Method for preparing n-hydroxybenzamidine compound

Abstract

Provided in the present invention is a method for preparing an N-hydroxybenzamidine compound. Specifically, the present invention relates to a method for preparing a compound as represented by formula (I) and the use thereof in the prevention and / or treatment of post-stroke cerebral edema, hereditary angioedema (HAE) and diabetic macular edema (DME). The preparation method of the present invention has the advantages of readily available raw materials, a low cost, a high reaction safety, a high yield, a high product purity, etc., is suitable for industrial production, and has good application prospects.

Description

A method for preparing N-hydroxybenzomididine compounds Technical Field

[0001] This invention relates to a method for preparing a prodrug compound of formula (I) that is a selective inhibitor of plasma kallikrein (PKa), and the use of the compound of formula (I) in the prevention and / or treatment of cerebral edema after stroke, hereditary angioedema (HAE), and diabetic macular edema (DME). Background Technology

[0002] The KKS (Kallikrein-Kinin System), also known as the kininogen-kinin system, is an important regulatory system in the human body. This system mainly consists of kininogen, kinin, bradykinin (BK), bradykinin B1 receptor (B1R), bradykinin B2 receptor (B2R), and kininase. It is widely distributed in various tissues and organs, including the cardiovascular system, kidneys, pancreas, and central nervous system, and participates in functions such as blood pressure regulation, inflammation, cardiovascular homeostasis, pain transmission, cytokine release, glucose metabolism, smooth muscle contraction, and cell proliferation. The plasma KKS components are mainly synthesized in the liver and secreted into the bloodstream. Plasma prekallikrein (PPK), a precursor of serine proteases in the KKS signaling pathway, is converted by factor XIIa (FXIIa) into catalytically active plasma kallikrein (PKa), which participates in inflammatory responses. In the KKS system, PPK cleaves high-molecular-weight kininogen (HK) to produce bradykinin. BK is a potent inflammatory cytokine; it can bind to and activate B1R and B2R. Activation of both B1R and B2R induces calcium channel blockers (CCBs) in smooth muscle and epithelial cells. 2+ Increased concentrations promote the release of factors such as arachidic acid, NO, eprostol, tissue plasminogen activator (IPA), and glucose transporter 2 (GLU-2). These factors are associated with inflammation, angiogenesis, vasodilation, vascular permeability, and pain regulation.

[0003] Plasma kallikrein plays a role in a variety of inflammatory conditions and can have numerous effects in these conditions, such as hereditary angioedema (HAE), retinopathy or diabetic retinopathy, proliferative and nonproliferative retinopathy, diabetic macular edema (DME), ischemia-reperfusion injury in all types of situations associated with tissue and / or organ transplantation, surgery-induced brain injury, focal cerebral ischemia, global cerebral ischemia, gyromas-associated edema, spinal cord injury, pain, ischemia, focal cerebral ischemia, neurological and cognitive deficits, deep vein thrombosis, stroke, myocardial infarction, acquired angioedema, high-altitude cerebral edema, cytotoxic cerebral edema, osmotic cerebral edema, obstructive hydrocephalus, radiation-induced edema, lymphedema, and traumatic brain injury. Injuries, hemorrhagic stroke, intracerebral hemorrhage, hemorrhagic transformation of ischemic stroke, blood clotting disorders such as thrombosis, itching, inflammatory component disorders (such as multiple sclerosis), encephalitis, Alzheimer's disease, increased blood pressure associated with diabetes or hyperlipidemia, renal insufficiency, chronic kidney disease, heart failure, microalbuminuria, albuminuria, proteinuria, cerebral hemorrhage, deep vein thrombosis, coagulation after fibrinolytic therapy, angina, angioedema, sepsis, lupus, gout, psoriasis, inflammatory bowel disease, diabetes, diabetic complications, Parkinson's disease, amyotrophic lateral sclerosis (ALS) Kujjah disease, stroke, epilepsy, allergic edema such as airflow obstruction from chronic allergic sinusitis or perennial rhinitis, airflow obstruction from acute asthma, and other diseases.

[0004] Plasma kallikrein inhibitors are considered suitable for treating a wide range of conditions, especially edema formation in diseases such as edema associated with ischemia-reperfusion injury in stroke, retinopathy, or edema-associated diseases such as hereditary angioedema, macular edema, and cerebral edema.

[0005] Currently, the treatment of acute ischemic stroke mainly consists of general management and specific therapies. Specific therapies are crucial for treating ischemic stroke, primarily including improving cerebral blood circulation and neuroprotection. The earlier neuroprotection is initiated after a stroke, and the more effectively the progression of brain nerve death is slowed, the more effectively harmful substances can be prevented from damaging the brain, buying more time for the brain to heal. Currently, edaravone and butylphthalide are the mainstream neuroprotective drugs in domestic clinical practice. Others include citicoline, monosialotetrahexosylganglioside sodium, and guipizide, but overall, the treatment effect is not ideal, and they are still largely used as adjunctive therapies. There is an urgent need to develop new, effective, and safe anti-stroke drugs or drug combinations to meet the pressing clinical needs. Since cerebral ischemic injury is related to multiple mechanisms, including excessive free radical formation, excitatory amino acid toxicity, intracellular calcium overload, and inflammatory responses, regardless of the mechanism, the final outcome is always nerve cell death, functional impairment, and the formation of cerebral infarction foci. The kallikrein-kinin system (KKS) mediates thrombosis, vascular permeability, and blood pressure changes during cerebral ischemia. The kallikrein pathway is initiated by activation of coagulation factor XII (FXIIa), which cleaves plasma prokallikrein into plasma kallikrein (PK). Subsequently, PK acts on high-molecular-weight kininogen, inducing the release of the pro-inflammatory peptide hormone bradykinin (BK). BK binds to its endothelial bradykinin receptors 1 and 2 (B1R / B2R), initiating a cascade of inflammatory signals that lead to thrombin generation and intracellular influx of circulating immune cells, ultimately resulting in thrombosis and neuroinflammation. Inhibition of PK after stroke can significantly reduce cerebral thrombosis, stabilize the blood-brain barrier, and thus reduce the number of brain-infiltrating immune cells (Seminars in Immunopathology 2023, 45:389–410). At the same time, inhibition of PK can also reduce NMDA receptor-mediated excitotoxicity and ischemic neuronal death (Stroke 2014, Vol 45, Issue suppl_1, Abstract WP210).

[0006] Currently, conventional neuroprotective mechanisms offer limited clinical benefits. The KKS system is closely related to stroke-related hemorrhage, edema, inflammation, and neuroprotection. PK inhibition can significantly reduce stroke-induced intracerebral thrombosis, stabilize the blood-brain barrier, thereby reducing the number of brain-infiltrating immune cells. It can also inhibit NMDA receptor-mediated excitotoxicity and ischemic neuronal death, significantly reducing the extent of cerebral infarction.

