Pyrazolone compound as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a pyrazolone compound, a pharmaceutical composition containing the pyrazolone compound and application of the pyrazolone compound and the pharmaceutical composition. The pyrazolone compound is a compound shown in a general formula I or a tautomer, a raceme, a meso-raceme, an enantiomer, a diastereoisomer and pharmaceutically acceptable salt of the compound. The pyrazolone compound provided by the invention can inhibit a Z-type nucleic acid binding protein 1 (ZBP1) signal channel, and has an application prospect in treatment of diseases related to ZBP1 protein functions.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a pyrazolinone compound, its pharmaceutical composition, and its uses. Background Technology

[0002] Z-DNA binding protein 1 (ZBP1) was first discovered in 1999. Its N-terminus contains two functional Z-type nucleic acid binding domains, Zα1 and Zα2, which recognize and bind to Z-DNA and Z-RNA, respectively. [1] The central portion of the ZBP1 protein contains a receptor-interaction homology interaction motif (RHIM), enabling ZBP1 to homologously interact with other proteins (RIPK1, RIPK3, and TRIF) containing the RHIM domain. Its conserved C-terminus can interact with TBK1 and IRF3 to induce a type I interferon (IFN) response to immunostimulatory DNA. [2] Furthermore, the Z-DNA binding domain (ZBD) found in ZBP1 is also present in the editing enzyme dsRNA adenosine deaminase (ADAR1) of vertebrate and fish PKZ protein kinases, and in the E3L of poxviruses, and can typically convert B-DNA into Z-DNA and bind to it. Although they have limited similarity at the amino acid sequence level, they share similar binding patterns with Z-DNA, with many conserved residues involved in DNA binding.

[0003] ZBP1 is an interferon-induced cytoplasmic nucleic acid sensor that senses endogenous or exogenous Z-nucleotides. Upon activation, it recruits RIPK3 via its RHIM domain, leading to RIPK3 autophosphorylation and MLKL phosphorylation. Phosphorylated MLKL oligomerizes and translocates to the membrane, thereby disrupting membrane integrity and inducing inflammatory programmed cell death, including necrotizing apoptosis. [3] Another protein containing RHIM, RIPK1, can prevent ZBP1 from activating RIPK3, thereby inhibiting ZBP1-mediated inflammatory cell death. [4] Furthermore, ZBP1 can regulate the activation of the NLRP3 inflammasome through the RIPK3-caspase-8 axis to mediate pyroptosis and the secretion of IL-1β and IL-18. In the presence of both RIPK3 and RIPK1, RIPK3 can form a complex with RIPK1, FADD, and caspase-8, promoting apoptosis. [5] In the absence of RIPK3, ZBP1 binds to RIPK1, activating the NF-κB signaling pathway and mediating the secretion of IL-6 and TNF, forming an important line of defense against pathogen infection.

[0004] ZBP1 plays a crucial role in autoinflammatory diseases, regulating cell death, and acting as a pathogen sensor, representing a novel potential target and approach for infectious and autoinflammatory diseases. Nicholas Hubbard et al. reported in Nature that autoinflammatory diseases (AGS) caused by point mutations in the ZBD sequence of ADAR1 are driven by ZBP1 activation. [6] ZBP1 deficiency can rescue pathological responses caused by ADAR1 alterations. Studies by Richard de Reuver et al. have shown that ADAR1 is a negative regulator of ZBP1; ADAR1 can limit the formation and accumulation of endogenous immune-stimulating double-stranded RNA, further inhibiting spontaneous ZBP1 activation, thereby preventing autoimmune diseases. [7] Studies by Huipeng Jiao et al. have reached similar conclusions from different perspectives. ZBP1 regulates type I IFN expression through a novel mechanism, while ADAR1 inhibits the Z-RNA-dependent activation of ZBP1 in response to pathogenic type I IFN. [8] The above research demonstrates that abnormal ZBP1 activation-mediated programmed cell death is a crucial node in inflammatory autoinflammatory diseases. To avoid the production of such Z-type nucleic acids in the body, the host utilizes enzymes such as adenosine deaminase (ADAR) to perform post-transcriptional base modifications on RNA, thereby preventing endogenous pattern recognition receptors from sensing and recognizing Z-type nucleic acids. However, when ADAR1 is mutated, it can produce or introduce abnormal Z-type nucleic acids, leading to autoinflammatory immune diseases such as Aicardi-Goutières Syndrome 6 (AGS), systemic lupus erythematosus (SLE), and bilateral striatal necrosis (BSN).

[0005] ZBP1-mediated cell necrosis and inflammation play an important role in the body's immune defense and maintenance of homeostasis. Research on ZBP1 inhibitors holds promise for the treatment of infectious diseases, inflammatory diseases, and autoimmune diseases.

[0006] [1] Jiao , H. , Wachsmuth , L. , Kumari , S. , Schwarzer , R. , Lin , J. ,Eren , RO , Fisher , A. , Lane , R. , Young , GR , Kassiotis , G. , Kaiser , WJ , & Pasparakis , M. (2020). Z-nucleic acid sensing triggers ZBP1-dependentnecroptosis and inflammation. Nature, 580(7803), 391–395.

[0007] [2] Takaoka , A. , Wang , Z. , Choi , M. K. , Yanai , H. , Negishi , H. , Ban , T. , Lu , Y. , Miyagishi , M. , Kodama , T. , Honda , K. , Ohba , Y. , & Taniguchi , T. (2007). DAI (DLM-1 / ZBP1) is a cytosolic DNA sensor and an activator of the innateimmune response. Nature, 448(7152), 501–505.

[0008] [3] Yang , D. , Liang , Y. , Zhao , S. , Ding , Y. , Zhuang , Q. , Shi , Q. , Ai , T. , Wu , SQ , & Han , J. (2020). ZBP1 mediates interferon-inducednecroptosis. Cellular & molecular immunology, 17(4), 356–368.

[0009] [4] Newton, K., Wickliffe, K. E., Maltzman, A., Dugger, D. L.,Strasser, A., Pham, V. C., Lill, J. R., Roose-Girma, M., Warming, S., Solon,M., Ngu, H., Webster, J. D., & Dixit, V. M. (2016). RIPK1 inhibits ZBP1-driven necroptosis during development. Nature, 540(7631), 129–133.

[0010] [5] Kuriakose, T., & Kanneganti, T. D. (2018). ZBP1: Innate SensorRegulating Cell Death and Inflammation. Trends in immunology, 39(2), 123–134.

[0011] [6] Hubbard, N. W., Ames, J. M., Maurano, M., Chu, L. H., Somfleth,K. Y., Gokhale, N. S., Werner, M., Snyder, J. M., Lichauco, K., Savan, R.,Stetson, D. B., & Oberst, A. (2022). ADAR1 mutation causes ZBP1-dependentimmunopathology. Nature, 607(7920), 769–775.

[0012] [7] de Reuver, R., Verdonck, S., Dierick, E., Nemegeer, J., Hessmann,E., Ahmad, S., Jans, M., Blancke, G., Van Nieuwerburgh, F., Botzki, A., Vereecke, L., van Loo, G., Declercq, W., Hur, S., Vandenabeele, P., &Maelfait, J. (2022). ADAR1 prevents autoinflammation by suppressingspontaneous ZBP1 activation. Nature, 607(7920), 784–789.

[0013] [8] Jiao, H., Wachsmuth, L., Wolf, S., Lohmann, J., Nagata, M., Kaya, GG, Oikonomou, N., Kondylis, V., Rogg, M., Diebold, M., Tröder, SE, Zevnik, B., Prinz, M., Schell, C., Young, GR, Kassiotis, G., & Pasparakis, M. (2022). ADAR1 averts fatal type I interferon induction by ZBP1. Nature, 607(7920), 776–783. Summary of the Invention

[0014] The purpose of this invention is to provide a pyrazolinone compound, a pharmaceutical composition thereof, and its use, in order to solve the problems mentioned in the background art.

[0015] To achieve the above objectives, the present invention provides the following technical solution:

[0016] This invention provides a pyrazolinone compound, which is a pyrazolinone compound as shown in general formula I or an isomer of a pyrazolinone compound as shown in general formula I, or a pharmaceutically acceptable salt thereof;

[0017]

[0018] Among them, R 1 Selected from hydrogen, C(O)2Ra or C(O)NHR b ;

[0019] R 2 Selected from hydrogen, OR c ;

[0020] R 3 R 4 R 5 R 6 R 7 Each is independently selected from hydrogen, C1-C6 alkyl, halogen, halogen-substituted C1-C6 alkyl, and -C(O)2R. d Nitro, cyano, C1-C6 alkoxy, halogen-substituted C1-C6 alkoxy, -C(O)NHR e C3-C6 nitrogen-containing heteroaryl groups; wherein each of the C1-C6 alkyl groups is independently optionally divided by one or more R f replace;

[0021] R a R c R d Each is independently selected from hydrogen or C1~C6 alkyl groups;

[0022] R b R e Each is independently selected from hydrogen or hydroxyl;

[0023] R f Each is independently selected from C1-C6 alkyl groups that are hydrogen- or halogen-substituted;

[0024] The isomers include tautomers, racemates, mesomers, enantiomers, and diastereomers.

