Heterocyclic compounds for the treatment of cancer

Abstract

The present invention relates to a compound of formula (I) [Chemical Formula 1] TIFF2025524627000025.tif38170 (wherein R 1 to R 3 , M, A, Y and W are as described herein) and pharmaceutically acceptable salts thereof, as well as compositions containing the compound and methods of using the compound.

Description

Technical Field

[0001] The present invention relates to organic compounds useful for the treatment and / or prevention in mammals, specifically to the inhibition of CD73 useful for treating cancer.

[0002] Hyperactivation of the adenosine pathway impairs antitumor immunity and contributes to an immunosuppressive tumor microenvironment (TME) that limits the efficacy of immune checkpoint inhibitors. At the final stage of the adenosine pathway, the ecto-5'-nucleotidase (CD73) enzyme catalyzes the conversion of AMP to adenosine, which is recognized by adenosine receptors present on multiple immune cell types, resulting in the suppression of effector T cells and natural killer (NK) cells, the activation of regulatory T (Treg) and myeloid-derived suppressor cells (MDSC), and collectively other changes in the immune system that culminate in an immunosuppressive environment. CD73 is frequently overexpressed in cancer, and its upregulation is associated with poor clinical prognosis. Preclinical studies in various in vivo tumor models have demonstrated the restoration of immune cell function and inhibition of tumor growth upon genetic ablation or pharmacological inhibition of CD73. Therefore, alleviating the immunosuppressive TME by CD73 inhibition is thought to have therapeutic potential to restore antitumor immunity, enhance the efficacy of immunotherapy, and induce tumor regression. Considering the high and unmet need for effective cancer treatment, inhibition of CD73 activity by administration of small molecules (SM) is promising. The present disclosure describes the invention of novel small molecule CD73 inhibitors.

Summary of the Invention

[0003] The present invention relates to a novel compound of formula (I)

Chemical Formula

[0004] The compound of formula (I) exhibits good CD73 inhibition. In another embodiment, the compounds of the present invention showed excellent cancer cell inhibition. Furthermore, the compounds of formula (I) also exhibit good or improved stability, cytotoxicity, and solubility profiles in human hepatocytes.

Mode for Carrying Out the Invention

[0005] Definition The term "C 1-6 alkyl" refers to a saturated straight-chain or branched-chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. Particular "C 1-6 alkyl" groups are methyl, ethyl, and n-propyl.

[0006] The term "C 1-6 alkylene" refers to a straight-chain or branched saturated divalent hydrocarbon group of 1 to 6 carbon atoms or a divalent branched saturated divalent hydrocarbon group of 3 to 6 carbon atoms. C 1-6Examples of alkylene groups include methylene, ethylene, propylene, 2-methylpropylene, butylene, 2-ethylbutylene, pentylene, and hexylene.

[0007] The terms "halogen" and "halo" are used interchangeably herein and refer to fluoro, chloro, bromo, or iodo.

[0008] "C 3~7 The term "cycloalkyl" means a monovalent saturated monocyclic or bicyclic hydrocarbon group containing 3 to 7 ring carbon atoms. Bicyclic means consisting of two saturated carbon rings that commonly have one or more carbon atoms in common. Examples of monocyclic cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. Examples of bicyclic cycloalkyls are bicyclo[1.1.0]butyl, bicyclo[2.2.1]heptanyl, bicyclo[1.1.1]pentanyl, or bicyclo[2.2.2]octanyl.

[0009] "C 3-7 The term "cycloalkylene" means a divalent C 3-7 cycloalkyl group.

[0010] The terms "heterocyclic radical", "heterocyclic", "heterocycle", "heterocyclyl", or "heterocyclo" are used interchangeably and mean any mono-, bi-, tri-cyclic, spiro or bridged saturated, partially saturated, or unsaturated non-aromatic ring system having from 3 to 20 ring atoms, where the ring atoms are carbon and at least one atom in the ring or ring system is a heteroatom selected from nitrogen, sulfur, or oxygen. If any ring atom of the ring system is a heteroatom, the system is a heterocycle regardless of the point of attachment of the ring system to the remainder of the molecule. In one example, heterocyclyl contains from 3 to 11 ring atoms ("members"), the ring atoms are carbon, and at least one atom in the ring or ring system is a heteroatom selected from nitrogen, sulfur, or oxygen, and includes monocyclic, bicyclic, tricyclic, spiro, and bridged ring systems. In another example, heterocyclyl contains from 4 to 10, or from 5 to 10 ring atoms. In one example, heterocyclyl contains from 1 to 4 heteroatoms. In one example, heterocyclyl contains from 1 to 3 heteroatoms. In another example, heterocyclyl includes a 3- to 7-membered monocyclic ring having from 1 to 2, 1 to 3, or 1 to 4 heteroatoms selected from nitrogen, sulfur, or oxygen. In another example, heterocyclyl includes a 4- to 6-membered monocyclic ring having from 1 to 2, 1 to 3, or 1 to 4 heteroatoms selected from nitrogen, sulfur, or oxygen. In another example, heterocyclyl includes a 3-membered monocyclic ring. In another example, heterocyclyl includes a 4-membered monocyclic ring. In another example, heterocyclyl includes a 5- to 6-membered monocyclic ring. In some embodiments, heterocycloalkyl contains at least one nitrogen. In one example, a heterocyclyl group contains from 0 to 3 double bonds. Any nitrogen or sulfur heteroatom may optionally be oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom may optionally be quaternized (e.g., [NR4] + Cl - , [NR4] + OH -)). Examples of heterocycles include oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl, isoquinolinyl, tetrahydroisoquinolinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolidinonyl, 1,1-dioxoisothiazolyl, oxazolidinonyl, imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6,7-tetrahydrobenzo[d]imidazolyl, thiazinyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinonyl, pyrimidinedionyl, pyrimidine-2,4-dionyl, piperazinonyl, piperazinedionyl, pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.2] Examples include octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl, 1-azaspiro[4.5]decan-2-onyl, azaspiro[5.5]undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl, and 2,3,4a,5,7,7a-hexahydro-[1,4]dioxino[2,3-c]pyrrolyl.

[0011] The term "heterocyclylene" refers to a divalent heterocyclyl group.

