Pyrazolopyrimidine compound and pharmaceutical use thereof

Abstract

An NLRP3 inhibitor, a preparation method therefor and a pharmaceutical use thereof. Provided are an NLRP3 inhibitor of formula (I), a composition thereof and a use thereof. The compound can be used for treating or preventing diseases or conditions related to abnormal expression of an NLRP3 signaling pathway.

Description

Pyrazolopyrimidine compounds and their medical uses Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and relates to a pyrazolopyrimidine compound, specifically to an isomer of the compound or a pharmaceutically acceptable salt thereof. Furthermore, the present invention also discloses a preparation method and pharmaceutical use of the pyrazolopyrimidine compound, which can be used as an NLRP3 inhibitor to treat diseases related to abnormal expression of the NLRP3 signaling pathway. Background Art

[0002] NLRP3 belongs to the NOD-like receptor protein family and is the most studied intracellular pattern recognition receptor in recent years. After recognizing pathogen-associated molecular patterns (PAMPs) or host-derived danger signal molecular patterns (DAMPs), it recruits apoptosis-associated speck-like protein (ASC) and caspase-1 to assemble into inflammasomes, and simultaneously releases activated inflammatory cytokines IL-1β and IL-18, causing an inflammatory response.

[0003] Inflammation is the body's defensive response to stimuli, including both infectious and sterile inflammation. Inflammation manifests primarily as redness, swelling, heat, pain, and functional impairment, typically caused by increased permeability of vascular endothelial cells and infiltration of immune cells into the plasma. While the inflammatory response quickly resolves after tissue repair, excessive cytokine production can lead to an inflammatory storm, causing damage to the body. Inflammatory disorders are a key pathogenesis of numerous human diseases.

[0004] Abnormal NLRP3 inflammasome activation has been observed in many inflammatory diseases, and excessive production of IL-1β and IL-18 has also been involved in and promoted the occurrence and development of various diseases, including pyrogenic protein-associated periodic syndrome; sickle cell disease; autoimmune diseases such as systemic lupus erythematosus and psoriasis; liver diseases such as chronic liver disease, viral hepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis and alcoholic liver disease; inflammatory arthritis-related diseases such as gout, chondrocalcinosis, osteoarthritis and rheumatoid arthritis; kidney diseases such as hyperoxaluria, lupus nephritis, hypertensive nephropathy, hemodialysis-related Inflammation and diabetic nephropathy; neuroinflammatory diseases such as brain infection, acute injury and neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease; cardiovascular and metabolic diseases such as atherosclerosis, type I and type II diabetes and related complications (such as nephropathy, retinopathy), peripheral arterial disease, acute heart failure and hypertension; wound healing and scar formation; inflammatory skin diseases such as acne and hidradenitis suppurativa; asthma; sarcoidosis; age-related macular degeneration; cancer-related diseases such as myeloproliferative neoplasms, leukemia, myelodysplastic syndrome, myelofibrosis, lung cancer, colon cancer, etc.

[0005] Currently, no NLRP3 inhibitors are marketed. The first compound to enter clinical development, MCC950, had its trials terminated due to liver toxicity. However, it has been extensively investigated as a tool molecule for various indications and mechanisms of action. Compounds such as OLT-1177, Emlenoflast, RG6418, and DFV890 are currently in clinical development for the treatment of conditions such as acute gout attacks, knee osteoarthritis, Schnitzler syndrome, cryopyrin-associated periodic syndromes, ulcerative colitis, and Parkinson's disease. Diseases that are inflammatory or immune in nature are often difficult to diagnose or treat effectively. Most treatments involve symptomatic treatment, slowing disease progression, lifestyle changes, and, as a last resort, surgery. More effective and efficient approaches are urgently needed to treat these conditions.

[0006] Therefore, developing NLRP3 inflammasome inhibitors to target such diseases with inflammatory pathological features has certain therapeutic potential. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a small molecule inhibitor targeting NLRP3 with a novel structure, which has significant NLRP3 inhibitory activity and is used to prevent or treat diseases related to abnormal NLRP3 expression.

[0008] In order to solve the above technical problems, the technical solutions provided by the present invention are as follows:

[0009] In one aspect, the present invention provides a pyrazolopyrimidine compound having the following structural formula (I), a compound, an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0010] in, is selected from a single bond or a double bond;

[0011] m 1 、m 2 、m 3 、m 4 、m 5 Each independently selected from 1 or 2;

[0012] X and Y are each independently selected from C or N;

[0013] R 1 、R 5 Each is independently selected from the group consisting of absence, hydrogen, halogen, hydroxy, cyano, amino, C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl may be optionally further substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent;

[0014] R 2 、R 3 、R 4 Each independently selected from hydrogen, halogen, hydroxy, cyano, amino, C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl may be optionally further substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent;

[0015] or R 3 With R 4 Together with the carbon atoms to which they are connected, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0016] or R 3 With R 2 Together with the carbon atoms to which they are connected, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0017] R 1 、R 2 、R 3 、R 4 、R 5 At least one is a hydroxyl group;

[0018] R 6 Selected from hydrogen, halogen or C 1-6 Alkyl; the C 1-6 The alkyl group may be optionally further substituted with one or more selected from C 1-6substituted by an alkyl or halogen substituent;

[0019] L is a chemical bond, NR 7 , OR 7 or C(R 7 )2; R 7 Selected from hydrogen, C 1-6 Alkyl group or not present;

[0020] R 8 Selected from hydrogen, hydroxy, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl or 5-10 membered heteroaryl; said C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 The cycloalkyl or 5-10 membered heteroaryl may be optionally further substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent;

[0021] Ring B is selected from C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; said C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 The aryl or 5-10 membered heteroaryl may be optionally further substituted with one or more alkyl radicals selected from hydroxy, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylhydroxyl, -COOR 9 、-CH2COOR 9 or C 1-6 substituted by an alkylaminoalkyl substituent;

[0022] R 9 Selected from hydrogen or C 1-6 alkyl;

[0023] The 3-6 membered heterocyclic group, the 3-9 membered heterocyclic group, and the 5-10 membered heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0024] In some embodiments, the compound has the following formula (IA), a compound isomer or a pharmaceutically acceptable salt thereof:

[0025] Wherein, X and Y are each independently selected from C or N;

[0026] R 1 、R 2 、R 3、R 4 、R 5 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted halogenated C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkylamino, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted halogenated C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted halogenated C 2-6 Alkenyl, substituted or unsubstituted C 2-6 Alkynyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclyl, substituted or unsubstituted 5-10 membered heteroaryl, hydroxyl, cyano, amino, and R 1 、R 2 、R 3 、R 4 、R 5 At least one is a hydroxyl group; when C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 2-6 Alkenyl, halogenated C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 When there is at least one substituent on a cycloalkyl, 3-6 membered heterocyclic group, or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; or

[0027] R 3 With a close R 3 R 4 Together with the carbon atoms to which they are attached, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; R 3 With a close R 3 R 2 Together with the carbon atoms to which they are attached, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0028] R 6 Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl or halogen; when C 1-6 When there is at least one substituent on the alkyl group, the substituent is selected from one or more of the following groups: C 1-6 Alkyl or halogen;

[0029] L is a direct key, NR 7 , OR 7 or CR 7 ; R 7 Selected from H, C 1-6 Alkyl group or not present;

[0030] R 8 Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted halogenated C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted halogenated C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkynyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 5-10 membered heteroaryl, hydroxyl, cyano, amino; when C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 When there is at least one substituent on the cycloalkyl or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 Alkyl or halogen;

[0031] Ring B is selected from substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-9 membered heterocyclic group, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 membered heteroaryl; when C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 When there is at least one substituent on the aryl or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 Alkyl, hydroxyl, halogen, C 1-6 Halogenated alkyl, C 1-6 Alkylhydroxyl, -COOR 9 or C 1-6 Alkylaminoalkyl;

[0032] R 9 Selected from hydrogen or C 1-6 alkyl;

[0033] The heterocyclic group and the heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0034] In some embodiments, the compound has the following formula (IB), an isomer, or a pharmaceutically acceptable salt thereof:

[0035] wherein ring B is selected from C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; said C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 The aryl or 5-10 membered heteroaryl may be optionally further substituted with one or more alkyl radicals selected from hydroxy, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylhydroxyl, -CH2COOR 9 or C 1-6 substituted by an alkylaminoalkyl substituent;

[0036] X, Y, L, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 8 、R 9 The definition of is as described in the general formula (I).

[0037] In some embodiments, the compound has the following formula (I-1), a compound, an isomer, or a pharmaceutically acceptable salt thereof:

[0038] Wherein, X and Y are each independently selected from C or N;

[0039] R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted halogenated C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkylamino, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted halogenated C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkynyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclyl, substituted or unsubstituted 5-10 membered heteroaryl, hydroxyl, cyano, and R 1 、R 2 、R 3 、R 4 、R 5 At least one is a hydroxyl group; when C 1-6 Alkyl, C 1-6 Alkylamino, halogenated C 1-6Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 When there is at least one substituent on a cycloalkyl, 3-6 membered heterocyclic group, or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; or

[0040] R 3 With a close R 3 R 4 Together with the carbon atoms to which they are attached, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; R 3 With a close R 3 R 2 Together with the carbon atoms to which they are attached, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0041] R 6 Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl or halogen; when C 1-6 When there is at least one substituent on the alkyl group, the substituent is selected from one or more of the following groups: C 1-6 Alkyl or halogen;

[0042] L is selected from NR 7 , OR 7 or CR 7 ; R 7 is selected from H, methyl or absent;

[0043] Ring B is selected from substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-9 membered heterocyclyl, substituted or unsubstituted 5-10 membered heteroaryl; when C 3-8 When there is at least one substituent on a cycloalkyl, 3-9 membered heterocyclic group or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 Alkyl, hydroxyl, halogen, C 1-6 Halogenated alkyl, C 1-6 Alkylhydroxyl, -CH2COOEt or C 1-6 Alkylaminoalkyl;

[0044] The heterocyclic group and the heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0045] In some embodiments, the compound has the following formula (II), isomers, or pharmaceutically acceptable salts thereof:

[0046] Wherein, X and Y are each independently selected from C or N;

[0047] R 1 、R 5 are each independently selected from the group consisting of absence, hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3- 8-membered cycloalkyl, 3-6-membered heterocyclyl or 5-10-membered heteroaryl may be further optionally substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent;

[0048] R 2 、R 3 、R 4 Each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl may be optionally further substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent;

[0049] or R 3 With R 4 Together with the carbon atoms to which they are connected, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0050] or R 3 With R 2 Together with the carbon atoms to which they are connected, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0051] R 1 、R 2 、R 3 、R 4 、R5 At least one is a hydroxyl group;

[0052] L is selected from NR 7 , OR 7 or C(R 7 )2; R 7 is selected from hydrogen, methyl or absent;

[0053] Ring B is selected from C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; said C 3-8 Cycloalkyl, 3-9 membered heterocyclic group, C 6-10 The aryl or 5-10 membered heteroaryl may be optionally further substituted with one or more alkyl radicals selected from hydroxy, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkylhydroxyl, -CH2COOEt or C 1-6 substituted by an alkylaminoalkyl substituent;

[0054] The 3-6 membered heterocyclic group, the 3-9 membered heterocyclic group, and the 5-10 membered heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0055] In some embodiments, the compound has the following formula (IIA), a compound isomer or a pharmaceutically acceptable salt thereof:

[0056] Among them, R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted halogenated C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkylamino, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted halogenated C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkynyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclyl, substituted or unsubstituted 5-10 membered heteroaryl, hydroxyl, cyano, and R 1 、R 2 、R 3 、R 4 、R 5 At least one is a hydroxyl group; when C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 When there is at least one substituent on a cycloalkyl, 3-6 membered heterocyclic group, or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; or

[0057] R 3 With a close R 3 R 4 Together with the carbon atoms to which they are attached, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; R 3 With a close R 3 R 2 Together with the carbon atoms to which they are attached, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0058] R 6 Selected from hydrogen, C 1-6 Alkyl or halogen;

[0059] R 7 Selected from hydrogen or C 1-6 alkyl;

[0060] Ring B is selected from substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-8 membered heterocyclyl, substituted or unsubstituted 5-10 membered heteroaryl; when C 3-8 When there is at least one substituent on a cycloalkyl, 3-8 membered heterocyclic group or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 Alkyl, hydroxyl, halogen, C 1-6 Halogenated alkyl, C 1-6 Alkylhydroxyl, -CH2COOEt or C 1-6 Alkylaminoalkyl;

[0061] The heterocyclic group and the heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0062] In some embodiments, the compound has the following formula (II-1), a compound, an isomer, or a pharmaceutically acceptable salt thereof:

[0063] wherein n is selected from 0, 1, 2 or 3;

[0064] R 1 、R 2 、R 3 、R 4 、R 5Each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2- 6 alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl may be optionally further substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent;

[0065] or R 3 With R 4 Together with the carbon atoms to which they are connected, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0066] or R 3 With R 2 Together with the carbon atoms to which they are connected, they form C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl;

[0067] R 1 、R 2 、R 3 、R 4 、R 5 At least one is a hydroxyl group;

[0068] L is as defined in the general formula (II).

[0069] In some embodiments, the compound has the following formula (IIA-1), a compound isomer or a pharmaceutically acceptable salt thereof:

[0070] wherein n is selected from 1 or 2;

[0071] L is selected from O, NH or N(CH3);

[0072] R 3 Selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclic group or 5-10 membered heteroaryl, the C 1-6 Alkyl, C 1-6 Alkylamino, C1-6 Alkoxy, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl may be optionally further substituted by one or more halogen or C 1-6 substituted by an alkyl substituent.

[0073] In some embodiments, the compound has the following formula (IIA-1-1), an isomer, or a pharmaceutically acceptable salt thereof:

[0074] Among them, R 3 The definition of is as described in the general formula (IIA-1).

[0075] In some embodiments, R 3 Selected from hydrogen, halogenated C 1-6 Alkyl or halogen.

[0076] In some embodiments, the compound has the following formula (IIA-1-2), an isomer, or a pharmaceutically acceptable salt thereof:

[0077] Among them, R 3 The definition of is as described in the general formula (IIA-1).

[0078] In some embodiments, the R 3 Selected from hydrogen, halogen, cyano, methyl, cyclopropyl, trifluoromethyl, difluoromethyl, trifluoromethoxy, dimethylamino,

[0079] The present invention also provides a pyrazolopyrimidine compound, which is a compound, isomer or pharmaceutically acceptable salt thereof having the following structure:

[0080] On the other hand, the present invention provides a pharmaceutical composition comprising at least one compound represented by Formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, an isomer or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0081] In another aspect, the present invention provides a compound represented by Formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, an isomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the preparation of a medicament for preventing and / or treating a disease or condition associated with NLRP3.

[0082] In another aspect, the present invention also provides a compound represented by Formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, an isomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing and / or treating diseases or conditions associated with NLRP3.

[0083] In another aspect, the present invention also provides a method for preventing and / or treating a disease, comprising administering to a patient in need thereof an effective amount of a compound represented by Formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2), an isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof; the disease to be prevented and / or treated is a disease or condition associated with NLRP3.

[0084] In some embodiments, the NLRP3-related disease or disorder is selected from a cancer disease, an inflammatory disease, or a disease accompanied by an inflammatory response;

[0085] The cancer disease includes, but is not limited to, myeloproliferative neoplasms, myeloid leukemia, lung cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, bile duct cancer, oral cancer, head and neck cancer, mesothelioma, adrenocortical cancer, kidney cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, bone cancer, brain cancer, breast cancer, melanoma, pancreatic cancer, skin cancer, lymphoma, bladder cancer, small intestine cancer, soft tissue sarcoma, endometrial cancer, cervical cancer, osteosarcoma, prostate cancer or testicular cancer;

[0086] The inflammatory diseases or diseases accompanied by inflammatory responses include but are not limited to:

[0087] 1) Autoinflammatory diseases, such as chillin-associated periodic syndrome (CAPS), familial Mediterranean fever, Schnitzler syndrome, and mevalonate kinase deficiency (MKD);

[0088] 2) chronic pain, including neuropathic pain and non-neuropathic pain;

[0089] 3) Skin conditions, such as contact hypersensitivity, bullous pemphigoid, sunburn, contact dermatitis, seborrheic dermatitis, hidradenitis suppurativa (HS), diabetic (foot) ulcers, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, acne, and alopecia;

[0090] 4) Respiratory system diseases, such as chronic obstructive pulmonary disease (COPD), asthma, bronchitis, rhinitis, sinusitis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, sarcoidosis, adult respiratory distress syndrome, and pneumonia;

[0091] 5) Joint diseases, such as arthritis;

[0092] 6) Muscle diseases, such as polymyositis and myasthenia gravis;

[0093] 7) Cardiovascular diseases, such as hypertension, ischemia, reperfusion injury, vasculitis, and pericarditis;

[0094] 8) Blood diseases, such as sickle cell disease;

[0095] 9) Central nervous system diseases, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, brain injury, multiple sclerosis, and amyotrophic lateral sclerosis;

[0096] 10) Metabolic diseases, such as type 2 diabetes (T2D), atherosclerosis, obesity, and gout;

[0097] 11) Liver diseases, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease (AFLD), and alcoholic steatohepatitis (ASH);

[0098] 12) Kidney disease, such as acute kidney disease, hyperoxaluria, chronic kidney disease, nephrocalcinosis, glomerulonephritis, and diabetic nephropathy;

[0099] 13) Gastrointestinal diseases, such as inflammatory bowel disease and pancreatitis;

[0100] 14) Eye diseases, such as uveitis and allergic conjunctivitis;

[0101] 15) Graft-versus-host disease;

[0102] 16) Burns, sunburn, and mechanical injuries.