[0025] As a further improvement of the present invention

[0026] R 1 For hydrogen, C(O)2R a or C(O)NHR b ;

[0027] R 2 For hydrogen, OR c ;

[0028] When R 3 R 4 R 5 R 6 R 7 When the halogen-substituted C1-C6 alkyl group is a halogen-substituted C1-C6 alkyl group, the halogen-substituted C1-C6 alkyl group is a trifluoromethyl group;

[0029] When R 3 R 4R 5 R 6 R 7 When the halogen is halogen, the halogen includes fluorine, chlorine, and bromine;

[0030] When R 3 R 4 R 5 R 6 R 7 When the C1-C6 alkoxy group is halogen-substituted, the halogen-substituted C1-C6 alkoxy group is a trifluoromethoxy group;

[0031] When R 5 When the C3-C6 nitrogen-containing heteroaryl group is C3-C6, the C3-C6 nitrogen-containing heteroaryl group is tetrazolium.

[0032] As a further improvement of the present invention, the C1-C6 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, and tert-butyl; the C1-C6 alkoxy group is selected from methoxy, ethoxy, n-propyloxy, isopropyloxy, and tert-butyloxy.

[0033] As a further improvement of the present invention, the pharmaceutically acceptable salt includes organic or inorganic base salts, selected from ammonium salts, alkylammonium salts, ethylenediamine salts, cyclohexylamine salts, arginine salts, lysine salts, choline salts, sodium salts, potassium salts, calcium salts, and magnesium salts.

[0034] As a further improvement of the present invention, the pyrazolinone compound having one of the following structures, or isomers of pyrazolinone compounds having one of the following structures, and their pharmaceutically acceptable salts:

[0035]

[0036]

[0037] .

[0038] The present invention also provides a pharmaceutical composition comprising the pyrazolinone compounds described above, pharmaceutical excipients, and / or pharmaceutically acceptable carriers.

[0039] As a further improvement of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of one or a mixture of the pyrazolinone compound, its pharmaceutically acceptable crystal form, hydrate, solvate, prodrug, metabolite, and further comprises pharmaceutical excipients and / or a pharmaceutically acceptable carrier.

[0040] Pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions, including solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc., with oral pharmaceutical excipients such as disintegrants, solubilizers, solvents, and lubricants being preferred.

[0041] Pharmaceutically acceptable carriers are systems that can alter the way drugs enter the body and their distribution within the body, control the rate of drug release, and deliver drugs to target organs. These include microcapsules and microspheres, nanoparticles, and liposomes. When a drug composition is used for solid tumor diseases, the preferred composition is an active ingredient plus a carrier.

[0042] This invention also provides the use of the pyrazolone compounds or pharmaceutical compositions described above in the preparation of remedies for diseases related to ZBP1 protein function. The compounds of this invention can inhibit the ZBP1 signaling pathway and have promising applications in treating diseases related to ZBP1 protein function.

[0043] As a further improvement of the present invention, the diseases related to ZBP1 protein function include inflammation, autoimmune diseases, and infectious diseases.

[0044] The inflammatory diseases mentioned include atherosclerosis, non-alcoholic steatohepatitis (NASH), inflammatory bowel disease (IBD), acute respiratory distress syndrome (ARDS), ischemia-reperfusion injury, and sepsis.

[0045] The infectious diseases mentioned include bacterial and viral infections, particularly those related to influenza A virus (IAV), herpes simplex virus (HSV), Francisella, Candida albicans, Aspergillus fumigatus, and human immunodeficiency virus (HIV) infection.

[0046] The autoimmune diseases mentioned include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriasis, multiple sclerosis (MS), and autoimmune liver disease (ALD).

[0047] The present invention also provides the use of the pyrazolinone compounds or the pharmaceutical compositions described above in the preparation of Z-type nucleic acid binding protein inhibitors.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] Compared to existing ZBP1 inhibitor molecules, this invention features a simplified structure, reduced molecular complexity, and a simpler synthetic route. It utilizes the classic route of pyrazolone condensation, hydrazone reaction, and biphenyl coupling, making it suitable for industrial production. Its structural parameters (molecular weight approximately 500, hydrogen bond donor / acceptor ratio of 4 / 6, and lipid-water partition coefficient clogP in the 3-4 range) better conform to the "five rules of drug-likeness," indicating superior drug development potential. The pyrazolone compounds of this invention are inhibitors that directly target the Z-type nucleic acid binding protein (ZBP1), which senses viral Z-type nucleic acid and endogenous reverse transcription elements. ZBP1 inhibitors are specific sensors whose inhibitors can block only pathological necrosis and inflammation triggered by viral or endogenous nucleic acid abnormalities, preserving the host's basic defense capabilities against other pathogens. They hold promise for treating diseases that are currently unresponsive to treatments (such as some neuroinflammatory diseases and autoimmune diseases), providing new treatment options. ZBP1 inhibitors represent an innovative direction in drug development and have significant scientific value in the fields of medicinal chemistry and cell death. In research on autoimmune diseases and neurological diseases (such as amyotrophic lateral sclerosis and Alzheimer's disease), ZBP1 inhibitors have unique advantages, precisely targeting pathogenic signals. Attached Figure Description

[0050] Figure 1 These are the results of a Western-Blot experiment based on MEF cells;

[0051] Figure 2 A graph showing the colon length of mice after drug administration in a mouse colitis model;

[0052] Figure 3 This is the general structural formula for the pyrazolinone compounds of this invention. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] For experimental methods in this embodiment of the invention that do not specify specific conditions, follow conventional methods and conditions, or select according to the product instructions.

[0055] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or high-resolution mass spectrometry (HRMS). 1 H NMR and 13C10 NMR spectra were determined using a Bruker AV-300 NMR spectrometer with deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrometry (HDMS) was performed using a WaterQ-Tof micro mass spectrometer manufactured by Waters Corporation, USA. Chemical reactions were detected using a 0.25 mm GF254 thin-layer chromatography silica gel plate and observed using a ZF7 three-way UV analyzer.

[0056] Unless otherwise specified in the embodiments, the reaction is carried out in an air atmosphere.

[0057] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0058] Unless otherwise specified in the examples, the reaction temperature is room temperature of 20–30 °C.

[0059] In the examples, the purity of the compounds was determined by HPLC using an Agilent C18 (4.6 mm × 150 mm, 3.5 μm) reversed-phase column, with methanol / water (0.1% formic acid) mixed solvent as the mobile phase and a flow rate of 1 mL / min. The compounds were detected by UV absorption at 254 nm and 365 nm.

[0060] Example 1

[0061] Preparation of compound C1:

[0062] .

[0063] Step 1: Synthesize intermediates 1-2

[0064] Starting material 1-1 (3 g, 27.5 mmol) was dissolved in tetrahydrofuran, and N,N-carbonyldiimidazole (4.46 g, 27.5 mmol) was added. The mixture was heated under reflux for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched with dilute hydrochloric acid. The mixture was concentrated, extracted with ethyl acetate and water, and the organic layer was washed with saturated NaCl, dried over anhydrous Na2SO4, and dried under vacuum to obtain 1-2 in 85.3% yield. 1 H NMR (300 MHz, DMSO-d6) δ 11.62 (s, 1H), 7.28 (dd, J = 7.6, 2.1 Hz, 1H), 7.19 – 7.03 (m, 3H).

[0065] Step 2: Synthesize intermediates 1-3

[0066] Dissolve 1-2 (2.5 g, 18.51 mmol) in a mixed solvent of glacial acetic acid and water (1:1), and slowly add liquid bromine (3.05 g, 19.07 mmol) dropwise under ice bath conditions, stirring overnight at room temperature. After the reaction is complete, filter, wash with water, and dry under vacuum to obtain intermediate 1-3, with a yield of 53.6%. 1 H NMR (300 MHz, DMSO-d6) δ 11.83 (s, 1H), 7.60 (d, J= 1.9 Hz, 1H), 7.34 (dd, J = 8.3, 1.9 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H).

[0067] Step 3: Synthesize intermediates 1-4

[0068] 1-3 (2 g, 9.39 mmol) and 3-carboxyphenylboronic acid (2.03 g, 12.21 mmol) were dissolved in methanol, and an aqueous solution of sodium carbonate (1.99 g, 18.78 mmol) was added. Under nitrogen protection, 0.2 g of palladium dichloride of bis(triphenylphosphine) was added, and the mixture was heated at 100 °C for 4 h. After the reaction was complete, the mixture was cooled to room temperature, concentrated to remove volatile solvents, and extracted with saturated ammonium chloride and ethyl acetate. The organic layer was washed with saturated NaCl, dried over anhydrous Na₂SO₄, and vacuum dried. The resulting granules were purified by column chromatography to obtain 1-4 in 57.2% yield. 1 H NMR (300 MHz, DMSO-d6) δ 12.86 (s, 1H), 11.83 (s, 1H), 8.43 (s,1H), 8.24 (s, 1H), 8.05 – 7.95 (m, 1H), 7.61 (d, J = 2.0 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.34 (dd, J = 8.3, 1.9 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H).