[0012] The term "aryl" means a monocyclic or bicyclic ring system of a monovalent aromatic carbocyclic ring containing 6 to 10 carbon ring atoms. Examples of aryl moieties include phenyl and naphthyl. The term "arylene" means a divalent aryl group.

[0013] The term "heteroaryl" means any mono-, bi-, or tricyclic aromatic ring system containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and in an exemplary embodiment, at least one heteroatom is nitrogen. For example, Lang’s Handbook of Chemistry (Dean, J.A., ed.) 13 thPlease refer to ed.Table 7-2

[1985] . This definition includes any bicyclic group in which any of the above heteroaryl rings is fused to an aryl ring and the aryl ring or heteroaryl ring is attached to the remainder of the molecule. In one embodiment, heteroaryl includes a 5- to 6-membered monocyclic aromatic group in which one or more ring atoms are nitrogen, sulfur, or oxygen. In one embodiment, heteroaryl includes a 7- to 12-membered bicyclic aromatic group in which one or more ring atoms are nitrogen, sulfur, or oxygen. Exemplary heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[1,5-b]pyridazinyl, imidazol[1,2-a]pyrimidinyl, 1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[3,4-d]pyridazine, imidazo[1,5-a]pyrazine, imidazo[5,1-f][1,2,4]triazine, [1,2,4]triazolo[4,3-a]pyrazine, 1H-pyrazolo[3,4-c]pyridazine, 1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[4,3-d]pyrimidine, 1H-pyrazolo[3,4-c]pyridine, 1H-pyrazolo[4,3-c]pyridine and purinyl, as well as benzo-fused derivatives such as benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, indazolyl, and indolyl.

[0014] The term "heteroarylene" means a divalent heteroaryl group.

[0015] In certain embodiments, the heterocyclyl or heteroaryl group is attached at the position of a carbon atom of the heterocyclyl or heteroaryl group. For example, as a carbon-bonded heterocyclyl group, at the 2, 3, 4, 5, or 6 position of a pyridine ring, at the 3, 4, 5, or 6 position of a pyridazine ring, at the 2, 4, 5, or 6 position of a pyrimidine ring, at the 2, 3, 5, or 6 position of a pyrazine ring, at the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiophene, thiophene, pyrrole, or tetrahydropyrrole ring, at the 2, 4, or 5 position of an oxazole, imidazole, or thiazole ring, at the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole ring, at the 2 or 3 position of an aziridine ring, at the 2, 3, or 4 position of an azetidine ring, at the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline ring, or at the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline ring, the bonding arrangement is exemplified.

[0016] In certain embodiments, the heterocyclyl or heteroaryl group is N-bonded. For example, as a nitrogen-bonded heterocyclyl or heteroaryl group, aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, the bonding arrangement at the 1 position of 1H-indazole, at the 2 position of isoindole or isoindoline, at the 4 position of morpholine, and at the 9 position of carbazole or β-carboline.

[0017] In one embodiment, those skilled in the art can understand that keto-enol tautomerism exists for certain structures as shown below.

Chemical formula

[0018] Unless otherwise specified, the term "optionally substituted" means that a group may be unsubstituted or substituted with one or more (e.g., 0, 1, 2, 3, 4, or 5 or more, or any range variable thereof) substituents (the substituents may be the same or different) listed for that group. In one embodiment, the optionally substituted group has one substituent. In another embodiment, the optionally substituted group has two substituents. In another embodiment, the optionally substituted group has three substituents. In another embodiment, the optionally substituted group has four substituents. In another embodiment, the optionally substituted group has five substituents.

[0019] The term "protecting group" or "PG" refers to a group that selectively blocks a reaction site in a polyfunctional compound such that a chemical reaction can be carried out selectively at another unprotected reaction site in the meaning customarily associated with synthetic chemistry. The protecting group can be removed at an appropriate point. Exemplary protecting groups are amino protecting groups, carboxy protecting groups, or hydroxy protecting groups.

[0020] The term "pharmaceutically acceptable salt" means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid addition salts and base addition salts.

[0021] The term "pharmaceutically acceptable acid addition salt" refers to a pharmaceutically acceptable salt formed from an organic acid selected from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, including aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic acids.

[0022] The term "pharmaceutically acceptable basic addition salt" means a pharmaceutically acceptable salt formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperidine, piperazine, N-ethylpiperidine, and polyamine resins.

[0023] The term "pharmaceutically active metabolite" refers to a pharmacologically active product produced through the metabolism in vivo of a specific compound or its salt. After entering the body, most drugs serve as substrates for chemical reactions that can change their physical properties and biological effects. These metabolic conversions usually affect the polarity of the compounds of the present invention and change the way the drugs are distributed in and excreted from the body. However, in some cases, drug metabolism is required for a therapeutic effect.

[0024] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the present invention which, when administered to a subject, (i) treats or prevents a specific disease, symptom or disorder, (ii) attenuates, alleviates or eliminates one or more symptoms of a specific disease, symptom or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, symptom or disorder described herein. A therapeutically effective amount will vary depending on the compound, the state of the disease being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

[0025] The term "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient together with pharmaceutically acceptable additives, which is administered to a mammal, such as a human in need thereof.

[0026] The terms "pharmaceutically acceptable additive", "pharmaceutically acceptable carrier" and "therapeutically inert additive" are used interchangeably and mean any pharmaceutically acceptable ingredient used in the formulation of a medicament that has no therapeutic activity and is non-toxic to the subject being administered, such as a disintegrant, binder, filler, solvent, buffer, isotonic agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant.

[0027] Inhibitor of CD73 The present invention relates to (i) a compound of formula (I)

Chemical formula

[0028] A further embodiment of the present invention is (ii) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, C 1-6 alkyl or -L 1 -R 2 wherein L 1 is C 1-6 alkylene and R 2 is phenyl.

[0029] A further embodiment of the present invention is (iii) a compound of formula (I) according to (i) or (ii), or a pharmaceutically acceptable salt thereof, wherein R 1 is methyl, isopropyl or benzyl.

[0030] A further aspect of the present invention is (iv) a compound of formula (I) described in any one of (i) to (iii), or a pharmaceutically acceptable salt thereof, wherein R 3 is H or C 1-6 alkyl.

[0031] A further embodiment of the present invention is (v) a compound of formula (I) described in any one of (i) to (iv), wherein R 3 is H or methyl.

