Heterocyclic compounds for treatment of cancer

Abstract

The present invention relates to: compounds of formula (I) # imgabs0 # wherein R1 to R3, M, A, Y and W are as described herein, and pharmaceutically acceptable salts thereof; as well as compositions comprising the compounds and methods of using the compounds.

Description

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

[0002] Excessive activation of the adenosine pathway creates an immunosuppressive tumor microenvironment (TME) that impairs antitumor immunity and limits the efficacy of immune checkpoint inhibitors. In the final step of the adenosine pathway, the enzyme ecto-5'-nucleotidase (CD73) catalyzes the conversion of AMP to adenosine, which is recognized by adenosine receptors present in multiple immune cell types, leading to inhibition of effector T cells and natural killer (NK) cells, activation of regulatory T (Treg) and myeloid-derived suppressor cells (MDSC), and other changes in the immune system, which ultimately together create an immunosuppressive environment. CD73 is frequently overexpressed in cancer, and its upregulation is associated with poor clinical prognosis. Preclinical work in various in vivo tumor models has shown that genetic ablation or pharmacological inhibition of CD73 results in restoration of immune cell function and suppression of tumor growth. Therefore, it is conceivable that alleviating the immunosuppressive TME by inhibiting CD73 has the therapeutic potential to restore antitumor immunity and enhance the efficacy of immunotherapy-induced tumor regression. Given the increasing and unmet need for effective cancer treatments, inhibition of CD73 activity by administering small molecules (SMs) is promising. This disclosure describes the invention of novel small molecule CD73 inhibitors. Summary of the invention

[0003] The present invention relates to novel compounds having formula (I),

[0004]

[0005] in

[0006] M is NR 1 , where R 1 C 1-6 Alkyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl C 1-6 Alkyl or -L 1 -

[0007] R 2 ; where L 1 C 1-6 Alkylene, C 3-7 Cycloalkylene, heterocyclylene or heteroarylene; R 2 is optionally substituted phenyl, heteroaryl or benzoyl;

[0008] Y is NR 3 , where R 3 H, C1-6 Alkyl, aryl, heteroaryl, C 3-7 Cycloalkyl or C 3-7 ring

[0009] Alkyl C 1-6 alkyl;

[0010] A is CH or N;

[0011] W is CH or N;

[0012] or a pharmaceutically acceptable salt thereof.

[0013] The compound of formula (I) shows good CD73 inhibition. In another embodiment, the compound of the present invention shows excellent cancer cell inhibition. In addition, the compound of formula (I) also shows good or improved human hepatocyte stability, cytotoxicity, and solubility profiles. DETAILED DESCRIPTION

[0014] definition

[0015] The term "C 1-6 "Alkyl" means a saturated, straight-chain or branched alkyl group containing 1 to 6, especially 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. In particular, "C 1-6 "Alkyl" groups are methyl, ethyl and n-propyl.

[0016] The term “(C 1-6 ")Alkylene" means a straight 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. 1-6 ) Examples of the alkylene group include methylene, ethylene, propylene, 2-methylpropylene, butylene, 2-ethylbutylene, pentylene, hexylene.

[0017] The terms "halogen" and "halo" are used interchangeably herein to refer to fluorine, chlorine, bromine or iodine.

[0018] The term "C 3-7 "Cycloalkyl" means a monovalent saturated monocyclic or bicyclic hydrocarbon radical of 3 to 7 ring carbon atoms. Bicyclic means consisting of two saturated carbocyclic rings having one or more common carbon atoms. Examples of monocyclic cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. Examples of bicyclic cycloalkyl are bicyclo[1.1.0]butyl, bicyclo[2.2.1]heptyl, bicyclo[1.1.1]pentanyl or bicyclo[2.2.2]octyl.

[0019] The term "C 3-7 "Cycloalkylene" means a divalent C 3-7 Cycloalkyl groups.

