Pyrazolopyridine compound, composition and application thereof

Abstract

The invention discloses a pyrazolopyridine compound, a composition and application of the pyrazolopyridine compound, and belongs to the field of chemical medicines. The invention provides a pyrazolopyridine compound as shown in a formula (I), or pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotope variant thereof, which can be used for treating and preventing human diseases including virus infection and cancer. The compound is used for treating and / or preventing related diseases such as inflammatory diseases and symptoms, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer) and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine, and particularly relates to a pyrazolo pyridine compound, a composition and use thereof. BACKGROUND

[0002] Stimulator of interferon genes (STING), which exists mainly in the form of symmetric dimers on the endoplasmic reticulum membrane, is a transmembrane protein. STING encodes viruses and bacteria, which are major regulators of the innate immune response to infection, promoting innate immune signals. And as a sensor of bacterial and viral cytosolic DNA, it promotes the production of type I interferons (IFN-alpha and IFN-beta). The protein is a pattern recognition receptor (PRP) that can detect cytoplasmic nucleic acids and transmit signals to activate the type I interferon response. Activation of PRP leads to upregulation of genes involved in inflammatory responses, including type I interferons (also known as IFN or INF), pro-inflammatory cytokines and chemokines that inhibit pathogen replication and promote adaptive immunity.

[0003] Adapter protein STING (also known as TMEM173) has been identified as a central signaling molecule in the innate immune sensing pathway in response to cytosolic nucleic acids. STING is essential for the response to cytosolic DNA from pathogens or host-derived cytosolic DNA. The activation of STING by cyclic dinucleotides (CDN) produced in response to cytosolic DNA leads to the upregulation of IRF3 and NFKB pathways, resulting in the induction of interferon beta (INF-beta) and other cytokines.

[0004] A (STING) activator with excellent potency is disclosed in the patent document WO2022195462A1 of Pfizer, which has the following structure:

[0005]

[0006] This small molecule compound (AIF11) can activate the innate immune response (including the activation of type I INF and other cytokines), which can be an important strategy for the treatment and prevention of human diseases including viral infections and cancers. Such immunomodulatory strategies have the potential to identify compounds that can be used to treat diseases and conditions such as inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants.

[0007] However, the biological activity of the above-mentioned currently reported (STING) activators still needs to be further improved. SUMMARY

[0008] The present application provides a new class of pyrazolopyridine compounds, which have more optimal biological activity.

[0009] The present application provides a new class of pyrazolopyridine compounds, which have more optimal biological activity.

[0010] An object of the present application is to provide a pyrazolopyridine compound of the structure shown in general formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically labeled, prodrug thereof,

[0011]

[0012] In the formula:

[0013] R, R' are independently selected from substituted or unsubstituted C1-4alkyl, C3-6cycloalkyl, groups substituted on C1-4alkyl, C3-6cycloalkyl are independently selected from any one or more of halogen, hydroxyl, cyano, C1-4alkoxy;

[0014] X, Y, Z are independently selected from N, CR"; R" is H, substituted or unsubstituted C1-4alkyl, C3-6cycloalkyl, groups substituted on C1-4alkyl, C3-6cycloalkyl are independently selected from any one or more of halogen, hydroxyl, cyano, C1-4alkoxy.

[0015] In an embodiment of the present application, R, R' are independently selected from -(CH2) n -(CH2) n OCH3, n = 0, 1, 2, 3.

[0016] In an embodiment of the present application, X, Y, Z are independently selected from -N-, -CH- or -CCH3.

[0017] In an embodiment of the present application, preferably:

[0018] X is N, Y is N, Z is N;

[0019] or X is N, Y is CR", Z is N;

[0020] or X is CR", Y is N, Z is N;

[0021] or X is CR", Y is N, Z is CR";

[0022] or X is CR", Y is CR", Z is N.

[0023] In one embodiment of the present application, further: X is CR", Y is N, Z is N, or X is CR", Y is N, Z is CR", R' is preferably unsubstituted C1-4alkyl, C3-6cycloalkyl.

[0024] In one embodiment of the present application, the above-mentioned pyrazolopyridine compound can be specifically selected from:

[0025]

[0026] The present application provides a compound of general formula (I) or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate.

