Pyrazolopyrimidine compound and medical application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly relates to an NLRP3 inhibitor, a preparation method thereof and application of the NLRP3 inhibitor in medicine. The invention provides an NLRP3 inhibitor as shown in a formula (I) as well as a composition and application thereof. The NLRP3 inhibitor can be used for treating or preventing diseases or symptoms related to abnormal expression of an NLRP3 signal channel. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and relates to a pyrazolopyrimidine compound, specifically to an isomer of such a compound or a pharmaceutically acceptable salt thereof; further, the present invention also discloses a preparation method and a 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 one of the most studied intracellular pattern recognition receptors 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 an inflammasome, and at the same time releases the activated inflammatory cytokines IL-1β and IL-18, causing an inflammatory response.

[0003] Inflammation is the body's defensive response to stimuli, including infectious inflammation and sterile inflammation. Inflammation is mainly manifested as redness, swelling, heat, pain and dysfunction, usually caused by increased permeability of vascular endothelial cells and exudation of immune cells in plasma. After tissue repair, the inflammatory response will end quickly, but excessive cytokine production will lead to a cytokine storm, causing damage to the body. Inflammatory dysregulation is an important pathogenesis of many human diseases.

[0004] Abnormal activation of the NLRP3 inflammasome has been observed in many inflammations and inflammatory diseases, and the overproduction of IL-1β and IL-18 also participates in and promotes the occurrence and development of various diseases, including cryopyrin-associated periodic syndromes; 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; neuroinflammation-related diseases such as brain infections, acute injuries and neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease; cardiovascular and metabolic-related 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 syndromes, myelofibrosis, lung cancer, colon cancer, etc.

[0005] Currently, there are no marketed drugs for NLRP3 inhibitors. The earliest compound MCC950 that entered clinical research was terminated due to its liver toxicity, but it has been used as a tool molecule in a large number of literatures to explore different indications or mechanisms. Existing compounds such as OLT-1177, Emlenoflast, RG6418, and DFV890 are in the clinical research stage for the clinical treatment of acute gout attacks, knee osteoarthritis, Schnitzler syndrome, Cryopyrin-associated periodic syndrome, ulcerative colitis, Parkinson's disease, and other diseases. Diseases that are essentially inflammatory or immune are usually difficult to diagnose or treat efficiently. Most treatment methods include treating symptoms, slowing the progression of the disease, changing lifestyle, and surgery as a last resort. There is an urgent need for more efficient and effective means to treat such diseases.

[0006] Therefore, developing NLRP3 inflammasome inhibitors has certain therapeutic potential for treating such diseases with inflammatory pathological characteristics. 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, having significant NLRP3 inhibitory activity, for preventing or treating diseases related to abnormal NLRP3 expression.

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

[0009] On the one hand, the present invention provides a pyrazolopyrimidine compound, which is a compound having the following general formula (I), an isomer, or a pharmaceutically acceptable salt thereof:

[0010]

[0011] Wherein, is selected from a single bond or a double bond;

[0012] m 1 、m 2 、m 3 、m 4 、m 5 are each independently selected from 1 or 2;

[0013] X and Y are each independently selected from C or N;

[0014] R 1 、R 5 are 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, C2-6 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;

[0015] R 2 、R 3 、R 4 Each independently is 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; 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;

[0016] 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;

[0017] 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;

[0018] R 1 、R 2 、R 3 、R 4 、R 5 At least one is hydroxy;

[0019] R 6 Is selected from hydrogen, halogen, or C 1-6 Alkyl; the C 1-6The alkyl group may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen;

[0020] 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;

[0021] R 8 is 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; the 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;

[0022] Ring B is selected from C 3-8 cycloalkyl, 3-9 membered heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl; the C 3-8 cycloalkyl, 3-9 membered heterocyclic group, C 6-10 aryl or 5-10 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;

[0023] R 9 is selected from hydrogen or C 1-6 alkyl;

[0024] Wherein, the 3-6 membered heterocyclic group, 3-9 membered heterocyclic group, 5-10 membered heteroaryl contains at least one heteroatom, and the heteroatom is selected from N, O or S.

[0025] In some embodiments, the compound has a compound, isomer or pharmaceutically acceptable salt thereof of the following formula (IA):

[0026]

[0027] Wherein, X and Y are each independently selected from C or N;

[0028] 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, hydroxy, cyano, amino, and at least one of R 1 、R 2 、R 3 、R 4 、R 5 is hydroxy; 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

[0029] R 3 combines with an adjacent R 3 and the carbon atoms to which they are attached to form a C 4 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl; R 3-8 combines with an adjacent R 3 and the carbon atoms to which they are attached to form a C 3 and the adjacent R 2 3-8 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl;

[0030]

[0030] R 6 is selected from hydrogen, substituted or unsubstituted C 1-6 alkyl or halogen; when C 1-6When 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;

[0031] L is a direct bond, NR 7 、OR 7 or CR 7 ; R 7 is selected from H, C 1-6 alkyl or does not exist;

[0032] R 8 is 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 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 has at least one substituent, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen;

[0033] 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 aryl or 5-10 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;

[0034] R 9 is selected from hydrogen or C 1-6 alkyl;

[0035] The heterocyclic group and heteroaryl contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0036] In some embodiments, the compound is a compound, isomer, or pharmaceutically acceptable salt thereof of formula (IB) as follows:

[0037]

[0038] Wherein, ring B is selected from C 3-8 cycloalkyl, 3-9 membered heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl; the C 3-8 cycloalkyl, 3-9 membered heterocyclic group, C 6-10 aryl or 5-10 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;

[0039] X, Y, L, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 8 、R 9 are as defined in general formula (I).

[0040] In some embodiments, the compound is a compound, isomer, or pharmaceutically acceptable salt thereof of formula (I-1) as follows:

[0041]

[0042] Wherein, X and Y are each independently selected from C or N;

[0043] 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 3-6 membered heterocyclic group, substituted or unsubstituted 5-10 membered heteroaryl, hydroxy, cyano, and R 1, R 2 , R 3 , R 4 , R 5 At least one is a hydroxyl group; when there is at least one substituent on C 1-6 alkyl, C 1-6 alkylamine group, halogenated C 1-6 alkyl, C 1-6 alkoxy, halogenated 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

[0044] R 3 combines with an adjacent R 3 to form a C 4 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl together with the respective connected carbon atoms; R 3-8 combines with an adjacent R 3 to form a C 3 cycloalkyl, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl together with the respective connected carbon atoms; 2 3-8

[0045] R

[0046] 6 is selected from hydrogen, substituted or unsubstituted C 1-6 alkyl or halogen; when there is at least one substituent on C 1-6 alkyl, the substituent is selected from one or more of the following groups: C 1-6 alkyl or halogen;

[0047] L is selected from NR 7 , OR 7 or CR 7 ; R 7 is selected from H, methyl or absent;

[0047] Ring B is selected from substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 3- to 9-membered heterocyclic group, substituted or unsubstituted 5- to 10-membered heteroaryl; when there is at least one substituent on C 3-8 cycloalkyl, 3- to 9-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, hydroxyl, halogen, C 1-6 haloalkyl, C 1-6 alkylhydroxyl, -CH2COOEt or C 1-6 alkylamine alkyl;

[0048] The heterocyclic group and heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0049] In some embodiments, the compound has a compound, isomer or pharmaceutically acceptable salt thereof of the following formula (II):

[0050]

[0051] Wherein X and Y are each independently selected from C or N;

[0052] R 1 、R 5 are each independently selected from absent, 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 group; the 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 group may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen;

[0053] R 2 、R 3 、R 4 are 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 heterocyclic group or 5-10 membered heteroaryl group; the 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 group may optionally be further substituted by one or more substituents selected from C 1-6 alkyl or halogen;

[0054] Or R 3 and R 4 together with the carbon atoms to which they are attached form a C 3-8 cycloalkyl, 3-6 membered heterocyclic group or 5-10 membered heteroaryl group;

[0055] Or R 3 and R2 Together with the respective connected carbon atoms, form a C 3-8 cycloalkyl, 3- to 6-membered heterocyclic group, or 5- to 10-membered heteroaryl;

[0056] R 1 、R 2 、R 3 、R 4 、R 5 at least one is a hydroxyl group;

[0057] L is selected from NR 7 、OR 7 or C(R 7 )2; R 7 is selected from hydrogen, methyl, or absent;

[0058] 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 hydroxyl, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkylhydroxy, -CH2COOEt, or C 1-6 alkylaminoalkyl;

[0059] wherein, the 3- to 6-membered heterocyclic group, 3- to 9-membered heterocyclic group, and 5- to 10-membered heteroaryl contain at least one heteroatom, and the heteroatom is selected from N, O, or S.

[0060] In some embodiments, the compound has a compound, isomer, or pharmaceutically acceptable salt thereof of the following formula (IIA):

[0061]

[0062] 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 3-8Cycloalkyl, substituted or unsubstituted 3- to 6-membered heterocyclic group, substituted or unsubstituted 5- to 10-membered heteroaryl group, hydroxyl group, cyano group, and R 1 、R 2 、R 3 、R 4 、R 5 at least one of which is a hydroxyl group; when there is at least one substituent on the C 1-6 alkyl group, halogenated C 1-6 alkyl group, C 1-6 alkoxy group, halogenated C 1-6 alkoxy group, C 2-6 alkynyl group, C 3-8 cycloalkyl group, 3- to 6-membered heterocyclic group, 5- to 10-membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 alkyl group or halogen; or

[0063] R 3 combines with an adjacent R 3 and the R 4 connected to each of them to form a C 3-8 cycloalkyl group, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl group; R 3 combines with an adjacent R 3 and the R 2 connected to each of them to form a C 3-8 cycloalkyl group, 3- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl group;

[0064] R 6 is selected from hydrogen, C 1-6 alkyl group or halogen;

[0065] R 7 is selected from hydrogen or C 1-6 alkyl group;

[0066] ring B is selected from substituted or unsubstituted C 3-8 cycloalkyl group, substituted or unsubstituted 3- to 8-membered heterocyclic group, substituted or unsubstituted 5- to 10-membered heteroaryl group; when there is at least one substituent on the C 3-8 cycloalkyl group, 3- to 8-membered heterocyclic group or 5- to 10-membered heteroaryl group, the substituent is selected from one or more of the following groups: C 1-6 alkyl group, hydroxyl group, halogen, C 1-6 haloalkyl group, C 1-6 alkylhydroxyl group, -CH2COOEt or C 1-6 alkylaminoalkyl group;

[0067] The heterocyclic group and heteroaryl group contain at least one heteroatom, and the heteroatom is selected from N, O or S.

[0068] In some embodiments, the compound is a compound, isomer or pharmaceutically acceptable salt thereof of formula (II-1) as follows:

[0069]

[0070] wherein n is selected from 0, 1, 2 or 3;

[0071] 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 alkynyl, C 3-8 cycloalkyl, 3-6 membered heterocyclic group 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 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;

[0072] or R 3 and R 4 together with the carbon atoms to which they are attached form a C 3-8 cycloalkyl, 3-6 membered heterocyclic group or 5-10 membered heteroaryl;

[0073] or R 3 and R 2 together with the carbon atoms to which they are attached form a C 3-8 cycloalkyl, 3-6 membered heterocyclic group or 5-10 membered heteroaryl;

[0074] R 1 、R 2 、R 3 、R 4 、R 5 at least one is hydroxy;

[0075] L is defined as in general formula (II).

[0076] In some embodiments, the compound is a compound, isomer or pharmaceutically acceptable salt thereof of formula (IIA-1) as follows:

[0077]

[0078] Among them, n is selected from 1 or 2;

[0079] L is selected from O, NH or N(CH3);

[0080] R 3 is 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- to 6-membered heterocyclic group or 5- to 10-membered heteroaryl group, and 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 group may optionally be further substituted by one or more substituents selected from halogen or C 1-6 alkyl.

[0081] In some embodiments, the compound is a compound, isomer or pharmaceutically acceptable salt thereof having the following formula (IIA-1-1):

[0082]

[0083] wherein the definition of R 3 is as described in general formula (IIA-1).

[0084] In some embodiments, R 3 is selected from hydrogen, halo C 1-6 alkyl or halogen.

[0085] In some embodiments, the compound is a compound, isomer or pharmaceutically acceptable salt thereof having the following formula (IIA-1-2):

[0086]

[0087] wherein the definition of R 3 is as described in general formula (IIA-1).

[0088] In some embodiments, the R 3 is selected from hydrogen, halogen, cyano, methyl, cyclopropyl, trifluoromethyl, difluoromethyl, trifluoromethoxy, dimethylamino,

[0089] The present invention also provides a pyrazolopyrimidine compound, which is a compound, isomer or pharmaceutically acceptable salt thereof having the following structure:

[0090]

[0091]

[0092] On the other hand, the present invention provides a pharmaceutical composition comprising at least one compound, isomer or pharmaceutically acceptable salt thereof of formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, and at least one pharmaceutically acceptable carrier or excipient.

[0093] In yet another aspect, the present invention provides the use of a compound, isomer or pharmaceutically acceptable salt thereof of formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, or a pharmaceutical composition thereof, in the preparation of a medicament for preventing and / or treating NLRP3-related diseases or disorders.

[0094] In yet another aspect, the present invention further provides a compound, isomer or pharmaceutically acceptable salt thereof of formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, or a pharmaceutical composition thereof, for preventing and / or treating NLRP3-related diseases or disorders.

