Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitors for the treatment of hypophosphatasia

Abstract

A method for treating hypophosphatasia (HPP) is provided herein, comprising administering a therapeutically effective dose of an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor. A method for using an ENPP1 inhibitor to treat or prevent cartilage disease is disclosed herein. In some embodiments, the cartilage disease is hypophosphatasia. Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is an extracellular enzyme that hydrolyzes ATP to PPi and AMP in the extracellular space.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 493,684, filed on 31 March 2023, and U.S. Provisional Application No. 63 / 495,994, filed on 13 April 2023, both of which are incorporated herein by reference in their entirety.

[0002] A method for treating hypophosphatasia (HPP) is provided herein, comprising administering a therapeutically effective dose of an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor. [Background technology]

[0003] Hypophosphatasia (HPP) is a rare hereditary metabolic disorder caused by loss-of-function mutations in the ALPL gene, which encodes a tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Low TNSALP activity leads to extracellular accumulation of phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5-phosphate (PLP) in the blood or urine. PPi, an inhibitor of bone mineralization, is one of the key substrates involved in the pathogenesis of HPP. In HPP, excess PPi contributes to mineralization defects of bone (rickets or osteomalacia), premature loss of deciduous teeth, chronic bone and muscle pain, recurrent fractures, respiratory and muscle dysfunction, and periodontal defects. Symptoms range from fatal or severe in perinatal or infant-onset HPP to moderate or mild in childhood and adult-onset forms.

[0004] The incidence of severe HPP ranges from approximately 1 in 100,000 to 1 in 300,000 births. In certain populations, such as the Canadian Mennonites, the incidence is higher, reaching about 1 in 2,500 births.

[0005] Hypophosphatasia poses a significant burden to affected individuals and their families. Therefore, identifying compounds, compositions, and methods that can be used to treat or prevent hypophosphatasia would address a critical unmet need. [Overview of the project] [Means for solving the problem]

[0006] Methods using ENPP1 inhibitors to treat or prevent cartilage disease are disclosed herein. In some embodiments, the cartilage disease is hypophosphatasia. 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, HAP is the inorganic mineral component of calcium phosphate and is essential for bone mineralization. Since ENPP1 is a major source of PPi, inhibition of this enzyme is a promising therapeutic approach for the treatment of HPP. Normalizing extracellular PPi levels by targeting ENPP1 can result in significant relief from the severe symptoms of this debilitating disease. Targeted therapeutics that inhibit ENPP1 activity or neutralize PPi may provide effective interventions for HPP.

[0007] In some embodiments, hypophosphatasia is delayed-onset hypophosphatasia. In some embodiments, hypophosphatasia is childhood-onset hypophosphatasia.

[0008] In some embodiments, hypophosphatasia is prenatal hypophosphatasia. In some embodiments, hypophosphatasia is perinatal hypophosphatasia.

[0009] In some embodiments, this disorder is infantile hypophosphatasia.

[0010] In some embodiments, this condition is childhood hypophosphatasia.

[0011] In some embodiments, this condition is adult hypophosphatasia.

[0012] In some embodiments, the disease is any form of hypophosphatasia, where hypophosphatasia involves teeth and / or premature tooth loss.

[0013] In some embodiments, the disease is tooth-specific hypophosphatasia.

[0014] In some embodiments, a method for treating a subject with dental disease is provided by an agent that inhibits ENPP1.

[0015] In some embodiments, a method for treating a subject with periodontal disease is provided by an agent that inhibits ENPP1.

[0016] In some embodiments, a method for treating hypophosphatasia is provided by a drug that inhibits ENPP1.

[0017] In some embodiments, a method is provided for treating hypophosphatasia in a subject, comprising administering to the subject in need a therapeutically effective dose of a compound that inhibits ATP hydrolysis via ENPP1 inhibition.

[0018] In some embodiments, a method is provided for treating hypophosphatasia, comprising administering a therapeutically effective dose of an ENPP1 inhibitory compound to a subject in need thereof.

[0019] In some embodiments, the compound is a compound described in US2022 / 0135598A1, which is incorporated herein by reference. In some embodiments, the compound is a compound of formula I, [ka] or a pharmaceutically acceptable salt thereof, wherein ring C is a 5- to 6-membered heteroaryl, ring D is a C6-aryl or a 5- to 6-membered heteroaryl, and ring D is fused to ring C, A is hydrogen, C1-C6 alkyl, or C6-aryl, each of which is optionally substituted with halogen, G is a bond, -CH2-, or -CH2-CH2-, R a and R b are independently hydrogen or C1-C6 alkyl, or alternatively, R a and R b together with the atom to which they are attached form a C3-C6 cycloalkyl ring, or alternatively, either one of R a and R b and A together with the atom to which they are attached form a C4-C6 cycloalkyl ring, L is a bond, straight-chain or branched C1-C6 alkylene, or straight-chain or branched C2-C6 alkenylene, t is 0 or 1, provided that when t is 0, L is straight-chain or branched C2-C6 alkenylene, 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 2These are independently oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, adamantyl, 3-6 membered heterocyclyl, C6-aryl, 5-6 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 And each is independent, R 9 Replaced by choice, Alternatively, two R's 2 However, together with the atoms to which they are bonded, they form C5-C6 cycloalkyl, 5-6 membered heterocyclyl, C6-aryl, or 5-6 membered heteroaryl, and each independently, R 9 Replaced by choice, Each R 9 These are independently oxo, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-6 member heterocyclyl, C6-aryl, 5-6 member 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 selected from the group consisting of C1-C6 alkyl groups optionally substituted with oxo, -OH, or halogen, Each R c These are independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3-6 membered heterocyclyl, -(C1-C6 alkylene)3-6 membered heterocyclyl, -(C1-C6 alkylene)C6-aryl, 5-6 membered heteroaryl, -(C1-C6 alkylene)5-6 membered heteroaryl, C1-C6 haloalkyl, -(C1-C6 alkylene)OR 13 ,-(C1-C6 Alkilen)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 And each is independent, R d Replaced by optional selection, Each R dis 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 is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3- to 6-member heterocyclyl, or -(C1-C6 alkylene)3- to 6-member heterocyclyl, and each of R 10 , R 11 and R 12 is independently optionally substituted by oxo, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen, C1-C6 alkoxy, or C1-C6 alkyl optionally substituted by oxo, -OH, or halogen, or, R 11 and R 12 together with the atom(s) to which they are attached form a 3- to 6-member heterocyclyl optionally substituted by oxo, -OH, halogen, or C1-C6 alkyl optionally substituted by oxo, -OH, or halogen, each R 13 , R 14 , and R 15 is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or 3- to 6-member heterocyclyl, and each of R 10 , R 11 , and R 12 is independently optionally substituted by oxo, -OH, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen, or C1-C6 alkyl optionally substituted by oxo, -OH, or halogen, or, R 14 and R 15These, together with the atoms to which they are bonded, form 3- to 6-membered heterocyclines optionally substituted with oxo, -OH, halogen, or C1-C6 alkyl groups optionally substituted with oxo, -OH, or halogen. m is 0, 1, or 2. n is 0, 1, 2, 3, or 4. However, Z is -NR c If the expression is S(O)2CH3, -CONH2, or -C(O)OH, where G is a bond and t is 1, then L is not a bond. The compound is not 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl]-3-azetidine butanoic acid, 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl]-3-azetidine pentanoic acid, or N-((1-(6-fluorobenzo[d]oxazole-2-yl)azetidine-3-yl)methyl)-N-methylacetamide. [Brief explanation of the drawing]

[0020] [Figure 1] This disclosure demonstrates the effect of the compounds on mineralization in Saos-2 cells. [Figure 2] This shows the time-dependent effect of compound 76 on PPi levels in Sprague-Dawley rats 1, 4, and 8 hours after a single dose. [Figure 3] This study shows the time-dependent effect of compound 76 on PPi levels in mice one hour and eight hours after the final dose of a five-day continuous medication regimen.