[0032] A further embodiment of the present invention is a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of (vi)(i) to (v), wherein A is CH.

[0033] A further embodiment of the present invention is a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of (vii)(i) to (vi), wherein W is N.

[0034] A further embodiment of the present invention is a compound of formula (I) (wherein M is NR 1 and R 1 is C 1-6 alkyl or -L 1 -R 2 where L 1 is C 1-6 alkylene, R 2 is phenyl, Y is NR 3 and R 3 is H or C 1-6 alkyl, A is CH, W is N), or a pharmaceutically acceptable salt thereof.

[0035] A further embodiment of the present invention is a compound of formula (I) (wherein M is NR 1 and R 1 is methyl, isopropyl or benzyl, Y is NR 3 and R 3 is H or methyl, A is CH, W is N), or a pharmaceutically acceptable salt thereof.

[0036] Another aspect of the present invention is a compound of formula (I) selected from (x) the following 5-(5-Methyl-6-oxo-7H-imidazo[][4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; 5-(5,7-Dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; 5-(5-Isopropyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; and 5-(5-Benzyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; or a pharmaceutically acceptable salt thereof.

[0037] Another embodiment of the present invention relates to a method for preparing a compound according to any one of (i) to (x), which comprises the following steps (xi). a) Deprotection of the compound of formula (VII) by an acid or by hydrogenation

Chemical formula

Chemical formula

[0038] Another embodiment of the present invention is a compound according to any one of (i) to (x) or a pharmaceutically acceptable salt thereof for use as a therapeutic active substance.

[0039] Another embodiment of the present invention relates to a pharmaceutical composition comprising a compound according to any one of (i) to (x) and a pharmaceutically acceptable additive.

[0040] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) for treating (xiv) cancer.

[0041] Another embodiment of the present invention is the use according to (xv), which is the use described in (xiv), and the cancer is pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, or melanoma.

[0042] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) for inhibiting (xvi) CD73.

[0043] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) for preparing a medicament for treating or preventing (xvii) cancer, and the cancer is pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, melanoma, multiple myeloma, acute myeloid leukemia, or acute and chronic lymphoblastic leukemia.

[0044] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) for preparing a medicament as an (xviii) CD73 inhibitor.

[0045] Another embodiment of the present invention is a compound according to any one of (i) to (x) or a pharmaceutically acceptable salt thereof when produced according to the method of (xix) (xi).

[0046] Pharmaceutical Compositions and Administration Another embodiment provides a pharmaceutical composition or medicament containing a compound of the present invention and a therapeutically inert carrier, diluent or additive, and a method of using a compound of the present invention for preparing such a composition and medicament. In one example, a compound of formula (I) can be formulated into a herbal dosage form by mixing it with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used, at ambient temperature, at an appropriate pH, and at the desired degree of purity. The pH of the formulation mainly depends on the specific application and the concentration of the compound, but is preferably somewhere in the range of about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate buffer at pH 5. In another embodiment, a compound of formula (I) is sterile. The compound can be stored, for example, as a solid or amorphous composition, as a lyophilized formulation, or as an aqueous solution.

[0047] The composition is formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to medical practitioners. The "effective amount" of the compound to be administered is determined by such considerations and is the minimum amount necessary to inhibit the enzymatic activity of the CD73 protein when converting AMP to adenosine. In one example, the pharmaceutically effective amount of a compound of the present invention administered parenterally per dose ranges from about 0.01 to 100 mg / kg of the patient's body weight per day, or from about 0.1 to 50 mg / kg of the patient's body weight per day, and a typical initial range for the compound used is 0.3 to 30 mg / kg / day. In another embodiment, oral unit dosage forms such as tablets and capsules preferably contain from about 1 to about 1000 mg of a compound of the present invention.

[0048] The compounds of the present invention can be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intraluminal, intradermal, intrathecal and epidural, as well as intranasal, and, if desired for local treatment, intralesional administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0049] The compounds of the present invention can be administered in any convenient dosage form, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions can contain ingredients conventional in pharmaceutical preparations, for example, diluents, carriers, pH adjusters, sweeteners, bulking agents and additional active agents.

[0050] Typical formulations are prepared by mixing the compounds of the present invention with carriers or additives. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel, Howard C. et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004, Gennaro, Alfonso R. et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000, and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations can also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, flow promoters, processing aids, colorants, sweeteners, flavors, fragrances, diluents and other known additives for providing an aesthetically pleasing presentation of the drug (i.e., the compound of the present invention or its pharmaceutical composition) or for assisting in the manufacture of a pharmaceutical product (i.e., a pharmaceutical).

[0051] Examples of suitable oral dosage forms are tablets containing from about 0.1 to 500 mg of anhydrous lactose, from about 0.1 to 500 mg of croscarmellose sodium, from about 0.1 to 500 mg of polyvinylpyrrolidone (PVP) K30, and from about 0.1 to 500 mg of magnesium stearate, and from about 0.1 mg to 500 mg of the compound of the present invention. The powdered components are first mixed together and then mixed with a solution of PVP. The resulting composition can be dried, granulated, mixed with magnesium stearate, and compressed into tablet form using conventional equipment. Examples of aerosol formulations can be prepared, for example, by dissolving from 1 to 450 mg of the compound of the present invention in a suitable buffer solution, such as phosphate buffer, and adding salts, such as sodium chloride, as an isotonic agent if desired. The solution can be filtered, for example, using a 0.2 micron filter to remove impurities and contaminants.

[0052] Accordingly, one embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof. In a further embodiment, it includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or additive.

[0053] Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of cancer. Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of cancer.

[0054] The following embodiments illustrate typical compositions of the present invention but serve merely as representatives thereof.