[0027] In one aspect, the present application provides a use of the above-mentioned compound of general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically labeled, prodrug thereof in the preparation of a medicament for treating and / or preventing inflammatory diseases and conditions.

[0028] In another aspect, the present application provides a use of the above-mentioned compound of general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically labeled, prodrug thereof in the preparation of a medicament for treating and / or preventing lung cancer, bone cancer and other cell abnormal proliferation related diseases.

[0029] The present application also provides a preparation method of the compound of general formula (I).

[0030] The present application also provides a pharmaceutical composition containing the above-mentioned compound of general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically labeled, prodrug thereof, and a pharmaceutical excipient.

[0031] In one embodiment of the present application, the pharmaceutical excipient comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0032] The pharmaceutically acceptable carrier includes microspheres, nanoparticles and liposomes.

[0033] In one embodiment of the present application, the pharmaceutical composition dosage form includes injection solution, injection lyophilized powder, suspension, implant, embolism, capsule, tablet, pill and oral liquid.

[0034] Advantages:

[0035] The compounds of the present application can be used as an important strategy for treating and preventing human diseases, including viral infections and cancers, have significantly improved activity or selectivity compared to the positive control (WO2022195462 A1, Example AIF11), and can be used for treating and / or preventing inflammatory diseases and disorders, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants.

[0036] Among them, the cancer is lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal axis tumor, brain stem glioma or pituitary adenoma.

[0037] In addition, the compound or salt thereof of the present application has almost no side effects and has good drug metabolism and pharmacokinetics. Therefore, the compound designed in the present application has good development and application prospect. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be described in detail below in combination with examples.

[0039] In the present application, "substituted" means that one or more hydrogen atoms in a group are replaced by the same or different substituent.

[0040] In the present application, "C3-6cycloalkyl" refers to a three to eight-membered ring without heteroatom hybridization or with heteroatom hybridization. The heteroatoms include N, O, or S. Specifically, for example: cyclopropyl, cyclobutyl, oxetanyl, azetidinyl, etc.

[0041] In the present application, "administration" or "administration" of a compound to an individual means providing the compound of the present application to an individual in need of treatment.

[0042] The term "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0043] <Pharmaceutical composition>

[0044] As used herein, the term "pharmaceutical composition" refers to a composition comprising one or more of the compounds described herein, or a pharmaceutically acceptable isomer, racemate, hydrate, solvate, isotope, or salt thereof, formulated with a pharmaceutically acceptable carrier, which can further include other additives, and is approved by a government regulatory agency for manufacture or sale as part of a therapeutic regimen for the treatment of a disease in a mammal.

[0045] The compounds of the invention and their pharmaceutically acceptable pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., tablets, capsules, caplets, soft gels, or syrups); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for administration to pediatric subjects (e.g., solutions, syrups, suspensions, elixirs, powders for reconstitution into a suspension or solution, dispersible / effervescent tablets, chewable tablets, lollipops, popsicles, lozenges, oral strips, orally disintegrating tablets, orally disintegrating strips, and sprayable oral powders or granules); or in any other formulation described herein. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21stEdition, Gennaro, Ed., Lippencott Williams & Wilkins (2005) and The United States Pharmacopeia: The National Formulary (USP 36 NF31) published in 2013.

[0046] The compounds of the invention and their pharmaceutically acceptable salts, isomers, hydrates, solvates, or isotopes, as well as including tautomeric, polymorphic, stereoisomeric, and isotopically labeled forms.

[0047] The compounds of the invention and their pharmaceutically acceptable salts can be provided in dosage forms suitable for parenteral injection, including, but not limited to, physiologically acceptable, sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.

[0048] The compounds of the invention or their pharmaceutically acceptable salts can be provided in dosage forms suitable for topical administration, including, for example, as ointments, powders, suppositories, drops, sprays, and inhalers. The compounds of the invention of general formula (I) or their pharmaceutically acceptable salts as active ingredients are mixed in a sterile condition and with physiologically acceptable carriers and, optionally, preservatives, buffers, and, if necessary, propellants.