[0020] The terms "heterocyclic group", "heterocyclic", "heterocycle", "heterocyclyl" or "heterocyclyl" are used interchangeably and refer to any monocyclic, bicyclic, tricyclic, spirocyclic or bridged, saturated, partially saturated or unsaturated, non-aromatic ring system having 3 to 20 ring atoms, wherein 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 of the ring atoms of the ring system is a heteroatom, the system is a heterocycle, regardless of the point of attachment of the ring system to the rest of the molecule. In one example, the heterocyclyl includes 3-11 ring atoms ("members"), and includes monocyclic, bicyclic, tricyclic, spirocyclic and bridged ring systems, wherein the ring atoms are carbon, wherein at least one atom in the ring or ring system is a heteroatom selected from nitrogen, sulfur or oxygen. In other examples, the heterocyclyl includes 4-10 or 5-10 ring atoms. In one example, the heterocyclyl includes 1 to 4 heteroatoms. In one example, the heterocyclyl includes 1 to 3 heteroatoms. In another example, the heterocyclic group includes a 3- to 7-membered monocyclic ring having 1-2, 1-3 or 1-4 heteroatoms selected from nitrogen, sulfur or oxygen. In another example, the heterocyclic group includes a 4- to 6-membered monocyclic ring having 1-2, 1-3 or 1-4 heteroatoms selected from nitrogen, sulfur or oxygen. In another example, the heterocyclic group includes a 3-membered monocyclic ring. In another example, the heterocyclic group includes a 4-membered monocyclic ring. In another example, the heterocyclic group includes a 5- to 6-membered monocyclic ring. In some embodiments, the heterocycloalkyl group includes at least one nitrogen. In one example, the heterocyclic group includes 0 to 3 double bonds. Any nitrogen or sulfur heteroatom may be optionally oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom may be optionally quaternized (e.g., [NR4] + Cl - 、[NR4] + OH - ). Examples of heterocycles include oxirane, aziridinyl, thiopyranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, imidazolidinyl, piperidinyl, piperazinyl, isoquinolyl, tetrahydroisoquinolyl, morpholinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinyl, thiazinyl, thiooxanyl, homopiperazinyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepine 1,4-diazepanyl, ... Thiazepine 1,1-isothiazolidinyl, 1,1-dioxaisothiazolyl, oxazolidinyl, imidazolinyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6,7-tetrahydrobenzo[d]imidazolyl, thiazinyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dioxazinyl, Thiadiazinyl, imidazolinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolyl, thiopyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolyl, pyrazolidinyl, dithienyl, dithiolanyl, pyrimidinone, pyrimidinedione, pyrimidine-2,4-diacyl, piperazinone, piperazinediacyl, pyrazolinylimidazolinyl, 3-azabis[3.1.0]hexane 3,6-diazabicyclo[3.1.1]heptyl, 6-azabicyclo[3.1.1]heptyl, 3-azabicyclo[3.1.1]heptyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 2-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl, 2-azabicyclo[2.2.2]octyl, 8-azabicyclo[2.2.2]octyl, 7-oxo heterobicyclo[2.2.1]heptane, azaspiro[3.5]nonane, azaspiro[2.5]octanyl, azaspiro[4.5]decane, 1-azaspiro[4.5]decan-2-yl, azaspiro[5.5]undecyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindolyl, 1,1-dioxohexahydrothiopyranyl, and 2,3,4a,5,7,7a-hexahydro-[1,4]dioxadieno[2,3-c]pyrrolyl.

[0021] The term "heterocyclylene" refers to a divalent heterocyclyl group.

[0022] The term "aryl" refers to a monovalent aromatic carbocyclic monocyclic or bicyclic ring system containing 6 to 10 carbon ring atoms. Examples of aryl moieties include phenyl and naphthyl. The term "arylene" refers to a divalent aryl group.

[0023] The term "heteroaryl" refers to any monocyclic, bicyclic 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. See, for example, Lang's Handbook of Chemistry (Dean, JA ed.) 13th edition, Table 7-2

[1985] . This definition includes any bicyclic group, wherein any of the above heteroaryl rings is fused to an aryl ring, wherein the aryl ring or the heteroaryl ring is connected to the rest of the molecule. In one embodiment, heteroaryl includes 5-6 yuan monocyclic aromatic groups, wherein one or more ring atoms are nitrogen, sulfur or oxygen. In one embodiment, heteroaryl includes 7-12 yuan bicyclic aromatic groups, wherein one or more ring atoms are nitrogen, sulfur or oxygen. Exemplary heteroaryl groups include thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazolyl, tetrazolo[1,5-b]pyridazinyl, imidazo[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, benzofuranyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, indazolyl and indolyl.

[0024] The term "heteroarylene" refers to a divalent heteroaryl group.

[0025] In certain embodiments, a heterocyclyl group or a heteroaryl group is attached to a carbon atom of the heterocyclyl group or the heteroaryl group. By way of example, carbon-bonded heterocyclyl groups include the following bonding arrangements: 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, thiofuran, 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.

[0026] In certain embodiments, the heterocyclyl or heteroaryl group is N-linked. For example, nitrogen-bonded heterocyclyl or heteroaryl groups include the following bonding arrangements: position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindole, 1H-indazole; position 2 of isoindole or isoindolinone; position 4 of morpholine; and position 9 of carbazole or β-carboline.

[0027] In one embodiment, those skilled in the art will appreciate that certain structures have keto-enol tautomerism, as shown below:

[0028]

[0029] Unless otherwise indicated, the term "optionally substituted" means that the group may be unsubstituted or substituted with one or more (e.g., 0, 1, 2, 3, 4, or 5 or more, or any range derivable therein) substituents for the group, wherein the substituents may be the same or different. In an embodiment, the optionally substituted group has 1 substituent. In another embodiment, the optionally substituted group has 2 substituents. In another embodiment, the optionally substituted group has 3 substituents. In another embodiment, the optionally substituted group has 4 substituents. In another embodiment, the optionally substituted group has 5 substituents.

[0030] As used herein, the term "protecting group" or "PG" refers to a group that selectively blocks a reactive site in a multifunctional compound so that a chemical reaction selectively occurs on another unprotected reactive site that is usually associated with it in synthetic chemistry. The protecting group can be removed at an appropriate time point. Exemplary protecting groups are amino protecting groups, carboxyl protecting groups, or hydroxyl protecting groups.

[0031] The term "pharmaceutically acceptable salts" refers to salts that are not biologically or otherwise undesirable. "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts.

[0032] "Pharmaceutically acceptable acid addition salts" refer to those pharmaceutically acceptable salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the like, and organic acids, which may be selected from aliphatic, alicyclic, aromatic, aromatic aliphatic, heterocyclic, formic and sulfonic acid 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, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, and the like.

[0033] The term "pharmaceutical base addition salt" refers to those pharmaceutical salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary amines, secondary amines, tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine and polyamine resins).