[0095] In yet another aspect, the present invention further provides a method for preventing and / or treating a disease, comprising administering to a patient in need an effective amount of a compound, isomer or pharmaceutically acceptable salt thereof of formula (I), (IA), (IB), (I-1), (II), (IIA), (II-1), (IIA-1), (IIA-1-1) or (IIA-1-2) as described above, or a pharmaceutical composition thereof; the disease to be prevented and / or treated is an NLRP3-related disease or disorder.

[0096] In some embodiments, the NLRP3-related disease or disorder is selected from cancer diseases, inflammatory diseases or diseases accompanied by an inflammatory response;

[0097] The cancer diseases include, but are not limited to, myeloproliferative neoplasms, myeloid leukemia, lung cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, cholangiocarcinoma, oral cancer, head and neck cancer, mesothelioma, adrenocortical carcinoma, renal 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;

[0098] The inflammatory disease or the disease accompanied by an inflammatory response includes, but is not limited to:

[0099] 1) Auto-inflammatory diseases such as cryopyrin-associated periodic syndromes (CAPS), familial Mediterranean fever, Schnitzler syndrome, mevalonate kinase deficiency (MKD);

[0100] 2) Chronic pain, including neuropathic pain and non-neuropathic pain;

[0101] 3) Skin disorders such as contact hypersensitivity, bullous pemphigoid, sunburn, contact dermatitis, seborrheic dermatitis, hidradenitis suppurativa (HS), diabetic (foot) ulcer, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, acne, alopecia;

[0102] 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;

[0103] 5) Joint disorders such as arthritis;

[0104] 6) Muscle disorders such as polymyositis, myasthenia gravis;

[0105] 7) Cardiovascular disorders such as hypertension, local anemia, reperfusion injury, vasculitis, pericarditis;

[0106] 8) Blood diseases such as sickle cell disease;

[0107] 9) Central nervous system diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, brain injury, multiple sclerosis, amyotrophic lateral sclerosis;

[0108] 10) Metabolic diseases such as type 2 diabetes (T2D), atherosclerosis, obesity, gout;

[0109] 11) Liver diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH);

[0110] 12) Kidney diseases such as acute kidney disease, hyperoxaluria, chronic kidney disease, nephrocalcinosis, glomerulonephritis, diabetic nephropathy;

[0111] 13) Gastrointestinal disorders such as inflammatory bowel disease, pancreatitis;

[0112] 14) Eye disorders such as uveitis, allergic conjunctivitis;

[0113] 15) Graft-versus-host disease;

[0114] 16) Burns, sunburns, mechanical injuries.

[0115] In some embodiments, the neuropathic pain described herein includes central neuropathic pain and peripheral neuropathic pain;

[0116] The central neuropathic pain described herein includes, but is not limited to, neuropathic pain caused by spinal cord injury, pain after stroke, pain in multiple sclerosis, pain in syringomyelia, ischemic myelopathy pain, compressive myelopathy pain, post-radiation myelopathy pain, pain in Parkinson's disease, phantom limb pain, myelitis pain;

[0117] The peripheral neuropathic pain described herein 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;

[0118] The non-neuropathic pain described herein includes, but is not limited to, osteoarthritis pain, chronic low back pain, chronic visceral pain, cancer pain, fibromyalgia.

[0119] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commodity or its active ingredient.

[0120] The "compound" described in the present invention includes, but is not limited to, the following situations of the compound: free base, stereoisomer, geometric isomer, tautomer, isotope, pharmaceutically acceptable salt, solvate, hydrate, prodrug (ester), etc.

[0121] The "compound" described in the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise specified, all stereoisomers are included, such as enantiomers and diastereomers. Compounds containing asymmetric carbon atoms in the present invention can be isolated in optically pure form or in racemic form. The optically pure form can be obtained by resolution of the racemic mixture, synthesis using chiral starting materials or chiral reagents.

[0122] In the present invention, "isomers" refer to, unless otherwise specified, stereoisomers or tautomers. Unless otherwise specified, the term "stereoisomers" refers to compounds having the same chemical constitution but different spatial arrangements of atoms or groups. Stereoisomers include, but are not limited to, enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans isomers), and atropisomers. Any mixture of the resulting stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization. Unless otherwise specified, the term "tautomers" refers to structural isomers that can be interconverted through a low energy barrier and have different energies. If tautomerism is possible (e.g., in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as proton-transfer tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions through the reorganization of some bonding electrons.

[0123] In the present invention, "isotopes" refer to, unless otherwise specified, the compounds of the present invention can exist in isotopically labeled or enriched forms, containing one or more atoms whose atomic weights or mass numbers are different from those of the atoms found in the largest amounts in nature. Isotopes can be radioactive or non-radioactive isotopes. Isotopes commonly used for isotope labeling 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 S. These isotopically labeled compounds can be used to study the distribution of medicinal molecules in tissues, especially 3 H and 13 C, because they are easy to label and convenient to detect, and are more widely used. Certain heavy isotopes, such as deuterium ( 2Substitution at (H) can enhance metabolic stability, extend the half-life, and thus achieve the goal of reducing the dose to provide a therapeutic advantage. Isotope-labeled compounds generally start from the labeled starting materials and are synthesized using known synthetic techniques in the same way as non-isotope-labeled compounds.

[0124] In the present invention, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reliable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0125] "Pharmaceutically acceptable salts" in the present invention refer to salts of the compounds of the present invention, which are formed by the compounds with specific substituents found in the present invention and one of 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, hydroxyethanesulfonic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, subacetic acid, succinic acid, aminosulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin, tartaric acid, and p-toluenesulfonic acid; or when containing relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of 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 salts, ammonium, or organic amines. For example: alkali metal salts, alkaline earth metal salts, other metal salts, inorganic base salts, organic base salts, inorganic acid salts, lower alkanesulfonates, arylsulfonates, organic acid salts, amino acid salts, etc.

[0126] "Solvate" in the present invention is selected from hydrates, ethanolates, methanolates, acetonates, etherates, or isopropanolates.

[0127] The terms "direct bond" and "chemical bond" both refer to two adjacent atoms directly connected by a single bond.

[0128] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0129] The term "hydroxyl" refers to -OH; the term "cyano" refers to -CN; the term "amino" refers to -NH2.

[0130] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group composed of carbon atoms and hydrogen atoms, such as C 1-6Alkyl, including but 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).

[0131] The term "halo C 1-6 alkyl" means an alkyl in which one or more H 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.

[0132] The term "alkyl hydroxy" means a hydroxy group further substituting an alkyl as defined above, such as C 1-6 alkyl hydroxy, including but not limited to methyl hydroxy, ethyl hydroxy, propyl hydroxy or isopropyl hydroxy, etc.

[0133] The term "alkylamino" refers to an open-chain alkyl containing a nitrogen atom, such as C 1-6 alkylamino, including but not limited to methylamino, ethylamino, isopropylamino, dimethylamino, methylethylamino, diethylamino, etc.

[0134] The term "alkylaminoalkyl" refers to an alkyl further substituting an alkylamino 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.

[0135] The term "alkylaminoalkyl" refers to an alkyl further substituting an alkylamino as defined above, such as C 1-6 alkylaminoalkyl refers to C 1-6 alkylamino-C 1-6 alkyl.

[0136] The term "cycloalkyl" refers to a monocyclic alkyl composed of carbon atoms and hydrogen atoms, such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0137] The term "alkoxy" refers to a straight-chain or branched-chain alkyl connected through an oxygen atom, such as C1-6 Alkoxy groups, including but not limited to methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, sec-butoxy, tert-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy, neopentyloxy), hexyloxy (n-hexyloxy, 2-methylpentyloxy, 3-methylpentyloxy, 2,3-dimethylbutoxy, 2,2-dimethylbutoxy), etc.

[0138] The term "halo 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.

[0139] The term "alkenyl" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one double bond. The alkenyl group may contain 2 - 20 carbon atoms, preferably 2 - 10 carbon atoms (i.e., C 2-10 alkenyl), more preferably 2 - 8 carbon atoms (i.e., C 2-8 alkenyl), still more preferably 2 - 6 carbon atoms (i.e., C 2-6 alkenyl), 2 - 5 carbon atoms (i.e., C 2-5 alkenyl), 2 - 4 carbon atoms (i.e., C 2-4 alkenyl), 2 - 3 carbon atoms (i.e., C 2-3 alkenyl), 2 carbon atoms (i.e., C2 alkenyl). For example, "C 2-6 alkenyl" means that the group is 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 vinyl, 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, etc.

[0140] The term "alkynyl" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond. The alkynyl group may contain 2 - 20 carbon atoms, preferably 2 - 10 carbon atoms (i.e., C 2-10 alkynyl), more preferably 2 - 8 carbon atoms (C 2-8 alkynyl), still more preferably 2 - 6 carbon atoms (i.e., C 2-6 alkynyl), 2 - 5 carbon atoms (i.e., C 2-5 alkynyl), 2 - 4 carbon atoms (i.e., C 2-4 alkynyl), 2 - 3 carbon atoms (i.e., C 2-3 alkynyl), 2 carbon atoms (i.e., C2 alkynyl). For example, "C 2-6"Alkynyl" means that the group is 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, etc.

[0141] 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, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0142] The term "alicyclic ring" refers to a monocyclic alicyclic hydrocarbon in which a specific number of carbon atoms are connected to each other by single and double carbon-carbon bonds, preferably containing 3 to 6 carbon atoms. Non-limiting examples of alicyclic rings include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclohexene, etc.

[0143] The term "aromatic ring" refers to a fully carbon monocyclic or fused polycyclic ring of 6 to 16 carbon atoms having a fully conjugated π-electron system, including but not limited to benzene ring, naphthalene ring, anthracene ring, etc., preferably benzene ring.

[0144] The term "aryl" refers to a fully carbon monocyclic or fused polycyclic group of 6 to 16 carbon atoms having a fully conjugated π-electron system, including but not limited to phenyl, naphthyl, anthryl, etc., preferably phenyl.

[0145] The term "heterocycle" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic cyclic hydrocarbon, which is a non-aromatic structure, containing 3-20 ring atoms, wherein one, two, three or more ring atoms are selected from N, O or S, and the remaining ring atoms are C. Bicyclic or polycyclic heterocycles include spiro, fused and bridged heterocycles. Bicyclic or polycyclic "heterocycles" include cases where one ring is an aromatic ring and the other rings are non-aromatic rings, including spiro, fused and bridged heterocycles. Bicyclic heterocycles can contain one or more heteroatoms in one or two rings. In some embodiments, the heterocycle also includes a ring system in which the heterocycle as defined above is fused to one or more carbocyclic groups, wherein the attachment point is on the ring of the carbocycle or heterocycle; or, in some embodiments, the heterocycle also includes a ring system in which the heterocycle as defined above is fused to one or more aromatic / heteroaromatic rings, wherein the attachment point is on the ring of the aromatic / heteroaromatic ring or heterocycle; or, in some embodiments, a ring system in which the heterocycle as defined above is fused to one or more heterocycles as defined above, wherein the attachment point is on the ring of any heterocycle. In the above cases, the ring system of the heterocycle has the number of ring atoms of the fused ring system. In some embodiments, the heterocycle is optionally substituted, for example, unsubstituted (unsubstituted heterocycle) or substituted by 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 1 heteroatom include (but are not limited to) azetidine, oxetane and thietane. Exemplary 5-membered heterocycles containing 1 heteroatom include (but are not limited to) tetrahydrofuran, dihydrofuran, tetrahydrothiophene, dihydrothiophene, pyrrolidine, dihydropyrrole and 2,5-pyrrolidinedione. Exemplary 5-membered heterocycles containing 2 heteroatoms include (but are not limited to) dioxolane, oxathiolane, dithiolane and 2-oxazolidinone. Exemplary 5-membered heterocycles containing 3 heteroatoms include (but are not limited to) triazole, oxadiazole and thiadiazole. Exemplary 6-membered heterocycles containing 1 heteroatom include (but are not limited to) piperidine, tetrahydropyran, dihydropyridine and tetrahydrothiopyran. Exemplary 6-membered heterocycles containing 2 heteroatoms include (but are not limited to) piperazine, morpholine, thiomorpholine, etc. Exemplary 6-membered heterocycles containing 3 heteroatoms include (but are not limited to) triazane, oxadiazane, thiadiazane, oxathiazane and dioxazane. 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, oxocane and thiooctane. Exemplary 5-membered heterocycles fused to a C6 aryl ring (herein also referred to as 5,6-bicyclic heterocycles) include but are not limited to indoline, isoindoline, dihydrobenzofuran, dihydrobenzothiophene, benzoxazolinone. Exemplary 6-membered heterocycles fused to a C6 aryl ring (herein also referred to as 6,6-bicyclic heterocycles) include (but are not limited to) tetrahydroquinoline, tetrahydroisoquinoline.

[0146] The term "heterocyclic group" refers to a substituent generated on the basis of the foregoing heterocyclic definition. Exemplary 3-membered heterocyclic groups containing 1 heteroatom include (but are not limited to) aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include (but are not limited to) azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing 1 heteroatom include (but are not limited to) tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and 2,5-dioxopyrrolidinyl. Exemplary 5-membered heterocyclic groups containing 2 heteroatoms include (but are not limited to) dioxolanyl, oxathiolanyl, dithiolanyl, and 2-oxooxazolidinyl. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include (but are not limited to) triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing 1 heteroatom include (but are not limited to) piperidinyl, tetrahydropyranyl, dihydropyridyl, and tetrahydrothiopyranyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include (but are not limited to) piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing 3 heteroatoms include (but are not limited to) triazinyl, oxadiazinyl, thiadiazinyl, oxathiazinyl, and dioxazinyl. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include (but are not limited to) azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include (but are not limited to) azocanyl, oxocanyl, and thiocanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (herein also referred to as 5,6-bicyclic heterocyclic groups) include (but are not limited to) dihydroindolyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, and benzoxazolinone. Exemplary 6-membered heterocyclic groups fused to a C6 aryl ring (herein also referred to as 6,6-bicyclic heterocyclic groups) include (but are not limited to) tetrahydroquinolinyl and tetrahydroisoquinolinyl.