[0055] Composition A The compound of the present invention can be used as an active ingredient in a manner known per se for producing tablets of the following composition. 425 mg per tablet Active ingredient 200 mg Microcrystalline cellulose 155 mg Corn starch 25 mg 25 mg of talc 20 mg of hydroxypropylmethylcellulose

[0056] Composition B The compound of the present invention can be used as an active ingredient in a manner known per se for producing capsules of the following composition. 220.0 mg per capsule 100.0 mg of active ingredient 20.0 mg of corn starch 95.0 mg of lactose 4.5 mg of talc 0.5 mg of magnesium stearate

[0057] Indications and treatment methods The compound of the present invention inhibits the enzymatic activity of CD73 when converting AMP to adenosine. Therefore, the compound of the present invention is useful for reducing the adenosine level in the TME. The compound of the present invention is useful for promoting the immune-mediated killing of cancer cells that overexpress CD73, such as pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, melanoma, multiple myeloma, acute myeloid leukemia, or acute and chronic lymphoblastic leukemia. Alternatively, the compound of the present invention is useful for promoting the immune-mediated killing in cancer cells that depend on the adenosine pathway or in malignant solid tumors in which the adenosine pathway is enhanced by dysregulation or mutation of effector pathways such as EGFR-RAS-MAPK and PI3K-AKT-driven signaling for targeted therapy in pancreatic adenocarcinoma, non-small cell lung cancer, esophageal and gastric adenocarcinoma, etc. More broadly, the compound can be used for the treatment and prevention of all cancer types exhibiting an immunosuppressive TME.

[0058] Another embodiment includes a method of treating or preventing cancer in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a compound of formula (I), stereoisomer, tautomer, or a pharmaceutically acceptable salt thereof.

[0059] Synthesis The compounds of the present invention can be prepared by any conventional means. Suitable methods for synthesizing these compounds as well as their starting materials are shown in the following schemes and examples. All substituents, especially R 1 from R 3 to R

[0060] A general synthetic route for preparing the compounds of formula (I) is shown below.

[0061] Scheme 1

Chemical formula

[0062] Scheme 2 [Chemistry] Alternatively, the compound of formula (VIII) can be prepared according to Scheme 2. The compound of formula (IX) can be converted to the compound of formula (X) via a condensation reaction with a condensing reagent such as CDI or triphosgene in the presence of a base such as DIEA or TEA. The Suzuki coupling of the halide (X) and the boronic acid (VI) is achieved in the presence of a catalyst such as Pd(dppf)Cl2 and cataCXium-A-Pd-G3 and a base such as Cs2CO3 and K3PO4, and can provide the compound of formula (XI). The selective N-substitution reaction or metal-catalyzed coupling reaction (e.g., Buchwald-Hartwig amination, or Ullmann coupling) of the compound of formula (XI) with the halide R 1 X, or the Mitsunobu reaction of the compound of formula (XI) with the alcohol R 1 OH, or the Chan-Lam coupling of the compound of formula (XI) with the boronic acid R 1 B(OH)2 can afford the compound of formula (VII). Deprotection of the compound of formula (VII) by removing PG1 with an acid such as HCl or hydrogenation gives the final compound of formula (VIII).

[0063] Scheme 3 [Chemistry] Alternatively, the compound of formula (VIII) can be prepared according to Scheme 3. The compound of formula (X) can be subjected to a selective N-substitution reaction or metal-catalyzed coupling reaction (e.g., Buchwald-Hartwig amination, or Ullmann coupling) with the halide R 1 X, or the Mitsunobu reaction of the compound of formula (X) with the alcohol R 1 OH, or the reaction of the compound of formula (X) with the boronic acid R 1It can be converted to the compound of formula (V) by Chan-Lam coupling with B(OH)₂. The subsequent coupling of the halide (V) and the boronic acid (VI) can be achieved by Suzuki coupling conditions using catalysts such as Pd(dppf)Cl₂ and cataCXium-A-Pd-G3 and bases such as Cs₂CO₃ and K₃PO₄ to obtain the compound of formula (VII). Deprotection or hydrogenation of the compound of formula (VII) to remove PG1 with an acid such as HCl gives the final compound of formula (VIII).

[0064] The compounds of the present invention can be obtained as mixtures of diastereomers or enantiomers, which can be separated by methods well known in the art, such as (chiral) HPLC or SFC. In another embodiment, the compounds of formula (I) can be obtained according to the above scheme by using the corresponding chiral starting materials.

[0065] The present invention also relates to a method for the preparation of a compound of formula (I) comprising the following steps: a) Deprotection of the compound of formula (VII) by an acid or by hydrogenation

Chemical formula

Chemical formula

[0066] The compounds of formula (I) when produced by the above method are also an object of the present invention.

Examples

[0067] The present invention will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present invention.

[0068] Abbreviations The present invention will be more fully understood by referring to the following examples. However, these examples should not be construed as limiting the scope of the present invention.

[0069] The abbreviations used in this specification are as follows: ACN: Acetonitrile aq. Aqueous Boc2O: Di-tert-butyl dicarbonate CataCXium-A-Pd-G3 [(Di(1-adamantyl)-butylphosphine)-2-(2’-amino-1,1’-biphenyl)] palladium(II) methanesulfonate CDI: N,N’-Carbonyldiimidazole DCM: Dichloromethane DIPEA or DIEA: N,N-Diisopropylethylamine DMA: N,N-Dimethylacetamide Pd(dppf)Cl2 . DCM: [1,1’-Bis(diphenylphosphino)ferrocene] dichloropalladium(II), complex with dichloromethane EA or EtOAc: Ethyl acetate FA: Formic acid IC 50 : 50% inhibitory concentration LCMS: Liquid chromatography mass spectrometry MS: Mass spectrometry PE: Petroleum ether Preparative HPLC: Preparative high performance liquid chromatography Preparative TLC: Preparative thin layer chromatography rt: Room temperature RT: Retention time SFC: Supercritical fluid chromatography TFA: Trifluoroacetic acid TLC: Thin layer chromatography v / v: Volume ratio

[0070] General experimental conditions The intermediates and final compounds were purified using the following equipment: i) Biotage SP1 system and Quad12 / 25 cartridge module, ii) ISCO combi-flash chromatography equipment, silica gel brand and pore size: i) KP-SIL 60 Å, particle size: 40 - 60 μm; ii) CAS registration number: silica gel: 63231-67-4, particle size: 47 - 60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd., pore size: 200 - 300 or 300 - 400.

[0071] The intermediates and final compounds were purified by preparative HPLC on a reverse-phase column using an XBridge™ Prep-C18 (5 μm, OBD™ 30×100 mm) column, a SunFire™ Prep-C18 (5 μm, OBD™ 30×100 mm) column, a Phenomenex Synergi-C18 (10 μm, 25×150 mm) or a Phenomenex Gemini-C18 (10 μm, 25×150 mm). A Waters AutoP purification system (sample manager 2767, pump 2525, detector: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water; acetonitrile and 0.1% FA in water or acetonitrile and 0.1% TFA in water), or a Gilson-281 purification system (pump 322, detector: UV 156, solvent system: acetonitrile and 0.05% ammonium hydroxide in water; acetonitrile and 0.225% FA in water; acetonitrile and 0.05% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water), was used for Prep-HPLC on a reverse-phase column.