[0021] It will be recognized that some or all of the drawings are schematic diagrams for illustrative purposes only. [Modes for carrying out the invention]

[0022] definition "Alkyl" refers to a specified number of carbon atoms (i.e., C1-C1). 10 A C1-C20 alkyl group refers to and includes saturated linear and branched monovalent hydrocarbon structures and combinations thereof that have 1 to 10 carbon atoms. Certain alkyl groups have 1 to 20 carbon atoms (C1-C20). 20 Alkyl groups are defined as 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, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0023] As used herein, “alkenyl” has at least one olefin unsaturated moiety (i.e., at least one part of the formula C=C) and a specified number of carbon atoms (i.e., C2-C) 10 An alkenyl group refers to an unsaturated, linear or branched monovalent hydrocarbon chain or a combination thereof, having 2 to 10 carbon atoms (where C2 means 2 to 10 carbon atoms). An alkenyl group can be in a "cis" or "trans" configuration, or alternatively, an "E" or "Z" configuration. Certain alkenyl groups have 2 to 20 carbon atoms (where C2 means 2 to 20 carbon atoms). 20Alkenyls are those having 2 to 8 carbon atoms ("C2-C8 alkenyls"), 2 to 6 carbon atoms ("C2-C6 alkenyls"), or 2 to 4 carbon atoms ("C2-C4 alkenyls"). Examples of alkenyls include, but are not limited to, groups such as ethenyl (or vinyl), propa-1-enyl, propa-2-enyl (or allyl), 2-methylpropa-1-enyl, buta-1-enyl, buta-2-enyl, buta-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, and their homologs and isomers.

[0024] As used herein, "alkylene" refers to the same residue as alkyl, but is divalent. Specific alkylene groups have 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 alkylenes include, but are not limited to, groups such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and butylene (-CH2CH2CH2CH2-). As used herein, "alkenylene" refers to the same residue as alkenyl, but is divalent.

[0025] As used herein, "alkynyl" has at least one acetylene unsaturated moiety (i.e., at least one part of the formula C≡C) and a specified number of carbon atoms (i.e., C2-C) 10 A C2-C2 group refers to an unsaturated, linear or branched monovalent hydrocarbon chain or a combination thereof, having 2 to 10 carbon atoms. Certain alkynyl groups have 2 to 20 carbon atoms (C2-C2). 20Alkynnyl groups include those having 2 to 8 carbon atoms ("C2-C8 alkynyl"), those having 2 to 6 carbon atoms ("C2-C6 alkynyl"), or those having 2 to 4 carbon atoms ("C2-C4 alkynyl"). Examples of alkynyl groups include, but are not limited to, ethynyl (or acetylenyl), propa-1-inyl, propa-2-inyl (or propargyl), buta-1-inyl, buta-2-inyl, buta-3-inyl, and their homologs and isomers.

[0026] "Aryl" refers to, and includes, polyunsaturated aromatic hydrocarbon groups. Aryl groups may include additional fused rings (e.g., 1 to 3 rings), including further fused aryl, heteroaryl, cycloalkyl, and / or heterocyclyl rings. In one variation, the aryl group contains 6 to 14 cyclic carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and biphenyl.

[0027] "Carbonyl" refers to the C=O group.

[0028] "Cycloalkyl" is saturated, monounsaturated, or polyunsaturated but non-aromatic and has a specified number of carbon atoms (e.g., C1-C1). 10 The term C1 refers to a cyclic monovalent hydrocarbon structure (meaning 1 to 10 carbon atoms), and includes these. Cycloalkyls can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyl, but without an aryl group. In certain embodiments, cycloalkyls containing more than one ring may be condensed, spiro, or cross-linked, or a combination thereof. In certain embodiments, a cycloalkyl is a cyclic hydrocarbon having 3 to 13 cyclic carbon atoms. In certain embodiments, a cycloalkyl is a cyclic hydrocarbon having 3 to 8 cyclic carbon atoms ("C3-C8 cycloalkyl"). Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, and others.

[0029] "Halo" or "halogen" refers to elements in the Group 17 series having atomic numbers 9 to 85. In certain embodiments, halo groups include fluoro, chloro, bromo, and iodine. When a residue is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties bonded to it. For example, dihaloaryl, dihaloalkyl, and trihaloaryl refer to aryl and alkyl groups substituted with two ("di") or three ("tri") halo groups, which may be the same halo but are not necessarily the same; therefore, 4-chloro-3-fluorophenyl is within the range of dihaloaryl. An alkyl group that replaces each hydrogen with a halo group is called a "perhaloalkyl." In certain embodiments, a perhaloalkyl group is a trifluoroalkyl (-CF3). Similarly, "perhaloalkoxy" refers to an alkoxy group in which the halogen occupies the position of each H in the hydrocarbon that constitutes the alkyl moiety of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF3).

[0030] A "heteroaryl" refers to an unsaturated aromatic cyclic group having 1 to 10 cyclic carbon atoms and at least one cyclic heteroatom, including but not limited to nitrogen, oxygen, and sulfur, where the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Heteroaryl groups can be bonded to the rest of the molecule with cyclic carbons or cyclic heteroatoms. Heteroaryls may contain additional fused rings (e.g., 1 to 3 rings), including further fused aryl, heteroaryl, cycloalkyl, and / or heterocyclyl rings. Examples of heteroaryl groups include imidazolyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, thiophenyl, furanyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyloxazolyl, isoxazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidyl, pyridadinyl, pyrizinyl, indolyl, indazolyl, benzimidazolyl, pyrrolopyridinyl, pyrrolopyridadinyl, pyrrolopyridinyl This includes, but is not limited to, pyrazolopyridinyl, pyrazolopyridinyl, imidazopyridinyl, prinyl, benzofuranyl, flupyridinyl, benzoxazolyl, benzothiophenyl, benzothiazolyl, oxazolopyridinyl, thiazolopyridinyl, thienopyridinyl, quinolinyl, quinolonyl, naphthilidinyl, quinazolinyl, pyridopyridinyl, sinnolinyl, or pyridopyridazinyl.

[0031] A "heterocyclyl" refers to a saturated or unsaturated non-aromatic group having 1 to 10 cyclic carbon atoms and 1 to 4 cyclic heteroatoms such as nitrogen, sulfur, or oxygen, where the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Heterocyclyl groups may have a monocycle or multiple fused polycycles, but exclude heteroaryl groups. Heterocycles containing one or more rings may be fused, spiro, or bridging, or a combination thereof. In fused ring systems, one or more fused rings may be aryl or heteroaryl. Examples of heterocyclyl groups include, but are not limited to, azilidinyl, azetidinyl, oxetanyl, morpholinyl, thiomorpholinyl, azepanyltetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuranyl, and tetrahydrothiophenyl.

[0032] "Oxo" refers to a part, or O.

[0033] "Optionally substituted" means, unless otherwise specified, that a group may be unsubstituted or substituted by one or more substituents listed for that group, which may have the same or different substituents (e.g., 1, 2, 3, 4, or 5). In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In yet another embodiment, an optionally substituted group has three substituents. In yet another embodiment, an optionally substituted group has four substituents. In some embodiments, an optionally substituted group has 1-2, 2-5, 3-5, 2-3, 2-4, 3-4, 1-3, 1-4, or 1-5 substituents.

[0034] As used herein, the term “pharmaceutically acceptable” means a material that does not negate the biological activity or properties of a compound and is relatively non-toxic, i.e., a material such as a carrier or diluent that can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of a composition containing it.

[0035] "Pharmacologically acceptable salts" means salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. If a compound such as those disclosed herein contains a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of the desired base, either undiluted or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. If a compound such as those disclosed herein contains a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of the desired acid, either undiluted or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those 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, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Salts of amino acids such as alginates and salts of organic acids such as glucuronic acid or galacturonic acid are also mentioned. Certain compounds in this disclosure may contain both basic and acidic functional groups that enable the conversion of the compound into either a base addition salt or an acid addition salt. A list of suitable salts is provided in Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa., (1985) and Journal of Pharmaceutical Science, 66:2 (1977), which are incorporated herein by reference in their entirety.

[0036] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one compound, does not destroy its pharmacological activity, is generally safe and nontoxic, and is not biologically or otherwise undesirable when administered in a dose sufficient to deliver a therapeutic dose of the drug.

[0037] "Treatment" or "treating" is an approach to obtain beneficial or desired outcomes, 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 resulting from a disease or condition, and / or reducing the severity of the disease or condition); b) delaying or preventing the onset of one or more clinical symptoms associated with a 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., causing regression of clinical symptoms (e.g., improving the disease state, providing partial or overall recovery from the disease or condition, enhancing the effect of another medicine, slowing the progression of the disease, improving quality of life, and / or extending survival).

[0038] "Prevention" or "prevention" means any treatment of a disease or condition that prevents the development of clinical symptoms of the disease or condition. In some embodiments, the compound may be administered to subjects (including humans) who are at risk or have a family history of the disease or condition.

[0039] "Disease" is a state of health in which an animal is unable to maintain homeostasis, and if the disease does not improve, the animal's health continues to deteriorate.