[0007] Plasma kallikrein (PK) inhibitors are considered particularly suitable for the treatment of retinopathy, such as macular edema (DME) associated with diabetes and / or hypertension.

[0008] Proteomic analysis of vitreous samples from patients with diabetic retinal metastasis (DME) showed an 11.0-fold increase in PPK (plasma prekallikrein) and plasma kallikrein compared to control vitreous samples from patients with macular holes (p<0.0001) (Diabetes 2015, 64, 3588–3599). Studies have shown that continuous systemic administration of PKal inhibitors in diabetic mice can reduce retinal vascular permeability and retinal blood flow abnormalities (Diabetes 2011, 60, 590–1598). Further preclinical evidence suggests that plasma kallikrein (PK) may contribute to VEGF-mediated retinal edema (Investig. Ophthalmol. Vis. Sci. 2016, 57, 2390–2399). Current treatment strategies for DME are primarily limited to vascular endothelial growth factor (VEGF) inhibitors and laser photocoagulation. These treatments are not universally effective in all patients, and potential side effects persist in a significant proportion of patients. Currently, the main treatment for diabetic retinal muscular atrophy (DME) relies on anti-VEGF therapy, but some patients (approximately 50%) do not respond to VEGF therapy. This means that nearly 50% of patients do not receive effective treatment. Preclinical studies have shown that activation of the intraocular KKS can induce retinal vascular permeability, vasodilation, and retinal thickening. Proteomic analysis of the vitreous humor in DME-affected eyes indicates that the KKS and VEGF pathways are potentially independent biological pathways. Furthermore, proteins associated with DME in the vitreous humor are significantly more correlated with the KKS pathway than with the VEGF pathway. Preclinical experiments in diabetic animals have shown that inhibiting the KKS system is an effective way to reduce retinal vascular permeability (Invest Ophthalmol Vis Sci. 2016; 57(6):2390–2399).

[0009] (Z)-1-(4-(cyclopropylmethyl)benzyl)-N-(4-(N'-hydroxyaminoiminoyl)benzyl)-1H-pyrazole-4-carboxamide (I) is a prodrug of N-hydroxybenzomididine, a selective inhibitor of plasma kallikrein. In vivo, it can be reduced to the parent compound (II) by NADH-cytochrome b5 reductase (CN2024100315)(Molecules 2023,28,4713). It can be used to prevent and / or treat diabetic macular edema (DME), age-related macular degeneration (AMD), choroidal neovascularization, hereditary angioedema (HAE), and post-stroke cerebral edema.

[0010] This invention provides a method for preparing the compound of formula (I). This method has advantages such as readily available raw materials, low cost, high reaction safety, and high yield, and has excellent application prospects. The method also has good atom economy, yields a high-purity product, and is suitable for industrial production. Summary of the Invention

[0011] In one aspect, this invention provides a method for preparing the compound represented by formula (I).

[0012] It includes step a): the compound of formula (III) and the hydroxylamine salt are reacted in a solvent in the presence of a base to prepare the compound of formula (I).

[0013] In some embodiments of the present invention, the solvent in step a) is selected from ethanol, methanol or isopropanol; preferably, the solvent is selected from ethanol.

[0014] In some embodiments of the present invention, the hydroxylamine salt in step a) is selected from hydroxylamine hydrochloride and hydroxylamine sulfate; preferably, the hydroxylamine salt is selected from hydroxylamine hydrochloride.

[0015] In some embodiments of the present invention, the base in step a) is selected from triethylamine and N,N-diisopropylethylamine; preferably, the base is selected from triethylamine.

[0016] In some embodiments of the present invention, the molar ratio of the hydroxylamine salt to the compound of formula (III) in step a) is 3:1 to 5:1; preferably, the molar ratio of the hydroxylamine salt to the compound of formula (III) is 3:1.

[0017] In some embodiments of the present invention, the reaction temperature in step a) is selected from 50°C to 70°C; preferably, the reaction temperature is selected from 60°C to 65°C.

[0018] In some embodiments of the present invention, the method for preparing the compound represented by formula (I) according to the present invention includes step b) prior to step a): compound (IV) and compound (V) are reacted in a solvent in the presence of a condensing agent and a base to prepare compound (III).

[0019] In some embodiments of the present invention, the solvent in step b) is selected from DMF, DME, and dichloromethane; preferably, the solvent is selected from DMF.

[0020] In some embodiments of the present invention, the base in step b) is selected from triethylamine and N,N-diisopropylethylamine (DIEA); preferably, the base is selected from N,N-diisopropylethylamine.

[0021] In some embodiments of the present invention, the condensing agent in step b) is selected from EDCI / HOBT, HATU, and CDI; preferably, the condensing agent is selected from EDCI / HOBT.

[0022] In some embodiments of the present invention, the reaction temperature in step b) is room temperature, preferably 20-30°C.

[0023] In some embodiments of the present invention, the method for preparing the compound represented by formula (I) according to the present invention includes steps c1) and c2) before step b):

[0024] Step c1): Compound (VI) and compound (VII) react in a solvent in the presence of an acid to give the imine intermediate compound (VIII);

[0025] Step c2): Compound (V) is prepared by Wolff-Kishner reduction reaction in the presence of a base;

[0026] In some embodiments of the present invention, the solvent in step c1) is selected from ethanol, ethylene glycol, and diethylene glycol; preferably, the solvent is selected from ethylene glycol.

[0027] In some embodiments of the present invention, the acid in step c1) is selected from acetic acid, sulfuric acid, and p-toluenesulfonic acid; preferably, the acid is selected from acetic acid.

[0028] In some embodiments of the present invention, the molar ratio of the acid and the compound of formula (VI) in step c1) is 1:10.

[0029] In some embodiments of the present invention, the molar ratio of compound (VI) and compound (VII) in step c1) is 1:1 to 1:3; preferably, the molar ratio of compound (VI) and compound (VII) is 1:2.

[0030] In some embodiments of the present invention, the reaction temperature of step c1) is selected from 100°C to 120°C; preferably, the reaction temperature is selected from 100°C to 110°C.

[0031] In some embodiments of the present invention, the alkali in step c2) is selected from sodium hydroxide and potassium hydroxide; preferably, the alkali is selected from potassium hydroxide.

[0032] In some embodiments of the present invention, the reaction temperature of step c2) is selected from 130 to 140°C.

[0033] In some embodiments of the present invention, steps c1 and c2) are completed by a one-pot method, wherein the molar ratio of the compound of formula (VI) to the base is 1:5 to 1:8, preferably 1:6.

