A pyrazolopyridine compound, composition and uses thereof that acts on interferon gene-stimulating protein

Abstract

This invention discloses a pyrazolopyridine compound, a composition thereof, and its use in relation to interferon gene-stimulating proteins, belonging to the field of chemical medicine. This invention provides a pyrazolopyridine compound as shown in formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer, or isotopic variant thereof, which can be used to treat and prevent human diseases, including viral infections and cancer, and for the treatment and / or prevention of related diseases such as inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, and abnormal cell growth (including cancer).

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, specifically relating to a pyrazolopyridine compound, composition and use thereof that acts on interferon gene-stimulating protein. Background Technology

[0002] STING, a transmembrane protein, exists primarily as a symmetrical dimer on the endoplasmic reticulum membrane. Encoding viruses and bacteria, STING is a key regulator of the innate immune response to infection, promoting innate immune signaling. It also acts as a sensor for bacterial and viral cytosolic DNA, promoting the production of type I interferons (IFN-α and IFN-β). This protein is a pattern recognition receptor (PRP) that detects cytoplasmic nucleic acids and transmits signals activating the type I interferon response. PRP activation leads to the upregulation of genes involved in the inflammatory response, including type I interferons (also known as IFN or INF) that inhibit pathogen replication and promote adaptive immunity, as well as pro-inflammatory cytokines and chemokines.

[0003] The adaptor protein STING (also known as TMEM173) has been identified as a central signaling molecule in the innate immune sensing pathway in response to cytosol DNA. STING is crucial for responses to cytosol DNA from pathogens or host-derived cytosol DNA. Activation of STING by cyclic dinucleotides (CDNs) generated in response to cytosol DNA leads to upregulation of the IRF3 and NFκB pathways, resulting in the induction of interferon β (INF-β) and other cytokines.

[0004] Pfizer's patent document WO2022195462A1 discloses a (STING) activator with excellent efficacy, the structure of which is shown below:

[0005]

[0006] This small molecule compound can activate innate immune responses (including activation of type I INF and other cytokines), potentially serving as an important strategy for treating and preventing human diseases, including viral infections and cancer. 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 aforementioned (STING) activators needs further improvement. Summary of the Invention

[0008] This invention proposes a new class of pyrazolopyridine compounds with superior biological activity.

[0009] This invention relates to novel pyrazolopyridine compounds and their pharmaceutically usable salts, and their use in treating or improving diseases or conditions such as inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants.

[0010] One object of the present invention is to provide pyrazolopyridine compounds of general formula (I), or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, prodrugs, etc.

[0011]

[0012] In the formula:

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

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

[0015] In one embodiment of the present invention, R and R' are each independently selected from -(CH2). n CH3, -(CH2) n OCH3, n = 0, 1, 2, 3.

[0016] In one embodiment of the present invention, X, Y, and Z are each independently selected from -N-, -CH-, or -CCH3.

[0017] In one embodiment of the present invention, the following can be specifically selected: 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 CR”, Z is N; or X is CR”, Y is N, Z is CR”.

[0018] Further optimizations can be made where X is N, Y is N, and Z is N; or X is N, Y is CR”, and Z is N; or X is CR”, Y is N, and Z is N; or X is CR”, Y is CR”, and Z is N.

[0019] In one embodiment of the present invention, the above-mentioned pyrazolopyridine compounds may specifically be selected from:

[0020]

[0021] The present invention relates to compounds of general formula (I) or pharmaceutically acceptable salts thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate; and wherein the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, sulfonate, benzenesulfonate, and salicylate.

[0022] The present invention also provides a method for preparing compounds of general formula (I).

[0023] The present invention also provides pharmaceutical compositions comprising the above-described compound of general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, prodrug, and pharmaceutical excipient.

[0024] In one embodiment of the invention, the pharmaceutical excipient comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0025] Pharmaceutically acceptable carriers include microspheres, nanoparticles, and liposomes.

[0026] In one embodiment of the present invention, the dosage form of the pharmaceutical composition includes injection, lyophilized powder for injection, suspension, implant, embolization, capsule, tablet, pill and oral liquid.

