodies or antagonists include, but are not limited to, rituximab (RITUXAN® and MABTHERA®), ofatumumab (ARZERRA®), and bexarotene (TARGRETIN®).

[0156] Examples of additional active ingredients or raw materials (antineoplastic agents) for use in combination with or concurrently administered with the disclosed compounds include, but are not limited to, Toll-like receptor 4 (TLR4) antagonists, including aminoalkylglucosaminide phosphates (AGPs).

[0157] AGPs are known to be useful as vaccine adjuvants and immunostimulants in immunized animals to stimulate cytokine production, activate macrophages, promote innate immune responses, and increase antibody production. AGPs are synthetic ligands for TLR4. AGPs and their immunomodulatory effects via TLR4 are disclosed in patent publications, e.g., WO2006016997, WO2001090129, and / or U.S. Patent No. 6,113,918, and reported in the literature. Additional AGP derivatives are disclosed in U.S. Patents No. 7,129,219, 6,911,434, and 6,525,028. Some AGPs act as TLR4 agonists, while others are recognized as TLR4 antagonists.

[0158] An additional, non-limiting example of a further active ingredient or raw material (antineoplastic agent) for use in combination with or concurrently administered with the disclosed compound is an antibody against ICOS.

[0159] The CDR for mouse antibodies against human ICOS possessing agonist activity is shown in PCT / EP2012 / 055735 (WO2012131004). Antibodies against ICOS are also disclosed in WO2008137915, WO2010056804, EP1374902, EP1374901, and EP1125585.

[0160] Examples of additional active ingredients or raw materials (antineoplastic agents) for use in combination with or concurrently administered with the disclosed compounds are poly-ADP-ribose polymerase (PARP) inhibitors. Non-limiting examples of such inhibitors include niraparib, olaparib, rucaparib, and talazoparib.

[0161] The normal function of B cell maturation antigen (BCMA) is to promote cell survival through transduction of signaling from two known ligands: B cell activator (BAFF / BLyS) from the TNF family and proliferation-inducing ligand (APRIL). BCMA expression is constrained in B cells at later stages of differentiation and is expressed on germinal center B cells, hematopoietic plasmablasts, and long-lived plasma cells in the tonsils. BCMA is associated with multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), large B-cell lymphoma (LBCL), and chronic lymphocytic leukemia. BCMA is expressed at varying frequencies in various B-cell malignancies, including CLL and Waldenström macroglobulinemia (WM). The constrained, habitual tissue expression profile of BCMA, along with its upregulation and survival function in MM and other cancers, makes it an interesting target for therapeutic antibodies utilizing direct cytotoxic activity. BCMA inhibitors and other targeting agents, such as antibody-drug conjugates, may be used for the purposes of this invention. Berantamab mahodotin, an anti-BCMA antibody-drug conjugate, is disclosed in U.S. Patent No. 9,273,141.

[0162] Additional, non-limiting examples of further active ingredients or raw materials (antineoplastic agents) for use in combination with or concurrently administered with the disclosed compounds include STING modifiers, CD39 inhibitors, and A2a and A2a adenosine antagonists.

[0163] Selected antineoplastic agents that may be used in combination with the compound of formula (I) or its prodrug, or a pharmaceutically acceptable salt thereof include abalerix, abemaciclib, abiraterone, afatinib, aflibercept, aldoxorubicin, alectinib, alemtuzumab, arsenic trioxide, asparaginase, axitinib, AZD-9291, bellinostat, bendamustine, bevacizumab, blinatumomab, bosutinib, brentuximab vedotin, and others. Bazitaxel, cabozantinib, capecitabine, ceritinib, clofarabine, cobimetinib, crizotinib, daratumumab, dasatinib, degarelix, denosumab, dinutuximab, docetaxel, elotuzumab, entinostat, enzalutamide, epirubicin, eribulin, filgrastim, flumatinib, fulvestrant, fluquintinib, gemtuzumab ozogamicin, ibritumomab, ibrutinib, idelalisib, imatinib, irino Tecan, Ixabepirone, Ixazomib, Lenalidomide, Lenvatinib, Leucovorin, Mechloretamine, Necitumumab, Nelarabine, Netupitant, Nilotinib, Obinutuzumab, Olaparib, Omasetaxin, Osimertinib, Oxaliplatin, Paclitaxel, Palbociclib, Palonosetron, Panitumumab, Pegfilgrastim, Peginterferon alfa-2b, Pemetrexed, Plerixafor, Pomalidomide, Ponatinib, Plalat This list includes, but is not limited to, Lexart, Quizartinib, Radium-223, Ramucirumab, Regorafenib, Lorapitant, Lucaparib, Cipleucel-T, Sonidegib, Sunitinib, Tarimodine Laherparepbec, Tipiracil, Topotecan, Trabectedin, Trifluridine, Triptorelin, Uridine, Vandetanib, Velaparib, Vemurafenib, Venetoclax, Vincristine, Bismodegib, and Zoledronic Acid. Preferred antineoplastic agents include Venetoclax.

[0164] Preferred agonists include BCL2 targeters, e.g., venetoclax; tyrosine kinase inhibitors, e.g., those targeting FLT3 mutations (gilteritinib and midostaurin); sonic hedgehog inhibitors, glassegib; IDH1 or IHD2 mutation targeters, e.g., ivosidenib or enasidenib; NEDD8 targeters, e.g., pevonezistat; HDAC inhibitors, e.g., vorinostat or panobinostat; agonists targeting PRC2 complexes, e.g., tazemetostat (EZH2i) or MAK683 (EEDi); platinum-based antineoplastic agents, e.g., cisplatin; IO targeters, e.g., anti-CD47 (maglorimab), TIM-3 (sabatrimab), CTLA-4 (ipilimumab), and anti-PD-1 / PD-L1 (pembrolizumab); and P53 targeting drugs.

[0165] In one embodiment, the cancer treatment method of the claimed invention comprises the co-administration of a compound of formula (I) and / or a prodrug thereof, and / or a pharmaceutically acceptable salt thereof, with at least one antineoplastic agent selected from the group consisting of, for example, microtubule inhibitors, platinum coordination complexes, alkylating agents, antibiotic preparations, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signaling pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, cell cycle signaling inhibitors; proteasome inhibitors; and inhibitors of cancer metabolism.

[0166] Compounds of formula (I) or their prodrigs, and pharmaceutically acceptable salts thereof, may be administered concurrently with at least one other active agent known to be useful in the treatment of abnormal betahemoglobin disorders, such as sickle cell disease, sickle cell anemia, and betathalassemia.

[0167] An example of a further active ingredient or raw material to be used in combination with or administered simultaneously with the combination of the present invention is hydroxyurea.

[0168] The compounds of the present invention are prepared using conventional organic synthesis methods. A preferred synthesis route is described below in the following general reaction scheme. All starting materials are commercially available or can be easily prepared by those skilled in the art from commercially available starting materials.

[0169] The symbols used herein, as well as the conventions used in these processes, schemes, and examples, are consistent with those used in modern scientific literature, e.g., the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard one- or three-letter abbreviations are generally used to specify amino acid residues, which are assumed to be in L-configuration unless otherwise noted. Unless otherwise noted, all starting materials are obtained from commercial suppliers and used without further purification. Specifically, the following abbreviations may be used in the examples and throughout the specification: Ac (acetyl), Ac2O (acetic anhydride), CH3CN (acetonitrile), Boc(tert-butoxycarbonyl), Boc2O(di-tert-butyl dicarbonate), Cbz (benzyloxycarbonyl), DCE(1,2-dichloroethane), DCM (Dichloromethane), ATP (adenosine triphosphate), Bis-pinacolatodiborone (4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane), BSA (Bovine serum albumin), C18 (referring to an 18-carbon alkyl group on silicon in the HPLC stationary phase) Cy (cyclohexyl), DCM (Dichloromethane), DIEA (diisopropylethylamine), DIPEA (Hünig base, N-ethyl-N-(1-methylethyl)-2-propanamine), Dioxane (1,4-dioxane), DMAP (4-dimethylaminopyridine), DME (1,2-dimethoxyethane), DMEDA (N,N'-dimethylethylenediamine), DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), DPPA (Diphenylphosphoryl azide), EDC (N-(3-dimethylaminopropyl)-N'ethylcarbodiimide) hydrochloride, EDTA (ethylenediaminetetraacetic acid), HCl (ethyl acetate), EtOH (ethanol), Et2O (diethyl ether), HEPES (4-(2-hydroxyethyl)-1-piperazinylethanesulfonic 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), Hünig base (N,N-diisopropylethylamine), IPA (isopropyl alcohol), Indoline (2,3-dihydro-1H-indole), KHMDS (Potassium Hexamethyl Disilazide), LAH (Lithium Aluminum Hydrogen), LDA (Lithium Diisopropylamide), LHMDS (Lithium Hexamethyl Disilazide) MeOH (methanol), MTBE (methyl tert-butyl ether), mcM (micromoles), mCPBA (m-chloroperbezoic acid), NaHMDS (Sodium Hexamethyldisilazide), NCS (N-chlorosuccinimide), NBS (N-bromosuccinimide), PE (petroleum ether), Pd2(dba)3(Tris(dibenzylideneacetone)dipalladium(0), Pd(dppf)Cl2.DCM complex ([1,1'-bis(diphenylphosphin)ferrocene]dichloropalladium(II).dichloromethane complex), PyBOP (benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromotripyrrolidinophosphonium hexafluorophosphate), RPHPLC (Reverse-Phase High-Pressure Liquid Chromatography), RT (room temperature), Sat. (saturated), SFC (Supercritical Fluid Chromatography), SGC (Silica Gel Chromatography), SM (Starting materials), TLC (Thin-Layer Chromatography), TEA (triethylamine), TEMPO(2,2,6,6-tetramethylpiperidinyl 1-oxyl, free radical), TFA (trifluoroacetic acid), THF (tetrahydrofuran), and Ts-Cl(p-toluenesulfonyl chloride).

