Exonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitors for treatment of hypophosphoesterase disorders

Abstract

Provided herein are methods for treating hypophosphoesterase (HPP), the methods comprising administering a therapeutically effective amount of an inhibitor of exonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1).

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 493,684, filed March 31, 2023, and U.S. Provisional Application No. 63 / 495,994, filed April 13, 2023, both of which are hereby incorporated herein by reference in their entirety. Technical Field

[0003] This article provides a method for treating hypophospholipase syndrome (HPP) involving the administration of a therapeutically effective amount of an inhibitor of exonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1). Background Technology

[0004] Hypophosphatase disorder (HPP) is a rare, inherited metabolic disorder caused by loss-of-function mutations in the ALPL gene, which encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Low TNSALP activity leads to the extracellular accumulation of phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal phosphate (PLP) in the blood or urine. PPi is an inhibitor of bone mineralization and is one of the key substrates involved in the pathogenesis of HPP. In HPP, excessive PPi can lead to defects in bone mineralization (rickets or osteomalacia), premature loss of primary teeth, chronic bone and muscle pain, recurrent fractures, respiratory and muscle dysfunction, and periodontal defects. Symptoms range from fatal or severe symptoms in perinatal or infancy-onset HPP to mild or mild symptoms in childhood and adult-onset HPP.

[0005] The incidence of severe HPP ranges from approximately 1 in 100,000 to 1 in 300,000 live births. In some populations, the incidence is even higher, such as among Mennonites in Canada, where the incidence is as high as approximately 1 in 2,500 newborns.

[0006] Hypophosphatase syndrome places a heavy burden on affected individuals and their families. Therefore, identifying compounds, compositions, and methods that can be used to treat or prevent hypophosphatase syndrome will fill an important unmet need. Summary of the Invention

[0007] Disclosed herein are methods of treating or preventing a cartilage disease using an ENPP1 inhibitor. In some embodiments, the cartilage disease is hypophosphatasia. The ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is an extracellular enzyme that hydrolyzes ATP to PPi and AMP in the extracellular space. PPi acts as a potent inhibitor of hydroxyapatite (HAP) crystal growth, which is the inorganic mineral component of calcium phosphate and is essential for bone mineralization. Since ENPP1 is the major source of PPi, inhibition of this enzyme is a promising therapeutic approach to treat HPP. Normalization of extracellular PPi levels by targeting ENPP1 can significantly alleviate the severe symptoms of this debilitating disease. Targeted therapies that inhibit ENPP1 activity or neutralize PPi can lead to effective intervention for HPP.

[0008] In some embodiments, the hypophosphatasia is late-onset hypophosphatasia. In some embodiments, the hypophosphatasia is pediatric-onset hypophosphatasia.

[0009] In some embodiments, the hypophosphatasia is prenatal hypophosphatasia. In some embodiments, the hypophosphatasia is perinatal hypophosphatasia.

[0010] In some embodiments, the disease is infantile hypophosphatasia.

[0011] In some embodiments, the disease is childhood hypophosphatasia.

[0012] In some embodiments, the disease is adult hypophosphatasia.

[0013] In some embodiments, the disease is any form of hypophosphatasia with dental involvement and / or premature tooth loss.

[0014] In some embodiments, the disease is dental hypophosphatasia.

[0015] In some embodiments, there is provided a method of treating a subject having a dental condition with an agent that inhibits ENPP1.

[0016] In some embodiments, there is provided a method of treating a subject having a periodontal disease with an agent that inhibits ENPP1.

[0017] In some embodiments, there is provided a method of treating hypophosphatasia with an agent that inhibits ENPP1.

[0018] In some embodiments, there is provided a method of treating hypophosphatasia in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits the hydrolysis of ATP via ENPP1 inhibition.

[0019] In some embodiments, a method of treating hypophosphatasia is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits ENPP1.

[0020] In some embodiments, the compound is a compound described in US20220135598A1, which is incorporated by reference herein. In some embodiments, the compound has the formula I:

[0021] I

[0022] or a pharmaceutically acceptable salt thereof, wherein:

[0023] Ring C is a 5- to 6-membered heteroaryl;

[0024] Ring D is a C6-aryl or a 5- to 6-membered heteroaryl, wherein Ring D is fused to Ring C;

[0025] A is hydrogen, C1-C6alkyl, or C6-aryl, each of which is optionally substituted with halo;

[0026] G is a bond, -CH2-, or -CH2-CH2-;

[0027] R a and R b are independently hydrogen or C1-C6alkyl;

[0028] or R a and R b together with the atom to which they are attached form a C3-C6cycloalkyl ring;

[0029] or either of R a and R b together with A and the atoms to which they are attached form a C4-C6cycloalkyl ring;

[0030] L is a bond, straight or branched chain C1-C6alkylene, or straight or branched chain C2-C6alkenylene;

[0031] t is 0 or 1; provided that when t is 0, then L is straight or branched chain C2-C6alkenylene;

[0032] Z is -NR c S(O)2NH2, -NR c S(O)2CH3, -SO2NH2, -NR c C(O)CH3, -C(O)OH, -CONH2, -NR c CONH2, -CONH(OH), -B(OH)2, -P(O)(OH)2, -SO2OH, -NR cS(O)2CF3, -NR c S(O)2NHCH3, or -NR c CH2C6-aryl-S(O)2NH2.

[0033] each R 1 and R 2 is independently oxo, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, adamantyl, 3- to 6-membered heterocyclyl, C6-aryl, 5- to 6-membered heteroaryl, -CN, halogen, C1-C6alkoxy, C1-C6haloalkoxy, -OR 10 , -SR 10 , -S(O)2R 10 , -S(O)2NR 11 R 12 , -NR 10 S(O)2R 11 , -NR 11 R 12 , -C(O)R 10 , -NR 10 C(O)R 11 , -NR 10 C(O)NR 11 R 12 , -C(O)OR 10 , -C(O)ONR 11 R 12 , or -C(O)NR 11 R 12 , wherein each is independently optionally substituted with R 9 ;

[0034] or two R 2 together with the atom to which they are attached form a C5-C6cycloalkyl, 5- to 6-membered heterocyclyl, C6-aryl, or 5- to 6-membered heteroaryl, wherein each is independently optionally substituted with R 9 ;

[0035] each R 9 is independently selected from the group consisting of oxo, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, 3- to 6-membered heterocyclyl, C6-aryl, 5- to 6-membered heteroaryl, -CN, halogen, C1-C6alkoxy, C1-C6haloalkoxy, C1-C6haloalkyl, -OR 13 , -SR 13 , -S(O)2R 13 , -S(O)2NR 14 R 15 , -NR 13 S(O)2R 14 , -NR14 R 15 C(O)R 13 NR 13 C(O)R 14 NR 13 C(O)NR 14 R 15 C(O)OR 13 C(O)ONR 14 R 15 C(O)NR 14 R 15 and C1-C6 alkyl optionally substituted with oxo, -OH, or halogen;

[0036] each R c is independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3- to 6-membered heterocyclyl, -(C1-C6 alkylene)3- to 6-membered heterocyclyl, -(C1-C6 alkylene)C6-aryl, 5- to 6-membered heteroaryl, -(C1-C6 alkylene)5- to 6-membered heteroaryl, C1-C6 haloalkyl, -(C1-C6 alkylene)OR 13 , -(C1-C6 alkylene)SR 13 , -(C1-C6 alkylene)S(O)2R 13 , -(C1-C6 alkylene)S(O)2NR 14 R 15 , -(C1-C6 alkylene)NR 13 S(O)2R 14 , -(C1-C6 alkylene)NR 14 R 15 , -(C1-C6 alkylene)C(O)R 13 , -(C1-C6 alkylene)NR 13 C(O)R 14 , -(C1-C6 alkylene)NR 13 C(O)NR 14 R 15 , -(C1-C6 alkylene)C(O)OR 13 , -(C1-C6 alkylene)C(O)ONR 14 R 15 , or -(C1-C6 alkylene)-C(O)NR 14 R 15 , each of which is independently optionally substituted with R d ;

[0037] each R dindependently selected from the group consisting of halogen, -OH, oxo, -CN, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, -COOH, and C1-C6alkyl optionally substituted with -OH, halogen, CN, or oxo;

[0038] each R 10 , R 11 , and R 12 is independently hydrogen, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, -(C1-C6alkylene)C3-C6cycloalkyl, 3- to 6-membered heterocyclyl, or -(C1-C6alkylene)3- to 6-membered heterocyclyl, wherein R 10 , R 11 , and R 12 each is independently optionally substituted with oxo, C2-C6alkenyl, C2-C6alkynyl, -CN, halogen, C1-C6alkoxy, or C1-C6alkyl optionally substituted with oxo, -OH, or halogen;

[0039] or R 11 and R 12 , together with the atom to which they are attached, form a 3- to 6-membered heterocyclyl optionally substituted with oxo, -OH, halogen, or C1-C6alkyl optionally substituted with oxo, -OH, or halogen;

[0040] each R 13 , R 14 , and R 15 is independently hydrogen, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6haloalkyl, C3-C6cycloalkyl, or 3- to 6-membered heterocyclyl, wherein R 10 , R 11 , and R 12 each is independently optionally substituted with oxo, -OH, C2-C6alkenyl, C2-C6alkynyl, -CN, halogen, or C1-C6alkyl optionally substituted with oxo, -OH, or halogen;

[0041] or R 14 and R 15 , together with the atom to which they are attached, form a 3- to 6-membered heterocyclyl optionally substituted with oxo, -OH, halogen, or C1-C6alkyl optionally substituted with oxo, -OH, or halogen;

[0042] m is 0, 1, or 2; and

[0043] n is 0, 1, 2, 3, or 4;

[0044] provided that when Z is -NR cWhen S(O)2CH3, -CONH2, or -C(O)OH are involved, G is a bond and t is 1, then L is not a bond; and

[0045] The compound is not 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-3-azacyclobutanebutanoic acid, 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-3-azacyclobutanevalanoic acid, or N-((1-(6-fluorobenzo[d]oxazol-2-yl)azacyclobutane-3-yl)methyl)-N-methylacetamide. Attached Figure Description

[0046] Figure 1 The effects of the compounds disclosed herein on Saos-2 cell mineralization are demonstrated.