[0027] The present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels, and prodrugs in the preparation of medicaments for activating the STING signaling pathway.

[0028] The present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels, and prodrugs in the preparation of medicaments for treating viral infections and cancer-related diseases.

[0029] In one embodiment of the present invention, the viral infection and cancer-related diseases include inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach 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, cancers 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, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, tumors of the central nervous system (CNS), primary CNS lymphoma, spinal cord axis tumors, brainstem gliomas, or pituitary adenomas.

[0030] Beneficial effects:

[0031] The compounds of this invention can serve as an important strategy for treating and preventing human diseases, including viral infections and cancer. Compared with the positive control (compound AIF11 in WO2022195462 A1), they exhibit significantly improved activity or selectivity and can be used to treat and / or prevent inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants, showing great promise for development and application. Detailed Implementation

[0032] The technical solution of the present invention will be described in detail below with reference to the embodiments.

[0033] In this invention, "substituted" means that one or more hydrogen atoms in a group are replaced by the same or different substituents.

[0034] In this invention, "C3-6 cycloalkyl" refers to three- to eight-membered rings with or without heteroatom hybridization. Heteroatoms include N, O, or S. Specific examples include cyclopropyl, cyclobutyl, oxocyclobutyl, and azircyclobutyl.

[0035] In this invention, "administering" or "giving" an individual compound refers to providing the compound of this invention to an individual in need of treatment.

[0036] The term "pharmaceutically acceptable salt" refers to a salt that, within reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without excessive toxicity, activating effects, anaphylactic reactions, etc., and has a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0037] As used herein, the term "pharmaceutical composition" means a composition comprising one or more of the compounds described herein or their pharmaceutically acceptable isomers, racemates, hydrates, solvates, isotopes or salts, formulated together with a pharmaceutically acceptable carrier, and may also contain other additives, and is manufactured or sold with the approval of a government regulatory agency as part of a treatment regimen for treating diseases in mammals.

[0038] The compounds of the present invention and their pharmaceutically acceptable pharmaceutical compositions can be formulated for oral administration, for example, in unit dosage forms (e.g., tablets, capsules, pouches, soft capsules, or syrups); topically (e.g., as creams, gels, lotions, or ointments); intravenously (e.g., as sterile solutions without emboli and in solvent systems suitable for intravenous use); to pediatric subjects (e.g., solutions, syrups, suspensions, elixirs, powders for reconstitution into suspensions or solutions, dispersible / effervescent tablets, chewable tablets, lollipops, popsicles, tablets, oral strips, oral disintegrating tablets, oral disintegrating strips, and spray oral powders or granules); or in any other formulation form described herein. The routine procedures and ingredients used for selecting and preparing suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippencott Williams & Wilkins (2005) and The United States Pharmacopeia: The National Formulary (USP 36NF31), published in 2013.

[0039] The present invention includes the compounds and their pharmaceutically acceptable salts, isomers, hydrates, solvates or isotopes, as well as forms including tautomers, polymorphs, stereoisomers and isotopically labeled forms.

[0040] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, and excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0041] The compounds of this invention or pharmaceutically acceptable salts thereof can be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays, and inhalers. The compounds of general formula (I) of this invention or pharmaceutically acceptable salts thereof, as active ingredients, are mixed under sterile conditions with a physiologically acceptable carrier and optionally with preservatives, buffers, and propellants, if necessary.

[0042] The pharmaceutical compositions of the present invention comprise a compound of general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient, and diluent. In preparing the pharmaceutical compositions, the compound of general formula (I) or a pharmaceutically acceptable salt thereof is typically 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.

[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The technical solutions of the present invention will be described in detail below with reference to the embodiments.

[0044] The following examples are illustrative and not limiting of the synthesis of compounds of general formula (I). All temperatures are in degrees Celsius. Unless otherwise stated, all evaporations were performed under reduced pressure. Unless otherwise stated, reagents were purchased from commercial suppliers and used without further purification. The structures of the final products, intermediates, and starting materials were confirmed by standard analytical methods, such as elemental analysis and spectroscopic characterization, such as MS and NMR. Abbreviations used are conventional abbreviations in the art.