[0069] Step 4: Synthesize intermediates 1-5

[0070] Mix 1-4 (1.3 g, 5.10 mmol), glacial acetic acid, and concentrated hydrochloric acid, heat under reflux for 6 h, and concentrate under reduced pressure after the reaction is complete to obtain 1-5, with a yield of 51.8%. 1H NMR (300 MHz, DMSO-d6) δ 8.16 (t, J = 1.7 Hz,1H), 7.94 (dt, J = 7.7, 1.5 Hz, 1H), 7.79 (dt, J = 7.7, 1.5 Hz, 1H), 7.58 (t,J = 7.7 Hz, 1H), 6.82 – 6.71 (m, 2H), 6.57 (dd, J = 7.0, 2.2 Hz, 1H).

[0071] Step 5: Synthesize intermediates 1-6

[0072] Dissolve 1-5 (0.5 g, 2.18 mmol) in 4 mL of hydrochloric acid. Under ice bath conditions, add 160 mg, 2.32 mmol, of sodium nitrite aqueous solution dropwise. Stir for 20 min, then add triethylamine to adjust the pH to 8-9. Add ethyl acetoacetate (0.28 mL, 2.17 mmol) and stir until the reaction is complete. Add 1 M hydrochloric acid to adjust the pH to 1-2, and a yellow solid precipitates. Filter to obtain the crude product, prepare sintered sand, and purify by column chromatography to obtain 1-6, with a yield of 73.1%. 1 H NMR (300 MHz, DMSO-d6) δ 12.88 (s, 1H),8.18 (s, 1H), 8.04 (d, J = 7.0 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.58 (d, J= 7.3 Hz, 2H), 7.22 – 6.97 (m, 2H), 4.42 – 4.28 (m, 2H), 2.56 (s, 3H), 1.35 –1.26 (m, 3H).

[0073] Step 6: Synthesize compound C1

[0074] Compounds 1-6 (200 mg, 0.54 mmol), 1-7 (95 mg, 0.55 mmol), and sodium acetate (52 mg, 0.63 mmol) were dissolved in glacial acetic acid and heated and stirred at 95 °C for 6 h. After the reaction was complete, the mixture was cooled to room temperature and stirred until no more solid precipitated. The solid was filtered, dried, and the crude product was prepared into a slurry. The slurry was purified by column chromatography to obtain compound C1 in 56.7% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.77 (s, 1H), 13.06 (s, 1H), 9.69 (s, 1H), 8.15 (s, 1H), 7.98 (d,J = 7.7 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.73 (s, 2H), 7.64 (t, J = 7.9 Hz,2H), 7.28 – 7.07 (m, 3H), 2.35 (s, 3H), 2.28 (s, 3H), 2.24 (s, 3H).

[0075] Example 2

[0076] Preparation of compound C2:

[0077] .

[0078] Step 1: Synthesize intermediate 2-2

[0079] Compounds 2-1a (1.61 g, 7.49 mmol) and 2-1b (1.5 g, 8.98 mmol) were dissolved in a mixed solvent of dioxane and water (2:1). An aqueous solution of sodium carbonate (1.19 g, 11.23 mmol) was added. Under nitrogen protection, tetrakis(triphenylphosphine)palladium (70 mg, 0.22 mmol) was added, and the mixture was heated overnight at 100 °C. After the reaction was complete, the mixture was cooled to room temperature, concentrated to remove volatile solvents, and extracted with water and dichloromethane. The organic layer was washed with saturated NaCl, dried over anhydrous Na₂SO₄, and vacuum dried. The resulting granules were purified by column chromatography to obtain 2-2 in 35.9% yield. 1 H NMR (300 MHz, DMSO-d6) δ 8.43 (s, 1H), 8.30– 8.12 (m, 3H), 8.03 (dd, J = 13.9, 7.6 Hz, 2H), 7.76 (t, J = 8.0 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 3.88 (s, 3H).

[0080] Step 2: Synthesize intermediates 2-3

[0081] 2-2 (250 mg, 1.03 mmol) was dissolved in ethanol, and ammonium chloride solution (28 mg, 0.52 mmol) was added, followed by iron powder (230 mg, 4.13 mmol). The mixture was heated to reflux. After the reaction was complete, diatomaceous earth was filtered, concentrated, and extracted with ethyl acetate and water. The organic layer was washed with saturated NaCl, dried over anhydrous Na2SO4, and vacuum dried. The resulting shavings were purified by column chromatography to obtain 2-3 in 64.2% yield. 1 H NMR (300 MHz, DMSO-d6) δ 8.05 (t, J = 1.8 Hz, 1H), 7.91 – 7.75 (m, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 6.86 – 6.80 (m,1H), 6.79 – 6.71 (m, 1H), 6.59 – 6.47 (m, 1H), 5.19 (s, 2H), 3.82 (s, 3H).

[0082] Step 3: Synthesize compound C2

[0083] Dissolve 2-4 (300 mg, 1.41 mmol) in methanol. Under ice bath conditions, add 4 M hydrochloric acid and stir. Add sodium nitrite aqueous solution (98 mg, 1.42 mmol) dropwise and stir for 1 h. Then add aminosulfonic acid (22 mg, 0.23 mmol) and stir for 1 h. After that, add sodium bicarbonate to adjust the pH to 7-8, add 2-5 (280 mg, 1.41 mmol), and stir overnight at room temperature. After the reaction is complete, filter the solid and wash with 4 M hydrochloric acid and methanol to give an orange-red solid compound C2 in 48.6% yield. 1 H NMR (300MHz, DMSO-d6) δ 8.26 (s, 1H), 7.99 (d, J = 8.7 Hz, 3H), 7.76 – 7.53 (m, 6H), 7.21 (d, J = 8.2 Hz, 1H), 2.31 (s, 3H), 2.27 (s, 3H), 2.23 (s, 3H).

[0084] Example 3

[0085] Preparation of compound C3

[0086]

[0087]

[0088] Step 1: Synthesize intermediate 3-1

[0089] 2-2 (200 mg, 0.78 mmol) was dissolved in a methanol / tetrahydrofuran / water (1:1:1) mixture. Under ice bath conditions, lithium hydroxide (65 mg, 1.56 mmol) was added, and the mixture was heated to 60 °C until the reaction solution became clear. The solution was concentrated, and the pH was adjusted with dilute hydrochloric acid. A white solid precipitated and was filtered to obtain 3-1, with a yield of 85.9%. 1 H NMR (300 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.49 (s, 1H), 8.34 – 8.19 (m, 3H), 8.07 (t, J = 8.8 Hz, 2H), 7.82 (t, J = 8.0Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H).

[0090] Step 2: Synthesize intermediate 3-2

[0091] The synthesis method is the same as step 2 in Example 2, except that intermediate 2-2 is replaced by 3-1, and the yield is 56.4%. 1 HNMR (500 MHz, DMSO-d6) δ 12.86 (s, 1H), 8.05 – 7.97 (m, 2H), 7.66 (dt, J =7.5, 1.6 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.17 – 7.09 (m, 3H), 6.65 (ddd, J= 5.2, 3.8, 1.6 Hz, 1H).

[0092] Step 3: Synthesize compound C3

[0093] The synthesis method is the same as step 3 in Example 2, except that intermediate 2-3 is replaced by 3-2, and the yield is 43.5%. 1 HNMR (300 MHz, DMSO-d6) δ 13.36 (s, 1H), 8.25 (s, 1H), 7.99 (dd, J = 14.0, 6.1Hz, 3H), 7.66 (ddd, J = 14.7, 9.4, 2.7 Hz, 4H), 7.56 (d, J = 5.0 Hz, 2H), 7.20 (d, J = 8.3 Hz, 1H), 3.91 (s, 3H), 2.30 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H).

[0094] Example 4

[0095] Preparation of compound C4:

[0096]

[0097] Step 1: Synthesize intermediate 4-2

[0098] The synthesis steps for 4-2 from 4-1a and 4-1b are as described in step 3 of Example 1, with a yield of 53.7%. 1 H NMR (300 MHz, DMSO-d6) δ 7.90 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.65– 7.35 (m, 6H), 3.45 (s, 3H).

[0099] Step 2: Synthesize intermediate 4-3

[0100] Similar to step 2 in Example 2, except that intermediate 2-2 is replaced with 4-2, and the yield is 53.5%. 1 H NMR (500MHz, DMSO-d6) δ 7.93 – 7.88 (m, 2H), 7.85 – 7.78 (m, 3H), 7.66 (dd, J = 7.5,1.6 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.20 (dd, J = 7.5, 1.6 Hz, 1H), 3.52(s, 3H).

[0101] Step 3: Synthesize compound C4

[0102] The synthesis method is the same as step 3 in Example 2, except that intermediate 2-3 is replaced by 4-3, and the yield is 42.8%. 1 HNMR (300 MHz, DMSO-d6) δ 13.84 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.73 (dd, J= 17.2, 7.6 Hz, 4H), 7.66 – 7.51 (m, 3H), 7.44 (t, J = 7.9 Hz, 1H), 7.31 (t,J = 7.9 Hz, 2H), 3.55 (s, 3H), 2.42 (s, 3H), 2.36 (s, 3H), 2.32 (s, 3H).