[0147] The term "heteroaromatic ring" refers to an aromatic monocyclic, bicyclic or polycyclic ring system having a 5- to 16-membered structure, preferably a 5- to 14-membered structure, a 5- to 12-membered structure, a 5- to 10-membered structure, a 5- to 8-membered structure, more preferably a 5- to 6-membered structure, wherein one, two, three or more ring atoms are heteroatoms and the remaining atoms are carbon, and the heteroatoms are independently selected from O, N or S, and the number of heteroatoms is preferably one, two or three. Bicyclic or polycyclic heteroaromatic rings include fused 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, thiadiazole, triazine, phthalazine, quinoline, isoquinoline, pteridine, purine, indole, isoindole, indazole, benzofuran, benzothiophene, benzopyridine, benzopyrimidine, benzopyrazine, benzimidazole, benzophthalazine, 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.

[0148] The term "heteroaryl" refers to a substituent generated on the basis of the foregoing 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, pyrimidyl, pyrazinyl, pyridazinyl, thiadiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuryl, benzothienyl, benzopyridyl, benzopyrimidyl, benzopyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidyl, [1,2,4]triazolo[1,5-a]pyridinyl.

[0149] The term "pharmaceutical composition" refers to a mixture composed of one or more compounds of the present application or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the present application to an organism.

[0150] In the present invention, "a", "one", "the", "at least one" and "one or more" can be used interchangeably. Thus, for example, a mixture composed of "a" pharmaceutically acceptable excipient can be interpreted as meaning that the pharmaceutical composition includes "one or more" pharmaceutically acceptable excipients.

[0151] The term "pharmaceutically acceptable excipient" refers to those excipients that have no significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0152] 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, etc.

[0153] 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, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

[0154] The term "treatment" generally refers to obtaining the desired pharmacological and / or physiological effect. This effect can be therapeutic depending on partially or completely stabilizing or curing the disease and / or the side effects caused by the disease. "Treatment" as used herein encompasses any treatment of a patient's disease, including: (a) inhibiting the symptoms of the disease, i.e., preventing its progression; or (b) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

[0155] The term "effective amount" means the amount of the compound of the present application that (i) treats or prevents a specific disease, condition or disorder, (ii) alleviates, improves 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 the compound of the present application 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 the present disclosure.

[0156] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0157] Based on the target design of NLRP3 inhibitors, the present invention has developed pyrazolopyrimidine compounds with a completely new structure. Relevant biological tests have shown that the compounds of the present invention exhibit significant inhibitory activity against IL-1β expression and high selectivity for NLRP3 inflammasome in THP-1 and PBMC cells. In addition, the compounds of the present invention have low hERG toxicity, good metabolic stability and pharmacokinetic properties; moreover, the compounds of the present invention have no obvious inhibitory effect on a variety of cytochrome P450 (CYP450) enzymes and show good safety in long-term toxicity experiments in rats; in a mouse inflammation model, the compounds of the present invention can significantly reduce the levels of cytokines IL-1β, TNF-α and IL-6 in serum, having great clinical application prospects. In addition, the compound synthesis route provided by the present invention is novel, safe, environmentally friendly and has good production feasibility. Description of the Drawings

[0158] Figure 1 : Effect of Compound 2 in Example 26 on cytokine IL-1β in serum of LPS-induced mice.

[0159] Figure 2 : Effect of Compound 2 in Example 26 on cytokine TNF-α in serum of LPS-induced mice.

[0160] Figure 3 : Effect of Compound 2 in Example 26 on cytokine IL-6 in serum of LPS-induced mice.

[0161] Figure 4 : Effect of Compound 4 hydrochloride in Example 27 on cytokine IL-1β in serum of LPS-induced mice.

[0162] Figure 5 : Effect of Compound 4 hydrochloride in Example 27 on cytokine TNF-α in serum of LPS-induced mice.

[0163] Figure 6 : Effect of Compound 4 hydrochloride in Example 27 on cytokine IL-6 in serum of LPS-induced mice.

[0164] Figure 7 : Effect of continuous intragastric administration of Compound 4 hydrochloride in Example 28 for 28 days on the body weight of male SD rats.

[0165] Figure 8 : Effect of continuous intragastric administration of Compound 4 hydrochloride in Example 28 for 28 days on the body weight of female SD rats. Detailed Description of the Invention

[0166] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the protection scope 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 protection scope of the present invention.

[0167] 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.

[0168] The reaction starting materials and common intermediates involved in the embodiments of the present invention can be purchased commercially or prepared in-house. For the starting materials and common intermediates that need to be prepared in-house, the preparation process is described in detail as follows.

[0169] 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 API 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.). According to the quality inspection report provided by the API supplier, the purity (NMR) was ≥95.0%, and the optical rotation was -6.7° (c=0.45 g / 100 mL, CHCl3).

[0170] The following abbreviations are used in the examples:

[0171] 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): isopropanol; 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 glucuronosyltransferase; In the examples of the present invention, x mL×y means repeating y times, each time x mL. For example, extracting with EA (80 mL×3) means extracting with 80 mL of EA each time and repeating 3 times.

[0172] Preparation of Intermediate 1-2

[0173]

[0174] Step 1: Synthesis of 2,4-Dichloro-5-(dimethoxymethyl)pyrimidine (1-2)

[0175] To the flask, add compound 1-1 (500.0 mg, 2.82 mmol), TsOH·H2O (32.3 mg, 0.17 mmol), trimethyl orthoformate (1.08 g, 10.18 mmol) in sequence, add MeOH (6 mL), and react at 65 °C for 16 hours. After the reaction is completed, the reaction mixture is concentrated under reduced pressure. The residue is purified by column chromatography (EA: Petroleum ether = 1:5) to obtain Intermediate 1-2 (439.0 mg), which is a colorless liquid with a yield of 73.5%.

[0176] LC-MS (m / z): 223.0 [M+H] + 。

[0177] 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)

[0178]

[0179] Step 1: Synthesis of 4-Chloro-2-methoxy-6-methylaniline (1-4)

[0180] To the flask, add compound 1-3 (5.00 g, 21.14 mmol), methylboronic acid (2.54 g, 42.43 mmol), K2CO3 (8.77 g, 63.45 mmol), Pd(dppf)Cl2 (774.0 mg, 1.06 mmol) in sequence. Then add dioxane (60 mL) and water (12 mL), and react at 100 °C for 16 hours under nitrogen protection. After the reaction is completed, dilute the reaction mixture with water (50 mL), extract with EA (80 mL × 3), combine the organic phases, wash with saturated NaCl (80 mL), dry over anhydrous Na2SO4, and filter. Concentrate the filtrate under reduced pressure. Purify the residue by column chromatography (EA: petroleum ether = 1:15) to obtain compound 1-4 (3.48 g), which is a yellow solid with a yield of 95.7%.

[0181] LC-MS (m / z): 172.0 [M+H] + 。

[0182] Step 2: Synthesis of (4-chloro-2-methoxy-6-methylphenyl)hydrazine (1-5)

[0183] Add compound 1-4 (1.87 g, 10.91 mmol) to the flask, add concentrated hydrochloric acid (13 mL), dissolve NaNO2 (1.13 g, 16.38 mmol) in distilled water (13 mL) and add it at 0 °C. After reacting for 0.5 hours, add a solution of SnCl2·2H2O (4.93 g, 21.85 mmol) in concentrated hydrochloric acid (13 mL) at 0 °C and react for 1 hour. After the reaction is completed, filter the reaction mixture, adjust the pH of the filter cake to 8 - 9 with NaOH solution (3N), dilute with water (30 mL), extract with DCM (60 mL × 3), combine the organic phases, wash with saturated NaCl (50 mL), dry over anhydrous Na2SO4, filter, and concentrate the filtrate under reduced pressure to obtain compound 1-5 (1.58 g), which is a yellow solid with a yield of 77.6%.

[0184] LC-MS (m / z): 187.0 [M+H] + 。

[0185] Step 3: Synthesis of 2-chloro-4-(2-(4-chloro-2-methoxy-6-methylphenyl)hydrazino)-5-(dimethoxymethyl)pyrimidine (1-6)

[0186] To the flask were successively added 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). THF (8 mL) was added, and the reaction was carried out at room temperature for 16 hours under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (EA: petroleum ether = 1:5) to obtain Compound 1-6 (439.0 mg) as a yellow solid with a yield of 56.7%.

[0187] LC-MS (m / z): 373.0 [M+H] + 。

[0188] Step 4: Synthesis of 6-chloro-2-(4-chloro-2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (1-7)

[0189] To the flask were successively added Compound 1-6 (339.0 mg, 0.91 mmol) and TsOH·H2O (172.8 mg, 0.91 mmol). ACN (6 mL) was added, and the reaction was carried out at 70 °C for 2 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (EA: petroleum ether = 1:2) to obtain Compound 1-7 (235.0 mg) as a yellow solid with a yield of 73.0%.

[0190] LC-MS (m / z): 309.0 [M+H] + 。

[0191] 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)

[0192] To the flask were successively added 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). n-Butanol (9 mL) was added, and the reaction was carried out by microwave at 100 °C for 1 hour. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH = 10:1), and then lyophilized to obtain Target Compound 1 (250.0 mg) as a yellow solid with a yield of 89.0%.

[0193] LC-MS (m / z): 387.0 [M+H] + 。

[0194] 11H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.42 (s, 1H), 8.16 (s, 1H), 7.21 (d, J = 1.6 Hz, 1H), 7.13 (d, J = 1.6 Hz, 1H), 4.08 - 3.93 (m, 1H), 3.74 (s, 3H), 3.06 - 2.92 (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).

[0195] 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)

[0196]

[0197] To a flask was added Compound 1 (120.0 mg, 0.31 mmol), and ultradry DCM (5 mL) was added. Under nitrogen protection, a DCM solution of BBr3 (1.6 mL, 3.10 mmol, 2 M) was added at 0 °C, and the reaction was carried out at 0 °C for 1 hour. After the reaction was completed, 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 aqueous solution = 85%) to obtain the target compound 2 (69.1 mg), which was a yellow solid with a yield of 60.1%.

[0198] LC-MS (m / z): 373.0 [M + H] + .

[0199] 1 1H NMR (400 MHz, DMSO-d6) δ 9.08 (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).

[0200] 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)

[0201]

[0202] Step 1: Synthesis of 4-bromo-2-methoxy-6-methylaniline (3-2)

[0203] Dissolve 3-1 (22.00 g, 160.30 mmol) in ACN (220 mL). Slowly add NBS (34.25 g, 192.40 mmol) under an ice-water bath. After addition, keep the temperature and continue the reaction for 10 minutes. Concentrate the reaction solution under reduced pressure to remove most of the solvent. Dilute with water (100 mL), extract twice with DCM / MeOH (10:1, 100 mL). Combine the organic phases, wash with saturated NaCl (100 mL), dry over anhydrous Na2SO4, filter, concentrate, and purify by column chromatography (EA: petroleum ether = 4:96) to obtain compound 3-2 (22.34 g), which is a dark brown solid with a yield of 64.5%.

[0204] LC-MS (m / z): 216.0 / 218.0 [M+H] + 。

[0205] Step 2: Synthesis of 4-bromo-2-methoxy-6-methylphenylhydrazine (3-3)

[0206] Under an ice-salt bath, dissolve 3-2 (5.00 g, 23.15 mmol) in concentrated hydrochloric acid (25 mL), keep the internal temperature below 0 °C, slowly add a solution of NaNO2 (2.40 g, 34.72 mmol) in water (25 mL). After keeping the temperature and continuing the reaction for 20 minutes, keep the internal temperature below 5 °C, slowly add a solution of SnCl2·2H2O (10.40 g, 46.30 mmol) in concentrated hydrochloric acid (25 mL), and keep the temperature and continue the reaction for 1 hour. Filter the reaction solution. After rinsing the filter cake with a small amount of water, dilute with DCM (50 mL), adjust to alkaline with saturated aqueous Na2CO3, separate the layers. Extract the aqueous phase with DCM (50 mL), combine the organic phases, wash with saturated NaCl (50 mL), dry over anhydrous Na2SO4, filter, and concentrate under reduced pressure to obtain the crude product of compound 3-3 (3.98 g), which is a dark brown oily liquid.

[0207] Step 3: Synthesis of 4-(2-(4-bromo-2-methoxy-6-methylphenyl)hydrazino)-2-chloro-5-(dimethoxymethyl)pyrimidine (3-4)

[0208] 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), TEA (7.16 mL, 51.67 mmol) was added, and the mixture was reacted 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), which was a brown solid with a two-step yield of 31.6%.