[0040] "Subject" refers to an animal, such as a mammal (including a human), that has been or will be the subject of treatment, observation, or experimentation. The methods described herein may be useful in human therapeutic and / or veterinary applications. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0041] In this specification, the terms “therapeutic effective dose” or “effective dose” of a compound or any pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analog described herein mean an amount sufficient to, when administered to a subject, produce a therapeutic effect and provide a therapeutic benefit such as improvement of symptoms or delay of disease progression. For example, a therapeutic effective dose may be an amount sufficient to reduce the symptoms of a disease or condition of hypophosphatasia. The therapeutic effective dose may vary depending on the subject, the disease or condition being treated, the subject’s weight and age, the severity of the disease or condition, and the mode of administration, and can be easily determined by those skilled in the art.

[0042] As used herein, the terms "NPP" or "ENPP" refer to ectonucleotide pyrophosphatase / phosphodiesterase.

[0043] As used herein, the terms “change,” “deletion,” “mutation,” or “spontaneous mutation” refer to mutations in intracellular genes that affect the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes. Mutations included in this disclosure may be any mutation in intracellular genes that results in enhancement or disruption of the function, activity, expression or conformation of the encoded polypeptide, including the complete absence of expression of the encoded protein, and may include, for example, missense and nonsense mutations, insertions, deletions, frameshifts, and premature termination. Mutations included in this disclosure may, but are not limited to, alter the splicing of mRNA (splice site mutations) or cause a shift in the reading frame (frameshift).

[0044] The methods described herein may be applied to cell populations in vivo or ex vivo. “In vivo” means within a living organism, such as an animal or a human. In this context, the methods described herein may be used therapeutically in an organism. “Ex vivo” means outside a living organism. Examples of ex vivo cell populations include biological samples, including in vitro cell cultures and fluid or tissue samples obtained from organisms. Such samples may be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the compounds and compositions described herein may be used for a variety of purposes, including therapeutic and experimental purposes. For example, the compounds and compositions described herein may be used ex vivo to determine the optimal schedule and / or dosage of the compounds disclosed herein for a given indication, cell type, organism, and other parameters. Information gathered from such use may be used in a clinical setting for experimental purposes or to establish protocols for in vivo treatment. Other ex vivo uses in which the compounds and compositions described herein may be suitable are described below or will be apparent to those skilled in the art. Selected compounds may be further characterized to investigate their safety or tolerable dosage in human or non-human subjects. Such properties can be investigated using methods generally known to those skilled in the art.

[0045] As used herein, "HPP" refers to hypophosphatasia.

[0046] Treatment method A method for treating or preventing cartilage disease is provided herein, comprising administering a therapeutically effective amount of an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor to a subject in need thereof. In a particular embodiment, a method for treating or preventing hypophosphatasia is provided, comprising administering a therapeutically effective amount of an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor to a subject in need thereof.

[0047] Hypophosphatasia (HPP) is a hereditary metabolic disorder characterized by low activity of tissue-nonspecific alkaline phosphatase (TNAP). This low activity of TNAP leads to a systematic accumulation of its substrates, namely inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and pyridoxal-5'-phosphate (PLP), a cofactor for several enzymes, which accounts for much of the disease's musculoskeletal and systemic characteristics. HPP is characterized by a wide range of symptoms and severity, from intrauterine death to dental complications in children and adults only, or asymptomatic carriers of ALPL mutations. Due to the disease's associated rarity, lack of awareness of HPP among physicians, and the absence of pathological symptoms, particularly in mild adult forms, its diagnosis and management remain considerably delayed.

[0048] The specification also provides a method for treating or preventing hypophosphatasia, comprising administering a therapeutically effective amount of a composition comprising an ENPP1 inhibitor and a pharmaceutically acceptable carrier to a subject in need thereof.

[0049] In some embodiments, therapeutically active doses of an ENPP1 inhibitor treat hypophosphatasia in a subject in need by restoring normal circulating PPi levels.

[0050] In some embodiments, therapeutically active ENPP1 inhibitors treat hypophosphatasia in subjects requiring it by modulating the phosphate / pyrophosphate (Pi / PPi) ratio.

[0051] In some embodiments, therapeutically active doses of an ENPP1 inhibitor treat hypophosphatasia in a subject in need by promoting mineralization.

[0052] In some embodiments, the subjects suffer from prenatal hypophosphatasia.

[0053] In some embodiments, the subjects suffer from perinatal hypophosphatasia.

[0054] In some embodiments, the subjects suffer from infantile hypophosphatasia.

[0055] In some embodiments, the subjects are children suffering from hypophosphatasia.

[0056] In some embodiments, the subjects are adults suffering from hypophosphatasia.

[0057] In some embodiments, the subjects suffer from tooth-specific hypophosphatasia.

[0058] In some embodiments, the subject suffers from periodontal disease.

[0059] In some embodiments, the subject has a change in the ALPL gene. In some embodiments, the change in the ALPL gene is a mutation. In some embodiments, the subject has a change in a gene that can regulate TNSALP activity. In some embodiments, the change in the gene that can regulate TNSALP activity is a change in RUNX2.

[0060] In some embodiments, a therapeutically effective dose of the ENPP1 inhibitor is administered daily. In some embodiments, a therapeutically effective dose of the ENPP1 inhibitor is administered two to three times a day.

[0061] In some embodiments, a therapeutically effective dose of the ENPP1 inhibitor is formulated for oral administration.

[0062] In some embodiments, a therapeutically effective dose of the ENPP1 inhibitor is formulated for subcutaneous administration.

[0063] In some embodiments, a therapeutically effective dose of the ENPP1 inhibitor is formulated for intraperitoneal administration.

[0064] compound Compounds that inhibit ENPP1 are provided herein, and such compounds may be used in methods and compositions disclosed herein, such as for the treatment or prevention of cartilage diseases. Such compounds may be useful in the treatment of hypophosphatasia.

[0065] In certain embodiments, the ENPP1 inhibitor is a compound described in US2022 / 0135598A1. In certain embodiments, the compound is a compound of formula I. [ka] or a pharmaceutically acceptable salt thereof, in the formula, Ring C is a 5-6 member heteroaryl, Ring D is a C6-aryl or 5-6 membered heteroaryl, and ring D is condensed with ring C. A is hydrogen, a C1-C6 alkyl group, or a C6-aryl group, each of which can be optionally substituted with a halogen. G is a bond, -CH2-, or -CH2-CH2-, R a and R b These are independently hydrogen or a C1-C6 alkyl group. Alternatively, R a and R b These, together with the atoms to which they are bonded, form a C3-C6 cycloalkyl ring. Alternatively, R a and R bAny one of these, along with A, together with the atom to which they are bonded, forms a C4-C6 cycloalkyl ring. L is a bond, a linear or branched C1-C6 alkylene, or a linear or branched C2-C6 alkenylene. t is either 0 or 1, where if t is 0, L is a linear or branched C2-C6 alkenylene. 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 It is CH2C6-aryl-S(O)2NH2. Each R 1 and R 2 These are independently oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, adamantyl, 3-6 membered heterocyclyl, C6-aryl, 5-6 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 And each is independent, R9 Replaced by choice, Alternatively, two R's 2 However, together with the atoms to which they are bonded, they form C5-C6 cycloalkyl, 5-6 membered heterocyclyl, C6-aryl, or 5-6 membered heteroaryl, and each independently, R 9 Replaced by choice, Each R 9 These are independently oxo, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-6 member heterocyclyl, C6-aryl, 5-6 member 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 selected from the group consisting of C1-C6 alkyl groups optionally substituted with oxo, -OH, or halogen, Each R c These are independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3-6 membered heterocyclyl, -(C1-C6 alkylene)3-6 membered heterocyclyl, -(C1-C6 alkylene)C6-aryl, 5-6 membered heteroaryl, -(C1-C6 alkylene)5-6 membered heteroaryl, C1-C6 haloalkyl, -(C1-C6 alkylene)OR 13 ,-(C1-C6 Alkilen)SR 13 -(C1-C6 alkylene)S(O)2R13 -(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 And each is independent, R d Replaced by optional selection, Each R d These are independently selected from the group consisting of halogens, -OH, oxo, -CN, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -COOH, and C1-C6 alkyls optionally substituted with -OH, halogen, CN, or oxo. Each R 10 , R 11 , and R 12 R is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, -(C1-C6 alkylene)C3-C6 cycloalkyl, 3-6 membered heterocyclyl, or -(C1-C6 alkylene)3-6 membered heterocyclyl. 10 , R 11 and R 12 Each of these is independently optionally substituted with an oxo, a C2-C6 alkenyl, a C2-C6 alkynyl, a -CN, a halogen, a C1-C6 alkoxy, or a C1-C6 alkyl optionally substituted with an oxo, -OH, or halogen. Alternatively, R 11 and R 12These, together with the atom(s) to which they bond, form a 3- to 6-membered heterocycline optionally substituted with an oxo, -OH, halogen, or a C1-C6 alkyl group optionally substituted with an oxo, -OH, or halogen. Each R 13 , R 14 , and R 15 R is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or 3-6 member heterocyclyl. 10 , R 11 , and R 12 Each of these is independently optionally substituted with an oxo, -OH, C2-C6 alkenyl, C2-C6 alkynyl, -CN, halogen, or a C1-C6 alkyl which is optionally substituted with an oxo, -OH, or halogen. Alternatively, R 14 and R 15 These, together with the atoms to which they are bonded, form 3- to 6-membered heterocyclines optionally substituted with oxo, -OH, halogen, or C1-C6 alkyl groups optionally substituted with oxo, -OH, or halogen. m is 0, 1, or 2. n is 0, 1, 2, 3, or 4. However, Z is -NR c If the expression is S(O)2CH3, -CONH2, or -C(O)OH, where G is a bond and t is 1, then L is not a bond.