[0047] Figure 2 The time-dependent effects of compound 76 on PPi levels in Sprague-Dawley rats at 1, 4, and 8 hours after a single dose were demonstrated.

[0048] Figure 3 The time-dependent effects of compound 76 on mouse PPi levels were demonstrated at 1 and 8 hours after the last dose of a 5-day series of administrations.

[0049] It should be recognized that some or all of the diagrams are schematic representations for illustrative purposes. Detailed Implementation

[0050] definition

[0051] "Alkyl" refers to and includes saturated straight-chain and branched monovalent hydrocarbon structures and combinations thereof, having a specified number of carbon atoms (e.g., C1-C1). 10 This refers to one to ten carbon atoms. Specific alkyl groups are alkyl groups having 1 to 20 carbon atoms (“C1-C2”). 20 Alkyl groups are alkyl groups having 1 to 8 carbon atoms (“C1-C8 alkyl”), 3 to 8 carbon atoms (“C3-C8 alkyl”), 1 to 6 carbon atoms (“C1-C6 alkyl”), 1 to 5 carbon atoms (“C1-C5 alkyl”), or 1 to 4 carbon atoms (“C1-C4 alkyl”). Examples of alkyl groups include, but are not limited to, homologues and isomers of groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0052] As used herein, “alkenyl” refers to an unsaturated straight-chain or branched monovalent hydrocarbon chain or combination thereof, having at least one alkene unsaturated site (e.g., having at least one part of the formula C=C) and having a specified number of carbon atoms (e.g., C2-C). 10 This refers to two to ten carbon atoms. The alkenyl group can be in a "cis" or "trans" configuration, or alternatively, an "E" or "Z" configuration. Specific alkenyl groups include those with 2 to 20 carbon atoms ("C2-C"). 20 Alkenyl groups are groups having 2 to 8 carbon atoms (“C2-C8 alkenyl”), 2 to 6 carbon atoms (“C2-C6 alkenyl”), or 2 to 4 carbon atoms (“C2-C4 alkenyl”). Examples of alkenyl groups include, but are not limited to, groups such as: vinyl (ethenyl or vinyl), propenyl, propenyl (or allyl), 2-methylpropenyl, butenyl, butenyl, butenyl, butenyl, butenyl, 1,3-dienyl, 2-methylbutenyl, 1,3-dienyl, their homologues and isomers, etc.

[0053] As used herein, “alkylene” refers to a residue that is identical to an alkyl group but has a divalent oxidation state. Specific alkylene groups include alkylene groups having 1 to 6 carbon atoms (“C1-C6 alkylene”), 1 to 5 carbon atoms (“C1-C5 alkylene”), 1 to 4 carbon atoms (“C1-C4 alkylene”), or 1 to 3 carbon atoms (“C1-C3 alkylene”). Examples of alkylene groups include, but are not limited to, groups such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), etc. As used herein, “alkenylene” refers to a residue that is identical to an alkenyl group but has a divalent oxidation state.

[0054] As used herein, "alkynyl" refers to an unsaturated straight-chain or branched monovalent hydrocarbon chain or combination thereof, having at least one alkynyl unsaturated site (e.g., having at least one part of the formula C≡C) and having a specified number of carbon atoms (e.g., C2-C). 10 This refers to two to ten carbon atoms. Specific alkynyl groups include those with 2 to 20 carbon atoms ("C2-C"). 20 An alkynyl group is defined as an alkynyl group having 2 to 8 carbon atoms (“C2-C8 alkynyl”), 2 to 6 carbon atoms (“C2-C6 alkynyl”), or 2 to 4 carbon atoms (“C2-C4 alkynyl”). Examples of alkynyl groups include, but are not limited to, groups such as ethynyl, propynyl, propynyl-2-alkynyl (or propynyl), butynyl, butynyl, butynyl-2-alkynyl, butynyl-3-alkynyl, their homologues and isomers, etc.

[0055] "Aryl" refers to and includes polyunsaturated aromatic hydrocarbon groups. Aryl groups may contain additional fused rings (e.g., 1 to 3 rings), and additionally include fused aryl, heteroaryl, cycloalkyl, and / or heterocyclic rings. In one variant, the aryl group contains 6 to 14 ring carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, etc.

[0056] "Carbonyl" refers to the group C=O.

[0057] "Cycloalkyl" refers to and includes cyclic monovalent hydrocarbon structures that can be fully saturated, monounsaturated, or polyunsaturated, but not aromatic, and have a specified number of carbon atoms (e.g., C1-C1). 10 (This refers to one to ten carbon atoms). A cycloalkyl group can consist of one ring (such as cyclohexyl) or multiple rings (such as adamantyl), but does not include aryl groups. In some embodiments, a cycloalkyl group containing more than one ring can be fused, spirocyclic, bridged, or a combination thereof. In some embodiments, a cycloalkyl group is a cyclic hydrocarbon having 3 to 13 ring carbon atoms. In some embodiments, a cycloalkyl group is a cyclic hydrocarbon having 3 to 8 ring carbon atoms (“C3-C8 cycloalkyl”). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, borneol, etc.

[0058] "Halogen" or "halogen" refers to Group 17 elements with atomic numbers 9 to 85. In some embodiments, halogenated groups include fluorine, chlorine, bromine, and iodine. When a residue is substituted with more than one halogen, the residue can be designated by using a prefix corresponding to the number of halogen moieties attached, for example, dihaloaryl, dihaloalkyl, trihaloaryl, etc., refer to aryl and alkyl groups substituted with two ("di") or three ("tri") halogenated groups, which can be, but are not necessarily, the same halogen; thus, 4-chloro-3-fluorophenyl falls within the range of dihaloaryl. An alkyl group in which every hydrogen atom is substituted with a halogenated group is called a "perhaloalkyl". In some embodiments, the perhaloalkyl group is trifluoroalkyl (-CF3). Similarly, "perhaloalkoxy" is an alkoxy group in which each H atom in a hydrocarbon constituting the alkyl moieties of the alkoxy group is substituted with a halogen. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF3).

[0059] "Heteroaryl" refers to and includes unsaturated aromatic cyclic groups having 1 to 10 cyclic carbon atoms and at least one cyclic heteroatom, including but not limited to heteroatoms such as nitrogen, oxygen, and sulfur, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the remainder of the molecule at a cyclic carbon or cyclic heteroatom. Heteroaryl groups may contain additional fused rings (e.g., 1 to 3 rings), and additionally include fused aryl, heteroaryl, cycloalkyl, and / or heterocyclic rings. Examples of heteroaryl groups include, but are not limited to, imidazole, pyrrole, pyrazolyl, 1,2,4-triazolyl, thiophene, furanyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, oxazolyl, isoxazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, indolyl, inzolyl, benzimidazole, pyrrolopyridyl, pyrrolopyridazinyl, pyrrolopyrimidinyl, pyrazolopyridyl, pyrazolopyrimidinyl, imidazolepyridyl, purine, benzofuranyl, furanolpyridyl, benzooxazolyl, benzothiophene, benzothiazolyl, oxazolopyridyl, thiazopyridyl, thiophenepyridyl, quinolinyl, quinoloneyl, naphridyl, quinazolinyl, pyridinolylpyrimidinyl, cenolinyl, or pyridinolpyridazinyl, etc.

[0060] "Heterocyclic" or "heterocyclic group" refers to a saturated or unsaturated non-aromatic group having 1 to 10 ring carbon atoms and 1 to 4 ring heteroatoms (such as nitrogen, sulfur, or oxygen), wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Heterocyclic groups can have a single ring or multiple fused rings, but do not include heteroaryl groups. Heterocyclic groups containing more than one ring can be fused, spirocyclic, bridged, or any combination thereof. In fused ring systems, one or more fused rings can be aryl or heteroaryl. Examples of heterocyclic groups include, but are not limited to, aziridinyl, azirrocyclobutyl, oxacyclobutyl, morpholinyl, thiomorpholinyl, azirrocycloheptyl, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazine, pyrrolidinyl, thiazolinyl, thiazoalkyl, tetrahydrofuranyl, tetrahydrothiophene, etc.

[0061] "Oxytochemical" refers to the partial = O.

[0062] Unless otherwise stated, "optionally substituted" means that the group may be unsubstituted or substituted with one or more of the substituents listed for the group (e.g., 1, 2, 3, 4, or 5), wherein the substituents may be the same or different. 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 yet another embodiment, the optionally substituted group has four substituents. In some embodiments, the optionally substituted group has 1 to 2, 2 to 5, 3 to 5, 2 to 3, 2 to 4, 3 to 4, 1 to 3, 1 to 4, or 1 to 5 substituents.

[0063] As used herein, the term “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, i.e., that the material can be administered to an individual without causing undesirable biological effects or interacting with any component of a composition containing the material in a harmful manner.

[0064] "Pharmaceutically acceptable salt" means a salt comprising an active compound prepared with a relatively non-toxic acid or base, depending on the specific substituents present on the compound described herein. When the compound disclosed herein contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound in its neutral form with a sufficient amount of the desired base, either purely or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compound disclosed herein contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound in its neutral form with a sufficient amount of the desired acid, either purely or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, phosphoric acid, partially neutralized phosphoric acid, sulfuric acid, partially neutralized sulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Also included are amino acid salts (such as arginine salts) and organic acid salts (such as glucuronic acid or galacturonic acid). Certain specific compounds of this disclosure may contain both basic and acidic functional groups, allowing the compound to be converted into a basic or acid addition salt. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., (1985) and Journal of Pharmaceutical Science, 66:2 (1977), each of which is incorporated herein by reference in its entirety.