[0103] Example 5

[0104] Preparation of compound C5:

[0105] Compound C4 (155 mg, 0.36 mmol) was dissolved in dichloromethane, and boron tribromide (0.11 mL, 1.09 mmol) was slowly added dropwise under ice bath conditions. The reaction was carried out at -20 °C for 2 h. After the reaction was completed, methanol was added to quench the reaction, and the solution was concentrated, prepared into sand, and purified by column chromatography to obtain compound C5 in 67.6% yield. 1 H NMR (300 MHz, DMSO-d6) δ 13.69 (s,1H), 9.51 (s, 1H), 7.63 (dt, J = 18.3, 5.6 Hz, 3H), 7.53 (d, J = 7.3 Hz, 2H), 7.44 (t, J = 7.3 Hz, 2H), 7.36 (d, J = 7.1 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 4.8 Hz, 2H), 2.28 (s, 3H), 2.22 (s, 3H), 2.18 (s, 3H).

[0106] Example 6

[0107] Preparation of compound C6:

[0108]

[0109] Step 1: Synthesize intermediate 6-2

[0110] The synthesis steps are the same as step 5 in Example 1, except that intermediates 1-5 are replaced by 6-1, and the yield is 75.9%. 1 HNMR (300 MHz, Chloroform-d) δ 12.97 (s, 1H), 7.74 – 7.66 (m, 3H), 7.59 – 7.47(m, 6H), 4.46 (dq, J = 12.3, 7.1 Hz, 2H), 2.65 (d, J = 25.9 Hz, 3H), 1.50 (t,J = 7.1 Hz, 3H).

[0111] Step 2: Synthesize compound C6

[0112] The synthesis steps are the same as step 6 in Example 1, except that intermediates 1-6 are replaced by 6-2, and the yield is 81.5%. 1HNMR (300 MHz, DMSO-d6) δ 7.89 (s, 1H), 7.73 (d, J = 7.8 Hz, 3H), 7.62 (dd, J= 8.7, 6.1 Hz, 2H), 7.52 (p, J = 7.8 Hz, 4H), 7.46 – 7.37 (m, 1H), 7.20 (d, J= 8.2 Hz, 1H), 2.30 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H).

[0113] Example 7

[0114] Preparation of compound C7:

[0115]

[0116] Step 1: Synthesize intermediate 7-2

[0117] The synthesis steps are the same as step 3 in Example 1, except that intermediates 1-3 are replaced with 7-1a, and the yield is 68.3%. 1 HNMR (500 MHz, DMSO-d6) δ 12.81 (s, 1H), 8.02 – 7.76 (m, 3H), 7.52 (dd, J =7.5, 1.6 Hz, 1H), 6.98 (dt, J = 7.5, 1.6 Hz, 1H), 6.79 (t, J = 7.5 Hz, 1H), 6.60 (t, J = 7.5 Hz, 1H), 3.26 (s, 3H).

[0118] Step 2: Synthesize intermediate 7-3

[0119] Similar to step 2 in Example 2, except that intermediate 2-2 is replaced with 7-2, and the yield is 58.4%. 1 H NMR (300MHz, DMSO-d6) δ 12.82 (s, -1H), 8.09 (t, J = 1.8 Hz, 1H), 7.92 (dt, J = 7.7,1.5 Hz, 1H), 7.76 (dt, J = 7.7, 1.6 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 6.91(t, J = 7.7 Hz, 1H), 6.79 – 6.69 (m, 1H), 6.53 (dd, J = 7.6, 1.6 Hz, 1H), 3.28 (s, 3H).

[0120] Step 3: Synthesize intermediate 7-4

[0121] The synthesis steps are the same as step 5 in Example 1, except that intermediates 1-5 are replaced with 7-3, and the yield is 79.2%. 1 HNMR (300 MHz, Chloroform-d) δ 13.04 (s, 1H), 8.33 (d, J = 5.7 Hz, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.84 (dd, J = 16.4, 7.7 Hz, 1H), 7.68 (d, J = 8.0 Hz,1H), 7.54 (s, 1H), 7.32 – 7.09 (m, 2H), 4.47 – 4.29 (m, 2H), 3.47 (s, 3H),2.57 (d, J = 21.9 Hz, 3H), 1.40 (t, J = 6.9 Hz, 3H).

[0122] Step 4: Synthesize compound C7

[0123] The synthesis steps are the same as step 6 in Example 1, except that intermediates 1-6 are replaced by 7-4, and the yield is 86.3%. 1 HNMR (300 MHz, DMSO-d6) δ 13.75 (s, 1H), 8.18 (d, J = 1.9 Hz, 1H), 8.01 (d, J= 7.7 Hz, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.77 (dd, J = 8.2, 1.6 Hz, 1H), 7.71– 7.59 (m, 3H), 7.37 (t, J = 7.9 Hz, 1H), 7.28 (dd, J = 7.7, 1.7 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 3.45 (s, 3H), 2.32 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H).

[0124] Example 8

[0125] Preparation of compound C8:

[0126] The synthesis steps are the same as step 6 in Example 1, except that intermediates 1-7 are replaced with phenylhydrazine hydrochloride, and the yield is 67.9%. 1H NMR (300 MHz, DMSO-d6) δ 13.81 (s, 1H), 12.88 (s, 1H), 9.77 (s, 1H), 8.21(t, J = 1.8 Hz, 1H), 8.02 (t, J = 9.0 Hz, 3H), 7.92 – 7.85 (m, 1H), 7.80 (dd,J = 6.7, 3.1 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.9 Hz, 2H), 7.37 – 7.15 (m, 3H), 2.42 (s, 3H).

[0127] Example 9

[0128] Preparation of compound C9:

[0129] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-methylphenylhydrazine hydrochloride, and the yield was 62.8%. 1 H NMR (300 MHz, DMSO-d6) δ 13.75 (s, 1H), 9.71 (s, 1H), 8.13 (s, 1H), 7.97 (d, J = 7.4 Hz, 1H), 7.86 – 7.68 (m, 4H), 7.62 (t, J = 7.6 Hz, 1H), 7.34(t, J = 7.8 Hz, 1H), 7.21 – 7.10 (m, 2H), 7.04 (d, J = 7.5 Hz, 1H), 2.36 (s,3H), 2.34 (s, 3H).

[0130] Example 10

[0131] Preparation of compound C10:

[0132] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-methylphenylhydrazine hydrochloride, and the yield was 59.2%. 1H NMR (300 MHz, DMSO-d6) δ 13.73 (s, 1H), 13.00 (s, 1H), 9.67 (s,1H), 8.16 – 8.10 (m, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.86 – 7.76 (m, 3H), 7.71(dd, J = 6.6, 3.2 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.26 (d, J = 8.1 Hz, 2H), 7.16 (q, J = 3.9, 3.4 Hz, 2H), 2.33 (s, 3H), 2.31 (s, 3H).

[0133] Example 11

[0134] Preparation of compound C11:

[0135] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-fluorophenylhydrazine hydrochloride, and the yield was 61.8%. 1 H NMR (300 MHz, DMSO-d6) δ 13.67 (s, 1H), 13.03 (s, 1H), 9.75 (s, 1H), 8.14 (d, J = 1.9 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.84 – 7.67 (m, 4H), 7.62(t, J = 7.7 Hz, 1H), 7.50 (q, J = 7.8 Hz, 1H), 7.21 – 7.10 (m, 2H), 7.05 (td,J = 8.4, 2.6 Hz, 1H), 2.34 (s, 3H).

[0136] Example 12

[0137] Preparation of compound C12:

[0138] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-chlorophenylhydrazine hydrochloride, and the yield was 58.5%. 1H NMR (300 MHz, DMSO-d6) δ 13.68 (s, 1H), 13.00 (s, 1H), 9.70 (d, J =45.1 Hz, 1H), 8.15 (s, 1H), 8.06 – 7.88 (m, 3H), 7.82 (d, J = 7.8 Hz, 1H), 7.73 (dd, J = 7.4, 2.4 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.50 (t, J = 8.2Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.25 – 7.08 (m, 2H), 2.35 (s, 3H).

[0139] Example 13

[0140] Preparation of compound C13:

[0141] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-bromophenylhydrazine hydrochloride, and the yield was 63.8%. 1 H NMR (300 MHz, DMSO-d6) δ 13.66 (s, 1H), 12.61 (s, 1H), 9.75 (s, 1H), 8.14 (s, 2H), 8.01 – 7.90 (m, 2H), 7.81 (d, J = 7.7 Hz, 1H), 7.71 (d, J = 7.1Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 6.2 Hz, 2H), 7.21 – 7.10 (m,2H), 2.34 (s, 3H).

[0142] Example 14

[0143] Preparation of compound C14:

[0144] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-trifluoromethylphenylhydrazine hydrochloride, and the yield was 52.8%. 1H NMR (300 MHz, DMSO-d6) δ 13.68 (s, 1H), 12.54 (s, 1H), 9.75 (s,1H), 8.29 (s, 1H), 8.23 ​​(d, J = 8.3 Hz, 1H), 8.14 (t, J = 1.8 Hz, 1H), 8.02 –7.92 (m, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.77 – 7.66 (m, 2H), 7.66 – 7.52 (m,2H), 7.24 – 7.09 (m, 2H), 2.36 (s, 3H).