[0045] Preparation of intermediate a-7: 4-bromo-N-benzyl-1-methyl-4,5-dihydro-1H-pyrazole[4,3-c]pyridine-6-carboxamide

[0046]

[0047] Step 1: Synthesis of (Z)-1-methyl-5-((2-methyl-5-oxazol-4(5H)-ylidene)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester (a-2)

[0048]

[0049] At room temperature, ethyl 5-formyl-1-methyl-1H-pyrazole-4-carboxylate (a-1, 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, followed by another 5 mL of acetic anhydride and stirring. The mixture was heated to 100 °C and refluxed. During heating, the white turbid suspension turned into a clear yellow solution, and after 10 minutes, it turned into a brown solution. After 1 hour, the reaction mixture was cooled to room temperature. A saturated aqueous solution of sodium bicarbonate was added dropwise with magnetic stirring until bubbling stopped. The aqueous layer was extracted with 4 x 1000 mL of dichloromethane:isopropanol = 3:1 (V / V) and 2 x 1500 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The resulting dark brown residue was dissolved in approximately 50 mL of DCM. Methyl tert-butyl ether (approximately 50 mL) was added dropwise to this solution to give a pale yellow solid (Z)-1-methyl-5-((2-methyl-5-oxoxazo-4(5H)-ylidene)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester (a-2, 142.1 g, 98%). MS-ESI (m / z): 264.09 [M+l] + .

[0050] Step 2: Synthesis of 1-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[4,3-c]pyridine-6-carboxylic acid (a-3)

[0051]

[0052] Intermediate a-2 (141.5 g, 538.1 mmol) was dissolved in methanol (538 mL, 1 M), and potassium carbonate (168 g, 1076.2 mmol) was added. The mixture was heated to 70 °C and stirred for 16 hours. After the reaction was complete, the mixture 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 acidified to pH 1 with concentrated hydrochloric acid. A yellowish-brown solid precipitate was formed and filtered off under reduced pressure. The filtrate was then diluted with MeOH / MTBE at a ratio of 1:1 and filtered again under reduced pressure. The compound 1-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[4,3-c]pyridine-6-carboxylic acid (a-3) (95.5 g, 92%) was given as a yellowish-brown solid. MS-ESI (m / z): 193.05 [M+l] + .

[0053] Step 3: Synthesis of methyl 1-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[4,3-c]pyridine-6-carboxylate (a-4)

[0054]

[0055] 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 and reacted for 17 hours. After cooling to room temperature, a white microcrystalline solid precipitated. The reaction mixture was filtered under reduced pressure and the filter cake was washed with water. The first batch was collected, and the filtrate was diluted with 50 mL of LACN, 50 mL of MTBE, and 100 mL of ethanol, and then allowed to stand at 0 °C. After 2 hours, the white microcrystalline solid precipitated from the solution was collected by vacuum filtration and combined with the previous batch 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+l] + Step 4: Synthesis of methyl 4-bromo-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxylate (a-5)

[0056]

[0057] Intermediate a-4 (100.5 g, 372.2 mmol) was dissolved in acetonitrile (55 mL), and pyridine (45.0 mL, 558.3 ​​mmol) was added all at once, followed by dropwise addition of trifluoroformic anhydride (94.0 mL, 558.3 ​​mmol). After adding 50 mL, the solution changed from yellow to red (although still turbid), and after adding the remaining trifluoromethanesulfonic anhydride, the reaction turned yellow again and began to clarify. 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 reacting for 1 hour, the reaction mixture was slowly poured into a conical flask containing 2000 mL of saturated NaHCO3 while stirring. After gas production stopped, 8000 mL of ethyl acetate was added to the mixture in a separatory funnel, and the layers were allowed to separate. The aqueous layer was discarded. The organic layer was then washed once with sodium thiosulfate for decolorization, 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. The turbid solution was left to stand overnight at 0 °C, after which the product precipitated and was filtered under vacuum to give a yellowish-brown solid of 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+l] + .