[0065] "Pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" means an excipient, carrier, or adjuvant that can be administered to a subject together with at least one compound without destroying the pharmacological activity of the compound, and is generally safe and non-toxic, and does not produce biological or other adverse effects when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0066] "Treatment" or "treating" is a method used to obtain a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes may include one or more of the following: a) suppressing a disease or condition (e.g., reducing one or more symptoms caused by the disease or condition and / or alleviating the severity of the disease or condition); b) slowing or halting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and / or preventing or delaying the spread of the disease or condition (e.g., metastasis)); and / or c) alleviating the disease, i.e., resulting in the resolution of clinical symptoms (e.g., improving the disease state, providing partial or complete relief of the disease or condition, enhancing the effect of another medication, delaying the progression of the disease, improving quality of life, and / or prolonging survival).

[0067] "Prevention" or "preventing" means any treatment of a disease or condition that results in the absence of clinical symptoms of that disease or condition. In some embodiments, the compound may be administered to subjects (including humans) who are at risk of the disease or condition or who have a family history of the disease or condition.

[0068] "Disease" is a health condition in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not improved.

[0069] "Subject" refers to an animal, such as a mammal (including a human), that has been or will be used as the subject of treatment, observation, or experimentation. The methods described herein can be used for human therapeutics and / or veterinary applications. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0070] The term "therapeutic effective amount" or "effective amount" for a compound or its pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analogue described herein means an amount sufficient to produce a therapeutic effect when administered to a subject, providing therapeutic benefits such as improvement of symptoms or slowing disease progression. For example, a therapeutic effective amount may be an amount sufficient to reduce symptoms of a disease or condition of hypophosphatase. A therapeutic effective amount can vary based on one or more of the following, which can be readily determined by a person skilled in the art: the subject and the disease or condition being treated, the subject's weight and age, the severity of the disease or condition, and the method of administration.

[0071] As used herein, the terms “NPP” or “ENPP” refer to exonucleotide pyrophosphatase / phosphodiesterase.

[0072] As used herein, the terms “alteration,” “defect,” “variation,” or “mutation” refer to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation), or conformation of the polypeptide it encodes. The mutations covered by this disclosure can be any mutation in a gene in a cell that results in enhancement or disruption of the function, activity, expression, or conformation of a polypeptide, including complete loss of expression of the protein, and can include, for example, missense and nonsense mutations, insertions, deletions, frameshifts, and premature termination. However, this is not limited to these; the mutations covered by this disclosure can alter mRNA splicing (splice site mutation) or cause a shift in the reading frame (frameshift).

[0073] The methods described herein can be applied to in vivo or ex vivo cell populations. “In vivo” means within a living individual, such as an animal or human. In this context, the methods described herein can be used therapeutically in an individual. “Ex vivo” means outside a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples, including liquid or tissue samples obtained from an individual. Such samples can be obtained by methods well known in the art. Exemplary biological liquid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the compounds and compositions described herein can be used for a variety of purposes, including therapeutic and experimental purposes. For example, the compounds and compositions described herein can be used ex vivo to determine the optimal administration regimen and / or dosage of the disclosed compounds for a given indication, cell type, individual, and other parameters. Information gathered from such uses can be used for experimental purposes or in clinical settings to establish regimens for in vivo treatments. Other ex vivo uses of the compounds and compositions described herein may be suitable for are described below or will become apparent to those skilled in the art. Selected compounds can be further characterized to examine safety or tolerable doses in human or non-human subjects. Such properties can be examined using methods well known to those skilled in the art.

[0074] As used in this article, "HPP" refers to hypophospholipase syndrome.

[0075] Treatment

[0076] This article provides methods for treating or preventing cartilage diseases, comprising administering a therapeutically effective amount of an exonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor to a subject in need. In some embodiments, methods for treating or preventing hypophosphatase syndrome are provided, comprising administering a therapeutically effective amount of an exonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor to a subject in need.

[0077] Hypophosphatase syndrome (HPP) is a hereditary metabolic disorder characterized by low activity of tissue-specific alkaline phosphatase (TNAP). This low TNAP activity leads to the systemic accumulation of its substrates, inorganic pyrophosphate (PPi) (a potent mineralization inhibitor) and pyridoxal phosphate (PLP) (a cofactor for many enzymes), which largely explains the musculoskeletal and systemic features of the disease. HPP is characterized by a wide range of clinical manifestations and severity, ranging from intrauterine death to dental complications occurring only in children and adults or asymptomatic carriers of ALPL mutations. Due to the relative rarity of the disease, lack of physician awareness of HPP, and the lack of characteristic symptoms (especially in the mild adult form), its diagnosis and treatment remain significantly delayed.

[0078] This article also provides a method for treating or preventing hypophospholipase syndrome, which involves administering to a subject in need a therapeutically effective amount of a composition comprising an ENPP1 inhibitor and a pharmaceutically acceptable carrier.

[0079] In some embodiments, a therapeutic amount of an ENPP1 inhibitor treats hypophosphatase in subjects who require this treatment by restoring normal circulating PPi levels.

[0080] In some embodiments, a therapeutic amount of an ENPP1 inhibitor treats hypophosphatase in subjects who require this treatment by modulating the phosphate / pyrophosphate (Pi / PPi) ratio.

[0081] In some embodiments, a therapeutic amount of an ENPP1 inhibitor treats hypophosphatase in subjects who require this treatment by promoting mineralization.

[0082] In some embodiments, the subject has prenatal hypophospholipase syndrome.

[0083] In some embodiments, the subjects had perinatal hypophosphatase syndrome.

[0084] In some embodiments, the subjects had infantile hypophosphatase syndrome.

[0085] In some embodiments, the subjects had childhood hypophosphodiesterase syndrome.

[0086] In some embodiments, the subjects had adult-onset hypophosphodiesterase syndrome.

[0087] In some embodiments, the subjects had dental hypophosphodiesterase syndrome.

[0088] In some embodiments, the subjects had periodontal disease.

[0089] In some embodiments, the subject's ALPL gene is altered. In some embodiments, the alteration of the ALPL gene is a mutation. In some embodiments, the subject's gene is altered in a way that can regulate TNSALP activity. In some embodiments, the gene alteration that can regulate TNSALP activity is an alteration of RUNX2.

[0090] In some embodiments, a therapeutically effective amount of an ENPP1 inhibitor is administered daily. In some embodiments, a therapeutically effective amount of an ENPP1 inhibitor is administered two to three times daily.

[0091] In some embodiments, a therapeutically effective amount of an ENPP1 inhibitor is formulated for oral administration.

[0092] In some embodiments, a therapeutically effective amount of an ENPP1 inhibitor is formulated for subcutaneous administration.

[0093] In some embodiments, a therapeutically effective amount of an ENPP1 inhibitor is formulated for intraperitoneal administration.

[0094] compound

[0095] This document provides compounds that inhibit ENPP1, wherein such compounds can be used in the methods and compositions disclosed herein, such as for the treatment or prevention of cartilage diseases. Such compounds can also be used to treat hypophosphatase syndrome.

[0096] In some embodiments, the ENPP1 inhibitor is a compound described in US20220135598A1. In some embodiments, the compound is a compound of formula I:

[0097] I

[0098] Or its pharmaceutically acceptable salt, wherein:

[0099] The ring C is a 5- to 6-membered heteroaryl group;

[0100] Ring D is a C6-aryl or a 5- to 6-membered heteroaryl group, wherein ring D is fused with ring C;

[0101] A is hydrogen, C1-C6 alkyl or C6-aryl, wherein each is optionally substituted with a halogen;

[0102] G represents a bond, -CH2- or -CH2-CH2-;

[0103] R a and R b Independently hydrogen or C1-C6 alkyl;

[0104] Or R a and R bTogether with the atoms they are attached to, they form C3-C6 cyclic alkyl rings;

[0105] Or R a and R b Any of them, along with A and the atoms they are attached to, form a C4-C6 cyclic alkyl ring;

[0106] L represents a bond, a straight-chain or branched C1-C6 alkylene group, or a straight-chain or branched C2-C6 alkenyl group;

[0107] t is 0 or 1; the condition is that when t is 0, L is a straight-chain or branched C2-C6 alkenyl group.

[0108] Z is -NR c S(O)2NH2、-NR c S(O)2CH3, -SO2NH2, -NR c C(O)CH3, -C(O)OH, -CONH2, -NR c CONH2, -CONH(OH), -B(OH)2, -P(O)(OH)2, -SO2OH, -NR c S(O)2CF3、-NR c S(O)2NHCH3 or -NR c CH2C6-aryl-S(O)2NH2.