[0209] LC-MS (m / z): 419.0 [M+H] + 。

[0210] Step 4: Synthesis of 2-(4-bromo-2-methoxy-6-methylphenyl)-6-chloro-2H-pyrazolo[3,4-d]pyrimidine (3-5)

[0211] Compound 3-4 (3.05 g, 7.30 mmol) was dissolved in ACN (30 mL), TsOH·H2O (1.39 g, 7.30 mmol) was added, and the reaction solution was reacted at 70 °C for 1 hour. After the reaction solution was 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), which was a brown solid with a yield of 99.9%.

[0212] LC-MS (m / z): 355.0 [M+H] + 。

[0213] 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)

[0214] 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), DIPEA (3.82 mL, 21.90 mmol) was added, and the mixture was reacted at 100 °C for 1 hour. After the reaction solution was 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), which was an off-white solid with a yield of 84.4%.

[0215] LC-MS (m / z): 431.0 / 433.0 [M+H] + 。

[0216] 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)

[0217] Compound 3-6 (0.20 g, 0.46 mmol) was dissolved in DCM (10 mL). Under an ice-water bath, a DCM solution of BBr3 (2.0 M, 1.2 mL) was added, and the mixture was 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 NaHCO3 (20 mL) and saturated NaCl (20 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (MeOH:DCM = 15:85), and then further purified by reverse-phase chromatography (ACN: 0.1% formic acid aqueous solution = 85%) to obtain the target compound 3 (25.8 mg), which was a white solid with a yield of 13.3%.

[0218] LC-MS (m / z): 417.0 / 419.0 [M+H] + 。

[0219] 1 1H NMR (400 MHz, DMSO-d6) δ 9.06 (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 - 2.63 (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).

[0220] 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)

[0221]

[0222] Step 1: Synthesis of 2-bromo-6-methoxy-4-(trifluoromethyl)aniline (4-2)

[0223] Compound 4-1 (1.00 g, 5.23 mmol) was dissolved in ACN (20 mL). While maintaining the internal temperature below 10°C, NBS (977.5 mg, 5.49 mmol) was slowly added under an ice-water bath, and the mixture was 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 obtain compound 4-2 (1.15 g), which was a brown solid with a yield of 81.6%.

[0224] LC-MS (m / z): 270.0 / 272.0 [M+H] + 。

[0225] Step 2: Synthesis of 2-methoxy-6-methyl-4-(trifluoromethyl)aniline (4-3)

[0226] Add 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) into a flask in sequence. Add dioxane (20 mL) and water (2 mL). Under nitrogen protection, react at 100 °C for 16 hours. Cool the reaction solution to room temperature, dilute it with water (50 mL), extract with EA (50 mL × 2), combine the organic phases, wash with saturated NaCl (50 mL), dry over anhydrous Na2SO4, filter, concentrate under reduced pressure, and purify by column chromatography (EA: petroleum ether = 1:9) to obtain compound 4-3 (568.0 mg), which is a green oily liquid with a yield of 65.1%.

[0227] LC-MS (m / z): 206.0 [M+H] + 。

[0228] Step 3: Synthesis of 2-methoxy-6-methyl-4-(trifluoromethyl)phenylhydrazine (4-4)

[0229] Under an ice-salt bath, dissolve compound 4-3 (568.0 mg, 2.77 mmol) in concentrated hydrochloric acid (5 mL), keep the internal temperature below 0 °C, slowly add dropwise a water (5 mL) solution of NaNO2 (286.5 mg, 4.15 mmol), continue the reaction while keeping warm for 40 minutes, then keep the internal temperature below 0 °C, slowly add dropwise a concentrated hydrochloric acid (3 mL) solution of SnCl2·2H2O (1.25 g, 5.34 mmol), and continue the reaction while keeping warm for 1 hour. Filter the reaction solution, wash the filter cake with a small amount of water, then dilute it with EA (20 mL), adjust to alkaline with saturated aqueous Na2CO3 solution, separate the layers, extract the aqueous phase with EA (20 mL), combine the organic phases, wash with saturated NaCl (20 mL), dry over anhydrous Na2SO4, filter, and concentrate under reduced pressure to obtain compound 4-4 (135.0 mg), which is an off-white solid with a yield of 22.1%.

[0230] Step 4: Synthesis of 2-chloro-5-(dimethoxymethyl)-4-(2-(2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)hydrazino)pyrimidine (4-5)

[0231] 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), TEA (0.25 mL, 1.84 mmol) was added, and the mixture was reacted 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 with a yield of 35.3%.

[0232] LC-MS (m / z): 407.0 [M+H] + 。

[0233] Step 5: Synthesis of 6-chloro-2-(2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-2H-pyrazolo[3,4-d]pyrimidine (4-6)

[0234] Compound 4-5 (88.0 mg, 0.22 mmol) was dissolved in ACN (5 mL), TsOH·H2O (41.1 mg, 0.22 mmol) was added, and the reaction solution was reacted at 70 °C for 1 hour. After the reaction solution was 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 oily liquid with a yield of 79.7%.

[0235] LC-MS (m / z): 343.0 [M+H] + 。

[0236] 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)

[0237] 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), DIPEA (0.09 mL, 0.52 mmol) was added, and the mixture was reacted at 100 °C for 1 hour. After the reaction solution was 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.

[0238] LC-MS (m / z): 421.0 [M+H] + 。

[0239] 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)

[0240] Compound 4 - 7 (128.0 mg) was dissolved in DCM (5 mL). A DCM solution of BBr3 (2.0 M, 0.5 mL) was added under an ice - water bath, and the mixture was 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 aqueous solution = 85%) to obtain the target compound 4 (41.3 mg), which is a white solid. The two - step yield was 47.0%, and the e.e.% > 99.9%.

[0241] LC - MS (m / z): 407.0[M + H] + 。

[0242] 1 1H NMR (400 MHz, 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), 2.80 - 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).

[0243] 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

[0244]

[0245] Compound 4 (3.22 g) was dissolved in MeOH (40 mL), and an EA solution of HCl (4.0 M, 10 mL) was added. After reacting at room temperature for 1 hour, it was concentrated and dried to obtain the hydrochloride of compound 4 (3.47 g), which is a yellow solid with a yield of 98.9%.

[0246] LC - MS (m / z): 407.0[M + H] + 。

[0247] 11H NMR (600 MHz, DMSO-d6) δ 11.09 (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 = 2.0 Hz, 1H), 7.25 (d, J = 2.0 Hz, 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).

[0248] 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)

[0249]

[0250] 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)

[0251] To a flask was added Compound 3-6 (100.0 mg, 0.23 mmol), dioxane (2 mL) and water (0.4 mL). Then, 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 added successively. The reaction was carried out under a nitrogen atmosphere at 100 °C for 16 hours. After the reaction was completed, the reaction mixture was diluted with water (10 mL) and extracted with EA (30 mL × 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 obtain Compound 5-1 (77.0 mg) as a yellow solid with a yield of 90.6%.

[0252] LC-MS (m / z): 367.0 [M+H] + .

[0253] 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)

[0254] Compound 5-1 (77.0 mg, 0.21 mmol) was added to a flask, and ultradry DCM (3 mL) was added. Under a nitrogen atmosphere, BBr3 (1.1 mL, 2 M in DCM) was added at 0 °C, and the reaction was carried out at 0 °C for 1 hour. After the reaction was completed, 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 aqueous solution = 85%) to obtain the target compound 5 (10.2 mg), which was a white solid with a yield of 13.8%.

[0255] LC-MS (m / z): 353.0 [M+H] + 。

[0256] 1 1H NMR (400 MHz, DMSO-d6) δ 10.20 - 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).

[0257] 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)

[0258]

[0259] 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)

[0260] Weigh out 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) separately, add dioxane (2.5 mL), then weigh out anhydrous K2CO3 solid (115.3 mg, 0.83 mmol) and dissolve it in water (1 mL) to form a solution, and add this solution to the reaction system. Protect it by replacing with nitrogen, heat to 90 °C and react for 16 hours. Stop heating and let it stand to cool. Add water (50 mL) to the reaction solution, then add EA (40 mL) for extraction. Separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure and purify by column chromatography (DCM:MeOH = 8:1) to obtain compound 6-1 (70.0 mg), which is a brown oil, with a yield of 85.4%.

[0261] LC-MS (m / z): 393.0 [M+H] + 。

[0262] 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)

[0263] First, dissolve compound 6-1 (70.0 mg, 0.178 mmol) in DCM (3 mL), then slowly add dropwise a DCM solution of BBr3 (2 M, 0.5 mL), and then continue to react at room temperature for 1 hour. Slowly add dropwise methanol (2 mL) to quench, then use a rotary evaporator to evaporate the solvent to dryness. Add saturated aqueous NaHCO3 solution (30 mL), then add EA (70 mL) for extraction. Separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure. The obtained crude brown-yellow solid is purified by preparative HPLC, and after freeze-drying, the target compound 6 (10.2 mg) is obtained, which is a white solid, with a yield of 15.1%.

[0264] LC-MS (m / z): 379.0 [M+H] + 。

[0265] 11H NMR (400 MHz, DMSO-d6) δ 9.88 (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 - 2.62 (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).

[0266] 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)

[0267]

[0268] Weigh out Compound 3 (60.0 mg, 0.15 mmol), ethynyltributyltin (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) respectively, add anhydrous DMF (2.5 mL), displace and protect with nitrogen, seal, and heat to 120 °C for reaction for 16 hours. Stop heating and let it stand to cool. Weigh KF (300.0 mg) and dissolve it in water (4 mL) to prepare a solution, add this solution to the reaction system, then add EA (15 mL), and stir at room temperature for 2 hours. Filter to remove insoluble substances, collect the filtrate, separate the organic phase, extract the aqueous phase with EA (30 mL) once again, combine the organic phases, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, purify the obtained solid crude product by preparative HPLC, and after lyophilization, obtain Target Compound 7 (5.5 mg), which is a light yellow solid, with a yield of 10.5%.

[0269] LC-MS (m / z): 363.0 [M + H] + .

[0270] 11H NMR (400 MHz, DMSO-d6) δ 10.36 (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).

[0271] 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)

[0272]

[0273] Compound 3 (60.0 mg, 0.15 mmol) was dissolved in ultradry DMF (4 mL), and then tributyl(prop-1-yn-1-yl)stannane (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) were added successively. The reaction was carried out at 120 °C for 16 h under a nitrogen atmosphere. After the reaction was completed, an aqueous KF solution was added and stirred at room temperature for 2 h. The reaction mixture was diluted with water (10 mL), extracted with EA (30 mL × 3), and 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 target compound 8 (19.9 mg) was obtained as a white solid by purification by reverse-phase chromatography (ACN: 0.1% aqueous formic acid solution = 85%), with a yield of 36.9%.

[0274] LC-MS (m / z): 377.0 [M+H] + .

[0275] 11H NMR (400 MHz, DMSO-d6) δ 10.60 - 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), 2.86 - 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).

[0276] 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)

[0277]

[0278] Step 1: Synthesis of (2-methoxy-6-methylphenyl)hydrazine (9-1)

[0279] First, weigh out compound 3-1 (1.37 g, 9.98 mmol), control the temperature with an ice-water bath, and dissolve it in concentrated hydrochloric acid (6 mL). Then weigh out NaNO2 (1.03 g, 14.97 mmol) and dissolve it in water (6 mL) to form a solution, and add this solution dropwise into the reaction system. After that, continue to control the temperature with an ice-water bath and react for 25 minutes. Then weigh out SnCl2·2H2O (4.50 g, 19.96 mmol) and dissolve it in concentrated hydrochloric acid (5 mL), and then add it dropwise into the reaction system. After that, continue to control the temperature with an ice-water bath and react for 2 hours. Add water (50 mL) to the reaction solution, then dropwise add sodium hydroxide aqueous solution (4 M, 35 mL) to adjust the pH value to 9. Add DCM (80 mL) for extraction, separate the organic phase, wash the organic phase with water (70 mL) once, add anhydrous Na2SO4 for drying, filter, and concentrate under reduced pressure to obtain the crude product of compound 9-1 (1.16 g), which is a red oil.

[0280] LC-MS (m / z): 153.0 [M + H] + 。

[0281] Step 2: Synthesis of 2-chloro-5-(dimethoxymethyl)-4-(2-(2-methoxy-6-methylphenyl)hydrazino)pyrimidine (9-2)

[0282] Weigh 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) separately, dissolve them in anhydrous THF (6 mL), and react overnight at room temperature. Add water (70 mL) to the reaction solution, then add EA (80 mL) for extraction. Separate the organic phase, wash the organic phase with water (50 mL) once, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 5:1) to obtain compound 9-2 (420.0 mg), which is a red oil, with a yield of 62.9%.

[0283] LC-MS (m / z): 339.0 [M+H] + 。

[0284] Step 3: Synthesis of 6-chloro-2-(2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (9-3)

[0285] Weigh compound 9-2 (420.0 mg, 1.24 mmol) and TsOH·H2O (259.4 mg, 1.36 mmol) separately, add ACN (6 mL), and react at 70 °C for 2 hours. Add water (50 mL) to the reaction solution, then add EA (50 mL) for extraction. Separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 1:1) to obtain compound 9-3 (175.0 mg), which is a white solid, with a yield of 51.4%.

[0286] LC-MS (m / z): 275.0 [M+H] + 。

[0287] 1 1H-NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.05 (s, 1H), 7.51 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 7.7 Hz, 1H), 3.73 (s, 3H), 1.97 (s, 3H).

[0288] 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)

[0289] Weigh out 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) separately, add n-butanol (3.5 mL), displace and protect with nitrogen, heat to 100 °C and react for 2 hours. Stop heating, let it stand and cool, concentrate under reduced pressure. The obtained crude product is purified by column chromatography (DCM:CH3OH = 8:1) to obtain compound 9-4 (270.0 mg), which is a white solid.