[0058] Step 5: Synthesis of 4-bromo-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxylic acid (a-6)

[0059]

[0060] 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] + .

[0061] Step 6: Synthesis of 4-bromo-N-(2,4-dimethoxybenzyl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (a-7)

[0062]

[0063] 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] + .

[0064] Preparation of intermediate b-5: 4-(benzyloxy)-5-bromo-3-methyl-1-propyl-1H-pyrazole

[0065]

[0066] Step 1: Preparation of 1-propyl-3-methyl-1H-pyrazole-4-yl formate (b-2)

[0067]

[0068] 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)

[0069]

[0070] 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] + .

[0071] Step 3: Preparation of 1-propyl-4-(benzyloxy)-3-methyl-1H-pyrazole (b-4)

[0072]

[0073] 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] + .

[0074] Step 4: Preparation of 4-(benzyloxy)-5-bromo-3-methyl-1-propyl-1H-pyrazole (b-5)

[0075]

[0076] NBS (0.93 g, 5.1 mmol) was added to a solution of 1-propyl-4-(benzyloxy)-3-methyl-1H-pyrazole (b-4, 1.05 g, 4.6 mmol) in THF (20 mL), and the mixture was stirred at room temperature for 2 hours. The mixture was then concentrated, and the residue was purified by column chromatography to give compound (b-5) (928 mg, 65%).

[0077] MS-ESI (m / z): 309.01 [M+l] + .

[0078] Preparation of intermediate c-1: 2-(4-(phenoxy)-3-methyl-1-propyl-1H-2 2 2-(4-(4-(phenoxy)-3-methyl-1-propyl-1H-22 2-(4-(phenoxy)-3-methyl-1-propyl-1H-2-tetrazol)

[0079]

[0080] Intermediate 4-(benzyloxy)-5-bromo-3-methyl-1-propyl-1H-pyrazole (b-5) (90.0 mg, 0.29 mmol), 5-bromo-2H-tetrazole (b-6) (43.0 mg, 0.29 mmol), and potassium carbonate (56 mg, 0.6 mmol) were added to DMF (5 mL), followed by N,N'-dimethylethylenediamine (81.5 mg, 0.4 mmol), and the mixture was stirred 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 layers were combined, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound (c-1) (35 mg, 32.0%). MS-ESI (m / z): 377.09 [M+l] + .

[0081] Using the corresponding intermediates to replace the b-6 synthesis raw material, intermediates c-2 to c-7 were synthesized following a similar procedure to c-1 (see Table 1).

[0082] Table 1: Structural information of intermediates c-2 to c-7

[0083]

[0084] Example 1: Preparation of 4-(2-(4-hydroxy-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazol-5-yl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide

[0085]

[0086] Step 1: Add Pd(OAc)2 (46.0 mg, 0.2 mmol), PCy3HBF4 (100.0 mg, 0.27 mmol), CuI (258.0 mg, 1.37 mmol) to a solution of 4-bromo-N-(2,4-dimethoxybenzyl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (a-7, 148.0 mg, 0.43 mmol), 2-(4-(phenoxy)-3-methyl-1-propyl-1H-2 2 2 2-(4-(4-(phenoxy)-3-methyl-1-propyl-1H-2 2 2-(4-(phenoxy)-3-methyl-1-propyl-1H-2-tetrazolium) in 1,4-dioxane (2 mL) to a solution of 4-bromo-N-(2,4-dimethoxybenzyl)-1-methyl-1H-2-tetrazolium (c-1, 256.0 mg, 0.68 mmol) in 1,4-dioxane (2 mL). The mixture was stirred at 140 °C for 18 hours under N2 conditions with CO3 (220.0 mg, 0.68 mmol). The mixture was then filtered, the filter cake was washed with DCM / MeOH (V / V = 20 / 1), the filtrate was concentrated, and the residue was purified by preparative TLC to give compound ac-1 (94.0 mg, 39%). MS-ESI (m / z): 563.29 [M+l] + .