[0109] Each R 1 and R 2 Independently oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, adamantyl, 3- to 6-membered heterocyclic, C6-aryl, 5- to 6-membered heteroaryl, -CN, halogen, C1-C6 alkoxy, C1-C6 haloalkoxy, -OR 10 -SR 10 -S(O)2R 10 -S(O)2NR 11 R 12 -NR 10 S(O)2R 11 -NR 11 R 12 -C(O)R 10 -NR 10 C(O)R 11 -NR 10 C(O)NR 11 R 12 -C(O)OR 10 -C(O)ONR 11 R 12 or -C(O)NR 11 R 12Each of them can be independently and optionally controlled by R 9 replace;

[0110] Or two Rs 2 Together with the atoms to which they are attached, they form C5-C6 cycloalkyl, 5- to 6-membered heterocyclic, C6-aryl, or 5- to 6-membered heteroaryl groups, wherein each is optionally independently controlled by R. 9 replace;

[0111] Each R 9 Independently selected from the group consisting of: oxo, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 6-membered heterocyclic, C6-aryl, 5- to 6-membered heteroaryl, -CN, halogen, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 haloalkyl, -OR 13 -SR 13 -S(O)2R 13 -S(O)2NR 14 R 15 -NR 13 S(O)2R 14 -NR 14 R 15 -C(O)R 13 -NR 13 C(O)R 14 -NR 13 C(O)NR 14 R 15 -C(O)OR 13 -C(O)ONR 14 R 15 -C(O)NR 14 R 15 And C1-C6 alkyl groups optionally substituted with oxo, -OH or halogen;

[0112] Each R c Independently, it can be hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3- to 6-membered heterocyclic, -(C1-C6 alkylene)C3- to 6-membered heterocyclic, -(C1-C6 alkylene)C6-aryl, 5- to 6-membered heteroaryl, -(C1-C6 alkylene)5- to 6-membered heteroaryl, C1-C6 haloalkyl, -(C1-C6 alkylene)OR 13 -(C1-C6 alkylene)SR 13 -(C1-C6 alkylene)S(O)2R 13 -(C1-C6 alkylene)S(O)2NR 14 R 15 -(C1-C6 alkylene)NR 13 S(O)2R14 -(C1-C6 alkylene)NR 14 R 15 -(C1-C6 alkylene)C(O)R 13 -(C1-C6 alkylene)NR 13 C(O)R 14 -(C1-C6 alkylene)NR 13 C(O)NR 14 R 15 -(C1-C6 alkylene)C(O)OR 13 -C1-C6 alkylene)C(O)ONR 14 R 15 or -(C1-C6 alkylene)-C(O)NR 14 R 15 Each of them can be independently and optionally processed by R d replace;

[0113] Each R d Independently selected from the group consisting of: halogen, -OH, oxo, -CN, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -COOH and C1-C6 alkyl optionally substituted with -OH, halogen, CN or oxo.

[0114] Each R 10 R 11 and R 12 Independently, it is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3- to 6-membered heterocyclic group, or -(C1-C6 alkylene)3- to 6-membered heterocyclic group, wherein RR 10 R 11 and R 12 Each of them may be independently and optionally substituted with an oxo, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen, C1-C6 alkoxy or optionally substituted with an oxo, -OH or halogen-substituted C1-C6 alkyl group;

[0115] Or R 11 and R 12 Together with the atoms to which they are attached, they form 3 to 6-membered heterocyclic groups, which may optionally be substituted with oxo, -OH, halogen or C1-C6 alkyl groups substituted with oxo, -OH or halogen;

[0116] Each R 13 R 14 and R 15Independently, it is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or a 3- to 6-membered heterocyclic group, wherein R 10 R 11 and R 12 Each of them may be independently and optionally substituted with an oxo, -OH, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen or optionally substituted with an oxo, -OH or halogen C1-C6 alkyl group;

[0117] Or R 14 and R 15 Together with the atoms to which they are attached, they form 3 to 6-membered heterocyclic groups, which may optionally be substituted with oxo, -OH, halogen or C1-C6 alkyl groups substituted with oxo, -OH or halogen;

[0118] m is 0, 1, or 2; and

[0119] n can be 0, 1, 2, 3, or 4;

[0120] The condition is when Z is -NR c When S(O)2CH3, -CONH2, or -C(O)OH are involved, G is a bond and t is 1, then L is not a bond.

[0121] In some embodiments, the compound is not 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-3-azacyclobutanebutanoic acid, 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-3-azacyclobutanevalanoic acid, or N-[[1-(6-fluoro-2-benzoxazolyl)-3-azacyclobutane]methyl]-N-methylacetamide.

[0122] In some embodiments, A is hydrogen. In some embodiments, A is a C1-C6 alkyl group. In some embodiments, A is methyl. In some embodiments, A is a C1-C6 alkyl group optionally substituted with a halogen. In some embodiments, A is -CF3. In some embodiments, A is a C6-aryl group. In some embodiments, A is phenyl.

[0123] In some embodiments, G is a key. In some embodiments, G is -CH2-. In some embodiments, G is -CH2-CH2-.

[0124] In some embodiments, R a and R b Independently hydrogen or C1-C6 alkyl. In some embodiments, R a and R bBoth are hydrogen. In some embodiments, R a and R b Either of them is hydrogen, and the other is a C1-C6 alkyl group. In some embodiments, R a and R b Both are C1-C6 alkyl groups. In some embodiments, R a and R b Either of them is hydrogen, and the other is methyl. In some embodiments, R a and R b Either of them is hydrogen, and the other is ethyl. In some embodiments, R a and R b Both are methyl groups.

[0125] In some embodiments, R a and R b Together with the atoms they are attached to, they form C3-C6 cycloalkyl rings. In some embodiments, R a and R b Together with the atoms they are attached to, they form a cyclopropyl ring.

[0126] In some embodiments, R a and R b Any of these, along with A and the atoms they are attached to, form a C4-C6 cycloalkyl group. In some embodiments, R a and R b Any of these atoms, along with A and the atoms they are attached to, form a cyclobutyl ring. In some embodiments, R a and R b Any of these atoms, along with A and the atoms they are attached to, form a cyclopentyl ring. In some embodiments, R a and R b Any one of them, along with A and the atoms they are attached to, forms a cyclohexyl ring.

[0127] In some embodiments, R a and R b Any of these, along with A and the atoms they are attached to, form a C4-C6 cycloalkyl ring. In such cases, R a Or R b The other component is hydrogen. In some embodiments, R a and R b Any of the atoms in R, along with A and the atoms they are attached to, form a cyclobutyl ring. In such cases, R a Or R b The other component is hydrogen. In some embodiments, R a and R bAny of the atoms in R, along with A and the atoms they are attached to, form a cyclopentyl ring. In such cases, R a Or R b The other component is hydrogen. In some embodiments, R a and R b Any of the atoms in R, along with A and the atoms they are attached to, form a cyclohexyl ring. In such cases, R a Or R b The other one is hydrogen.

[0128] In some embodiments, G is a bond, -CH2- Or -CH2-CH2-; and R a and R b Any of these, along with A and the atoms they are attached to, form a C4-C6 cycloalkyl ring. In some embodiments, G is -CH2-; and R a and R b Any of these atoms, along with A and the atoms they are attached to, form a cyclobutyl ring. In some embodiments, G is -CH2-; and R... a and R b Any of these atoms, along with A and the atoms they are attached to, form a cyclopentyl ring. In some embodiments, G is -CH2-CH2-; and R... a and R b Any one of them, along with A and the atoms they are attached to, forms a cyclohexyl ring.

[0129] In some embodiments, L is a bond or a C1-C6 alkylene or C2-C6 alkenylene group. The C1-C6 alkylene or C2-C6 alkenylene group can be straight-chain or branched. In some embodiments, L is a bond. In some embodiments, L is a C1-C6 alkylene group. In some embodiments, L is -CH2-. In some embodiments, L is -C2H4-. In some embodiments, L is -C3H6-. In some embodiments, L is -CH(CH3)-. In some embodiments, L is -C(CH3)2-. In some embodiments, L is -CH(CH3)-CH2-. In some embodiments, L is -CH2-CH(CH3)-. In some embodiments, L is -C(CH3)2-CH2-. In some embodiments, L is -CH2-C(CH3)2-. In some embodiments, L is -CH(C2H5)-CH2-.

[0130] In some embodiments, L is a C2-C6 alkenyl group. The C2-C6 alkenyl group can be straight-chain or branched. In some embodiments, L is -CH=CH-. In some embodiments, L is -CH2-CH=CH-.

[0131] In some embodiments, t is 0 or 1. In some embodiments, t is 0. In some embodiments, t is 1.

[0132] In some embodiments, when t is 0, then R a and R b It does not exist, and L is a C2-C6 alkenyl group. In some embodiments, when t is 0, then R a and R b It does not exist, and L is -CH=CH-.

[0133] In some embodiments, when t is 1, then R a and R b It exists, and L is a bond or a C1-C6 alkylene group. In some embodiments, when t is 1, then R a and R b It exists, and L is the key. In some embodiments, when t is 1, then R a and R b It exists, and L is a C1-C6 alkylene group. In some embodiments, when t is 1, then R a and R b It exists, and L is -CH2-. In some embodiments, when t is 1, then R a and R b It exists, and L is -CH2-CH2-. Where R... a and R b It is independently selected from hydrogen or C1-C6 alkyl.

[0134] In some embodiments, when t is 1, then R a and R b It exists, and R a and R b Any of these, along with A and the atoms they are attached to, form a C4-C6 cycloalkyl ring. In such cases, R a Or R b The other component is hydrogen. In some embodiments, when t is 1, then R a and R b It exists, and R a and R b Any of the atoms in R, along with A and the atoms they are attached to, form a cyclobutyl ring. In such cases, R a Or R b The other one is hydrogen.

[0135] In some embodiments, Z is selected from -NR c SO2NH2, -NR c S(O)2CH3, -SO2NH2, -NR cC(O)CH3, -C(O)OH, -CONH2, -NR c CONH2, -CONH(OH), -B(OH)2, -P(O)(OH)2, -SO2OH, -NR c S(O)2CF3、-NR c S(O)2NHCH3 or -NR c CH2C6-aryl-S(O)2NH2. In some embodiments, Z is -NR. c S(O)₂NH₂. In some embodiments, Z is -NR. c S(O)₂CH₃. In some embodiments, Z is -SO₂NH₂. In some embodiments, Z is -NR. c C(O)CH3. In some embodiments, Z is -C(O)OH. In some embodiments, Z is -CONH2. In some embodiments, Z is -NR. c CONH2. In some embodiments, Z is -CONH(OH). In some embodiments, Z is -B(OH)2. In some embodiments, Z is -P(O)(OH)2. In some embodiments, Z is -SO2OH. In some embodiments, Z is -NR. c S(O)2CF3. In some embodiments, Z is -NR. c S(O)₂NHCH₃. In some embodiments, Z is -NR. c CH2C6-aryl-S(O)2NH. .. In some embodiments, R c Selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C6 haloalkyl. In some embodiments, R c methyl. In some embodiments, R c It is ethyl. In some embodiments, R c It is isopropyl. In some embodiments, R... c It is n-propyl. In some embodiments, R... c It is cyclopropyl. In some embodiments, R c It is cyclobutyl. In some embodiments, R c It is cyclopentyl. In some embodiments, R c for In some embodiments, R c for In some embodiments, R c for .