[0103] In some embodiments, the neuropathic pain includes central neuropathic pain and peripheral neuropathic pain;

[0104] The central neuralgia includes but is not limited to spinal cord injury neuralgia, post-stroke pain, multiple sclerosis pain, syringomyelia pain, ischemic myelopathy pain, compressive myelopathy pain, post-radiation myelopathy pain, Parkinson's disease pain, phantom limb pain, and myelitis pain.

[0105] The peripheral neuropathic pain includes, but is not limited to, postherpetic neuralgia, HIV neuropathy, diabetic peripheral neuropathy, chronic pain after trauma / surgery, neuropathy after chemotherapy / radiotherapy, trigeminal neuralgia, glossopharyngeal neuralgia, residual limb pain, toxic contact neuropathy, sciatica, and dorsal root neuralgia;

[0106] The non-neuropathic pain includes, but is not limited to, osteoarthritis pain, chronic low back pain, chronic visceral pain, cancer pain, and fibromyalgia.

[0107] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0108] The "compounds" described in the present invention include but are not limited to the following forms of compounds: free base, stereoisomers, geometric isomers, tautomers, isotopes, pharmaceutically acceptable salts, solvates, hydrates, prodrugs (esters), etc.

[0109] The "compounds" described herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. Compounds of the present invention containing asymmetric carbon atoms may be isolated in optically pure or racemic forms. Optically pure forms may be obtained by resolution of racemic mixtures or by synthesis using chiral starting materials or reagents.

[0110] In the present invention, "isomer" refers to, unless otherwise specified, stereoisomers or tautomers. Unless otherwise specified, the term "stereoisomer" refers to a compound with the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include, but are not limited to, enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, and atropisomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization. Unless otherwise specified, the term "tautomer" refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be reached. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations.Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0111] As used herein, "isotope" means, unless otherwise specified, that the compounds of the present invention may exist in an isotopically labeled or enriched form containing one or more atoms whose atomic mass or mass number differs from the atomic mass or mass number of the largest atom found in nature. Isotopes may be radioactive or non-radioactive. Isotopes commonly used as isotope labels are: hydrogen isotopes, including but not limited to 2 H and 3 H; Carbon isotopes: including but not limited to 13 C and 14 C; Chlorine isotopes: including but not limited to 35 Cl and 37 Cl; Fluorine isotopes: including but not limited to 18 F; Iodine isotopes: including but not limited to 123 I and 125 I; Nitrogen isotopes: including but not limited to 13 N and 15 N; oxygen isotopes: including but not limited to 15 O. 17 O and 18 O; Sulfur isotopes: including but not limited to 35 These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues, especially 3 H and 13 C, because they are easy to label and detect, they are more widely used. Some heavy isotopes, such as deuterium ( 2H) substitution can enhance metabolic stability and prolong half-life, thereby achieving the goal of reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques similar to those used for synthesizing non-isotope-labeled compounds.

[0112] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0113] The "pharmaceutically acceptable salt" in the present invention refers to a salt of the compound of the present invention, wherein the compound having a specific substituent discovered by the present invention is reacted with 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, magnesium, ammonium or organic amine salts. Examples include alkali metal salts, alkaline earth metal salts, other metal salts, inorganic base salts, organic base salts, inorganic acid salts, lower alkane sulfonates, aryl sulfonates, organic acid salts, amino acid salts, and the like.

[0114] The "solvate" in the present invention is selected from hydrate, ethanolate, methanolate, acetoneate, etherate or isopropanolate.

[0115] The terms "direct bond" and "chemical bond" both refer to two adjacent atoms being directly connected by a single bond.

[0116] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0117] The term "hydroxy" refers to -OH; the term "cyano" refers to -CN; and the term "amino" refers to -NH2.

[0118] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, such as C 1-6Alkyl groups include, but are not limited to, methyl, ethyl, propyl (including: 1-propyl or n-propyl, 2-propyl or isopropyl), butyl (including: 1-butyl or n-butyl, 2-methyl-1-propyl or isobutyl, 2-methyl-1-propyl or isobutyl, 1-methylpropyl or sec-butyl, 1,1-dimethylethyl or tert-butyl), pentyl (1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl), hexyl (1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl).

[0119] The term "halogenated C 1-6 "Alkyl" refers to an alkyl group in which one or more H atoms are replaced by one or more halogen atoms (such as fluorine, chlorine, bromine or iodine), including but not limited to -CF3, -CH2Cl, -CH2CF3, -CHCl2, -CCl3, etc.

[0120] The term "alkylhydroxy" refers to a hydroxy group further substituted with an alkyl group as defined above, such as C 1-6 The alkylhydroxyl group includes, but is not limited to, methylhydroxyl, ethylhydroxyl, propylhydroxyl, isopropylhydroxyl and the like.

[0121] The term "alkylamino" refers to an open-chain alkyl group containing a nitrogen atom, such as C 1-6 Alkylamino groups include, but are not limited to, methylamino, ethylamino, isopropylamino, dimethylamino, methylethylamino, diethylamino, and the like.

[0122] The term "alkylaminoalkyl" refers to an alkyl group further substituted with an alkylamino group as defined above, such as C 1-8 Alkylamino-C 1-8 Alkyl- or C 1-6 Alkylamino-C 1-6 Alkyl-, including but not limited to methylaminomethyl, ethylaminomethyl, ethylaminoethyl, isopropylaminomethyl or propylaminomethyl, etc.

[0123] The term "alkylaminoalkyl" refers to an alkyl group further substituted with an alkylamino group as defined above, such as C 1-6 Alkylaminoalkyl, refers to C 1-6 Alkylamino-C 1- 6 alkyl.

[0124] The term "cycloalkyl" refers to a monocyclic alkyl group composed of carbon atoms and hydrogen atoms, such as C 3-8 Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0125] The term "alkoxy" refers to a straight or branched chain alkyl group attached through an oxygen atom, such as C 1-6 Alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, sec-butoxy and tert-butoxy), pentoxy (including n-pentoxy, isopentoxy and neopentoxy), hexoxy (n-hexyloxy, 2-methylpentoxy, 3-methylpentoxy, 2,3-dimethylbutoxy and 2,2-dimethylbutoxy), etc.

[0126] The term "halogenated C 1-6 "Alkoxy" refers to an alkoxy group in which one or more H atoms are replaced by one or more halogen atoms (such as fluorine, chlorine, bromine or iodine), including but not limited to -OCF3, -OCH2Cl, -OCH2CF3, -OCHCl2, -OCCl3, etc.

[0127] The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. The alkenyl group may contain 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms (i.e., C 2-10 Alkenyl), further preferably 2-8 carbon atoms (ie C 2-8 Alkenyl), more preferably 2-6 carbon atoms (ie C 2-6 alkenyl), 2-5 carbon atoms (ie C 2-5 alkenyl), 2-4 carbon atoms (ie C 2-4 alkenyl), 2-3 carbon atoms (ie C 2-3 alkenyl), 2 carbon atoms (i.e., C2 alkenyl), for example, "C 2-6 The term "alkenyl" refers to an alkenyl group, and the number of carbon atoms in the carbon chain is between 2 and 6 (i.e., 2, 3, 4, 5, or 6). Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1,3-butadien-1-yl, 1,3-butadien-2-yl, and the like.

[0128] The term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. The alkynyl group may contain 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms (i.e., C 2-10 Alkynyl), further preferably 2-8 carbon atoms (C 2-8 Alkynyl), more preferably 2-6 carbon atoms (ie C 2-6 Alkynyl), 2-5 carbon atoms (ie C 2-5 Alkynyl), 2-4 carbon atoms (ie C 2-4 Alkynyl), 2-3 carbon atoms (ie C 2-3 alkynyl), 2 carbon atoms (i.e., C2 alkynyl), for example, "C2-6 The term "alkynyl" refers to an alkynyl group, and the number of carbon atoms in the carbon chain is between 2 and 6 (i.e., 2, 3, 4, 5, or 6). Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, 1,3-butadiynyl, 1-pentynyl, 3-methyl-1-butynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 3-methyl-1-pentynyl, 4-methyl-1-pentynyl, 3,3-dimethyl-1-butynyl, 3-ethyl-1-butynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 3-methyl-1,4-pentadiynyl, 1,5-hexadiynyl, and the like.

[0129] The term "cycloalkyl" refers to a monocyclic or bicyclic alkyl group composed of carbon atoms and hydrogen atoms, such as C 3-8 Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0130] The term "aliphatic ring" refers to a monocyclic aliphatic hydrocarbon containing a specific number of carbon atoms interconnected by carbon-carbon single and double bonds, preferably containing 3-6 carbon atoms. Non-limiting examples of aliphatic rings include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cyclohexene.

[0131] The term "aromatic ring" refers to an all-carbon monocyclic ring or fused polycyclic ring of 6-16 carbon atoms with a completely conjugated π-electron system, including but not limited to a benzene ring, a naphthalene ring, an anthracene ring, etc., preferably a benzene ring.

[0132] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 6 to 16 carbon atoms with a completely conjugated π-electron system, including but not limited to phenyl, naphthyl, anthracenyl, etc., preferably phenyl.

[0133] The term "heterocycle" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic hydrocarbon ring that is non-aromatic and contains 3-20 ring atoms, wherein 1, 2, 3, or more ring atoms are selected from N, O, or S, and the remaining ring atoms are C. Bicyclic or polycyclic heterocycles include spirocyclic, fused, and bridged heterocycles. Bicyclic or polycyclic "heterocycles" include those in which one ring is aromatic and the other rings are non-aromatic, including spirocyclic, fused, and bridged heterocycles. Bicyclic heterocycles may contain one or more heteroatoms in one or both rings. In some embodiments, heterocycles also include ring systems in which a heterocycle as defined above is fused to one or more carbocyclyl groups, wherein the point of attachment is on the ring of the carbocycle or heterocycle; or, in some embodiments, heterocycles also include ring systems in which a heterocycle as defined above is fused to one or more aromatic / heteroaromatic rings, wherein the point of attachment is on the ring of the aromatic / heteroaromatic or heterocycle; or, in some embodiments, a heterocycle as defined above is fused to one or more heterocycles as defined above, wherein the point of attachment is on the ring of either heterocycle. In these cases, the number of ring members of the heterocycle is the number of ring atoms in the ring system after fusion. In some embodiments, heterocycles are optionally substituted, e.g., unsubstituted (unsubstituted heterocycle) or substituted with one or more substituents (substituted heterocycle). Exemplary 3-membered heterocycles containing 1 heteroatom include, but are not limited to, aziridine, oxirane, and thiirane. Exemplary 4-membered heterocycles containing one heteroatom include, but are not limited to, azetidine, oxetane, and thietane. Exemplary 5-membered heterocycles containing one heteroatom include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, dihydrothiophene, pyrrolidine, dihydropyrrole, and 2,5-pyrrolidinedione. Exemplary 5-membered heterocycles containing two heteroatoms include, but are not limited to, dioxolane, oxathiolane, dithiolane, and 2-oxoxazolidine. Exemplary 5-membered heterocycles containing three heteroatoms include, but are not limited to, triazole, oxadiazole, and thiadiazole. Exemplary 6-membered heterocycles containing one heteroatom include, but are not limited to, piperidine, tetrahydropyran, dihydropyridine, and tetrahydrothiopyran. Exemplary 6-membered heterocycles containing two heteroatoms include, but are not limited to, piperazine, morpholine, thiomorpholine, and the like. Exemplary 6-membered heterocycles containing 3 heteroatoms include, but are not limited to, triazinane, oxadiazinane, thiadiazinane, oxathiazinane, and dioxazinane. Exemplary 7-membered heterocycles containing 1 heteroatom include, but are not limited to, azepane, oxepane, and thiepane. Exemplary 8-membered heterocycles containing 1 heteroatom include, but are not limited to, azocane, oxepane, and thiecane. Exemplary 5-membered heterocycles fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocycles) include, but are not limited to, indoline, isoindoline, dihydrobenzofuran, dihydrobenzothiophene, and benzoxazolinone. Exemplary 6-membered heterocycles fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocycles) include, but are not limited to, tetrahydroquinoline and tetrahydroisoquinoline.

[0134] The term "heterocyclyl" refers to a substituent derived from the above definition of heterocycle. Exemplary three-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiirane. Exemplary four-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thiirane. Exemplary five-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and 2,5-dioxopyrrolidinyl. Exemplary five-membered heterocyclyls containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, dithiolanyl, and 2-oxoxazolidinyl. Exemplary five-membered heterocyclyls containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and tetrahydrothiopyranyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazinyl, oxadiazinyl, thiadiazinyl, oxathiazinyl, and dioxazinyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxepanyl, and thiepanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl) include, but are not limited to, dihydroindolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, and benzoxazolinyl. Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl) include, but are not limited to, tetrahydroquinolinyl and tetrahydroisoquinolinyl.

[0135] The term "heteroaromatic ring" refers to an aromatic monocyclic, bicyclic, or polycyclic ring system containing 5-16 members, preferably 5-14 members, 5-12 members, 5-10 members, 5-8 members, and more preferably 5-6 members, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N, or S, and the number of heteroatoms being preferably 1, 2, or 3. Bicyclic or polycyclic heteroaromatic rings include fused-ring heteroaromatic rings. Examples of heteroaromatic rings include, but are not limited to, furan, thiophene, oxazole, thiazole, isoxazole, oxadiazole, thiadiazole, pyrrole, pyrazole, imidazole, triazole, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, thiodiazole, triazine, phthalazine, quinoline, isoquinoline, pteridine, purine, indole, isoindole, indazole, benzofuran, benzothiophene, benzopyridine, benzopyrimidine, benzopyrazine, benzimidazole, benzo Phthalazine, pyrrolo[2,3-b]pyridine, imidazo[1,2-a]pyridine, pyrazolo[1,5-a]pyridine, pyrazolo[1,5-a]pyrimidine, imidazo[1,2-b]pyridazine, [1,2,4]triazolo[4,3-b]pyridazine, [1,2,4]triazolo[1,5-a]pyrimidine, [1,2,4]triazolo[1,5-a]pyridine.

[0136] The term "heteroaryl" refers to a substituent derived from the above definition of heteroaromatic ring. Examples of heteroaryl include, but are not limited to, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, Benzopyridinyl, benzopyrimidinyl, benzopyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl.

[0137] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

[0138] In the present invention, "a", "an", "the", "at least one" and "one or more" are used interchangeably. Thus, for example, a mixture comprising "a" pharmaceutically acceptable excipient composition can be interpreted as indicating that the pharmaceutical composition includes "one or more" pharmaceutically acceptable excipients.

[0139] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0140] The pharmaceutical compositions of the present invention can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0141] The administration routes of the compounds of the present invention, their prodrugs, isomers, solvates or pharmaceutically acceptable salts thereof or their pharmaceutical compositions include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0142] The term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be therapeutic in terms of partial or complete stabilization or cure of a disease and / or side effects caused by the disease. As used herein, "treat" encompasses any treatment of a patient's disease, including: (a) suppressing the symptoms of the disease, i.e., arresting its progression; or (b) relieving the symptoms of the disease, i.e., causing regression of the disease or its symptoms.

[0143] The term "effective amount" means an amount of a compound of the present invention that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and this disclosure.