[0145] Example 15

[0146] Preparation of compound C15:

[0147] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with methyl 3-carboxylate phenylhydrazine hydrochloride, and the yield was 68.2%. 1 H NMR (300 MHz, DMSO-d6) δ 13.72 (s, 1H), 13.08 (s, 1H), 8.58 (t,J = 2.0 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.16 (t,J = 1.9 Hz, 1H), 7.99 (d,J = 7.7 Hz, 1H), 7.82 (t, J = 6.8 Hz, 2H), 7.73 (dd, J = 7.4, 2.5 Hz, 1H), 7.63 (td, J = 7.9, 4.2 Hz, 2H), 7.26 – 7.10 (m, 2H), 3.91 (s, 3H), 2.37 (s, 3H).

[0148] Example 16

[0149] Preparation of compound C16:

[0150] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-carboxylic acid phenylhydrazine hydrochloride, with a yield of 59.1%. 1H NMR (300 MHz, DMSO-d6) δ 13.71 (s, 1H), 13.06 (s, 2H), 9.72 (s,1H), 8.54 (t, J = 2.0 Hz, 1H), 8.22 – 8.16 (m, 1H), 8.14 (d, J = 1.9 Hz, 1H),8.01 – 7.93 (m, 1H), 7.85 – 7.75 (m, 2H), 7.72 (dd, J = 7.0, 2.7 Hz, 1H),7.60 (dt, J = 10.8, 7.8 Hz, 2H), 7.23 – 7.08 (m, 2H), 2.36 (s, 3H).

[0151] Example 17

[0152] Preparation of compound C17:

[0153] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-nitrophenylhydrazine hydrochloride, and the yield was 63.6%. 1 H NMR (300 MHz, DMSO-d6) δ 13.66 (s, 1H), 13.02 (s, 1H), 9.79 (s,1H), 8.79 (t, J = 2.2 Hz, 1H), 8.37 (dd, J = 8.2, 2.3 Hz, 1H), 8.15 (t, J =1.7 Hz, 1H), 8.09 – 7.95 (m, 2H), 7.81 (t, J = 7.8 Hz, 1H), 7.77 – 7.68 (m,2H), 7.64 (t, J = 7.6 Hz, 1H), 7.24 – 7.09 (m, 2H), 2.37 (s, 3H).

[0154] Example 18

[0155] Preparation of compound C18:

[0156] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-cyanophenylhydrazine hydrochloride, and the yield was 56.2%. 1H NMR (300 MHz, DMSO-d6) δ 13.55 (s, 1H), 12.92 (s, 1H), 9.68 (s,1H), 8.24 – 8.10 (m, 2H), 8.05 (t, J = 1.8 Hz, 1H), 7.88 (d, J = 7.7 Hz, 1H),7.72 (d, J = 7.7 Hz, 1H), 7.67 – 7.48 (m, 4H), 7.13 – 7.01 (m, 2H), 2.26 (s,3H).

[0157] Example 19

[0158] Preparation of compound C19:

[0159] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 3-fluoromethoxyphenylhydrazine hydrochloride, and the yield was 56.2%. 1 H NMR (300 MHz, DMSO-d6) δ 13.66 (s, 1H), 12.67 (s, 1H), 9.90 –9.67 (m, 1H), 8.14 (s, 1H), 7.97 (d, J = 8.3 Hz, 3H), 7.81 (d, J = 7.6 Hz,1H), 7.72 (d, J = 7.4 Hz, 1H), 7.61 (q, J = 7.7 Hz, 2H), 7.26 – 7.09 (m, 3H),2.35 (s, 3H).

[0160] Example 20

[0161] Preparation of compound C20:

[0162]

[0163] Step 1: Synthesize intermediate 20-1

[0164] 1-5 (1 g, 4.36 mmol) was dissolved in methanol, and thionyl chloride (3.16 mL, 43.6 mmol) was slowly added dropwise under ice bath conditions. The mixture was heated under reflux for 5 h under nitrogen protection. After the reaction was complete, the volatile solvent was removed by concentration, and the mixture was extracted with ethyl acetate and water. The extraction was repeated multiple times with ethyl acetate. The organic phases were combined and washed with saturated NaCl, dried over anhydrous Na₂SO₄, dried under vacuum, and precipitated by column chromatography to obtain intermediate 20-1 in 86.2%. 1H NMR (300 MHz, DMSO-d6) δ 8.10(s, 1H), 7.92 – 7.83 (m, 1H), 7.79 – 7.70 (m, 1H), 7.54 (t, J = 7.7 Hz, 1H), 6.79 – 6.67 (m, 2H), 6.55 (dd, J = 5.7, 3.6 Hz, 1H), 3.86 (s, 3H), 2.52 –2.46 (m, 1H).

[0165] Step 2: Synthesize intermediate 20-2

[0166] The synthesis steps are the same as step 5 in Example 1, except that intermediates 1-5 are replaced with 20-1, and the yield is 81.5%. 1 HNMR (300 MHz, DMSO-d6) δ 12.57 (s, 1H), 8.11 (s, 1H), 7.95 (d, J = 7.7 Hz,1H), 7.78 (d, J = 7.7 Hz, 1H), 7.61 (dd, J = 7.7, 2.7 Hz, 2H), 7.19 – 7.04(m, 2H), 4.44 – 4.03 (m, 2H), 3.85 (s, 3H), 2.43 (s, 3H), 1.31 (dt, J = 10.0,7.0 Hz, 3H).

[0167] Step 3: Synthesize intermediate 20-3

[0168] The synthesis steps are the same as step 6 in Example 1, except that intermediates 1-6 are replaced with 20-2, and the yield is 61.5%. 1 HNMR (300 MHz, DMSO-d6) δ 13.76 (s, 1H), 9.72 (s, 1H), 8.15 (s, 1H), 7.98 (d,J = 7.8 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.71 (s, 2H), 7.65 (t, J = 7.8 Hz, 2H), 7.18 (q, J = 7.4, 6.6 Hz, 3H), 3.89 (s, 3H), 2.33 (s, 3H), 2.27 (s, 3H), 2.23 (s, 3H).

[0169] Step 4: Synthesize compound C20

[0170] 11.2 g of potassium hydroxide was dissolved in 40 mL of methanol and stirred for 10 min. 9.6 g of hydroxylamine hydrochloride was added to 48 mL of methanol and stirred for 10 min. The mixture was then stirred for 1 h, filtered, and the filtrate was collected. An appropriate amount of the filtrate was used to dissolve 20-3 (100 mg, 0.22 mmol), sonicated for 10 s, and concentrated to remove volatile solvents. After evaporation to dryness, a small amount of water was added, and the pH was adjusted to 5-6 under ice bath conditions to precipitate a solid. The solid was filtered and dried to obtain compound C20, with a yield of 85.7%. 1 H NMR (300 MHz, DMSO-d6) δ 11.29 (s, 1H), 9.11 (s, 1H), 7.95 (s, 1H), 7.78 (d, J = 7.7 Hz,1H), 7.71 (q, J = 4.7, 3.9 Hz, 3H), 7.66 – 7.60 (m, 1H), 7.56 (t, J = 7.8 Hz,1H), 7.25 – 7.09 (m, 3H), 2.32 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H).

[0171] Example 21

[0172] Preparation of compound C21:

[0173] Compound C1 (60 mg, 0.14 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 77.14 mg, 0.20 mmol), was dissolved in DMF (N,N-dimethylformamide). Triethylamine (283 μL, 2.03 mmol) was added and the mixture was stirred for 1 h. Then, ammonium chloride (72.53 mg, 1.36 mmol) was added and the mixture was stirred overnight at room temperature. After the reaction was complete, the volatile solvent was removed by concentration, and the mixture was extracted with ethyl acetate and water. The organic layer was washed with saturated NaCl, dried over anhydrous Na2SO4, and vacuum dried. The resulting granules were purified by column chromatography to give compound C21 in 53.7% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.76(s, 1H), 9.63 (s, 1H), 8.05 (d, J = 7.9 Hz, 2H), 7.89 (d, J = 7.6 Hz, 1H), 7.71 (q, J = 2.8 Hz, 3H), 7.66 – 7.61 (m, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.40(s, 1H), 7.24 – 7.10 (m, 3H), 2.33 (s, 3H), 2.27 (s, 3H), 2.23 (s, 3H).

[0174] Example 22

[0175] Preparation of compound C22:

[0176] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 2-fluorophenylhydrazine hydrochloride, and the yield was 52.5%. 1 H NMR (300 MHz, DMSO-d6) δ 13.56 (s, 1H), 12.99 (s, 1H), 9.65 (s, 1H), 8.11 – 8.04 (m, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.69(dd, J = 6.5, 3.2 Hz, 1H), 7.63 – 7.48 (m, 2H), 7.48 – 7.36 (m, 2H), 7.31(td, J = 8.8, 4.6 Hz, 1H), 7.13 (q, J = 3.9 Hz, 2H), 2.28 (s, 3H).

[0177] Example 23

[0178] Preparation of compound C23

[0179] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 2-chlorophenylhydrazine hydrochloride, and the yield was 56.7%. 1H NMR (300 MHz, DMSO-d6) δ 13.72 – 13.42 (m, 1H), 13.00 (s, 1H), 9.67(s, 1H), 8.11 (s, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.83 – 7.70 (m, 2H), 7.70 –7.59 (m, 2H), 7.58 – 7.53 (m, 1H), 7.53 – 7.45 (m, 2H), 7.16 (d, J = 6.0 Hz,2H), 2.31 (s, 3H).

