Use of NLRP3 inhibitor for treating cerebrovascular diseases

Abstract

Use of an NLRP3 inhibitor for treating cerebrovascular diseases. Specifically, the present invention relates to use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cerebrovascular diseases, wherein each substituent is as defined in the description.

Description

Use of an NLRP3 inhibitor for the treatment of cerebrovascular diseases Technical Field

[0001] This disclosure relates to the use of an NLRP3 inhibitor in the treatment of cerebrovascular diseases and belongs to the pharmaceutical field. Background Technology

[0002] Stroke is the second leading cause of death worldwide and a leading cause of permanent disability. The molecular and cellular mechanisms by which stroke leads to neuronal degeneration are complex and not fully understood, but involve bioenergy depletion, acidosis, excitotoxicity, oxidative stress, and inflammation, leading to cell necrosis or apoptosis. Post-stroke inflammation is a complex process involving the activation of innate local immune responses in glial cells and the recruitment of circulating leukocytes to the affected brain tissue. Activated glial cells and leukocytes produce a variety of pro-inflammatory mediators, including complement anaphylatoxins, cytokines, chemokines, and prostaglandins. Recent findings have provided insights into a newly discovered inflammatory mechanism mediated by a multi-protein complex called the inflammasome, leading to the death of neurons and glial cells in cerebral ischemia. Studies of the inflammasome complex in peripheral tissues have shown that it amplifies the production and secretion of pro-inflammatory cytokines, as well as apoptotic and pyroptotic cell death.

[0003] Recent studies have shown that ischemic conditions increase the levels of NLRP1 and NLRP3 inflammasome proteins, as well as IL-1β and IL-18, in primary cortical neurons. Similarly, the levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β, and IL-18 are also elevated in ipsilateral brain tissue of brain I / R mice and stroke patients (D Yang-Wei Fann et al., Cell Death Dis 4, e790.).

[0004] PCT / CN2024 / 088250 discloses a new class of NLRP3 inhibitors, and the applications of this class of inhibitors are currently being explored. Summary of the Invention

[0005] This disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cerebrovascular diseases.

[0006] Among them, R 1 Selected from hydrogen, methyl, or difluoromethyl;

[0007] R 2 Each is independently selected from hydrogen, halogen, and C. 1-6 alkyl;

[0008] R 3 and R 4 Each is independently selected from hydrogen or halogen;

[0009] R 5 Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0010] R 6 Selected from C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0011] R 7 Selected from fluorine;

[0012] m and p are each independently selected from 0, 1, 2, and 3;

[0013] n is independently selected from 1, 2, and 3.

[0014] This disclosure also provides a method for treating cerebrovascular diseases by administering (optionally a therapeutically effective amount) of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject.

[0015] This disclosure, in another aspect, provides a compound of formula (I) for treating cerebrovascular diseases, or a pharmaceutically acceptable salt thereof.

[0016] This disclosure also provides a method for treating cerebrovascular diseases by administering a subject a compound of formula (I) or a pharmaceutically acceptable salt thereof and reperfusion therapy.

[0017] Another aspect of this disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a reperfusion therapy drug (e.g., a drug that dissolves thrombi or reduces thrombus formation) in the preparation of a medicament for treating cerebrovascular diseases.

[0018] Another aspect of this disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is used in combination with a reperfusion therapy agent (e.g., an agent that dissolves thrombi or reduces thrombus formation) for the treatment of cerebrovascular diseases.

[0019] Another aspect of this disclosure provides a reperfusion therapy drug (e.g., a drug that dissolves or reduces thrombus formation) for use in combination with a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment of cerebrovascular diseases.

[0020] In some implementations, the R 1 Selected from hydrogen.

[0021] In some implementations, the R 2 Each is independently selected from hydrogen.

[0022] In some implementations, the R 3 and R 4 Each is independently selected from hydrogen.

[0023] In some implementations, the R 5 Each is independently selected from hydrogen.

[0024] In some implementations, the R 6 Selected from methyl or ethyl.

[0025] In some implementations, the R 6 It is a methyl group.

[0026] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is compound 1 or a pharmaceutically acceptable salt thereof.

[0027] In some implementations, the cerebrovascular disease described in this disclosure is ischemic cerebrovascular disease.

[0028] In some embodiments, the cerebrovascular disease described in this disclosure is stroke (e.g., hemorrhagic stroke, ischemic stroke).

[0029] In some implementations, the cerebrovascular disease described in this disclosure is ischemic stroke.

[0030] In some embodiments, the cerebrovascular disease described in this disclosure is the necrosis or softening of local brain tissue (e.g., nerve cells, glial cells, and connecting fibers).