[0034] In some embodiments of the present invention, the method for preparing the compound represented by formula (I) according to the present invention includes step d) prior to step c1): preparing the compound of formula (VI) by hydrolysis of the compound of formula (IX) in a solvent in the presence of a base.

[0035] In some embodiments of the present invention, the solvent in step d) is selected from ethanol or methanol; preferably, the solvent is selected from methanol.

[0036] In some embodiments of the present invention, in step d), the alkali is selected from sodium hydroxide and potassium hydroxide; preferably, the alkali is selected from sodium hydroxide.

[0037] In some embodiments of the present invention, the molar ratio of the compound of formula (IX) and the base in step d) is 1:2 to 1:3, preferably 1:2.

[0038] In some embodiments of the present invention, the reaction temperature in step d) is 60°C.

[0039] In some embodiments of the present invention, the method for preparing the compound represented by formula (I) according to the present invention includes step e) prior to step d): the compound of formula (X) and the compound of formula (XI) are prepared by hydrolysis in a solvent in the presence of a base to obtain the compound of formula (IX).

[0040] In some embodiments of the present invention, the solvent in step e) is selected from DMF, DMSO, and DMA; preferably, the solvent is selected from DMF.

[0041] In some embodiments of the present invention, the molar ratio of compound (X) and compound (XI) in step e) is 1:1 to 1:1.5; preferably, the molar ratio of compound (X) and compound (XI) is 1:1.3.

[0042] In some embodiments of the present invention, the alkali in step e) is selected from sodium carbonate, potassium carbonate, and cesium carbonate; preferably, the alkali is selected from potassium carbonate.

[0043] In some embodiments of the present invention, the molar ratio of the compound of formula (X) and the base in step e) is 1:2 to 1:4, preferably 1:3.

[0044] In some embodiments of the present invention, the reaction temperature in step e) is room temperature, preferably 20-30°C.

[0045] In some embodiments of the present invention, the method for preparing the compound represented by formula (I) according to the present invention includes step f) prior to step e): the compound of formula (XII) is prepared by radical bromination in a solvent in the presence of NBS or AIBN to obtain the compound of formula (X).

[0046] In some embodiments of the present invention, the solvent in step f) is selected from carbon tetrachloride, 1,2-dichloroethane, and chlorobenzene; preferably, the solvent is selected from chlorobenzene.

[0047] In some embodiments of the present invention, the molar ratio of the compound of formula (XII) and NBS in step f) is 1:1 to 1:1.5; preferably, the molar ratio of the compound of formula (XII) and NBS is 1:1.4.

[0048] In some embodiments of the present invention, the molar ratio of the compound of formula (XII) and AIBN in step f) is 1:0.05 to 1:0.2; preferably, the molar ratio of the compound of formula (XII) and AIBN is 1:0.1.

[0049] In some embodiments of the present invention, the reaction temperature in step f) is selected from 70°C to 90°C; preferably, the reaction temperature is selected from 70°C to 75°C.

[0050] In some embodiments of the present invention, during the post-reaction treatment in step f), dibromo byproducts are removed by adding DIEA and diethyl phosphite, wherein the molar ratio of the compound of formula (XII) to DIEA and diethyl phosphite is 1:1.2:1.1 to 1:1.6:1.4; preferably, the molar ratio of the compound of formula (XII) to DIEA and diethyl phosphite is 1:1.5:1.3.

[0051] In some embodiments of the present invention, the method for preparing the compound represented by formula (I) according to the present invention includes step g) prior to step f): preparing the compound of formula (XII) by Friedel-Crafts reaction in toluene in the presence of aluminum trichloride.

[0052] In some embodiments of the present invention, the molar ratio of the compound of formula (XIII) and aluminum trichloride in step g) is 1:1 to 1:1.2; preferably, the molar ratio of the compound of formula (XIII) and aluminum trichloride is 1:1.

[0053] In some embodiments of the present invention, the reaction temperature for adding aluminum trichloride in step g) is selected from -10℃ to 10℃, preferably 0℃ to 5℃, and the reaction temperature is controlled at 20℃ to 30℃ after the addition is completed.

[0054] Another aspect of the present invention provides a method for preparing a compound of formula (I), comprising the following steps:

[0055] Step g): Compound (XII) was prepared by Friedel-Crafts reaction in toluene in the presence of aluminum trichloride, yielding compound (XIII);

[0056] Step f): Compound (X) is prepared by radical bromination in a solvent in the presence of NBS or AIBN to obtain compound (X);

[0057] Step e): Compound (X) and compound (XI) are prepared by hydrolysis in a solvent in the presence of a base to obtain compound (IX);

[0058] Step d): Compound (IX) is prepared by hydrolysis in a solvent in the presence of a base to obtain compound (VI);

[0059] Step c): Compound (VI) and compound (VII) are reacted in a solvent in the presence of an acid to give imine intermediate compound (VIII). Compound (VIII) is not separated and is prepared by Wolff-Kishner reduction reaction in the presence of a base to give compound (V).

[0060] Step b): Compound (IV) and compound (V) are reacted in a solvent in the presence of a condensing agent and a base to prepare compound (III);

[0061] Step a): Compound (I) is prepared by addition reaction of compound (III) with hydroxylamine salt in a solvent in the presence of a base.

[0062] In some embodiments of the present invention, according to step g), wherein,

[0063] The molar ratio of the compound of formula (XIII) to aluminum trichloride is 1:1 to 1:1.2; preferably, the molar ratio of the compound of formula (XIII) to aluminum trichloride is 1:1; and / or

[0064] The reaction temperature is selected from -10℃ to 10℃; preferably, the reaction temperature is selected from 0℃ to 5℃.

[0065] In some embodiments of the present invention, according to step f), wherein,

[0066] The solvent is selected from carbon tetrachloride, 1,2-dichloroethane, and chlorobenzene; preferably, the solvent is selected from chlorobenzene; and / or

[0067] The molar ratio of the compound of formula (XII) to NBS is 1:1 to 1:1.5; preferably, the molar ratio of the compound of formula (XII) to NBS is 1:1.4; and / or

[0068] The molar ratio of the compound of formula (XII) to AIBN is 1:0.05 to 1:0.2; preferably, the molar ratio of the compound of formula (XII) to AIBN is 1:0.1; and / or

[0069] The reaction temperature is selected from 70℃ to 90℃; preferably, the reaction temperature is selected from 70℃ to 75℃; and / or

[0070] In the post-reaction treatment, dibromo byproducts are removed by adding DIEA and diethyl phosphite. The molar ratio of the compound of formula (XII) to DIEA and diethyl phosphite is 1:1.2:1.1 to 1:1.6:1.4. Preferably, the molar ratio of the compound of formula (XII) to DIEA and diethyl phosphite is 1:1.5:1.3.