[0066] In some embodiments, the compound is not 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl]-3-azetidinebutanoic acid, 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl]-3-azetidinepentanoic acid, or acetamide, N-[1-(6-fluoro-2-benzoxazolyl)-3-azetidinyl]methyl]-N-methyl.

[0067] 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.

[0068] In some embodiments, G is a bond. In some embodiments, G is -CH2-. In some embodiments, G is -CH2-CH2-.

[0069] In some embodiments, R a and R b R is independently hydrogen or a C1-C6 alkyl group. In some embodiments, R a and R b Both are hydrogen. In some embodiments, R a and R b One 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. In some embodiments, R a and R b One of them is hydrogen and the other is methyl. In some embodiments, R a and R b One of them is hydrogen and the other is ethyl. In some embodiments, R a and R b Both are methyl.

[0070] In some embodiments, R a and R b These, together with the atoms to which they are bonded, form a C3-C6 cycloalkyl ring. In some embodiments, R a and R bThese atoms, together with the atoms to which they bond, form a cyclopropyl ring.

[0071] In some embodiments, R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a C4-C6 cycloalkyl group. In some embodiments, R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a cyclobutyl ring. In some embodiments, R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a cyclopentyl ring. In some embodiments, R a and R b One of these, along with A, together with the atom to which they are bonded, forms a cyclohexyl ring.

[0072] In some embodiments, R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a C4-C6 cycloalkyl ring, and in such a case, R a or R b The other is hydrogen. In some embodiments, R a and R b Any one of them, along with A, together with the atom to which they are bonded, forms a cyclobutyl ring, and in such a case, R a or R b The other is hydrogen. In some embodiments, R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a cyclopentyl ring, and in such a case, R a or R b The other is hydrogen. In some embodiments, R a and R bAny one of these, along with A, together with the atom to which they are bonded, forms a cyclohexyl ring, and in such a case, R a or R b The other is hydrogen.

[0073] In some embodiments, G is a bond, -CH2- or -CH2-CH2-, and R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a C4-C6 cycloalkyl ring. In some embodiments, G is -CH2- and R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a cyclobutyl ring. In some embodiments, G is -CH2- and R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a cyclopentyl ring. In some embodiments, G is -CH2-CH2- and R a and R b One of these, along with A, together with the atom to which they are bonded, forms a cyclohexyl ring.

[0074] In some embodiments, L is a bond or a C1-C6 alkylene or C2-C6 alkenylene. The C1-C6 alkylene or C2-C6 alkenylene may be linear or branched. In some embodiments, L is a bond. In some embodiments, L is a C1-C6 alkylene. 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-.

[0075] In some embodiments, L is a C2-C6 alkenylene. The C2-C6 alkenylene can be linear or branched. In some embodiments, L is -CH=CH-. In some embodiments, L is -CH2-CH=CH-.

[0076] In some embodiments, t is 0 or 1. In some embodiments, t is 0. In some embodiments, t is 1.

[0077] In some embodiments, when t is 0, R a and R b It does not exist, and L is a C2-C6 alkenylene. In some embodiments, when t is 0, R a and R b It does not exist, and L is -CH=CH-.

[0078] In some embodiments, when t is 1, R a and R bThere exists, and L is a bond or C1-C6 alkylene. In some embodiments, when t is 1, R a and R b exists, and L is a bond. In some embodiments, when t is 1, R a and R b It exists, and L is a C1-C6 alkylene. In some embodiments, when t is 1, R a and R b It exists, and L is -CH2-. In some embodiments, when t is 1, R a and R b There exists and L is -CH2-CH2-. Here, R a and R b This is independently selected from hydrogen or C1-C6 alkyl groups.

[0079] In some embodiments, when t is 1, R a and R b It exists, R a and R b Any one of these, along with A, together with the atom to which they are bonded, forms a C4-C6 cycloalkyl ring, and in such a case, R a or R b The other is hydrogen. In some embodiments, when t is 1, R a and R b It exists, R a and R b Any one of them, as well as A, together with the atom to which they are bonded, form a cyclobutyl ring, and in such a case, R a or R b The other is hydrogen.

[0080] In some embodiments, Z is -NR c SO2NH2, -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, -NRc S(O)2CF3, -NR c S(O)2NHCH3, or -NR c Selected from CH2C6-aryl-S(O)2NH2. In some embodiments, Z is -NR c It is S(O)2NH2. In some embodiments, Z is -NR c It is S(O)2CH3. In some embodiments, Z is -SO2NH2. In some embodiments, Z is -NR c It is C(O)CH3. In some embodiments, Z is -C(O)OH. In some embodiments, Z is -CONH2. In some embodiments, Z is -NR c In some embodiments, Z is 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 It is S(O)2CF3. In some embodiments, Z is -NR c It is S(O)2NHCH3. In some embodiments, Z is -NR c It is CH2C6-aryl-S(O)2NH. In some embodiments, R c R is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C6 haloalkyl. In some embodiments, R c is methyl. In some embodiments, R c is ethyl. In some embodiments, R c is isopropyl. In some embodiments, R c is n-propyl. In some embodiments, R c is cyclopropyl. In some embodiments, R c is cyclobutyl. In some embodiments, R c is cyclopentyl. In some embodiments, R c teeth, [ka] In some embodiments, R c teeth, [ka] In some embodiments, R c teeth, [ka] That is the case.

[0081] In some embodiments, R c R d It is a -(C1-C6 alkylene)C3-C6 cycloalkyl that is optionally substituted. In some embodiments, R c R d It is a 3- to 6-membered heterocycline that has been optionally substituted. In some embodiments, R c R d It is a -(C1-C6 alkylene) 3-6 member heterocycline that is optionally substituted with R. In some embodiments, c R d It is a -(C1-C6 alkylene)C6-aryl that is optionally substituted. In some embodiments, R c R d It is a 5-6 member heteroaryl that is optionally substituted with R. In some embodiments, c R d It is a -(C1-C6 alkylene) 5-6 member heteroaryl that is optionally substituted. In some embodiments, R c is -(C1-C6 alkylene) OR 13 In some embodiments, R c is -(C1-C6 Alkilen)SR 13 In some embodiments, R c is -(C1-C6 alkylene)S(O)2R 13 In some embodiments, R c is -(C1-C6 alkylene)S(O)2NR 14 R 15 In some embodiments, Rc is -(C1-C6 alkylene)NR 13 S(O)2R 14 In some embodiments, R c is -(C1-C6 alkylene)NR 14 R 15 In some embodiments, R c is -(C1-C6 alkylene)C(O)R 13 In some embodiments, R c is -(C1-C6 alkylene)NR 13 C(O)R 14 In some embodiments, R c is -(C1-C6 alkylene)NR 13 C(O)NR 14 R 15 In some embodiments, R c is -(C1-C6 alkylene)C(O)OR 13 In some embodiments, R c is -(C1-C6 alkylene)C(O)ONR 14 R 15 In some embodiments, R c is -(C1-C6 alkylene)-C(O)NR 14 R 15 That is the case.

[0082] In some embodiments, any R c The base -C1-C6 alkylene is R d It may be replaced by any option.

[0083] In some embodiments, any R c The -C1-C6 alkylene group is either linear or branched.

[0084] In some embodiments, R c methyl, ethyl, isopropyl, n-propyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, [ka] Selected from the group consisting of, in the formula, the dashed line indicates a joining point.

[0085] In some embodiments, Z is [ka] Selected from the group consisting of, in the formula, the dashed line indicates a joining point.