[0031] In some embodiments, the reperfusion therapy described in this disclosure is selected from intravenous thrombolysis or mechanical thrombectomy.

[0032] In some implementations, the intravenous thrombolysis described in this disclosure uses drugs that dissolve thrombi or reduce thrombus formation.

[0033] In some embodiments, the thrombolytic or thrombus-reducing drug described in this disclosure is a tissue plasminogen activator (tPA).

[0034] In some embodiments, the compound of formula (I) or its pharmaceutically acceptable salt is administered at a dose independent of the patient's body weight or surface area (fixed dose), for example, 0.01 mg to 1000 mg, specifically 0.1 mg to 500 mg, 1 mg to 400 mg, 5 mg to 200 mg, 10 mg to 150 mg, for example, selected from: 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, etc. mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 1 40mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg , 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 28 5mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg , 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 43 0mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg, 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg.

[0035] In some implementations, the compound of formula (I) or its pharmaceutically acceptable salt is administered at the following frequencies: once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw)), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (Bid), or three times a day (tid) over a period of time.

[0036] In some implementations, the duration of administration is from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about one year, from about one year to about two years, or from about two years to about four years, or longer.

[0037] In some embodiments, the compound of formula (I) or its pharmaceutically acceptable salt is administered via parenteral administration or oral administration.

[0038] In some embodiments, the compound of formula (I) or its pharmaceutically acceptable salt is administered orally.

[0039] This disclosure also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0040] Terminology Explanation

[0041] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0042] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as having an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of “including but not limited to.”

[0043] The terms “subject” and “patient” refer to mammals, especially primates, and particularly humans, especially patients who require relevant treatment.

[0044] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "giving" and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving" and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0045] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical testing methods commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0046] An "effective amount" includes the amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0047] "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 20 carbon atoms. Alkyl groups containing 1 to 6 carbon atoms are also included. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and their various branched isomers.

[0048] The term "halogen" refers to fluorine, chlorine, bromine, or iodine. Attached Figure Description

[0049] Figure 1. Average cerebral infarction area (%) in tMCAO rats. Detailed Implementation

[0050] The present disclosure will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0051] Test conditions of the instruments used in the experiment:

[0052] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE NEO 500M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard. The spatial configurations of the optical isomers (isomers) of the compounds were further confirmed by measuring single-crystal parameters.

[0053] HPLC determinations were performed using a Waters ACQUITY ultra high performance LC, Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or Agilent 1200LC high performance liquid chromatograph (ACQUITY UPLC BEH C18 1.7UM 2.1×50MM column, Ultimate XB-C18 3.0×150mm column, or Xtimate C18 2.1×30mm column).

[0054] MS measurements were performed using a Waters SQD2 mass spectrometer in positive / negative ion mode, with a mass scan range of 100–1200.

[0055] Chiral HPLC analysis was performed using Chiralpak IC-3 100×4.6mm ID, 3µm, Chiralpak AD-3 150×4.6mm ID, 3µm, Chiralpak AD-3 50×4.6mm ID, 3µm, Chiralpak AS-3 150×4.6mm ID, 3µm, Chiralpak AS-3 100×4.6mm ID, 3µm, ChiralCel OD-3 150×4.6mm ID, 3µm, Chiralcel OD-3 100×4.6mm ID, 3µm, ChiralCel OJ-H 150×4.6mm ID, 5µm, and Chiralcel OJ-3 150×4.6mm ID, 3µm columns.

[0056] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0057] Rapid column purification systems use either the Combiflash Rf150 (TELEDYNE ISCO) or Isolara One (Biotage).

[0058] Normal column chromatography generally uses Yantai Huanghai silica gel of 100-200 mesh, 200-300 mesh or 300-400 mesh as the carrier, or Changzhou Santai pre-filled ultrapure normal phase silica gel column (40-63μm, 60, 12g, 25g, 40g, 80g or other specifications).

[0059] Reversed-phase column chromatography typically uses Changzhou Sante pre-packed ultrapure C18 silica gel columns (20-45μm). 40g, 80g, 120g, 220g or other sizes).

[0060] The high-pressure column purification system uses Waters AutoP, in conjunction with the Waters XBridge BEH C18 OBD Prep Column. 5μm, 19mm×150mm or Atlantis T3OBD Prep Column, 5μm, 19mm×150mm.

[0061] Chiral preparation columns used were DAICL CHIRALPAK IC (250 mm × 30 mm, 10 μm) or Phenomenex-Amylose-1 (250 mm × 30 mm, 5 μm).