[0290] LC-MS (m / z): 353.0 [M+H] + 。

[0291] 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)

[0292] Dissolve the crude product of compound 9-4 (270.0 mg) in DCM (6 mL), and then slowly add dropwise the DCM solution of BBr3 (2 M, 1.5 mL) at room temperature. After that, continue to react at room temperature for 2 hours. Control the temperature with an ice-water bath, slowly add dropwise methanol (5 mL) to the reaction solution to quench the reaction, concentrate under reduced pressure, add saturated NaHCO3 aqueous solution (30 mL), then add DCM / CH3OH (8:1, 50 mL) for extraction. Separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, and purify by column chromatography (DCM:CH3OH = 8:1) to obtain the target compound 9 (116.0 mg), which is a brown-yellow solid, and the two-step yield is 55.3%.

[0293] LC-MS (m / z): 339.0 [M+H] + 。

[0294] 1 1H-NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.06 (s, 1H), 8.39 (s, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.06 (s, 1H), 6.90 (d, J = 8.0, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.07 - 3.93 (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).

[0295] 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)

[0296]

[0297] Step 1: Synthesis of 2-Bromo-4-fluoro-6-methoxyaniline (10-2)

[0298] First, weigh 1.20 g (8.50 mmol) of Compound 10-1 and dissolve it in ACN (10 mL). Protect it by purging with nitrogen and control the temperature with an ice-water bath. Weigh NBS (1.58 g, 8.93 mmol) and add it in batches, then continue to react for 1 hour under the ice-water bath. Add saturated NaHCO3 aqueous solution (20 mL) to the reaction solution, then add water (50 mL), and stir well. Extract with EA (60 mL), separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 5:1) to obtain Compound 10-2 (930.0 mg), which is a yellow oil, with a yield of 49.7%.

[0299] LC-MS (m / z): 220.0 / 222.0 [M+H] + 。

[0300] Step 2: Synthesis of 4-Fluoro-2-methoxy-6-methylaniline (10-3)

[0301] Weigh 930.0 mg (4.23 mmol) of Compound 10-2, MeB(OH)2 (505.9 mg, 8.45 mmol), and Pd(dppf)Cl2 (175.0 mg, 0.24 mmol) respectively, and add them to anhydrous dioxane (8 mL). Weigh anhydrous K2CO3 solid (1.75 g, 12.68 mmol) and dissolve it in water (5 mL) to form a solution, and add this solution to the reaction system. Protect it by purging with nitrogen and heat to 100 °C for reaction for 16 hours. Stop heating and let it stand to cool. Add water (50 mL) to the reaction solution, then add EA (50 mL) for extraction, separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 5:1) to obtain Compound 10-3 (463.0 mg), which is a pale yellow solid product, with a yield of 70.6%.

[0302] LC-MS (m / z): 156.0 [M+H] + 。

[0303] 1H-NMR (400 MHz, DMSO-d6) δ 6.61 (dd, J = 12.0 Hz, 2.8 Hz, 1H), 6.45 (dd, J = 12.0 Hz, 2.8 Hz, 1H), 4.24 (s, 2H), 3.76 (s, 3H), 2.07 (s, 3H).

[0304] Step 3: Synthesis of (4-Fluoro-2-methoxy-6-methylphenyl)hydrazine (10-4)

[0305] First, weigh out compound 10-3 (460.0 mg, 2.96 mmol), control the temperature with an ice-water bath, and dissolve it in concentrated hydrochloric acid (3 mL). Then weigh out NaNO2 (306.8 mg, 4.45 mmol) and dissolve it in water (2 mL) to prepare a solution, and add this solution dropwise into the reaction system. After that, continue to control the temperature with an ice-water bath and react for 10 minutes. Then weigh out SnCl2·2H2O (1.34 g, 5.93 mmol) and dissolve it in concentrated hydrochloric acid (5 mL), and add it dropwise into the reaction system. Then continue to control the temperature with an ice-water bath and react for 2 hours. Add water (20 mL) to the reaction solution, then add sodium hydroxide aqueous solution (0.5 M, 10 mL) to adjust the pH value to 9. Add DCM (50 mL) for extraction, separate the organic phase, wash the organic phase with water (30 mL) once, add anhydrous Na2SO4 for drying, filter, and concentrate under reduced pressure to obtain the crude product of compound 10-4 (300 mg), which is a red oil.

[0306] LC-MS (m / z): 171.0 [M+H] + 。

[0307] Step 4: Synthesis of 2-Chloro-5-(dimethoxymethyl)-4-(2-(4-fluoro-2-methoxy-6-methylphenyl)hydrazino)pyrimidine (10-5)

[0308] First, weigh out compound 10-4 (300 mg), compound 1-2 (393.2 mg, 1.76 mmol), and TEA (535.1 mg, 5.29 mmol) and dissolve them in anhydrous tetrahydrofuran (6 mL), and react at room temperature for 16 hours. Add water (50 mL) to the reaction solution, then add EA (50 mL) for extraction, separate the organic phase, wash the organic phase with water (30 mL) once, add anhydrous Na2SO4 for drying, filter, and concentrate under reduced pressure. Purify by column chromatography (PE:EA = 5:1) to obtain compound 10-5 (360.0 mg), which is a red solid with a yield of 57.2%.

[0309] LC-MS (m / z): 357.0 [M+H] + 。

[0310] Step 5: Synthesis of 6-chloro-2-(4-fluoro-2-methoxy-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (10-6)

[0311] Weigh out compound 10-5 (360.0 mg, 1.01 mmol) and TsOH·H2O (211.1 mg, 1.11 mmol) separately, add ACN (6 mL), heat to 70 °C and react for 2 hours. Stop heating and let it cool down by standing. Add water (70 mL) to the reaction solution, then add EA (60 mL) for extraction. Separate the organic phase, add anhydrous Na2SO4 for drying, filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 1:1) to obtain compound 10-6 (134.0 mg), which is a white solid with a yield of 45.3%.

[0312] LC-MS (m / z): 293.0 [M+H] + 。

[0313] 1 1H-NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.04 (s, 1H), 7.13 (dd, J = 12.0, 2.4 Hz, 1H), 6.97 (dd, J = 8.0, 2.8 Hz, 1H), 3.75 (s, 3H), 1.97 (s, 3H).

[0314] 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)

[0315] Weigh out 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) separately, add n-butanol (3.5 mL), displace and protect with nitrogen, heat to 100 °C and react for 2 hours. Stop heating and let it cool down by standing. Directly dry the solvent by rotary evaporation, and purify the obtained crude product by silica gel column chromatography (DCM:MeOH = 8:1) to obtain the crude product of compound 10-7 (220.0 mg).

[0316] LC-MS (m / z): 371.0 [M+H] + 。

[0317] 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)

[0318] The crude product of compound 10-7 (220.0 mg) was dissolved in DCM (3 mL), and the DCM solution of BBr3 (2 M, 2 mL) was added dropwise at room temperature. Then the reaction was continued at room temperature for 1 hour. The temperature was controlled with an ice-water bath, and methanol (4 mL) was added dropwise to the reaction solution to quench the reaction. Then the solvent was removed by rotary evaporation. Saturated aqueous NaHCO3 solution (30 mL) was added, and then extraction was carried out 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), which was an off-white solid with a two-step yield of 44.3%.

[0319] LC-MS (m / z): 357.0 [M+H] + 。

[0320] 1 1H-NMR (400 MHz, DMSO-d6) δ 10.54 (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).

[0321] 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)

[0322]

[0323] Referring to Steps 2 - 5 of Example 1, compound 1-4 in Step 2 was replaced with compound 67-1, and other intermediate materials and preparation methods were the same as those in Example 1. The target compound 67 (395.1 mg) was prepared from compound 67-1 (2.00 g), which was a white solid with a total four-step reaction yield of 8.5%.

[0324] LC-MS (m / z): 371.0 [M+H] + 。

[0325] 11H NMR (400 MHz, DMSO-d6) δ 9.12 (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 - 2.75 (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).

[0326] 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)

[0327]

[0328] Step 1: Synthesis of (R)-1-methylpiperidin-3-ol (48-2)

[0329] Dissolve 48-1 (1.01 g, 5.00 mmol) in anhydrous THF (10 mL). Add LiAlH4 (380.0 mg, 10.00 mmol) under an ice-water bath and react at 40 °C for 3 hours. Quench the reaction mixture with saturated aqueous Na2CO3 until no gas is evolved, filter, wash the filter cake with MeOH, and concentrate the filtrate to obtain 48-2 (357.0 mg) with a yield of 62.0%.

[0330] LC-MS (m / z): 116.0 [M+H] + .

[0331] 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)

[0332] 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 reacting at a constant temperature for 0.5 h, 1 - 7 (100.0 mg, 0.32 mmol) was added, and then it was immediately placed at 50 °C for reaction for 0.5 h. After the reaction solution cooled to room temperature, it was quenched with saturated ammonium chloride aqueous solution, diluted with water (20 mL), and extracted with EA (20 mL×2). The organic phases were combined, washed with saturated brine (20 mL), filtered, concentrated, and purified by column chromatography (MeOH:DCM:TEA = 15:84:1) to obtain 48 - 3 (30.0 mg) with a yield of 24.2%.

[0333] LC - MS (m / z): 388.0 [M + H] + 。

[0334] 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)

[0335] 48 - 3 (30.0 mg, 0.08 mmol) was dissolved in DCM (2 mL). A DCM solution of BBr3 (2.0 M, 0.5 mL) was added, and the mixture was reacted at room temperature for 2 h. The reaction solution was quenched with MeOH under an ice - water bath, concentrated, and purified by two thin - layer chromatography (MeOH:DCM = 1:10) to obtain the target compound 48 (4.6 mg) with a yield of 15.9%.

[0336] LC - MS (m / z): 374.0 [M + H] + 。

[0337] 1 1H NMR (400 MHz, DMSO - d6) δ 10.70 (s, 1H), 9.36 (s, 1H), 8.73 (s, 1H), 6.97 (d, J = 2.2 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 5.10 (tt, J = 8.4, 4.0 Hz, 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).

[0338] 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)

[0339]

[0340] Step 1: Synthesis of 4-chloro-2-(difluoromethoxy)-1-nitrobenzene (60-2)

[0341] Dissolve 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) in anhydrous DMF (20 mL), and react at 100 °C for 5 hours under nitrogen protection. After the reaction solution is cooled to room temperature, adjust it to pH less than 5 with dilute hydrochloric acid, dilute with water (30 mL), extract with EA (30 mL × 2). After combining the organic phases, wash with water (30 mL × 3) and saturated brine (30 mL), dry over anhydrous Na2SO4, filter, concentrate, and purify by column chromatography (PE:EA = 70:30) to obtain 60-2 (2.18 g), with a yield of 84.6%.

[0342] Step 2: Synthesis of 4-chloro-2-(difluoromethoxy)aniline (60-3)

[0343] Dissolve 60-2 (2.18 g, 9.75 mmol) in ethanol (20 mL), add an aqueous solution (10 mL) of NH4Cl (4.70 g, 87.78 mmol) and iron powder (4.92 g, 87.78 mmol), and react at 80 °C for 1 hour. After the reaction solution is cooled to room temperature, filter the reaction solution, dilute the filtrate with EA (50 mL), wash with water (20 mL × 2) and saturated brine (20 mL), dry over anhydrous Na2SO4, filter, concentrate, and purify by column chromatography (PE:EA = 65:35) to obtain the crude product of 60-3 (1.94 g).

[0344] LC-MS (m / z): 194.0 [M+H] + 。

[0345] Step 3: Synthesis of 2-bromo-4-chloro-6-(difluoromethoxy)aniline (60-4)

[0346] Dissolve the crude product of 60-3 (1.94 g) in acetonitrile (20 mL), and slowly add NBS (1.91 g, 10.72 mmol) in an ice-water bath. After addition, continue to keep the reaction warm for 0.5 hour. Dilute the reaction solution with EA (30 mL), wash successively with saturated aqueous NaHCO3 solution (20 mL), water (20 mL) and saturated brine (20 mL), dry with anhydrous Na2SO4, filter, concentrate, and purify by column chromatography (PE:EA = 95:5) to obtain 60-4 (2.10 g), with a two-step yield of 79.1%.

[0347] LC-MS (m / z): 274.0 [M+H] + 。

[0348] Step 4: Synthesis of 4-chloro-2-(difluoromethoxy)-6-methylaniline (60-5)

[0349] Add 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) to a mixed solvent of dioxane (30 mL) and water (3 mL). React at 100 °C for 16 hours under nitrogen protection. After the reaction solution is cooled to room temperature, filter the reaction solution, concentrate it, and purify it by column chromatography (PE:EA = 90:10) to obtain 60-5 (1.23 g) with a yield of 76.8%.

[0350] LC-MS (m / z): 208.0 [M+H] + 。

[0351] Step 5: Synthesis of (4-chloro-2-(difluoromethoxy)-6-methylphenyl)hydrazine hydrochloride (60-6)

[0352] Dissolve 60-5 (1.23 g, 5.95 mmol) in concentrated hydrochloric acid (6 mL). Dropwise add an aqueous solution (6 mL) of NaNO2 (616.2 mg, 8.93 mmol) at 0 °C and keep the reaction at this temperature for 1 hour. Continue to dropwise add a mixed solution of SnCl2·2H2O (2.69 g, 11.91 mmol) and concentrated hydrochloric acid (6 mL) at 5 °C. After addition, gradually raise the temperature to room temperature and react for 1 hour. Filter, wash the filter cake with a small amount of water and EA, and dry to obtain the crude product of 60-6 (704.0 mg), which is directly used in the next step.