[0034] The term "pharmaceutically active metabolite" refers to a pharmacologically active product produced by the metabolism of a specific compound or its salt in the body. After entering the human body, most drugs are substrates for chemical reactions that may change their physical properties and biological effects. These metabolic transformations, which usually affect the polarity of the compounds of the present invention, change the way the drug is distributed in the body and excreted from the body. However, in some cases, drug metabolism is necessary for therapeutic effects.

[0035] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the invention that, when administered to a subject, (i) treats or prevents a particular disease, disorder or condition, (ii) reduces, ameliorates or eliminates one or more symptoms of a particular disease, disorder or condition, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, disorder or condition as described herein. The therapeutically effective amount depends on the compound, the disease state 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 medical or veterinary doctor and other factors.

[0036] The term "pharmaceutical composition" means a mixture or solution comprising a therapeutically effective amount of an active drug ingredient together with a pharmaceutically acceptable excipient for administration to a mammal (eg, a human) in need thereof.

[0037] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," and "therapeutically inert excipient" are used interchangeably and refer to any pharmaceutical ingredient in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant used to formulate a drug product.

[0038] CD73 inhibitors

[0039] The present invention relates to (i) a compound of formula (I),

[0040]

[0041] in

[0042] M is NR 1 , where R 1 C 1-6 Alkyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl C 1-6 Alkyl or -L 1 -R 2 ; where L 1 C 1-6 Alkylene, C 3-7 Cycloalkylene, heterocyclylene or heteroarylene; R 2 is optionally substituted phenyl, heteroaryl or benzoyl;

[0043] Y is NR 3 , where R 3 H, C 1-6 Alkyl, aryl, heteroaryl, C 3-7 Cycloalkyl or C 3-7 Cycloalkyl C 1-6 alkyl;

[0044] A is CH or N;

[0045] W is CH or N;

[0046] or a pharmaceutically acceptable salt thereof.

[0047] A further embodiment of the present invention is (ii) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to (i), wherein C 1-6 Alkyl or -L 1 -R 2 ; where L 1 C 1-6 Alkylene; R 2 It is phenyl.

[0048] A further embodiment of the present invention is (iii) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to (i) or (ii), wherein R 1 is methyl, isopropyl or benzyl.

[0049] A further embodiment of the present invention is (iv) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of (i) to (iii), wherein R 3 H or C 1-6 alkyl.

[0050] A further embodiment of the present invention is (v) a compound of formula (I) according to any one of (i) to (iv), wherein R 3 It is H or methyl.

[0051] A further embodiment of the present invention is (vi) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of (i) to (v), wherein A is CH.

[0052] A further embodiment of the present invention is (vii) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of (i) to (vi), wherein W is N.

[0053] A further embodiment of the present invention is (viii) a compound of formula (I), wherein M is NR 1 , where R 1 C 1-6 Alkyl or -L 1 -R 2 ; where L 1 C 1-6 Alkylene; R 2 is phenyl; Y is NR 3 , where R 3 H or C 1-6 alkyl;

[0054] A is CH;

[0055] W is N;

[0056] or a pharmaceutically acceptable salt thereof.

[0057] A further embodiment of the present invention is (ix) a compound of formula (I), wherein M is NR 1 , where R 1 is methyl, isopropyl or benzyl;

[0058] Y is NR 3 , where R 3 is H or methyl;

[0059] A is CH;

[0060] W is N;

[0061] or a pharmaceutically acceptable salt thereof.

[0062] Another embodiment of the present invention is (x) a compound of formula (I) selected from the following compounds:

[0063] 5-(5-methyl-6-oxo-7H-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione;

[0064] 5-(5,7-dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione;

[0065] 5-(5-isopropyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione; and

[0066] 5-(5-Benzyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione;

[0067] or a pharmaceutically acceptable salt thereof.

[0068] Another embodiment of the present invention relates to (xi) a method for preparing a compound according to any one of (i) to (x), the method comprising the following steps:

[0069] a) treating a compound of formula (VII) with an acid or by hydrogenation

[0070] Deprotection to obtain a compound of formula (VIII)

[0071] Among them PG 1 R is methyl, tert-butyl, TBS, ethoxymethyl or benzyl. 1 , R 3 , A and W are as defined in any one of (i) to (ix); the acid in step a) is HCl.

[0072] Another embodiment of the present invention is (xii) a compound or a pharmaceutically acceptable salt according to any one of (i) to (x) for use as a therapeutically active substance.

[0073] Another embodiment of the present invention is (xiii) a pharmaceutical composition comprising the compound according to any one of (i) to (x) and a pharmaceutically acceptable excipient.

[0074] Another embodiment of the present invention is (xiv) use of the compound according to any one of (i) to (x) for treating cancer.

[0075] Another embodiment of the present invention is (xv) the use according to (xiv), wherein the cancer is pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer or melanoma.

[0076] Another embodiment of the present invention is (xvi) use of the compound according to any one of (i) to (x) for inhibiting CD73.

[0077] Another embodiment of the present invention is (xvii) use of a compound according to any one of (i) to (x) 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.

[0078] Another embodiment of the present invention is (xviii) use of the compound according to any one of (i) to (x) for preparing a drug as a CD73 inhibitor.

[0079] Another embodiment of the present invention is (xix) a compound or a pharmaceutically acceptable salt according to any one of (i) to (x), which is prepared according to the method of (xi).