[0071] In some embodiments of the present invention, according to step e), wherein,

[0072] The solvent is selected from DMF, DMSO, and DMA; preferably, the solvent is selected from DMF; and / or

[0073] The molar ratio of the compound of formula (X) to the compound of formula (XI) is 1:1 to 1:1.5; preferably, the molar ratio of the compound of formula (X) to the compound of formula (XI) is 1:1.3; and / or

[0074] The alkali is selected from sodium carbonate, potassium carbonate, and cesium carbonate; preferably, the alkali is selected from potassium carbonate, and / or

[0075] The molar ratio of the compound of formula (X) to the base is 1:2 to 1:4, preferably 1:3; and / or

[0076] The reaction temperature is room temperature, preferably 20-30℃.

[0077] In some embodiments of the present invention, according to step d) of the present invention, wherein,

[0078] The solvent is selected from ethanol and methanol; preferably, the solvent is selected from methanol; and / or

[0079] The alkali is selected from sodium hydroxide and potassium hydroxide; preferably, the alkali is selected from sodium hydroxide; and / or

[0080] The molar ratio of the compound of formula (IX) to the base is 1:2 to 1:3, preferably 1:2; and / or

[0081] The reaction temperature is 60℃.

[0082] In some embodiments of the present invention, according to step c), wherein,

[0083] The solvent is selected from ethanol, ethylene glycol, and diethylene glycol; preferably, the solvent is selected from ethylene glycol; and / or

[0084] The acid is selected from acetic acid, sulfuric acid, and p-toluenesulfonic acid; preferably, the acid is selected from acetic acid; and / or

[0085] The molar ratio of the acid to the compound of formula (VI) is 1:10; and / or

[0086] The molar ratio of compound (VI) to compound (VII) is 1:1 to 1:3; preferably, the molar ratio of compound (VI) to compound (VII) is 1:2 and / or

[0087] The reaction temperature of compound (VI) with compound (VII) is selected from 100°C to 120°C; preferably, the reaction temperature is selected from 100°C to 110°C; and / or

[0088] The temperature for the Wolff-Kishner reduction reaction is selected from 130–140 °C; and / or

[0089] The alkali is selected from sodium hydroxide and potassium hydroxide; preferably, the alkali is selected from potassium hydroxide; and / or

[0090] The molar ratio of the compound of formula (VI) to the base is 1:5 to 1:8, preferably 1:6.

[0091] In some embodiments of the present invention, according to step b), wherein...

[0092] The solvent is selected from DMF, DME, and dichloromethane; preferably, the solvent is selected from DMF; and / or

[0093] The base is selected from triethylamine and N,N-diisopropylethylamine; preferably, the base is selected from N,N-diisopropylethylamine; and / or

[0094] The condensing agent is selected from EDCI / HOBT, HATU, and CDI; preferably, the condensing agent is selected from EDCI / HOBT; and / or

[0095] The reaction temperature is room temperature, preferably 20-30℃.

[0096] In some embodiments of the present invention, according to step a), wherein,

[0097] The solvent is selected from ethanol, methanol, or isopropanol; preferably, the solvent is selected from ethanol; and / or

[0098] The hydroxylamine salt is selected from hydroxylamine hydrochloride and hydroxylamine sulfate; preferably, the hydroxylamine salt is selected from hydroxylamine hydrochloride; and / or

[0099] The base is selected from triethylamine and N,N-diisopropylethylamine; preferably, the base is selected from triethylamine; and / or

[0100] The molar ratio of the hydroxylamine salt to the compound of formula (III) is 3:1 to 5:1; preferably, the molar ratio of the hydroxylamine salt to the compound of formula (III) is 3:1; and / or

[0101] The reaction temperature is selected from 50℃ to 70℃; preferably, the reaction temperature is selected from 60℃ to 65℃.

[0102] Another aspect of the present invention provides the use of the compound of formula (I) prepared by the preparation method according to the present invention in the preparation of a medicament for the prevention and / or treatment of post-stroke cerebral edema, hereditary angioedema (HAE) or diabetic macular edema (DME).

[0103] Technical effect

[0104] The method for preparing compound (I) provided by this invention not only uses inexpensive and readily available toluene as a starting material, but also does not involve expensive reagents in the preparation process of compound (I); the entire route has a higher yield, better purity of the final product, and is simpler, safer, and more suitable for industrial production. Attached Figure Description

[0105] Figure 1: The 1H NMR spectrum of compound (I).

[0106] Figure 2: Inhibitory effect of compound (I) on carrageenan-induced paw edema in rats.

[0107] Figure 3: Effect of compound (I) on the AUC of carrageenan-induced toe edema in rats.

[0108] Figure 4: Effect of compound (I) on the mNSS score of rat ischemia-reperfusion neurobehavioral studies.

[0109] Figure 5: Effect of compound (I) on the volume of ischemic-reperfusion cerebral infarction in rats.

[0110] Figure 6: Effect of compound (I) on CA-1-induced retinal thickness in rats. Detailed Implementation

[0111] The present invention is further illustrated below by means of embodiments. The embodiments provided in this application are for illustrative purposes only and are not intended to limit the present invention. Modifications, alterations, and variations made within the scope of protection of the present invention are all within the scope of protection of the present invention.

[0112] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts were expressed in 10⁻¹⁰ increments. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker Ascend-400 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.

[0113] The LC-MS determination was performed using an Agilent 1290-6490 mass spectrometer (manufacturer: Agilent).

[0114] The determination by high performance liquid chromatography (HPLC) was performed using an Agilent 1260 liquid chromatograph (Agilent TC-C18 250×4.6mm 5μm column).

[0115] In the following embodiments, the abbreviations are explained as follows:

[0116] AIBN: Azobisisobutyronitrile;

[0117] DCE: Dichloroethane;

[0118] DMF: N,N-dimethylformamide;

[0119] DIEA: N,N-Diisopropylethylamine

[0120] DMSO: Dimethyl sulfoxide;

[0121] DMA: N,N-dimethylacetamide;

[0122] DME: Dimethyl ethylene diether

[0123] CDI: 1,1'-carbonyldiimidazole;

[0124] EA: Ethyl acetate

[0125] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0126] HOBT: 1-Hydroxybenzotriazole;

[0127] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0128] NBS: N-bromosuccinimide;

[0129] MTBE: Methyl tert-butyl ether

[0130] MS: Mass Spectrometry

[0131] ESI: Electrospray Ionization

[0132] 1 1H NMR: Proton NMR spectrum

[0133] LC-MS: Liquid Chromatography-Mass Spectrometry

[0134] TEA: Triethylamine

[0135] Example 1: Synthesis of cyclopropyl (4-methylcyclohexyl-2,4-dien-1-yl) ketone (XII)

[0136] Synthesis example 1:

[0137] Under a nitrogen atmosphere and at room temperature, toluene (125 mL) and aluminum trichloride (63.7 g, 0.478 mol, 1.0 eq) were added to a three-necked flask and stirred to suspend the mixture. The temperature was lowered to 0-5 °C, and then cyclopropionyl chloride (XIII) (purchased from Maclean's) (50.0 g, 0.478 mol, 1.0 eq, diluted with 75 mL of toluene) was slowly added dropwise, keeping the temperature below 5 °C (0-5 °C). After the addition was complete, the reaction was maintained between 20-30 °C and stirred under a nitrogen atmosphere for 1 hour. The reaction mixture was then slowly poured into ice water (500 mL), keeping the temperature below 10 °C. After quenching, the mixture was filtered through a diatomaceous earth filter. The liquid and liquid phases were separated and combined. The organic phases were washed once with saturated NaHCO3 aqueous solution (500 mL), and then washed once with saturated NaCl aqueous solution (500 mL). The mixture was concentrated under vacuum at 50 ± 5 °C until no obvious fraction remained. Then, chlorobenzene (250 mL) was added and the mixture was concentrated under vacuum until no obvious fraction remained, yielding 77.1 g of a yellow solid (XII) compound, which was used directly in the next reaction. HPLC purity: 99.06%.

[0138] Synthesis example 2:

[0139] Under a nitrogen atmosphere and at room temperature, toluene (1125 mL) and aluminum trichloride (573 g, 4.30 mol, 1.0 eq) were added to a three-necked flask and stirred to suspend the mixture. The temperature was lowered to 0-5 °C, and then cyclopropionyl chloride (XIII) (purchased from Maclean's) (450 g, 4.30 mol, 1.0 eq, diluted with 675 mL of toluene) was slowly added dropwise, keeping the temperature below 5 °C (0-5 °C). After the addition was complete, the reaction was maintained at 20-30 °C with stirring under a nitrogen atmosphere for 1 hour. The reaction mixture was then slowly poured into ice water (4.5 L), keeping the temperature below 10 °C. After quenching, the mixture was filtered through a diatomaceous earth filter. The liquid and liquid phases were separated and combined. The organic phases were washed once with 4.5 L of saturated NaHCO3 aqueous solution, and then washed once again with 4.5 L of saturated NaCl aqueous solution. The mixture was concentrated under vacuum at 50 ± 5 °C until no obvious fraction remained. Then, 2.25 L of chlorobenzene was added and the mixture was concentrated under vacuum until no obvious fraction remained, yielding 663.7 g of a yellow solid (XII) compound (yield: 96.2%), which was used directly in the next reaction. HPLC purity: 98.92%.

[0140] Example 2: Synthesis of (4-(bromomethyl)phenyl)(cyclopropyl)methyl ketone (X)

[0141] Synthesis example 1:

[0142] At room temperature, DCE (35 mL), compound (XII) (5.0 g, 31.2 mmol, 1.0 eq), NBS (2.0 g, 11.2 mmol, 0.36 eq), and AIBN (513 mg, 3.12 mmol, 0.1 eq) were added to the reaction vessel and stirred until insoluble. The temperature was raised to 70–75 °C, and NBS (2.0 g, 11.2 mmol, 0.36 eq), NBS (2.1 g, 11.8 mmol, 0.38 eq), and NBS (1.1 g, 6.24 mmol, 0.20 eq) were added in three batches under controlled temperature. While the temperature rise was controllable with the batch addition, the product purity remained unchanged, but the dibromine impurity gradually increased with increasing NBS content. After the reaction, HLPC analysis showed that the product was 73.9% and the dibromine impurity was 10.1%. After the reaction was complete, diethyl phosphite (431 mg, 3.12 mmol, 0.1 eq) was added dropwise and stirred for 22 hours. HLPC analysis showed that the dibromine impurity decreased to about 1%, and the product also decreased to 71.7%.

[0143] Synthesis example 2:

[0144] At room temperature, chlorobenzene (560 mL), compound (XII) (80.0 g, 0.5 mol, 1.0 eq), NBS (124.6 g, 0.7 mol, 1.4 eq), and AIBN (8.21 g, 50 mmol, 0.1 eq) were added to a reaction vessel and stirred until insoluble. The temperature was raised to 70–75 °C, and the reaction was maintained at this temperature for 2–3 hours. Then, NBS (8.9 g, 50 mmol, 0.1 eq) was added, and the reaction was maintained at this temperature for another 2 hours. 67% of the product was formed, and the remaining 3% of the starting material (XII) compound had a dibromine impurity of 17.9%. The reaction was stopped, and the mixture was filtered. The residue was washed with chlorobenzene (160 mL), and the filtrate was collected. The organic phase was washed twice with water (800 mL × 2). The obtained organic phase was dried with anhydrous Na₂SO₄, filtered, and the residue was washed with chlorobenzene (160 mL). The filtrate was collected. DIEA (96.9 g, 0.75 mol, 1.5 eq) was added to the filtrate, and diethyl phosphite (89.8 g, 0.65 mol, 1.3 eq) was added dropwise while maintaining the temperature at 20–30 °C. The mixture was stirred for 2 hours while maintaining the temperature at 20–30 °C. The results showed that there were no dibromo impurities within the detection limit, and 85% of the product was present. Water (800 mL) was added dropwise while maintaining the temperature at 20–30 °C. After the addition was complete, the mixture was stirred for 30–60 minutes, separated, and the organic phase was collected. The organic phase was washed once with water (400 mL), collected, dried with anhydrous Na₂SO₄, filtered, and the residue was washed with chlorobenzene (160 mL). The filtrate was collected and concentrated to give 158.2 g of a deep red liquid compound (X) (purity: 56.4%, yield: 74.7%), with a purity of 85.43% by HPLC (254 nm).

[0145] Synthesis example 3:

[0146] At room temperature, chlorobenzene (3.5 L), compound (XII) (500.0 g, 3.12 mol, 1.0 eq), NBS (777.4 g, 4.37 mol, 1.4 eq), and AIBN (50.9 g, 0.31 mol, 0.1 eq) were added to a reaction vessel and stirred until insoluble. The temperature was raised to 70–75 °C, and the reaction was maintained at this temperature for 2 hours. 69.7% of the product was formed, with 3.5% of the starting material (XII) remaining, and 17% containing dibromo impurities. The reaction was stopped, and the mixture was filtered. The residue was washed with chlorobenzene (1 L), and the filtrate was collected. The organic phase was washed twice with water (5 L × 2). The resulting organic phase was dried with anhydrous Na₂SO₄, filtered, and the residue was washed with chlorobenzene (1 L). The filtrate was collected. DIEA (604.8 g, 4.68 mol, 1.5 eq) was added to the filtrate, and diethyl phosphite (559.7 g, 4.06 mol, 1.3 eq) was added dropwise while maintaining the temperature at 20–30 °C. The mixture was stirred at 20–30 °C for 2 hours. The results showed that there were no dibromo impurities within the detection limit, and 82.6% of the product was present. Water (5 L) was added dropwise while maintaining the temperature at 20–30 °C. After the addition was complete, the mixture was stirred for 30–60 minutes, separated, and the organic phase was collected. The organic phase was washed once with water (2.5 L), collected, dried with anhydrous Na₂SO₄, filtered, and the residue was washed with chlorobenzene (1 L). The filtrate was collected and concentrated to give 1023.0 g of a dark red liquid compound of formula (X) (purity: 49.2%, yield: 67.45%), with a purity of 82.60% by HPLC (254 nm).