[0136] In some embodiments, R c For optional R dSubstituted -(C1-C6 alkylene)C3-C6 cycloalkyl. In some embodiments, R c For optional R d Replaced 3- to 6-membered heterocyclic groups. In some embodiments, R c For optional R d Substituted -(C1-C6 alkylene)3 to 6-membered heterocyclic groups. In some embodiments, R c For optional use by R d Substituted -(C1-C6 alkylene)C6-aryl. In some embodiments, R c For optional R d Substituted 5- to 6-membered heteroaryl groups. In some embodiments, R c For optional R d Substituted -(C1-C6 alkylene) 5- to 6-membered heteroaryl. In some embodiments, R c -(C1-C6 alkylene)OR 13 In some embodiments, R c -(C1-C6 alkylene)SR 13 In some embodiments, R c -(C1-C6 alkylene)S(O)2R 13 In some embodiments, R c -(C1-C6 alkylene)S(O)2NR 14 R 15 In some embodiments, R c -(C1-C6 alkylene)NR 13 S(O)2R 14 In some embodiments, R c -(C1-C6 alkylene)NR 14 R 15 In some embodiments, R c -(C1-C6 alkylene)C(O)R 13 In some embodiments, R c -(C1-C6 alkylene)NR 13 C(O)R 14 In some embodiments, R c -(C1-C6 alkylene)NR 13 C(O)NR 14 R 15 In some embodiments, R c -(C1-C6 alkylene)C(O)OR 13 In some embodiments, R c -(C1-C6 alkylene)C(O)ONR 14 R 15In some embodiments, R c -(C1-C6 alkylene)-C(O)NR 14 R 15 .

[0137] In some embodiments, any R c The -C1-C6 alkylene group can optionally be via R d replace.

[0138] In some embodiments, any R c The -C1-C6 alkylene groups are either straight-chain or branched.

[0139] In some embodiments, R c Choose from the group consisting of: methyl, ethyl, isopropyl, n-propyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0140] In some embodiments, Z is selected from the group consisting of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0141] In some embodiments, Z is -NR c SO2NH2.

[0142] In some embodiments, Z is -NHSO2NH2. In some embodiments, Z is... In some embodiments, Z is In some embodiments, Z is In some embodiments, Z is In some embodiments, Z is In some embodiments, Z is In some embodiments, Z is In some embodiments, Z is .

[0143] In some embodiments, Z is -NHS(O)2CH3.

[0144] In some embodiments, Z is -SO2NH2.

[0145] In some embodiments, Z is -NHC(O)CH3.

[0146] In some embodiments, Z is -C(O)OH.

[0147] In some embodiments, Z is -CONH2.

[0148] In some embodiments, Z is -NHCONH2.

[0149] In some embodiments, Z is -CONH(OH).

[0150] In some embodiments, Z is -B(OH)2.

[0151] In some embodiments, Z is -P(O)(OH)2.

[0152] In some embodiments, Z is -S(O)2OH.

[0153] In some embodiments, Z is -NR c S(O)2CF3.

[0154] In some embodiments, Z is -NR c S(O)2NHCH3.

[0155] In some embodiments, Z is -NR c CH2C6-aryl-S(O)2NH2.

[0156] In some embodiments, Z is -NHCH2C6-aryl-S(O)2NH2.

[0157] In some embodiments, Z is -N(CH3)CH2C6-aryl-S(O)2NH2.

[0158] In some embodiments, G, R a R b L, t, and Z are selected together from the following groups: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0159] In some embodiments, when R a and R b When any of the atoms in L and Z together form a C4-C6 cycloalkyl ring, along with A and the atoms to which they are attached, L and Z are selected from the group consisting of the following: , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0160] In some embodiments, when R a and R b When any of the atoms in L and Z together form a C4-C6 cycloalkyl ring, along with A and the atoms to which they are attached, L and Z are selected from the group consisting of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0161] In some embodiments, when R a and R b When either of the following, along with A and the atoms to which they are attached, forms a cyclobutyl ring (which forms an azirospiroheptane ring with aziridine), then L and Z are selected from the group consisting of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0162] In some embodiments, ring C is optionally routed via R 1 The substituted 5- to 6-membered heteroaryl group. In embodiments, ring C is optionally substituted via R. 1 The substituted 5-membered heteroaryl group. In some embodiments, ring C is optionally substituted with R. 1 A substituted 6-membered heteroaryl group. In some embodiments, the ring C is selected from the group consisting of: imidazole, pyrazole, pyrrole, pyridine, pyrimidine, pyridinone, pyrimidinone, pyridazine, pyridazinone, and triazine, wherein each of these may optionally be substituted with R 1 Replacement. In some embodiments, R 1 Selected from hydrogen, oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, 3- to 6-membered heterocyclic, C6-aryl, 5- to 6-membered heteroaryl, -CN, -CONR 11 R 12 or -NR 11 R 12 Each of these can optionally be R 9 Replacement. In some embodiments, R 1It is selected from hydrogen, oxo, methyl, ethyl, vinyl, propynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, oxacyclobutyl, tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidinyl, phenyl, -CONH2, -NH2, -CHF2, -CF3, -Cl and -CN; wherein each of these may optionally be further substituted by pyridyl, -F, -CF3 or -CHF2.

[0163] In some embodiments, ring D is a C6-aryl or a 5- to 6-membered heteroaryl, each of which may optionally be R- 2 Replacement; wherein ring D is fused with ring C. In some embodiments, ring D is optionally via R 2 Substituted C6-aryl; wherein ring D is fused to ring C. In some embodiments, ring D is optionally via R 2 A substituted 5- to 6-membered heteroaryl group; wherein ring D is fused with ring C. In some embodiments, ring D is optionally substituted with R 2 A substituted 5-membered heteroaryl group; wherein ring D is fused with ring C. In some embodiments, ring D is optionally substituted with R 2 A substituted 6-membered heteroaryl group; wherein ring D is fused with ring C. In some embodiments, ring D is selected from the group consisting of: optionally via R 2 Substituted phenyl, pyrrole, pyrazole, imidazole, pyridine, thiophene, and pyrimidine; wherein each of these is fused with a ring C. In some embodiments, R 2 Selected from hydrogen, halogen, oxo, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, -OR 10 -CN and C6-aryl; wherein each of these may optionally be R 9 Replacement. In some embodiments, R 2 Selected from hydrogen, fluorine, bromine, chlorine, oxo, methyl, ethyl, isopropyl, methoxy, ethoxy, propoxy, phenyl, 4-methoxyphenyl, -CN, -OCH2F, -OCH2OCH3, -(OCH2CH2)morpholinyl, 4-hydroxycyclohexyl, -CF3, cyclopropyl and phenyl.

[0164] In some embodiments, R 2 Any two of these, together with the atoms to which they are attached, form a C5-C6 cycloalkyl, a 5- to 6-membered heterocyclic group, a C6-aryl, or a 5- to 6-membered heteroaryl, wherein each of these may optionally be R 9 Replace; in some embodiments, R 2 Any two of them together with the atoms to which they are attached form imidazole, dioxolane, and dihydrodioxane, wherein each of these may optionally be substituted with a methyl group.

[0165] In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2.

[0166] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0167] In some embodiments, rings C, D, and R 1 and R 2 Choose a group consisting of the following items:

[0168] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and , , , , and The wavy line indicates the connection point.

[0169] In some embodiments, rings C, D, and R 1 and R 2 Choose a group consisting of the following items:

[0170] , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and The wavy line indicates the connection point.

[0171] It should be understood that for A, G, R a R b L, Z, ring C, ring D, R 1 R 2 R c Each description of m, n, and t can be independently compared with the pairs A, G, and R. a R b L, Z, ring C, ring D, R 1 R 2 R c Each combination of descriptions of m, n, and t is listed as if each combination were specific and separate.

[0172] In some embodiments, a compound of formula II is provided:

[0173] II

[0174] or its salts, wherein A, R a R b L, Z, ring C, ring D, R 1 R 2 m and n are each described independently as detailed in this article.

[0175] In some embodiments, a compound of formula III is provided:

[0176] III

[0177] Or its salts, wherein L, Z, ring C, ring D, R 1 R 2 m and n are each described independently as detailed in this article.

[0178] In some embodiments, a compound of formula IV is provided:

[0179] IV

[0180] or its salt, wherein:

[0181] X is N or CR 1 ;

[0182] Y is N or CR 1 The condition is that neither X nor Y is N; and

[0183] A, G, R a R b L, Z, ring D, R 1 R 2 n and t are each described independently as detailed in this article.

[0184] In some embodiments, a compound of any one of formulas IV-1 to IV-11 is provided:

[0185] IV-1 IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10 IV-11

[0186] or its salt, wherein

[0187] X1, X2, and X3 are independently N and NR. 2 or CR 2 ;

[0188] The condition is that any one of X1, X2, and X3 is NR. 2 And the others are N or CR 2 ;and

[0189] A, G, R a R b L, Z, R 1 R 2 t and t are described independently as detailed in this article.

[0190] In some embodiments, a compound of formula V is provided:

[0191] V

[0192] or its salt, wherein

[0193] X is N or CR 1 ;

[0194] Y is N or CR 1 The condition is that neither X nor Y is N; and

[0195] L, Z, ring D, R 1 R 2 Each of them, n, is described independently as detailed in this article.