[0062] XRPD (X-ray Powder Diffraction) was used for analysis: measurements were performed using a BRUKER D8 X-ray diffractometer. Specific data collected included: Cu anode (40 kV, 40 mA), Cu-Kα1 rays. Kα2 rays Kβ rays Scanning mode: θ / 2θ, scanning range (2θ range): 3°~45°.

[0063] DSC stands for Differential Scanning Calorimetry: Measurements were performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter with a heating rate of 10℃ / min. The specific temperature range was referenced from the corresponding spectra (mostly 25-250℃), and the nitrogen purging rate was 50mL / min.

[0064] TGA is thermogravimetric analysis: the test was performed using a METTLER TOLEDO TGA 2 thermogravimetric analyzer, with a heating rate of 10℃ / min, and the specific temperature range was referenced from the corresponding spectrum (mostly 30-350℃). The nitrogen purging rate was 50mL / min.

[0065] DVS stands for Dynamic Moisture Adsorption: The detection method is SMSDVS Advantage, with humidity changing from 50% to 95% to 0% to 95% to 50% at 25℃, in 10% increments (the final step is 5%) (the specific humidity range is subject to the corresponding spectrum; the methods listed here are the most commonly used). The judgment criterion is Tmax 360min, and dm / dt not greater than 0.002%.

[0066] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0067] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0068] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0069] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: n-hexane / ethyl acetate system, B: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0070] Example 1

[0071] 5-(4-(((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)amino)-7,8-dihydro-5H-pyran[3,4-d]pyridazine-1-yl)-2,3-dihydrobenzofuran-4-ol

[0072] first step

[0073] tert-butyl((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)carbamate 1b

[0074] A solution of tert-butyl((3R,5R)-5-fluoropiperidin-3-yl)carbamate 1a (5.0 g, 22.91 mmol) in dichloroethane (120 mL) was mixed with an aqueous formaldehyde solution (30%, 3.34 mL, 34.36 mmol), stirred at room temperature for 1 hour, and then sodium triacetoxyborohydride (12.14 g, 57.27 mmol) and acetic acid (0.2 mL) were added under ice bath conditions. The reaction mixture was stirred overnight at room temperature. The solvent was removed by vacuum concentration, and water (30 mL) was added to the remaining solution. The pH was adjusted to approximately 9 with ammonia, and the mixture was extracted with dichloromethane (50 mL × 2). The organic phases were combined, washed with saturated brine (30 mL), and dried over anhydrous sodium sulfate. The solution was concentrated under vacuum to give compound 1b (5.1 g, 95% yield). MS m / z (ESI): 233.4 [M+H]+.

[0075] Step 2

[0076] (3R,5R)-5-fluoro-1-methylpiperidin-3-amine 1c

[0077] Compound 1b (5.1 g, 21.95 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (15 mL) was added at room temperature. The mixture was stirred for 2 hours. The reaction solution was concentrated under reduced pressure to obtain crude trifluoroacetate 1c (5.9 g, 110% yield), which was used directly in the next reaction.

[0078] MS m / z (ESI): 133.4 [M+H] + .

[0079] Step 3

[0080] 1-(3,6-dihydro-2H-pyran-4-yl)pyrrolidine 1bb

[0081] Tetrahydro-4H-pyran-4-one 1aa (1.0 g, 10 mmol) and potassium carbonate (0.14 g, 1 mmol) were mixed in tetrahydropyrrole (0.71 g, 10 mmol). The mixture was stirred at 0 °C until fully reacted. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under vacuum to obtain crude compound 1bb (1.35 g), which could be used in the next step without further purification.

[0082] ES-MS m / z = 154.1 [M+H] + .

[0083] Step 4

[0084] 1,4-Dichloro-7,8-dihydro-5H-pyran[3,4-d]pyrazine 1cc

[0085] Compound 1bb (305 mg, 2 mmol) and 3,6-dichlorotetraazine (0.2 g, 1.32 mmol) were mixed in dichloromethane (5 mL). The mixture was stirred at 0 °C until fully reacted, diluted with water (5 mL), and extracted with dichloromethane (5 mL × 3). The liquid and liquid phases were separated, washed with brine (5 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum to obtain the crude product, which was purified by rapid column chromatography (eluent: 0-20% ethyl acetate in petroleum ether) to give compound 1cc (24 mg, yield 18.3%).

[0086] MS m / z(ESI): 205.0 [M+H] + .

[0087] 1 H NMR (400MHz, CDCl3): δppm 4.70 (s, 2H), 4.05 (t, J = 5.5Hz, 2H), 2.53 (t, J = 6.0Hz, 2H).