[0072] For SFC chiral separation, the intermediate was separated using a chiral column (Daicel chiralpak IC, 5 μm, 30×250 mm), AS (10 μm, 30×250 mm) or AD (10 μm, 30×250 mm), with a Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar80 preparative SFC, solvent system: CO2 and IPA (0.5% TEA in IPA) or CO2 and MeOH (0.1% NH3·H2O in MeOH), back pressure 100 bar, and detection ultraviolet light at 254 or 220 nm.

[0073] The LC / MS spectra of the compounds were obtained using LC / MS (Waters™ Alliance 2795 - Micromass ZQ, Shimadzu Alliance 2020 - Micromass ZQ or Agilent Alliance 6110 - Micromass ZQ), and the LC / MS conditions were as follows (run time 3 minutes or 1.5 minutes). Acidic condition I: A: 0.1% TFA in H2O, B: 0.1% TFA in acetonitrile, Acidic condition II: A: 0.0375% TFA in H2O, B: 0.01875% TFA in acetonitrile, Basic condition I: A: 0.1% NH3·H2O in H2O, B: acetonitrile, Basic condition II: A: 0.025% NH3·H2O in H2O, B: acetonitrile, Neutral condition: A: H2O, B: acetonitrile.

[0074] Mass spectrum (MS): Generally, only the ions indicating the parent mass are reported, and unless otherwise stated, the mass ions cited are the positive mass ions (MH) + is.

[0075] The NMR spectra were obtained using a Bruker Avance 400 MHz or 500 MHz.

[0076] The microwave-assisted reaction was carried out on a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were conducted under an argon or nitrogen atmosphere. Reagents were used as received from commercial suppliers without further purification, unless otherwise specified.

[0077] Preparation Example The following examples are intended to illustrate the meaning of the present invention but in no way represent a limitation within the scope of the meaning of the present invention.

[0078] Example 1 5-(5-Methyl-6-oxo-7H-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione [Chemical formula] The title compound was synthesized according to the following scheme: [Chemical formula]

[0079] Step (a): Preparation of 6-chloro-N 4 -methyl-pyridazine-3,4-diamine (Compound 1.2) To a solution of 4-bromo-6-chloro-pyridazin-3-amine (Compound 1.1, 5.0 g, 24.0 mmol) in 1-butanol (20 mL), DIEA (8.4 mL, 48.0 mmol) and methylamine (2 M in THF, 24.0 mL, 48.0 mmol) were added under a N2 atmosphere to obtain a brown solution. The reaction mixture was stirred at 80 °C for 16 h, then concentrated and purified by perp-HPLC to obtain Compound 1.2 (3.5 g). MS: calculated value 159.0 [(M+H) + , measured value 159.3 [(M+H) + .

[0080] Step (b): Preparation of 3-chloro-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.3) 6-Chloro-N 4 -Methyl-pyridazine-3,4-diamine (Compound 1.2, 200.0 mg, 1.26 mmol) in THF (4 mL) was added with CDI (250.0 mg, 1.54 mmol) at room temperature. The reaction mixture was stirred at room temperature for 5 h, then concentrated, and the obtained residue was purified by preparative HPLC to obtain Compound 1.3 (80.0 mg). MS: calculated value 185.0 [(M+H) + , measured value 185.0 [(M+H) + .

[0081] Step (c): Preparation of 3-(2,4-dimethoxypyrimidin-5-yl)-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.5) To a solution of 3-chloro-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.3, 80.0 mg, 0.43 mmol) in 1,4-dioxane (2 mL) and water (0.4 mL), 2,4-dimethoxypyrimidine-5-boronic acid (Compound 1.4, 87.7 mg, 0.48 mmol), Cs2CO3 (423.6 mg, 1.3 mmol) and Pd(dppf)Cl2 . DCM (35.4 mg, 0.04 mmol) were added under N2. The obtained mixture was stirred at 80 °C for 4 h, then quenched with water and extracted 3 times with EA. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative HPLC to obtain Compound 1.5 (20.0 mg, yield 16.0%). MS: calculated value 289.1 [(M+H) + , measured value 289.1 [(M+H) + .

[0082] Step (d): Preparation of 5-(5-methyl-6-oxo-7H-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 1) To a solution of 3-(2,4-dimethoxypyrimidin-5-yl)-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.5, 20.0 mg, 0.07 mmol) in methanol (0.2 mL) was added 2M HCl (0.5 mL, 1.0 mmol). After stirring at 60 °C for 1 hour, a precipitate formed, and the solid was collected by filtration, washed with MeOH, and lyophilized to give Example 1 (2.22 mg). MS: calculated 261.1 [(M+H) + , measured 261.0 [(M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ = 12.7 (s, 1H), 12.05 - 11.92 (m, 1H), 11.78 (brs, 1H), 8.36 (brs, 1H), 8.05 (s, 1H), 3.37 (s, 3H).

[0083] Example 2 5-(5,7-Dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione

Chemical formula

Chemical formula

[0084] Step (a): Preparation of 3-chloro-5,7-dimethyl-imidazo[4,5-c]pyridazin-6-one (Compound 2.1) To a solution of 3-chloro-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.3, 300.0 mg, 1.63 mmol) in DMF (3 mL) was added sodium hydride (60% dispersion in mineral oil, 130.0 mg, 3.25 mmol) at 0 °C. The reaction mixture was stirred for an additional 30 minutes, then iodomethane (346.0 mg, 2.44 mmol) was added at 0 °C. The resulting mixture was stirred at room temperature for 1 hour, then quenched with water and extracted three times with EA. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: 0% to 20% MeOH in DCM) to give Compound 2.1 (220.0 mg, yield 68.2%). MS: calculated value 199.0 [(M+H) + , measured value 199.0 [(M+H) + .