[0144] Compared with the prior art, the present invention has the following beneficial effects:

[0145] Based on the target design of NLRP3 inhibitors, the present invention has developed a novel structural pyrazolopyrimidine compound. Relevant biological experiments have shown that the compound of the present invention exhibits significant IL-1β expression inhibition activity in both THP-1 and PBMC cells, high selectivity for the NLRP3 inflammasome, low hERG toxicity, good metabolic stability and pharmacokinetic properties; in addition, the compound of the present invention has no significant inhibitory effect on multiple cytochrome P450 (CYP450) enzymes and has shown good safety in long-term toxicity experiments in rats; in a mouse inflammation model, the compound of the present invention can significantly reduce the levels of cytokines IL-1β, TNF-α and IL-6 in serum, and has great clinical application prospects. In addition, the compound provided by the present invention has a novel synthetic route that is safe, environmentally friendly and has good production feasibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1: Effects of compound 2 in Example 26 on the cytokine IL-1β in mouse serum induced by LPS.

[0147] Figure 2: Effect of compound 2 in Example 26 on the cytokine TNF-α in mouse serum induced by LPS.

[0148] Figure 3: Effects of compound 2 in Example 26 on the cytokine IL-6 in mouse serum induced by LPS.

[0149] Figure 4: Effects of the hydrochloride of compound 4 in Example 27 on the cytokine IL-1β in mouse serum induced by LPS.

[0150] Figure 5: Effect of the hydrochloride of compound 4 in Example 27 on the cytokine TNF-α in mouse serum induced by LPS.

[0151] Figure 6: Effect of the hydrochloride of compound 4 in Example 27 on the cytokine IL-6 in mouse serum induced by LPS.

[0152] Figure 7: Effects of continuous oral administration of Compound 4 hydrochloride in Example 28 for 28 days on the body weight of SD male rats.

[0153] Figure 8: Effects of continuous oral administration of Compound 4 hydrochloride in Example 28 for 28 days on the body weight of SD female rats. DETAILED DESCRIPTION

[0154] The following are specific embodiments of the present invention to further describe the technical solution of the present invention, but the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0155] In addition, all operations involving raw materials that are easily oxidized or hydrolyzed are carried out under nitrogen protection. Unless otherwise specified, the raw materials used in the present invention are commercially available raw materials and can be used directly without further purification.

[0156] The reaction starting materials and common intermediates involved in the embodiments of the present invention can be purchased commercially or prepared in-house. If the starting materials and common intermediates need to be prepared in-house, the preparation process is described in detail below.

[0157] Compound 1-8: (R)-1-Methylpiperidin-3-amine was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. According to the quality inspection report provided by the raw material supplier, the purity (NMR) was 97%, and the optical rotation was 9.8417° (c = 1.0 g / 100 mL, CHCl3); Compound (S)-1-Methylpiperidin-3-amine was purchased from Leyan Reagent (Shanghai Haohong Biotechnology Co., Ltd.), and according to the quality inspection report provided by the raw material supplier, the purity (NMR) was ≥95.0%, and the optical rotation was -6.7° (c = 0.45 g / 100 mL, CHCl3).

[0158] The following abbreviations are used in the examples:

[0159] TsOH·H2O: p-toluenesulfonic acid monohydrate; MeOH(CH3OH): methanol; MeB(OH)2: methylboric acid; Pd(dppf)Cl2: [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride; K2CO3: potassium carbonate; Dioxane: dioxane; H2O: water; NaNO2: sodium nitrite; SnCl2·2H2O: stannous chloride dihydrate; HCl: hydrochloric acid; TEA: triethylamine; THF: tetrahydrofuran; ACN: acetonitrile; DIPEA :N,N-diisopropylethylamine; n-BuOH: n-butanol; BBr3: boron tribromide; DCM: dichloromethane; NBS: N-bromosuccinimide; CuI: cuprous iodide; CsF: cesium fluoride; DMF: N,N-dimethylformamide; LiAlH4: lithium aluminum hydride; NaH: sodium hydride; Na2CO3: sodium carbonate; Fe: iron powder; NH4Cl: ammonium chloride; EtOH: ethanol; IPA(i-PrOH): isopropyl alcohol; NaBH3CN: sodium cyanoborohydride; (HCHO) n: paraformaldehyde; EA: ethyl acetate; NaCl: sodium chloride; NaHCO3: sodium bicarbonate; Na2SO4: sodium sulfate; KF: potassium fluoride; PE: petroleum ether; LC-MS: liquid chromatography-mass spectrometry; NADPH: reduced coenzyme II; UDPGA: uridine diphosphate glucuronic acid; PB: phosphate buffer; CYP3A4: cytochrome P450 3A4 enzyme; UGT: uridine diphosphate glucuronyltransferase; in the embodiments of the present invention, x mL×y means repeated y times, x mL each time, for example, extraction with EA (80 mL×3) means extraction with 80 mL of EA each time, repeated 3 times.

[0160] Preparation of intermediate 1-2

[0161] Step 1: Synthesis of 2,4-dichloro-5-(dimethoxymethyl)pyrimidine (1-2)

[0162] Compound 1-1 (500.0 mg, 2.82 mmol), TsOH·H₂O (32.3 mg, 0.17 mmol), and trimethyl orthoformate (1.08 g, 10.18 mmol) were added to a vial, followed by MeOH (6 mL). The mixture was allowed to react at 65°C for 16 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (EA:petroleum ether = 1:5) to afford intermediate 1-2 (439.0 mg) as a colorless liquid in a 73.5% yield.

[0163] LC-MS (m / z): 223.0 [M+H] + .

[0164] Example 1: Synthesis of (R)-2-(4-chloro-2-methoxy-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (target compound 1)

[0165] Step 1: Synthesis of 4-chloro-2-methoxy-6-methylaniline (1-4)

[0166] Compound 1-3 (5.00 g, 21.14 mmol), methylboronic acid (2.54 g, 42.43 mmol), K2CO3 (8.77 g, 63.45 mmol), and Pd(dppf)Cl2 (774.0 mg, 1.06 mmol) were added to a vial in sequence. Dioxane (60 mL) and water (12 mL) were then added. The mixture was reacted at 100°C under nitrogen for 16 hours. After completion of the reaction, the reaction mixture was diluted with water (50 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated NaCl (80 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (EA:petroleum ether = 1:15) to afford compound 1-4 (3.48 g) as a yellow solid in a 95.7% yield.

[0167] LC-MS (m / z): 172.0 [M+H] + .

[0168] Step 2: Synthesis of (4-chloro-2-methoxy-6-methylphenyl)hydrazine (1-5)

[0169] Compound 1-4 (1.87 g, 10.91 mmol) was added to a flask, followed by concentrated hydrochloric acid (13 mL). NaNO₂ (1.13 g, 16.38 mmol) was dissolved in distilled water (13 mL) at 0°C and added. After 0.5 hour of reaction, a solution of SnCl₂·2H₂O (4.93 g, 21.85 mmol) in concentrated hydrochloric acid (13 mL) was added at 0°C and allowed to react for 1 hour. After completion of the reaction, the reaction mixture was filtered, and the filter cake was adjusted to a pH of 8-9 with 3N NaOH solution. Water (30 mL) was added for dilution and extraction with DCM (60 mL x 3). The combined organic phases were washed with saturated NaCl (50 mL), dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to afford compound 1-5 (1.58 g) as a yellow solid in a 77.6% yield.

[0170] LC-MS (m / z): 187.0 [M+H] + .

[0171] Step 3: Synthesis of 2-chloro-4-(2-(4-chloro-2-methoxy-6-methylphenyl)hydrazino)-5-(dimethoxymethyl)pyrimidine (1-6)

[0172] Compound 1-5 (387.3 mg, 2.08 mmol), compound 1-2 (463.0 mg, 2.08 mmol), and TEA (631.5 mg, 6.24 mmol) were added to a vial, followed by THF (8 mL). The mixture was allowed to react at room temperature under a nitrogen atmosphere for 16 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (EA:petroleum ether = 1:5) to afford compound 1-6 (439.0 mg) as a yellow solid in a 56.7% yield.

[0173] LC-MS (m / z): 373.0 [M+H] + .

[0174] Step 4: Synthesis of 6-chloro-2-(4-chloro-2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (1-7)

[0175] Compound 1-6 (339.0 mg, 0.91 mmol) and TsOH·H2O (172.8 mg, 0.91 mmol) were added to the vial, followed by ACN (6 mL). The mixture was allowed to react at 70°C for 2 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (EA:petroleum ether = 1:2) to afford compound 1-7 (235.0 mg) as a yellow solid in a 73.0% yield.

[0176] LC-MS (m / z): 309.0 [M+H] + .

[0177] Step 5: Synthesis of (R)-2-(4-chloro-2-methoxy-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (target compound 1)

[0178] Compound 1-7 (225.0 mg, 0.73 mmol), compound 1-8 (99.8 mg, 0.87 mmol), and DIPEA (470.3 mg, 3.64 mmol) were added to a vial, followed by n-butanol (9 mL). The mixture was microwaved at 100°C for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH = 10:1) and lyophilized to afford the title compound 1 (250.0 mg) as a yellow solid in an 89.0% yield.

[0179] LC-MS (m / z): 387.0 [M+H] + .

[0180] 1H NMR(400MHz,DMSO-d6)δ9.08(s,1H),8.42(s,1H),8.16(s,1H),7.21(d,J=1 .6Hz,1H),7.13(d,J=1.6Hz,1H),4.08-3.93(m,1H),3.74(s,3H),3.06-2.9 2(m,2H),2.78-2.64(m,1H),2.28(s,3H),2.15-2.01(m,1H),1.99(s,3H),1 .90-1.80(m,1H),1.79-1.67(m,1H),1.64-1.49(m,1H),1.43-1.30(m,1H).

[0181] Example 2: Synthesis of (R)-5-chloro-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 2)

[0182] Compound 1 (120.0 mg, 0.31 mmol) and ultra-dry DCM (5 mL) were added to the vial. Under nitrogen, a solution of BBr in DCM (1.6 mL, 3.10 mmol, 2 M) was added at 0°C. The mixture was allowed to react for 1 hour at 0°C. After completion, the reaction was quenched with an appropriate amount of MeOH in an ice-water bath. The filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography (ACN:0.1% formic acid in water = 85%) to afford the title compound 2 (69.1 mg) as a yellow solid in a 60.1% yield.

[0183] LC-MS (m / z): 373.0 [M+H] + .

[0184] 1 H NMR(400MHz,DMSO-d6)δ9.08(s,1H),8.41(s,1H),8.16(s,1H),7.22-7.05(m,1H),7.00-6.86(m,2H),4.13-3.95(m,1H),3.13-2.95(m,1H), 2.86-2.71(m,1H),2.33(s,3H),2.21-2.03(m,2H),1.97(s,3H),1.91- 1.82(m,1H),1.79-1.69(m,1H),1.65-1.51(m,1H),1.45-1.31(m,1H).

[0185] Example 3: Synthesis of (R)-5-bromo-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 3)

[0186] Step 1: Synthesis of 4-bromo-2-methoxy-6-methylaniline (3-2)

[0187] 3-1 (22.00 g, 160.30 mmol) was dissolved in ACN (220 mL). NBS (34.25 g, 192.40 mmol) was slowly added in an ice-water bath. After addition, the reaction was continued for 10 minutes while incubating. The reaction solution was concentrated under reduced pressure to remove most of the solvent, diluted with water (100 mL), and extracted twice with DCM / MeOH (10:1, 100 mL). The combined organic phases were washed with saturated NaCl (100 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (EA:petroleum ether = 4:96) to afford compound 3-2 (22.34 g) as a dark brown solid in a 64.5% yield.

[0188] LC-MS (m / z): 216.0 / 218.0 [M+H] + .

[0189] Step 2: Synthesis of 4-bromo-2-methoxy-6-methylphenylhydrazine (3-3)

[0190] In an ice-salt bath, 3-2 (5.00 g, 23.15 mmol) was dissolved in concentrated hydrochloric acid (25 mL). The internal temperature was maintained below 0°C, and a solution of NaNO2 (2.40 g, 34.72 mmol) in water (25 mL) was slowly added dropwise. The reaction was continued for 20 minutes. Then, a solution of SnCl2·2H2O (10.40 g, 46.30 mmol) in concentrated hydrochloric acid (25 mL) was slowly added dropwise, maintaining the internal temperature below 5°C, and the reaction was continued for 1 hour. The reaction solution was filtered, the filter cake was rinsed with a small amount of water, diluted with DCM (50 mL), and adjusted to alkaline with saturated aqueous Na2CO3. The layers were separated, and the aqueous phase was extracted with DCM (50 mL). The organic phases were combined, washed with saturated NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product of compound 3-3 (3.98 g) as a dark brown oily liquid.

[0191] Step 3: Synthesis of 4-(2-(4-bromo-2-methoxy-6-methylphenyl)hydrazino)-2-chloro-5-(dimethoxymethyl)pyrimidine (3-4)

[0192] The crude product of compound 3-3 (3.98 g) and compound 1-2 (3.84 g, 17.23 mmol) were dissolved in THF (40 mL), and TEA (7.16 mL, 51.67 mmol) was added. The mixture was allowed to react at room temperature for 16 hours. The reaction solution was directly concentrated and purified by column chromatography (EA:petroleum ether = 35:65) to obtain compound 3-4 (3.05 g) as a brown solid, with a two-step yield of 31.6%.

[0193] LC-MS (m / z): 419.0 [M+H] + .

[0194] Step 4: Synthesis of 2-(4-bromo-2-methoxy-6-methylphenyl)-6-chloro-2H-pyrazolo[3,4-d]pyrimidine (3-5)

[0195] Compound 3-4 (3.05 g, 7.30 mmol) was dissolved in ACN (30 mL), and TsOH·H2O (1.39 g, 7.30 mmol) was added. The reaction mixture was incubated at 70°C for 1 hour. After the reaction mixture cooled to room temperature, it was concentrated and purified by column chromatography (EA:petroleum ether = 1:1) to obtain compound 3-5 (2.82 g) as a brown solid in a 99.9% yield.

[0196] LC-MS (m / z): 355.0 [M+H] + .

[0197] Step 5: Synthesis of (R)-2-(4-bromo-2-methoxy-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (3-6)

[0198] Compound 3-5 (2.82 g, 7.30 mmol) and compound 1-8 (1.00 g, 8.76 mmol) were dissolved in n-butanol (20 mL), and DIPEA (3.82 mL, 21.90 mmol) was added. The mixture was reacted at 100°C for 1 hour. After the reaction mixture cooled to room temperature, it was concentrated under reduced pressure and purified by column chromatography (MeOH:DCM = 5:95) to obtain compound 3-6 (2.66 g) as an off-white solid with a yield of 84.4%.

[0199] LC-MS (m / z): 431.0 / 433.0 [M+H] + .

[0200] Step 6: Synthesis of (R)-5-bromo-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 3)

[0201] Compound 3-6 (0.20 g, 0.46 mmol) was dissolved in DCM (10 mL). A 2.0 M solution of BBr in DCM (1.2 mL) was added under an ice-water bath and allowed to react at room temperature for 1 hour. The reaction solution was quenched with MeOH, diluted with DCM (20 mL), washed with saturated aqueous NaHCO (20 mL) and saturated NaCl (20 mL), dried over anhydrous NaSO, filtered, concentrated under reduced pressure, and purified by column chromatography (MeOH:DCM = 15:85) and then by reverse phase chromatography (ACN:0.1% aqueous formic acid = 85%) to obtain the target compound 3 (25.8 mg) as a white solid in a 13.3% yield.

[0202] LC-MS (m / z): 417.0 / 419.0 [M+H] + .

[0203] 1 H NMR(400MHz,DMSO-d6)δ9.06(s,1H),8.40(s,1H),7.17-6.99(m,3H),4.04-3.92(m,1H),3.01-2.90(m,1H),2.69-2.6 3(m,1H),2.22(s,3H),2.05-1.88(m,5H),1.88-1.80(m,1H),1.75-1.67(m,1H),1.62-1.48(m,1H),1.40-1.27(m,1H).

[0204] Example 4: Synthesis of (R)-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-5-(trifluoromethyl)phenol (target compound 4)

[0205] Step 1: Synthesis of 2-bromo-6-methoxy-4-(trifluoromethyl)aniline (4-2)

[0206] Compound 4-1 (1.00 g, 5.23 mmol) was dissolved in ACN (20 mL), maintaining the internal temperature below 10°C. NBS (977.5 mg, 5.49 mmol) was slowly added in an ice-water bath and allowed to react at room temperature for 1 hour. The reaction solution was diluted with EA (50 mL), washed with aqueous NaHCO₃ (30 mL), water (20 mL), and saturated NaCl (20 mL), dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (EA:petroleum ether = 7:93) to afford compound 4-2 (1.15 g) as a brown solid in an 81.6% yield.

[0207] LC-MS (m / z): 270.0 / 272.0 [M+H] + .