[0196] In some embodiments, a compound of any one of formulas V-1 to V-11 is provided:

[0197] V-1 V-2 V-3 V-4 V-5 V-6 V-7 V-8 V-9 V-10 V-11

[0198] or its salt, wherein

[0199] X1, X2, and X3 are independently N and NR. 2 or CR 2 ;

[0200] The condition is that any one of X1, X2, and X3 is NR. 2 And the others are N or CR 2 ;and

[0201] L, Z, R 1 and R 2 Each is described independently as detailed in this article.

[0202] In some embodiments, a compound of formula VI is provided:

[0203] VI

[0204] or its salt, wherein

[0205] Y1 is N or NR 1 ;

[0206] Y2 represents N and NR 1 or CR 1 The condition is that either Y1 or Y2 is NR.1 And the other is not NR 1 ;and

[0207] A, G, R a R b L, Z, R 1 R 2 t and t are described independently as detailed in this article.

[0208] In some embodiments, a compound of formula VII is provided:

[0209] VII

[0210] or its salt, wherein

[0211] Z1 is N or CR 1 ;and

[0212] A, G, R a R b L, Z, R 1 R 2 t and t are described independently as detailed in this article.

[0213] In some embodiments, a compound of formula VIII is provided:

[0214] VIII

[0215] or its salts, wherein A, G, R a R b L, Z, R 1 R 2 t and t are described independently as detailed in this article.

[0216] In some embodiments, a compound of formula IX is provided:

[0217] IX

[0218] or its salt, wherein

[0219] Y1 is N or NR 1 ;

[0220] Y2 represents N and NR 1 or CR 1 The condition is that either Y1 or Y2 is NR. 1 And the other is not NR 1 ;and

[0221] L, Z, R 1 and R 2 Each is described independently as detailed in this article.

[0222] In some embodiments, a compound of formula X is provided:

[0223] X

[0224] or its salt, wherein

[0225] Z1 is N or CR 1 ;and

[0226] L, Z, R 1 and R 2 Each is described independently as detailed in this article.

[0227] In some embodiments, a compound of formula XI is provided:

[0228] XI

[0229] or its salts, wherein L, Z, R 1 and R 2 Each is described independently as detailed in this article.

[0230] In some embodiments, a compound of formula XII is provided:

[0231] XII

[0232] or its salt, wherein,

[0233] X is N or CR 1 ;

[0234] X4 and X5 are independently N or CR 2 The condition is that neither of them is N; and

[0235] A, G, R a R b L, Z, R 1 R 2 t and t are described independently as detailed in this article.

[0236] In some embodiments, a compound of any one of formulas XII-1 to XII-6 is provided:

[0237] XII-1 XII-2 XII-3 XII-4 XII-5 XII-6

[0238] or its salts, wherein A, G, R a R b L, Z, R 1 R 2 R c t and t are described independently as detailed in this article.

[0239] In some embodiments, a compound of formula XIII is provided:

[0240] XIII

[0241] or its salt, wherein

[0242] X is N or CR 1 ;

[0243] X4 and X5 are independently N or CR 2 The condition is that neither of them is N.

[0244] And L, Z, R 1 and R 2 Each is described independently as detailed in this article.

[0245] In some embodiments, a compound of any one of formulas XIII-1 to XIII-9 is provided:

[0246] XIII-1 XIII-2 XIII-3 XIII-4 XIII-5 XIII-6 XIII-7 XIII-8 XIII-9

[0247] or its salts, wherein L, Z, R 1 R 2 and R c Each is described independently as detailed in this article.

[0248] Salts of the compounds mentioned herein, such as pharmaceutically acceptable salts, are also provided. This disclosure also includes any or all stereochemical forms of the said compounds, including any enantiomers or diastereomers, as well as any tautomers or other forms.

[0249] In some embodiments, the compound is selected from Table 1. It should be understood that individual enantiomers and diastereomers are included in the general compound structures shown in Table 1. Specific synthetic methods for preparing the compounds in Table 1 are provided in US20220135598A1.

[0250] Table 1

[0251] Compound No. Structure Compound No. Structure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

[0252] Reagent test kit

[0253] This document also provides a kit comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analog thereof, and suitable packaging. In one embodiment, the kit further includes instructions for use. In one aspect, the kit comprises a compound of the present disclosure or a pharmaceutically acceptable salt thereof, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analog thereof, and a label and / or instructions for using the compound to treat an indication including the diseases or conditions described herein.

[0254] This document also provides articles comprising, in suitable containers, the compounds described herein or their pharmaceutically acceptable salts, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs. Containers may be vials, wide-mouth bottles, ampoules, pre-filled syringes, and intravenous bags.

[0255] Pharmaceutical Compositions and Administration Methods

[0256] The compounds described herein are typically administered in the form of pharmaceutical compositions. Therefore, pharmaceutical compositions comprising one or more of the compounds described herein or their pharmaceutically acceptable salts, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs, and one or more pharmaceutically acceptable carriers selected from carriers, adjuvants, and excipients are also provided herein. Suitable pharmaceutically acceptable carriers may include, for example, inert solid diluents and fillers, diluents (including sterile aqueous solutions and various organic solvents), permeation enhancers, solubilizers, and adjuvants. Such compositions are prepared in a manner well known in the pharmaceutical industry. See, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa., 17th edition (1985); and Modern Pharmaceuticals, Marcel Dekker, Inc., 3rd edition (GS Banker & CT Rhodes, editors).

[0257] The pharmaceutical composition can be administered in single or multiple doses. It can be administered by various methods, including, for example, rectal, sublingual, intranasal, and transdermal routes. In some embodiments, the pharmaceutical composition can be administered via intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, or as an inhaler.

[0258] One mode of administration is parenteral, such as by injection. The pharmaceutical compositions described herein may be contained in forms for administration by injection, including, for example, aqueous or oil suspensions or emulsions containing sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, glucose, or sterile aqueous solutions, and similar drug carriers.

[0259] Oral administration may be another route of administration for the compounds described herein. Administration may be via, for example, capsules or enteric-coated tablets. In the preparation of pharmaceutical compositions comprising at least one of the compounds described herein or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analogue thereof, the active ingredient is typically diluted with an excipient and / or encapsulated in a carrier, which may be a capsule, sac, paper, or other container. When the excipient acts as a diluent, it may be in the form of a solid, semi-solid, or liquid material that can act as a mordant, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft gelatin capsules and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

[0260] Some examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, astragalus gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulation may also contain lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methylparaben and propylparaben; sweeteners; and flavoring agents.

[0261] Compositions comprising at least one of the compounds described herein or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analogue thereof may be formulated using methods known in the art to provide a rapid, sustained, or delayed release of the active ingredient upon administration to a subject. Controlled-release drug delivery systems for oral administration include osmotic pump systems and dissolution systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled-release systems are given in U.S. Patent Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345. Another formulation for use with the methods disclosed herein employs a transdermal delivery device (“patch”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds described herein in controlled amounts. The construction and use of transdermal patches for delivering pharmaceutical agents are well known in the art. See, for example, U.S. Patent Nos. 5,023,252, 4,992,445, and 5,001,139. Such patches can be configured for continuous, pulsed, or on-demand drug delivery.

[0262] To prepare solid compositions (such as tablets), a major active ingredient may be mixed with a pharmaceutical excipient to form a homogeneous mixture of the compound described herein or its pharmaceutically acceptable salts, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs. When these preformation compositions are referred to as homogeneous, the active ingredient can be uniformly dispersed throughout the composition, making it easy to further fractionate the composition into equally effective unit dosage forms, such as tablets, pills, and capsules.

[0263] The tablets or pills of the compounds described herein may be coated or otherwise compounded to provide a dosage form with the advantage of prolonged action or protection from the acidic conditions of the stomach. For example, the tablets or pills may comprise an internal dose component and an external dose component, the latter being coated over the former. The two components may be separated by an enteric coating layer, which resists disintegration in the stomach and allows the internal component to be delivered intact into the duodenum or to be released with a delay. Various materials may be used for such an enteric coating or coating, including a variety of polymeric acids and mixtures of polymeric acids with such materials as shellac, hexadecyl alcohol, and cellulose acetate.

[0264] Compositions for inhalation or inhalation may comprise solutions and suspensions or mixtures thereof in pharmaceutically acceptable aqueous or organic solvents, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. In some embodiments, these compositions are administered via the oral or nasal route for local or systemic effects. In other embodiments, compositions in pharmaceutically acceptable solvents may be nebulized using an inert gas. The nebulized solution may be inhaled directly from a nebulizer, or the nebulizer may be attached to a mask plug or intermittent positive pressure ventilation machine. The solution, suspension, or powder composition may preferably be administered orally or nasally from a device that delivers the formulation in a suitable manner.

[0265] Dosage

[0266] For any given subject, the specific dose level of the compounds in this application will depend on a variety of factors, including the activity of the particular compound used, the age, weight, general health, sex, diet, time of administration, route of administration and excretion rate, drug combination, and the severity of the specific disease. For example, the dose may be expressed as milligrams (mg / kg) of the compound described herein per kilogram of the subject's body weight. A dose between about 0.1 and 150 mg / kg may be suitable. In some embodiments, about 0.1 to 100 mg / kg may be suitable. In other embodiments, a dose between 0.5 and 60 mg / kg may be suitable. Standardization based on the subject's weight is particularly useful when adjusting the dose among subjects with significant differences in body size, such as when using the drug in children and adults, or when converting an effective dose for non-human subjects (e.g., dogs) to a dose suitable for human subjects.

[0267] The daily dose can also be described as the total amount of the compound described herein administered per dose or per day. The daily dose of the compound of Formula I can be from about 1 mg to 4,000 mg, about 2,000 to 4,000 mg / day, about 1 to 2,000 mg / day, about 1 to 1,000 mg / day, about 10 to 500 mg / day, about 20 to 500 mg / day, about 50 to 300 mg / day, about 75 to 200 mg / day, or about 15 to 150 mg / day.