[0088] Step 5

[0089] 1-Chloro-N-((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)-7,8-dihydro-5H-pyrano[3,4-d]pyrazine-4-amine1d-2

[0090] The crude trifluoroacetate of compound 1c (5.9 g, 21.94 mmol), compound 1cc (4.5 g, 21.94 mmol), tris(dibenzylacetone)dipalladium (0.8 g, 0.88 mmol), cesium carbonate (25.02 g, 76.79 mmol), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (2.73 g, 4.39 mmol) were mixed in dry toluene (120 mL) and stirred overnight at 110 °C under a nitrogen atmosphere. After cooling the reaction solution to room temperature, it was filtered, and the residue was washed with ethyl acetate (20 mL × 3). The filtrates were combined. The filtrate was concentrated under vacuum to obtain the crude product, which was then purified by rapid column chromatography. The purified product was further separated by SFC (column: ChiralPak AY, 250×20mm ID, 5μm, mobile phase: A: CO2; B: MeOH + 0.1% NH3H2O, gradient: B 40%; flow rate: 40mL / min; temperature: 35℃).

[0091] Compound 1d-2 (280 mg, yield 4.2%).

[0092] 1 H NMR (400MHz, DMSO) δ5.89(d,J=7.8Hz,1H),4.93(d,J=47.1Hz,1H),4.51-4.43(m,1H),4.41(s,2H),3.87(t,J=5.6Hz,2H ),2.96-2.83(m,2H),2.60(t,J=5.4Hz,2H),2.19(s,3H),2.18-2.04(m,2H),1.87(t,J=10.1Hz,1H),1.76-1.57(m,1H).

[0093] 19 F NMR (377MHz, DMSO) δ-180.76 (s).

[0094] MS m / z (ESI): 301.3 [M+H] + .

[0095] Step 6

[0096] 5-Bromo-4-(methoxymethoxy)-2,3-dihydrobenzofuran 1f

[0097] 5-Bromo-2,3-dihydrobenzofuran-4-ol 1e (5.9 g, 27.44 mmol) was dissolved in dichloromethane (60 mL). N,N-diisopropylethylamine (9.1 mL, 54.87 mmol) and chloromethyl ether (MOMCl) (2.87 g, 35.67 mmol) were added dropwise under ice bath conditions. The reaction was allowed to proceed to completion at room temperature. The solution was diluted with dichloromethane (200 mL), and the diluted solution was concentrated under vacuum to obtain a crude product. Purification was performed by rapid column chromatography to give compound 1f (6.05 g, 76.6% yield).

[0098] 1HNMR (400MHz, CDCl3), δ7.18(d,1H),6.38(d,1H),5.05(s,2H),4.48(t,2H),3.50(s,3H),3.24(t,2H).

[0099] Step 7

[0100] 1g of 2-(4-(methoxymethoxy)-2,3-dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane

[0101] Under a nitrogen atmosphere, compound 1f (6.05 g, 23.35 mmol) was dissolved in acetonitrile (100 mL). Pinara-borane (5.08 mL, 35.03 mmol), triethylamine (9.74 mL, 70.05 mmol), and Pd(dppf)Cl2 (1.73 g, 2.34 mmol) were slowly added at room temperature, and the reaction temperature was raised to 80 °C. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under vacuum to obtain the crude product. This crude product was purified by rapid column chromatography to give compound 1 g (5.7 g, yield 71.76%).

[0102] 1HNMR (400MHz, DMSO), δ7.42(d,1H),6.54(d,1H),5.03(s,2H),4.55(t,2H),3.45(s,3H),3.19(t,2H),1.26(s,12H).

[0103] Step 8

[0104] N-((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)-1-(4-(methoxymethoxy)-2,3-dihydrobenzofuran-5-yl)-7,8-dihydro-5H-pyran[3,4-d]pyridazine-4-amine1h

[0105] Under a nitrogen atmosphere, compound 1d-2 (270 mg, 0.90 mmol) was dissolved in 1,4-dioxane (10 mL) and water (1 mL). Cesium carbonate (731 mg, 2.24 mmol), (1,1'-bis(diphenylphosphine)ferrocene)palladium dichloride (65 mg, 0.09 mmol), and 1 g of compound (329 mg, 1.08 mmol) were added at room temperature. The reaction was heated to 95 °C and stirred. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the filtrate was diluted with ethyl acetate (50 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered again. The filtrate was concentrated under vacuum to obtain the crude product, which was purified by rapid column chromatography to give compound 1h (260 mg, 65% yield).

[0106] MS m / z (ESI): 445.4 [M+H] + .