[0170] All references to ether refer to diethyl ether, and brine refers to a saturated aqueous solution of NaCl.

[0171] Synthesis scheme Those skilled in the art will recognize that if a substituent described herein is not compatible with the synthetic method described herein, the substituent may be protected with a suitable protecting group that is stable under the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to obtain the desired intermediate or target compound. Suitable protecting groups, and methods for protecting and deprotecting different substituents using such suitable protecting groups, are well known to those skilled in the art, and examples can be found in T. Greene and P. Wuts, Protecting Groups in Organic Synthesis (4th edition), John Wiley & Sons, NY (2006). In some examples, substituents may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is useful as an intermediate compound or is a desirable substituent in the target compound.

[0172] The prodrug of formula 16 in scheme 1 may be a single enantiomer or a racemic compound, depending on whether chiral cleavage is performed. The dicyanopyridine nuclear intermediate 6 can be readily prepared from 2-cyanoacetamide 1. The prodrug containing the intermediate can be prepared by starting with a substituted mandelic acid, e.g., commercially available 4-hydroxymandelic acid 7. N A variety of protecting and leaving group scenarios can be imagined that could enable conversion to the two electrophilic partner 13.

[0173] [ka] Compounds that do not possess the function of a prodrug, such as 17, can be prepared from intermediate 11 as shown in Scheme 2. At this stage, 17 can be divided as needed to obtain a single enantiomer 18.

[0174] [ka] Instead of chiral resolution, an asymmetric pathway can be used to obtain the chiral amide 22 or its corresponding prodrug 16, as detailed in Scheme 3. A single stereocenter can be established by asymmetric reduction of the ketoamide intermediate 19 through various methods described in the literature.

[0175] [ka]

[0176] [ka]

[0177] [ka]

[0178] Specific compounds of the present invention are prepared in the Examples section.

[0179] [Examples] [Example 1] Method A (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid Step 1: 5-(4-hydroxyphenyl)-2,2-dimethyl-1,3-dioxolan-4-one

[0180] [ka] 2-hydroxy-2-(4-hydroxyphenyl)acetic acid (1000 g, 5.947 mol) was added to 16 L JLR (container A) along with dichloromethane (DCM) (5000 mL), and stirring was started. 2,2-dimethoxypropane (2925 mL, 23.8 mol) was then added to the reaction mixture. The reaction mixture was cooled to 0°C. When the reaction temperature reached 0-5°C, BF3·OEt2 complex (45.2 mL, 0.357 mol) was added by pipette over 2 minutes. The addition was completed using DCM. The reaction mixture was then stirred at 0°C for 4 hours.

[0181] In a separate reaction vessel (vessel B), 5 L of saturated NaHCO3 aqueous solution was added, and the internal temperature of the vessel was set to 0°C. The reaction mixture from vessel A was added to the saturated sodium bicarbonate solution in vessel B by vacuum feed. After the addition was complete, the suspension was stirred for 10 minutes and then warmed to 20°C. The organic layer was separated into a 20 L carboy. The aqueous layer was extracted with an additional 1.2 L of DCM, and this organic layer was combined with the organic layer in the 20 L carboy. The combined organic layers were returned to JLR and washed with brine (3 L). The organic layers were separated again. The next day, the DCM was distilled to the minimum stirring volume. The remaining reaction mixture was concentrated to dryness using a rotary evaporator. The remaining off-white solid was then dried under vacuum to obtain 5-(4-hydroxyphenyl)-2,2-dimethyl-1,3-dioxolan-4-one (720 g, 58.1% yield) as an off-white solid. 1 HNMR (400 MHz, CDCl3) δ ppm 7.28 - 7.24 (m, 2H), 6.83 - 6.78 (m, 2H), 5.37 (s, 1H), 1.75 (s, 3H), 1.70 - 1.67 (m, 3H).

[0182] Step 2: 4-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)phenylmethanesulfonate

[0183] [ka] Into a 16 L JLR (vessel A), a solution of 5-(4-hydroxyphenyl)-2,2-dimethyl-1,3-dioxolan-4-one (720.0 g, 3458 mmol) dissolved in dichloromethane (DCM) (4390 mL) was added and the reaction mixture was cooled to 0 °C. Methanesulfonyl chloride (323 mL, 4150 mmol) was then added to the reaction mixture via an addition funnel over about 5 min, and the addition funnel was rinsed with 100 mL of DCM to complete the transfer into the reactor. Triethylamine (723 mL, 5187 mmol) was slowly added using an addition funnel over about 43 min, and the addition funnel was rinsed with 100 mL of DCM to complete the transfer into the reactor. The reaction mixture was stirred at 0 °C for 2 hours.

[0184] To another reaction vessel (vessel B), 4390 mL of saturated aqueous NaHCO3 was added and the internal temperature of the vessel was set to 0 °C. Vessel B was placed under reduced pressure to facilitate a gentle vacuum feed of the reaction mixture from vessel A into vessel B. Vessel A was rinsed with 300 mL of DCM and this wash solution was also transferred to vessel B. Stirring was stopped and 500 mL of DI water was added to vessel B via a spray ball to wash down the walls of the reactor. Stirring was restarted and the internal temperature was set to 20 °C. Stirring was interrupted after 30 min and the biphasic mixture was left overnight at 20 °C. The initial temperature was set to 10 °C and the vessel was placed under vacuum. After 30 min, the internal temperature was raised to 25 °C. Thereafter, over 1.5 hours, the volume was decreased from 1.5 to 2 L and the vacuum was released. The organic layer in vessel B was drained, the reactor was rinsed with DCM to complete the transfer. The remaining organic layer was concentrated to dryness on a rotary evaporator and further dried under high vacuum to obtain 4-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)phenyl methanesulfonate (689.67 g, 69.7% yield) as an off-white solid. LCMS m / z = 304.2 [M+H2O] + . 1HNMR (400 MHz, CDCl3) δ ppm 7.61 - 7.56 (m, 2H), 7.39 - 7.35 (m, 2H), 5.44 (s, 1H), 3.18 (s, 3H), 1.76 (s, 3H), 1.72 (s, 3H).

[0185] Step 3: 4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate

[0186] [ka] 689.67 g (2409 mmol) of 4-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)phenylmethanesulfonate was added to 16 L JLR with 2400 mL of methanol, and the solution was stirred. The jacket temperature was set to 0°C. 1377 mL (9636 mmol) of ammonia solution in 7 M MeOH was added to the reaction mixture over 47 minutes. The reaction mixture was stirred at the same temperature for 7.5 hours, after which the jacket temperature was raised to 10°C. An additional 250 mL (1750 mmol) of 7 M ammonia in methanol was added, and the reaction mixture was stirred at the same temperature for 16.5 hours. The reaction mixture was then filtered through a pan filter. The collected solid was washed with additional methanol and dried to obtain 544.86 g (92% yield) of 4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate as a white solid. LCMS m / z = 268.1 [M+Na] + . 1 HNMR (400 MHz, DMSO-d6) δ ppm 7.64 - 7.54 (m, 3H), 7.40 - 7.32 (m, 3H), 6.29 (d, J = 4.4 Hz, 1H), 5.03 (d, J = 4.4 Hz, 1H), 3.38 (s, 3H).

[0187] Step 4: 4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate

[0188] [ka] 545 g, 2222 mmol of 4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate and 4331 mL of dichloromethane (DCM) were added to a 16 L JLR (container A). Stirring was started and the jacket temperature was set to 0°C. After 25 minutes, mesyl chloride (216 mL, 2778 mmol) was added to the reaction mixture. Triethylamine (465 mL, 3333 mmol) was then slowly added over 35 minutes using an addition funnel. After the addition was complete, the jacket temperature was raised to 20°C over 40 minutes. The reaction mixture was stirred at the same temperature for 19 hours, but some starting material remained. The jacket temperature was reset to 0°C. An additional 30 mL, 385.8 mmol of mesyl chloride was added to the reaction mixture along with 70 mL of DCM to complete the addition. An additional amount of triethylamine (70 mL, 501.7 mmol) was then added. After the addition was complete, the jacket temperature was set to 22°C and the reaction mixture was stirred for 1 hour.

[0189] In a separate reaction vessel (vessel B), saturated sodium bicarbonate aqueous solution (2166 mL) was added, and the jacket temperature was set to 0°C. The reaction mixture from vessel A was vacuum-fed into vessel B at a rate that controlled the exothermic and gas release. Once the transfer was complete, the jacket temperature was raised to 20°C over 30 minutes, and the reaction mixture was stirred at this temperature for a further 30 minutes. The reaction mixture was then filtered through a pan filter. The collected solid material was washed with water (550 mL) and dried overnight in a pan filter under vacuum. The solid was then further dried overnight in a vacuum oven at 45°C to obtain 4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (530.36 g, 73.8% yield) as a white solid. LCMS m / z = 346.1 [M+Na] + . 1HNMR (400 MHz, DMSO-d6) δ ppm 7.88 (s, 1H), 7.63 - 7.58 (m, 2H), 7.45 - 7.41 (m, 2H), 5.92 (s, 1H), 3.42 (s, 3H), 3.27 (s, 3H).