[0086] In some embodiments, Z is -NR c It is SO2NH2.

[0087] In some embodiments, Z is -NHSO2NH2. In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] It is.

[0088] In some embodiments, Z is -NHS(O)2CH3.

[0089] In some embodiments, Z is -SO2NH2.

[0090] In some embodiments, Z is -NHC(O)CH 3. It is.

[0091] In some embodiments, Z is -C(O)OH.

[0092] In some embodiments, Z is -CONH2.

[0093] In some embodiments, Z is -NHCONH2.

[0094] In some embodiments, Z is -CONH(OH).

[0095] In some embodiments, Z is -B(OH)2.

[0096] In some embodiments, Z is -P(O)(OH)2.

[0097] In some embodiments, Z is -S(O)2OH.

[0098] In some embodiments, Z is -NR c S(O)2CF3.

[0099] In some embodiments, Z is -NR c S(O)2NHCH3.

[0100] In some embodiments, Z is -NR c CH2C6 - aryl - S(O)2NH2.

[0101] In some embodiments, Z is -NHCH2C6-aryl-S(O)2NH2.

[0102] In some embodiments, Z is -N(CH3)CH2C6-aryl-S(O)2NH2.

[0103] In some embodiments, G, R a , R b , L, t and Z are all

Chemical formula

[0104] In some embodiments, if any one of R a and R b , and A together with the atom to which they are attached form a C4-C6 cycloalkyl ring, then L and Z together

Chemical formula

[0105] In some embodiments, if any one of R a and R b , and A together with the atom to which they are attached form a C4-C6 cycloalkyl ring, then L and Z together

Chemical formula

[0106] In some embodiments, if any one of R a and R b , and A together with the atom to which they are attached form a cyclobutyl ring (which forms an azaspiroheptane ring together with azetidine), then L and Z together

Chemical formula

[0107] In some embodiments, ring C is R 1 It is a 5-6 member heteroaryl that is optionally substituted with R. In the embodiment, ring C is R 1 It is a 5-membered heteroaryl substituted with R. In some embodiments, the ring C is R 1 It is a 6-membered heteroaryl that is optionally substituted with R. In some embodiments, ring C is selected from the group consisting of imidazole, pyrazole, pyrrole, pyridine, pyrimidine, pyridone, pyrimidone, pyridazine, pyridazinon, and triazine, which are respectively R 1 It is optionally replaced by R. In some embodiments, 1 These include hydrogen, oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-6 member heterocyclyl, C6-aryl, 5-6 member heteroaryl, -CN, and -CONR. 11 R 12 , or -NR 11 R 12 These are selected from, and each of them is R 9 It is optionally replaced by R. In some embodiments, 1 These are selected from hydrogen, oxo, methyl, ethyl, ethylene, propynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, oxetanyl, tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidyl, phenyl, -CONH2, -NH2, -CHF2, -CF3, -Cl, and -CN, which are optionally further substituted with pyridyl, -F, -CF3, or -CHF2, respectively.

[0108] In some embodiments, ring D is a C6-aryl or a 5-6 membered heteroaryl, and these are R 2 It is optionally substituted, and ring D is condensed into ring C. In some embodiments, ring D is R 2A C6-aryl is optionally substituted, and ring D is condensed with ring C. In some embodiments, ring D is R 2 A 5-6 member heteroaryl is optionally substituted, and ring D is condensed with ring C. In some embodiments, ring D is R 2 A 5-membered heteroaryl is optionally substituted, and ring D is condensed with ring C. In some embodiments, ring D is R 2 A 6-membered heteroaryl is optionally substituted, and ring D is condensed with ring C. In some embodiments, ring D is R 2 A selection is made from the group consisting of phenyl, pyrrole, pyrazole, imidazole, pyridine, thiophene, and pyrimidine, which are optionally substituted and condensed to ring C. In some embodiments, R 2 These include hydrogen, halogen, oxo, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, and -OR 10 Selected from R, -CN, and C6-aryl, these are R 9 It is optionally replaced by R. In some embodiments, 2 The elements are selected from hydrogen, fluorine, bromine, chloro, oxo, methyl, ethyl, isopropyl, methoxy, ethoxy, propoxy, phenyl, 4-methoxyphenyl, -CN, -OCH2F, -OCH2OCH3, -(OCH2CH2)morpholine, 4-hydroxycyclohexyl, -CF3, cyclopropyl, and phenyl.

[0109] In some embodiments, R 2 Any two of these, together with the atom they bond to, form a C5-C6 cycloalkyl, a 5-6 membered heterocyclyl, a C6-aryl, or a 5-6 membered heteroaryl, and these are each R 9 It is optionally replaced by R. In some embodiments, 2 Any two of these, together with the atom they bond to, form imidazole, dioxol, and dihydrodioxin, which are each optionally substituted with methyl atoms.

[0110] In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2.

[0111] 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.

[0112] In some embodiments, ring C, ring D, R 1 , and R 2 together [ka] [ka] [ka] Selected from the group consisting of, where the dashed line in the formula indicates a joining point.

[0113] In some embodiments, ring C, ring D, R 1 , and R 2 together [ka] [ka] [ka] [ka] [ka] Selected from the group consisting of, where the dashed line in the formula indicates a joining point.

[0114] A, G, R a , R b , L, Z, ring C, ring D, R 1, R 2 , R c The explanations for m, n, and t are A, G, and R. a , R b , L, Z, ring C, ring D, R 1 , R 2 , R c The descriptions of m, n, and t may be combined independently, and it should be understood that each combination is equivalent to a specific and individual enumeration.

[0115] In some embodiments, the compound of formula II is provided. [ka] or a salt thereof, where A, R a , R b , L, Z, ring C, ring D, R 1 , R 2 m and n are each described in detail independently herein.

[0116] In some embodiments, the compound of formula III is provided. [ka] or a salt thereof, where L, Z, ring C, ring D, R 1 , R 2 m and n are each described in detail independently herein.

[0117] In some embodiments, the compound of formula IV is provided. [ka] or a salt thereof, in the formula, X is N or CR 1 And, Y is N or CR 1 And, however, at the same time, both X and Y are not N. A, G, R a , R b , L, Z, ring D, R 1 , R 2n, and t are each described in detail independently herein.

[0118] In some embodiments, the provided compound is one of the compounds from formulas IV-1 to IV-11. [ka] or a salt thereof, in the formula, X1, X2, and X3 are independently N, NR 2 , or CR 2 And, However, if any one of X1, X2, or X3 is NR 2 The other is N or CR 2 And, A, G, R a , R b L, Z, R 1 , R 2 , and t are each described in detail independently herein.

[0119] In some embodiments, a compound of formula V is provided. [ka] or a salt thereof, in the formula, X is N or CR 1 And, Y is N or CR 1 And, however, at the same time, both X and Y are not N. L, Z, ring D, R 1 , R 2 , and n are each described in detail independently herein.

[0120] In some embodiments, the provided compound is one of the compounds from formulas V-1 to V-11. [ka] [ka] or a salt thereof, in the formula, X1, X2, and X3 are independently N, NR 2 , or CR 2 And, However, if any one of X1, X2, or X3 is NR 2 The other is N or CR 2 And, L, Z, R 1 , and R 2 Each of these is described in detail separately herein.

[0121] In some embodiments, the compound of formula VI is provided. [ka] or a salt thereof, in the formula, Y1 is N or NR 1 And, Y2 is N, NR 1 , or CR 1 However, if either Y1 or Y2 is NR 1 And the other is NR 1 It is unexpected, A, G, R a , R b L, Z, R 1 , R 2 , and t are each described in detail independently herein.

[0122] In some embodiments, the compound of formula VII is provided. [ka] or a salt thereof, in the formula, Z1 is N or CR 1 And, A, G, R a , R b L, Z, R 1 , R 2 , and t are each described in detail independently herein.

[0123] In some embodiments, the compound of formula VIII is provided. [ka] or a salt thereof, where A, G, R a , R b L, Z, R 1 , R 2 , and t are each described in detail independently herein.

[0124] In some embodiments, the compound of formula IX is provided. [ka] or a salt thereof, in the formula, Y1 is N or NR 1 And, Y2 is N, NR 1 , or CR 1 However, if either Y1 or Y2 is NR 1 And the other is NR 1 It is unexpected, L, Z, R 1 , and R 2 Each of these is described in detail separately herein.

[0125] In some embodiments, a compound of formula X is provided. [ka] or a salt thereof, in the formula, Z1 is N or CR 1 And, L, Z, R 1 , and R 2 Each of these is described in detail separately herein.