[0180] Example 24

[0181] Preparation of compound C24:

[0182] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 2-bromophenylhydrazine hydrochloride, and the yield was 62.5%. 1 H NMR (300 MHz, DMSO-d6) δ 13.62 (s, 1H), 13.07 (s, 1H), 9.69 (s, 1H), 8.17 – 8.10 (m, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.82 (t, J = 8.0 Hz, 2H), 7.75(dd, J = 6.2, 3.7 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 4.4 Hz,2H), 7.50 – 7.38 (m, 1H), 7.18 (q, J = 3.2, 2.5 Hz, 2H), 2.33 (s, 3H).

[0183] Example 25

[0184] Preparation of compound C25:

[0185] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 2-trifluoromethylphenylhydrazine hydrochloride, and the yield was 46.3%. 1H NMR (300 MHz, DMSO-d6) δ 13.60 (s, 1H), 13.10 (s, 1H), 9.71 (s,1H), 8.14 (s, 1H), 7.97 (t, J = 7.2 Hz, 2H), 7.91 – 7.73 (m, 4H), 7.72 – 7.58(m, 2H), 7.19 (d, J = 4.9 Hz, 2H), 2.33 (s, 3H).

[0186] Example 26

[0187] Preparation of compound C26:

[0188] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 2-carboxylic acid phenylhydrazine hydrochloride, with a yield of 45.8%. 1 H NMR (300 MHz, DMSO-d6) δ 12.72 (s, 1H), 11.54 (s, 1H), 9.44 (s,1H), 8.20 (d, J = 1.9 Hz, 1H), 7.93 (dd, J = 8.8, 3.8 Hz, 3H), 7.82 (d, J =7.7 Hz, 1H), 7.60 (t, J = 7.6 Hz, 2H), 7.52 (dd, J = 7.9, 1.6 Hz, 1H), 7.07(t, J = 7.9 Hz, 1H), 7.01 – 6.85 (m, 2H), 2.23 (s, 3H).

[0189] Example 27

[0190] Preparation of compound C27:

[0191] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 2-trifluoromethoxyphenylhydrazine hydrochloride, with a yield of 43.9%. 1H NMR (300 MHz, DMSO-d6) δ 13.62 (s, 1H), 13.09 (s, 1H), 9.73(s, 1H), 8.15 (d, J = 2.1 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 7.7Hz, 1H), 7.76 (dd, J = 6.3, 3.3 Hz, 1H), 7.61 (tq, J = 11.9, 6.1, 5.4 Hz, 5H), 7.26 – 7.12 (m, 2H), 2.35 (s, 3H).

[0192] Example 28

[0193] Preparation of compound C28:

[0194] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-fluorophenylhydrazine hydrochloride, and the yield was 61.5%. 1 H NMR (300 MHz, DMSO-d6) δ 13.70 (s, 1H), 13.03 (s, 1H), 9.73 (s, 1H), 8.14 (d, J = 2.0 Hz, 1H), 8.03 – 7.88 (m, 3H), 7.81 (d, J = 7.6 Hz, 1H), 7.71(dd, J = 7.0, 2.8 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.30 (t, J = 8.9 Hz,2H), 7.21 – 7.07 (m, 2H), 2.33 (s, 3H).

[0195] Example 29

[0196] Preparation of compound C29:

[0197] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-chlorophenylhydrazine hydrochloride, and the yield was 63.8%. 1H NMR (300 MHz, DMSO-d6) δ 13.69 (s, 1H), 12.99 (s, 1H), 9.74 (s, 1H), 8.15 (t, J = 1.8 Hz, 1H), 7.98 (dd, J = 8.6, 3.7 Hz, 3H), 7.82 (d, J = 7.7Hz, 1H), 7.72 (dd, J = 7.1, 2.6 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.57 –7.46 (m, 2H), 7.16 (d, J = 7.0 Hz, 2H), 2.34 (s, 3H).

[0198] Example 30

[0199] Preparation of compound C30:

[0200] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-bromophenylhydrazine hydrochloride, and the yield was 71.6%. 1 H NMR (300 MHz, DMSO-d6) δ 13.66 (s, 1H), 12.99 (s, 1H), 9.71 (s, 1H), 8.11 (d, J = 1.8 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.83– 7.74 (m, 1H), 7.69 (dd, J = 7.2, 2.6 Hz, 1H), 7.66 – 7.55 (m, 3H), 7.20 –7.08 (m, 2H), 2.31 (s, 3H).

[0201] Example 31

[0202] Preparation of compound C31:

[0203] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-trifluoromethylphenylhydrazine hydrochloride, and the yield was 52.5%. 1H NMR (300 MHz, DMSO-d6) δ 13.72 (s, 1H), 13.11 (s, 1H), 9.81 (s,1H), 8.26 – 8.14 (m, 3H), 8.02 (d, J = 7.7 Hz, 1H), 7.86 (d, J = 8.7 Hz, 3H),7.76 (dd, J = 7.4, 2.3 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.28 – 7.13 (m,2H), 2.40 (s, 3H).

[0204] Example 32

[0205] Preparation of compound C32:

[0206] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with methyl 4-carboxylate phenylhydrazine hydrochloride, and the yield was 56.2%. 1 H NMR (300 MHz, DMSO-d6) δ 13.64 (s, 1H), 13.16 – 12.85 (m, 1H), 9.75 (s, 1H), 8.14 (s, 1H), 8.03 (dt, J = 18.6, 8.9 Hz, 5H), 7.80 (d, J = 7.7Hz, 1H), 7.75 – 7.54 (m, 2H), 7.14 (p, J = 7.7, 6.9 Hz, 2H), 3.83 (s, 3H), 2.32 (s, 3H).

[0207] Example 33

[0208] Preparation of compound C33:

[0209] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-carboxylic acid phenylhydrazine hydrochloride, with a yield of 53.9%. 1H NMR (300 MHz, DMSO-d6) δ 13.68 (s, 1H), 12.97 (s, 1H), 9.72 (s,1H), 8.13 (t, J = 1.8 Hz, 1H), 7.96 (dd, J = 8.6, 3.7 Hz, 3H), 7.80 (d, J =7.7 Hz, 1H), 7.71 (dd, J = 7.1, 2.6 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.55 –7.44 (m, 2H), 7.15 (d, J = 7.0 Hz, 2H), 2.33 (s, 3H).

[0210] Example 34

[0211] Preparation of compound C34:

[0212] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-nitrophenylhydrazine hydrochloride, and the yield was 62.2%. 1 H NMR (300 MHz, DMSO-d6) δ 13.59 (s, 1H), 9.78 (s, 1H), 8.30 (d, J =9.0 Hz, 2H), 8.18 (d, J = 9.0 Hz, 2H), 8.11 (s, 1H), 7.95 (d, J = 7.7 Hz,1H), 7.78 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.60 (t, J = 7.7 Hz,1H), 7.20 – 7.06 (m, 2H), 2.34 (s, 3H).

[0213] Example 35

[0214] Preparation of compound C35:

[0215] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-cyanophenylhydrazine hydrochloride, and the yield was 55.8%. 1H NMR (300 MHz, DMSO-d6) δ 13.61 (s, 1H), 12.96 (s, 1H), 9.78 (s,1H), 8.12 (d, J = 8.1 Hz, 3H), 7.97 (d, J = 7.6 Hz, 1H), 7.94 – 7.85 (m, 2H), 7.80 (d, J = 7.7 Hz, 1H), 7.70 (dd, J = 7.5, 2.3 Hz, 1H), 7.62 (t, J = 7.7Hz, 1H), 7.23 – 7.07 (m, 2H), 2.34 (s, 3H).

[0216] Example 36

[0217] Preparation of compound C36:

[0218] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-methoxyphenylhydrazine hydrochloride, and the yield was 48.2%. 1 H NMR (300 MHz, DMSO-d6) δ 13.74 (s, 1H), 13.06 (s, 1H), 9.69 (s,1H), 8.13 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.86 – 7.76 (m, 3H), 7.72 (dd, J= 6.5, 3.3 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.22 – 7.10 (m, 2H), 7.02 (d, J= 9.1 Hz, 2H), 3.78 (s, 3H), 2.33 (s, 3H).

[0219] Example 37

[0220] Preparation of compound C37:

[0221] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-7 were replaced with 4-trifluoromethoxyphenylhydrazine hydrochloride, with a yield of 42.5%. 1H NMR (300 MHz, DMSO-d6) δ 13.79 (s, 1H), 13.17 (s, 1H), 9.84(s, 1H), 8.22 (s, 1H), 8.19 – 8.10 (m, 2H), 8.06 (d, J = 7.6 Hz, 1H), 7.90(d, J = 7.6 Hz, 1H), 7.86 – 7.78 (m, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.57 (d,J = 8.7 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 2.44 (s, 3H).

[0222] Example 38

[0223] Preparation of compound C38:

[0224]

[0225]

[0226] Step 1: Synthesize intermediate 38-2

[0227] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-6 were replaced with ethyl acetoacetate and 1-7 were replaced with 4-cyanophenylhydrazine hydrochloride, with a yield of 68.5%. 1 H NMR (300 MHz, DMSO-d6) δ 7.98 (dd, J =10.7, 3.7 Hz, 2H), 7.92 – 7.83 (m, 2H), 3.78 (s, 2H), 2.34 (s, 3H).