[0190] Step 5: 4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate

[0191] [ka] 8 L of tetrahydrofuran (THF) was added to a 16 L JLR (container A) with the internal temperature set to -5°C, and stirring was started. Next, sodium hydride (60% by weight) (203.45 g, 5.087 mol) was added to the reaction vessel using a powder addition funnel. The container and funnel were rinsed into the reactor with 1 L of THF to complete the transfer. Tetrabenzyl pyrophosphate (1471 g, 2.732 mol) was added to the reaction mixture using a powder addition funnel. The container and funnel were rinsed into the reactor with 0.5 L of THF to complete the transfer. The reaction temperature was set to -3°C, and an additional 2.5 L of THF was added to the reaction mixture. 736 g, 2276 mol of 4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate was added to the reaction mixture over 30 minutes, while the reaction mixture was aerated during the addition. The addition was completed by rinsing the dimesylate container with an additional 3 L of THF, bringing the total THF in the reactor to 15 L. The stirring speed was increased to 300 rpm, and the reaction jacket was warmed to 25°C over 30 minutes. The reaction mixture was then stirred at the same temperature for 2 hours.

[0192] The reaction was quenched in two parts. 6 L of saturated citric acid solution was added to another reaction vessel (vessel B) set to an internal temperature of 0°C. 8.5 L of the reaction mixture from vessel A was added to this solution in vessel B over 30 minutes. During the addition, the jacket temperature of vessel B was adjusted to -15°C. After the addition was complete, the jacket temperature of vessel B was raised to 10°C. After stirring at 10°C for 1 hour, the mixture was filtered through a pan filter (using two sheets of sharkskin filter paper). After completely draining from vessel B, 6 L of saturated citric acid solution was added again, and the temperature was adjusted to -15°C. The quenching procedure was repeated by adding the remaining reaction mixture from vessel A to vessel B over 35 minutes. The temperature of vessel B was adjusted again to 10°C. Vessel A was rinsed with 500 mL of THF, and this rinse solution was added to vessel B. The jacket temperature of container B was adjusted to 25°C, and the mixture was held at the same temperature for 25 minutes. The reaction mixture was filtered through the same pan filter used previously. Tert-butyl methyl ether (TBME) was added to the pan filter to aid filtration, but the improvement, if any, was slight. After 50 minutes, the material in the pan filter was transferred back into the reaction vessel by vacuum feed. 8 L of TBME was added to the reactor, and the mixture was filtered again through the pan filter. As filtration progressed, the solid mixture became a paste and required scraping to facilitate the process. After 1.5 hours, all contents from the container were added to the pan filter, and the reaction vessel was rinsed with an additional 1.5 L of TBME. This rinse was added to the pan filter, and the filtration was left overnight. After overnight vacuum filtration, the isolated solid was transferred to a glass drying tray. The tray was placed in a vacuum oven at 25°C overnight.

[0193] After drying overnight in a vacuum oven, the solid material was transferred to a 12 L three-necked flask and suspended in 8 L of water. The mixture was vigorously mixed for 1 hour using an overhead mechanical stirrer and then filtered through a stainless steel pan filter with three layers of sharkskin filter paper. An additional 2 L of water was used to complete the transfer. The filtered cake was washed twice with 2 L of water, followed by 4 L of TBME. The solid was then dried overnight under vacuum in the pan filter. The solid was transferred from the pan filter to two baking dishes and further dried for approximately 48 hours in an unheated vacuum oven with nitrogen bleeding. The solids were combined to obtain 4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (1060 g, 80%) as a white solid. LCMS m / z = 584.3 [M+H] + . 1 HNMR (400 MHz, DMSO-d6) δ ppm 10.51 (d, J = 9.4 Hz, 1H), 7.67 - 7.61 (m, 2H), 7.48 - 7.43 (m, 2H), 7.41 - 7.31 (m, 8H), 7.28 - 7.23 (m, 2H), 6.11 (s, 1H), 5.10 - 4.85 (m, 4H), 3.41 (s, 3H), 3.29 (s, 3H).

[0194] Step 6: Ammonium 3,5-dicyano-4-ethyl-6-hydroxypyridine-2-oleate

[0195] [ka] To a stirred solution of 2-cyanoacetamide (300 g, 3.571 mol) and ammonia (25% by weight in water, 618 mL, 7.142 mol) in 750 mL of water cooled to 0°C, propionaldehyde (128 mL, 1.785 mol) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours. The precipitated solid was collected by filtration, washed with ice-cold water (2 × 500 mL), followed by cold methanol (300 mL), and dried to obtain ammonium 3,5-dicyano-4-ethyl-6-hydroxypyridine-2-oleate (150 g, 39%) as an off-white solid. LCMS m / z = 188.0 [MH] - . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.35 (s, 1H), 7.1 (br s, 4H), 2.48 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H).

[0196] Step 7: 2,6-Dichloro-4-ethylpyridine-3,5-Dicarbonitrile

[0197] [ka] N,N-dimethylaniline (150 mL, 1601 mmol) was added dropwise to a stirred suspension of ammonium 3,5-dicyano-4-ethyl-6-hydroxypyridine-2-oleate (150 g, 697 mmol) in POCl3 (750 mL, 8046 mmol) cooled to 0°C. The reaction mixture was heated at 120°C for 6 hours. The progress of the reaction was monitored by TLC (TLC system, 10% siRNA in hexane, Rf: 0.6, detection: UV). The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was diluted with ice-cold water and stirred for 10 minutes. The precipitated solid was filtered and dried. The solid was dissolved in dichloromethane (2 L) and washed with saturated sodium bicarbonate solution (1 L), water (1.5 L), and brine solution (1 L). The organic layer was dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a yellow solid. The solid material was pulverized with diethyl ether (500 mL), filtered, and dried to obtain 2,6-dichloro-4-ethylpyridine-3,5-dicarbonitric acid (130 g, 571 mmol, 82% yield) as a yellow solid. LCMS m / z = 224.1 [MH] - . 1 H NMR (400 MHz, CDCl3) δ ppm 3.13 (q, J = 7.6 Hz, 2H), 1.42 (t, J = 7.6 Hz, 3H).

[0198] Step 8: 2-(dimethylamino)-4-ethyl-6-mercaptopyridine-3,5-dicarbonitrine

[0199] [ka] 2,6-dichloro-4-ethylpyridine-3,5-dicarbonitric acid (130.0 g, 575 mmol) and DMF (1300 mL) were placed in a 3 L three-necked flask equipped with an overhead steering and temperature probe, and stirred to form a reddish solution. The reaction flask was placed in an ice bath, and the solution was stirred until the internal temperature reached approximately 3°C. A solution of dimethylamine in THF (288 mL, 575 mmol) was added using an addition funnel at a rate that kept the internal temperature below 5°C. Triethylamine (80 mL, 575 mmol) was added dropwise, maintaining the internal temperature below 7°C. The mixture turned dark purple towards the end of the triethylamine addition. Potassium thioacetate (164 g, 1438 mmol) was added, and the cooling bath was removed. The mixture was stirred at RT for 2 hours and poured into a mixture of cold 1N HCl solution (1150 mL, 1150 mmol) and water (2600 mL). The mixture was stirred for approximately 30 minutes, and the precipitated solid was filtered off. The filtered cake was washed with several parts of water (total 1 L) and dried overnight on a Buchner funnel. The orange solid was transferred to a 3 L three-necked flask with an overhead steering. Ethyl acetate (1200 mL) was added, and the slurry was stirred for approximately 30 minutes. The solid was filtered off. The cake was slurried with 200 mL of RINKAN and dried by suction on a Buchner funnel. The slurriing / drying process was repeated two more times to obtain 2-(dimethylamino)-4-ethyl-6-mercaptopyridine-3,5-dicarbonitride (115.57 g, 87% yield) as a bright yellow solid. LCMS m / z = 233.0 [M+H] + . 1 HNMR: (400 MHz, DMSO-d6) δ ppm 14.05 - 9.86 (m, 1H), 3.29 (s, 6H), 2.68 (q, J = 7.3 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H).

[0200] Step 9: 4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate

[0201]

change

[0202] Step 10: (R)-4-(2-((Bis(benzyloxy)phosphoryl)amino)-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate

[0203]

Chemical formula

[0204] Step 11: (R)-(2-((3,5-Dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramidic acid

[0205] [Chemical formula] (R)-4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((3,5-dicyano6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate (200.0 g, 278 mmol) and dichloromethane (2000 mL) were added to a 12 L three-necked round-bottom flask equipped with a mechanical overhead stirrer to obtain a homogeneous orange solution. To this solution, a solution of iodotrimethylsilane (122 g, 611 mmol) in dichloromethane (200 mL) was added dropwise over 50 minutes using an addition funnel at room temperature. LC-MS analysis of aliquots quenched in MeOH / MeCN 25 minutes after stirring following the addition of iodotrimethylsilane indicated the desired product and 4% mono-benzyl phosphate ester. An additional 1.660 mL of iodotrimethylsilane (12.19 mmol) was added, and the pale orange suspension was stirred for 20 minutes. After a total of 2 hours of stirring, methanol (200 mL, 4946 mmol) was added dropwise over 28 minutes using an additive funnel. An additional 200 mL of dichloromethane was added, and stirring was continued. Further dilution with 200 mL of dichloromethane was necessary to aid stirring. After stirring for 15 minutes, an additional 200 mL of dichloromethane was added (the total amount of dichloromethane was 2800 mL). After the methanol addition was complete, the mixture was mechanically stirred for 2 hours and 45 minutes. The suspension was divided and filtered through a funnel with six disposable polypropylene filters attached to a polyethylene frit disc. The solid was repeatedly rinsed with dichloromethane until no red / pink color was visible in the filtrate. The collected white solid was dried under vacuum in the funnel, transferred to a single mortar, and slowly ground into a fine white powder with high fluidity. The solid was placed in a vacuum oven without heating for 14 hours to obtain (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid (123.4 g, 82%) as a white solid. LCMS m / z = 540.0 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ ppm 11.55 (br s, 2H), 9.74 (br d, J = 9.3 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 5.79 (br s, Chiral HPLC: 98.9% (R)-enantiomer.