[0147] Example 3: Synthesis of methyl 1-(4-(cyclopropylcarbonyl)benzyl)-1H-pyrazole-4-carboxylate (XI)

[0148] Synthesis example 1:

[0149] Compound (X) (actual content of crude product treated with diethyl phosphite, equivalent to 85.0 g, 355.5 mmol, 1.0 eq), DMF (425 mL), methyl 1H-pyrazole-4-carboxylate (XI) (64.8 g, 462.2 mmol, 1.3 eq), and K2CO3 (147.2 g, 1066.5 mmol, 3.0 eq) were added sequentially to the reaction vessel. The mixture was stirred until it did not dissolve completely. The reaction was carried out at room temperature (20-30 °C) for 15 hours, yielding 89.62% of the product compound (IX), with 0.3% of the starting compound (X) remaining. Water (1275 mL) was added to the reaction solution, and the mixture was stirred for 40-60 min. The precipitated solid showed good properties. After centrifugation, the filter cake was washed twice with water (145 mL × 2). Methyl tert-butyl ether (595 mL) was added to the filter cake, and the mixture was stirred at 20-30 °C for 2-3 hours, followed by centrifugation. The filter cake was washed with methyl tert-butyl ether (145 mL) and dried in a vacuum drying oven at 40 ± 5 °C to give 91.25 g of a pale yellow solid of compound (IX) (yield: 86.1%). HPLC (254 nm) purity: 98.65%. MS (ESI) m / z: 299.2 [M+H] + .

[0150] Synthesis example 2:

[0151] Compound (X) (actual content of crude product treated with diethyl phosphite, equivalent to 500.0 g, 2.09 mol, 1.0 eq), DMF (2.5 L), methyl 1H-pyrazole-4-carboxylate (XI) (380.8 g, 2.72 mol, 1.3 eq), and K2CO3 (865.3 g, 6.27 mol, 3.0 eq) were added sequentially to the reaction vessel. The mixture was stirred until it did not dissolve completely. The reaction was carried out at room temperature (20-30℃) for 20 hours, yielding 82.86% of the product compound (IX). No reactant compound (X) remained within the detection limit. Water (7.5 L) was added to the reaction solution, and the mixture was stirred for 40-60 min. The precipitated solid showed good properties. After centrifugation, the filter cake was washed twice with water (850 mL × 2). Methyl tert-butyl ether (3.5 L) was added to the filter cake, and the mixture was stirred at 20-30℃ for 2-3 hours, followed by centrifugation. The filter cake was washed with methyl tert-butyl ether (850 mL) and dried in a vacuum drying oven at 40 ± 5 °C to give 530.03 g of a pale yellow solid of compound (IX) (yield: 85.07%, first three steps yield: 56.99%). HPLC (254 nm) purity: 99.06%. MS (ESI) m / z: 299.2 [M+H] + .

[0152] Example 4: Synthesis of 1-(4-(cyclopropylcarbonyl)benzyl)-1H-pyrazole-4-carboxylic acid (VI)

[0153] Synthesis example 1:

[0154] Compound (IX) with a purity of 98.65% (85.0 g, 284.92 mmol, 1.0 eq) and MeOH (425 mL) were added to a reaction vessel and stirred until dissolved. NaOH (22.78 g, 569.84 mmol, 2.0 eq) was added to 106 mL of water and stirred until dissolved. The mixture was cooled to 40-50 °C, and the dissolved NaOH aqueous solution was added to the reaction vessel. The mixture was kept at 60 °C and stirred for 1 hour, resulting in the formation of product compound (VI) with a purity of 97.70%. After the reaction was completed and cooled to room temperature, the reaction solution was concentrated and diluted with water (1.3 L). The aqueous phase was extracted once with EA (680 mL), and the aqueous phase was collected. 4 mol / L HCl aqueous solution (149 mL) was slowly added to the resulting aqueous phase to adjust the pH to 2, precipitating a white solid. The solid was filtered, and the filter cake was washed with water (85 mL). The filter cake was dried in a vacuum drying oven at 50 ± 5 °C to obtain 70.2 g of a white solid of compound (VI) (yield: 91.1%). HPLC (254 nm) purity: 99.39%. MS (ESI) m / z: 271.3 [M+H] + .

[0155] Synthesis example 2:

[0156] Compound (IX) with a purity of 98.81% (530.0 g, 1.78 mol, 1.0 eq) and MeOH (2650 mL) were added to a reaction vessel and stirred until dissolved. NaOH (142.4 g, 3.56 mol, 2.0 eq) was added to 663 mL of water and stirred until dissolved. The mixture was cooled to 40-50 °C, and the dissolved NaOH aqueous solution was added to the reaction vessel. The mixture was kept at 60 °C and stirred for 1 hour, resulting in the formation of product compound (VI) with a purity of 98.44%. After the reaction was completed, the mixture was cooled to room temperature, concentrated, and diluted with water (8 L). The aqueous phase was extracted once with EA (4.2 L), and the aqueous phase was collected. A 4 mol / L HCl aqueous solution (928 mL) was slowly added to the resulting aqueous phase to adjust the pH to 2, resulting in the precipitation of a white solid. The solid was filtered, and the filter cake was washed with water (530 mL). The filter cake was dried in a vacuum drying oven at 50 ± 5 °C to obtain 440 g of a white solid of compound (VI) (yield: 91.8%). HPLC (254 nm) purity: 99.71%. MS (ESI) m / z: 271.3 [M+H] + .