[0085] Step (b): Preparation of 5-(5,7-dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 2) In Step (c), 5-(5,7-dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 2) was prepared in the same manner as in Example 1 by replacing 3-chloro-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.3) with 3-chloro-5,7-dimethyl-imidazo[4,5-c]pyridazin-6-one (Compound 2.1). 10.6 mg of Example 2 was obtained. MS: calculated value 275.1 [(M+H) + , measured value 275.2 [(M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ = 11.45 (s, 1H), 8.13 (s, 1H), 7.86 (s, 1H), 3.42 (s, 3H), 3.35 (s, 3H).

[0086] Example 3 5-(5-Isopropyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione [Chemical formula] The title compound was synthesized according to the following scheme: [Chemical formula]

[0087] Step (a): Preparation of 6-chloro-N 4 -isopropyl-pyridazine-3,4-diamine (Compound 3.1) To a solution of 4-bromo-6-chloro-pyridazine-3-amine (Compound 1.1, 5.0 g, 23.99 mmol) in n-BuOH (20 mL), DIEA (8.4 mL, 47.98 mmol) and isopropylamine (2.41 g, 40.78 mmol) were added at room temperature. The resulting mixture was stirred at 120 °C for 12 hours. After cooling to room temperature, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: 50% to 100% EA in PE) to obtain Compound 3.1 (1.2 g). 1 1H NMR (400 MHz, DMSO-d6) δ = 6.33 (s, 1H), 6.10 (s, 2H), 5.97 (br d, J = 7.2 Hz, 1H), 3.72 - 3.61 (m, 1H), 1.17 (d, J = 6.4 Hz, 6H).

[0088] Step (b): Preparation of 3-chloro-5-isopropyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 3.2) 6-chloro-N 4 -isopropyl-pyridazine-3,4-diamine (Compound 3.1, 0.50 g, 2.68 mmol) in THF (20 mL) was added CDI (531.1 mg, 3.28 mmol) at room temperature. The resulting mixture was stirred at room temperature for 5 hours and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: 50% to 75% EA in PE) to obtain Compound 3.2 (400.0 mg, yield 68.4%). MS: calculated value 213.0, 215.0 [M+H] + Measured value 213.2, 215.2 [M+H] + .

[0089] Step (c): Preparation of 3-chloro-5-isopropyl-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 3.3) To a solution of 3-chloro-5-isopropyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 3.2, 350.0 mg, 1.65 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 131.7 mg, 3.29 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 30 minutes, then iodomethane (350.4 mg, 2.47 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then quenched by the gradual addition of aqueous saturated NH4Cl (20 mL) and extracted three times with EA (20 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (PE / EA = 1 / 1) to give Compound 3.3 (130.0 mg). MS: calculated 227.1, 229.1 [(M+H) + , measured 227.1, 229.1 [(M+H) + .

[0090] Step (d): Preparation of 3-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 3.4) To a solution of 3-chloro-5-isopropyl-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 3.3, 130.0 mg, 0.57 mmol) in 1,4-dioxane (2 mL) and water (0.4 mL) were added 2,4-dimethoxypyrimidine-5-boronic acid (Compound 1.4, 116.06 mg, 0.63 mmol), Cs2CO3 (560.6 mg, 1.72 mmol) and Pd(dppf)Cl2 .DCM (46.9 mg, 0.06 mmol) was added under N2. The resulting mixture was stirred at 80 °C for 12 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: 50% to 80% EA in PE) to give compound 3.4 (180.0 mg). MS: calcd 331.1 [(M+H) + , found 331.1 [(M+H) + .

[0091] Step (e): Preparation of 5-(5-isopropyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 3) To a solution of 3-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 3.4, 91.7 mg, 0.28 mmol) in methanol (0.2 mL) was added 2M HCl (2.0 mL, 4.0 mmol). The resulting mixture was stirred at 60 °C for 1 h, then aqueous ammonia (1 mL) and water (2 mL) were added. The formed solid was collected by filtration, washed with MeOH and lyophilized to give Example 3 (38.16 mg). MS: calcd 303.1 [(M+H) + , found 303.1 [(M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ = 11.47 (s, 1H), 8.13 (s, 1H), 7.96 (s, 1H), 4.63 - 4.56 (m, 1H), 3.41 (s, 3H), 1.44 (d, J = 6.8 Hz, 6H).

[0092] Example 4 5-(5-Benzyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione

Chemical formula

Chemical formula

[0093] Step (a): Preparation of 3-chloro-7-methyl-5H-imidazo[4,5-c]pyridazin-6-one (Compound 4.2) 6-chloro-N 3 To a solution of THF (30 mL) containing 6-chloro-N-methyl-pyridazine-3,4-diamine (Compound 4.1, 3.0 g, 18.92 mmol) were added CDI (3.75 g, 23.13 mmol) and DIEA (6.6 mL, 37.83 mmol). The resulting mixture was stirred at 80 °C for 12 h and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: 0% to 30% MeOH in DCM) to give Compound 4.2 (2.9 g). MS: calculated 185.0 [(M+H) + , measured 185.0 [(M+H) + .

[0094] Step (b): Preparation of 3-(2,4-di-tert-butoxypyrimidin-5-yl)-7-methyl-5H-imidazo[4,5-c]pyridazin-6-one (Compound 4.4) To a solution of 1,4-dioxane (30 mL) and water (5 mL) containing (2,4-di-tert-butoxypyrimidin-5-yl)boronic acid (Compound 4.3, 2.61 g, 9.75 mmol) were added 3-chloro-7-methyl-5H-imidazo[4,5-c]pyridazin-6-one (Compound 4.2, 1.5 g, 8.13 mmol), Cs2CO3 (7.94 g, 24.38 mmol) and Pd(dppf)Cl2 . DCM (0.66 g, 0.81 mmol) under N2. The resulting mixture was stirred at 80 °C for 12 h. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: 20% to 100% EA in PE) to give Compound 4.4 (900.0 mg). MS: calculated 373.2 [(M+H) + , measured 373.1 [(M+H) + .

[0095] Step (c): Preparation of 5-benzyl-3-(2,4-di-tert-butoxypyrimidin-5-yl)-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 4.5) To a solution of 3-(2,4-di-tert-butoxypyrimidin-5-yl)-7-methyl-5H-imidazo[4,5-c]pyridazin-6-one (Compound 4.4, 100.0 mg, 0.27 mmol) in THF (5 mL) was added NaH (60% dispersion in mineral oil, 21.5 mg, 0.54 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 30 minutes and then benzyl bromide (0.04 mL, 0.32 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then quenched by the gradual addition of aqueous saturated NH4Cl (20 mL) and extracted three times with EA (20 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC to give Compound 4.5 (40.0 mg, yield 32.2%). MS: calculated value 463.2 [(M+H) + , measured value 463.2 [(M+H) + .