[0268] When administered orally, the total daily dose for human subjects may be 1 mg to 1,000 mg, about 1,000 to 2,000 mg / day, about 10 to 500 mg / day, about 50 to 300 mg / day, about 75 to 200 mg / day, or about 100 to 150 mg / day.

[0269] The compound or combination thereof may be administered once, twice, three or four times daily using any of the suitable modes described above. Furthermore, the application or treatment may continue for several days; for example, a treatment cycle typically lasts at least 7, 14, or 28 days. Between cycles, the treatment cycle may be alternated with rest periods of approximately 1 to 28 days (typically approximately 7 or 14 days). In other embodiments, the treatment cycle may also be continuous.

[0270] In certain embodiments, the method includes administering to a subject an initial daily dose of about 1 to 800 mg of the compound described herein, and increasing the dose in increments until clinical efficacy is achieved. Increments of about 5, 10, 25, 50, or 100 mg may be used to increase the dose. The dose may be increased daily, every other day, twice a week, or once a week.

[0271] Compound Synthesis

[0272] The compounds can be prepared using the methods disclosed in US20220135598A1 (incorporated in its entirety by reference), with conventional modifications thereof, as will be apparent from the disclosure herein and methods well known in the art. In addition to the teachings herein, conventional and well-known synthetic methods may also be used. Reagents may be purchased from commercial sources, such as from Sigma Aldrich or other chemical suppliers, if available.

[0273] Example

[0274] The following examples illustrate specific embodiments of this disclosure. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques that work well in the practice of this disclosure and can therefore be considered to constitute a specific mode of practice therein. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of this disclosure and still obtaining the same or similar results.

[0275] Bioassay

[0276] The following examples describe biological assays for testing the ENPP1 inhibitors disclosed herein.

[0277] Example 1: ENPP1 inhibition assay using ATP substrate

[0278] The ability of the disclosed compounds to inhibit ENPP1 was tested in a biochemical ENPP1 inhibition assay using ATP as a substrate. A stock solution of the test compound was prepared in DMSO, and then serially diluted 3-fold to 10 concentrations in 384-well plates (ProxiPlate-384 Plus; PE# 6008280) using a TECAN EVO200 liquid processor. 30 nL of the stock solution was transferred to each well using an Echo550 dispenser. DMSO was used as a solvent control. 5 µL of a solution containing 120 pM ENPP1 enzyme (R&D# 6136-EN-10), 2 mM MgCl2, 1 mM TCEP pH 7.0, 50 mM Tris HCl, and 0.005% Tween-80 was added to the assay plate and incubated at RT for 30 min. A mixture of 5 µL containing 300 nM ATP (Promega#V915B), 2 mM MgCl2, 1 mM TCEP pH7.0, 50 mM Tris HCl, and 0.005% Tween 80 was added to all wells. The final concentrations of ENPP1 enzyme and ATP were 60 pM and 300 nM, respectively, for a total volume of 10 µL. The plate was incubated at RT for 90 min. The reaction was terminated by adding 5 µL of a stop solution containing 20 mM EDTA, 50 mM Tris-HCl, 0.1% Prionex, 6 nM AMP / GMP HiLyte647 tracer (BellBrook Labs# 3020-10K), and 6 nM AMP / GMP antibody-Tb to all wells, and the plate was incubated for an additional 120 min. The resulting signal was measured using an Envision instrument. The remaining activity (%) was calculated using the reading conversion rate (CR) according to the following formula:

[0279] Remaining activity (%) = 100 x (CR) 样品 -CR LC ) / (CR HC -CR LC )

[0280] IC is calculated using the floating upper and lower limits method with XLFit (Formula 201). 50 value.

[0281] Some of the compounds disclosed in Table 1 were tested in the above assays and showed IC50 values ​​of less than 3 µM. 50 Table A shows the IC50 values ​​of some compounds. 50 The data is presented as a range, where “+++” < 100 nM, 100 nM ≤ “++” < 3000 nM, and “+” ≥ 3000 nM.

[0282] Table A

[0283] Compound No. ENPP1 IC 50 ]]> 76 ++ 79 +++ 87 ++ 105 + 123 +++ 125 +++ 126 +++ 127 +++ 131 +++ 134 +++ 135 +++

[0284] Example 2: Mineralization determination

[0285] The ability of the compounds disclosed herein to regulate human bone-associated cell mineralization was investigated. Saos-2 cells (ATCC CAT#HTB-85) were seeded in 24-well plates at a seeding density of 3.5 × 10⁶ cells / well. 5 Cells were placed per well and allowed to grow to 100% confluence over the next 1-2 days. Once fully confluenced, the medium was replaced with fresh medium supplemented with 1x ascorbic acid (Sigma # A4544, “AA”), 1x β-glycerophosphate (Sigma # G9422, “BGP”), and different concentrations of ENPP1 inhibitor. The day the mineralizing component was added was considered Day 1. On Day 3, the medium was gently aspirated without washing, and then fresh medium, mineralizing component, and ENPP1 inhibitor at concentrations of 1 and 10 µM were added. On Day 5, the medium was gently aspirated, and the cells were washed twice with PBS. The cells were then fixed with ice-cold 70% ethanol at 4°C for 1 hour. The cells were washed once with PBS and stained with Alizarin Red by gentle rotation for 30 minutes at room temperature. The stained cells were washed three times with water by gentle rotation for 10 minutes each at room temperature. After imaging the cells, dye extraction reagent was added to each well, and the cells were incubated at room temperature for 30 minutes. Alizarin Red was quantified by measuring absorbance at 405 nm using an Envision instrument.

[0286] like Figure 1 As shown, the compounds disclosed herein possess the ability to regulate mineralization. "AA+BGP" represents 1x ascorbic acid + 1x β-glycerophosphate. Compounds 131 and 134 exhibited higher mineralization levels at 1 µM than at 10 µM. Compounds 135 and 76 showed similar mineralization levels at both 1 µM and 10 µM.

[0287] Example 3: Effects of compounds on PPi levels in WT animals

[0288] The effects of the exemplary compounds of this disclosure on PPi levels in wild-type animals were investigated. Compound 76 was administered orally at a single dose of 100 mg / kg to 6- to 8-week-old female SD rats (n=3) and 100 mg / kg to 6- to 8-week-old C57BL / 6J mice (n=6), twice daily for 5 consecutive days. Plasma samples were collected from rats before administration and at 1, 4, and 8 hours after administration, and from mice at 1 and 8 hours after the last administration. The samples were placed in heparinized SST tubes and centrifuged immediately (7000 rpm x 10 min). For PPi analysis, platelet removal was performed by immediately centrifuging plasma with a Spin-X column (0.22 μM, Costa 8160) (7000 rpm x 2 min) and immediately aliquoting / freezing the eluted plasma on dry ice. Prior to PPi analysis, plasma samples were deproteinized using a 10 kD cutoff column (Amicon #UFC501024) (4°C). In the presence of adenosine monophosphate (APS; Sigma# A5508), PPi from filtered plasma was first converted to ATP by ATP sulfatase (R&D# 7175-AS-020). The reaction solution was incubated at 37°C for 30 min, followed by incubation at 90°C for 10 min to inactivate ATP sulfatase. The resulting ATP was then quantified using a Bactiter Glo assay (Promega G8230; Madison, WI, USA). To calculate the endogenous ATP present in plasma, a blank reaction was performed on each sample using heat-inactivated ATP sulfatase. The luminescence signal from this reaction was subtracted from the total luminescence signal to calculate the plasma PPi level.

[0289] Compound 76 was able to reduce PPi levels in rats in a time-dependent manner following a single dose (1, 4, and 8 hours after administration). Figure 2 As shown), and reduced PPi levels in mice at 1 and 8 hours after the last dose of a 5-day continuous administration regimen (as shown). Figure 3 (As shown).

[0290] Example 4: Mineralization assay of TNAP KO MC3T3-E1-C4 cells

[0291] The compounds disclosed herein were tested in a mineralization assay of TNAP-knockout MC3T3-E1-C4 cells. The osteoblast cell line (MC3T3-E1) established from the skull of C57BL / 6 mice is capable of synthesizing extracellular matrix (collagen) and differentiating into osteoblasts and osteocytes in vitro. To induce mineralization, cells were cultured for 5 days in αMEM containing 50 μg / ml ascorbic acid, followed by supplementation with 2.5 mM NaPO4 or 5 mM β-glycerophosphate, as previously described (Liu et al., 2014, Bone. 67: 81–94). After 5 days, the culture medium was gently aspirated without washing, and fresh culture medium, mineralization components, and different concentrations of ENPP1 inhibitors were added. After 7 days, the culture medium was gently aspirated, and the cells were washed twice with PBS. The cells were then fixed with ice-cold 70% ethanol at 4°C for 1 hour. The cells were washed once with PBS and stained with Alizarin Red by gentle rotation for 30 minutes at room temperature. The stained cells were gently swirled with water three times for 10 minutes each time at room temperature. After imaging the cells, dye extraction reagent was added to each well, and the cells were incubated at room temperature for 30 minutes. Alizarin Red was quantified by measuring absorbance at 405 nm using an Envision instrument.

[0292] Example 5: Determination of ENPP1 in TNAP KO MC3T3-E1-C4

[0293] Cells were treated with different concentrations of the ENPP1 inhibitor disclosed herein and incubated for 5 days. After 5 days, the monolayer of cells was washed twice with cold PBS, and 150 μL of lysis buffer was added to each well. Cells were lysed using the freeze-thaw method, centrifuged at 13.3 rpm for 10 min at 4°C, and the supernatant was collected. The samples were incubated with assay buffer (100 mM Tris (pH 9.0), 500 mM NaCl, 5 mM MgCl2, 0.05% Triton X-100). 2 mM pNP-TMP (Sigma# T4510) was added to each well as a substrate. The production of p-nitrophenol was analyzed kinetically by measuring the absorbance at 405 nm using a Molecular Devices (San Jose, CA, USA) SpectraMax M2 microplate reader. The activity was calculated as the change in absorbance over time (mOD / min).