[0206] The X-ray powder diffraction (XRPD) pattern of the free acid matrix of Example 1 is shown in Figure 7, and a summary of the diffraction angles and d-intervals is shown in Table III below.

[0207] [Table 1]

[0208] The differential scanning calorimetry (DSC) thermogram of this free acid matrix is ​​identical to that obtained with the previous DSC equipment and is shown in Figure 8. The experiment was conducted in a lightly compressed aluminum pan using a heating rate of 10°C / min to a final temperature of 300°C. The compound showed a simple single melting event on DSC, with an onset temperature of 166.6°C, a peak temperature of 173.8°C, and a melting enthalpy of 68 J / g, followed by thermal decomposition above 200°C. The compound showed a negligible weight loss by TGA before the decomposition event. Those skilled in the art will understand that the endothermic onset temperature, peak temperature, and enthalpy may vary depending on the experimental conditions.

[0209] The thermogravimetric analysis (TGA) thermogram of this free acid matrix is ​​identical to that obtained with the previous TGA equipment and is shown in Figure 9. The experiment was carried out in an open aluminum pan with N2 purging and a heating rate of 10°C / min to a final temperature of 200°C. The compound showed a weight loss of 1.3% at 190°C before the decomposition event.

[0210] Method B (via mirror-selective pathway) (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid Step 1: (S)-4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate

[0211] [ka] A 500 mL round-bottom flask was heated with a heat gun for approximately 5 minutes while being purged with nitrogen. After cooling to room temperature, NaH (60% dispersion in mineral oil, 1.089 g, 27.2 mmol) and THF (120 mL) were added to the flask. The mixture was cooled to 0°C, and tetrabenzyl diphosphate (7.99 g, 14.85 mmol) was added, followed by (S)-4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (4.0 g, 12.37 mmol) being added gradually over approximately 3 minutes. The reaction mixture was stirred at 0°C under a nitrogen balloon. After 2 hours at 0°C, LC-MS analysis showed no remaining starting material. The reaction mixture was carefully poured into 200 mL of 10% citric acid aqueous solution and vigorously stirred. The resulting precipitate was filtered, rinsed with water (3 × 50 mL), followed by Et2O (3 × 50 mL), and dried to a constant weight under high vacuum to obtain (S)-4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (6.57 g, 11.26 mmol, 91% yield) as a white solid. LCMS m / z = 584.0 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 10.48 (br s, 1H), 7.69 - 7.57 (m, 2H), 7.48 - 7.41 (m, 2H), 7.41 - 7.22 (m, 10H), 6.11 (s, 1H), 5.13 - 4.96 (m, 3H), 4.96 - 4.88 (m, 1H), 3.40 (s, 3H), 3.28 (s, 3H). Chiral SFC: 100% ee.

[0212] Step 2: (R)-4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate

[0213] [ka] To a solution of 2-(dimethylamino)-4-ethyl-6-mercaptopyridine-3,5-dicarbonitride (480 mg, 2.066 mmol) in THF (20 mL), NaH (83 mg, 2.066 mmol) was added at 0°C. The resulting mixture was stirred at 0°C for 30 min, and then added dropwise at 0°C to a solution of (S)-4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (1266 mg, 2.170 mmol) in DCM (1 mL). The mixture was warmed to room temperature, stirred for 1 hour, and quenched with ammonium chloride. The aqueous layer was extracted with DCM and dehydrated with sodium sulfate. The residue was ground with MeOH to obtain (R)-4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate (908 mg, 1.198 mmol, 58% yield) as an off-white solid. LCMS m / z = 720.2 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 10.56 (d, J = 10.8 Hz, 1H), 7.67 - 7.56 (m, 2H), 7.48 - 7.39 (m, 2H), 7.37 - 7.29 (m, 8H), 7.29 - 7.23 (m, 2H), 5.81 (s, 1H), 5.09 - 4.86 (m, 4H), 3.39 (s, 3H), 3.27 (s, 6H), 2.76 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H), Approximately 25% contaminated with starting mesylate. Chiral HPLC: 99.3% ee.

[0214] Method C (Enantiomer-selective synthesis of glycine salts) (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide glycine salt

[0215] Step 1: (S)-4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate

[0216] [ka] To a 1 L JLR equipped with overhead steering, H2O (400 mL) was added followed by 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane (10.8 g, 51.4 mmol), and the mixture was stirred for 3 min. The jacket temperature was set to 20 °C. A solution prepared from beta-nicotinamide adenine dinucleotide phosphate disodium salt (NADP+, disodium) (500 mg), ketoreductase enzyme (1.50 g) and H2O (15 mL) was then added to the JLR, and the resulting solution was stirred for 5 min. 4-(2-Amino-2-oxoacetyl)phenylmethanesulfonate (50.0 g, 206 mmol), H2O (100 mL) and then isopropanol (63.4 mL, 822 mmol) were added, and the reaction was heated to 30 °C. After stirring for 24 h, the reaction was cooled to 0 °C and 1N aqueous NaOH solution (25.0 mL) was added. The resulting slurry was kept at 0 °C for 23 h, and then the precipitated solid was filtered off by vacuum filtration. The isolated solid was washed twice with H2O (250 mL each) and then with tert-butyl methyl ether (250 mL). After drying under vacuum, the desired product (S)-4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate was isolated as a white to off-white solid (45.0 g, 184 mmol, 89% yield). LCMS m / z = 246.0 [M+H] + . 1 H NMR: (400 MHz, DMSO-d6) δ ppm 7.54 - 7.49 (m, 2H), 7.42 (br s, 1H), 7.33 - 7.28 (m, 2H), 7.21 (br s, 1H), 6.13 (d, J = 4.9 Hz, 1H), 4.89 (d, J = 4.9 Hz, 1H), 3.37 (s, 3H). Chiral HPLC: >99% ee.

[0217] Step 2: (S)-4-(2-Amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate

[0218]

Chemical Structure

[0219] Step 3: (S)-4-(2-((dimethoxyphosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate

[0220] [ka] In a 1 L JLR flushed with nitrogen and equipped with overhead steering, tetrahydrofuran (200 mL) was added, followed by (S)-4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (40 g, 124 mmol). The mixture was then stirred for approximately 5 minutes, and the jacket temperature was set to 20°C. The slurry was then cooled to 0°C, and chlorodimethyl phosphate (20 mL, 186 mmol) was added to the container. After stirring for 1 hour, the reaction temperature was maintained at <5°C while slowly adding a 1 M solution of lithium tert-butoxide in tetrahydrofuran (272 mL, 272 mmol). The reaction was stirred for approximately 1 hour, and HPLC analysis determined it to be incomplete. Additional chlorodimethyl phosphate (6.3 mL, 58 mmol) was added to the reaction, followed by the slow addition of a 1 M solution of lithium tert-butoxide in tetrahydrofuran (37.1 mL, 37.1 mmol). The reaction was stirred for 30 minutes, then quenched by slowly adding 40 mL of 10% citric acid aqueous solution (w / w), while maintaining the reaction temperature <5°C. The reaction was stirred for approximately 15 minutes, then an additional 80 mL of 10% citric acid aqueous solution (w / w) was added while maintaining the reaction temperature <5°C. The temperature was raised to 20°C and maintained at that temperature for approximately 30 minutes. The reaction mixture was then cooled to 0°C and maintained for approximately 14 hours. The reaction was concentrated to a total volume of approximately 300 mL by vacuum distillation. Isopropanol (380 mL) was added to the reaction, and the reaction was stirred at 20°C for 2 hours. The reaction was concentrated to a total volume of approximately 520 mL by vacuum distillation, then H2O (80 mL) and isopropanol (80 mL) were added, and the reaction was cooled to 0°C. After stirring for 19 hours, the product slurry was transferred to a filter dryer. The mother liquor was removed by filtration using nitrogen pressure. The reactor was rinsed with H2O (400 mL), then transferred to a filter dryer to wash the isolated solid. Nitrogen pressure was used to push the washing through the product filtration cake. The reactor was rinsed with isopropanol (400 mL), then transferred to a filter dryer to wash the isolated solid. Nitrogen pressure was used to push the washing through the product filtration cake. The product solid was then washed with tert-butyl methyl ether (200 mL). The isolated solid was then removed. The solid was dried under nitrogen for 23 hours, and then dried under vacuum at 20°C for 21 hours to obtain the desired product, (S)-4-(2-((dimethoxyphosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate, as a white solid (35.1 g, 81 mmol, 66% yield). LCMS m / z = 454.1 [M+Na] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.28 (br s, 1H), 7.60 - 7.68 (m, 2H), 7.44 - 7.51 (m, 2H), 6.07 (s, 1H), 3.67 (d, J = 12 Hz, 3H), 3.57 (d, J = 12 Hz, 3H), 3.43 (s, 3H), 3.30 (s, 3H). Chiral HPLC: >99% ee.