[0096] Step (d): Preparation of 5-(5-benzyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 4) To a solution of 5-benzyl-3-(2,4-di-tert-butoxypyrimidin-5-yl)-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 4.5, 40.0 mg, 0.09 mmol) in methanol (0.2 mL) was added 2M HCl (1.0 mL, 2.0 mmol). The mixture was stirred at room temperature for 1 hour and then aqueous ammonia (1 mL) and water (2 mL) were added to the reaction mixture. The solid formed was collected by filtration, washed with MeOH, and lyophilized to give Example 4 (15.99 mg). MS: calculated value 351.1 [(M+H) + , measured value 351.2 [(M+H) + . 11H NMR (400 MHz, DMSO-d6) δ = 11.56 - 11.31 (m, 2H), 8.13 (d, J = 6.0 Hz, 1H), 7.85 (s, 1H), 7.35 - 7.28 (m, 5H), 5.10 (s, 2H), 3.46 (s, 3H).

[0097] Biological Examples Example 5: CD73 Biochemical Assay Using an Echo 555 liquid handler (Labcyte), serial dilutions (1:3) of the compound were prepared in the corresponding wells of a 384-well plate. 25 μL of the enzyme working solution [0.2 nM recombinant CD73 protein (purchased from R&D Systems, Inc.), 25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.01% BSA, 0.01% Brij-35 nonionic surfactant] was added to the assay plate and incubated with the compound at room temperature for 15 minutes. After adding 25 μL of the AMP working solution (10 μM AMP, 25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.01% BSA, 0.01% Brij-35) to the assay plate, the reaction was incubated at room temperature for 10 minutes. Then, 10 μL of the malachite green A solution was added to each well for a 10-minute incubation. 10 μL of the malachite B solution was added to each well and then incubated for an additional 30 minutes. Finally, the absorbance values were read at 620 nM using an Envision plate reader (PerkinElmer). The percent inhibition was calculated using the formula {% Inhibition = 100 × [I - (X - MIN) / (MAX - MIN)]}. Where X is equal to the well signal, Max is equal to the signal of the DMSO control, and MIN is equal to the signal without the cell control. [Table 1]

[0098] Example 6: CD73 Cell Assay Using an Echo 555 liquid handler (Labcyte), serial dilutions (1:3) of the compounds were prepared in the corresponding wells of a 384-well plate. 40 μL of MDA-MB-231 cells (ATCC, HTB-26, breast cancer, final concentration 20,000 cells / mL) suspended in assay buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.01% BSA, 0.01% Brij-35) were added to the corresponding wells of the plate. After incubation with the compounds for 30 minutes, 40 μL of AMP working solution (10 μM AMP, 25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.01% BSA, 0.01% Brij-35) was added to each well of the assay plate. The assay plate was then incubated at 37 °C for 45 minutes in a 5% CO2 incubator (Thermo Fisher Scientific). After the reaction was complete, 50 μL of the supernatant was collected and transferred to a new 384-well plate. 10 μL of malachite A was added to each well of the assay plate and incubated for 10 minutes. Then, 10 μL of malachite B was added to each corresponding well of the plate and incubated for 30 minutes. Finally, the absorbance values were read at 620 nM using an Envision plate reader. Calculated inhibition rate using the formula {% inhibition = 100 × [I - (X - MIN) / (MAX - MIN)]}. Where X is equal to the well signal, Max is equal to the signal of the DMSO control, and MIN is equal to the signal without cell control.

Table 2

[0099] Example 7: CD73 LC / MS Assay The purpose of this assay is to identify and characterize inhibitors of CD73 enzyme activity. Using an Echo 555 liquid handler (Labcyte), serial dilutions (1:3) of compounds were prepared in the corresponding wells of a 384-well plate. 12.5 μL of enzyme working solution (containing recombinant CD73 protein, 10 mM Tris pH 7.5, 100 mM NaCl, 0.01% BSA, 0.2 mM octyl glucoside) was added to the assay plate and incubated with the compounds for 15 minutes at room temperature. After adding 15 μL of AMP working solution (containing 10 mM Tris pH 7.5, 100 mM NaCl, 0.01% BSA, 0.2 mM octyl glucoside regardless of the presence or absence of NaH2PO4), the assay was incubated for 10 minutes at room temperature. The reaction was stopped by adding 75 μL of stop solution (5% TCA in H2O containing 250 nM 13C5-adenosine) to each well for a 10-minute incubation. After centrifugation, 75 μL of the mixture was transferred to a new 384-well plate for LC / MS analysis.

[0100] Samples from the 384-well plate were loaded onto the autosampler deck and then injected into the ADDA-LC-MS / MS. The aqueous mobile phase is 0.1% formic acid in water. The organic mobile phase is 0.1% formic acid in acetonitrile. The flow rate was maintained at 0.8 mL / min using a Shimadzu pump. The column is an ACE 5 Phenyl, 50×2.1 mm. The analysis was performed on a SCIEX triple quadrupole mass spectrometer operating in positive ion mode. The effluent from the HPLC column was introduced directly into electrospray ionization (ESI). Multiple reaction monitoring (MRM) was used to determine the analyte and internal standard (IS) responses. The MRM for adenosine is 268.1 / 136.1 and the MRM for 13C5-adenosine (IS) is 273.2 / 136.2. The data was calculated using the peak area ratio (PAR) semi-quantification method.