[0049] The pharmaceutical composition of the present application comprises a compound of general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient, diluent. In preparing the pharmaceutical composition, generally, the compound of general formula (I) or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable carrier, excipient or diluent. The content of the compound of general formula (I) or a pharmaceutically acceptable salt thereof can be 0.01-1000 mg, for example, 0.05-800 mg, 0.1-500 mg, 0.01-300 mg, 0.01-200 mg, 0.05-150 mg, 0.05-50 mg, etc.

[0050] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The technical solutions of the present application will be described in detail in conjunction with the examples.

[0051] The following examples are used to illustrate but not limit the synthetic method of the compound of general formula (I). The temperature is in Celsius. If not otherwise specified, all evaporations are carried out under reduced pressure. If not otherwise specified, otherwise the reagents are purchased from commercial suppliers and used without further purification. The structure of the final product, intermediate and raw material is confirmed by standard analytical methods, for example, elemental analysis, spectral characteristic analysis, for example, MS, NMR. The abbreviations used are the conventional abbreviations in the art.

[0052] Preparation of intermediate a-7: 4-bromo-N-benzyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3- c]pyridine-6-carboxamide

[0053]

[0054] First step: synthesis of (Z)-ethyl 1-methyl-5-((2-methyl-5-oxazol-4(5H)-ylidene)methyl)-1H- pyrazole-4-carboxylate (a-2)

[0055]

[0056] At room temperature, 5-formyl-l-methyl-lH-pyrazole-4-carboxylic acid ethyl ester (a-l, 100.0 g, 547.7 mmol) and N-acetylglycine (96.3 g, 822.5 mmol) were dissolved in acetic anhydride (150 mL, 4 M), potassium acetate (9.09 g, 88.0 mmol) was added and an additional 5 mL of acetic anhydride was added and stirred. The reaction was heated to 100 °C and refluxed. During the heating, the white turbid suspension turned into a clear yellow solution and after 10 minutes into a brown solution. After 1 hour, the reaction was cooled to room temperature. Under magnetic stirring, saturated aqueous sodium bicarbonate solution was added dropwise until the effervescence stopped. The aqueous layer was extracted with 4*1000 mL dichloromethane: isopropanol = 3: 1 (V / V) and 2*1500 mL dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The obtained dark brown residue was dissolved in about 50 mL DCM. To this, methyl tert-butyl ether (about 50 mL) was added dropwise to obtain a light yellow solid (Z)-l-methyl-5-((2-methyl-5-oxazol-4(5H)-ylidene)methyl)-lH-pyrazole-4-carboxylic acid ethyl ester (a-2, 142.1 g, 98%). MS-ESI (m / z): 264.09 [M+l] + .

[0057] Second step: synthesis of l-methyl-4-oxo-4,5-dihydro-lH-pyrazolo[4,3-c]pyridine-6- carboxylic acid (a-3)

[0058]

[0059] Intermediate a-2 (141.5 g, 538.1 mmol) was dissolved in methanol (538 mL, 1 M), potassium carbonate (168 g, 1076.2 mmol) was added, heated to 70 °C and stirred for 16 hours. After the reaction was complete, it was filtered under reduced pressure and the filter cake was washed with methanol (1000 mL MeOH) and methyl tert-butyl ether (MTBE). The solid filter cake was suspended in water and concentrated hydrochloric acid was added to acidify to pH 1. A yellow-brown solid precipitated, which was filtered off under reduced pressure, after which the filtrate was diluted with MeOH / MTBE = 1 : 1 and filtered again under reduced pressure. Compound l-methyl-4-oxo-4,5-dihydro-lH-pyrazolo[4,3-c]pyridine-6-carboxylic acid (a-3) was obtained as a yellow-brown solid (95.5 g, 92%). MS-ESI (m / z): 193.05 [M+l] + .