[0126] In some embodiments, the compound of formula XI is provided. [ka] or a salt thereof, where L, Z, R1 and R 2 These are each described in detail independently in this specification.

[0127] In some embodiments, the compound of formula XII is provided. [ka] or a salt thereof, in the formula, X is N or CR 1 And, X4 and X5 are independently N or CR 2 However, both are not N at the same time. A, G, R a , R b L, Z, R 1 , R 2 , and t are each described in detail independently herein.

[0128] In some embodiments, a compound is provided which is one of the compounds of formulas XII-1 to XII-6. [ka] or a salt thereof, where A, G, R a , R b L, Z, R 1 , R 2 , R c , and t are each described in detail independently herein.

[0129] In some embodiments, the compound of formula XIII is provided. [ka] or a salt thereof, in the formula, X is N or CR 1 And, X4 and X5 are independently N or CR 2 However, both are not N at the same time. L, Z, R 1 , and R 2These are each described in detail independently in this specification.

[0130] In some embodiments, the provided compound is one of the compounds from formulas XIII-1 to XIII-9. [ka] or a salt thereof, where L, Z, R 1 , R 2 , and R c These are each described in detail independently in this specification.

[0131] Also provided are salts of the compounds referred to herein, e.g., pharmaceutically acceptable salts. The disclosure also includes any or all of the stereochemical forms, including any enantiomer or diastereomer forms, and any tautomers or other forms of the compounds described.

[0132] In some embodiments, the compound is selected from Table 1. It is understood that the 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 described in US2022 / 0135598A1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 [Table 1-42] [Table 1-43] [Table 1-44] [Table 1-45] [Table 1-46] [Table 1-47] [Table 1-48] [Table 1-49] [Table 1-50]

[0133] kit This specification also provides kits comprising the compounds of the Disclosure, or pharmaceutically acceptable salts thereof, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs, and suitable packaging. In one embodiment, the kit further includes instructions for use. In one embodiment, the kit comprises the compounds of the Disclosure, or pharmaceutically acceptable salts thereof, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs, and labels and / or instructions for the use of the compounds in the treatment of indications, including diseases or conditions described herein.

[0134] This specification also provides manufactured articles containing the compounds described herein or their pharmaceutically acceptable salts, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs in suitable containers. The containers may be vials, bottles, ampoules, pre-loaded syringes, and intravenous bags.

[0135] Pharmaceutical composition and dosage form The compounds provided herein are typically administered in the form of pharmaceutical compositions. Accordingly, 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 vehicles selected from carriers, adjuvants, and excipients are also provided herein. Suitable pharmaceutically acceptable vehicles include, for example, inert solid diluents and fillers, diluents containing sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers, and adjuvants. Such compositions are prepared by methods well known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GSBanker & CTRhodes, Eds.).

[0136] The pharmaceutical composition may be administered in single or multiple doses. The pharmaceutical composition may be administered by various methods, including, for example, rectally, orally, intranasally, and transdermally. In certain embodiments, the pharmaceutical composition may be administered by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant.

[0137] One mode of administration is, for example, parenteral administration by injection. Forms into which the pharmaceutical compositions described herein may be incorporated for administration by injection include, for example, sesame oil, corn oil, cottonseed oil, or peanut oil, as well as aqueous or oily suspensions or emulsions with elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles.

[0138] Oral administration may be another route for administering the compounds described herein. Administration may be carried out, for example, via capsules or enteric-coated tablets. When preparing a pharmaceutical composition comprising at least one of the compounds described herein, or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analog thereof, the active ingredient is usually diluted with an excipient and / or encapsulated in a carrier, which may be in the form of a capsule, pouch, paper, or other container. If the excipient functions as a diluent, it may be in the form of a solid, semi-solid, or liquid material acting as a vehicle, carrier, or medium for the active ingredient. Thus, compositions may be in the form of tablets, pills, powders, licks, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in a liquid medium), or ointments, containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injection solutions, and sterile packaging powders.

[0139] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. In addition, the formulation may contain lubricants, wetting agents, emulsifiers and suspending agents such as talc, magnesium stearate, and mineral oil, preservatives such as methyl and propyl hydroxybenzoates, sweeteners, and flavorings.

[0140] Compositions comprising at least one compound described herein, or a pharmaceutically acceptable salt thereof, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analog thereof, can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a subject by using procedures known in the art. Controlled-release drug delivery systems for oral administration include osmotic pump systems and dissolution systems comprising polymer-coated reservoirs or drug polymer matrix formulations. Examples of controlled-release systems are shown in U.S. Patents 3,845,770, 4,326,525, 4,902,514, and 5,616,345. Another formulation for use in the manner disclosed herein uses transdermal delivery devices ("patches"). 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 drug delivery 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 may be constructed for continuous, pulsating, or on-demand delivery of the drug.

[0141] To prepare solid compositions such as tablets, the main active ingredient may be mixed with a pharmaceutically acceptable excipient to form a solid preliminary formulation composition containing a homogeneous mixture of the compound described herein, or its pharmaceutically acceptable salts, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs. When these preliminary formulation compositions are referred to as homogeneous, the active ingredient may be uniformly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules.

[0142] Tablets or pills of the compounds described herein may be coated or otherwise formulated to provide a dosage form that offers the benefit of long-term action or to protect from the acidic conditions of the stomach. For example, a tablet or pill may consist of an inner dosing component and an outer dosing component, the outer dosing component being in the form of an envelope above the inner dosing component. The two components are separated by an enteric coating, which can resist disintegration in the stomach and allow the inner component to pass through the duodenum intact or delay its release. A variety of materials, including many high molecular weight acids and mixtures of high molecular weight acids with materials such as shellac, cetyl alcohol, and cellulose acetate, can be used for such enteric coatings or coatings.

[0143] Compositions for inhalation or inhalation may include solutions and suspensions in pharmaceutically acceptable aqueous solvents or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may include suitable pharmaceutically acceptable excipients as described herein. In some embodiments, compositions are administered orally or via nasal respiratory routes for topical or systemic effects. In other embodiments, compositions in pharmaceutically acceptable solvents may be sprayed using an inert gas. The sprayed solution may be inhaled directly from a spraying device, or the spraying device may be attached to a face mask, embolus, or intermittent positive airway pressure (CPAP) respirator. The solution, suspension, or powder composition may preferably be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0144] dosage The specific dose levels of the compounds of this application for any particular subject will depend on a variety of factors, including the activity of the particular compound used in the subject receiving treatment, age, weight, overall health, sex, diet, administration time, route of administration, and excretion rate, drug combinations, and the severity of the particular disease. For example, the dosage may be expressed as milligrams (mg / kg) of the compound described herein per kilogram of the subject's body weight. Doses of about 0.1 to 150 mg / kg may be appropriate. In some embodiments, about 0.1 to 100 mg / kg may be appropriate. In other embodiments, doses of 0.5 to 60 mg / kg may be appropriate. Normalizing according to the subject's body weight is particularly useful when adjusting dosages between subjects of significantly different sizes, such as when using drugs in both children and adults, or when converting effective dosages in non-human subjects such as dogs to dosages suitable for human subjects.

[0145] The daily dose may 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 may be 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.

[0146] When administered orally, the total daily dose for human subjects may range from 1 mg to 1,000 mg, approximately 1,000 to 2,000 mg / day, approximately 10 to 500 mg / day, approximately 50 to 300 mg / day, approximately 75 to 200 mg / day, or approximately 100 to 150 mg / day.

[0147] The compound or composition of the present application may be administered once, twice, three times, or four times daily using any preferred form described above. The administration or treatment with the compound may also be continued for several days; for example, treatment would typically last for at least 7, 14, or 28 days for one treatment cycle. Treatment cycles may alternate periodically with rest periods of approximately 1 to 28 days, typically about 7 or 14 days. Treatment cycles may also be continuous in other embodiments.

[0148] In certain embodiments, the method comprises administering an initial daily dose of about 1 to 800 mg of the compound described herein and increasing the dose until clinical efficacy is achieved. The dose may be increased using increments of about 5, 10, 25, 50, or 100 mg. The dosage may be increased daily, every other day, twice a week, or once a week.

[0149] Compound Synthesis The compounds may be prepared using the method disclosed in US2022 / 0135598A1, which is incorporated in whole by reference, and its customary modifications, which will be apparent given the disclosure herein and methods well known in the art. In addition to the teachings herein, conventional and well known synthetic methods may be used. Reagents, where available, may be commercially available from, for example, Sigma Aldrich or other chemical suppliers. [Examples]

[0150] The following embodiments are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that function well in the implementation of the Disclosure and can therefore be considered to constitute a particular form for implementation. However, those skilled in the art will understand that many modifications can be made in light of the Disclosure to the specific embodiments disclosed, and that similar or equivalent results can still be obtained without departing from the spirit and scope of the Disclosure.