[0107] Step 9

[0108] 5-(4-(((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)amino)-7,8-dihydro-5H-pyran[3,4-d]pyridazine-1-yl)-2,3-dihydrobenzofuran-4-ol

[0109] At room temperature, a 1,4-dioxane solution (4M, 10 mL) of hydrogen chloride was added to a tetrahydrofuran solution (15 mL) containing 260 mg (0.59 mmol) of compound 1 h, and the mixture was stirred until the reaction was complete. After concentration, the residue was adjusted to pH ≈ 9 with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (20 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid column chromatography to give compound 1 (137 mg, yield 58%).

[0110] MS m / z (ESI): 401.4 [M+H] + .

[0111] 1HNMR(400MHz,DMSO)δ10.15(s,1H),7.00(d,J=8.4Hz,1H),6.35(d,J=8.4Hz,1H) ,5.66(d,J=7.6Hz,1H),4.95(d,J=47.4Hz,1H),4.57(t,J=8.8Hz,3H),4.48(s,2H ),3.76(t,J=5.6Hz,2H),3.14(t,J=8.8Hz,2H),2.99-2.86(m,2H),2.52-2.51(m ,2H),2.21(s,3H),2.17-2.08(m,2H),1.91(t,J=10.0Hz,1H),1.79-1.62(m,1H).

[0112] 19 F NMR (377MHz, DMSO) δ-180.65 (s).

[0113] Test Example 1: Determination of NLRP3 inflammasome inhibitory activity in THP1-Null cells

[0114] 1. Experimental instruments and reagents

[0115] 1.1 Experimental Apparatus

[0116] Plate reader:PerkinElmer 2104EnVision Multilabel Plate Readers

[0117] 1.2 Experimental Reagents

[0118] 2. Experimental Design

[0119] Day 1: THP1-Null cells were seeded in 96-well plates at 10 × 10⁶ cells / well. 4 Cells / 180 μL of hygromycin B- and Normocin-free medium / well. Add 20 μL of PMA (final concentration 100 nM) to each well and incubate at 37°C in 5% CO2 for 24 hours.

[0120] Day 2: Dilute the compound with phenol red-free RPMI 1640 medium containing 25 mM HEPES and 0.5% DMSO. Remove the medium and pretreat cells with 160 μL of medium containing the compound, incubating at 37°C for 1 hour in 5% CO2. Then prepare LPS in phenol red-free RPMI 1640 medium containing 25 mM HEPES. Add 20 μL of 9 μg / mL LPS (final concentration 1 μg / mL) to each well and incubate at 37°C for 3.5 hours in 5% CO2. Add 20 μL of 50 mM ATP (final concentration 5 mM) to the compound-treated wells and high control wells, and add 20 μL of medium to the low control wells, incubating at 37°C for 0.5 hours in 5% CO2. Transfer 160 μL of the supernatant to a new plate and store at -80°C.

[0121] Day 3: The supernatant was used for IL-1β release assay in THP1-Null cells according to the manufacturer's instructions.

[0122] (1) Take 16 μL of IL1β standard (Std 0-Std 7) and add it to each standard well, or take 16 μL of the sample to be tested and add it to each sample well.

[0123] (2) Add 4 μL of premixed IL1β antibody working solution to all wells.

[0124] (3) Seal the plate and incubate overnight at room temperature.

[0125] (4) Read the ratio of fluorescence wavelengths 665 / 620 using the Envision instrument.

[0126] 3. Experimental Results

[0127] Activation of the NLRP3 inflammasome leads to the release of the inflammatory cytokine IL-1β, while dysregulation of NLRP3 inflammasome activation drives the development of many diseases. The data in the table show that the compounds disclosed in this paper have NLRP3 inhibitory activity.

[0128] Table 1. Tests on the inhibitory activity of the compounds disclosed in this paper against NLRP3.

[0129] Test Example 2. Toxicity assay of NLRP3 inhibitor in HepG2 cells

[0130] 1. Experimental instruments and reagents

[0131] 1.1 Experimental Apparatus

[0132] PerkinElmer 2104 EnVision Multilabel Plate Readers

[0133] Multidrop TM Pico 8 Digital Dispenser

[0134] 1.2 Experimental Reagents

[0135] Compound A: Compound 50 prepared according to the method of Example 45 of WO2022135567A.

[0136] 2. Experimental Design

[0137] Day 1: HepG2 cells were seeded in 384-well plates at a density of 2 × 10⁶ cells / well. 3 Cells / 50 μL DMEM medium containing 10% FBS / well, cultured overnight at 37°C with 5% CO2.