[0060] Third step: synthesis of methyl l-methyl-4-oxo-4,5-dihydro-lH-pyrazolo[4,3-c]pyridine-6- carboxylate (a-4)

[0061]

[0062] Intermediate a-3 (95.0 g, 492.0 mmol) was dissolved in 40 mL of methanol and concentrated sulfuric acid (984 mmol, 50 mL) was added dropwise. The reaction was heated to 70 °C for 17 hours, after which the reaction was cooled to room temperature. Upon completion of the reaction, a white microcrystalline solid precipitated. The reaction mixture was filtered under reduced pressure and the filter cake was washed with water. This first crop was collected, then the filtrate was diluted with 50 mL ACN, 50 mL MTBE, and 100 mL ethanol, then allowed to stand at 0 °C. After 2 hours, the white microcrystalline precipitated from the solution was collected by vacuum filtration and combined with the previous crop to provide methyl 1-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[4,3-c]pyridine-6-carboxylate (a-4) (101.1 g, 99.3%) as a white solid. MS-ESI (m / z): 208.06 [M+1] + Fourth Step: Synthesis of methyl 4-bromo-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxylate (a-5)

[0063]

[0064] Intermediate a-4 (100.5 g, 372.2 mmol) was dissolved in acetonitrile (55 mL), pyridine (45.0 mL, 558.3 mmol) was added in one portion, and trifluoroacetic anhydride (94.0 mL, 558.3 mmol) was added dropwise. After 50 mL was added, the solution changed from yellow to red (although still hazy), and upon addition of the remaining trifluoromethanesulfonic anhydride, the reaction turned yellow again and began to clear. After 45 minutes, lithium bromide (4076.4 g, 46.9 mol) and trifluoroacetic acid (910 mL, 10.3 mol) were added to the reaction mixture to produce an orange suspension. After 1 hour the reaction mixture was slowly poured into a conical flask containing 2000 mL of saturated NaHCO3while stirring. After the gas evolution ceased, 8000 mL of ethyl acetate was added to the mixture in a separatory funnel, allowed to separate, and the aqueous layer was discarded. The organic layer was then washed once with sodium thiosulfate to decolorize, and the two layers were separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting brown oil was dissolved in 100 mL of dichloromethane, and 100 mL of acetonitrile and 100 mL of acetone were added. This hazy solution was allowed to stand at 0 °C overnight, after which the product had precipitated and was collected by vacuum filtration as a yellow-brown solid, methyl 4-bromo-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxylate (a-5) (79.4 g, 79%). MS-ESI (m / z): 269.99 [M+1] + .

[0065] Step 5: Synthesis of 4-bromo-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxylic acid (a-6)

[0066]

[0067] Intermediate a-5 (79.0 g, 292.5 mmol) was added to a flask containing 400 mL tetrahydrofuran and 160 mL water. Lithium hydroxide (10.5 g, 438.6 mmol) was added to the solution at room temperature and the mixture was stirred for 2 hours until the reaction was complete. The reaction mixture was acidified to pH 1 with concentrated hydrochloric acid, at which point the solution became turbid. The resulting acidic suspension was placed at 0 °C for 1 hour, after which the product was observed to have precipitated. Filtration gave a white, semi-crystalline solid compound, 4-bromo-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxylic acid (a-6) (68.2 g, 90%). MS-ESI (m / z): 255.93 [M+l] + .

[0068] Step 6: Synthesis of 4-bromo-N-(2,4-dimethoxybenzyl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (a-7)

[0069]

[0070] A first portion of triethylamine (147.8 mL, 1052.3 mmol) was added to a suspension of intermediate a-6 (68.0 g, 350.4 mmol) dissolved in DMF (70 mL), followed by benzylamine (37.5 g, 350.4 mmol), which resulted in a clear solution. 1-Propylphosphine anhydride (307.8 mL, 50% in ethyl acetate, 526.1 mmol) was added to the solution, and the mixture was stirred for 30 minutes until the reaction was complete. The solution was diluted with 150 mL of ethyl acetate under magnetic stirring and then filtered under reduced pressure. The solid was washed with ethyl acetate and dried to provide a white solid compound 4-bromo-N-benzyl-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (a-7) (97.8 g, 81%). MS-ESI (m / z): 345.06 [M+l] + .