[0228] Step 2: Synthesize intermediate 38-3

[0229] 38-2 (300 mg, 1.51 mmol) and 5 M sodium hydroxide (2.66 mL, 13.56 mmol) were dissolved in a mixed solvent of DMSO and ethanol (1:1). Under ice bath conditions, 30% hydrogen peroxide (1.4 mL, 13.56 mmol) was slowly added dropwise, and the mixture was heated to 60 °C for 3 h. After the reaction was complete, the mixture was cooled to room temperature, the reaction was quenched with water, filtered, the precipitate was collected, dried under vacuum, and purified by column chromatography to obtain 38-3 in 63.5% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.07 – 7.96 (m, 2H),7.91 (d, J = 9.1 Hz, 2H), 7.17 (d, J = 7.3 Hz, 2H), 3.77 (s, 2H), 2.35 (s,3H).

[0230] Step 3: Synthesize compound C38

[0231] The synthesis method is the same as step 3 in Example 2, except that intermediate 2-4 is replaced by 38-3 and intermediate 2-3 is replaced by 1-5, with a yield of 33.9%. 1 H NMR (300 MHz, DMSO-d6) δ 13.70 (s, 1H), 12.99 (s, 1H),9.74 (s, 1H), 8.21 – 7.90 (m, 7H), 7.81 (d, J = 7.7 Hz, 1H), 7.73 (dd, J =7.3, 2.7 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.16 (d, J = 7.3 Hz, 2H), 2.36(s, 3H).

[0232] Example 39

[0233] Preparation of compound C39:

[0234] The synthesis steps were the same as step 4 in Example 20, except that intermediate 20-3 was replaced with compound C31, and the yield was 41.8%. 1 H NMR (300 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.06 (s, 1H), 8.15 (s, 2H), 8.04 (s, 2H), 7.94 (s, 1H), 7.82 (d, J = 8.8 Hz, 3H), 7.71 (s, 1H), 7.61 (d,J = 9.0 Hz, 1H), 7.15 (s, 2H), 2.36 (s, 3H).

[0235] Example 40

[0236] Preparation of compound C40:

[0237]

[0238] Step 1: Synthesize intermediate 40-1

[0239] 38-2 (200 mg, 1.00 mmol), trimethyl azidosilane (TMSN3, 334 μL, 5.00 mmol), and tetrabutylammonium fluoride (TBAF, 760 μL, 1.50 mmol) were dissolved in toluene and heated and stirred at 100 °C for 6 h under nitrogen protection. After the reaction was complete, the volatile solvent was removed by concentration, the pH was adjusted to about 5 by adding 1 M hydrochloric acid, and the mixture was extracted with ethyl acetate and water. The organic phase was washed with saturated NaCl, dried over anhydrous Na2SO4, vacuum dried, and then purified by column chromatography to obtain intermediate 40-1 in 66.5% yield. 1 H NMR (500 MHz, DMSO-d6) δ 13.01 (s, 1H), 8.29 – 8.21 (m, 2H), 7.77 – 7.72 (m, 2H), 3.79 (s, 2H), 2.33 (s, 3H).

[0240] Step 2: Synthesize compound C40

[0241] The synthesis method is the same as step 3 in Example 2, except that intermediate 2-4 is replaced by 40-2 and intermediate 2-3 is replaced by intermediate 1-5, with a yield of 37.1%. 1 H NMR (300 MHz, DMSO-d6) δ 13.70 (s, 1H), 13.06 (s, 1H),9.75 (s, 1H), 8.14 (t, J = 7.5 Hz, 5H), 7.97 (d, J = 7.7 Hz, 1H), 7.81 (d, J= 7.7 Hz, 1H), 7.71 (dd, J = 7.3, 2.5 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.22– 7.10 (m, 2H), 2.36 (s, 3H).

[0242] Example 41

[0243] Preparation of compound C41:

[0244] The synthesis steps were the same as in Example 21, except that compound C38 was used instead of C1, and the yield was 48.2%. 1H NMR (300MHz, DMSO-d6) δ 13.74 (s, 1H), 9.72 (s, 1H), 8.13 – 7.95 (m, 7H), 7.92 (d, J= 7.7 Hz, 1H), 7.81 – 7.66 (m, 2H), 7.58 (t, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.36 (s, 1H), 7.19 (p, J = 7.2 Hz, 2H), 2.38 (s, 3H).

[0245] Example 42

[0246] Preparation of compound C42:

[0247]

[0248]

[0249] Step 1: Synthesize intermediate 42-1

[0250] The synthesis steps were the same as step 6 in Example 1, except that intermediates 1-6 were replaced with 20-2 and intermediates 1-7 were replaced with 4-carboxylic acid phenylhydrazine hydrochloride, with a yield of 58.8%. 1 H NMR (300 MHz, DMSO-d6) δ 13.69 (s, 1H), 12.78(s, 1H), 9.78 (s, 1H), 8.15 (s, 1H), 8.02 (dd, J = 18.3, 9.3 Hz, 5H), 7.84(d, J = 7.8 Hz, 1H), 7.72 (dd, J = 7.3, 2.4 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.23 – 7.10 (m, 2H), 2.35 (s, 3H).

[0251] Step 2: Synthesize intermediate 42-2

[0252] The synthesis steps are the same as step 4 in Example 20, except that intermediate 42-1 is used instead of 20-3, and the yield is 43.2%. 1HNMR (300 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.03 (s, 1H), 8.16 (s, 1H), 8.08 –7.93 (m, 3H), 7.85 (d, J = 8.4 Hz, 3H), 7.72 (s, 1H), 7.66 (d, J = 8.1 Hz,1H), 7.17 (s, 2H), 3.89 (s, 3H), 2.36 (s, 3H).

[0253] Step 3: Synthesize compound C42

[0254] The synthesis steps are the same as step 4 in Example 20, except that intermediate 42-2 is used instead of 20-3, and the yield is 41.6%. 1 HNMR (300 MHz, DMSO-d6) δ 13.57 (s, 1H), 11.12 (d, J = 18.1 Hz, 1H), 9.58 (s,1H), 8.94 (s, 1H), 7.90 (t, J = 10.0 Hz, 3H), 7.81 (s, 1H), 7.72 (d, J = 8.4Hz, 1H), 7.61 (dd, J = 19.6, 7.4 Hz, 3H), 7.43 (t, J = 7.7 Hz, 1H), 7.03 (d,J = 9.6 Hz, 2H), 2.22 (s, 3H).

[0255] Example 43

[0256] The inhibitory activity of the compound against ZBP1 was determined using fluorescence polarization (FP) assays.

[0257] This experiment reflects the competitive binding ability of the compound and the fluorescent probe d(CGCGCG)2-FAM to the ZBP1 protein by measuring changes in fluorescence polarization values.

[0258] Experimental Step 1: The ZBP1 plasmid (constructed by GenScript) was transformed into BL21(DE3) Escherichia coli, and then amplified in LB medium containing 50 µg / mL kanamycin (37 °C). When the OD value reached 0.6, 0.5 mM IPTG was added, and protein expression was induced at 37 °C for 6 hours. The bacterial cells were collected by centrifugation for 15 minutes (4000 rpm), resuspended in lysis buffer (20 mM HEPES, 500 mM NaCl, pH 7.4), and sonicated. The supernatant was collected by centrifugation at 10000 rpm for 20 minutes. The filtered supernatant was then added to a nickel column using an AKTA pure25 (GE Healthcare, Life Sciences) instrument. Impurities were washed with buffer containing 10 mM imidazole, and the target protein was eluted with buffer containing 500 mM imidazole. The collected ZBP1 protein eluent was concentrated in an ultrafiltration centrifuge tube (3 kDa). The concentrated sample was then subjected to molecular sieve chromatography using a Superdex 75 gel filtration column. The molecular weight and purity of the bands were confirmed by SDS-PAGE electrophoresis. Finally, the concentrated ZBP1 protein was aliquoted and stored at -80°C.

[0259] Experimental Step 2: Using a 384-well black plate (Corning, catalog number 3575), 20 μL each of serially diluted compounds (33.33, 16.67, 8.33, 4.17, 2.08, 1.04, 0.52, 0.26 μM), 150 nM ZBP1 protein, and 5 nM fluorescent probe d(CGCGCG)2-FAM (GenScript) were added to each well, resulting in a final volume of 60 μL per well. The plate was incubated at room temperature for 30 minutes, and then detected using a SpectraMax Multi-Mode Microplate Reader (Molecular Devices). The excitation wavelength was 485 nm, and the emission wavelength was 535 nm. The polarization value (FP) was measured. Inhibition (%) was calculated using the formula: Inhibition (%) = [1 − (FP) / (1 - (FP) / ... / 1 ​​- (FP) / 1 - (FP) / 1 - (FP) / 1 - (FP) / 1 - (FP / 1 - (FP) / 1 - (FP) / 1 - (FP) / 1 - (FP / 1 - (FP) / 1 - (FP) / 1 - (FP) / 1 - (FP / 1 - (FP) / 1 - (FP) / 1 - (FP) / 1 - (FP) / test -FP blank ) / (FP negative -FP blank The inhibition rate of the compound was calculated by multiplying the result by 100. The data was then processed using GraphPad Prism 7.0 to obtain the compound's IC50. 50 value.