[0138] Day 2: via Multidrop TM The Pico 8 Digital Dispenser was used to add serially diluted compounds to 50 μL of cell culture medium, resulting in final compound concentrations of 150, 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.069, and 0.023 μM. The DMSO concentration was less than 0.3%. Cells were incubated at 37°C in a 5% CO2 incubator for 2 days.

[0139] Day 4: Add 25 μL of the assay kit (Celltiter Glo assay kit) to each well and shake (in the dark) for 3 min. Incubate at room temperature in the dark for 10 min, then read the plate using the Envision instrument.

[0140] 3. Experimental Results

[0141] Table 2. Cytotoxicity test of the disclosed compounds against HepG2 cells.

[0142] Compared to compound A, compound 1 exhibits weaker cytotoxicity against HepG2.

[0143] Test Example 3: Inhibition of hERG potassium ion channels by compound

[0144] 1. Experimental materials and instruments

[0145] Compound A: Compound 50 prepared according to the method of Example 45 of WO2022135567A.

[0146] 2. Cell lines and cell culture

[0147] HEK293 cell line (catalog number: K1236) stably expressing the hERG ion channel was purchased from Invitrogen. This cell line was cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acid solution, 100 U / mL penicillin-streptomycin solution, 25 mM HEPES, 5 μg / mL blastomycin, and 400 μg / mL genimycin. When the cell density reached 40%–80% of the culture dish bottom area, the cells were digested with trypsin and passaged three times per week. Before experiments, cells were cultured at a density of 5 × 10⁵ cells in 6 cm culture dishes, induced with 1 μg / mL doxycycline for 48 hours, then digested and seeded on slides for subsequent manual patch-clamp experiments.

[0148] 3. Solution preparation

[0149] 1) Extracellular fluid (in mM): 132 sodium chloride, 4 potassium chloride, 3 calcium chloride, 0.5 magnesium chloride, 11.1 glucose and 10 HEPES (pH adjusted to 7.35 with sodium hydroxide).

[0150] 2) Intracellular fluid (in mM): 140 potassium chloride, 2 magnesium chloride, 10 EGTA, 5 magnesium ATP and 10 HEPES (pH adjusted to 7.35 using potassium hydroxide).

[0151] 4. Preparation of the solution of the compound to be tested

[0152] 1) The test compound was dissolved in DMSO and prepared into a stock solution with a final concentration of 10 mM.

[0153] 2) The stock solution was serially diluted with DMSO at a ratio of 1:3 to prepare three other intermediate concentration solutions with concentrations (mM) of 3.33, 1.11 and 0.37.

[0154] 3) Before the experiment, the intermediate solution of the test compound gradient was diluted again with extracellular fluid at a ratio of 1:1000 to prepare a series of working solutions with final concentrations (μM): 10, 3.33, 1.11, and 0.37. The 30 μM working solution was prepared by diluting the 10 mM stock solution 333.33 times. The content of DMSO in the working solutions was 0.1-0.3% (volume ratio).

[0155] 4) Five working solutions at different concentration gradients (30, 10, 3.33, 1.11, and 0.37 μM) were used to determine the potential inhibitory effect of the compound on hERG channels, and to fit dose-response curves and calculate IC50. 50 .

[0156] 5. Experimental Design

[0157] 1) Place the small glass slide containing HEK293 cells in the culture dish into the perfusion tank of the micromanipulation table.

[0158] 2) Using an Olympus IX51, IX71, or IX73 inverted microscope, position the appropriate cells in the center of the field of view. Use a ×10 objective lens to locate the tip of the glass electrode and center it in the field of view. Then, use the micromanipulator to move the electrode down while adjusting the coarse focus knob to slowly bring the electrode closer to the cells.

[0159] 3) When you get close to the cell, switch to a 40x objective lens for observation. Use the micromanipulator to fine-tune the setting so that the electrode gradually gets closer to the cell surface.

[0160] 4) Apply negative pressure to form a seal with a resistance higher than 1G between the electrode tip and the cell membrane.

[0161] 5) In voltage clamping mode, the instantaneous capacitive current C fast Compensation is then performed. Short bursts of negative pressure are then repeatedly applied to rupture the membrane, eventually forming a whole-cell recording pattern.

[0162] 6) Under the condition that the membrane potential is clamped at -60mV, the slow capacitive current C slow The cell membrane capacitance (Cm) and input membrane resistance (Ra) are compensated separately.

[0163] 7) After the cells stabilize, change the clamping voltage to -90mV, set the sampling frequency to 20kHz, and the filtering frequency to 10kHz. The leakage current detection conditions are: clamping voltage changed to -80mV, time duration 500ms.