[0080] Pharmaceutical compositions and administration

[0081] Another embodiment provides a pharmaceutical composition or medicine containing the compounds of the invention and a therapeutic inert carrier, diluent or excipient, and methods for preparing such compositions and medicines using the compounds of the invention. In one example, the compound of formula (I) can be formulated as a galenical administration form by mixing with a physiologically acceptable carrier (i.e., a carrier that is nontoxic to the recipient at the dosage and concentration used) at ambient temperature at an appropriate pH and desired purity. The pH of the formulation depends primarily on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the compound of formula (I) is formulated in an acetate buffer at pH 5. In another embodiment, the 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.

[0082] The composition is formulated, metered, and administered in a manner consistent with good medical practice. Factors to be considered in this context 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 delivery of the agent, the method of administration, the timing of administration, and other factors known to practitioners. The "effective amount" of the compound to be administered will be affected by such considerations and is the minimum amount required to inhibit the enzymatic activity of the CD73 protein to convert AMP to adenosine. In one example, the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.01 to 100 mg / kg of patient body weight per day, alternatively in the range of about 0.1 to 50 mg / kg of patient body weight, typically with an initial range of 0.3 to 30 mg / kg / day of the compound used. In another embodiment, oral unit dosage forms such as tablets and capsules preferably contain about 1 to about 1000 mg of the compound of the invention.

[0083] 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, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and (if necessary for local treatment) intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0084] The compounds of the present invention can be administered in any convenient administration form, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain conventional components of pharmaceutical preparations, for example, diluents, carriers, pH adjusters, sweeteners, fillers and other active agents.

[0085] Typical formulations are prepared by mixing the compounds of the invention with carriers or excipients. Suitable carriers and excipients are well known to those skilled in the art and are described in, for example, 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 formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents, flavoring agents, diluents and other known additives to provide an aesthetically pleasing display of a drug (e.g., a compound of the present invention or a pharmaceutical composition thereof) or to facilitate the preparation of a pharmaceutical product (e.g., a drug).

[0086] An example of a suitable oral dosage form is a tablet containing about 0.1 mg to 500 mg of the compound of the present invention and about 0.1 mg to 500 mg of anhydrous lactose, about 0.1 mg to 500 mg of cross-linked sodium carboxymethylcellulose, about 0.1 mg to 500 mg of polyvinyl pyrrolidone (PVP) K30 and about 0.1 mg to 500 mg of magnesium stearate. The powdered ingredients are first mixed together and then mixed with the PVP solution. The resulting composition can be dried, granulated, mixed with magnesium stearate and compressed into tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound of the present invention (e.g., 1 to 450 mg) in a suitable buffer solution (e.g., phosphate buffer) and adding a penetration enhancer (e.g., a salt such as sodium chloride) if necessary. The solution can be filtered, for example, using a 0.2 micron filter to remove impurities and contaminants.

[0087] Thus, embodiments include pharmaceutical compositions comprising a compound of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof. Further embodiments include a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0088] Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in treating cancer. Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in treating cancer.

[0089] The following examples illustrate typical compositions of the present invention but are intended to be representative only.

[0090] Composition A

[0091] The compounds of the invention can be used as active ingredients in a manner known per se to produce tablets of the following composition:

[0092]

[0093] Composition B

[0094] The compounds of the invention can be used as active ingredients in a manner known per se to produce capsules of the following composition:

[0095]

[0096] Indications and treatments

[0097] The compounds of the present invention inhibit the enzymatic activity of CD73 to convert AMP into adenosine. Therefore, the compounds of the present invention can be used to reduce adenosine levels in TME. The compounds of the present invention can be used to promote 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 compounds of the present invention can be used to promote immune-mediated killing of cancer cells that rely on adenosine pathways or cancer cells in malignant solid tumors, wherein the adenosine pathway is enhanced by the deregulation or mutation of signal transduction driven by effector pathways such as EGFR-RAS-MAPK, PI3K-AKT, so as to be used for targeted therapy of pancreatic adenocarcinoma, non-small cell lung cancer, esophageal cancer and gastric adenocarcinoma, etc. More broadly, the compound can be used to treat and prevent all cancer types that exhibit immunosuppressive TME.

[0098] Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of formula (I), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof.

[0099] synthesis

[0100] The compounds of the present invention may be prepared by any conventional method. Suitable methods for synthesizing these compounds and their starting materials are provided in the following schemes and examples. Unless otherwise indicated, all substituents, especially R 1 To R 3 , M, A, Y and W are as defined above. In addition, unless otherwise specifically stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to those of ordinary skill in the art of organic chemistry.

[0101] The general synthetic route for preparing compounds of formula (I) is shown below.

[0102] Solution 1

[0103]

[0104] Where X is a halogen; each PG 1 is independently an oxygen protecting group (such as methyl, tert-butyl, TBS, ethoxymethyl or benzyl). The compound of formula (VIII) can be prepared according to Scheme 1. The dihalogenated aniline (II) and the amine R 1Selective aromatic nucleophilic substitution of NH2(III) or metal-catalyzed coupling reactions (such as Buchwald-Hartwig amination or Ullmann coupling) give compounds of formula (IV). Compounds of formula (IV) can be converted to compounds of formula (V) via condensation reactions with condensation reagents (such as CDI or triphosgene) in the presence of a base (such as DIEA or TEA). Subsequent coupling of halide (V) with organoboronic 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 provide compounds of formula (VII). Deprotection of compounds of formula (VII) with an acid (such as HCl) or hydrogenation removes PG. 1 , to obtain the final compound of formula (VIII).