[0208] Step 2: Synthesis of 2-methoxy-6-methyl-4-(trifluoromethyl)aniline (4-3)

[0209] Compound 4-2 (1.14 g, 4.25 mmol), MeB(OH)2 (0.51 g, 8.50 mmol), K2CO3 (1.47 g, 10.62 mmol), and Pd(dppf)Cl2 (115.5 mg, 0.21 mmol) were added to a vial in sequence. Dioxane (20 mL) and water (2 mL) were added, and the mixture was reacted at 100°C under nitrogen for 16 hours. The reaction solution was cooled to room temperature, diluted with water (50 mL), and extracted with EA (50 mL x 2). The organic phases were combined, washed with saturated NaCl (50 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (EA:petroleum ether = 1:9) to obtain compound 4-3 (568.0 mg) as a green oily liquid with a yield of 65.1%.

[0210] LC-MS (m / z): 206.0 [M+H] + .

[0211] Step 3: Synthesis of 2-methoxy-6-methyl-4-(trifluoromethyl)phenylhydrazine (4-4)

[0212] Compound 4-3 (568.0 mg, 2.77 mmol) was dissolved in concentrated hydrochloric acid (5 mL) under an ice-salt bath. A solution of NaNO2 (286.5 mg, 4.15 mmol) in water (5 mL) was slowly added dropwise, maintaining the internal temperature below 0°C. The reaction was continued for 40 minutes. A solution of SnCl2·2H2O (1.25 g, 5.34 mmol) in concentrated hydrochloric acid (3 mL) was slowly added dropwise, maintaining the internal temperature below 0°C, and the reaction was continued for 1 hour. The reaction solution was filtered, the filter cake was rinsed with a small amount of water, diluted with EA (20 mL), and adjusted to alkalinity with saturated aqueous Na2CO3. The layers were separated, and the aqueous phase was extracted with EA (20 mL). The organic phases were combined, washed with saturated NaCl (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 4-4 (135.0 mg) as an off-white solid with a yield of 22.1%.

[0213] Step 4: Synthesis of 2-chloro-5-(dimethoxymethyl)-4-(2-(2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)hydrazino)pyrimidine (4-5)

[0214] Compound 4-4 (135.0 mg, 0.61 mmol) and compound 1-2 (136.8 mg, 0.61 mmol) were dissolved in THF (50 mL), and TEA (0.25 mL, 1.84 mmol) was added. The mixture was allowed to react at room temperature for 16 hours. The reaction solution was directly concentrated and purified by column chromatography (EA:petroleum ether = 35:65) to obtain compound 4-5 (88.0 mg) as a yellow solid in a 35.3% yield.

[0215] LC-MS (m / z): 407.0 [M+H] + .

[0216] Step 5: Synthesis of 6-chloro-2-(2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-2H-pyrazolo[3,4-d]pyrimidine (4-6)

[0217] Compound 4-5 (88.0 mg, 0.22 mmol) was dissolved in ACN (5 mL), and TsOH·H2O (41.1 mg, 0.22 mmol) was added. The reaction mixture was incubated at 70°C for 1 hour. After the reaction mixture cooled to room temperature, it was concentrated and purified by column chromatography (EA:petroleum ether = 30:70) to obtain compound 4-6 (59.0 mg) as a yellow oil in a 79.7% yield.

[0218] LC-MS (m / z): 343.0 [M+H] + .

[0219] Step 6: Synthesis of (R)-2-(2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (4-7)

[0220] Compound 4-6 (59.0 mg, 0.17 mmol) and compound 1-8 (23.6 mg, 0.21 mmol) were dissolved in n-butanol (3 mL), and DIPEA (0.09 mL, 0.52 mmol) was added. The mixture was reacted at 100°C for 1 hour. After the reaction solution cooled to room temperature, it was concentrated under reduced pressure and purified by column chromatography (MeOH:DCM = 5:95) to obtain compound 4-7 (128.0 mg) as an off-white solid.

[0221] LC-MS (m / z): 421.0 [M+H] + .

[0222] Step 7: Synthesis of (R)-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-5-(trifluoromethyl)phenol (target compound 4)

[0223] Compound 4-7 (128.0 mg) was dissolved in DCM (5 mL). A 2.0 M solution of BBr in DCM (0.5 mL) was added under an ice-water bath and allowed to react at room temperature for 1 hour. The reaction solution was quenched with MeOH, concentrated under reduced pressure, and purified by reverse-phase chromatography (ACN:0.1% formic acid in water = 85%) to afford the title compound 4 (41.3 mg) as a white solid with a two-step yield of 47.0% and an ee% >99.9%.

[0224] LC-MS (m / z): 407.0 [M+H] + .

[0225] 1 H NMR(400MHz,DMSO-d6)δ9.10(s,1H),8.48(s,1H),7.23(s,1H),7.20-7.13(m,2H),4.08-3.94(m,1H),3.06-2.98(m,1H),2.8 0-2.70(m,1H),2.30(s,3H),2.21-1.99(m,5H),1.91-1.82(m,1H),1.79-1.69(m,1H),1.65-1.50(m,1H),1.45-1.29(m,1H).

[0226] Example 5: Synthesis of (R)-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-5-(trifluoromethyl)phenol (target compound 4) hydrochloride

[0227] Compound 4 (3.22 g) was dissolved in MeOH (40 mL), and HCl in EA solution (4.0 M, 10 mL) was added. After reacting at room temperature for 1 hour, the mixture was concentrated and dried to obtain the hydrochloride of compound 4 (3.47 g) as a yellow solid with a yield of 98.9%.

[0228] LC-MS (m / z): 407.0 [M+H] + .

[0229] 1H NMR(600MHz,DMSO-d6)δ11.09(brs,1H),10.55-10.35(m,1H),9.30-9.15(m, 1H),8.72-8.56(m,1H),8.10-7.70(m,1H),7.31(d,J=2.0Hz,1H),7.25(d,J= 2.0Hz,1H),4.32-4.24(m,1H),3.65-3.54(m,1H),3.41-3.33(m,1H),2.92-2 .74(m,5H),2.08(s,3H),2.07-2.01(m,1H),1.95-1.76(m,2H),1.50(m,1H).

[0230] Example 6: Synthesis of (R)-3,5-dimethyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 5)

[0231] Step 1: Synthesis of (R)-2-(2-methoxy-4,6-dimethylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (5-1)

[0232] Compound 3-6 (100.0 mg, 0.23 mmol) was added to a vial, followed by dioxane (2 mL) and water (0.4 mL). MeB(OH)2 (27.8 mg, 0.46 mmol), K2CO3 (96.2 mg, 0.70 mmol), and Pd(dppf)Cl2 (17.0 mg, 0.02 mmol) were then added sequentially. The reaction mixture was reacted at 100°C under a nitrogen atmosphere for 16 hours. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with EA (30 mL x 3). The combined organic phases were washed with saturated NaCl (20 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH = 10:1) to afford compound 5-1 (77.0 mg) as a yellow solid in a 90.6% yield.

[0233] LC-MS (m / z): 367.0 [M+H] + .

[0234] Step 2: Synthesis of (R)-3,5-dimethyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 5)

[0235] Compound 5-1 (77.0 mg, 0.21 mmol) was added to a vial, followed by ultra-dry DCM (3 mL). BBr (1.1 mL, 2 M in DCM) was added under a nitrogen atmosphere at 0°C and allowed to react for 1 hour. After completion, the reaction was quenched with an appropriate amount of methanol in an ice-water bath. The filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography (ACN:0.1% formic acid in water = 85%) to afford the title compound 5 (10.2 mg) as a white solid in a 13.8% yield.

[0236] LC-MS (m / z): 353.0 [M+H] + .

[0237] 1 H NMR(400MHz,DMSO-d6)δ10.20-9.65(m,1H),9.05(s,1H),8.34(s,1H),7.11- 6.93(m,1H),6.69(s,1H),6.65(s,1H),4.09-3.92(m,1H),3.04-2.87(m,1H), 2.79-2.62(m,1H),2.27(s,3H),2.24(s,3H),2.06-1.96(m,2H),1.93(s,3H) ,1.88-1.80(m,1H),1.76-1.67(m,1H),1.62-1.48(m,1H),1.41-1.27(m,1H).

[0238] Example 7: Synthesis of (R)-5-cyclopropyl-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 6)

[0239] Step 1: Synthesis of (R)-2-(4-cyclopropyl-2-methoxy-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (6-1)

[0240] Compound 3-6 (90.0 mg, 0.21 mmol), cyclopropylboronic acid (53.7 mg, 0.63 mmol), and Pd(PPh3)4 (12.1 mg, 0.01 mmol) were weighed separately and dioxane (2.5 mL) was added. Anhydrous K2CO3 solid (115.3 mg, 0.83 mmol) was dissolved in water (1 mL) to prepare a solution. This solution was added to the reaction system, purged with nitrogen, and heated to 90°C for 16 hours. Heating was stopped and the reaction mixture was allowed to cool. Water (50 mL) was added to the reaction solution, followed by extraction with EA (40 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 8:1) to obtain compound 6-1 (70.0 mg) as a brown oil in an 85.4% yield.

[0241] LC-MS (m / z): 393.0 [M+H] + .

[0242] Step 2: Synthesis of (R)-5-cyclopropyl-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 6)

[0243] Compound 6-1 (70.0 mg, 0.178 mmol) was first dissolved in DCM (3 mL), and then a 2M solution of BBr in DCM (0.5 mL) was added dropwise. The reaction was then continued at room temperature for 1 hour. Methanol (2 mL) was added dropwise to quench the reaction, and the solvent was removed by rotary evaporation. Saturated aqueous NaHCO₃ (30 mL) was added, and then EA (70 mL) was added for extraction. The organic phase was separated, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude brown solid was purified by preparative HPLC and lyophilized to obtain the target compound 6 (10.2 mg) as a white solid in a 15.1% yield.

[0244] LC-MS (m / z): 379.0 [M+H] + .

[0245] 1H NMR(400MHz,DMSO-d6)δ9.88(s,1H),9.04(s,1H),8.33(s,1H),7.02(s,1H) ,6.58(s,1H),6.54(s,1H),4.09-3.89(m,1H),2.96-2.89(m,1H),2.73-2.6 2(m,1H),2.23(s,3H),1.93(s,3H),1.92-1.81(m,4H),1.78-1.66(m,1H),1 .63-1.47(m,1H),1.43-1.28(m,1H),1.06-0.89(m,2H),0.77-0.58(m,2H).

[0246] Example 8: Synthesis of (R)-5-ethynyl-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 7)

[0247] Compound 3 (60.0 mg, 0.15 mmol), ethynyltri-n-butyltin (181.2 mg, 0.57 mmol), CsF (43.7 mg, 0.29 mmol), CuI (5.5 mg, 0.03 mmol), and Pd(dppf)Cl2 (10.5 mg, 0.01 mmol) were weighed separately and added to anhydrous DMF (2.5 mL). The mixture was purged with nitrogen, sealed, and heated to 120°C for 16 hours. Heating was stopped and the mixture was allowed to cool. KF (300.0 mg) was weighed and dissolved in water (4 mL) to prepare a solution. This solution was added to the reaction system, followed by EA (15 mL) and stirred at room temperature for 2 hours. Filter and remove insoluble matter, collect the filtrate, separate the organic phase, extract the aqueous phase once with EA (30 mL), combine the organic phases, add anhydrous Na2SO4 to dry, filter, and concentrate under reduced pressure. The resulting crude solid product is purified by preparative HPLC and lyophilized to obtain the target compound 7 (5.5 mg) as a brown solid with a yield of 10.5%.

[0248] LC-MS (m / z): 363.0 [M+H] + .

[0249] 1H NMR(400MHz,DMSO-d6)δ10.36(s,1H),9.06(s,1H),8.41(s,1H),7.15-7.01(m,1H),6.97(s,2H),4.27(s,1H),4.02-3.90(m,1H),2.96- 2.84(m,1H),2.65-2.58(m,1H),2.18(s,3H),1.98(s,3H),1.95-1.76(m,3H),1.73-1.64(m,1H),1.61-1.46(m,1H),1.39-1.26(m,1H).

[0250] Example 9: Synthesis of (R)-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-5-(prop-1-yn-1-yl)phenol (target compound 8)

[0251] Compound 3 (60.0 mg, 0.15 mmol) was dissolved in ultra-dry DMF (4 mL), followed by the addition of tributylstannane (189.3 mg, 0.57 mmol), CsF (43.8 mg, 0.29 mmol), CuI (5.5 mg, 0.03 mmol), and Pd(dppf)Cl2 (10.7 mg, 0.01 mmol). The reaction was allowed to react at 120°C under a nitrogen atmosphere for 16 hours. After completion of the reaction, aqueous KF solution was added and stirred at room temperature for 2 hours. The reaction mixture was diluted with water (10 mL) and extracted with EA (30 mL x 3). The combined organic phases were washed with saturated NaCl (20 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The resulting mixture was purified by reverse phase chromatography (ACN:0.1% formic acid in water = 85%) to afford the target compound 8 (19.9 mg) as a white solid in a 36.9% yield.

[0252] LC-MS (m / z): 377.0 [M+H] + .

[0253] 1H NMR(400MHz,DMSO-d6)δ10.60-9.99(m,1H),9.07(s,1H),8.41(s,1H),7.26 -7.07(m,1H),6.90-6.85(m,2H),4.10-3.98(m,1H),3.13-2.96(m,1H),2.8 6-2.75(m,1H),2.33(s,3H),2.24-2.09(m,2H),2.06(s,3H),1.95(s,3H),1 .90-1.81(m,1H),1.80-1.70(m,1H),1.69-1.49(m,1H),1.46-1.31(m,1H).

[0254] Example 10: Synthesis of (R)-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 9)

[0255] Step 1: Synthesis of (2-methoxy-6-methylphenyl)hydrazine (9-1)

[0256] Compound 3-1 (1.37 g, 9.98 mmol) was weighed and dissolved in concentrated hydrochloric acid (6 mL) in an ice-water bath. NaNO₂ (1.03 g, 14.97 mmol) was dissolved in water (6 mL) to prepare a solution. This solution was added dropwise to the reaction system, followed by a continuous ice-water bath for 25 minutes. SnCl₂·2H₂O (4.50 g, 19.96 mmol) was dissolved in concentrated hydrochloric acid (5 mL) and added dropwise to the reaction system, followed by a continuous ice-water bath for 2 hours. Water (50 mL) was added to the reaction solution, followed by a 4 M aqueous sodium hydroxide solution (35 mL) dropwise to adjust the pH to 9. DCM (80 mL) was added for extraction, and the organic phase was separated and washed once with water (70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to yield crude compound 9-1 (1.16 g) as a red oil.

[0257] LC-MS (m / z): 153.0 [M+H] + .

[0258] Step 2: Synthesis of 2-chloro-5-(dimethoxymethyl)-4-(2-(2-methoxy-6-methylphenyl)hydrazino)pyrimidine (9-2)

[0259] The crude product of compound 9-1 (300.0 mg, 1.97 mmol), compound 1-2 (439.6 mg, 1.97 mmol), and TEA (598.3 mg, 5.91 mmol) were weighed and dissolved in anhydrous THF (6 mL). The mixture was allowed to react overnight at room temperature. Water (70 mL) was added to the reaction solution, followed by extraction with EA (80 mL). The organic phase was separated and washed once with water (50 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 5:1) to obtain compound 9-2 (420.0 mg) as a red oil in a 62.9% yield.

[0260] LC-MS (m / z): 339.0 [M+H] + .

[0261] Step 3: Synthesis of 6-chloro-2-(2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (9-3)

[0262] Compound 9-2 (420.0 mg, 1.24 mmol) and TsOH·H2O (259.4 mg, 1.36 mmol) were weighed separately, ACN (6 mL) was added, and the mixture was heated to 70°C for 2 hours. Water (50 mL) was added to the reaction solution, followed by extraction with EA (50 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain compound 9-3 (175.0 mg) as a white solid in a 51.4% yield.

[0263] LC-MS (m / z): 275.0 [M+H] + .

[0264] 1 H-NMR (400MHz, DMSO-d6) δ9.55(s,1H),9.05(s,1H),7.51(t,J=8.1Hz,1H),7.17(d,J=8.4Hz,1H),7.07(d,J=7.7Hz,1H),3.73(s,3H),1.97(s,3H).