[0157] Example 5: Synthesis of (Z)-1-(4-(cyclopropyl(2-(methoxycarbonyl)hydrazine)methyl)benzyl)-1H-pyrazole-4-carboxylic acid (VIII) and 1-(4-(cyclopropylmethyl)benzyl)-1H-pyrazole-4-carboxylic acid (V)

[0158] Synthesis example 1:

[0159] At room temperature, ethylene glycol (150 mL), compound (VI) (30.0 g, 111 mmol, 1.0 eq), and methyl hydrazide (VII) (purchased from Maclean's) (19.9 g, 222 mmol, 2.0 eq) were added to a reaction vessel. The mixture was stirred until it did not dissolve completely. AcOH (668 mg, 11.1 mmol, 0.1 eq) was added dropwise. After the addition was complete, the temperature was maintained at 100 °C for 2 hours. 21.61% of the starting compound (VI) remained (at 230 nm). After 6 hours of reaction, 19.18% of the starting compound (VI) remained (at 230 nm). The reaction was stopped, and the temperature was lowered to no more than 50 °C. KOH (AR, 85%, 43.9 g, 666 mmol, 6.0 eq) was added to the reaction vessel. The mixture was stirred until it did not dissolve completely. The reaction mixture was heated to 130–140 °C and stirred until dissolved. The mixture was kept at this temperature and stirred for 18 hours. 97.49% of the product compound (V) was formed, while only 0.03% (230 nm) of the reactant compound (VI) remained. The reaction was stopped, cooled to 60–70 °C, diluted with 600 mL of water, and cooled to room temperature. A 4 mol / L HCl aqueous solution was slowly added to the aqueous phase to adjust the pH to 5.45, resulting in the precipitation of a white solid. The solid was filtered, and the filter cake was washed with 40 mL of water. The filter cake was dried in a vacuum drying oven at 55 ± 5 °C to obtain 25.40 g of the white solid compound (V) (yield: 89.3%). HPLC (230 nm) purity: 98.76%. MS (ESI) m / z: 257.2 [M+H]. + .

[0160] Synthesis example 2:

[0161] At room temperature, ethylene glycol (100 mL), compound (VI) (20.0 g, 74 mmol, 1.0 eq), and methyl hydrazide (VII) (13.2 g, 148 mmol, 2.0 eq) were added to a reaction vessel. The mixture was stirred until it did not dissolve completely. AcOH (448 mg, 7.4 mmol, 0.1 eq) was added dropwise. After the addition was complete, the temperature was maintained at 100 °C for 3 hours. 19.65% of the reactant compound (VI) remained (210 nm). The reaction was stopped, and the temperature was lowered to no more than 50 °C. KOH (AR, 85%, 29.2 g, 444 mmol, 6.0 eq) was added to the reaction vessel. The mixture was stirred until it did not dissolve completely. The reaction temperature was raised to 130–140 °C and stirred until dissolved. The mixture was kept at this temperature and stirred for 20 hours. 97.61% of the product compound (V) was formed, and only 0.13% of the reactant compound (VI) remained (210 nm). Stop the reaction, cool to 60℃-70℃, dilute with water (400mL), cool to room temperature, and divide into two batches.

[0162] A batch of MTBE (100 mL) was added, and the pH was adjusted to 5.50 by slowly adding 4 mol / L HCl aqueous solution. The mixture was filtered through diatomaceous earth, and the filter cake was washed with MTBE (20 mL). After standing, the mixture was separated, and the organic phase was collected. The aqueous phase was extracted with MTBE (50 mL), and the mixture was separated. The organic phases were combined, washed with NaHCO3 aqueous solution (60 mL, 0.015 eq. NaHCO3), and collected. The organic phase was concentrated at 50 °C until no obvious fraction remained. MTBE (20 mL) / n-heptane (40 mL) was added and stirred, filtered, and the filter cake was washed with MTBE / n-heptane = 1:2 (20 mL). The filter cake was dried in a vacuum drying oven at 55 ± 5 °C to obtain 7.99 g of a pale yellow solid of compound (V) (yield: 84.3%). HPLC (210 nm) purity: 99.51%. MS (ESI) m / z: 257.2 [M+H] + .

[0163] Another batch was treated with 100 mL of MTBE. The pH was adjusted to 5.00 by slowly adding 4 mol / L HCl aqueous solution. The mixture was filtered through diatomaceous earth. The filter cake was washed with 20 mL of MTBE, allowed to stand, and separated. The organic phase was collected, and the aqueous phase was extracted with 50 mL of MTBE. The organic phases were combined and washed with 60 mL of NaHCO3 aqueous solution (0.015 eq. NaHCO3). The organic phase was collected and concentrated at 50 °C until no obvious fraction remained. 20 mL of MTBE / 40 mL of n-heptane was added and stirred. The mixture was filtered, and the filter cake was washed with 20 mL of MTBE / n-heptane at a ratio of 1:2. The filter cake was dried in a vacuum drying oven at 55 ± 5 °C to obtain 8.26 g of a pale yellow solid (V) compound (yield: 87.2%). HPLC (210 nm) purity: 99.09%. MS (ESI) m / z: 257.2 [M+H] + .

[0164] Example 6: Synthesis of N-(4-cyanobenzyl)-1-(4-(cyclopropylmethyl)benzyl)-1H-pyrazole-4-carboxamide (III)

[0165] Synthesis example 1:

[0166] Compound (V) (6.0 g, 23.4 mmol, 1.0 eq), 4-(aminomethyl)benzonitrile hydrochloride (IV) (purchased from Aladdin) (4.74 g, 28.1 mmol, 1.2 eq), HOBT (3.80 g, 28.1 mmol, 1.2 eq), EDCI (5.39 g, 28.1 mmol, 1.2 eq), DMF (36 mL), and DIEA (9.07 g, 70.2 mmol, 3.0 eq) were added sequentially to a reaction vessel and reacted at room temperature (20–30 °C) for 15 hours. The reaction mixture was then added dropwise to 180 mL of water under stirring, causing a solid to precipitate. The mixture was stirred for 3 hours, filtered, and the filter cake was washed with 30 mL of water. The filter cake was then dried in a vacuum drying oven at 55 ± 5 °C. The crude product had a purity of 98.33% and contained 0.72% HOBT residue. Then, 24 mL of a mixed solvent (EA / DMSO = 7 / 1) was added to the dried crude product, and the mixture was stirred at 60 °C for 30 minutes until dissolved. The mixture was then cooled to room temperature (20-30 °C) with stirring, and a solid precipitated. Stirring was continued for 1 hour. The mixture was filtered, and the filter cake was first washed once with EA (10 mL), then once with water (10 mL). The filter cake was dried in a vacuum drying oven at 55 ± 5 °C to obtain 6.60 g of a white solid of compound (III) (yield: 76.1%), with an HPLC (210 nm) purity of 99.34%. MS (ESI) m / z: 371.3 [M+H]. + .