[0105] Solution 2

[0106]

[0107] 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 condensation reagent (such as CDI or triphosgene) in the presence of a base (such as DIEA or TEA). The Suzuki coupling of the halide (X) with the organic boronic acid (VI) can be 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) to provide a compound of formula (XI). The compound of formula (XI) and the halide R 1 Selective N-substitution reaction of X or metal-catalyzed coupling reaction (such as Buchwald-Hartwig amination or Ullmann coupling), or the compound of formula (XI) with alcohol R 1 Mitsunobu reaction of OH, or the reaction of formula (XI) with an organic boronic acid R 1 Chan-Lam coupling of B(OH)2 can give the compound of formula (VII). Deprotection of the compound of formula (VII) is performed by acid (such as HCl) or hydrogenation to remove PG 1 , to obtain the final compound of formula (VIII).

[0108] Solution 3

[0109]

[0110] Alternatively, compounds of formula (VIII) can be prepared according to Scheme 3. Compounds of formula (X) can be prepared by reacting a halide R 1Selective N-substitution reaction of X or metal-catalyzed coupling reaction (such as Buchwald-Hartwig amination or Ullmann coupling), or the reaction of a compound of formula (X) with an alcohol R 1 Mitsunobu reaction of OH, or the reaction of formula (X) with an organic boronic acid R 1 Chan-Lam coupling of B(OH)2 is converted to the compound of formula (V). The subsequent coupling of the halide (V) with the organoboronic acid (VI) can be achieved by Suzuki coupling conditions using a catalyst (such as Pd(dppf)Cl2 and cataCXium-A-Pd-G3) and a base (such as Cs2CO3 and K3PO4) to obtain the compound of formula (VII). Deprotection of the compound of formula (VII) with an acid (such as HCl) or hydrogenation to remove PG 1 , to obtain the final compound of formula (VIII).

[0111] The compounds of the present invention can be obtained as diastereomers or mixtures of enantiomers, which can be separated by methods well known in the art, for example, (chiral) HPLC or SFC. In another embodiment, compounds of formula (I) can be obtained according to the above scheme using the corresponding chiral starting materials.

[0112] The present invention also relates to a method for preparing a compound of formula (I), the method comprising the following steps:

[0113] a) treating a compound of formula (VII) with an acid or by hydrogenation Deprotection to obtain a compound of formula (VIII) in

[0114] In step a), the acid may be, for example, HCl.

[0115] A compound of formula (I), which is prepared according to the above process, is also an object of the present invention.

[0116] Examples

[0117] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention.

[0118] abbreviation

[0119] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention.

[0120] The abbreviations used in this article are as follows:

[0121] ACN: Acetonitrile

[0122] aq. water-based

[0123] Boc2O: di-tert-butyl dicarbonate

[0124] CataCXium-A-Pd-G3 [(Bis(1-adamantyl)-butylphosphine)-2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate

[0125] CDI: N,N'-Carbonyldiimidazole

[0126] DCM: Dichloromethane

[0127] DIPEA or DIEA: N,N-diisopropylethylamine

[0128] DMA: N,N-dimethylacetamide

[0129] Pd(dppf)Cl2·DCM: [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) chloride complex with dichloromethane

[0130] EA or EtOAc: Ethyl acetate

[0131] FA: Formic acid

[0132] IC 50 : Half inhibitory concentration

[0133] LCMS: Liquid chromatography-mass spectrometry

[0134] MS: mass spectrometry

[0135] PE: Petroleum Ether

[0136] prep-HPLC: Preparative High Performance Liquid Chromatography

[0137] prep-TLC: Preparative Thin Layer Chromatography

[0138] rt: room temperature

[0139] RT: retention time

[0140] SFC: Supercritical Fluid Chromatography

[0141] TFA: trifluoroacetic acid

[0142] TLC: Thin layer chromatography

[0143] v / v: volume ratio

[0144] General experimental conditions

[0145] Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and Quad 12 / 25 Cartridge module. ii) ISCO combi-flash chromatograph. Silica gel brand and pore size: i) KP-SIL 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 Ocean Chemical Co., Ltd., pore size: 200-300 or 300-400.

[0146] Intermediates and final compounds were purified by preparative HPLC on a reverse phase column using XBridge TM Prep-C18 (5μm, OBDTM 30×100mm) column, SunFire TM Prep-C18(5μm,OBD TM 30×100mm) chromatographic column, Phenomenex Synergi-C18 (10μm, 25×150mm) or Phenomenex Gemini-C18 (10μm, 25×150mm). 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 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).

[0147] For SFC chiral separation, the intermediates were separated by chiral columns (Daicel chiralpak IC, 5 μm, 30×250 mm), AS (10 μm, 30×250 mm) or AD (10 μm, 30×250 mm), using Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar 80 preparative SFC, solvent system: CO2 and IPA (0.5% TEA in IPA solution) or CO2 and MeOH (0.1% NH3·H2O in MeOH solution), back pressure 100 bar, UV detection at 254 or 220 nm.

[0148] Using LC / MS (Waters TM The LC / MS spectra of the compounds were obtained using an Agilent Alliance 2795-Micromass ZQ, Shimadzu Alliance2020-Micromass ZQ or Agilent Alliance 6110-Micromass ZQ. The LC / MS conditions were as follows (run time 3 or 1.5 min):

[0149] Acidic conditions I: A: 0.1% TFA solution in H2O; B: 0.1% TFA solution in acetonitrile;

[0150] Acidic conditions II: A: 0.0375% TFA solution in H2O; B: 0.01875% TFA solution in acetonitrile;

[0151] Basic conditions I: A: 0.1% NH3·H2O solution in H2O; B: acetonitrile;

[0152] Basic conditions II: A: 0.025% NH3·H2O solution in H2O; B: acetonitrile;

[0153] Neutral conditions: A: H2O; B: acetonitrile.