[0221] Step 4: (R)-4-(1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-((dimethoxyphosphoryl)amino)-2-oxoethyl)phenylmethanesulfonate

[0222] [ka] 338 mL of H₂O and 5.18 g of sodium carbonate (48.9 mmol) were added to a 1 L JLR with an overhead steering chamber that had been flushed with nitrogen, and the mixture was stirred at 20°C. The walls of the reaction vessel were rinsed with additional H₂O (42.0 mL), and the mixture was stirred for 10 minutes. 170 mL of acetone was added, followed by 2-(dimethylamino)-4-ethyl-6-mercaptopyridine-3,5-dicarbonitride (22.7 g of 98.0 mmol), and then additional acetone (42.0 mL). The mixture was stirred for approximately 1 hour, and then (S)-4-(2-((dimethoxyphosphoryl)amino)-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (42.2 g of 98.0 mmol) was added all at once. The reaction was stirred for 2 hours, and then the precipitated solid was collected by vacuum filtration. The isolated solid was washed twice with H2O (126 mL each), then twice with isopropanol (210 mL each), then with tert-butyl methyl ether (420 mL), and blow-dried under a nitrogen stream for 15 hours. The product filtration cake was then re-slurried with tert-butyl methyl ether (336 mL). After mixing for approximately 10 minutes, the solid was filtered and dried, and rinsed with an additional tert-butyl methyl ether (126 mL). After drying under vacuum, the desired product (R)-4-(1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-((dimethoxyphosphoryl)amino)-2-oxoethyl)phenylmethanesulfonate was collected to obtain an off-white solid (50.6 g, 88.0 mmol, 90% yield). LCMS m / z = 568.1 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ ppm 10.33 - 10.43 (m, 1H), 7.55 - 7.66 (m, 2H), 7.36 - 7.48 (m, 2H), 5.81 (br s, 1H), 3.64 (d, J = 12 Hz, 3H), 3.52 (d, J = 12 Hz, 3H), 3.42 (s, 3H), 3.37 (s, 6H), 2.77 (q, J = 8 Hz, 2H), 1.21 (t, J = 8 Hz, 3H). Chiral HPLC: >99% ee.

[0223] Step 5: (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide DMF hemisolvate

[0224] [ka] Dichloromethane (250 mL) and (R)-4-(1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-((dimethoxyphosphoryl)amino)-2-oxoethyl)phenylmethanesulfonate (65.0 g, 115 mmol) were added to a 2 L JLR purged with nitrogen, and the container wall was then washed with additional dichloromethane (100 mL) to produce a homogeneous yellowish-brown solution. After cooling this solution to 0°C, iodotrimethylsilane (42.1 mL, 309 mmol) was added while maintaining an internal temperature <5°C. The resulting brown reaction mixture was stirred at 0°C for 1 hour. HPLC analysis of the reaction mixture indicated an incomplete reaction, and therefore additional iodotrimethylsilane (3.12 mL, 22.9 mmol) was added. After stirring for 13 minutes, the reaction was quenched by adding approximately 50% (91 mL) of the DMF quench solution while maintaining an internal temperature <5°C (Note: The DMF quench solution was prepared by adding H2O (3.30 mL, 183 mmol) to anhydrous DMF (179 mL)). The reaction mixture was kept at 0°C for 3 minutes, and then the crystallized product was seeded into (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramic acid DMF hemi-solvate (165 mg) which had been slurryed in dichloromethane (6.50 mL), and kept at 0°C for approximately 30 minutes. The dilute slurry was then further diluted by gradually adding dichloromethane (650 mL), and then stirred at 0°C for approximately 30 minutes. The remaining approximately 50% (91 mL) of the DMF quench solution was slowly added while maintaining an internal temperature of <5°C. Once the addition was complete, the jacket temperature was raised to 20°C at a rate of 2°C / min and maintained at 20°C for approximately 45 minutes. The reaction slurry was further diluted with dichloromethane (650 mL) and stirred for 12 hours. The precipitated solid was collected by vacuum filtration and then washed twice with dichloromethane (325 mL each).The solid was re-slurried with ethyl acetate (325 mL) at 20°C for approximately 2.5 hours, isolated by vacuum filtration, and then dried under vacuum to obtain (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramic acid DMF hemi-solvate (58.4 g, 101 mmol, 89% yield) as a white to off-white product. ¹H NMR showed a product:DMF ratio of 1:0.8. LCMS m / z = 540.0 [M+H] + . 1 ¹H NMR (400 MHz, DMSO-d6) δ ppm: 10.77 - 12.19 (br s, 1 H), 9.75 (br d, J = 9.5 Hz, 1 H), 7.96 (s, 0.8 H, DMF), 7.58 - 7.65 (m, 2 H), 7.31 - 7.46 (m, 2 H), 5.80 (br s, 1 H), 3.42 (s, 3 H), 3.37 (m, 6 H), 2.90 (s, 2.8 H, DMF), 2.74 (m, 4.8 H, including DMF), 1.21 (t, J = 7.5 Hz, 3 H). Chiral HPLC: >99% ee.

[0225] Step 6: (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide glycine salt

[0226] [ka] The process was carried out in a pair of 16L JLRs, each equipped with overhead steering, using containers A and B. Micronized glycine (71.5 g, 0.952 mol), dichloromethane (9 L), and methanol (896.5 mL) were placed in container A, and the same filling was repeated in container B. The mixture was stirred at 25°C. Next, a slurry of (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramic acid DMF hemi-solvate (137.5 g, 0.238 mol) in ethyl acetate (1.1 L) was placed in container A for at least 15 minutes. The slurry container was rinsed with ethyl acetate (550 mL), and this rinse solution was then transferred to the reactor. The filling of the slurry into container B and rinsing with ethyl acetate were repeated. The crystals in containers A and B were seeded into (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide glycine salt (5.5 g). The process of slowly adding (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide DMF hemi-solvate (137.5 g, 0.238 mol) in ethyl acetate (1.1 L), rinsing the container with ethyl acetate (550 mL), and transferring the rinse solution to containers A and B was repeated three times in each reactor after seeding. The slurry was stirred overnight at 25°C to complete the reaction. The slurry mixtures from both containers A and B were emptied into a stainless steel pan filter equipped with filter paper. The mother liquor was removed by filtration using vacuum pressure. Containers A and B were each rinsed with ethyl acetate (3.3 L), then transferred to the pan filter to wash and filter the isolated solids. A second rinse of containers A and B with ethyl acetate was repeated. The washing solution was pushed through the product filtration cake using vacuum pressure.The isolated solid was dried at 50°C under vacuum until <1% of (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide glycine salt (1101 g, 1.791 mol, 92.5% yield) was obtained as a white solid by LOD analysis. 1H NMR showed a product:glycine ratio of 1:1. LCMS m / z = 540.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.41 (br d, J = 7.50 Hz, 1 H), 7.66 (br d, J = 8.0 Hz, 2 H), 7.36 (br d, J = 8.0 Hz, 2H), 5.80 (s, 1 H), 3.48 (s, 2 Chiral HPLC: >99% ee.

[0227] The crystalline compound of Example 1, glycine compound versus (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid (1:1).

[0228] The X-ray powder diffraction (XRPD) pattern of this material is shown in Figure 1, and a summary of the diffraction angles and d-intervals is shown in Table I below. XRPD analysis was performed on a PANanalytical X'Pert Pro Diffractometer on a Si zero background wafer using an X'celerator® RTMS (Real-time Multistrip) detector. The acquisition conditions were Cu K αRadiation, wavelength (λ): 1.5405980 Å, generator voltage: 45 kV, generator current: 40 mA, step size: 0.0167°²θ. Incident beam configuration: 10 mm programmable divergent slit, 0.02 rad Soller slit, anti-scattering slit (0.5°), and 10 mm beam mask. Diffraction beam configuration: included a 10 mm programmable anti-scattering slit assembly (X'celerator module) and a 0.02 rad Soller slit.

[0229] [Table 2]

[0230] The differential scanning calorimetry (DSC) thermogram of this material was recorded under a 40 mL / min N2 purge using a TA Instruments Discovery Differential Scanning Calorimeter equipped with an autosampler and refrigeration system, and is shown in Figure 2. The experiment was carried out in a lightly pressed aluminum pan using a heating rate of 10 °C / min to a final temperature of 200 °C. The compound showed a simple single melting event in DSC with an onset temperature of 183.6 °C, a peak temperature of 188.8 °C, and an enthalpy of fusion of 64 J / g. The determination of the enthalpy of fusion is not reliable due to immediate thermal decomposition after melting. The compound showed a negligible weight loss by TGA before the decomposition event. Those skilled in the art will recognize that the onset temperature, peak temperature, and enthalpy of endothermic reactions may vary depending on experimental conditions.

[0231] The thermogravimetric analysis (TGA) thermogram of this material was recorded using a TA Instruments Discovery Thermogravimetric Analyzer and is shown in Figure 3. The experiment was carried out in an open aluminum pan under N2 purging and at a heating rate of 10°C / min to a final temperature of 200°C. The compound showed a 0.3% weight loss at 160°C before the decomposition event.

[0232] Each of the general points in the above paragraph relating to XRPD, DSC, and TGA is applicable to each of the XRPD, DSC, and TGA analyses performed in this application. Furthermore, in all of the XRPD data reported in this case, the data has an accuracy of plus or minus 0.2.