[0071] Preparation of intermediate b-5: 3-(4-(benzyloxy)-3-methyl-1H-pyrazol-1-yl)prop-1-ol

[0072]

[0073] Step 1: Preparation of 1-propyl-3-methyl-1H-pyrazole-4-yl formate (b-2)

[0074]

[0075] 3-chloroperoxybenzoic acid (34.8 g, 172 mmol) was added to a solution of b-1 (26.0 g, 172 mmol) in CHCl3 (500 mL) at 10 °C, and the mixture was stirred at 25–30 °C for 40 hours. The reaction was then filtered, and the filtrate was concentrated under vacuum. The crude residue was purified by column chromatography to provide a yellow semi-solid compound, 1-propyl-3-methyl-1H-pyrazole-4-yl formate (b-2) (28.7 g), which was used without further purification. MS-ESI (m / z): 169.07 [M+l] + Step 2: Preparation of 1-propyl-3-methyl-1H-pyrazole-4-ol (b-3)

[0076]

[0077] NaHCO3 (15.8 g, 189 mmol) was added to a solution of 1-propyl-3-methyl-1H-pyrazole-4-yl formic acid (b-2) (28.7 g, 173 mmol) in MeOH (200 mL) and H2O (27 mL) at 15 °C and stirred for 5 hours. The reaction mixture was filtered, washed with MeOH, and concentrated under vacuum. The crude residue was purified by column chromatography to give 1-propyl-3-methyl-1H-pyrazole-4-ol (b-3) (15.0 g, 63% after two steps). MS-ESI (m / z): 141.08 [M+l] + .

[0078] Step 3: Preparation of 1-propyl-4-(benzyloxy)-3-methyl-1H-pyrazole (b-4)

[0079]

[0080] Benzyl bromide (13.7 mL, 115.3 mmol) was added to a solution of 1-propyl-3-methyl-1H-pyrazole-4-ol (b-3) (14.5 g, 104.8 mmol) and K₂CO₃ (21.7 g, 157 mmol) in DMF (242 mL) at 15 °C. The mixture was heated to 50 °C and stirred for 20 hours. The reaction was then cooled to room temperature, slowly poured into ice water (500 mL), and diluted with EtOAc (400 mL). The phases were then separated, and the aqueous phase was extracted with EtOAc (300 mL x 2), washed with water (300 mL x 2) and brine (300 mL x 2), and concentrated under vacuum. The crude residue was purified by column chromatography to give 1-propyl-4-(benzyloxy)-3-methyl-1H-pyrazole (b-4) (19.7 g, 80%). MS-ESI (m / z): 231.17 [M+l] + .

[0081] Step 4: Preparation of (4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)boronic acid (b-5)

[0082]

[0083] n-BuLi (1.4 mL, 3.5 mmol, 2.5 M in hexane) was added dropwise to intermediate b-4 (298.0 mg, 1.29 mmol) in a THF-free solution (6.0 mL) at -65 °C (internal temperature) to maintain an internal temperature below -60 °C, and the mixture was stirred for 1.5 h. Then, triisopropyl borate (3.0 mL, 13.0 mmol) was added to the reaction, the reaction was removed from the cold bath, gradually warmed to room temperature, and stirred for 16 h. The reaction was quenched with H₂O (5 mL), the phases were separated, and the aqueous phase was extracted with EtOAc (5 mL x 3). The combined organic phases were dried over Na₂SO₄, filtered, and concentrated under vacuum to provide (4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)boronic acid (b-5) (244 mg, 70%) as a grayish-white oily solid, which was used without further purification. MS-ESI (m / z): 275.19 [M+l] + .

[0084] Preparation of intermediate c-6: (4-(benzyloxy)-1-(2-methoxyethyl)-3-methyl-1H-pyrazol-5-yl)boronic acid

[0085] Step 1: Preparation of 1-(2-methoxyethyl)-3-methyl-1H-pyrazole-4-carboxaldehyde (c-2)

[0086]

[0087] NaH (190 mg, 4.70 mmol) was added to a solution of 1-(2-hydroxyethyl)-3-methyl-1H-pyrazole-4-carboxaldehyde (c-1) (611 mg, 3.94 mmol) in THF (13 mL) at 0 °C. The mixture was heated to 20 °C and stirred for 15 minutes. Then, a solution of iodomethane (655 mg, 4.62 mmol) in THF (2 mL) was added dropwise to the reaction, and the mixture was stirred at 20 °C for 1 hour. The reaction was quenched with H₂O (5 mL) and the phases were separated. The aqueous phase was extracted with EtOAc (5 mL x 3), the organic layer was washed with brine (20 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to provide the title compound 1-(2-methoxyethyl)-3-methyl-1H-pyrazole-4-carboxaldehyde (c-2) (602.3 mg, 91%) as a yellow oil. MS-ESI (m / z): 169.14 [M+l] + .