[0154] Mass spectra (MS): Usually only ions representing the parent mass are reported, and unless otherwise stated, the mass ion quoted is the positive mass ion (MH) + .

[0155] NMR spectra were obtained using a Bruker Avance 400 MHz or 500 MHz.

[0156] Microwave-assisted reactions were performed in a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were performed under argon or nitrogen atmosphere. Unless otherwise stated, reagents were purchased from commercial suppliers as received without further purification.

[0157] Preparation Example

[0158] The following examples are intended to illustrate the meaning of the present invention, but are by no means intended to limit the meaning of the present invention:

[0159] Example 1

[0160] 5-(5-methyl-6-oxo-7H-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione

[0161]

[0162] The title compound was synthesized according to the following scheme:

[0163]

[0164] Step (a): 6-chloro-N 4 Preparation of -methyl-pyridazine-3,4-diamine (Compound 1.2)

[0165] 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) was added DIEA (8.4 mL, 48.0 mmol) and methylamine (2M in THF, 24.0 mL, 48.0 mmol) under N2 atmosphere to give a brown solution. The reaction mixture was stirred at 80 °C for 16 hours, then concentrated and purified by preparative HPLC to give compound 1.2 (3.5 g). MS: calculated value 159.0 [(M+H + ]; measured value 159.3[(M+H) + ].

[0166] Step (b): Preparation of 3-chloro-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.3)

[0167] At room temperature, 6-chloro-N 4 -methyl-pyridazine-3,4-diamine (compound 1.2, 200.0 mg, 1.26 mmol) in THF (4 mL) was added CDI (250.0 mg, 1.54 mmol), the reaction mixture was stirred at room temperature for 5 hours, then concentrated, and the resulting residue was purified by preparative HPLC to give compound 1.3 (80.0 mg). MS: calculated value 185.0 [(M+H) + ]; measured value 185.0[(M+H) + ].

[0168] Step (c): Preparation of 3-(2,4-dimethoxypyrimidin-5-yl)-5-methyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 1.5)

[0169] 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) was added 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) under N2. The resulting mixture was stirred at 80°C for 4 hours, 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 preparative HPLC to give compound 1.5 (20.0 mg, 16.0% yield). MS: calculated value 289.1 [(M+H) + ]; measured value 289.1[(M+H) + ].

[0170] Step (d): Preparation of 5-(5-methyl-6-oxo-7H-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 1)

[0171] 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 value 261.0[(M+H) + ]. 1 H NMR (400MHz, DMSO-d6) δ = 12.7 (s, 1H), 12.05-11.92 (m, 1H), 11.78 (br s, 1H), 8.36 (br s, 1H), 8.05 (s, 1H), 3.37 (s, 3H).

[0172] Example 2

[0173] 5-(5,7-Dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione

[0174]

[0175] The title compound was synthesized according to the following scheme:

[0176]

[0177] Step (a): Preparation of 3-chloro-5,7-dimethyl-imidazo[4,5-c]pyridazin-6-one (Compound 2.1)

[0178] 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 another 30 minutes, and 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, 68.2% yield). MS: calculated value 199.0 [(M+H) + ]; measured value 199.0[(M+H) + ].

[0179] Step (b): Preparation of 5-(5,7-dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 2)

[0180] 5-(5,7-Dimethyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione (Example 2) was prepared in a similar manner to 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) in step (c). 10.6 mg of Example 2 were obtained. MS: Calculated value 275.1 [(M+H) + ]; measured value 275.2[(M+H) + ]. 1 H NMR (400MHz, DMSO-d6) δ = 11.45 (s, 1H), 8.13 (s, 1H), 7.86 (s, 1H), 3.42 (s, 3H), 3.35 (s, 3H)

[0181] Example 3

[0182] 5-(5-Isopropyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione

[0183]

[0184] The title compound was synthesized according to the following scheme:

[0185]

[0186] Step (a): 6-chloro-N 4 - Preparation of isopropyl-pyridazine-3,4-diamine (Compound 3.1)

[0187] 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) was added DIEA (8.4 mL, 47.98 mmol) and isopropylamine (2.41 g, 40.78 mmol) 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 give compound 3.1 (1.2 g). 1 H NMR (400MHz, DMSO-d6) δ = 6.33 (s, 1H), 6.10 (s, 2H), 5.97 (br d, J = 7.2Hz, 1H), 3.72-3.61 (m, 1H), 1.17 (d, J = 6.4Hz, 6H).

[0188] Step (b): Preparation of 3-chloro-5-isopropyl-7H-imidazo[4,5-c]pyridazin-6-one (Compound 3.2)

[0189] At room temperature, 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). 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 give compound 3.2 (400.0 mg, 68.4% yield). MS: calculated value 213.0, 215.0 [M+H] + Measured value 213.2,215.2[M+H] +

[0190] Step (c): Preparation of 3-chloro-5-isopropyl-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 3.3)

[0191] 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, and 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 slowly adding saturated aqueous 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 values ​​227.1, 229.1 [(M+H) + ]; measured values ​​227.1,229.1[(M+H) + ].

[0192] Step (d): Preparation of 3-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-7-methyl-imidazo[4,5-c]pyridazin-6-one (Compound 3.4)

[0193] 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) under N2 was 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). The resulting mixture was stirred at 80 °C for 12 hours. 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: calculated value 331.1 [(M+H + ]; measured value 331.1[(M+H) + ].