[0233] Step 7: (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide glycine monohydrate

[0234] [ka] (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramic acid DMF hemi-solvate (1.5 g, 2.54 mmol) and ethyl acetate (37.5 mL) were added to a 50 mL JLR equipped with overhead steering. The reactor jacket was heated to 50 °C at 1 °C / min while stirring, and maintained at 50 °C for 95 minutes. A solution of 2 M glycine in water (2.67 mmol glycine) was loaded into a fitted 10 mL dosing unit. 2 M glycine in water (1.33 mL) was added to the reaction mixture over 90 minutes. After filling, the mixture was maintained for 60 minutes, and then cooled to 5 °C at 0.25 °C / min. The reaction mixture was maintained at 5 °C for 60 minutes. The reaction mixture was then programmed overnight to heat to 50°C at 1°C / min, maintain at 50°C, cool to 5°C at 0.25°C / min, and maintain at 5°C for 60 minutes. This temperature cycle was completed twice. The reaction mixture was maintained at 5°C for 4.5 days and then filtered through a disposable filter funnel. The wet cake of the reaction solid was rinsed twice with ethyl acetate (2 × 9 mL) and filtered. The solid was left overnight in a vacuum oven at 25°C to dry and produced (R)-(2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid hydrate glycine salt (1.55 g, 2.45 mmol) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.34 (s, 1 H), 7.65 (br d, J = 7.5 Hz, 2 H), 7.36 (br d, J = 7.5 Hz, 2 H), 5.79 (br s, 1 H), 3.40 (s, 6 H), 3.35 (br Chiral HPLC: >99% ee.

[0235] The X-ray powder diffraction (XRPD) pattern of the monohydrated glycinate of Example 1 is shown in Figure 4, and a summary of the diffraction angles and d-intervals is shown in Table II below.

[0236] [Table 3]

[0237] A differential scanning calorimetry (DSC) thermogram of this hydrated glycinate material, identical to that obtained with previous DSC equipment, is shown in Figure 5. The experiment was conducted in a lightly pressed aluminum pan using a heating rate of 10°C / min to a final temperature of 250°C. The compound exhibits a broad endothermic dehydration event from 40 to 140°C, followed by a rapid endothermic melting with an onset temperature of 175.5°C, a peak temperature of 179.3°C, and an enthalpy of fusion of 52 J / g. The determination of the enthalpy of fusion is unreliable due to the immediate thermal decomposition after melting. Those skilled in the art will recognize that the onset temperature, peak temperature, and enthalpy of endothermic melting may vary depending on the experimental conditions.

[0238] The thermogravimetric analysis (TGA) thermogram of this hydrated glycinate material, identical to that obtained with previous TGA equipment, is shown in Figure 6. The experiment was carried out in an open aluminum pan under N2 purging and at a heating rate of 10°C / min to a final temperature of 250°C. The compound exhibited a 3.0% weight loss from 40 to 140°C and showed monohydrate form before the decomposition event.

[0239] [Example 2] (R)-4-(2-amino-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate Step 1: 4-(2-amino-2-oxoacetyl)phenylmethanesulfonate

[0240] [ka] To a stirred suspension of 4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate (50 g, 204 mmol) in acetonitrile (3000 mL), manganese dioxide (248 g, 2854 mmol) was added in a single packing over 1 minute at room temperature under nitrogen. The reaction mixture was heated at 70 °C for 16 hours, then at 80 °C for 48 hours. The reaction mixture was cooled to room temperature, filtered through celite, and the celite bed was washed with acetonitrile (2000 mL). The filtrate was concentrated under reduced pressure to obtain 44 g of the desired crude product as an off-white solid. Analysis of the material by UPLC MS indicated an area under the curve of 46.67% corresponding to the product mass and an area under the curve of 48.26% corresponding to the starting material mass. The crude product / starting material mixture was subjected to the oxidation reaction using PCC described below.

[0241] PCC (18.53 g, 86 mmol) was added in a single packing at room temperature under nitrogen by mixing with celite to a stirred solution containing a mixture of 4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate and 4-(2-amino-2-oxoacetyl)phenylmethanesulfonate (42.0 g, 86 mmol) in tetrahydrofuran (THF) (2.4 L). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered through celite, and the celite bed was washed with (2000 mL) of THF. The filtrate was concentrated under vacuum to obtain the crude product as a dark brown solid. The crude product was dissolved in a mixture of 100 mL of methanol and 150 mL of DCM and absorbed on 400 g of silica (60-120 mesh). The resulting material was filtered through 4 kg of silica (230-400 mesh), and the silica bed was washed with (3000 mL) of ethyl acetate. The filtrate was concentrated under vacuum to obtain 4-(2-amino-2-oxoacetyl)phenylmethanesulfonate (26 g, 52%) as an off-white solid. LCMS m / z = 244.0 [M+H] + . 1HNMR (400 MHz, DMSO-d6) δ ppm 8.37 (br s, 1H), 8.13-8.09 (m, 2H), 8.06 (br s, 1H), 7.58-7.54 (m, 2H), 3.49 (s, 3H).

[0242] Step 2: (S)-4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate

[0243] [ka] 4-(2-amino-2-oxoacetyl)phenylmethanesulfonate (5.0 g, 20.56 mmol) and isopropanol (10 mL) were added to a 500 mL round-bottom flask equipped with a large stirring bar. To the resulting mixture, 80 mL of a 1.25 mg / mL solution of beta-nicotinamide adenine dinucleotide phosphate disodium salt (NADP+, disodium) (100 mg, 20.56 mmol) in 0.1 M KPi buffer at pH 7.0 was added, followed by SynBio ketoreductase enzyme Seq ID S00000617 (300 mg, 20.56 mmol). The resulting slurry was vigorously stirred at room temperature. After a total of 46 hours, the aqueous layer was saturated with solid KCl, and the mixture was diluted with siRNA (200 mL). The formed emulsion, and therefore the mixture, was filtered through a Celite® pad and thoroughly rinsed with a large amount of SiO2 (3 × 75 mL) to obtain a clear, separate layer. The organic layer was washed with brine, and the combined aqueous layer was back-extracted with SiO2 (2 × 75 mL). The combined organic matter was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was dried to a constant weight under high vacuum to obtain (S)-4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate (4.40 g, 87% yield) as a white solid. LCMS m / z = 246.0 [M+H] + . 1HNMR: (400 MHz, DMSO-d6) δ ppm 7.54 - 7.49 (m, 2H), 7.42 (br s, 1H), 7.33 - 7.28 (m, 2H), 7.21 (br s, 1H), 6.13 (d, J = 4.9 Hz, 1H), 4.89 (d, J = 4.9 Hz, 1H), 3.37 (s, 3H). Chiral HPLC: >99% ee.

[0244] Step 3: (S)-4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate

[0245] [ka] To a cold (0°C) suspension of (S)-4-(2-amino-1-hydroxy-2-oxoethyl)phenylmethanesulfonate (4.3 g, 17.53 mmol) in dichloromethane (DCM) (40 mL), Ms-Cl (1.639 mL, 21.04 mmol) was added over approximately 1 minute, followed by TEA (3.67 mL, 26.3 mmol) over approximately 3 minutes. After 2 hours, LC-MS showed approximately 15% of the starting material (no further development after 3 hours), therefore the reaction mixture was cooled again to 0°C, and the additional Ms-Cl (0.410 mL, 5.26 mmol) and TEA (1.222 mL, 8.77 mmol) were added. The ice bath was removed, and the reaction was warmed to room temperature. After an additional 30 minutes, the reaction mixture was quenched with saturated NaHCO3 aqueous solution (50 mL) and vigorously stirred. The resulting suspension / emulsion was filtered, and the solid was sequentially rinsed with water (3 × 30 mL) and diethyl ether (3 × 30 mL). It was then dried to a constant weight under high vacuum to obtain (S)-4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (4.44 g, 78% yield) as a slightly off-white solid. LCMS m / z = 324.0 [M+H] + . 1HNMR (400 MHz, DMSO-d6) δ ppm 7.86 (s, 1H), 7.63 - 7.54 (m, 3H), 7.41 (d, J = 7.8 Hz, 2H), 5.91 (s, 1H), 3.41 (s, 3H), 3.26 (s, 3H).

[0246] Step 4: (R)-4-(2-amino-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate

[0247] [ka] To a suspension of 2-(dimethylamino)-4-ethyl-6-mercaptopyridine-3,5-dicarbonitride (1.437 g, 6.19 mmol) in ethyl acetate (50 mL), iPr2EtN (1.350 mL, 7.73 mmol) was added. The orange suspension was stirred at room temperature. After 30 minutes, a suspension of (S)-4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (2.0 g, 6.19 mmol) in ethyl acetate (30 mL) was added, and the resulting orange suspension was stirred at room temperature. After 4 hours, the inside of the flask was scraped with a spatula to initiate product precipitation. The resulting suspension was stirred for an additional 30 minutes and then cooled to 0°C. The precipitate was collected using a filter funnel and rinsed sequentially with cold (0°C) SiO (2 × 20 mL), water (2 × 10 mL), cold (0°C) SiO (2 × 20 mL), and then diethyl ether (2 × 10 mL). The solid was dried under high vacuum for 48 hours to obtain (R)-4-(2-amino-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate (1.37 g, 48.2% yield) as an off-white solid. LCMS m / z = 460.2 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ ppm 7.96 (s, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.42 - 7.31 (m, 3H), 5.66 (s, 1H), 3.39 (s, 3H), 3.33 (s, 6H), 2.75 (q, J = 7.8 Hz, 2H), 1.20 (t, J = 7.8 Hz, 3H).