[0101] Example 8: Cell Proliferation Assay The purpose of this assay is to characterize the efficacy of an inhibitor of CD73 in the rescue of adenosine-mediated inhibition of T cell proliferation. CD4+ or CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMC, HemaCare) by immunomagnetic negative selection using the EasySep™ Isolation Kit (STEMCELL Technologies) according to the supplier's protocol. CD4+ or CD8+ T cells were pelleted by centrifugation at 300 g for 10 minutes at room temperature and resuspended in PBS. CellTrace™ Violet staining solution (Invitrogen) was added at 1:2,000, protected from light, and incubated at 37 °C for 20 minutes. Then, complete culture medium [RPMI-1640 (Gibco), 10% fetal bovine serum (Gibco), 2 mM GlutaMAX (Gibco) and 1 mM sodium pyruvate (Gibco), 100 U / mL penicillin-streptomycin (Gibco) and MEM non-essential amino acids (NEAA) cell culture supplement (1:100, Gibco)] was added, mixed, and incubated at 37 °C for 5 minutes. The cells were then pelleted by centrifugation at 300 g for 10 minutes at room temperature and resuspended in fresh pre-warmed complete culture medium. 50 μL of cells were seeded per well in a 96-well U-bottom plate. 50 μL of medium containing CD3 / CD28 beads was added to each well and incubated overnight at 37 °C in a 5% CO2 incubator. 50 μL of medium containing the compound was added to the cells. 50 μL of medium containing AMP and EHNA hydrochloride (Sigma-Aldrich) were added to the cells at final concentrations of 200 μM and 5 μM, respectively. The cells were incubated at 37 °C for 72 hours in a 5% CO2 incubator. Then, 200 μL of PBS was added to each well and the cells were centrifuged at 300 g for 10 minutes at 4 °C. The supernatant was discarded. 50 μL of Human TruStain FcX™ (Fc receptor blocking solution, BioLegend) diluted 1:100 in PBS was added to each well, gently mixed, and incubated at 4 °C for 20 minutes.50 μL of staining solution (BioLegend) was added to each well, gently mixed, and incubated at 4 °C for 30 minutes. The cells were centrifuged at 300 g for 10 minutes at 4 °C, and the supernatant was discarded. The cell pellet was washed with 250 μL of cell staining buffer and centrifuged at 300 g for 10 minutes at 4 °C. The supernatant was discarded, and the cells were resuspended in 60 μL of cell staining buffer and analyzed by flow cytometry.

[0102] Example 9: T cell cytokine release function assay The purpose of this assay is to characterize the efficacy of inhibitors of CD73 in the rescue of adenosine-mediated inhibition of T cell cytokine release function. CD4+ or CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic negative selection using the EasySep™ Isolation Kit (STEMCELL Technologies) according to the supplier's protocol. The CD4+ or CD8+ T cells were then pelleted by centrifuging the cells at 300 g for 10 minutes at room temperature and resuspended in fresh pre-warmed complete culture medium. 50 μL of cells were seeded per well in a 96-well U-bottom plate. 50 μL of medium containing CD3 / CD28 beads was added to each well and incubated overnight at 37 °C in a 5% CO2 incubator. 50 μL of medium containing the compound was added to the cells. 50 μL of medium containing AMP and EHNA hydrochloride (Sigma-Aldrich) was added to the cells at final concentrations of 200 μM and 5 μM, respectively. The cells were incubated at 37 °C for 72 hours in a 5% CO2 incubator. 50 μL of supernatant was collected, and the levels of IL2 and IFN gamma were determined using an ELISA-MSD kit (Meso Scale Discovery).

Claims

1. A compound of formula (I) 【Chemical 1】 wherein M is NR １ where R １ is C １－６ alkyl, C ３－７ cycloalkyl, C ３－７ cycloalkyl C １－６ alkyl, or -L １ -R ２ where L １ is C １－６ alkylene, C ３－７ cycloalkylene, heterocyclylene or heteroarylene, and R ２ is optionally substituted phenyl, heteroaryl or benzoyl, Y is NR ３ wherein R ３ is H, C １－６ alkyl, aryl, heteroaryl, C ３－７ cycloalkyl or C ３－７ cycloalkyl C １－６ alkyl, A is CH or N, W is CH or N), or a pharmaceutically acceptable salt thereof.

2. R １ is C １－６ alkyl or -L １ -R ２ and L １ is C １－６ alkylene and R ２ is phenyl, the compound according to claim 1.

3. R １ The compound according to claim 1 or 2, wherein R is methyl, isopropyl or benzyl.

4. R ３ is H or C １－６ The compound according to any one of claims 1 to 3, wherein the alkyl is an alkyl group.

5. R ３ The compound according to any one of claims 1 to 4, wherein R is H or methyl.

6. The compound according to any one of claims 1 to 5, wherein A is CH.

7. The compound according to any one of claims 1 to 6, wherein W is N.

8. M is NR １ where R １ is C １－６ alkyl or -L １ -R ２ where L １ is C １－６ alkylene and R ２ is phenyl Y is NR ３ wherein R ３ is H or C １－６ alkyl, The compound according to claim 1, wherein A is CH and W is N, or a pharmaceutically acceptable salt thereof.

9. M is NR １ wherein １ R is methyl, isopropyl or benzyl, Y is NR ３ wherein R ３ is H or methyl, The compound according to claim 8, wherein A is CH and W is N, or a pharmaceutically acceptable salt thereof.

10. 5-(5-Methyl-6-oxo-7H-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; 5-(5,7-Dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; 5-(5-Isopropyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; and 5-(5-Benzyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; a compound selected therefrom, or a pharmaceutically acceptable salt thereof.

11. The following steps: a) Deprotection of the compound of formula (VII) by treatment with an acid or by hydrogenation 【Chemical 2】 to obtain the compound of formula (VIII) 【Chemical Formula 3】 A process for the preparation of a compound according to any one of claims 1 to 10, comprising any of the above steps. (wherein, PG １ is methyl, tert-butyl, TBS, ethoxymethyl or benzyl, R １ , R ３ , A and W are defined as in any one of claims 1 to 9, and the acid in step a) is HCl)

12. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, for use as a therapeutic active substance.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 and a pharmaceutically acceptable additive.

14. Use of a compound according to any one of claims 1 to 10 for the treatment of cancer.

15. Use according to claim 14, wherein the cancer is pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer or melanoma.

16. Use of a compound according to any one of claims 1 to 10 for inhibiting CD73.

17. ​ Use of a compound according to any one of claims 1 to 10 for preparing a medicament for treating or preventing cancer, wherein the cancer is pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, melanoma, multiple myeloma, acute myeloid leukemia, or acute and chronic lymphoblastic leukemia.

18. Use of a compound according to any one of claims 1 to 10 for preparing a medicament as a CD73 inhibitor.

19. A compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof when produced according to the method described in claim 11.

20. The invention as described above.

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