[0353] Step 6: Synthesis of 2-chloro-4-(2-(4-chloro-2-(difluoromethoxy)-6-methylphenyl)hydrazino)-5-(dimethoxymethyl)pyrimidine (60-7)

[0354] Add the crude product of 60-6 (704.0 mg) and 2,4-dichloro-5-(dimethoxymethyl)pyrimidine (606.2 mg, 2.72 mmol) to THF (10 mL). Add TEA (1.88 mL, 13.59 mmol) and react at room temperature for 16 hours. Filter the reaction solution, wash the filter cake with EA, concentrate the filtrate, and purify it by column chromatography (PE:EA = 65:35) to obtain 60-7 (497.0 mg) with a two-step yield of 24.6%.

[0355] LC-MS (m / z): 409.0 [M+H]+ .

[0356] Step 7: Synthesis of 6-chloro-2-(4-chloro-2-(difluoromethoxy)-6-methylphenyl)-2H-pyrazolo[3,4-d]pyrimidine (60-8)

[0357] Dissolve 60-7 (497.0 mg, 1.21 mmol) and TsOH·H2O (231.0 mg, 1.21 mmol) in ACN (5 mL), and react at 70 °C for 0.5 h. After the reaction solution is cooled to room temperature, concentrate it and purify it by column chromatography (PE:EA:TEA = 65:34:1) to obtain 60-8 (302.0 mg) with a yield of 72.3%.

[0358] LC-MS (m / z): 345.0 [M+H] + .

[0359] 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)

[0360] Add 60-8 (118.0 mg, 0.34 mmol), 1-8 (46.9 mg, 0.41 mmol) and DIPEA (0.12 mL, 0.68 mmol) to isopropanol (2 mL), and react at 80 °C for 0.5 h. After the reaction solution is cooled to room temperature, concentrate it and purify it by column chromatography (MeOH:DCM = 3:97), and then purify it by reverse-phase preparation (ACN: 0.1% formic acid aqueous solution = 85%) to obtain the target compound 60 (98.4 mg) with a yield of 68.0%.

[0361] LC-MS (m / z): 423.0 [M+H] + .

[0362] 1 1H NMR (400 MHz, DMSO-d6) δ 9.14 (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 - 2.97 (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).

[0363] 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)

[0364]

[0365] 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)

[0366] Dissolve 1-7 (221.0 mg, 0.72 mmol) in n-butanol (2 mL), and successively add (R)-1-methylpyrrolidin-3-amine (65.0 mg, 0.65 mmol) and DIPEA (420.0 mg, 3.75 mmol). React under microwave at 100 °C for 1 hour. After the reaction solution is cooled to room temperature, concentrate the reaction solution under reduced pressure and purify it by column chromatography (DCM:MeOH = 10:1) to obtain 61-1 (181.0 mg) with a yield of 75.1%.

[0367] LC-MS (m / z): 373.0 [M+H] + 。

[0368] 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)

[0369] Add 61-1 (181.0 mg, 0.49 mmol) to a flask, add ultradry DCM (5 mL), and add a DCM solution of BBr3 (2.5 mL, 2.0 M) under an ice-water bath and nitrogen protection. Keep the reaction warm for 1 hour. Quench the reaction of the reaction solution with an appropriate amount of MeOH, concentrate it under reduced pressure, and purify the residue by reverse-phase preparation (ACN: 10 mmol / L aqueous NH4HCO3 solution = 95%) to obtain the target compound 61 (75.9 mg) with a yield of 43.6%.

[0370] LC-MS (m / z): 359.0 [M+H] + 。

[0371] 11H NMR (400 MHz, DMSO-d6) δ 9.08 (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).

[0372] 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)

[0373]

[0374] Step 1: Synthesis of tert-butyl (1-methylazetidin-3-yl)carbamate (62-2)

[0375] Dissolve 62-1 (300.0 mg, 1.74 mmol) in MeOH (6 mL), successively add paraformaldehyde (261.9 mg, 8.72 mmol) and a drop of AcOH, add sodium cyanoborohydride (328.4 mg, 5.23 mmol) under an ice-water bath, and react at 35 °C for 3 hours. Dilute the reaction solution with water (10 mL), extract with EA (20 mL × 3), combine the organic phases, wash with saturated NaHCO3 (20 mL) and saturated brine (20 mL), dry with anhydrous Na2SO4, filter, and concentrate under reduced pressure to obtain the crude product of 62-2 (285.0 mg), which is directly used in the next step.

[0376] LC-MS (m / z): 187.0 [M + H] + .

[0377] Step 2: Synthesis of 1-methylazetidin-3-amine hydrochloride (62-3)

[0378] Dissolve the crude product of 62-2 (285.0 mg) in EA (5 mL), add an EA solution of HCl (5 mL, 4.0 M), and react at room temperature for 2 hours. After the reaction is completed, concentrate under reduced pressure to obtain 62-3 (131.0 mg), and the two-step yield is 61.3%.

[0379] 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)

[0380] 62 - 3 (131.0 mg, 1.07 mmol) was dissolved in isopropanol (6 mL). 1 - 7 (150.0 mg, 0.49 mmol) and DIPEA (984.0 mg, 7.61 mmol) were added successively, and the reaction was carried out at 80 °C for 1 hour. After the reaction solution was cooled to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH = 10:1) to obtain 62 - 4 (259.0 mg) with a yield of 67.5%.

[0381] LC-MS (m / z): 359.0 [M + H] + 。

[0382] 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)

[0383] 62 - 4 (259.0 mg, 0.72 mmol) and anhydrous DCM (5 mL) were added to a flask. A solution of BBr3 in DCM (3.7 mL, 2.0 M) was added under an ice-water bath and nitrogen protection, and the reaction was carried out for 1 hour. The reaction was quenched with an appropriate amount of MeOH under the ice-water bath, and concentrated under reduced pressure. The residue was purified by reverse-phase preparation (ACN: 0.1% formic acid aqueous solution = 85%) to obtain the target compound 62 (4.8 mg) with a yield of 1.9%.

[0384] LC-MS (m / z): 345.0 [M + H] + 。

[0385] 1 1H NMR (400 MHz, DMSO-d6) δ 9.16 (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 = 8.0 Hz, 2H), 3.24 (t, J = 7.2 Hz, 2H), 2.45 (s, 3H), 2.02 (s, 3H).

[0386] 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)

[0387]

[0388] 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)

[0389] Dissolve 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) in isopropanol (3 mL), and react at 80 °C for 3 hours under nitrogen protection. After cooling, concentrate the reaction solution and purify it by column chromatography (DCM:MeOH = 85:15) to obtain compound 63-1 (99.0 mg) with a yield of 76.3%.

[0390] LC-MS (m / z): 401.0 [M+H] + 。

[0391] 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)

[0392] Dissolve 63-1 (99.0 mg, 0.25 mmol) in DCM (2 mL), add a DCM solution of BBr3 (0.5 mL, 2 M) under an ice-water bath, and react at room temperature for 1.5 hours. Dropwise add methanol (2 mL) to quench the reaction, concentrate, and purify by column chromatography (DCM:MeOH = 85:15) to obtain the target compound 63 (44.8 mg) with a yield of 45.0%.

[0393] LC-MS (m / z): 387.0 [M+H] + 。

[0394] 1 1H NMR (400 MHz, DMSO-d6) δ 9.08 (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 = 7.2 Hz, 3H).

[0395] 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)

[0396]

[0397] 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)

[0398] Dissolve 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) in isopropanol (3 mL), and react at 80 °C for 3 hours under nitrogen protection. Stop heating, let it stand and cool, concentrate the reaction solution, and purify it by column chromatography (DCM:MeOH = 85:15) to obtain 64-1 (70.0 mg) with a yield of 55.8%.

[0399] LC-MS (m / z): 388.0 [M+H] + 。

[0400] 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)

[0401] Dissolve 64-1 (70.0 mg, 0.18 mmol) in DCM (3 mL), add a DCM solution of BBr3 (1.5 mL, 2 M) under an ice-water bath, and react at room temperature for 1 hour. Dropwise add methanol (2 mL) to quench the reaction, concentrate, purify by column chromatography (DCM:MeOH = 85:15), and then purify by reverse-phase chromatography (ACN: 0.1% formic acid aqueous solution = 85%) to obtain the target compound 64 (24.5 mg) with a yield of 36.3%.

[0402] LC-MS (m / z): 374.0 [M+H] + 。

[0403] 1 1H NMR (400 MHz, DMSO-d6) δ 10.17 (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).

[0404] 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)

[0405]

[0406] 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)

[0407] 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 the reaction solution cools to room temperature, concentrate it under reduced pressure and purify it by column chromatography (MeOH:DCM = 10%) to obtain the crude product of 65-1 (1.50 g).

[0408] LC-MS (m / z): 421.0 [M+H] + .

[0409] 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)

[0410] Dissolve the crude product of 65-1 (1.50 g) in DCM (20 mL), add a DCM solution of BBr3 (8.92 mL, 2.0 M) in an ice-water bath, and react at room temperature for 4 hours. Quench the reaction solution with MeOH, concentrate it under reduced pressure, and purify the residue by column chromatography (MeOH:DCM = 20%) to obtain the target compound 65 (856.3 mg), with a yield of 71.9% and e.e.% = 99.23%.

[0411] LC-MS (m / z): 407.0 [M+H] + .

[0412] 11H NMR (600 MHz, DMSO-d6) δ 11.20 - 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).

[0413] 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)

[0414]

[0415] Step 1: Synthesis of 3-Fluoro-2-methoxy-4-(trifluoromethyl)aniline (66-2)

[0416] Dissolve 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (14.00 g, 44.30 mmol) and K2CO3 (9.20 g, 66.45 mmol) in anhydrous ACN (350 mL), add 66-1 (9.40 g, 66.45 mmol), and react at 75 °C for 16 hours. Concentrate the reaction solution under reduced pressure, dissolve it in EA (100 mL), filter, wash the filtrate with hydrochloric acid solution (2N, 100 mL × 3) and saturated brine (50 mL × 2), dry over anhydrous Na2SO4, filter, concentrate the filtrate under reduced pressure, and purify the residue by column chromatography (EA:PE = 20:80) to obtain 66-2 (4.90 g) with a yield of 52.9%.

[0417] LC-MS (m / z): 210.0 [M+H] + .

[0418] Step 2: Synthesis of 6-Bromo-3-fluoro-2-methoxy-4-(trifluoromethyl)aniline (66-3)

[0419] 66-2 (4.70 g, 22.47 mmol) was dissolved in DMF (40 mL), NBS (4.20 g, 23.60 mmol) was added, and the mixture was reacted at room temperature for 1 h. 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, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (EA:PE = 20:80) to obtain 66-3 (3.10 g) with a yield of 47.9%.

[0420] LC-MS (m / z): 288.0 / 290.0 [M+H] + 。

[0421] Step 3: Synthesis of 3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)aniline (66-4)

[0422] 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 mixed solvent of dioxane and water (5:1, 90 mL), and reacted at 90 °C for 16 h under nitrogen protection. The reaction solution was concentrated, dissolved in EA (100 mL), filtered, the filtrate was washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (EA:PE = 10:90) to obtain 66-4 (2.10 g) with a yield of 87.4%.

[0423] LC-MS (m / z): 224.0 [M+H] + 。

[0424] Step 4: Synthesis of (3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)hydrazine hydrochloride (66-5)

[0425] Under an ice-salt bath, 66-4 (270.0 mg, 1.21 mmol) was dissolved in concentrated hydrochloric acid (2 mL), and a solution of NaNO2 (125.2 mg, 1.81 mmol) in water (1 mL) was slowly added dropwise at 0 °C. After maintaining the temperature and continuing the reaction for 1 h, a solution of SnCl2·2H2O (546.0 mg, 2.42 mmol) in concentrated hydrochloric acid (1 mL) was slowly added dropwise, and the reaction was continued while maintaining the temperature for 2 h. The reaction solution was filtered, the filter cake was rinsed with a small amount of water, and then dried under reduced pressure to obtain the crude product of 66-5 (200.0 mg).

[0426] Step 5: Synthesis of 2-chloro-5-(dimethoxymethyl)-4-(2-(3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)hydrazino)pyrimidine (66-6)

[0427] The crude product of 66-5 (200.0 mg) and 1-2 (182.8 mg, 0.76 mmol) were dissolved in ACN (3 mL), TEA (0.41 mL, 2.91 mmol) was added, and the reaction was carried out 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 obtain 66-6 (150.0 mg), and the two-step yield was 28.9%.

[0428] LC-MS (m / z): 425.0 [M+H] + 。

[0429] Step 6: Synthesis of 6-chloro-2-(3-fluoro-2-methoxy-6-methyl-4-(trifluoromethyl)phenyl)-2H-pyrazolo[3,4-d]pyrimidine (66-7)

[0430] 66-6 (150.0 mg, 0.35 mmol) was dissolved in ACN (2 mL), TsOH·H2O (67.2 mg, 0.35 mmol) was added, and the reaction solution was reacted at 80 °C for 1 hour. After the reaction solution was cooled to room temperature, it was diluted with EA (10 mL), washed with an aqueous solution of NaHCO3 (10 mL), washed with saturated brine (10 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (EA:PE = 30:70) to obtain 66-7 (70.0 mg), and the yield was 55.0%.