[0248] [Example 3] (2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid

[0249] [ka] To a solution of 4-(2-((bis(benzyloxy)phosphoryl)amino)-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate (77.34 g, 107 mmol) in acetonitrile (1 L), iodotrimethylsilane (34 mL, 247 mmol) was added at 0°C, and the mixture was warmed to room temperature. The reaction mixture was stirred at room temperature for 30 min. The reaction was quenched with 1.2 L of 10% sodium metabisulfite. The formed precipitate and the concentrated mixture were stirred. The mixture was filtered, and the collected solid was washed with 1 L of water. It was dried overnight under vacuum in a filter funnel. The resulting solid was suspended in 2 L of ether and stirred for 30 min. The mixture was filtered, and the collected solid was dried to obtain (2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-(4-((methylsulfonyl)oxy)phenyl)acetyl)phosphoramide acid (32 g, 59.3 mmol, 55.2% yield) as an off-white solid. LCMS m / z = 540.0 [M+H] + . 1¹H NMR (400 MHz, DMSO-d6) δ ppm 9.48 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 5.81 (s, 1H), 3.40 (s, 3H), 3.34 (s, 6H), 2.74 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H) (No protons were detected from the two phosphates).

[0250] [Example 4] 4-(2-amino-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate

[0251] [ka] To a yellow suspension of 2-(dimethylamino)-4-ethyl-6-mercaptopyridine-3,5-dicarbonitride (2.0 g, 8.61 mmol) in ethyl acetate (50 mL), DIEA (1.880 mL, 10.76 mmol) and 4-(2-amino-1-((methylsulfonyl)oxy)-2-oxoethyl)phenylmethanesulfonate (2.78 g, 8.61 mmol) were added sequentially and simultaneously. The mixture was stirred at room temperature for 24 hours. The solid was filtered and rinsed with ethyl acetate, water, and further ethyl acetate. The solid was dried to obtain 3.50 g of a pale yellow solid. The solid material was suspended in 30 mL of water and stirred for 1 hour. The solid was filtered, rinsed with water, and dried to obtain 4-(2-amino-1-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridine-2-yl)thio)-2-oxoethyl)phenylmethanesulfonate (3.09 g, 76%) as a pale yellow solid. LCMS m / z = 460.0 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ ppm 7.97 (s, 1H), 7.65 - 7.59 (m, 2H), 7.41 - 7.34 (m, 3H), 5.65 (s, 1H), 3.40 (s, 3H), 3.33 (s, 6H), 2.75 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H).

[0252] Biological data DNMT1 Scintillation Proximity Assay (SPA) - Assay A (Full-length Human DNMT1) This assay used scintillation proximity technology in a signal-increasing format to evaluate the potency of the compound. Full-length human DNMT1, hemimethylated double-stranded DNA. * Activity was monitored using tritiated SAM. Assay plates were prepared according to the following parameters, with 500 nL of 11pt, 3-fold serial dilutions of the compound pressed into 96-well Costar plates (#3884). Assay buffer mix consisting of the following was prepared on the day of the assay: 20 mM Tris pH 7.5, 1 mM DTT, 1 mM EDTA, and 5% glycerol. A 2× enzyme mix consisting of 30 nM DNMT1 protein (full-length human DNMT, homemade) in assay buffer was then prepared. Finally, a 2× substrate mix was prepared, which consisted of 160 nM 40-mer hemimethylated DNA in assay buffer. * , 0.48 μM 3 It consisted of H-SAM and 2.92μM cold SAM. 3H-SAM is added last. Quenching (1 mM SAH) was performed in bulk and the mixture was frozen at -20°C until use. 10 μL of 2× substrate mix was added to the whole plate using a multichannel electronic pipette. The plate was shaken for at least 10 seconds between additions to ensure mixing. Next, 20 μL of 2× quench mix was added to column 12 using a multichannel electronic pipette (shake the plate). Using a multichannel electronic pipette, 10 μL of 2× enzyme mix was added to the full plate, starting in column 11 and moving to column 10 (column 12 continued to avoid the carryover from before quenching). The plate was covered and incubated in a shaker for 30 minutes. At the end of the incubation period, 20 μL of quench mix was added to all wells except column 12 (shaking the plate), followed by 20 μL of 3 mg / mL PerkinElmer PEI PVT SPA beads (Cat. #RPNQ0097) diluted with DNase-free water, and the plate was shaken for at least 30 minutes. The plate was sealed with a clear seal and centrifuged at 500 rpm for 1 minute. The plate was read with MicroBeta (PerkinElmer, 3 I read about H (1 min / well).

[0253] Data were analyzed up to Vi / Vo using Microsoft Excel, and fitted using GraFit. Responses were normalized to non-inhibitory (DMSO) and pre-quenching controls within each plate. Dose-response curves were constrained to 0 Y. min The analysis was performed using a 3-parameter logistic fit, and the results were converted to IC. 50 It was expressed as a value.

[0254] Final assay conditions: 20 mM Tris (Hampton Research - HR-937-06), pH 7.5, 1 mM DTT (Invitrogen - P2325), 1 mM EDTA (Invitrogen - AM9260G), 5% glycerol (Teknova - G1796), 0.02% Pluronic F127 (Life Technologies - P6866), 15 nM DNMT1 (full-length human DNMT1 - GSK homemade), 240 nM 3 H-SAM (PerkinElmer - NET155H001MC), 1460 nM cold SAM (New England Biolabs - B9003S), and 80 nM 40-mer hemimethylated DNA oligonucleotide (Integrated DNA Technologies - custom), 1 mM SAH (Sigma A9384), and 1 mg / mL PEI PVT SPA Beads (PerkinElmer RPNQ0097) resuspended in water. * 40-mer me-DNA oligomeric double strand: 5'-CCTCTTCTAACTGCCAT(Me-dC)GATCCTGATAGCAGGTGCATGC-3' 5'-GCATGCACCTGCTATCAGGATCGATGGCAGTTAGAAGAGG-3'

[0255] DNMT1 Scintillation Proximity Assay (SPA) - Assay B (Human Sectioned DNMT1 (601-1600)) This assay utilized scintillation proximity technology in a signal-increasing format to evaluate the potency of the compound. Activity was monitored using human cleaved DNMT1 (601-1600), a single hemimethylated CpG site oligonucleotide, and tritiated SAM. Assay plate preparation consisted of the following parameters: 10 mM compound (11-point, 3-fold serial dilution) was pressed into a Griener white LV 384-well plate (#784075) at a rate of 100 nL per well (100× in 100% DMSO). The assay buffer mix consisting of the following was prepared on the day of the assay: base buffer: (500 mM Hepes, pH 8, 1 M MgCl2, prepared in advance and stored as stock at room temperature), 10% NP40-Surfact AMPS, 10% Ultrapure BSA 50 mg / ml, and 2 M DTT (DL-Dithiolthreitol). A 2× enzyme mix consisting of DNMT1 protein (cleaved human DNMT1-601-1600, homemade at a stock concentration of 16.876 μM) was then prepared and added to the assay buffer mix. Finally, a 2× substrate mix was prepared, consisting of 1 mM 40-mer hemimethylated DNA oligonucleotide, 12.5 μM 3H-SAM (adenosyl-L-methionine-S-[methyl-3H] specific activity 55-85 Ci / mmol), and a 32 mM solution of S-adenosyl-L-methionine (which was diluted to 1 mM in nuclease-free H2O before being added to the substrate mix), and added to the assay buffer mix (3H-SAM was added last). 5 μL of the assay buffer mix was dispensed into column 18 only using a Thermo Multidrop combi. Next, 5 μL of 2× enzyme mix was dispensed into columns 1-17 and 19-24 using a Thermo Multidrop combi. Then, 5 μL of 2× substrate mix was dispensed into the entire plate using a Thermo Multidrop combi. The plates were stacked and incubated for 40 minutes with a cover plate on top of the upper plate.The quench mix was prepared at approximately the 25-minute mark of the incubation step and consisted of a 32 mM solution of S-adenosyl-L-methionine & PerkinElmer PEI PS imaging beads (Cat. #RPNQ0098) (10 mg / ml) in nuclease-free H2O. The quench mix was vortexed before use to obtain the beads in solution. After 40 minutes of incubation, 10 μL of the quench mix was dispensed into the whole plate using a Thermo Multidrop combi. The plate was sealed with a clear seal, centrifuged at 1000 rpm / 1 min, and dark-adapted for 30 minutes. The plate was read using Viewlux (PerkinElmer, 613 nm emission filter, 300 sec double exposure (total reading time 10 min.)).

[0256] Data were analyzed using the Abase database. Responses were standardized to non-inhibitory (DMSO) and low-inhibitory controls within each plate. Dose-response curves were analyzed using 4-parameter logistic fit, and the results were obtained using pIC. 50 It was expressed as a value.

[0257] Final assay conditions: 50 mM HEPES (Teknova - H1035), pH 8.0, 2 mM MgCl2 (Sigma - M1028), 1 mM DTT (Sigma - D5545), 0.01% NP40 surfactant Amps (Themo Scientific - 28324), 0.01% BSA (Ambion - AM2618), 40 nM DNMT1 (cleaved human DNMT1 (601-1600 - GSK homemade)), 100 nM 3H-SAM (American Radiolabeled Chemicals Inc - ART 0288), 900 nM cold SAM (New England Biolabs - B9003S), and 200 nM 40-mer hemimethylated DNA oligonucleotide (Integrated DNA Technologies - 43334514).