[0265] Step 4: Synthesis of (R)-2-(2-methoxy-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (9-4)

[0266] Compound 9-3 (171.0 mg, 0.62 mmol), compound 1-8 (23.6 mg, 0.21 mmol) (85.3 mg, 0.75 mmol), and DIPEA (321.8 mg, 2.49 mmol) were weighed separately, n-butanol (3.5 mL) was added, the atmosphere was replaced with nitrogen, and the mixture was heated to 100°C for 2 hours. Heating was stopped, the mixture was allowed to cool, and the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:CH3OH=8:1) to obtain compound 9-4 (270.0 mg) as a white solid.

[0267] LC-MS (m / z): 353.0 [M+H] + .

[0268] Step 5: Synthesis of (R)-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 9)

[0269] The crude product of compound 9-4 (270.0 mg) was dissolved in DCM (6 mL), and a 2M solution of BBr in DCM (1.5 mL) was added dropwise at room temperature. The reaction was then continued at room temperature for 2 hours. An ice-water bath was used to control the temperature. Methanol (5 mL) was added dropwise to the reaction solution to quench the reaction. The solution was concentrated under reduced pressure, and saturated aqueous NaHCO (30 mL) was added. The solution was then extracted with DCM / CH OH (8:1, 50 mL). The organic phase was separated, dried over anhydrous Na 2 SO 4 , filtered, concentrated under reduced pressure, and purified by column chromatography (DCM:CH 3 OH = 8:1) to obtain the target compound 9 (116.0 mg) as a brown solid in a two-step yield of 55.3%.

[0270] LC-MS (m / z): 339.0 [M+H] + .

[0271] 1 H-NMR (400MHz, DMSO-d6) δ10.00(s,1H),9.06(s,1H),8.39(s,1H),7.23(t,J= 8.0Hz,1H),7.06(s,1H),6.90(d,J=8.0,1H),6.82(d,J=8.0Hz,1H),4.07-3.9 3(m,1H),3.04-2.93(m,1H),2.78-2.63(m,1H),2.26(s,3H),2.09-1.99(m,5H ),1.90-1.79(m,1H),1.77-1.67(m,1H),1.64-1.47(m,1H),1.43-1.28(m,1H).

[0272] Example 11: Synthesis of (R)-5-fluoro-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 10)

[0273] Step 1: Synthesis of 2-bromo-4-fluoro-6-methoxyaniline (10-2)

[0274] Compound 10-1 (1.20 g, 8.50 mmol) was dissolved in ACN (10 mL) and replaced with nitrogen. The temperature was controlled in an ice-water bath. NBS (1.58 g, 8.93 mmol) was added portionwise and the reaction continued in an ice-water bath for 1 hour. Saturated aqueous NaHCO₃ (20 mL) was added to the reaction solution, followed by water (50 mL) and thorough stirring. EA (60 mL) was added for extraction. The organic phase was separated, dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 5:1) to afford compound 10-2 (930.0 mg) as a yellow oil in a 49.7% yield.

[0275] LC-MS (m / z): 220.0 / 222.0 [M+H] + .

[0276] Step 2: Synthesis of 4-fluoro-2-methoxy-6-methylaniline (10-3)

[0277] Compound 10-2 (930.0 mg, 4.23 mmol), MeB(OH)2 (505.9 mg, 8.45 mmol), and Pd(dppf)Cl2 (175.0 mg, 0.24 mmol) were weighed separately and added to anhydrous dioxane (8 mL). Anhydrous K2CO3 solid (1.75 g, 12.68 mmol) was dissolved in water (5 mL) to prepare a solution. This solution was added to the reaction system, purged with nitrogen, and heated to 100°C for 16 hours. Heating was stopped and the mixture was allowed to cool. Water (50 mL) was added to the reaction solution, followed by extraction with EA (50 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 5:1) to obtain compound 10-3 (463.0 mg) as a pale yellow solid in a 70.6% yield.

[0278] LC-MS (m / z): 156.0 [M+H] + .

[0279] 1H-NMR (400MHz, DMSO-d6) δ6.61(dd,J=12.0Hz,2.8Hz,1H),6.45(dd,J=12.0Hz,2.8Hz,1H),4.24(s,2H),3.76(s,3H),2.07(s,3H).

[0280] Step 3: Synthesis of (4-fluoro-2-methoxy-6-methylphenyl)hydrazine (10-4)

[0281] First, compound 10-3 (460.0 mg, 2.96 mmol) was weighed and dissolved in concentrated hydrochloric acid (3 mL) in an ice-water bath. NaNO₂ (306.8 mg, 4.45 mmol) was then dissolved in water (2 mL) to prepare a solution. This solution was added dropwise to the reaction system, followed by a continuous ice-water bath for 10 minutes. SnCl₂·2H₂O (1.34 g, 5.93 mmol) was then dissolved in concentrated hydrochloric acid (5 mL) and added dropwise to the reaction system, followed by a continuous ice-water bath for 2 hours. Water (20 mL) was added to the reaction solution, followed by a 0.5 M aqueous sodium hydroxide solution (10 mL) to adjust the pH to 9. DCM (50 mL) was added for extraction, and the organic phase was separated and washed once with water (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain crude compound 10-4 (300 mg) as a red oil.

[0282] LC-MS (m / z): 171.0 [M+H] + .

[0283] Step 4: Synthesis of 2-chloro-5-(dimethoxymethyl)-4-(2-(4-fluoro-2-methoxy-6-methylphenyl)hydrazino)pyrimidine (10-5)

[0284] Compound 10-4 (300 mg), compound 1-2 (393.2 mg, 1.76 mmol), and TEA (535.1 mg, 5.29 mmol) were weighed and dissolved in anhydrous tetrahydrofuran (6 mL). The mixture was reacted at room temperature for 16 hours. Water (50 mL) was added to the reaction solution, followed by extraction with EA (50 mL). The organic phase was separated and washed once with water (30 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 5:1) to obtain compound 10-5 (360.0 mg) as a red solid in a 57.2% yield.

[0285] LC-MS (m / z): 357.0 [M+H] + .

[0286] Step 5: Synthesis of 6-chloro-2-(4-fluoro-2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (10-6)

[0287] Compound 10-5 (360.0 mg, 1.01 mmol) and TsOH·H2O (211.1 mg, 1.11 mmol) were weighed separately, ACN (6 mL) was added, and the reaction was heated to 70°C for 2 hours. Heating was stopped and the mixture was allowed to cool. Water (70 mL) was added to the reaction solution, followed by extraction with EA (60 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain compound 10-6 (134.0 mg) as a white solid in a 45.3% yield.

[0288] LC-MS (m / z): 293.0 [M+H] + .

[0289] 1 H-NMR (400MHz, DMSO-d6) δ9.55(s,1H),9.04(s,1H),7.13(dd,J=12.0,2.4Hz,1H),6.97(dd,J=8.0,2.8Hz,1H),3.75(s,3H),1.97(s,3H).

[0290] Step 6: Synthesis of (R)-2-(4-fluoro-2-methoxy-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (10-7)

[0291] Compound 10-6 (134.0 mg, 0.46 mmol), compound 1-8 (62.7 mg, 0.55 mmol), and DIPEA (236.7 mg, 1.83 mmol) were weighed separately, and n-butanol (3.5 mL) was added. The mixture was purged with nitrogen and heated to 100°C for 2 hours. Heating was stopped and the mixture was allowed to cool. The solvent was removed directly using a rotary evaporator. The crude product was purified by silica gel column chromatography (DCM:MeOH = 8:1) to obtain crude compound 10-7 (220.0 mg).

[0292] LC-MS (m / z): 371.0 [M+H] + .

[0293] Step 7: Synthesis of (R)-5-fluoro-3-methyl-2-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 10)

[0294] The crude compound 10-7 (220.0 mg) was dissolved in DCM (3 mL), and a 2M solution of BBr in DCM (2 mL) was added dropwise at room temperature. The reaction was then continued at room temperature for 1 hour. An ice-water bath was used to control the temperature, and methanol (4 mL) was added dropwise to the reaction solution to quench the reaction. The solvent was then removed by rotary evaporation. Saturated aqueous NaHCO₃ (30 mL) was added, and the mixture was extracted with DCM / MeOH (8:1, 50 mL). The organic phase was separated, dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 8:1) to obtain the target compound 10 (93.9 mg) as an off-white solid with a two-step yield of 44.3%.

[0295] LC-MS (m / z): 357.0 [M+H] + .

[0296] 1 H-NMR(400MHz,DMSO-d6)δ10.54(s,1H),9.06(s,1H),8.39(s,1H),7.06(s,1H),6.81-6.54(m,2H),4.04-3.90(m,1H),2.98-2.89 (m,1H),2.71-2.61(m,1H),2.21(s,3H),1.97(s,3H),2.05-1.77(m,3H),1.76-1.65(m,1H),1.60-1.48(m,1H),1.40-1.27(m,1H).

[0297] Example 12: Synthesis of (R)-2-(4-chloro-2,6-dimethylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (target compound 67)

[0298] Referring to steps 2-5 of Example 1, compound 1-4 in step 2 was replaced by compound 67-1. The other intermediate materials and preparation methods were the same as in Example 1. Compound 67-1 (2.00 g) was used to prepare the target compound 67 (395.1 mg) as a white solid. The total yield of the four-step reaction was 8.5%.

[0299] LC-MS (m / z): 371.0 [M+H] + .

[0300] 1H NMR(400MHz,DMSO-d6)δ9.12(s,1H),8.53(s,1H),7.40(s,2H),7.31-7.20(m,1H),4.23-3.86(m,1H),3.14-2.98(m,1H),2.86-2.7 5(m,1H),2.35(s,3H),2.23-2.07(m,2H),1.96(s,6H),1.91-1.82(m,1H),1.80-1.71(m,1H),1.66-1.51(m,1H),1.46-1.31(m,1H).

[0301] Example 13: Synthesis of (R)-5-chloro-3-methyl-2-(6-(1-methylpiperidin-3-yl)oxy)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 48)

[0302] Step 1: Synthesis of (R)-1-methylpiperidin-3-ol (48-2)

[0303] 48-1 (1.01 g, 5.00 mmol) was dissolved in anhydrous THF (10 mL). LiAlH4 (380.0 mg, 10.00 mmol) was added under an ice-water bath and allowed to react at 40°C for 3 hours. The reaction was quenched with saturated aqueous Na2CO3 until no gas was generated, filtered, and the filter cake was washed with MeOH. The filtrate was concentrated to afford 48-2 (357.0 mg) in a 62.0% yield.

[0304] LC-MS (m / z): 116.0 [M+H] + .

[0305] Step 2: Synthesis of (R)-2-(4-chloro-2-methoxy-6-methylphenyl)-6-((1-methylpiperidin-3-yl)oxy)-2H-pyrazolo[3,4-d]pyrimidine (48-3)

[0306] 48-2 (73.7 mg, 0.64 mmol) was dissolved in anhydrous THF (3 mL). NaH (38.4 mg, 60% w / w, 0.96 mmol) was added under an ice-water bath. After incubation for 0.5 h, 1-7 (100.0 mg, 0.32 mmol) was added and immediately incubated at 50°C for 0.5 h. The reaction mixture was cooled to room temperature and quenched with saturated aqueous ammonium chloride, diluted with water (20 mL), and extracted with EA (20 mL x 2). The combined organic phases were washed with saturated brine (20 mL), filtered, concentrated, and purified by column chromatography (MeOH:DCM:TEA = 15:84:1) to afford 48-3 (30.0 mg) in a 24.2% yield.

[0307] LC-MS (m / z): 388.0 [M+H] + .

[0308] Step 3: Synthesis of (R)-5-chloro-3-methyl-2-(6-(1-methylpiperidin-3-yl)oxy)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 48)

[0309] 48-3 (30.0 mg, 0.08 mmol) was dissolved in DCM (2 mL), and a 2.0 M solution of BBr in DCM (0.5 mL) was added. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was quenched with MeOH in an ice-water bath, concentrated, and purified by two thin-layer chromatography cycles (MeOH:DCM = 1:10) to afford the target compound 48 (4.6 mg) in a 15.9% yield.

[0310] LC-MS (m / z): 374.0 [M+H] + .

[0311] 1 H NMR (400MHz, DMSO-d6) δ10.70(s,1H),9.36(s,1H),8.73(s,1H),6.97(d,J=2.2Hz,1H),6.94(d,J=2.2Hz,1H),5.10(tt,J=8.4,4.0Hz ,1H),3.00-2.90(m,1H),2.60-2.52(m,1H),2.23-2.10(m,4H),2.11-1.98(m,2H),1.96(s,3H),1.83-1.71(m,1H),1.65-1.37(m,2H).

[0312] Example 14: Synthesis of (R)-2-(4-chloro-2-(difluoromethoxy)-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (target compound 60)

[0313] Step 1: Synthesis of 4-chloro-2-(difluoromethoxy)-1-nitrobenzene (60-2)

[0314] 60-1 (2.00 g, 11.53 mmol), anhydrous Na2CO3 (1.83 g, 17.29 mmol), and sodium difluorochloroacetate (3.52 g, 23.05 mmol) were dissolved in anhydrous DMF (20 mL) and reacted at 100°C under nitrogen for 5 hours. The reaction solution was cooled to room temperature and adjusted to pH <5 with dilute hydrochloric acid. The solution was diluted with water (30 mL) and extracted with EA (30 mL × 2). The combined organic phases were washed with water (30 mL × 3) and saturated brine (30 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (PE:EA = 70:30) to afford 60-2 (2.18 g) in a yield of 84.6%.

[0315] Step 2: Synthesis of 4-chloro-2-(difluoromethoxy)aniline (60-3)

[0316] 60-2 (2.18 g, 9.75 mmol) was dissolved in ethanol (20 mL), and an aqueous solution of NH4Cl (4.70 g, 87.78 mmol) (10 mL) and iron powder (4.92 g, 87.78 mmol) were added. The mixture was allowed to react at 80°C for 1 hour. After the reaction mixture was cooled to room temperature, it was filtered and the filtrate was diluted with EA (50 mL), washed with water (20 mL × 2) and saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (PE:EA = 65:35) to give crude 60-3 (1.94 g).

[0317] LC-MS (m / z): 194.0 [M+H] + .

[0318] Step 3: Synthesis of 2-bromo-4-chloro-6-(difluoromethoxy)aniline (60-4)

[0319] The crude product 60-3 (1.94 g) was dissolved in acetonitrile (20 mL) and NBS (1.91 g, 10.72 mmol) was slowly added in an ice-water bath. After the addition was complete, the reaction was continued for 0.5 h. The reaction solution was diluted with EA (30 mL) and washed sequentially with saturated aqueous NaHCO₃ (20 mL), water (20 mL), and saturated brine (20 mL). It was then dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by column chromatography (PE:EA = 95:5) to afford 60-4 (2.10 g), with a two-step yield of 79.1%.

[0320] LC-MS (m / z): 274.0 [M+H] + .

[0321] Step 4: Synthesis of 4-chloro-2-(difluoromethoxy)-6-methylaniline (60-5)

[0322] Compound 60-4 (2.10 g, 7.71 mmol), methylboronic acid (924.8 mg, 15.41 mmol), Pd(dppf)Cl2 (281.9 mg, 0.39 mmol), and anhydrous K2CO3 (2.66 g, 19.27 mmol) were added to a mixture of dioxane (30 mL) and water (3 mL). The mixture was reacted at 100°C under nitrogen for 16 hours. After cooling to room temperature, the reaction solution was filtered, concentrated, and purified by column chromatography (PE:EA = 90:10) to afford compound 60-5 (1.23 g) in a 76.8% yield.

[0323] LC-MS (m / z): 208.0 [M+H] + .

[0324] Step 5: Synthesis of (4-chloro-2-(difluoromethoxy)-6-methylphenyl)hydrazine hydrochloride (60-6)

[0325] Compound 60-5 (1.23 g, 5.95 mmol) was dissolved in concentrated hydrochloric acid (6 mL). A solution of NaNO2 (616.2 mg, 8.93 mmol) in water (6 mL) was added dropwise at 0°C and the mixture was kept warm for 1 hour. A mixture of SnCl2·2H2O (2.69 g, 11.91 mmol) and concentrated hydrochloric acid (6 mL) was then added dropwise at 5°C. After addition, the mixture was gradually warmed to room temperature and allowed to react for 1 hour. The mixture was filtered, and the filter cake was washed with a small amount of water and EA, then dried to afford crude 60-6 (704.0 mg), which was used directly in the next step.

[0326] Step 6: Synthesis of 2-chloro-4-(2-(4-chloro-2-(difluoromethoxy)-6-methylphenyl)hydrazino)-5-(dimethoxymethyl)pyrimidine (60-7)

[0327] The crude product 60-6 (704.0 mg) and 2,4-dichloro-5-(dimethoxymethyl)pyrimidine (606.2 mg, 2.72 mmol) were added to THF (10 mL), followed by TEA (1.88 mL, 13.59 mmol). The mixture was allowed to react at room temperature for 16 hours. The reaction mixture was filtered, the filter cake rinsed with EA, and the filtrate concentrated. The product was purified by column chromatography (PE:EA = 65:35) to afford 60-7 (497.0 mg) in a two-step yield of 24.6%.