[0151] Biological assays The following examples illustrate biological assays for testing the ENPP1 inhibitors of this disclosure.

[0152] Example 1: ENPP1 Inhibition Assay Using an ATP Substrate The ability of the compounds disclosed herein to inhibit ENPP1 was tested using a biochemical ENPP1 inhibition assay with ATP as the substrate. The stock solution of the test compound was prepared in DMSO and then serially diluted to 10 concentrations by 3-fold dilution in a 384-well plate (ProxiPlate-384 Plus, PE#6008280) using a TECAN EVO200 liquid handler. 30 nL of the stock solution was transferred to the 384-well plate using an Echo550 dispenser. DMSO was used as the vehicle control. A 5 μL 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 room temperature for 30 minutes. A 5 μL mixture containing 300 nM ATP (Promega #V915B), 2 mM MgCl2, 1 mM TCEP pH 7.0, 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, in a total volume of 10 μL. The plate was incubated at room temperature for 90 minutes. The reaction was stopped by adding a 5 μL 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 a further 120 minutes. The generated signals were measured using an Envision instrument. The residual activity (%) was calculated using the reading conversion ratio (CR) according to the following formula: Residual activity (%)=100×(CR sample -CR LC ) / (CR HC -CR LC )

[0153] By using XLFit (Equation 201), both the lower and upper parts are made to float, IC 50 The value was calculated.

[0154] A specific compound disclosed in Table 1 was tested in the above assay, and an IC of less than 3 μM was obtained. 50 This showed IC for a specific compound. 50 Table A shows the data as a range, where "+++" < 100 nM, 100 nM ≤ "++" < 3000 nM, and "+" ≥ 3000 nM. [Table A]

[0155] Example 2: Mineralization assay We investigated the ability of the compounds disclosed herein to regulate mineralization of human bone-related cells. Saos-2 cells (ATCC catalog #HTB-85) were used in 3.5 × 10⁶ cells. 5Cells were plated in 24-well plates at a seeding density of cells / well and grown to 100% confluence over the following 1-2 days. Once the cells were fully confluent, the medium was replaced with fresh medium supplemented with 1× ascorbic acid (Sigma#A4544, "AA"), 1× β-glycerophosphate (Sigma#G9422, "BGP"), and various concentrations of ENPP1 inhibitors. The day on which the mineralization components were added was considered day 1. On day 3, the medium was gently aspirated without washing, followed by the addition of fresh medium, mineralization components, and ENPP1 inhibitors at concentrations of 1 and 10 μM. On day 5, the medium was gently aspirated, and the cells were washed twice with PBS. The cells were then fixed in ice-cold 70% ethanol for 1 hour at 4°C. The cells were washed once with PBS and stained with Alizarin Red by gentle swishing for 30 minutes at room temperature. The stained cells were washed three times with water by gentle swishing for 10 minutes 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 a wavelength of 405 nm using an Envision instrument.

[0156] As shown in Figure 1, the compounds of this disclosure have the ability to modulate mineralization. "AA+BGP" represents 1× ascorbic acid + 1× β-glycerophosphate. Compounds 131 and 134 showed higher mineralization levels at 1 μM than at 10 μM, respectively. Compounds 135 and 76 showed similar mineralization levels at both 1 μM and 10 μM.

[0157] Example 3: Effect of compounds on PPi levels in WT animals The effects of the exemplary compounds of this disclosure on PPi levels in wild-type animals were investigated. Compound 76 was orally administered as a single dose of 100 mg / kg to 6-8 week old female SD rats (n=3) and to 6-8 week old C57BL / 6J mice (n=6) at 100 mg / kg for 5 days via BID. Plasma samples were collected in heparin SST tubes from rats before administration and at 1, 4, and 8 hours after administration, and from mice at 1 and 8 hours after the final administration, and immediately centrifuged (7000 rpm × 10 min). For PPi analysis, platelet removal was performed immediately by centrifuging the plasma on a Spin-X column (0.22 μM, Costar 8160) (7000 rpm × 2 min), and the eluted plasma was immediately parvotted / frozen on dry ice. Prior to PPi analysis, plasma samples were deproteinized using a 10kD cutoff column (Amicon#UFC501024) (4°C). PPi from filtered plasma was initially converted to ATP using ATP sulfurylase (R&D#7175-AS-020) in the presence of adenosine phosphosulfate (APS; Sigma#A5508). The reaction mixture was incubated at 37°C for 30 minutes, followed by incubation at 90°C for 10 minutes to inactivate the ATP sulfurylase. The resulting ATP was then quantified using Bactiter Glo detection reagent (Promega G8230, Madison, WI, USA). To account for endogenous ATP present in plasma, a blank reaction was performed for each sample with thermally inactivated ATP sulfurylase. Plasma PPi levels were calculated by subtracting the luminescence signal from this reaction from the total luminescence signal.

[0158] Compound 76 can reduce PPi levels in a time-dependent manner in rats after a single dose (1, 4, and 8 hours later), as shown in Figure 2, and in mice at 1 and 8 hours after the final dose of a 5-day continuous administration regimen, as shown in Figure 3.

[0159] Example 4: Mineralization assay in TNAP KO MC3T3-E1-C4 cells The compounds disclosed herein are tested in a mineralization assay in TNAP knockout MC3T3-E1-C4 cells. The osteoblast cell line (MC3T3-E1) established from C57BL / 6 mouse cranial vaults (skulls) has the ability to synthesize extracellular matrix (collagen) and differentiate into osteoblasts and osteocytes in vitro. To induce mineralization, cells are grown for 5 days in αMEM containing 50 μg / ml ascorbate, followed by supplementation with 2.5 mM NaPO4 or 5 mM β-glycerophosphate as described above (Liu et al. 2014, Bone. 67:81-94). After 5 days, the medium is gently aspirated without washing, followed by the addition of fresh medium, mineralization components, and ENPP1 inhibitors at different concentrations. After 7 days, the medium is gently aspirated, and the cells are washed twice with PBS. The cells are then fixed at 4°C for 1 hour with ice-cold 70% ethanol. Wash the cells once with PBS and stain them with alizarin red by gentle rotation at room temperature for 30 minutes. Wash the stained cells three times with water by gentle rotation at room temperature for 10 minutes each. After imaging the cells, add the dye extraction reagent to each well and incubate the cells at room temperature for 30 minutes. Alizarin red is quantified by measuring the absorbance at a wavelength of 405 nm using an Envision instrument.

[0160] Example 5: ENPP1 assay in TNAP KO MC3T3-E1-C4 cells Cells are treated with varying concentrations of the ENPP1 inhibitor of this disclosure and incubated for 5 days. After 5 days, the cell monolayer is washed twice with cold PBS and 150 μl of lysis buffer is added to each well. Cells are lysed using the freeze-thaw method and centrifuged at 13.3 rpm for 10 minutes at 4°C, and the supernatant is collected. Samples are 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) is added to each well as a substrate. p-nitrophenol production is kinetically analyzed by measuring absorbance at 405 nm using a Molecular Devices (San Jose, CA, USA) SpectraMax M2 plate reader. Activity is calculated as the change in absorbance over time (mOD / min).

[0161] Example 6: Effect of the compound on the survival of Akp- / - mice Akp- / - mice (n=17-20) are orally treated with the ENPP1 inhibitors of this disclosure using a QD regimen or vehicle, initiated within 2 days postnatal and continued until at least 25 days of age. Body weight is measured daily, and survival rates are analyzed at the end of the study. Plasma PPi concentrations are measured at different time points after the last dose (e.g., 0, 12, 24, 48, and 96 hours). Bone abnormalities are assessed by radiography 24 hours after the last dose on the day of the study.

[0162] Example 7: Akp2 + / - Effects of compounds on mice Akp2 + / - Mice (n=15) were orally treated with the ENPP1 inhibitor of this disclosure daily with QD 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 dose. Bone abnormalities were assessed by X-ray 24 hours after the last dose on the test day.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0164] The disclosures described herein as illustrative may be preferably carried out in the absence of any or more elements, limitations, or restrictions 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 are for illustrative purposes only and are not limiting; there is no intention to exclude any equivalent or part thereof of the illustrated and described features, although it is acknowledged that various modifications are possible within the scope of this disclosure.