[0258] Solubility of solid compounds in fasting-simulated intestinal fluid The solubility of solid compounds in fasting-simulated intestinal fluid (FaSSIF) was determined at pH 6.5 after equilibration at room temperature for 4 hours (using the procedure described in Sou, T.; Bergstrom, CAS Automated assays for thermodynamic (equilibrium) solubility determination. Drug Discovery Today: Technologies 2018, pp. 27, 11-19). 1 ml of FaSSIF buffer (3 mM sodium taurocholate, 0.75 mM lecithin in sodium phosphate buffer, pH 6.5) was added to 1 mg of the solid compound, manually weighed in a 4 ml vial. The resulting suspension was shaken at 900 rpm for 4 hours at room temperature, and then transferred to a Multiscreen HTS, 96-well solubility filter plate to separate the residual solid and filtrate. The compound concentrate in the filtrate was quantified by HPLC-UV using a single-point calibration of the compound at a known concentration in DMSO. Three sets of internal standards with known solubility (atovaquone, nimeslide, and warfarin, at 2, 20, and 140 μg / ml, respectively) were tested simultaneously with the compounds to evaluate process suitability. The dynamic range of the assay was 1–1000 μg / ml.

[0259] result

[0260] [Table 4] TIFF2026136150000045.tif59170

[0261] Pharmacokinetic Studies: All studies were conducted in accordance with GSK policies regarding the care, welfare, and treatment of experimental animals and reviewed by the GSK Animal Experimentation Committee or the ethical review process of the institution where the research was conducted. Pharmacokinetic studies were conducted using a non-crossover design, with non-fasted male Wistar Han rats (n=3 / administration route) in Example 2 and non-fasted male Sprague-Dawley rats (n=2 / administration route) in Reference Compound 4. Compounds were prepared as solutions in 5% DMA / 15% Solutol and administered via IV injection and forced oral administration (PO) over 60 minutes. Blood samples were collected continuously at multiple time points from before administration to 24 hours after administration, and samples were quantified by LC / MS / MS. The area under the dose-normalized curve (DNAUC) obtained from the PO administration interval was compared with the DNAUC from the IV route to determine [(PO DNAUC / IV DNAUC)]. *

[100] , the percentage bioavailability was calculated.

[0262] In vivo mouse studies - Methods: SKM-1 cells (3.8 × 10⁶) suspended in 50% Matrigel (BD Biosciences) / 50% Dulbecco's phosphate-buffered saline (DPBS). 6 ) 8-11 week old female NOD.CB17-Prkdc <scid>The implants were placed in 1NCrCrrl mice. Tumor size was measured with digital calipers, and the size was determined according to the efficacy or PK / PD study, at 219 or 1076 mm, respectively. 3 Mice were randomized into stratified blocks to receive treatment based on their mean tumor volume (P > 0.9085). GSK4172239A was mixed weekly in sterile water. Mice were measured twice a week for body weight and tumor size. Administration was started on the day of randomization. Animals were administered 22, 67, or 200 mg / kg of Example 1 twice daily (BID) by forced oral administration (PO). During the study, ≥2,500 mm in two consecutive measurements. 3 The maximum tumor load was not exceeded. In the PK / PD study, tumors, blood, and bone marrow were collected 2 hours after the 20th dose (day 10). In PK, blood was mixed with water 50:50, while tumors were homogenized in sterile water at a 1:4 dilution (Omni handheld homogenizer). Both samples were precipitated with acetonitrile, and the concentrations in Example 4 were determined by HPLC-MS / MS (Waters Acquity uPLC, Sciex API5000). SKM-1 tumor and mouse bone marrow samples were evaluated using a comprehensive DNA methylation (5-methylcytosine) LC-MS / MS assay. DNA was isolated using the Quick-DNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. For each sample, DNA Degradase Plus (Zymo Research) was added up to 1,250 ng of DNA according to the manufacturer's instructions to release individual nucleosides from the genomic DNA. Degradase-treated DNA (10 μl) was combined with 190 μl of acetonitrile / water / ammonium hydroxide (90:10:0.1) solution containing standards labeled with 100 ng / ml 2'-deoxycytidine-13C,15N2 (Toronto Research Chemicals) and 10 ng / ml 5-methyl-2'-deoxycytidine-13C,15N2 (Toronto Research Chemicals). HPLC-MS / MS method was optimized to quantify 2'-deoxycytidine and 5-methyl-2'-deoxycytidine. The samples and labeled standards were measured using a Waters Acquity UPLC on an Acquity BEH amide, 1.7 μm, 2.1 × 50 mm. 2 Separation was performed by HILIC (Hydrophilic Interaction Liquid Chromatography) using a column, followed by MS / MS analysis on a Sciex API5000 using cation turbospray ionization. The concentrations of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine were determined using standard curves generated from pure 2'-deoxycytidine (Sigma-Aldrich) and 5-methyl-2'-deoxycytidine (Santa Cruz Biotechnology). The concentration of 5-methylcytosine was standardized relative to the total cytosine concentration to determine the percentage of 5-methylcytosine. Values ​​from treated samples were standardized relative to the vehicle control.

[0263] In vivo mouse study - Results: Example 1 demonstrates in vivo activity. Example 1 was evaluated in immunodeficient mice carrying subcutaneous SKM-1 human AML (acute myeloid leukemia) xenografts. The animals were administered Example 1 at doses of 22, 67, or 200 mg / kg twice daily (BID) by forced oral administration (PO). Tumors, blood, and bone marrow were collected 2 hours after the 20th dose (10 days, n=5 animals per group) to examine pharmacokinetic (PK) and pharmacodynamic (PD) changes. Pharmacokinetic evaluation revealed that drug exposure (measured as Example 4 of the racemic active portion) was dose-proportional and approximately equivalent in blood and tumors. Furthermore, the reduction in DNA methylation resulting from the mechanism of DNMT1 inhibition was comprehensively evaluated using a 5-methylcytosine assay based on LC-MS / MS. Comprehensive DNA methylation was reduced in all doses of Example 1 compared to the vehicle, with the greatest changes observed in 49% of tumors and 47% of bone marrow in the 200 mg / kg group. In subsequent studies, tumor volume was measured twice weekly for ≥4 weeks during treatment to examine antitumor efficacy (n=10 animals per group). The vehicle group contained 9 animals on the final day, and when Example 1 was compared to the vehicle on day 22, it induced dose-dependent reduction in tumor volume due to tumor growth inhibition, ranging from a mean of 35% in the 22 mg / kg group to significant regression in the 200 mg / kg group.< / scid>

Claims

1. Equation (I) 【Chemistry 1】 Compounds thereof or their prodrugs, or pharmaceutically acceptable salts thereof.

2. Formula (II) 【Chemistry 2】 Compounds thereof or their prodrugs, or pharmaceutically acceptable salts thereof.

3. Formula (IV) 【Transformation 3】 A compound that is a prodrug of, or a pharmaceutically acceptable salt thereof.

4. Formula (V) 【Chemistry 4】 A compound that is a prodrug of, or a pharmaceutically acceptable salt thereof.

5. The prodrug according to claim 4, wherein the salt is a glycine salt.

6. The glycine salt according to claim 5, wherein the salt is anhydrous.

7. The glycine salt according to claim 6, wherein the salt is an anhydrous crystalline glycine salt.

8. The anhydrous crystalline glycine salt according to claim 7, characterized in that it substantially exhibits the XRPD pattern shown in Table 1, wherein the data in the table is plus or minus 0.

2. Table 1

9. The anhydrous crystalline glycine salt according to claim 7, characterized in that it substantially exhibits the XRPD pattern shown in Figure 1.

10. The anhydrous crystalline glycine salt according to claim 7, characterized by exhibiting an XRPD pattern having a typical diffraction peak.

11. A pharmaceutical composition comprising a compound or prodrug according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

12. A compound or prodrug according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, for use in drug therapy.

13. A compound or prodrug according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, for use in treating diseases associated with inappropriate DNMT1 activity.

14. Use of a compound or prodrug according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, for the manufacture of a pharmaceutical for use in treating diseases associated with inappropriate DNMT1 activity.

15. A method for treating a disease associated with inappropriate DNMT1 activity, comprising the step of administering a compound or prodrug according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, to a human patient in need.

16. The method according to claim 15, wherein the disease associated with inappropriate DNMT1 activity is cancer, a precancerous syndrome, or a beta-hemoglobin disorder.

17. The method according to claim 16, wherein the cancer is myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), colorectal cancer (CRC), lymphoma (e.g., non-Hodgkin lymphoma, melanoma, kidney cancer, gastric cancer, non-small cell lung cancer (NSCLC), or breast cancer).

18. The method according to claim 16, wherein the beta-abnormal hemoglobin disorder is sickle cell disease, sickle cell anemia, or beta-thalassemia.

19. A combination comprising a compound or prodrug according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, and one or more other activators.

20. A method for treating sickle cell disease, sickle cell anemia, or beta-thalassemia, comprising the step of administering a prodrug according to any one of claims 4 to 10, or a pharmaceutically acceptable salt thereof, to a human patient in need thereof.

21. A method for treating myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), colorectal cancer (CRC), non-Hodgkin lymphoma (NHL), melanoma, or breast cancer, comprising the step of administering a prodrug according to any one of claims 4 to 10, or a pharmaceutically acceptable salt thereof, to a human patient in need thereof.

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