[0194] 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)

[0195] 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 hour, then ammonia (1 mL) and water (2 mL) were added. The solid formed was collected by filtration, washed with MeOH, and lyophilized to give Example 3 (38.16 mg). MS: Calculated 303.1 [(M+H) + ]; measured value 303.1[(M+H) + ]. 1 HNMR (400MHz, 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 = 6.8Hz, 6H).

[0196] Example 4

[0197] 5-(5-Benzyl-7-methyl-6-oxo-imidazo[4,5-c]pyridazin-3-yl)-1H-pyrimidine-2,4-dione

[0198]

[0199] The title compound was synthesized according to the following scheme:

[0200]

[0201] Step (a): Preparation of 3-chloro-7-methyl-5H-imidazo[4,5-c]pyridazin-6-one (Compound 4.2)

[0202] To 6-chloro-N 3 -Methyl-pyridazine-3,4-diamine (compound 4.1, 3.0 g, 18.92 mmol) was added to a solution of THF (30 mL) with 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 hours 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 value 185.0 [(M+H) + ]; measured value 185.0[(M+H) + ].

[0203] 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)

[0204] To a solution of (2,4-di-tert-butoxypyrimidin-5-yl)boronic acid (compound 4.3, 2.61 g, 9.75 mmol) in 1,4-dioxane (30 mL) and water (5 mL) was 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 hours. 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 value 373.2 [(M+H) + ]; measured value 373.1[(M+H) + ].

[0205] 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)

[0206] 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 slowly adding saturated aqueous NH4Cl (20 mL) solution 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, 32.2% yield). MS: calculated value 463.2 [(M+H) + ]; measured value 463.2[(M+H) + ].

[0207] 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)

[0208] 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 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) + ]. 1 H NMR (400MHz, DMSO-d6) δ = 11.56–11.31 (m, 2H), 8.13 (d, J = 6.0Hz, 1H), 7.85 (s, 1H), 7.35–7.28 (m, 5H), 5.10 (s, 2H), 3.46 (s, 3H).

[0209] Biological Examples

[0210] Example 5: CD73 biochemical assay

[0211] Compound serial dilutions (1:3) were prepared into the corresponding wells of a 384-well plate using an Echo 555 liquid handler (Labcyte). 25 μL of 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 detergent] was added to the assay plate and incubated with the compound at room temperature for 15 minutes. After 25 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 the assay plate, the reaction solution was incubated at room temperature for 10 minutes. Then 10 uL of malachite green A solution was added to each well and incubated for 10 minutes. 10 uL of malachite B solution was added to each well and then incubated for another 30 minutes. Finally, the absorbance value at 620 nM was read on an Envision microplate reader (PerkinElmer). The percentage inhibition was calculated using the formula {% inhibition = 100 x [I-(X-MIN) / (MAX-MIN)]}, where X equals the well signal, Max equals the DMSO control signal, and MIN equals the cell-free control signal.

[0212] Table 1: Enzyme IC of the compounds of the present invention against CD73 50 value

[0213] Instance Number <![CDATA[IC 50 (μM)]]> 1 0.622 2 0.254 3 0.267

[0214] Example 6: CD73 cell assay

[0215] Compound serial dilutions (1:3) were prepared in the corresponding wells of 384-well plates using Echo 555 liquid handler (Labcyte). 40 μL MDA-MB-231 cells (ATCC, HTB-26, breast cancer, final concentration of 20,000 cells / mL) suspended in assay buffer (25mM Tris-HCl pH 7.5, 5mM MgCl2, 0.01% BSA, 0.01% Brij-35) were added to the corresponding wells of the plate. After incubation with the compound for 30 minutes, 40 μL AMP working solution (10 μM AMP, 25mMTris-HCl pH 7.5, 5mM MgCl2, 0.01% BSA, 0.01% Brij-35) were 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 is complete, 50 μL of supernatant is collected and transferred to a new 384-well plate. 10 μL of Malachite A is added to each well of the assay plate and incubated for 10 minutes. Then 10 μL of Malachite B is added to each corresponding well of the plate and incubated for 30 minutes. Finally, the absorbance value at 620 nM is read on the Envision microplate reader. The inhibition percentage is calculated by using the formula {% inhibition = 100 × [I-(X-MIN) / (MAX-MIN)]}, where X equals the well signal, Max equals the DMSO control signal, and MIN equals the cell-free control signal.

[0216] Table 2: Cellular IC of the compounds of the present invention against CD73 50 value

[0217] Instance Number <![CDATA[IC 50 (μM)]]> 1 0.074 2 0.156 3 0.027 4 0.107

[0218] Example 7: CD73 LC / MS Assay

[0219] The purpose of this assay is to identify and characterize inhibitors of CD73 enzymatic activity. Compound serial dilutions (1:3) were prepared into the corresponding wells of a 384-well plate using an Echo 555 liquid handler (Labcyte). 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 compound 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, with or without NaH2PO4), the assay was incubated at room temperature for 10 minutes. The reaction was terminated by adding 75 μL of stop solution (5% TCA in H2O, containing 250 nM 13C5-adenosine) to each well and incubating for 10 minutes. After centrifugation, 75 μL of the mixture was transferred to a new 384-well plate for LC / MS analysis.