[0431] LC-MS (m / z): 361.0 [M+H] + 。

[0432] 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)

[0433] 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), DIPEA (50.2 mg, 0.39 mmol) was added, and the reaction was carried out at 80 °C for 3 hours. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure, and the residue was purified by column chromatography (MeOH:DCM = 10:90) to obtain the crude product of 66-8 (85.0 mg).

[0434] LC-MS (m / z): 439.0 [M+H]+ 。

[0435] 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)

[0436] Dissolve the crude product of 66-8 (85.0 mg) in DCM (2 mL), add a DCM solution of BBr3 (0.49 mL, 2.0 M) under an ice-water bath, and place it at room temperature for reaction for 1 hour. Quench the reaction solution with MeOH under an ice-water bath, concentrate it under reduced pressure, and purify the residue by reverse-phase preparation (ACN: 10 mmol / L aqueous NH4HCO3 solution = 85%) to obtain the target compound 66 (21.7 mg) with a yield of 26.3%.

[0437] LC-MS (m / z): 425.0 [M+H] + 。

[0438] 1 1H NMR (600 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.45 (s, 1H), 7.11 (s, 1H), 6.94 (d, J = 6.0 Hz, 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).

[0439] Example 21: Inhibitory Effect of the Compounds of the Present Invention on the Release of IL-1β in THP-1 Cells Induced by PMA

[0440] THP-1 cells were purchased from Wuhan Punosai Life Science Co., Ltd. (product number CL-0233), and the complete medium used was RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin double antibody (Pen-Strep), and a final concentration of 0.05 mM β-mercaptoethanol. Cultivate the cells according to the supplier's instructions and maintain the cell density in the logarithmic phase before the experiment. Adjust the cell density to 1×10 5cells / mL, after induction with 100 ng / mL phorbol 12-myristate 13-acetate (PMA) for 16 h, it was stimulated with 100 ng / mL lipopolysaccharide (LPS) for 3 h. The compound of the present invention was dissolved in dimethyl sulfoxide (DMSO) and diluted 3-fold with the culture medium to the required concentration and added to a 96-well plate respectively. After 1 h, sodium nigericin with a final concentration of 10 μM was added to the culture plate containing the drug and incubated for 1 h. The cell-free supernatant was collected, and the IL-1β level was evaluated according to the instructions of the ELISA detection kit (Invitrogen). The vehicle was blank. EC 50 Fitted by the four-parameter method of log(agonist) vs. response-Variable slope using GraphPad software. The results are shown in Table 1.

[0441] Table 1 Inhibitory activity of the compound on the release of IL-1β in THP-1 cells

[0442]

[0443]

Note

[0444] The experimental results show that the compound of the present invention can significantly inhibit the pyroptosis of human-derived THP-1 cells induced by PMA and the expression of IL-1β in these cells.

[0445] Example 22: Inhibitory effect of the compound of the present invention on the release of IL-1β in PBMC cells

[0446] Healthy human peripheral blood mononuclear cells (PBMC) were purchased from Miaoshun Biology (product number PB010C), and the complete culture medium used was RPMI 1640 medium containing FBS (10%) and Pen-Strep (1%). After inoculating PBMC at 1×10 5 cells / well into a 96-well plate, it was cultured overnight in an incubator, and then stimulated with LPS (final concentration 100 ng / mL) for 3 h. The derivative of the present invention was dissolved in DMSO and diluted 3-fold with the culture medium to the required concentration and added to the 96-well plate for continued culture for 1 h, and then adenosine triphosphate (ATP) with a final concentration of 5 mM was added for continued culture for 1 h. The cell-free supernatant was collected, and the IL-1β level was evaluated according to the instructions of the ELISA detection kit (Invitrogen). The vehicle was blank. IC 50 Fitted by the four-parameter method of log(agonist) vs. response-Variable slope using GraphPad software. The results are shown in Table 2.

[0447] Table 2 Inhibitory Activity of Compounds on IL-1β Release in PBMC Cells

[0448] Compound <![CDATA[IC 50 (nΜ)]]> 2 5.9 3 2.4 4 14.8 5 6.9 6 12.2

[0449] Example 23: Inhibitory Effect of Compounds on hERG Ion Channels

[0450] The inhibitory effect of compounds on human hERG ion channels stably expressed in HEK293 cells was tested using the traditional patch clamp method. The compounds were formulated at a concentration of 10 μM. Each cell served as its own control. The compounds were perfused using a perfusion system utilizing their own gravity. After the current was stabilized in each cell, the magnitude of the hERG current before and after the addition of the compound was compared, and the blocking effect of the compound on the hERG current was calculated. The results are shown in Table 3.

[0451] Table 3 Blocking Effect of Compounds on hERG Current

[0452] Compound hERG% @ 10 μM 2 9.3％ 4 27.7％

[0453] Example 24: Detection of Metabolic Stability of Test Compounds

[0454] 1. Preparation of Experimental Materials

[0455] 1.1 Incubation Buffer

[0456] Weigh a certain amount of anhydrous sodium dihydrogen phosphate into a centrifuge tube, add an appropriate amount of ultrapure water, vortex and mix well to prepare a sodium dihydrogen phosphate solution with a concentration of 0.1 mol / L for standby; weigh a certain amount of anhydrous disodium hydrogen phosphate into a centrifuge tube, add an appropriate amount of ultrapure water, vortex and mix well to prepare a disodium hydrogen phosphate solution with a concentration of 0.1 mol / L for standby; mix the sodium dihydrogen phosphate solution (0.1 mol / L) and the disodium hydrogen phosphate solution (0.1 mol / L) in a reagent bottle at a ratio of 19:81 (v:v), mix well to obtain a PB buffer solution with a concentration of 100 mmol / L (the concentration is based on the phosphate ion concentration), and store it at 2 - 8 °C for standby.

[0457] 1.2 Initiating Factors (Mixed Solution of NADPH and UDPGA)

[0458] Accurately weigh an appropriate amount of NADPH into a 1.5 mL centrifuge tube, add an appropriate amount of PB solution, vortex to mix evenly, prepare an 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 to mix evenly, prepare a UDPGA solution with a concentration of 40 mmol / L, place it on ice for later use; respectively transfer the same volume of NADPH and UDPGA solutions into the same centrifuge tube, vortex to mix evenly, prepare a mixed solution of NADPH and UDPGA (containing 20 mmol / L of NADPH and UDPGA), place it on ice for later use.

[0459] 1.3 Positive substrate working solution (mixture of testosterone and 7-hydroxycoumarin)

[0460] Accurately weigh an appropriate amount of testosterone reference substance, dissolve it in DMSO, vortex to mix evenly to make it dissolve, prepare a 20 mmol / L testosterone stock solution, store it in a -20 °C refrigerator; accurately weigh an appropriate amount of 7-hydroxycoumarin reference substance, dissolve it in DMSO, vortex to mix evenly to make it dissolve, prepare a 20 mmol / L 7-hydroxycoumarin stock solution, store it in a -20 °C refrigerator; respectively transfer the same volume of testosterone stock solution and 7-hydroxycoumarin stock solution into the same 1.5 mL centrifuge tube, dilute it with 50% methanol-water to a 20 μmol / L mixed substrate working solution, place it on ice for later use.

[0461] 1.4 Test substrate working solution

[0462] Accurately weigh an appropriate amount of the test substance reference substance, dissolve it in DMSO, vortex to mix evenly to make it dissolve, prepare a 10 mmol / L test substance stock solution, store it in a -20 °C refrigerator for later use; transfer an appropriate amount of the test substance stock solution into a 1.5 mL centrifuge tube, dilute it with a 50% methanol-water diluent to a 20 μmol / L substrate working solution, place it on ice for later use.

[0463] 2 Experimental methods

[0464] The experimental design is divided into three groups, namely the positive control group (PC), the negative control group (NC), and the experimental group. The test substance is incubated with human, rat, or mouse liver microsomes for a certain time under the conditions of NADPH and UDPGA (using the absence of NADPH and UDPGA as the negative control and setting a positive control), add a termination solution to terminate the reaction, use an LC-MS / MS instrument to detect the remaining amount of the test substance in the sample, and compare the concentrations of the test substance at different reaction times with that at T0 to obtain the stability of the test substance 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.

[0465] 2.1 Experimental grouping

[0466] Experimental group: The test substance was incubated with liver microsomes for 60 min under the conditions of NADPH and UDPGA.

[0467] Negative control group (NC): The test substance was incubated with liver microsomes for 60 min without any coenzymes.

[0468] Positive control group (PC): The probe substrates of CYP3A4 and UGT, testosterone and 7-hydroxycoumarin, were incubated with liver microsomes for 60 min under the conditions of NADPH and UDPGA.

[0469] 2.2 Experimental procedures

[0470] (1) Take an appropriate amount of liver microsomes of the required species and thaw them on ice, then gently shake and mix well.

[0471] (2) Experimental group and negative control group (NC): Take an appropriate amount of PB buffer, add an appropriate amount of the working solution of the test substance substrate, and then transfer an appropriate amount of liver microsomes into a 1.5 mL centrifuge tube, pipette 20 - 30 times to mix well, ensuring that the final concentration of liver microsomes is 1 mg / mL (if there are special requirements, the concentration of liver microsomes can be adjusted to 0.5 mg / mL).

[0472] (3) Positive control group (PC): Take an appropriate amount of PB buffer, add an appropriate amount of the working solution of the positive substrate, and then transfer an appropriate amount of liver microsomes into a 1.5 mL centrifuge tube, pipette 20 - 30 times to mix well, ensuring that the final concentration of liver microsomes is 1 mg / mL.

[0473] (4) Experimental group: Aliquot 90 μL of the solution in (2) respectively, pre-incubate in a 37 °C water bath for 5 min, then add 10 μL of the starting factor at 20 mmol / L to initiate the reaction. After reaching the set incubation times, add an appropriate amount of pre-cooled methanol solution containing internal standard to terminate the reaction.

[0474] (n = 2)

[0475] (5) Negative control group (NC): Aliquot 90 μL of the solution in (2), pre-incubate in a 37 °C water bath for 5 min, then add 10 μL of PB buffer. After reaching the set incubation times, add an appropriate amount of pre-cooled methanol solution containing internal standard to terminate the reaction. (n ≥ 2)

[0476] (6) Positive control group (PC): Aliquot 90 μL of the solution in (3), pre-incubate in a 37 °C water bath for 5 min, then add 10 μL of the starting factor at 20 mmol / L to initiate the reaction. After reaching the set incubation times, add an appropriate amount of pre-cooled methanol solution containing internal standard to terminate the reaction. (n = 2)

[0477] (7) The prepared sample above was vortexed at 2500 rpm for 1 min, centrifuged at 17000 g and 4 °C for 10 min, and the supernatant was taken for LC-MS / MS detection.

[0478] 3.3 Data processing and analysis

[0479] The stability samples were quantified using a standard curve to obtain the concentration of the test substance at each time point, and the percentage of the remaining amount of the parent compound relative to the amount of the parent compound before incubation (0 min) was calculated; alternatively, the percentage of the remaining amount of the parent compound relative to the amount of the parent compound before incubation (0 min) was calculated by the ratio of the peak area of the test substance to the peak area of the internal standard. The data were calculated according to the following formula: Parent remaining percentage (%of 0 min) = T 60 Amount of parent compound / Amount of parent compound at T0 × 100%; T 60 : Incubation time point of 60 min; T0: Incubation time point of 0 min. The results are shown in Table 4.

[0480] Table 4 In vitro metabolic stability of compounds in liver microsomes

[0481]

[0482] Example 25: Pharmacokinetic evaluation of some compounds in rats

[0483] Male SD rats, after fasting overnight (with free access to water), were divided into an intravenous (IV) administration group and an oral (PO) administration group. In the IV group, blood was collected from the orbital venous plexus at 2 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration, approximately 0.3 mL of blood was collected from each sample, anticoagulated with disodium ethylenediaminetetraacetate (EDTA-2Na), placed on ice after collection, and centrifuged at 4 °C for 10 min within 1 hour to separate plasma, which was stored at -20 °C for further analysis. In the PO group, 0.3 mL of blood was collected from the orbital venous plexus at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration, and the treatment method was the same as that of the IV group. The plasma concentration of the prototype drug was determined by LC-MS / MS method, and the pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.1 based on the blood drug concentration data at different time points. The results are shown in Table 5.

[0484] Table 5 Pharmacokinetic test results of some compounds in rats

[0485]

[0486] The experimental results show that the compounds of the present invention have good pharmacokinetic properties in SD rats, including good oral bioavailability, oral exposure, maximum plasma concentration, half-life, etc.

[0487] Example 26: Inhibitory effect of Compound 2 on cytokines in a mouse inflammation model

[0488] Female C57BL / 6 mice aged 6 - 8 weeks were randomly divided into groups of 5 each, namely the Control group, the Model group, and the Compound 2 group. The Compound 2 group was given Compound 2 (10 mg / kg) by gavage once a day for three consecutive days. The Control group and the Model group were given the corresponding volume of vehicle every day. 1 h after the gavage on the third day, the mice in the Model group and the Compound 2 group were intraperitoneally injected with LPS (5 mg / kg), and the mice in the Control group were intraperitoneally injected with the corresponding volume of sodium chloride injection. Blood was collected from each group of mice 2 h after the intraperitoneal injection of LPS. After standing at room temperature for 1 h, the blood was centrifuged at 4°C and 5000 rpm for 10 min, and the serum was collected. Subsequently, cytokines IL-1β, TNF-α, and IL-6 were detected by ELISA. The experimental results are shown in Figure 1 、 Figure 2 and Figure 3 , where *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

[0489] Experimental conclusion: The compounds of the present invention can significantly reduce the levels of cytokines IL-1β, TNF-α, and IL-6 in the serum of LPS-induced mice.