[0088] Steps two through five:

[0089] Using the corresponding intermediate c-2 instead of the starting material b-1, intermediate (4-(benzyloxy)-1-(2-methoxyethyl)-3-methyl-1H-pyrazol-5-yl)boronic acid (c-6) was synthesized following a similar procedure to b-5. MS-ESI (m / z): 291.14 [M+l] + Preparation of intermediate d-2: tert-butyl 5-bromo-2H-tetrazole-2-carboxylate

[0090]

[0091] 5-Bromo-2H-tetrazole (d-1, 1.48 g, 10 mmol) was added to a solution of di-tert-butyl dicarbonate (2.61 g, 12 mmol) and 4-dimethylaminopyridine (183 mg, 1.5 mmol) in anhydrous acetonitrile (10 mL) at 30 °C. The reaction mixture was stirred for 2.5 h and then cooled to 25 °C. The mixture was concentrated. The residue was purified by column chromatography (hexane / EtOAc = 10:1) to give tert-butyl 1H-pyrrole-1-carboxylate (d-2) (2.02 g, 81%). MS-ESI (m / z): 248.94 [M+l] + .

[0092] Intermediates d-3 to d-11 were synthesized using the corresponding intermediates instead of the d-1 synthesis raw materials, following a similar procedure to d-2 (see Table 1).

[0093] Table 1: Structural information of intermediates d-3 to d-11

[0094]

[0095]

[0096] Preparation of intermediate e-1: 5-(4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole

[0097]

[0098] The reaction vessel containing (4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazole-5-yl)boronic acid (b-5, 500.5 mg, 1.82 mmol), tert-butyl 5-bromo-2H-tetrazole-2-carboxylate (d-2, 551.2 mg, 2.25 mmol), K3PO4 (834 mg, 3.93 mmol), and cataCXium A Pd G3 (53 mg, 0.073 mmol) in DMF (8 mL) and H2O (2 mL) was protected with nitrogen, heated to 80 °C, and stirred for 22 hours. The reaction mixture was then diluted with H2O (20 mL), and the phases were separated. The aqueous phase was extracted with ethyl acetate (20 mL × 4). The combined organic phases were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under vacuum. DCM (20 mL) and HCl (1.8 mL, 7.2 mmol, 4 M in 1,4-dioxane) were added to the above residue, and the reaction mixture was stirred for 3 hours. After the reaction was completed, the reaction mixture was diluted with H2O (10 mL), and the pH was adjusted to neutral by adding NaOH aqueous solution. The phases were separated, and the aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography to give 5-(4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole (e-1) (440.7 mg, 81%). MS-ESI (m / z): 299.14 [M+l]+.

[0099] Using the corresponding intermediates to replace the b-5 synthesis raw material, intermediates e-2 to e-12 were synthesized following a similar procedure to e-1 (see Table 2).

[0100] Table 2: Structural information of intermediates e-2 to e-12

[0101]

[0102]

[0103] Example 1: Preparation of 4-(5-(4-hydroxy-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole-2-yl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide

[0104]

[0105] Step 1: The intermediates 4-bromo-N-benzyl-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (a-7) (100.1 mg, 0.29 mmol), 5-(4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole (e-1) (77.7 mg, 0.26 mmol), and potassium carbonate (54 mg, 0.59 mmol) were added to DMF (5 mL), followed by N,N'-dimethylethylenediamine (80.9 mg, 0.39 mmol) and stirring at 120 °C for 2–3 hours. The reaction was quenched with water, and the mixture was extracted with ethyl acetate (5 mL * 3). The organic layer was combined and concentrated under reduced pressure to give intermediate 4-(5-(4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazol-2-yl)-N-benzyl-1-methyl-1H-pyrazino[4,3-c]pyridine-6-carboxamide (ae-1) (84.2 mg, 52.0%). MS-ESI (m / z): 563.21 [M+l] + .