[0328] LC-MS (m / z): 409.0 [M+H] + .

[0329] Step 7: Synthesis of 6-chloro-2-(4-chloro-2-(difluoromethoxy)-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (60-8)

[0330] 60-7 (497.0 mg, 1.21 mmol) and TsOH·H2O (231.0 mg, 1.21 mmol) were dissolved in ACN (5 mL) and reacted at 70°C for 0.5 h. The reaction solution was cooled to room temperature, concentrated, and purified by column chromatography (PE:EA:TEA = 65:34:1) to afford 60-8 (302.0 mg) in a 72.3% yield.

[0331] LC-MS (m / z): 345.0 [M+H] + .

[0332] Step 8: Synthesis of (R)-2-(4-chloro-2-(difluoromethoxy)-6-methylphenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (target compound 60)

[0333] Compound 60-8 (118.0 mg, 0.34 mmol), 1-8 (46.9 mg, 0.41 mmol), and DIPEA (0.12 mL, 0.68 mmol) were added to isopropanol (2 mL) and reacted at 80°C for 0.5 h. The reaction mixture was cooled to room temperature, concentrated, and purified by column chromatography (MeOH:DCM = 3:97), followed by reverse phase preparative purification (ACN:0.1% aqueous formic acid = 85%) to afford the target compound 60 (98.4 mg) in a 68.0% yield.

[0334] LC-MS (m / z): 423.0 [M+H] + .

[0335] 1 H NMR(400MHz,DMSO-d6)δ9.14(s,1H),8.53(s,1H),7.54-7.48(m,1H),7.48-7.44(m,1H),7.31-7.03(m,2H),4.08-3.96(m,1H),3.09-2.9 7(m,1H),2.81-2.66(m,1H),2.30(s,3H),2.16-1.97(m,5H),1.91-1.80(m,1H),1.80-1.70(m,1H),1.65-1.50(m,1H),1.45-1.26(m,1H).

[0336] Example 15: Synthesis of (R)-5-chloro-3-methyl-2-(6-((1-methylpyrrolidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 61)

[0337] Step 1: Synthesis of (R)-2-(4-chloro-2-methoxy-6-methylphenyl)-N-(1-methylpyrrolidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (61-1)

[0338] 1-7 (221.0 mg, 0.72 mmol) was dissolved in n-butanol (2 mL), and (R)-1-methylpyrrolidin-3-amine (65.0 mg, 0.65 mmol) and DIPEA (420.0 mg, 3.75 mmol) were added sequentially. The mixture was microwaved at 100°C for 1 hour. After cooling to room temperature, the reaction solution was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 61-1 (181.0 mg) in a 75.1% yield.

[0339] LC-MS (m / z): 373.0 [M+H] + .

[0340] Step 2: Synthesis of (R)-5-chloro-3-methyl-2-(6-((1-methylpyrrolidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 61)

[0341] To the vial was added 61-1 (181.0 mg, 0.49 mmol) and ultra-dry DCM (5 mL). In an ice-water bath under nitrogen, a solution of BBr in DCM (2.5 mL, 2.0 M) was added and the mixture was incubated for 1 hour. The reaction was quenched with an appropriate amount of MeOH and concentrated under reduced pressure. The residue was purified by reverse phase preparative purification (ACN: 10 mmol / L aqueous NH4HCO3 solution = 95%) to afford the target compound 61 (75.9 mg) in a 43.6% yield.

[0342] LC-MS (m / z): 359.0 [M+H] + .

[0343] 1 H NMR(400MHz,DMSO-d6)δ9.08(s,1H),8.41(s,1H),7.54-7.36(m,1H),6.98-6.88(m,2H),4.49-4.35(m,1H),3.07-2 .94(m,1H),2.84-2.71(m,1H),2.71-2.59(m,2H),2.40(s,3H),2.30-2.15(m,1H),1.97(s,3H),1.92-1.75(m,1H).

[0344] Example 16: Synthesis of 5-chloro-3-methyl-2-(6-((1-methylazetidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 62)

[0345] Step 1: Synthesis of tert-butyl (1-methylazetidin-3-yl)carbamate (62-2)

[0346] Compound 62-1 (300.0 mg, 1.74 mmol) was dissolved in MeOH (6 mL), and paraformaldehyde (261.9 mg, 8.72 mmol) and a drop of AcOH were added sequentially. Sodium cyanoborohydride (328.4 mg, 5.23 mmol) was added under ice-water bath, and the mixture was allowed to react at 35°C for 3 hours. The reaction solution was diluted with water (10 mL) and extracted with EA (20 mL × 3). The organic phases were combined, washed with saturated NaHCO₃ (20 mL) and saturated brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain crude product 62-2 (285.0 mg), which was used directly in the next step.

[0347] LC-MS (m / z): 187.0 [M+H] + .

[0348] Step 2: Synthesis of 1-methylazetidine-3-amine hydrochloride (62-3)

[0349] The crude product 62-2 (285.0 mg) was dissolved in EA (5 mL), and a 4.0 M HCl solution in EA (5 mL) was added. The mixture was reacted at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to afford 62-3 (131.0 mg), with a two-step yield of 61.3%.

[0350] Step 3: Synthesis of 2-(4-chloro-2-methoxy-6-methylphenyl)-N-(1-methylazetidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (62-4)

[0351] Compound 62-3 (131.0 mg, 1.07 mmol) was dissolved in isopropanol (6 mL), and compound 1-7 (150.0 mg, 0.49 mmol) and DIPEA (984.0 mg, 7.61 mmol) were added sequentially. The mixture was allowed to react at 80°C for 1 hour. After the reaction mixture cooled to room temperature, the reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (DCM:MeOH = 10:1) to afford compound 62-4 (259.0 mg) in a 67.5% yield.

[0352] LC-MS (m / z): 359.0 [M+H] + .

[0353] Step 4: Synthesis of 5-chloro-3-methyl-2-(6-((1-methylazetidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenol (target compound 62)

[0354] To a vial was added 62-4 (259.0 mg, 0.72 mmol) and anhydrous DCM (5 mL). A solution of BBr in DCM (3.7 mL, 2.0 M) was added under nitrogen in an ice-water bath and allowed to react for 1 hour. The reaction was quenched with an appropriate amount of MeOH under ice-water bath and concentrated under reduced pressure. The residue was purified by reverse phase preparative purification (ACN:0.1% aqueous formic acid = 85%) to afford the target compound 62 (4.8 mg) in a 1.9% yield.

[0355] LC-MS (m / z): 345.0 [M+H] + .

[0356] 1 H NMR(400MHz,DMSO-d6)δ9.16(s,1H),8.50(s,1H),7.86-7.76(m,1H),7.04-6.95(m,2H),4 .63-4.49(m,1H),3.84(t,J=8.0Hz,2H),3.24(t,J=7.2Hz,2H),2.45(s,3H),2.02(s,3H).

[0357] Example 17: Synthesis of (S)-5-chloro-2-(6-(((1-ethylpyrrolidin-2-yl)methyl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-3-methylphenol (target compound 63)

[0358] Step 1: Synthesis of (S)-2-(4-chloro-2-methoxy-6-methylphenyl)-N-((1-ethylpyrrolidin-2-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (63-1)

[0359] 1-7 (100.0 mg, 0.32 mmol), (S)-(1-ethylpyrrolidin-2-yl)methanamine (50.0 mg, 0.39 mmol), and DIPEA (167.2 mg, 1.30 mmol) were dissolved in isopropanol (3 mL) and reacted at 80°C under nitrogen for 3 hours. After cooling, the reaction solution was concentrated and purified by column chromatography (DCM:MeOH = 85:15) to obtain compound 63-1 (99.0 mg) in a 76.3% yield.

[0360] LC-MS (m / z): 401.0 [M+H] + .

[0361] Step 2: Synthesis of (S)-5-chloro-2-(6-(((1-ethylpyrrolidin-2-yl)methyl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-3-methylphenol (target compound 63)

[0362] Compound 63-1 (99.0 mg, 0.25 mmol) was dissolved in DCM (2 mL). A 2M solution of BBr in DCM (0.5 mL) was added under an ice-water bath and allowed to react at room temperature for 1.5 hours. The reaction mixture was quenched by dropwise addition of methanol (2 mL), concentrated, and purified by column chromatography (DCM:MeOH = 85:15) to afford the target compound 63 (44.8 mg) in a 45.0% yield.

[0363] LC-MS (m / z): 387.0 [M+H] + .

[0364] 1 H NMR(400MHz,DMSO-d6)δ9.08(s,1H),8.43(s,1H),7.26(s,1H),6.99-6.90(m,2H),3.63-3.53(m,1H),3.36-3.17(m,2H) ,3.16-2.93(m,2H),2.59-2.53(m,1H),2.47-2.39(m,1H),2.05-1.86(m,4H),1.82-1.63(m,3H),1.14(t,J=7.2Hz,3H).

[0365] Example 18: Synthesis of 5-chloro-2-(6-(((1R,2R)-2-hydroxycyclohexyl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-3-methylphenol (target compound 64)

[0366] Step 1: Synthesis of (1R,2R)-2-((2-(4-chloro-2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexan-1-ol (64-1)

[0367] 1-7 (100.0 mg, 0.32 mmol), (1R,2R)-2-aminocyclohexan-1-ol (44.7 mg, 0.39 mmol), and DIPEA (167.2 mg, 1.30 mmol) were dissolved in isopropanol (3 mL) and reacted at 80°C under nitrogen for 3 hours. Heating was stopped, the mixture was allowed to cool, and the reaction solution was concentrated and purified by column chromatography (DCM:MeOH = 85:15) to afford compound 64-1 (70.0 mg) in a 55.8% yield.

[0368] LC-MS (m / z): 388.0 [M+H] + .

[0369] Step 2: Synthesis of 5-chloro-2-(6-(((1R,2R)-2-hydroxycyclohexyl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-3-methylphenol (target compound 64)

[0370] Compound 64-1 (70.0 mg, 0.18 mmol) was dissolved in DCM (3 mL). A 2M solution of BBr in DCM (1.5 mL) was added under an ice-water bath and allowed to react at room temperature for 1 hour. The reaction mixture was quenched by dropwise addition of methanol (2 mL), concentrated, and purified by column chromatography (DCM:MeOH = 85:15), followed by reverse-phase chromatography (ACN:0.1% aqueous formic acid = 85%) to afford the target compound 64 (24.5 mg) in a 36.3% yield.

[0371] LC-MS (m / z): 374.0 [M+H] + .

[0372] 1 H NMR(400MHz,DMSO-d6)δ10.17(brs,1H),9.05(s,1H),8.38(s,1H),7.05-6.80(m,3H),4.69(s,1H),3.71-3.62(m, 1H),3.49-3.38(m,1H),2.10-2.00(m,1H),1.98(s,3H),1.94-1.87(m,1H),1.70-1.60(m,2H),1.33-1.16(m,4H).

[0373] Example 19: Synthesis of (S)-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-5-(trifluoromethyl)phenol (target compound 65)

[0374] Step 1: Synthesis of (S)-2-(2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (65-1)

[0375] Dissolve 4-6 (1.00 g, 2.92 mmol) and (S)-1-methylpiperidin-3-amine (433.2 mg, 3.79 mmol) in isopropanol (15 mL), add DIPEA (1.02 mL, 2.84 mmol), and react at 80°C for 3 hours. After cooling to room temperature, the reaction solution was concentrated under reduced pressure and purified by column chromatography (MeOH:DCM = 10%) to afford crude product 65-1 (1.50 g).

[0376] LC-MS (m / z): 421.0 [M+H] + .

[0377] Step 2: Synthesis of (S)-3-methyl-2-(6-(1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-5-(trifluoromethyl)phenol (target compound 65)

[0378] The crude product 65-1 (1.50 g) was dissolved in DCM (20 mL). A solution of BBr in DCM (8.92 mL, 2.0 M) was added under an ice-water bath and allowed to react at room temperature for 4 hours. The reaction solution was quenched with MeOH and concentrated under reduced pressure. The residue was purified by column chromatography (MeOH:DCM = 20%) to obtain the target compound 65 (856.3 mg) in a 71.9% yield and 99.23% ee.

[0379] LC-MS (m / z): 407.0 [M+H] + .

[0380] 1 H NMR(600MHz,DMSO-d6)δ11.20-10.48(brs,1H),9.09(s,1H),8.47(s,1H),7.24(s,1H),7.18(s,1H),7.15-7.07(brs,1H),4.08-3.91(m,1H), 3.03-2.90(m,1H),2.73-2.63(m,1H),2.23(s,3H),2.07(s,3H),2.02- 1.79(m,3H),1.75-1.67(m,1H),1.62-1.50(m,1H),1.42-1.30(m,1H).

[0381] Example 20: Synthesis of (R)-2-fluoro-5-methyl-6-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-3-(trifluoromethyl)phenol (target compound 66)

[0382] Step 1: Synthesis of 3-fluoro-2-methoxy-4-(trifluoromethyl)aniline (66-2)

[0383] 1-Trifluoromethyl-1,2-benzimidooxy-3(1H)-one (14.00 g, 44.30 mmol) and K2CO3 (9.20 g, 66.45 mmol) were dissolved in anhydrous ACN (350 mL), and compound 66-1 (9.40 g, 66.45 mmol) was added. The mixture was allowed to react at 75°C for 16 hours. The reaction mixture was concentrated under reduced pressure, dissolved in EA (100 mL), and filtered. The filtrate was washed with hydrochloric acid (2N, 100 mL × 3) and saturated brine (50 mL × 2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (EA:PE = 20:80) to afford compound 66-2 (4.90 g) in a 52.9% yield.

[0384] LC-MS (m / z): 210.0 [M+H] + .

[0385] Step 2: Synthesis of 6-bromo-3-fluoro-2-methoxy-4-(trifluoromethyl)aniline (66-3)

[0386] 66-2 (4.70 g, 22.47 mmol) was dissolved in DMF (40 mL), and NBS (4.20 g, 23.60 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. The reaction solution was quenched with sodium thiosulfate solution (200 mL), extracted with EA (100 mL × 3), washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (EA:PE = 20:80) to obtain 66-3 (3.10 g) in a yield of 47.9%.

[0387] LC-MS (m / z): 288.0 / 290.0 [M+H] + .

[0388] Step 3: Synthesis of 3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)aniline (66-4)

[0389] Compound 66-3 (3.10 g, 10.76 mmol), methylboronic acid (1.90 g, 32.29 mmol), Pd(dppf)Cl2 (393.8 mg, 0.54 mmol), and K2CO3 (4.50 g, 32.29 mmol) were dissolved in a mixture of dioxane and water (5:1, 90 mL) and reacted at 90°C under nitrogen for 16 hours. The reaction solution was concentrated, dissolved in EA (100 mL), and filtered. The filtrate was washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (EA:PE = 10:90) to afford compound 66-4 (2.10 g) in an 87.4% yield.

[0390] LC-MS (m / z): 224.0 [M+H] + .

[0391] Step 4: Synthesis of (3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)hydrazine hydrochloride (66-5)

[0392] In an ice-salt bath, 66-4 (270.0 mg, 1.21 mmol) was dissolved in concentrated hydrochloric acid (2 mL). A solution of NaNO2 (125.2 mg, 1.81 mmol) in water (1 mL) was slowly added dropwise at 0°C. After the reaction was continued for 1 hour, a solution of SnCl2·2H2O (546.0 mg, 2.42 mmol) in concentrated hydrochloric acid (1 mL) was slowly added dropwise. The reaction was continued for 2 hours. The reaction mixture was filtered, and the filter cake was rinsed with a small amount of water and dried under reduced pressure to obtain crude product 66-5 (200.0 mg).

[0393] Step 5: Synthesis of 2-chloro-5-(dimethoxymethyl-4-(2-(3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)hydrazino)pyrimidine (66-6)

[0394] The crude product of 66-5 (200.0 mg) and 1-2 (182.8 mg, 0.76 mmol) were dissolved in ACN (3 mL), and TEA (0.41 mL, 2.91 mmol) was added. The mixture was allowed to react at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (EA:PE = 20:80) to afford 66-6 (150.0 mg) with a two-step yield of 28.9%.

[0395] LC-MS (m / z): 425.0 [M+H] + .