[0087] Step 2: The reaction vessel containing N-benzyl-4-(2-(4-(benzyloxy)-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole-5-yl)-1-methyl-1H-pyrazine[4,3-c]pyridine-6-carboxamide (ac-1) (56.3 mg, 1.0 mmol), wet Pd / C (10%, 226 mg, 0.20 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-(2-(4-hydroxy-3-methyl-1-propyl-1H-pyrazol-5-yl)-2H-tetrazole-5-yl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-6-carboxamide (Example 1) (18.0 mg, 47%). MS-ESI (m / z): 383.07 [M+l] + .

[0088] 1H NMR(400MHz,DMSO-d6)δ8.81(s,1H),8.49(s,1H),7.99(br s,1H),7.85(br s,1H),7.69(s,1H),4.54-4.43(m,2H),4.31(s,3H),2.14(s,3H),1.87-1.71(m,2H),0.88(t,J=7.4Hz,3H).

[0089] Following the synthesis method of Example 1, intermediate a-7 was synthesized with intermediates c-2 to c-7 respectively to obtain Examples 2-7 (structural information is shown in Table 2).

[0090] Table 2

[0091]

[0092]

[0093]

[0094] Example 8 Bioactivity Test

[0095] Pyrazolopyridine activators were screened on THP-1 cells and THP-1ISG cells that primarily express STING.

[0096] Mechanism of measurement: STING mainly exists in symmetrical dimer form 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, STING activation represents potential therapeutic opportunities. 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.

[0097] Experimental methods:

[0098] IRF3 phosphorylation assay:

[0099] STING activation leads to TBK1 recruitment and phosphorylation of the IRF3 transcription factor, which then induces 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, followed by luminescence signal generation 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.

[0100] Interferon-β induction assay:

[0101] 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:

[0102] The activity evaluation results are shown in Table 3 below:

[0103] Table 3 shows the IRF3 phosphorylation and interferon-β induction activities of the series of compounds.

[0104]

[0105] The structure of positive control A is as follows:

[0106] Positive control B is AIF11 in WO2022195462 A1, with the following specific structure:

[0107] 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, the novel pyrazolopyridine compounds of general formula (I) of this invention were all highly efficient STING activators. Among them, some compounds were significantly superior to the positive control, while others achieved activity comparable to the positive control.

[0108] 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. Pyrazolopyridine compounds having the structure shown in general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, prodrugs: In the formula: R and R' are independently selected from substituted or unsubstituted C1-4 alkyl and C3-6 cycloalkyl groups, respectively. The substituted groups on the C1-4 alkyl and C3-6 cycloalkyl groups are independently selected from any one or more of the following: halogen, hydroxyl, cyano, and C1-4 alkoxy. X, Y, and Z are independently selected from N and CR”, respectively; R” is H, substituted or unsubstituted C1-4 alkyl, C3-6 cycloalkyl, and the substituted groups on the C1-4 alkyl and C3-6 cycloalkyl are independently selected from any one or more of the following: halogen, hydroxyl, cyano, C1-4 alkoxy.

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, 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 CR", and Z is N.

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, The compounds specifically include:

4. The pyrazolopyridine compound according to any one of claims 1-3, or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug, characterized in that, The pharmaceutically acceptable salt is an inorganic or organic salt. The inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate. The organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, benzenesulfonate, and salicylate.

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

6. Use of the pyrazolopyridine compound of any one of claims 1-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug thereof in the preparation of a medicament for treating viral infections and cancer-related diseases.

7. The use according to claim 6, characterized in that, The viral infections and cancer-related diseases mentioned include inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach 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, cancers 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, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, tumors of the central nervous system, primary CNS lymphoma, spinal cord axis tumors, brainstem gliomas, or pituitary adenomas.

8. A pharmaceutical composition, characterized in that, The product contains a pyrazolopyridine compound as described in any one of claims 1-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, prodrug, and pharmaceutical excipient.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical excipient contains excipients, diluents, or pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 8, characterized in that, The dosage forms of the pharmaceutical composition include injections, lyophilized powder for injection, suspensions, implants, embolic agents, capsules, tablets, pills, and oral liquids.

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