[0164] 8) The hERG current testing method is as follows: A depolarization command voltage of 4.8 seconds is applied to depolarize the membrane potential from -80mV to +30mV. Then, a repolarization voltage of 5.2 seconds is applied instantaneously to reduce the membrane potential to -50mV to remove channel inactivation, thereby allowing the hERG tail current to be observed. The peak value of the tail current is the magnitude of the hERG current.

[0165] 9) The hERG currents used to detect the test compounds were continuously recorded for 120 seconds before drug administration to assess the stability of hERG current generation in the test cells. Only stable cells within the acceptable range of the evaluation criteria were allowed to proceed to the subsequent compound detection.

[0166] Testing the inhibitory effect of the test compound on hERG current: First, the hERG current measured in extracellular fluid containing 0.1% DMSO was used as the baseline. After the hERG current stabilized for at least 5 minutes, the solution containing the test compound was sequentially perfused around the cells from low to high concentration. After each perfusion, approximately 5 minutes were allowed for the compound to fully act on the cells while simultaneously recording the hERG current. Once the recorded current stabilized, the last 5 hERG current values ​​were recorded, and their average was taken as the final current value at the specific concentration. After testing the compound, 150 nM of dofilad (positive control) was added to the same cell to completely inhibit its current, serving as a positive control for that cell. Simultaneously, the positive control compound dofilad was detected synchronously before and after the test drug experiment using the same patch-clamp system to ensure the reliability and sensitivity of the entire detection system.

[0167] 6. Data Analysis

[0168] 1) After injecting blank solvent or compound gradient solution, calculate the average of the five consecutive current values ​​obtained after stabilization, and use these averages as the "tail current magnitude". 空白 "and tail current magnitude" 化合物 ".

[0169] 2) The current suppression percentage is calculated using the following formula.

[0170] 3) The dose-response curve was fitted using Graphpad Prism 8.0 software and the IC was calculated. 50 value.

[0171] 7. Experimental Results

[0172] Table 3. Effects of the compounds disclosed herein on hERG potassium ion channels IC50 50 The result of the value

[0173] Compared to compound A, compound 1 disclosed herein has a weaker inhibitory effect on hERG potassium ion channels.

[0174] Example 4. Efficacy of drugs in rats with acute cerebral ischemia-reperfusion (tMCAO)

[0175] 4.1 Information on experimental animals and materials

[0176] Male Sprague-Dawley rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), 6-7 weeks old, weighing 190-210g.

[0177] The manufacturer of Stera 50 (Cat#8WSEA) is Vic, a French company.

[0178] The manufacturer of Xylazine Injection (Cat#20220430) is Jilin Huamu Animal Health Products Co., Ltd.

[0179] TTC (2,3,5-triphenylchlorotetrazolium, Cat#BCCF4219) is manufactured by Sigma.

[0180] Compound 1, The preparation method is described in Example 1.

[0181] 4.2 Experimental Design

[0182] The dosage and administration regimen for this independent experiment are shown in Table 4.

[0183] Table 4. Experimental design for the efficacy study of the test drug in tMCAO rats " / " indicates not applicable; "iv" indicates intravenous administration.

[0184] 4.3 Experimental Procedure

[0185] Sprague-Dawley rats were acclimatized for one week before the experiment began. Rats were weighed and randomly grouped. During the experiment, rats were anesthetized with a combination of 50 mg / kg (im) and xylazine (8 mg / kg, ip). The rats were then fixed in a supine position, and the right common carotid artery was exposed. The external and internal carotid arteries were separated. The internal carotid artery was clamped with an arterial clamp. A small incision was made between the two lines of the external carotid artery, and a suture plug was inserted from the incision toward the internal carotid artery. The suture plug was gently secured with a suture, the arterial clamp was released, and the suture plug was gently pushed inward toward the carotid artery until the suture plug was marked (approximately 18 mm ± 0.5 mm). The suture plug was then secured at the external carotid artery, and the rat's neck was sutured. After 90 minutes of ischemia, the suture plug was removed to the external carotid artery, restoring blood supply from the common carotid to the internal carotid artery. Cerebral blood flow in the ischemic area was tested using a laser speckle flow imaging system. A 50% decrease in cerebral blood flow (rCBF ≥ 50%) was considered a successful model. The rats were kept at 37°C post-operatively until they regained consciousness. The drugs were administered intravenously to rats 30 minutes after ischemia, 5 hours after reperfusion, and 22 hours after reperfusion. 24 hours after reperfusion, the rats were euthanized, and brain tissue was collected for TTC staining to calculate the infarct area.