[0106] Step 2: The reaction vessel containing 4-(5-(4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole-2-yl)-N-benzyl-1-methyl-1H-pyrazino[4,3-c]pyridine-6-carboxamide (ae-1) (56.3 mg, 1.0 mmol), wet Pd / C (10%, 230 mg, 0.22 mmol), and NEt3 (1.0 mL, 7.2 mmol) in MeOH (10 mL) and THF (10 mL) was stirred at 20 °C for 2 hours under H2 (15 psi, balloon). The reaction was then filtered through a diatomaceous earth mat, and the filtrate was concentrated under vacuum and purified by column chromatography to give 4-(5-(4-hydroxy-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole-2-yl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (Example 1) (20.3 mg, 53%). MS-ESI (m / z): 383.11 [M+l] + .

[0107] 1H NMR (400MHz, DMSO-d6) δ8.78(d,J=0.8Hz,1H),8.44(d,J=1.0Hz,1H),8.19(s,1H),7.99(br s,1H),7.81(br s,1H),4.64-4.43(m,2H),4.36(s,3H),2.21(s,3H),1.87-1.71(m,2H),0.89(t,J=7.2Hz,3H).

[0108] Following the synthesis method of Example 1, intermediate a-7 was synthesized with intermediates e-2 to e-12 to obtain Examples 2-12 (structural information is shown in Table 3).

[0109] Table 3

[0110]

[0111]

[0112]

[0113]

[0114] Example 13 Bioactivity Test

[0115] We screened for the activity of pyrazolidine activators on THP-1 cells and THP-1ISG cells that primarily express STING.

[0116] Mechanism of Assay: STING exists primarily as a symmetrical dimer on the endoplasmic reticulum membrane and is a transmembrane protein. STING activation leads to the recruitment of TBK1 and phosphorylation of the IRF3 transcription factor, upregulation of the IRF3 and NFκB pathways, thereby inhibiting pathogen replication and promoting the upregulation of adaptive immune interferon β (INF-β). Therefore, activation of STING activity indicates potential therapeutic potential. By activating STING, the innate immune response is activated, and STING activators have the potential to treat a wide range of diseases related to viral infections and cancer.

[0117] Experimental methods:

[0118] IRF3 phosphorylation assay:

[0119] STING activation leads to TBK1 recruitment and phosphorylation of the IRF3 transcription factor, subsequently inducing type I interferon. THP-1 cells (InvivoGen) were cultured in RPMI medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, and 0.5% Pen-Strep. 10 4Cells were seeded in 96-well plates and incubated overnight at 37°C and 5% CO2. Serially diluted test compound (finally 0.5% DMSO) was added to the cells and incubated for another 3 hours. After incubation, the plates were centrifuged at 2000 rpm for 5 min. Cells were then lysed in 100 μl of RIPA buffer and vortexed at room temperature for 30 min. 25 μl of the lysate was then transferred to clear polystyrene High Bind plates previously coated with mouse anti-human IRF-3 capture antibody (BD Pharmigen) and incubated at 4°C for 16 h. The plates were then washed and incubated with rabbit anti-phosphorylated IRF3 detection antibody (Cell Signaling Technologies) at room temperature for 1.5 h. Finally, HRP-linked secondary antibody (Cell Signaling Technologies) was added and incubated for 30 min, and a luminescent signal was generated using Glo Substrate reagent (R&D Systems). The signal was measured using a Perkin-Elmer Envision microplate reader. Data were normalized to “% effect” using a positive control STING agonist and a negative control DMSO, which are known to maximize phosphorylated IRF3 signal.

[0120] Interferon-β induction assay:

[0121] THP-1Lucia™ ISG cells (InvivoGen) express the secreted luciferase "Lucia" reporter gene, controlled by an IRF-inducible complex promoter consisting of five interferon-responsive elements. THP-1Lucia™ ISG cells were cultured in RPMI medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, and 0.5% Pen-Strep. Hygromycin B and Zeocin were present to maintain stable transfection. 10 4 Cells were seeded in 96-well plates and incubated overnight at 37°C and 5% CO2. 50 μL of serially diluted test compound was added to the culture medium, and the plates were incubated for another 24 hours. After incubation, the plates were centrifuged at 2000 rpm for 10 min. 50 μL of cell culture supernatant from each well was transferred to a white, opaque 96-well plate. One packet of QUANTI-Luc was prepared in 25 mL of endotoxin-free water. TM (InvivoGen) powder was added, and 100 μL of the prepared warm QUANTI-Luc solution was added to each well containing supernatant. The luminescence signal was measured using a Perkin-Elmer Envision microplate reader. Data were normalized to "% effect" using the positive control STING agonist and the negative control DMSO, both known to maximize luciferase signal. Results:

[0122] The activity evaluation results are shown in Table 3 below:

[0123] Table 3 shows the IRF3 phosphorylation and interferon-β induction activities of the series of compounds.

[0124]

[0125] The positive control is AIF11 in WO2022195462 A1, and its structure is as follows:

[0126] IRF3 phosphorylation and interferon-β induction activity experiments were conducted on the compounds in this series. The results showed that, within the same test batch, all the novel pyrazolopyridine compounds of general formula (I) of this invention were highly efficient STING activators. Among them, some compounds improved their adaptor protein STING agonist activity by optimizing the heterocyclic structure linked to the pyrazolopyridine backbone, resulting in significantly superior biological activity compared to the positive control; and other compounds also achieved activity levels comparable to the positive control.

[0127] For compounds of general formula (I) of this invention, the linking and substituent groups have a significant impact on the pharmacodynamic properties of the compounds. Although this disclosure has been described in some detail and some specificity has been used with respect to several described embodiments, it is not intended to limit it to any such details or embodiments or any particular embodiment, but should be interpreted with reference to the appended claims in order to provide the broadest possible interpretation of such claims in light of the prior art, and thus effectively cover the intended scope of this disclosure.

Claims

1. A pyrazolopyridine compound having the structure of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically-labeled, prodrug thereof: wherein: R, R' are independently selected from substituted or unsubstituted C1-4alkyl, C3-6cycloalkyl, C1-4alkyl, C3-6cycloalkyl substituted with groups independently selected from one or more of halogen, hydroxyl, cyano, C1-4alkoxy; X, Y, Z are independently selected from N, CR"; R" is H, substituted or unsubstituted C1-4alkyl, C3-6cycloalkyl, C1-4alkyl, C3-6cycloalkyl substituted with groups independently selected from one or more of halogen, hydroxyl, cyano, C1-4alkoxy. X is N, Y is N, Z is N; or X is N, Y is CR", Z is N; or X is CR", Y is N, Z is N; or X is CR", Y is N, Z is CR"; or X is CR", Y is CR", Z is N; R' is unsubstituted C1-4alkyl, C3-6cycloalkyl when X is CR", Y is N, Z is N, or X is CR", Y is N, Z is CR". The compound specifically includes:

2. The pyrazolopyridine compound or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug according to claim 1, characterized in that, The pharmaceutically acceptable salt is inorganic salt or organic salt, the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate.

5. Use of the pyrazolopyridine compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically-labeled, prodrug thereof of any one of claims 1-4 in the manufacture of a medicament for activating STING signaling pathway.

3. The pyrazolopyridine compound or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug according to claim 1, characterized in that, 6. Use of the pyrazolopyridine compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically-labeled, prodrug thereof of any one of claims 1-4 in the manufacture of a medicament for treating viral infection and cancer-related diseases.

4. The pyrazolo pyridine compound or pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically labeled, prodrug thereof according to any one of claims 1 to 3, characterized in that, ​ ​ ​ 7. Use according to claim 6, characterized in that, The viral infection and cancer-related diseases include inflammatory diseases and disorders, allergic diseases, autoimmune diseases, infectious diseases, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasm of the central nervous system, primary CNS lymphoma, spinal axis tumors, brain stem glioma, or pituitary adenoma.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the pyrazolo pyridine compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotopically labeled, prodrug, and pharmaceutical adjuvant of any one of claims 1 to 4.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical adjuvant comprises an excipient, diluent, or pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 8, wherein, The dosage form of the pharmaceutical composition comprises an injection solution, a freeze-dried injection for injection, a suspension, an implant, an embolus, a capsule, a tablet, a pill, and an oral solution.

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