[0165] Therefore, although this disclosure has been specifically disclosed through certain embodiments, optional features, modifications, improvements, and variations may be reused by those skilled in the art, and such modifications, improvements, and variations should be understood to be within the scope of this disclosure. The materials, methods, and examples provided herein are illustrative and not intended as limitations on the scope of this disclosure.

[0166] Specific embodiments are described broadly and generally herein. Each of the more narrowly defined species and subgenera also forms part of this disclosure. This includes a comprehensive specification with provisos or negative limitations that remove any subject matter from a genus, regardless of whether the removed material is specifically described herein.

[0167] All publications, patent applications, patents, and other references described herein are expressly incorporated by reference to the same extent that each is incorporated by reference individually. In case of any conflict, this specification shall prevail, including definitions.

[0168] While this disclosure has been described in conjunction with the embodiments described above, it should be understood that the foregoing description and examples are illustrative and not intended to limit the scope of this disclosure. Other aspects, advantages, and variations within the scope of this disclosure will be obvious to those skilled in the art to whom this disclosure relates.

Claims

1. A method for treating or preventing hypophosphatasia in a subject, wherein a therapeutically effective amount of a compound of formula I is given to the subject in need. 【Transformation 38】 or administering a pharmaceutically acceptable salt thereof, in the formula, Ring C is a 5-6 member heteroaryl, Ring D is C ６ - It is an aryl or 5-6 member heteroaryl, and ring D is condensed with ring C. A is hydrogen, C １ -C ６ Alkyl, or C ６ - These are aryl compounds, and each of them can be optionally substituted with a halogen. G is a bond, -CH ２ -, or -CH ２ -CH ２ - and R ａ and R ｂ are, independently, hydrogen or C １ -C ６ -alkyl, Alternatively, R ａ and R ｂ Together with the atoms they bond to, C ３ -C ６ Forming a cycloalkyl ring, Alternatively, R ａ and R ｂ Any one of these, along with A, together with the atom to which they are bonded, C ４ -C ６ Forming a cycloalkyl ring, L is a bonded, linear, or branched C １ -C ６ Alkylene, or linear or branched carbon dioxide ２ -C ６ It is alkenylene, t is either 0 or 1, except when t is 0, L is linear or branched C ２ -C ６ It is alkenylene, Z is -NR ｃ S(O) ２ NH ２ , -NR ｃ S(O) ２ CH ３ , -SO ２ NH ２ , -NR ｃ C(O)CH ３ , -C(O)OH, -CONH ２ , -NR ｃ CONH ２ , -CONH(OH), -B(OH) ２ , -P(O)(OH) ２ , -SO ２ OH, -NR ｃ S(O) ２ CF ３ , -NR ｃ S(O) ２ NHCH ３ , or -NR ｃ CH ２ C ６ -Ayl-S(O) ２ NH ２ And, Each R １ and R ２ is, independently, oxo, C １ -C ６ alkyl, C ２ -C ６ alkenyl, C ２ -C ６ alkynyl, C ３ -C ６ cycloalkyl, adamantyl, 3- to 6-membered heterocyclyl, C ６ -aryl, 5- to 6-membered heteroaryl, -CN, halogen, C １ -C ６ alkoxy, C １ -C ６ haloalkoxy, -OR １０ ,-SR １０ ,-S(O) ２ R １０ ,-S(O) ２ NR １１ R １２ ,-NR １０ S(O) ２ R 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 and is each independently optionally substituted by R ９ ​ Alternatively, two R's ２ However, together with the atoms they bond to, C ５ -C ６ Cycloalkyl, 5-6 membered heterocyclyl, C ６ - Forms an aryl or a 5-6 member heteroaryl, each independently, R ９ Replaced by choice, Each R ９ is independently oxo, C ２ -C ６ -alkenyl, C ２ -C ６ -alkynyl, C ３ -C ６ -cycloalkyl, 3- to 6-membered heterocyclyl, C ６ -aryl, 5- to 6-membered heteroaryl, -CN, halogen, C １ -C ６ -alkoxy, C １ -C6 haloalkoxy, C １ -C ６ -haloalkyl, -OR １３ -SR １３ -S(O) ２ R １３ -S(O) ２ NR １４ R １５ -NR １３ S(O) ２ R 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 is selected from the group consisting of C １ -C ６ -alkyl optionally substituted by oxo, -OH, or halogen, Each R ｃ These are, independently, hydrogen and C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, -(C １ -C ６ Alkylene C ３ -C ６ Cycloalkyl, 3-6 membered heterocyclyl, -(C １ -C ６ Alkilen) 3-6 member heterocycline, -(C) １ -C ６ Alkylene C ６ -aryl, 5-6 member heteroaryl, -(C １ -C ６ Alkylene) 5-6 member heteroaryl, C １ -C ６ Haloalkyl, -(C) １ -C ６ Alkylene OR １３ , - (C １ -C ６ Alkilen SR １３ , - (C １ -C ６ Alkylene S(O) ２ R １３ , - (C １ -C ６ Alkylene S(O) ２ NR １４ R １５ , - (C １ -C ６ Alkylene) NR １３ S(O) ２ R １４ , - (C １ -C ６ Alkylene) NR １４ R １５ , - (C １ -C ６ Alkylene C(O)R １３ , - (C １ -C ６ Alkylene) NR １３ C(O)R １４ , - (C １ -C ６ Alkylene) NR １３ C(O)NR １４ R １５ , - (C １ -C ６ Alkylene C(O)OR １３ , - (C １ -C ６ Alkylene C(O) ONR １４ R １５ , or -(C １ -C ６ Alkylene)-C(O)NR １４ R １５ And each is independent, R ｄ Replaced by optional selection, Each R ｄ These are, independently, halogen, -OH, oxo, -CN, and C. １ -C ６ Haloalkyl, C １ -C ６ Alkoxy, C １ -C ６ Haloalkoxys, -COOH, and C optionally substituted with -OH, halogen, CN, or oxo. １ -C ６ Selected from the group consisting of alkyl groups, Each R １０ , R １１ , and R １２ These are, independently, hydrogen and C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ６ Cycloalkyl, -(C １ -C ６ Alkylene C ３ -C ６ Cycloalkyl, 3-6 membered heterocyclyl, or -(C) １ -C ６ Alkilen) is a 3-6 member heterocycline, R １０ , R １１ and R １２ Each of these is independently oxo, C ２ -C ６ Alkenil, C ２ -C ６ Alkynyl, -CN, halogen, C １ -C ６ C optionally substituted with alkoxy, oxo, -OH, or halogen groups. １ -C ６ Optionally substituted by alkyl groups, Alternatively, R １１ and R １２ These, together with the atom(s) to which they bond, are C, optionally substituted with oxo, -OH, halogen, or oxo, -OH, or halogen. １ -C ６ They form 3- to 6-membered heterocyclines optionally substituted with alkyl groups. Each R １３ , R １４ , and R １５ These are, independently, hydrogen and C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C １ -C ６ Haloalkyl, C ３ -C ６ Cycloalkyl or 3-6 membered heterocyclyl, R １０ , R １１ , and R １２ Each of these is independently oxo, -OH, C ２ -C ６ Alkenil, C ２ -C ６ C optionally substituted with alkynyl, -CN, halogen, or oxo, -OH, or halogen. １ -C ６ Optionally substituted by alkyl groups, Alternatively, R １４ and R １５ These, together with the atoms to which they bond, are C, optionally substituted with oxo, -OH, halogen, or oxo, -OH, or halogen. １ -C ６ They form 3- to 6-membered heterocyclines optionally substituted with alkyl groups. m is 0, 1, or 2. n is 0, 1, 2, 3, or 4. However, Z is -NR ｃ S(O) ２ CH ３ , -CONH ２ , or -C(O)OH, where G is a bond and t is 1, then L is not a bond, The compound is not 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl]-3-azetidine butanoic acid, 1-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl]-3-azetidine pentanoic acid, or N-((1-(6-fluorobenzo[d]oxazole-2-yl)azetidine-3-yl)methyl)-N-methylacetamide, method.

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 administered chronically to the subject.

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

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

11. The method according to any one of claims 1 to 10, wherein the hypophosphatasia is selected from the group consisting of late-onset hypophosphatasia, prenatal hypophosphatasia, perinatal hypophosphatasia, infant hypophosphatasia, childhood hypophosphatasia, adult hypophosphatasia, tooth-limited hypophosphatasia, and hypophosphatasia with tooth involvement and / or early tooth loss.

12. The method according to claim 11, wherein the hypophosphatasia is adult hypophosphatasia.

13. The method according to claim 11, wherein the hypophosphatasia is delayed-onset hypophosphatasia.

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