[0490] Example 27: Inhibitory effect of Compound 4 hydrochloride on cytokines in a mouse inflammation model

[0491] Female C57BL / 6 mice aged 6 - 8 weeks were randomly divided into groups of 5 each, namely the Control group, the Model group, and the Compound 4 hydrochloride administration group (hereinafter referred to as the administration group). The administration group was given Compound 4 hydrochloride (10 mg / kg, 3 mg / kg, 1 mg / kg) by gavage once a day for three consecutive days. The Control group and the Model group were given the corresponding volume of vehicle every day. 1 h after the gavage on the third day, the mice in the Model group and the administration group were intraperitoneally injected with LPS (5 mg / kg), and the mice in the Control group were intraperitoneally injected with the corresponding volume of sodium chloride injection. Blood was collected from each group of mice 4 h after the intraperitoneal injection. After standing at room temperature for 1 h, the blood was centrifuged at 4°C and 5000 rpm for 10 min, and the serum was collected. Subsequently, cytokines IL-1β, TNF-α, and IL-6 were detected by ELISA. The experimental results are shown in Figure 4 、 Figure 5 and Figure 6, where *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

[0492] Example 28: Toxicity Investigation of Compound 4 Hydrochloride by Oral Gavage in SD Rats for 28 Consecutive Days

[0493] Eight male and eight female SD rats were randomly divided into a normal control group with 2 rats / sex, and low- and high-dose groups with 3 rats / sex each. The low- and high-dose groups were orally gavaged with 3 and 10 mg / mL (calculated as the base) of the drug solution, respectively, with a dosing volume of 10 mL / kg, and the dosing doses were 30 and 100 mg / kg, respectively. The normal control group was orally administered the corresponding vehicle water. During the experiment, the rats had free access to water and were continuously dosed for 28 days. On the day after the last dose, blood was collected to detect 23 routine blood routine indexes and biochemical indexes, and the rats were dissected to grossly observe the organs such as the heart, liver, spleen, lung, kidney, brain, gastrointestinal tract, thymus, testis (ovary), etc.

[0494] The experimental results showed that no obvious toxic reactions were observed in the animals of each dose group after dosing, and there was no obvious effect on the body weight of the animals. The experimental results are shown in Figure 7 and Figure 8 . Gross observation of the organs of the animals in each dose group showed no obvious pathological changes in the surface color, shape, size, texture, etc. of the organs. There were no obvious abnormalities in the blood routine and biochemical indexes compared with the normal group. The compound of the present invention has excellent safety.

[0495] Example 29: Inhibitory Effects of Compound 4 Hydrochloride on Cytochrome P450 Isoenzymes CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4-M, and CYP3A4-T

[0496] 1. Preparation of Phosphate Buffer (PB, 100 mM, pH 7.4)

[0497] Disodium hydrogen phosphate was ultrasonically dissolved in ultrapure water to prepare a 100 mM solution as Solution A. Potassium dihydrogen phosphate was ultrasonically dissolved in ultrapure water to prepare a 100 mM solution as Solution B. Solution A was slowly added to Solution B on a stirrer until the pH value reached 7.4. The phosphate buffer was stored at 4 °C for later use. The ultrapure water was prepared by a Sartorius ultrapure water machine.

[0498] 2. Preparation of Positive Control Solution

[0499] The positive control was prepared into a 10 mM DMSO stock solution, and the volume ratio of the organic solvent introduced into the test system with the test substance was 1.0%. The working solution concentration of the positive inhibitor and the final concentration in the reaction system are shown in Table 6 below.

[0500] Table 6

[0501]

[0502] 3. Preparation of Cocktail Substrate Stock Solution

[0503] The specific preparation of the substrate stock solution is shown in Table 7 below. After the stock solution is prepared, it is stored in a -20°C refrigerator. It is melted at room temperature before use.

[0504] Table 7

[0505]

[0506] 4. Preparation of Working Solution of Compound 4 Hydrochloride

[0507] Compound 4 hydrochloride is prepared into a 5 mM DMSO stock solution, and the volume ratio of the organic solvent introduced into the test system with the test substance is 1.0%. The concentration information of the working solution prepared from compound 4 hydrochloride and its final concentration in the reaction system is shown in Table 8 below.

[0508] Table 8

[0509]

[0510] 5. Preparation of Human Liver Microsome Mixture

[0511] Human liver microsomes are purchased from BIOIVT (product number: 452117, batch number: SEU), and the stock solution concentration is 20 mg / mL. The concentration information of the working solution prepared from human liver microsomes and its final concentration in the reaction system is shown in Table 9 below.

[0512] Table 9

[0513]

[0514] 6. Preparation of NADPH Cofactor Solution

[0515] Before the experiment, 13.14 mg of NADPH (Roche, product number 10107824001, batch number 71466624) is weighed and a 10 mM working solution is prepared with 1.545 mL of PB. The final concentration of NADPH in the test system is 1 mM.

[0516] 7. The entire incubation process was carried out in 1.5 mL centrifuge tubes. First, 2 μL of the working solution of compound 4 hydrochloride or the positive control was added to the corresponding centrifuge tubes, and 2 μL of the DMSO:MeOH (1:1) solvent was added to the inhibitor-free group. Then, 20 μL of the mixed substrate working solution and 158 μL of the liver microsome working solution were added, and the mixture was preheated in a 37 °C water bath for 5 minutes. After adding 20 μL of the NADPH cofactor to initiate the reaction, the incubation was continued at 37 °C for the corresponding time (see Table 10). The test samples were prepared in duplicate.

[0517] After the incubation was completed, 400 μL of the freezing termination solution (methanol containing 1 ng / mL LMTA) was added to terminate the reaction. After vortex mixing, the samples were centrifuged at 17000 g and 4 °C for 10 minutes. 180 μL of the supernatant was transferred to a 96-well plate for LC-MS / MS analysis.

[0518] The relative activity was calculated using the following formula: Relative activity (NC) = amount of metabolite generated in the test group or positive control group / average amount of metabolite generated in the blank negative control group; Inhibition rate = (1 - NC) × 100%. The experimental results are shown in Table 10.

[0519] Table 10 Inhibitory effects of compound 4 hydrochloride on CYP450 isoenzymes

[0520]

[0521] The experimental results showed that the compounds of the present invention did not show obvious direct inhibitory effects on the 7 main subtypes of human liver microsome CYP450 enzymes.

Claims

1. A pyrazolopyrimidine compound, which is a compound having the following structural formula (I), an isomer or a pharmaceutically acceptable salt thereof: in, is selected from a single bond or a double bond; m 1 、m 2 、m 3 、m 4 、m 5 Each independently selected from 1 or 2; X and Y are each independently selected from C or N; R 1 , R 5 are each independently selected from the group consisting of absence, 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, 3-6 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 The 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; R 2 , R 3 , R 4 are 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 The 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; or R 3 With 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; or R 3 With 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; R 1 , R 2 , R 3 , R 4 , R 5 At least one is a hydroxyl group; R 6 Selected from hydrogen, halogen or C 1-6 Alkyl; the C 1-6 The alkyl group may be further optionally substituted with one or more selected from C 1-6 substituted by an alkyl or halogen substituent; L is a chemical bond, NR 7 , OR 7 or C(R 7 )2; R 7 Selected from hydrogen, C 1-6 Alkyl or 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 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; 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 selected from hydroxy, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl hydroxyl, -COOR 9 、-CH2COOR 9 or C 1-6 substituted by an alkylaminoalkyl substituent; R 9 Selected from hydrogen or C 1-6 alkyl; 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.

2. The pyrazolopyrimidine compound according to claim 1, characterized in that A compound having the following general structural formula (IA), an isomer or a pharmaceutically acceptable salt thereof: 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 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 of them 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 the cycloalkyl, 3-6 membered heterocyclic group, or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: 1-6 Alkyl or halogen; or R 3 With a nearby 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 nearby 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; R 6 is 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; L is a direct key, NR 7 , OR 7 or CR 7 ; R 7 Selected from H, C 1-6 Alkyl or absent; R 8 is 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: 1-6 Alkyl or halogen; 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, the substituent is selected from one or more of the following groups: C 1-6 Alkyl, hydroxyl, halogen, C 1-6 Haloalkyl, C 1-6 Alkyl hydroxyl, -COOR 9 or C 1-6 Alkylaminoalkyl; R 9 Selected from hydrogen or C 1-6 alkyl; The heterocyclic group and the 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 having the following general structural formula (IB), an isomer or a pharmaceutically acceptable salt thereof: 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 selected from hydroxy, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl hydroxyl, -CH2COOR 9 or C 1-6 substituted by an alkylaminoalkyl substituent; X, Y, L, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , R 9 The definition is as stated in claim 1.

4. The pyrazolopyrimidine compound according to any one of claims 1 to 3, characterized in that A compound having the following general structural formula (II), an isomer or a pharmaceutically acceptable salt thereof: Wherein, X and Y are each independently selected from C or N; 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 The 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; R 2 , R 3 , R 4 are 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 The 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; or R 3 With 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; or R 3 With 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; 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, 3-9 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 selected from hydroxy, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkylhydroxyl, -CH2COOEt or C 1-6 substituted by an alkylaminoalkyl substituent; 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.

5. The pyrazolopyrimidine compound according to any one of claims 1 to 3, characterized in that A compound having the following general structural formula (IIA), an isomer or a pharmaceutically acceptable salt thereof: 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 of them 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 the cycloalkyl, 3-6 membered heterocyclic group, or 5-10 membered heteroaryl group, the substituent is selected from one or more of the following groups: 1-6 Alkyl or halogen; or R 3 With a nearby 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 nearby 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; 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 3-8 membered heterocyclyl, substituted or unsubstituted 5-10 membered heteroaryl; when C 3-8 When there is at least one substituent on the cycloalkyl, 3-8 membered heterocyclic or 5-10 membered heteroaryl, the substituent is selected from one or more of the following groups: 1-6 Alkyl, hydroxyl, halogen, C 1-6 Haloalkyl, C 1-6 Alkylhydroxyl, -CH2COOEt or C 1-6 Alkylaminoalkyl; The heterocyclic group and the 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 having the following general structural formula (II-1), an isomer or a pharmaceutically acceptable salt thereof: 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, 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 The 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; or R 3 With 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; or R 3 With 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; R 1 , R 2 , R 3 , R 4 , R 5 At least one is a hydroxyl group; L is defined as in claim 4.

7. The pyrazolopyrimidine compound according to any one of claims 1 to 4 and 6, characterized in that: A compound having the following general structural formula (IIA-1), an isomer or a pharmaceutically acceptable salt thereof: wherein n is selected from 1 or 2; L is selected from O, NH or N(CH3); 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 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 The cycloalkyl, 3-6 membered heterocyclyl or 5-10 membered heteroaryl may be optionally further substituted with one or more halogen or C 1-6 The alkyl group is substituted with an alkyl substituent.

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

9. The pyrazolopyrimidine compound according to claim 8, characterized in that: R 3 Selected from hydrogen, halogenated C 1-6 Alkyl or halogen.

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

11. The pyrazolopyrimidine compound according to any one of claims 1 to 8 and 10, characterized in that: R 3 is selected from hydrogen, halogen, cyano, methyl, cyclopropyl, trifluoromethyl, difluoromethyl, trifluoromethoxy, dimethylamino, 12. A pyrazolopyrimidine compound, which is a compound, an isomer or a 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 medicament for preventing or treating a disease associated with NLRP3.

15. The use according to claim 14, characterized in that: The NLRP3-related disease is cancer, 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 cancer, laryngeal cancer, esophageal cancer, bile duct cancer, oral cancer, head and neck cancer, mesothelioma, adrenocortical carcinoma, 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; The inflammatory diseases or diseases accompanied by inflammatory responses include, but are not limited to: 1) Autoinflammatory diseases, such as chillin-associated periodic syndrome (CAPS), familial Mediterranean fever, Schnitzler syndrome, and mevalonate kinase deficiency (MKD); 2) Chronic pain, including neuropathic pain and non-neuropathic pain; 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; 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, pneumonia; 5) Joint diseases, such as arthritis; 6) Muscle diseases, such as polymyositis and myasthenia gravis; 7) Cardiovascular diseases, such as hypertension, ischemia, reperfusion injury, vasculitis, pericarditis; 8) Blood diseases, 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, and 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 disease, such as acute kidney disease, hyperoxaluria, chronic kidney disease, nephrocalcinosis, glomerulonephritis, and diabetic nephropathy; 13) Gastrointestinal diseases, such as inflammatory bowel disease and pancreatitis; 14) Eye diseases, such as uveitis and allergic conjunctivitis; 15) Graft-versus-host disease; 16)Burns, sunburn, and mechanical injuries.

16. The use or method according to claim 15, characterized in that The neuropathic pain includes central neuralgia and peripheral neuropathic pain; 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. The peripheral neuropathic pain includes, but is not limited to, post-herpetic 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; The non-neuropathic pain includes, but is not limited to, osteoarthritis pain, chronic low back pain, chronic visceral pain, cancer pain, and fibromyalgia.

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