[0220] The samples in the 384-well plate were loaded onto the autosampler platform and then injected into the ADDA-LC-MS / MS. The aqueous mobile phase was 0.1% formic acid in water. The organic mobile phase was 0.1% formic acid in acetonitrile. The flow rate was maintained at 0.8 mL / min using a Shimadzu pump. The column was ACE 5Phenyl, 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 directly introduced into electrospray ionization (ESI). Multiple reaction monitoring (MRM) was used to determine the analyte and internal standard (IS) responses. The MRM for adenosine was 268.1 / 136.1, and the MRM for 13C5-adenosine (IS) was 273.2 / 136.2. The data were calculated using the peak area ratio (PAR) semi-quantitative method.

[0221] Example 8: T cell proliferation assay

[0222] The purpose of this assay is to characterize the potency of CD73 inhibitors in rescuing adenosine-mediated inhibition of T cell proliferation. TM CD4+ or CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMC, HemaCare) by immunomagnetic negative selection using the Isolation Kit (STEMCELL Technologies). CD4+ or CD8+ T cells were pelleted by centrifugation at 300 g for 10 min at room temperature and resuspended in PBS. CellTrace TMViolet staining solution (Invitrogen) and incubate at 37°C in the dark for 20 minutes. Complete medium [RPMI-1640 (Gibco), 10% fetal bovine serum (Gibco), 2mM GlutaMAX (Gibco) and 1mM sodium pyruvate (Gibco), 100U / mL penicillin-streptomycin (Gibco) and MEM non-essential amino acids (NEAA) cell culture supplement (1:100, Gibco)] was then added, mixed, and incubated at 37°C for 5 minutes. The cells were then pelleted by centrifugation at 300g for 10 minutes at room temperature and resuspended in fresh, preheated complete medium. 50 μL of cells were inoculated into each well of 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) at final concentrations of 200 μM and 5 μM, respectively, was added to the cells. The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. 200 μL of PBS was then added to each well and the cells were centrifuged at 300 g, 4°C for 10 minutes. The supernatant was discarded. 50 μL of Human TruStain FcX TM (Fc receptor blocking solution, BioLegend) 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 300g, 4°C for 10 minutes and the supernatant was discarded. The cell pellet was washed with 250 μL of cell staining buffer and centrifuged at 300g, 4°C for 10 minutes. The supernatant was discarded and the cells were resuspended in 60 μL of cell staining buffer and analyzed on a flow cytometer.

[0223] Example 9: T cell cytokine release function assay

[0224] The purpose of this assay is to characterize the potency of CD73 inhibitors in rescuing adenosine-mediated inhibition of cytokine release from T cells. TMSeparation kit (STEMCELL Technologies) separates CD4+ or CD8+T cells from peripheral blood mononuclear cells (PBMC) by immunomagnetic negative selection. Then the cells are precipitated by centrifugation of CD4+ or CD8+T cells at room temperature for 10 minutes at 300g, and resuspended in fresh, preheated complete medium. 50 μL cells are inoculated into each well of a 96-well U-shaped bottom plate. 50 μL of culture medium containing CD3 / CD28 beads is added to each well and incubated overnight at 37°C in a 5% CO2 incubator. 50 μL of culture medium containing compounds is added to the cells. 50 μL of culture medium containing AMP and EHNA hydrochloride (Sigma-Aldrich) is added to the cells at a final concentration of 200 μM and 5 μM, respectively. Cells are incubated at 37°C for 72 hours in a 5% CO2 incubator. 50 μL of supernatant is collected to determine the levels of IL2 and IFNγ using an ELISA-MSD kit (Meso Scale Discovery).

Claims

1. A compound of formula (I), in M is NR 1 , where R 1 C 1-6 Alkyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl C 1-6 Alkyl or -L 1 -R 2 ; where L 1 C 1-6 Alkylene, C 3-7 Cycloalkylene, heterocyclylene or heteroarylene; R 2 is optionally substituted phenyl, heteroaryl or benzoyl; Y is NR 3 , where R 3 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. The compound according to claim 1, wherein R 1 C 1-6 Alkyl or -L 1 -R 2 ;in L 1 C 1-6 Alkylene; R 2 It is phenyl.

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

4. A compound according to any one of claims 1 to 3, wherein R 3 H or C 1-6 alkyl.

5. A compound according to any one of claims 1 to 4, wherein R 3 It 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. The compound according to claim 1, wherein M is NR 1 , where R 1 C 1-6 Alkyl or -L 1 -R 2 ; where L 1 C 1-6 Alkylene; R 2 is phenyl; Y is NR 3 , where R 3 H or C 1-6 alkyl; A is CH; W is N; or a pharmaceutically acceptable salt thereof.

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

10. A compound selected from: 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.

11. A method for preparing a compound according to any one of claims 1 to 10, comprising any one of the following steps: a) treating a compound of formula (VII) with an acid or by hydrogenation Deprotection to obtain a compound of formula (VIII) Among them PG 1 R is methyl, tert-butyl, TBS, ethoxymethyl or benzyl. 1 , R 3 , A and W are as defined in any one of claims 1 to 9; the acid in step a) is HCl.

12. A compound or a pharmaceutically acceptable salt according to any one of claims 1 to 10 for use as a therapeutically active substance.

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

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

15. The 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. The use of a compound according to any one of claims 1 to 10 for the preparation of a medicament for the treatment or prevention of 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 the preparation of a medicament as a CD73 inhibitor.

19. A compound or pharmaceutically acceptable salt according to any one of claims 1 to 10, prepared according to the process of claim 11.

20. The invention as herein described.

Images