[0260] The experimental results are shown in Table 1. A: IC 50 ≤ 1 μM; B: 1 μM < IC 50 ≤ 10 μM; C: IC50 > 10 μM.

[0261] Table 1. Inhibitory activity (IC50) of the compounds of the present invention against ZBP1 protein 50 )

[0262] Compound numbering <![CDATA[ZBP1 inhibitory activity IC 50 (μM)]]> Compound numbering <![CDATA[ZBP1 inhibitory activity IC 50 (μM)]]> C1 B C22 B C2 B C23 B C3 C C24 B C4 C C25 B C5 C C26 B C6 C C27 B C7 B C28 B C8 B C29 B C9 B C30 A C10 B C31 B C11 B C32 B C12 B C33 A C13 B C34 A C14 B C35 A C15 B C36 B C16 A C37 B C17 B C38 A C18 B C39 A C19 B C40 A C20 A C41 B C21 B C42 A

[0263] As shown in the table above, the compound of this invention can inhibit the binding of ZBP1 to the nucleic acid probe d(CGCGCG)2-FAM and can be used as a small molecule inhibitor of ZBP1 protein.

[0264] Example 44

[0265] Mechanistic study of the inhibitory effect of some compounds on the ZBP1 signaling pathway in this invention

[0266] Western blotting experiments were used to investigate the effects of some compounds of this invention on the expression levels of downstream ZBP1 proteins.

[0267] Experimental procedure: MEF cells (InvivoGen) were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37 ℃ and 5% CO2, at a density of 5 × 10⁶ cells per well. 5 Cells were seeded into 6-well clear cell culture plates and cultured to 70% confluence. Interferon expression was induced for 24 h, followed by incubation with different concentrations of the test compound and CBL0137 (ZBP1 indirect agonist). Cells were then collected by centrifugation at 1000 rpm, washed three times with PBS, and lysed on ice for 20 min with IP lysis buffer, protease inhibitor, and phosphatase inhibitor. After centrifugation at 12000 rpm for 15 min, the supernatant was collected, and protein concentration was determined using a BCA (Thermo, Waltham, MA) kit. 5× Loading Buffer was added, and the mixture was incubated at 95 °C for 8 min. Samples were separated by SDS-PAGE (10%), run at 60 V for 30 min, then switched to 120 V. After gelation, the samples were transferred to a PVDF membrane (PerkinElmer, Northwalk, CT, USA) and run at 0.3 A using a transfer instrument. The PVDF membrane was then blocked with 5% BSA for 1.5 h, followed by overnight incubation with the corresponding primary antibody at 4 °C. After incubation, the PVDF membrane was washed three times with TBST (10 min each time), and then incubated with secondary antibody (DyLight 800 labeled) at room temperature for 1 h. After incubation, the PVDF membrane was washed three times again with TBST. Finally, the membrane was developed with ECL luminescence solution, and the bands were scanned using an Odyssey Infrared Imaging System (LI-COR; Lincoln, NE). The data were saved and analyzed.

[0268] The results are as follows Figure 1 As shown, C1 inhibits the CBL0137-induced ZBP1 necrotizing apoptosis signaling pathway and downregulates the phosphorylation of downstream proteins: Western blotting was used to detect the protein expression levels of ZBP1, RIPK3, p-RIPK3, MLKL, and p-MLKL in MEF cells, with CBL0137 as a negative control; Figure 1 It is evident that, in MEF cells, the C1 prepared in Example 1 can inhibit the phosphorylation of ZBP1 downstream proteins RIPK3 and MLKL, thereby inhibiting the ZBP1 signaling pathway.

[0269] The remaining 41 compounds exhibit the same inhibitory effect on the ZBP1 signaling pathway as in Example C1. The experimental principle is that CBL0137 can activate ZBP1 by inducing an increase in Z-type nucleic acids, thereby recruiting and phosphorylating RIPK3, and then inducing phosphorylation and oligomerization of MLKL, ultimately leading to necrosis and apoptosis. The pyrazolinone ZBP1 inhibitors provided by this invention can reduce the expression of p-RIPK3 and p-MLKL proteins.

[0270] Example 45

[0271] In vivo pharmacodynamic study based on a mouse colitis model induced by dextran sulfate sodium (DSS)

[0272] Experimental Procedure: Thirty SPF-grade C57BL / 6 female mice, aged 6-7 weeks and weighing 18-20g, were purchased and randomly divided into 5 groups (control group, DSS model group, 5-ASA (200 mg / kg) positive control group, and low- and high-dose administration groups of Example C1 (10 mg / kg and 20 mg / kg)), with 6 mice in each group. Mice in the model group, positive control group, and administration groups were given free access to 3% DSS solution for 7 days to induce acute colitis. The control group was given sterile distilled water during the same period. From day 1 to day 7 of model establishment, the positive control group and administration groups were administered the drug orally daily. During the modeling period, mouse weight, fecal characteristics, and fecal blood loss were recorded daily. On day 8, mice were euthanized by cervical dislocation and dissected. The colon tissue was completely separated, the intestinal contents were rinsed with pre-cooled PBS, dried with filter paper, and the colon length was measured. The colon tissue was stored in 4% paraformaldehyde and -80°C for subsequent experiments.

[0273] The results are as follows Figure 2As shown, compared with the solvent control group, the colon length of mice in the DSS model group was significantly shortened. However, intervention in the high-concentration group of Example C1 significantly alleviated the DSS-induced colon shortening, with a significantly increased colon length compared to the DSS model group. This indicates that Example C1 can treat acute inflammatory damage to the colonic mucosa induced by DSS disruption of the intestinal mucosal barrier and intestinal flora imbalance. In summary, the pyrazolone compounds provided by this invention can serve as effective ZBP1 inhibitors. Drugs containing these compounds as active ingredients can be used to prepare medications for treating clinical conditions related to ZBP1.

[0274] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0275] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A pyrazolinone compound, characterized in that, It is a pyrazolone compound as shown in general formula I or an isomer of a pyrazolone compound as shown in general formula I, or a pharmaceutically acceptable salt; Among them, R 1 Selected from hydrogen, C(O)2R a or C(O)NHR b ; R 2 Selected from hydrogen, OR c ; R 3 R 4 R 5 R 6 R 7 Each is independently selected from hydrogen, C1-C6 alkyl, halogen, halogen-substituted C1-C6 alkyl, and -C(O)2R. d Nitro, cyano, C1-C6 alkoxy, halogen-substituted C1-C6 alkoxy, -C(O)NHR e C3-C6 nitrogen-containing heteroaryl groups; wherein each of the C1-C6 alkyl groups is independently optionally divided by one or more R f replace; R a R c R d Each is independently selected from hydrogen or C1~C6 alkyl groups; R b R e Each is independently selected from hydrogen or hydroxyl; R f Each is independently selected from C1-C6 alkyl groups that are hydrogen- or halogen-substituted; The isomers include tautomers, racemates, mesomers, enantiomers, and diastereomers.

2. A pyrazolinone compound according to claim 1, characterized in that, R 1 For hydrogen, C(O)2R a or C(O)NHR b ; R 2 For hydrogen, OR c ; When R 3 R 4 R 5 R 6 R 7 When the halogen-substituted C1-C6 alkyl group is a halogen-substituted C1-C6 alkyl group, the halogen-substituted C1-C6 alkyl group is a trifluoromethyl group; When R 3 R 4 R 5 R 6 R 7 When the halogen is halogen, the halogen includes fluorine, chlorine, and bromine; When R 3 R 4 R 5 R 6 R 7 When the C1-C6 alkoxy group is halogen-substituted, the halogen-substituted C1-C6 alkoxy group is a trifluoromethoxy group; When R 5 When the C3-C6 nitrogen-containing heteroaryl group is C3-C6, the C3-C6 nitrogen-containing heteroaryl group is tetrazolium.

3. A pyrazolinone compound according to claim 1 or 2, characterized in that, The C1-C6 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, and tert-butyl; the C1-C6 alkoxy group is selected from methoxy, ethoxy, n-propyloxy, isopropyloxy, and tert-butyloxy.

4. A pyrazolinone compound according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts are selected from ammonium salts, alkylammonium salts, ethylenediamine salts, cyclohexylamine salts, arginine salts, lysine salts, choline salts, sodium salts, potassium salts, calcium salts, and magnesium salts.

5. A pyrazolinone compound according to claim 1 or 2, characterized in that, It includes pyrazolone compounds having one of the following structures, or isomers of pyrazolone compounds having one of the following structures, and their pharmaceutically acceptable salts: 。 6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the pyrazolone compounds of claims 1-5, pharmaceutical excipients, and / or pharmaceutically acceptable carriers.

7. Use of the pyrazolone compound of any one of claims 1-5 or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating diseases related to ZBP1 protein function.

8. The use according to claim 7, characterized in that, The diseases associated with ZBP1 protein function include inflammation, autoimmune diseases, and infectious diseases.

9. Use of the pyrazolone compound of any one of claims 1-5 or the pharmaceutical composition of claim 6 in the preparation of a Z-type nucleic acid binding protein inhibitor.

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