[0294] Example 6: Effects of compounds on the survival rate of Akp- / - mice

[0295] Akp- / - mice (n = 17-20) were treated orally with the ENPP1 inhibitor disclosed herein, either once daily or via a carrier, starting no later than 2 days after birth and continuing until at least 25 days of age. Body weight was measured daily, and survival was analyzed at the end of the study. Plasma PPi concentrations were measured at different time points after the last administration (e.g., 0, 12, 24, 48, and 96 hours). Skeletal abnormalities were assessed by X-ray 24 hours after the last administration on the study day.

[0296] Example 7: Compounds against Akp2 + / − Effects on mice

[0297] Akp2 + / − Mice (n = 15) received oral treatment with the ENPP1 inhibitor disclosed herein once daily for at least 25 days. Body weight was analyzed daily. Plasma PPi concentrations were measured at different time points (0, 12, 24, 48, and 96 hours) after the last administration. Skeletal abnormalities were assessed by X-ray 24 hours after the last administration on the study day.

[0298] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0299] The disclosure described herein may be practiced appropriately in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein have been used as terminology in the specification and are not intended to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications may be made within the scope of this disclosure.

[0300] Therefore, it should be understood that although this disclosure has been specifically disclosed through certain embodiments and optional features, modifications, improvements, and variations can be made by those skilled in the art, and such modifications, improvements, and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided herein are exemplary only and are not intended to be a limitation on the scope of this disclosure.

[0301] Specific embodiments have been described broadly and generally herein. Each of the more narrowly defined species and subgenus groups also forms part of this disclosure. This contains a general description, the preconditions or negative limitations of which are removed from any subject matter, whether or not the removed material is specifically described herein.

[0302] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety by reference as if they were individually incorporated by reference. In the event of any conflict, this specification (including definitions) shall prevail.

[0303] It should be understood that although this disclosure has been described in conjunction with the foregoing embodiments, the foregoing description and examples are intended to illustrate and not limit the scope of this disclosure. Other aspects, advantages, and modifications within the scope of this disclosure will be apparent to those skilled in the art to which this disclosure pertains.

Claims

1. A method for treating or preventing hypophosphatase syndrome in a subject, the method comprising administering a therapeutically effective amount of a compound of formula I to the subject in need: I Or its pharmaceutically acceptable salt, wherein: The ring C is a 5- to 6-membered heteroaryl group; Ring D is a C6-aryl or a 5- to 6-membered heteroaryl group, wherein ring D is fused with ring C; A is hydrogen, C1-C6 alkyl or C6-aryl, wherein each is optionally substituted with a halogen; G represents a bond, -CH2- or -CH2-CH2-; R a and R b Independently hydrogen or C1-C6 alkyl; Or R a and R b Together with the atoms they are attached to, they form C3-C6 cyclic alkyl rings; Or R a and R b Any of them, along with A and the atoms they are attached to, form a C4-C6 cyclic alkyl ring; L represents a bond, a straight-chain or branched C1-C6 alkylene group, or a straight-chain or branched C2-C6 alkenyl group; t is 0 or 1; the condition is that when t is 0, L is a straight-chain or branched C2-C6 alkenyl group. Z is -NR c S(O)2NH2、-NR c S(O)2CH3, -SO2NH2, -NR c C(O)CH3, -C(O)OH, -CONH2, -NR c CONH2, -CONH(OH), -B(OH)2, -P(O)(OH)2, -SO2OH, -NR c S(O)2CF3、-NR c S(O)2NHCH3 or -NR c CH2C6-aryl-S(O)2NH2. Each R 1 and R 2 Independently oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, adamantyl, 3- to 6-membered heterocyclic, C6-aryl, 5- to 6-membered heteroaryl, -CN, halogen, C1-C6 alkoxy, C1-C6 haloalkoxy, -OR 10 -SR 10 -S(O)2R 10 -S(O)2NR 11 R 12 -NR 10 S(O)2R 11 -NR 11 R 12 -C(O)R 10 -NR 10 C(O)R 11 -NR 10 C(O)NR 11 R 12 -C(O)OR 10 -C(O)ONR 11 R 12 or -C(O)NR 11 R 12 Each of them can be independently and optionally controlled by R 9 replace; Or two Rs 2 Together with the atoms to which they are attached, they form C5-C6 cycloalkyl, 5- to 6-membered heterocyclic, C6-aryl, or 5- to 6-membered heteroaryl groups, wherein each is optionally independently controlled by R. 9 replace; Each R 9 Independently selected from the group consisting of: oxo, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 6-membered heterocyclic, C6-aryl, 5- to 6-membered heteroaryl, -CN, halogen, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 haloalkyl, -OR 13 -SR 13 -S(O)2R 13 -S(O)2NR 14 R 15 -NR 13 S(O)2R 14 -NR 14 R 15 -C(O)R 13 -NR 13 C(O)R 14 -NR 13 C(O)NR 14 R 15 -C(O)OR 13 -C(O)ONR 14 R 15 -C(O)NR 14 R 15 And C1-C6 alkyl groups optionally substituted with oxo, -OH or halogen; Each R c Independently, it can be hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3- to 6-membered heterocyclic, -(C1-C6 alkylene)C3- to 6-membered heterocyclic, -(C1-C6 alkylene)C6-aryl, 5- to 6-membered heteroaryl, -(C1-C6 alkylene)5- to 6-membered heteroaryl, C1-C6 haloalkyl, -(C1-C6 alkylene)OR 13 -(C1-C6 alkylene)SR 13 -(C1-C6 alkylene)S(O)2R 13 -(C1-C6 alkylene)S(O)2NR 14 R 15 -(C1-C6 alkylene)NR 13 S(O)2R 14 -(C1-C6 alkylene)NR 14 R 15 -(C1-C6 alkylene)C(O)R 13 -(C1-C6 alkylene)NR 13 C(O)R 14 -(C1-C6 alkylene)NR 13 C(O)NR 14 R 15 -(C1-C6 alkylene)C(O)OR 13 -(C1-C6 alkylene)C(O)ONR 14 R 15 or -(C1-C6 alkylene)-C(O)NR 14 R 15 Each of them can be independently and optionally processed by R d replace; Each R d Independently selected from the group consisting of: halogen, -OH, oxo, -CN, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -COOH and C1-C6 alkyl optionally substituted with -OH, halogen, CN or oxo. Each R 10 R 11 and R 12 Independently, it is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3- to 6-membered heterocyclic group, or -(C1-C6 alkylene)3- to 6-membered heterocyclic group, wherein R 10 R 11 and R 12 Each of them may be independently and optionally substituted with an oxo, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen, C1-C6 alkoxy or optionally substituted with an oxo, -OH or halogen-substituted C1-C6 alkyl group; Or R 11 and R 12 Together with the atoms to which they are attached, they form 3 to 6-membered heterocyclic groups, which may optionally be substituted with oxo, -OH, halogen or C1-C6 alkyl groups substituted with oxo, -OH or halogen; Each R 13 R 14 and R 15 Independently, it is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or a 3- to 6-membered heterocyclic group, wherein R 10 R 11 and R 12 Each of them may be independently and optionally substituted with an oxo, -OH, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen or optionally substituted with an oxo, -OH or halogen C1-C6 alkyl group; Or R 14 and R 15 Together with the atoms to which they are attached, they form 3 to 6-membered heterocyclic groups, which may optionally be substituted with oxo, -OH, halogen or C1-C6 alkyl groups substituted with oxo, -OH or halogen; m is 0, 1, or 2; and n is 0, 1, 2, 3 or 4; The condition is when Z is -NR c When S(O)2CH3, -CONH2, or -C(O)OH are involved, G is a bond and t is 1, then L is not a bond; and The compound is not 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-3-azacyclobutanebutanoic acid, 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-3-azacyclobutanevalanoic acid, or N-((1-(6-fluorobenzo[d]oxazol-2-yl)azacyclobutane-3-yl)methyl)-N-methylacetamide.

2. The method according to claim 1, wherein the compound is selected from Table 1 or a pharmaceutically acceptable salt thereof.

3. The method according to claim 1 or 2, wherein the compound or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier.

4. The method according to any one of claims 1 to 3, wherein the compound or a pharmaceutically acceptable salt thereof is formulated for oral delivery.

5. The method according to any one of claims 1 to 3, wherein the compound or a pharmaceutically acceptable salt thereof is formulated for subcutaneous delivery.

6. The method according to any one of claims 1 to 3, wherein the compound or a pharmaceutically acceptable salt thereof is formulated for intraperitoneal delivery.

7. The method according to any one of claims 1 to 6, wherein the compound or a pharmaceutically acceptable salt thereof is acutely administered to the subject.

8. The method according to any one of claims 1 to 6, wherein the compound or a pharmaceutically acceptable salt thereof is chronically administered to the subject.

9. The method according to any one of claims 1 to 8, wherein the subject is a mammal.

10. The method of claim 9, wherein the mammal is a human.

11. The method according to any one of claims 1 to 10, wherein the hypophospholipase syndrome is selected from the group consisting of: late-onset hypophospholipase syndrome, prenatal hypophospholipase syndrome, perinatal hypophospholipase syndrome, infantile hypophospholipase syndrome, childhood hypophospholipase syndrome, adult hypophospholipase syndrome, dental hypophospholipase syndrome, and hypophospholipase syndrome with tooth involvement and / or premature tooth loss.

12. The method of claim 11, wherein the hypophosphodiesterase syndrome is adult-onset hypophosphodiesterase syndrome.

13. The method of claim 11, wherein the hypophosphatase syndrome is late-onset hypophosphatase syndrome.

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