[0186] 4.4 Data Analysis

[0187] Statistical analysis was performed using Graphpad Prism 9 software. The analysis was based on the raw data, and the results are expressed as mean ± SEM. One-way ANOVA and Dunnett's test were used for statistical analysis, with p < 0.05 considered statistically significant.

[0188] 4.5 Experimental Results

[0189] Table 5. Results of efficacy testing of the test drug in tMCAO rats

[0190] As shown in Figure 1, compared with the model group, compound 1 significantly improved the infarct area at a dose of 7.5 mg / kg, and the effect was even better at a dose of 15 mg / kg (**p<0.01, *p<0.05 vs Model).

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cerebrovascular diseases. in, R 1 Selected from hydrogen, methyl, or difluoromethyl; R 2 Each is independently selected from hydrogen, halogen, and C. 1-6 alkyl; R 3 and R 4 Each is independently selected from hydrogen or halogen; R 5 Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 6 Selected from C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 7 Selected from fluorine; m and p are each independently selected from 0, 1, 2, and 3; n is independently selected from 1, 2, and 3.

2. The use according to claim 1, wherein the R 1 Selected from hydrogen.

3. The use according to claim 1 or 2, wherein the R 2 Each is independently selected from hydrogen.

4. The use according to any one of claims 1 to 3, wherein the R 3 and R 4 Each is independently selected from hydrogen.

5. The R according to the use described in any one of claims 1 to 4 5 Each is independently selected from hydrogen.

6. The use according to any one of claims 1 to 5, wherein the R 6 Selected from methyl or ethyl, preferably methyl.

7. The use according to any one of claims 1 to 6, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is compound 1 or a pharmaceutically acceptable salt thereof.

8. The use according to any one of claims 1 to 7, wherein the cerebrovascular disease is an ischemic cerebrovascular disease.

9. The use according to any one of claims 1 to 7, wherein the cerebrovascular disease is stroke; preferably hemorrhagic stroke or ischemic stroke; most preferably ischemic stroke.

10. The use according to any one of claims 1 to 9, wherein the cerebrovascular disease is localized necrosis or softening of brain tissue (e.g., nerve cells, glial cells, and connecting fibers).

11. A method for treating cerebrovascular disease, comprising administering to a subject a compound of formula (I) or a pharmaceutically acceptable saline thereof and reperfusion therapy. in, R 1 Selected from hydrogen, methyl, or difluoromethyl; R 2 Each is independently selected from hydrogen, halogen, and C. 1-6 alkyl; R 3 and R 4 Each is independently selected from hydrogen or halogen; R 5 Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 6 Selected from C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 7 Selected from fluorine; m and p are each independently selected from 0, 1, 2, and 3; n is independently selected from 1, 2, and 3.

12. The method according to claim 11, wherein the R 1 Selected from hydrogen.

13. The method according to any one of claims 11 or 12, wherein the R 2 Each is independently selected from hydrogen.

14. The method according to any one of claims 11 to 13, wherein the R 3 and R 4 Each is independently selected from hydrogen.

15. The method according to any one of claims 11 to 14, wherein the R 5 Each is independently selected from hydrogen.

16. The method according to any one of claims 11 to 15, wherein the R 6 Selected from methyl or ethyl, preferably methyl.

17. The method according to any one of claims 11 to 16, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is compound 1 or a pharmaceutically acceptable salt thereof.

18. The method according to any one of claims 11 to 17, wherein the cerebrovascular disease is an ischemic cerebrovascular disease.

19. The method according to any one of claims 11 to 17, wherein the cerebrovascular disease is stroke; preferably hemorrhagic stroke or ischemic stroke; most preferably ischemic stroke.

20. The method according to any one of claims 11 to 19, wherein the cerebrovascular disease is localized brain tissue (e.g., nerve cells, glial cells, and connecting fibers) necrosis or softening.

21. The method according to any one of claims 11 to 20, wherein the reperfusion therapy is selected from intravenous thrombolysis or mechanical thrombectomy.

22. The method according to any one of claims 11 to 21, wherein the intravenous thrombolysis is performed using a drug that dissolves thrombi or reduces thrombus formation; preferably, the drug that dissolves thrombi or reduces thrombus formation is tissue plasminogen activator (tPA).

23. The use according to any one of claims 1 to 10 or the method according to any one of claims 11 to 22, wherein the dosage of the compound of formula (I) or its pharmaceutically acceptable salt is selected from 0.01 mg to 1000 mg, and the frequency of administration is selected from once a month, twice a month, three times a month, every other week, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, every other day, once a day, twice a day, or three times a day.

24. The use or method according to claim 23, wherein the compound of formula (I) or its pharmaceutically acceptable salt is administered by parenteral administration or oral administration, preferably oral administration.

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