[0396] Step 6: Synthesis of 6-chloro-2-(3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-2H-pyrazolo[3,4-d]pyrimidine (66-7)

[0397] 66-6 (150.0 mg, 0.35 mmol) was dissolved in ACN (2 mL), and TsOH·H2O (67.2 mg, 0.35 mmol) was added. The reaction mixture was incubated at 80°C for 1 hour. After the reaction mixture cooled to room temperature, it was diluted with EA (10 mL), washed with aqueous NaHCO3 (10 mL), washed with saturated brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (EA:PE = 30:70) to afford 66-7 (70.0 mg) in a 55.0% yield.

[0398] LC-MS (m / z): 361.0 [M+H] + .

[0399] Step 7: Synthesis of (R)-2-(3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-N-(1-methylpiperidin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (66-8)

[0400] 66-7 (70.0 mg, 0.19 mmol) and (R)-1-methylpiperidin-3-amine (28.8 mg, 0.25 mmol) were dissolved in isopropanol (2 mL), and DIPEA (50.2 mg, 0.39 mmol) was added. The mixture was allowed to react at 80°C for 3 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (MeOH:DCM = 10:90) to afford crude product 66-8 (85.0 mg).

[0401] LC-MS (m / z): 439.0 [M+H] + .

[0402] Step 8: Synthesis of (R)-2-fluoro-5-methyl-6-(6-((1-methylpiperidin-3-yl)amino)-2H-pyrazolo[3,4-d]pyrimidin-2-yl)-3-(trifluoromethyl)phenol (target compound 66)

[0403] The crude product 66-8 (85.0 mg) was dissolved in DCM (2 mL). A solution of BBr in DCM (0.49 mL, 2.0 M) was added under ice-water bath conditions and allowed to react at room temperature for 1 hour. The reaction solution was quenched with MeOH under ice-water bath conditions and concentrated under reduced pressure. The residue was purified by reverse phase preparative (ACN: 10 mmol / L NH4HCO3 aqueous solution = 85%) to afford the target compound 66 (21.7 mg) in a 26.3% yield.

[0404] LC-MS (m / z): 425.0 [M+H] + .

[0405] 1 H NMR(600MHz,DMSO-d6)δ9.07(s,1H),8.45(s,1H),7.11(s,1H),6.94(d,J=6.0Hz,1H),4.05-3.96(m,1H),3.01-2.95(m,1H),2 .76-2.65(m,1H),2.26(s,3H),2.08-1.95(m,5H),1.88-1.81(m,1H),1.76-1.68(m,1H),1.61-1.51(m,1H),1.41-1.29(m,1H).

[0406] Example 21: Inhibitory effect of the compounds of the present invention on IL-1β release in THP-1 cells after PMA-induced differentiation

[0407] THP-1 cells were purchased from Wuhan Punosai Life Science Co., Ltd. (Cat. No. CL-0233). The complete culture medium used was RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin (Pen-Strep), and a final concentration of 0.05 mM β-mercaptoethanol. Cells were cultured according to the supplier's instructions, and the cell density was maintained at the logarithmic phase before the experiment began. The cell density was adjusted to 1×10 5 Cells were cultured at a concentration of 100 ng / mL and phorbol 12-myristate 13-acetate (PMA) was added to induce for 16 hours, followed by stimulation with lipopolysaccharide (LPS) at a concentration of 100 ng / mL for 3 hours. The compound of the present invention was dissolved in dimethyl sulfoxide (DMSO), diluted 3-fold with culture medium to the required concentration and added to a 96-well plate. One hour later, nigericin sodium was added to the treated culture plate at a final concentration of 10 μM and incubated for 1 hour. The cell-free supernatant was collected and the IL-1β level was evaluated according to the instructions of the ELISA detection kit (Invitrogen). The solvent was blank. EC 50 The log(agonist) vs. response-variable slope four-parameter fitting method was used in GraphPad software. The results are shown in Table 1.

[0408] Table 1 Inhibitory activity of compounds on IL-1β release in THP-1 cells

Note

[0409] The experimental results show that the compound of the present invention can significantly inhibit the pyroptosis of human THP-1 cells after PMA-induced differentiation and the expression of IL-1β in the cells.

[0410] Example 22: Inhibitory effect of the compounds of the present invention on IL-1β release in PBMC cells

[0411] Healthy human peripheral blood mononuclear cells (PBMC) were purchased from Miaoshun Biotechnology (Cat. No. PB010C). The complete culture medium used was RPMI 1640 medium containing FBS (10%) and Pen-Strep (1%). PBMC were plated at 1×10 5 After inoculating cells / well into a 96-well plate, the cells were cultured overnight in an incubator and then stimulated for 3 hours by adding LPS (final concentration 100 ng / mL). The derivative of the present invention was dissolved in DMSO, diluted 3-fold with culture medium to the desired concentration and added to the 96-well plate for 1 hour. Adenosine triphosphate (ATP) was then added at a final concentration of 5 mM and cultured for 1 hour. The cell-free supernatant was collected and the IL-1β level was evaluated according to the instructions of the ELISA test kit (Invitrogen). The solvent was blank. IC 50 The log(agonist) vs. response-variable slope four-parameter fitting method was used in GraphPad software. The results are shown in Table 2.

[0412] Table 2 Inhibitory activity of compounds on IL-1β release in PBMC cells

[0413] Example 23: Inhibitory effect of compounds on hERG ion channels

[0414] Compounds were tested for their inhibitory effects on the human hERG ion channel stably expressed in HEK293 cells using conventional patch clamp techniques. Compounds were prepared at a 10 μM concentration. Each cell served as its own control. Compounds were perfused using a gravity-based perfusion system. After the current stabilized, the hERG current in each cell was compared before and after compound addition, and the blocking effect of the compound on the hERG current was calculated. The results are shown in Table 3.

[0415] Table 3 Blocking effect of compounds on hERG current

[0416] Example 24: Metabolic Stability Test of Test Compounds

[0417] 1. Preparation of Experimental Materials

[0418] 1.1 Incubation buffer

[0419] Weigh a certain amount of anhydrous sodium dihydrogen phosphate into a centrifuge tube, add an appropriate amount of ultrapure water, vortex mix, and prepare a sodium dihydrogen phosphate solution with a concentration of 0.1 mol / L for use; weigh a certain amount of anhydrous disodium hydrogen phosphate into a centrifuge tube, add an appropriate amount of ultrapure water, vortex mix, and prepare a sodium dihydrogen phosphate solution with a concentration of 0.1 mol / L for use; sodium dihydrogen phosphate solution (0.1 mol / L) and disodium hydrogen phosphate solution (0.1 mol / L) are mixed in a reagent bottle at a ratio of 19:81 (v:v), mix well, and obtain a PB buffer solution with a concentration of 100 mmol / L (concentration is based on phosphate ion concentration), which is stored at 2-8°C for use.

[0420] 1.2 Initiation factor (NADPH and UDPGA mixed solution)

[0421] Accurately weigh an appropriate amount of NADPH into a 1.5 mL centrifuge tube, add an appropriate amount of PB solution, vortex mix, prepare a NADPH solution with a concentration of 40 mmol / L, place it on ice for later use; accurately weigh an appropriate amount of UDPGA into a 1.5 mL centrifuge tube, add an appropriate amount of PB solution, vortex mix, prepare a UDPGA solution with a concentration of 40 mmol / L, place it on ice for later use; separately pipette the same volume of NADPH and UDPGA solution into the same centrifuge tube, vortex mix, prepare a mixed solution of NADPH and UDPGA (containing 20 mmol / L NADPH and UDPGA), place it on ice for later use.

[0422] 1.3 Positive substrate working solution (testosterone and 7-hydroxycoumarin mixture)

[0423] Accurately weigh an appropriate amount of testosterone reference substance, dissolve it in DMSO, vortex mix to dissolve it, prepare a 20 mmol / L testosterone stock solution, and store it in a -20 ° C refrigerator; accurately weigh an appropriate amount of 7-hydroxycoumarin reference substance, dissolve it in DMSO, vortex mix to dissolve it, prepare a 20 mmol / L 7-hydroxycoumarin stock solution, and store it in a -20 ° C refrigerator; respectively, transfer equal volumes of testosterone stock solution and 7-hydroxycoumarin stock solution into the same 1.5 mL centrifuge tube, dilute with 50% methanol water to a 20 μmol / L mixed substrate working solution, and place on ice for later use.

[0424] 1.4 Substrate working solution

[0425] Accurately weigh an appropriate amount of the test substance reference substance, dissolve it in DMSO, vortex mix to dissolve it, prepare a 10 mmol / L test substance stock solution, and place it in a -20°C refrigerator for use; transfer an appropriate amount of the test substance stock solution to a 1.5 mL centrifuge tube, dilute it with 50% methanol water diluent to a 20 μmol / L substrate working solution, and place it on ice for use.

[0426] 2 Experimental methods

[0427] The experimental design is divided into three groups: a positive control group (PC), a negative control group (NC), and an experimental group. The analyte is incubated with human, rat, or mouse liver microsomes under the conditions of NADPH and UDPGA for a certain period of time (and a negative control without NADPH and UDPGA is used, and a positive control is set). The reaction is terminated by adding the stop solution, and the remaining amount of the analyte in the sample is detected by LC-MS / MS instrumentation. The concentration of the test substance at different reaction times is compared with that at time T0 to obtain the stability of the analyte in liver microsomes. Generally, the final protein concentration of liver microsomes in the reaction system is 1.0 mg / mL. All incubations are carried out in a 37°C water bath.

[0428] 2.1 Experimental Grouping

[0429] Experimental group: The test substance was incubated with liver microsomes in the presence of NADPH and UDPGA for 60 min.

[0430] Negative control group (NC): The test substance was incubated with liver microsomes for 60 min without any coenzyme.

[0431] Positive control (PC): CYP3A4 and UGT probe substrates, testosterone and 7-hydroxycoumarin were incubated in liver microsomes in the presence of NADPH and UDPGA for 60 min.

[0432] 2.2 Experimental steps

[0433] (1) Take out an appropriate amount of liver microsomes of the species required for the experiment, thaw them on ice, and shake gently to mix.

[0434] (2) Experimental group and negative control group (NC): Take an appropriate amount of PB buffer, add an appropriate amount of the substrate working solution to be tested, and then transfer an appropriate amount of liver microsomes into a 1.5 mL centrifuge tube. Pipet and beat 20 to 30 times to mix them evenly, ensuring that the final concentration of liver microsomes is 1 mg / mL (if there are special needs, the liver microsome concentration can be adjusted to 0.5 mg / mL).

[0435] (3) Positive control group (PC): Take an appropriate amount of PB buffer, add an appropriate amount of positive substrate working solution, and then transfer an appropriate amount of liver microsomes into a 1.5 mL centrifuge tube. Pipet 20 to 30 times to mix them evenly to ensure that the final concentration of liver microsomes is 1 mg / mL.

[0436] (4) Experimental group: 90 μL of the solution in (2) was pipetted and aliquoted. After pre-incubation in a 37°C water bath for 5 min, 10 μL of 20 mmol / L initiator was added to initiate the reaction. After reaching the set incubation time, an appropriate amount of pre-cooled methanol solution containing the internal standard was added to terminate the reaction. (n=2)

[0437] (5) Negative control group (NC): 90 μL of the solution in (2) was transferred and aliquoted. After pre-incubation in a 37°C water bath for 5 min, 10 μL of PB buffer was added. After reaching the set incubation time, an appropriate amount of pre-cooled methanol solution containing the internal standard was added to terminate the reaction. (n ≥ 2)

[0438] (6) Positive control group (PC): 90 μL of the solution in (3) was transferred and aliquoted. After pre-incubation in a 37°C water bath for 5 min, 10 μL of 20 mmol / L initiator was added to initiate the reaction. After reaching the set incubation time, an appropriate amount of pre-cooled methanol solution containing the internal standard was added to terminate the reaction. (n=2)

[0439] (7) The sample prepared above was vortexed at 2500 rpm for 1 min, centrifuged at 17000 g at 4°C for 10 min, and the supernatant was collected for LC-MS / MS analysis.

[0440] 3.3 Data processing and analysis

[0441] Stability samples were quantified using a standard curve to determine the test substance concentration at each time point. The remaining parent was calculated as a percentage of the parent substance at the time of incubation (0 minutes). Alternatively, the remaining parent substance percentage at the time of incubation (0 minutes) was calculated by comparing the peak area of ​​the test substance to the peak area of ​​the internal standard. Data were calculated using the following formula: Parent remaining (% of 0 min) = T 60 Maternal weight / T0 maternal weight × 100%; T 60 T0: 60-min incubation time point; T1: 0-min incubation time point. The results are shown in Table 4.

[0442] Table 4 In vitro metabolic stability of compounds in liver microsomes

[0443] Example 25: Pharmacokinetic evaluation of some compounds in rats

[0444] Male SD rats were fasted overnight (with free access to water) and divided into either the tail vein (IV) or oral (PO) groups. For the IV group, blood samples (approximately 0.3 mL) were collected from the orbital venous plexus at 2, 10, 30, 1, 2, 4, 6, 8, and 24 hours after administration. These samples were anticoagulated with ethylenediaminetetraacetic acid disodium (EDTA-2Na) and placed on ice. Within 1 hour, plasma was separated by centrifugation at 4°C for 10 minutes and stored at -20°C until analysis. For the PO group, blood samples (0.3 mL) were collected from the orbital venous plexus at 5, 15, 30, 1, 2, 4, 6, 8, and 24 hours after administration and processed in the same manner as the IV group. Plasma unchanged drug concentrations were determined by LC-MS / MS. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.1 using plasma concentration data at different time points. The results are shown in Table 5.

[0445] Table 5 Pharmacokinetic test results of some compounds in rats

[0446] The experimental results show that the compound of the present invention has good pharmacokinetic properties in SD rats, including good oral bioavailability, oral exposure, maximum blood concentration and half-life.

[0447] Example 26: Inhibitory effect of compound 2 on cytokines in a mouse inflammation model

[0448] 6-8 week old female C57BL / 6 mice were randomly divided into 5 groups, each group consisting of control group, model group and compound 2 group. The compound 2 group was gavaged with compound 2 (10 mg / kg) once daily for three consecutive days, while the control and model groups were given the corresponding volume of vehicle every day. One hour after gavage on the third day, mice in the model and compound 2 groups were intraperitoneally injected with LPS (5 mg / kg), while mice in the control group were intraperitoneally injected with the corresponding volume of sodium chloride injection. Blood was collected from mice in each group 2 hours after intraperitoneal injection of LPS, and after standing at room temperature for 1 hour, the blood was centrifuged at 4°C and 5000 rpm for 10 minutes. Serum was then collected and tested for cytokines IL-1β, TNF-α and IL-6 by ELISA. The experimental results are shown in Figures 1, 2 and 3, where *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

[0449] Experimental conclusion: The compound of the present invention can significantly reduce the levels of cytokines IL-1β, TNF-α and IL-6 in the serum of mice induced by LPS.

[0450] Example 27: Inhibitory effect of compound 4 hydrochloride on cytokines in a mouse inflammation model

[0451] Six- to eight-week-old female C57BL / 6 mice were randomly divided into five groups: a control group, a model group, and a compound 4 hydrochloride-treated group (hereinafter referred to as the treatment group). The treatment groups were gavaged with compound 4 hydrochloride (10 mg / kg, 3 mg / kg, and 1 mg / kg) once daily for three consecutive days. The control and model groups were given the corresponding volume of vehicle daily. One hour after gavage on the third day, mice in the model and treatment groups were intraperitoneally injected with LPS (5 mg / kg), while mice in the control group were intraperitoneally injected with the corresponding volume of sodium chloride injection. Blood was collected from mice in each group 4 hours after the intraperitoneal injection. After standing at room temperature for 1 hour, the blood was centrifuged at 4°C and 5000 rpm for 10 minutes. Serum was collected and assayed for cytokines IL-1β, TNF-α, and IL-6 by ELISA. The experimental results are shown in Figures 4, 5, and 6. *P<0.05, **P<0.01, ***P<0.005, and ****P<0.001.

[0452] Example 28: Toxicity study of compound 4 hydrochloride administered orally to SD rats for 28 consecutive days

[0453] Eight male and eight female SD rats were randomly divided into a normal control group of 2 rats per sex, and a low-dose group and a high-dose group of 3 rats per sex. The low-dose group and the high-dose group were gavaged with 3 and 10 mg / mL (in terms of base) of the drug solution, respectively, with a dosing volume of 10 mL / kg and a dosing dose of 30 and 100 mg / kg, respectively. The normal control group was orally administered with the corresponding solvent water. During the experiment, the rats were allowed to drink water freely and the drug was administered continuously for 28 days. The day after the last dose, blood was collected and 23 routine blood and biochemical indicators were tested. The rats' hearts, livers, spleens, lungs, kidneys, brains, gastrointestinal tracts, thymuses, testicles (ovaries) and other organs were observed by dissection.

[0454] The experimental results showed that no significant toxic reactions were observed in the animals in any dose group after administration, and there was no significant effect on the animals' body weights. The experimental results are shown in Figures 7 and 8. Dissections of the animals in each dose group showed no significant pathological changes in the surface color, shape, size, or texture of their organs. Routine blood tests and biochemical parameters showed no significant abnormalities compared to those in the normal group. The compounds of this invention have an excellent safety profile.

[0455] Example 29: Inhibitory Effects of Compound 4 Hydrochloride on Cytochrome P450 Isoenzymes CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4-M, and CYP3A4-T

[0456] 1. Preparation of Phosphate Buffer (PB, 100 mM, pH 7.4)

[0457] Disodium hydrogen phosphate was ultrasonically dissolved in ultrapure water to a 100 mM concentration, which was used as Solution A. Potassium dihydrogen phosphate was ultrasonically dissolved in ultrapure water to a 100 mM concentration, which was used as Solution B. Solution A was stirred and slowly added to Solution B until the pH reached 7.4. The phosphate buffer was stored at 4°C until use. Ultrapure water was obtained from a Sartorius ultrapure water system.

[0458] 2. Preparation of Positive Control Solution

[0459] The positive control was prepared as a 10 mM DMSO stock solution and introduced into the test system with the test substance at a 1.0% volume ratio of organic solvent. The working solution concentrations of the positive inhibitors and the final concentrations in the reaction system are shown in Table 6 below.

[0460] Table 6

[0461] 3. Preparation of Cocktail Substrate Stock Solution

[0462] The specific preparation of the substrate stock solution is shown in Table 7 below. After preparation, the stock solution is stored in a refrigerator at -20°C. Thaw at room temperature before use.

[0463] Table 7

[0464] 4. Preparation of Compound 4 Hydrochloride Working Solution

[0465] Compound 4 hydrochloride was prepared as a 5 mM DMSO stock solution, and the organic solvent volume ratio introduced into the test system along with the test substance was 1.0%. The working solution concentration of Compound 4 hydrochloride and the final concentration in the reaction system are shown in Table 8 below.

[0466] Table 8

[0467] 5. Preparation of Human Liver Microsome Mixture

[0468] Human liver microsomes were purchased from BIOIVT (Cat. No. 452117, Batch No. SEU) with a stock solution concentration of 20 mg / mL. The working solution concentration of human liver microsomes and the final concentration in the reaction system are shown in Table 9 below.

[0469] Table 9

[0470] 6. Preparation of NADPH Cofactor Solution

[0471] Before the experiment, 13.14 mg of NADPH (Roche, product number 10107824001, batch number 71466624) was weighed and prepared with 1.545 mL of PB to prepare a working solution with a concentration of 10 mM. The final concentration of NADPH in the experimental system was 1 mM.

[0472] 7. The entire incubation process was performed in a 1.5 mL centrifuge tube. First, add 2 μL of compound 4 hydrochloride or positive control working solution to the corresponding centrifuge tube. For the no-inhibitor group, add 2 μL of DMSO:MeOH (1:1) solvent. Then, add 20 μL of mixed substrate working solution and 158 μL of liver microsome working solution. Preheat in a 37°C water bath for 5 minutes. After adding 20 μL of NADPH cofactor to initiate the reaction, continue incubation at 37°C for the corresponding time (see Table 10). Prepare the test samples in duplicate.

[0473] After incubation, the reaction was terminated by adding 400 μL of frozen stop solution (1 ng / mL LMTA in methanol). After vortexing, the sample was centrifuged at 17,000 g for 10 minutes at 4°C. 180 μL of the supernatant was transferred to a 96-well plate for LC-MS / MS analysis.

[0474] The relative activity was calculated using the following formula: relative activity (NC) = metabolite production in the test group or positive control group / average metabolite production in the blank negative control group; inhibition rate = (1-NC) x 100%. The experimental results are shown in Table 10.

[0475] Table 10 Inhibitory effect of compound 4 hydrochloride on CYP450 isoenzymes

[0476] The experimental results show that the compounds of the present invention do not show obvious direct inhibitory effects on the seven major subtypes of human liver microsomal CYP450 enzymes.

Claims

1. A pyrazolopyrimidine compound, which is a compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (I): Among them, Selected from a single bond or a double bond; m 1 、m 2 、m 3 、m 4 、m 5 Each independently selects from 1 or 2; X and Y are each independently selected from C or N; R 1 、R 5 each independently selected from absent, hydrogen, halogen, hydroxyl, cyano, amino, C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; the C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; R 2 、R 3 、R 4 are each independently selected from hydrogen, halogen, hydroxy, cyano, amino, C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl group; the C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl group may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; or R 3 and R 4 together with the respective connected carbon atoms form C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; or R 3 and R 2 together with the respective connected carbon atoms form C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; R 1 、R 2 、R 3 、R 4 、R 5 at least one of them is a hydroxyl group; R 6 selected from hydrogen, a halogen or C 1-6 alkyl; said C 1-6 alkyl may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; L is a chemical bond, NR 7 , OR 7 or C(R 7 )2; R 7 is selected from hydrogen, C 1-6 alkyl or is absent; R 8 selected from hydrogen, hydroxyl, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl or 5-10 membered heteroaryl; said C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl or 5-10 membered heteroaryl may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; Ring B is selected from C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl; the C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from hydroxy, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkylhydroxy, -COOR 9 、-CH2COOR 9 or C 1-6 alkylaminoalkyl; R 9 selected from hydrogen or C 1-6 alkyl; Wherein, the 3- to 6-membered heterocyclic group, 3- to 9-membered heterocyclic group, and 5- to 10-membered heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O, or S.

2. The pyrazolopyrimidine compound according to claim 1, wherein A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (IA): Wherein, X and Y are each independently selected from C or N; R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted halo C 1-6 alkyl, substituted or unsubstituted C 1-6 alkylamino, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted halo C 1-6 alkoxy, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted halo C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 3- to 6-membered heterocyclic group, substituted or unsubstituted 5- to 10-membered heteroaryl, hydroxyl, cyano, amino, and at least one of R 1 、R 2 、R 3 、R 4 、R 5 is hydroxyl; when there is at least one substituent on C 1-6 alkyl, halo C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo C 1-6 alkoxy, C 2-6 alkenyl, halo C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, 5- to 10-membered heteroaryl, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; or R 3 adjacent to an R 3 of R 4 together with their respective attached carbon atoms combine to form a C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, or 5- to 10-membered heteroaryl; R 3 adjacent to an R 3 of R 2 together with their respective attached carbon atoms combine to form a C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, or 5- to 10-membered heteroaryl; R 6 selected from hydrogen, substituted or unsubstituted C 1-6 alkyl or halogen; when there is at least one substituent on the C 1-6 alkyl, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; L is a direct bond, NR 7 , OR 7 or CR 7 ; R 7 is selected from H, C 1-6 alkyl or is absent; R 8 selected from hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted halo C 1-6 alkyl, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted halo C 1-6 alkoxy, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 5-10 membered heteroaryl, hydroxy, cyano, amino; when there is at least one substituent on C 1-6 alkyl, halo C 1-6 alkyl, C 1-6 alkoxy, halo C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 5-10 membered heteroaryl, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; Ring B is selected from substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 3- to 9-membered heterocyclic group, substituted or unsubstituted C 6-10 aryl, substituted or unsubstituted 5- to 10-membered heteroaryl; when C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl has at least one substituent, the substituent is selected from one or more of the following groups: C 1-6 alkyl, hydroxy, halogen, C 1-6 haloalkyl, C 1-6 alkyl hydroxy, -COOR 9 or C 1-6 alkylaminoalkyl; R 9 selected from hydrogen or C 1-6 alkyl; The heterocyclic group and heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O, or S.

3. The pyrazolopyrimidine compound according to claim 1, characterized in that, A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (IB): Among them, ring B is selected from C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl; the C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from hydroxy, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkylhydroxy, -CH2COOR 9 or C 1-6 alkylaminoalkyl; X, Y, L, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , R 9 are defined as described in claim 1.

4. The pyrazolopyrimidine compound according to any one of claims 1 to 3, characterized in that, A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (II): Wherein, X and Y are each independently selected from C or N; R 1 and R 5 are each independently selected from absent, hydrogen, halogen, hydroxy, cyano, C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-6 membered heterocyclic group or 5-10 membered heteroaryl; said C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkynyl, C 3- cycloalkyl, 3-6 membered heterocyclic group or 5-10 membered heteroaryl may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; R 2 、R 3 、R 4 are each independently selected from hydrogen, halogen, hydroxy, cyano, C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; the C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; or R 3 and R 4 together with the respective connected carbon atoms form C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; or R 3 and R 2 together with the respective connected carbon atoms form C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; R 1 、R 2 、R 3 、R 4 、R 5 at least one is a hydroxyl group; L is selected from NR 7 , OR 7 or C(R 7 )2; R 7 is selected from hydrogen, methyl or absent; Ring B is selected from C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl; the C 3-8 cycloalkyl, 3- to 9-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from hydroxy, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkylhydroxy, -CH2COOEt or C 1-6 alkylaminoalkyl; Wherein, the 3- to 6-membered heterocyclic group, 3- to 9-membered heterocyclic group, and 5- to 10-membered heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O, or S.

5. The pyrazolopyrimidine compound according to any one of claims 1 to 3, characterized in that, A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (IIA): Wherein, R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted halo C 1-6 alkyl, substituted or unsubstituted C 1-6 alkylamino, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted halo C 1-6 alkoxy, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 3- to 6-membered heterocyclic group, substituted or unsubstituted 5- to 10-membered heteroaryl, hydroxyl, cyano, and at least one of R 1 、R 2 、R 3 、R 4 、R 5 is hydroxyl; when there is at least one substituent on C 1-6 alkyl, halo C 1-6 alkyl, C 1- 6 alkoxy, halo C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, 5- to 10-membered heteroaryl, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen; or R 3 adjacent to an R 3 of R 4 together with their respective attached carbon atoms combine to form a C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, or 5- to 10-membered heteroaryl; R 3 adjacent to an R 3 of R 2 together with their respective attached carbon atoms combine to form a C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, or 5- to 10-membered heteroaryl; R 6 selected from hydrogen, C 1-6 alkyl or halogen; R 7 selected from hydrogen or C 1-6 alkyl; Ring B is selected from substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 3- to 8-membered heterocyclic group, substituted or unsubstituted 5- to 10-membered heteroaryl; when C 3-8 there is at least one substituent on the cycloalkyl, 3- to 8-membered heterocyclic group or 5- to 10-membered heteroaryl, the substituent is selected from one or more of the following groups: C 1-6 alkyl, hydroxy, halogen, C 1-6 haloalkyl, C 1-6 alkyl hydroxy, -CH2COOEt or C 1-6 alkylaminoalkyl; The heterocyclic group and heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O, or S.

6. The pyrazolopyrimidine compound according to any one of claims 1 to 4, characterized in that, A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (II-1): Wherein, n is selected from 0, 1, 2, or 3; R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, halogen, hydroxy, cyano, C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2- 6-ynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; the C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 -ynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen; or R 3 and R 4 together with the respective linked carbon atoms form a C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, or 5- to 10-membered heteroaryl; or R 3 and R 2 together with the respective linked carbon atoms form C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; R 1 、R 2 、R 3 、R 4 、R 5 at least one is a hydroxyl group; L is defined as described in claim 4.

7. The pyrazolopyrimidine compound according to any one of claims 1 to 4 and 6, characterized in that A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (IIA-1): Wherein, n is selected from 1 or 2; L is selected from O, NH, or N(CH3); R 3 selected from hydrogen, halogen, hydroxy, cyano, C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl, wherein the C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, C 2-6 alkynyl, C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl may optionally be further substituted by one or more substituents selected from halogen or C 1-6 alkyl.

8. The pyrazolopyrimidine compound according to any one of claims 1 to 7, characterized in that, A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (IIA-1-1): wherein, R 3 is defined as described in claim 7.

9. The pyrazolopyrimidine compound according to claim 8, wherein R 3 selected from hydrogen, halo-C 1-6 alkyl or halogen.

10. The pyrazolopyrimidine compound according to any one of claims 1 to 8, characterized in that, A compound, isomer or pharmaceutically acceptable salt thereof having the following general structural formula (IIA-1-2): wherein, R 3 is defined as described in claim 7.

11. The pyrazolopyrimidine compound according to any one of claims 1 to 8 and 10, characterized in that, R 3 selected from hydrogen, halogen, cyano, methyl, cyclopropyl, trifluoromethyl, difluoromethyl, trifluoromethoxy, dimethylamino, 12. A pyrazolopyrimidine compound, which is a compound, isomer or pharmaceutically acceptable salt thereof having the following structure:

13. A pharmaceutical composition comprising the compound, isomer, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, and at least one pharmaceutically acceptable carrier or excipient.

14. Use of the compound, isomer, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13 in the preparation of a drug for preventing or treating NLRP3-related diseases.

15. Use of the compound, isomer, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13 in preventing or treating NLRP3-related diseases.

16. A method for treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the compound, isomer, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13; the disease to be treated and / or prevented is an NLRP3-related disease or disorder.

17. The application according to claim 14 or 15, or the method according to claim 16, characterized in that The NLRP3-related disease is cancer, an inflammatory disease, or a disease accompanied by an inflammatory response; The cancer diseases include, but are not limited to, myeloproliferative neoplasms, myeloid leukemia, lung cancer, nasopharyngeal carcinoma, laryngeal cancer, esophageal cancer, cholangiocarcinoma, oral cancer, head and neck cancer, mesothelioma, adrenocortical carcinoma, kidney cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, bone cancer, brain cancer, breast cancer, melanoma, pancreatic cancer, skin cancer, lymphoma, bladder cancer, small intestine cancer, soft tissue sarcoma, endometrial cancer, cervical cancer, osteosarcoma, prostate cancer, or testicular cancer; The inflammatory diseases or diseases accompanied by an inflammatory response include, but are not limited to: 1) Auto-inflammatory diseases such as cryopyrin-associated periodic syndromes (CAPS), familial Mediterranean fever, Schnitzler syndrome, mevalonate kinase deficiency (MKD); 2) Chronic pain, including neuropathic pain and non-neuropathic pain; 3) Skin disorders such as contact hypersensitivity, bullous pemphigoid, sunburn, contact dermatitis, seborrheic dermatitis, hidradenitis suppurativa (HS), diabetic (foot) ulcers, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, acne, alopecia; 4) Respiratory system disorders such as chronic obstructive pulmonary disease (COPD), asthma, bronchitis, rhinitis, sinusitis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, sarcoidosis, adult respiratory distress syndrome, pneumonia; 5) Joint disorders such as arthritis; 6) Muscle disorders such as polymyositis, myasthenia gravis; 7) Cardiovascular disorders such as hypertension, ischemia, reperfusion injury, vasculitis, pericarditis; 8) Blood disorders such as sickle cell disease; 9) Central nervous system diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, brain injury, multiple sclerosis, amyotrophic lateral sclerosis; 10) Metabolic diseases such as type 2 diabetes (T2D), atherosclerosis, obesity, gout; 11) Liver diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH); 12) Kidney diseases such as acute kidney disease, hyperoxaluria, chronic kidney disease, nephrocalcinosis, glomerulonephritis, diabetic nephropathy; 13) Gastrointestinal disorders such as inflammatory bowel disease, pancreatitis; 14) Ocular disorders such as uveitis, allergic conjunctivitis; 15) Graft-versus-host disease; 16) Burns, sunburns, mechanical injuries.

18. The application or method according to claim 17, characterized in that, The neuropathic pain described above includes central neuropathic pain and peripheral neuropathic pain; The central neuropathic pain includes, but is not limited to, spinal cord injury neuropathic pain, post-stroke pain, multiple sclerosis pain, syringomyelia pain, ischemic myelopathy pain, compressive myelopathy pain, post-radiation myelopathy pain, Parkinson's disease pain, phantom limb pain, myelitis pain; The peripheral neuropathic pain includes, but is not limited to, postherpetic neuralgia, HIV neuropathy, diabetic peripheral neuropathy, chronic pain after trauma / surgery, neuropathy after chemo / radiotherapy, trigeminal neuralgia, glossopharyngeal neuralgia, stump pain, toxicant-induced neuropathy, sciatica, dorsal root neuralgia; The non-neuropathic pain includes, but is not limited to, osteoarthritis pain, chronic low back pain, chronic visceral pain, cancer pain, fibromyalgia.

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