[0167] Synthesis example 2:

[0168] Compound (V) (25.0 g, 97.5 mmol, 1.0 eq), 4-(aminomethyl)benzonitrile hydrochloride (IV) (19.7 g, 117 mmol, 1.2 eq), HOBT (15.8 g, 117 mmol, 1.2 eq), EDCI (22.4 g, 117 mmol, 1.2 eq), DMF (150 mL), and DIEA (37.8 g, 292.5 mmol, 3.0 eq) were added sequentially to a reaction vessel and reacted at room temperature (20–30 °C) for 18 hours. The reaction mixture was then added dropwise to stirred water (750 mL), causing a solid to precipitate. The mixture was stirred for 3 hours, filtered, and the filter cake was washed with water (125 mL). The filter cake was then dried in a vacuum drying oven at 55 ± 5 °C. The purity of the crude product was 96.15%, with 0.22% of the starting compound (V) remaining. Then, 375 mL of a mixed solvent (EtOH / H2O = 4:1) was added to the dried crude product, and the mixture was stirred at 60 °C for 30 minutes until dissolved. The mixture was then cooled to room temperature (20-30 °C) with stirring, and a solid precipitated. Stirring was continued for 1 hour. The mixture was filtered, and the filter cake was washed once with 50 mL of water. The filter cake was dried in a vacuum drying oven at 55 ± 5 °C to obtain 31.89 g of a white solid of compound (III) (yield: 88.3%), with a purity of 99.3% according to HPLC (210 nm). MS (ESI) m / z: 371.3 [M+H] + .

[0169] Example 7: Synthesis of (Z)-1-(4-(cyclopropylmethyl)benzyl)-N-(4-(N'-hydroxyaminoiminoyl)benzyl)-1H-pyrazole-4-carboxamide (I)

[0170] Synthesis example 1:

[0171] Compound (III) (6.0 g, 189 mmol, 1.0 eq), hydroxylamine hydrochloride (39.6 g, 567 mmol, 3.0 eq), EtOH (36 mL), and TEA (4.9 g, 48.6 mmol, 3.0 eq) were sequentially added to a reaction vessel, and the reaction was carried out at 60-65 °C for 3 hours. After cooling to room temperature (20-30 °C), the mixture was filtered, and the filter cake was washed with water (24 mL). The filter cake was then dried in a vacuum drying oven at 55±5 °C to obtain 5.77 g of white solid compound (I) (yield: 88.3%), with a purity of 99.28% by HPLC (210 nm).

[0172] MS(ESI) m / z: 404.3 [M+H] + .

[0173] Synthesis example 2:

[0174] Compound (III) (350.0 g, 945 mmol, 1.0 eq), hydroxylamine hydrochloride (198 g, 2.835 mol, 3.0 eq), EtOH (2100 mL), and TEA (285.8 g, 2.835 mol, 3.0 eq) were added sequentially to a reaction vessel, and the reaction was carried out at 60-65 °C for 2 hours. After cooling to room temperature (20-30 °C), the mixture was filtered, and the filter cake was washed with water (1.75 L). The filter cake was then dried in a vacuum drying oven at 55 ± 5 °C to obtain 335.2 g of white solid compound (I) (yield: 87.9%), with a purity of 99.537% by HPLC (210 nm).

[0175] MS(ESI) m / z: 404.3 [M+H] + .

[0176] 1 H NMR (400MHz, DMSO-d) 6 )δ9.56(s,1H),8.61(t,J=6.0Hz,1H),8.24(s,1H),7.90(s,1H),7.61(d,J=8.3Hz,2H),7.26(d,J=8.3Hz,2H),7.21(dd,J=17.5,8.3Hz ,4H),5.75(s,2H),5.30(s,2H),4.40(d,J=5.9Hz,2H),2.47(d,J=6.9Hz,2H),1.02–0.80(m,1H),0.55–0.31(m,2H),0.28–0.08(m,2H).

[0177] Biological tests

[0178] Test Example 1: Effect of compound (I) on hereditary angioedema (HAE)

[0179] The effect of compound (I) on hereditary angioedema (HAE) was determined using a carrageenan-induced rat paw edema model. Carrageenan-induced mouse / rat models have been widely used to test new anti-inflammatory drugs and to study inflammatory mechanisms. The development of edema induced by carrageenan injection leads to the immediate appearance of the main signs of acute local inflammation (edema, hyperalgesia, and erythema) after subcutaneous injection, due to the action of pro-inflammatory factors (bradykinin, histamine, tachykinin, complement, and reactive oxygen species and nitrogen). Carrageenan injection induces angioedema, with histamine, serotonin, and bradykinin (BK) being the first mediators involved in the early stages of the inflammatory response (0–1 hour), while prostaglandins and various cytokines (e.g., IL-1β, IL-6, IL-10, and TNF-α) are involved in this process, leading to dilation of postcapillary venules and resulting in inflammatory fluid and cellular exudation. The carrageenan-induced edema inflammatory response is typically quantified by an increase in paw size (edema). The experiment involved a single oral gavage administration of the test substance to evaluate whether it had an anti-acute inflammatory effect on carrageenan-induced paw edema in rats and whether it could alleviate edema.

[0180] After the acclimatization period, rats (6-8 week old male SD rats from Shanghai Silex Laboratory Animal Co., Ltd.) were divided into groups of 10 rats each based on their body weight and right hind paw volume: control group, model group, indomethacin (purchased from MCE) 5 mg / kg group, berotralstat (purchased from MCE) 10 mg / kg group, low-dose test substance (3 mg / kg) group, and high-dose test substance (9 mg / kg) group. After grouping, the indomethacin group animals were administered the drug via intraperitoneal injection (7.5 mg indomethacin dissolved in 6 mL of 0.1 M NaHCO3), while the other test substance groups animals were administered either the solvent control or the test substance orally by gavage. Weigh the required mass of the test substance according to the experimental concentration, and add it sequentially to 10% DMSO, 60% 1,2-propanediol, 10% polyoxyethylene castor oil, and 20% aqueous solution containing 20% ​​HP-β-CD. After each solvent is added, vortex and sonicate until completely dissolved before adding the next solvent. Twenty minutes after administration, all animals in all groups except the control group were injected with 0.1 mL of 2% carrageenan (purchased from Sigma) solution into the bottom of their right hind paw. The paw volume of the right hind paw was measured before and after the injection of carrageenan solution at 1 h, 2 h, 3 h, 5 h, and 8 h. The swelling inhibition rate (absolute increase), swelling (i.e., swelling change) value, and swelling value AUC (0-1 h, 0-2 h, 0-3 h) were calculated.

[0181] Figure 2 shows the inhibitory effect of the test substance on carrageenan-induced paw edema in rats, and Figure 3 shows the effect of the test substance on the AUC of carrageenan-induced paw edema in rats. The experimental results show that the compound of formula (I) has a significant alleviating effect on carrageenan-induced paw edema in rats, and has a dose-dependent effect.

[0182] Test Example 2: Effects of Compound (I) on Ischemic Stroke

[0183] Following stroke, the kinin-releasing enzyme-kinin system is activated, promoting vascular leakage, inflammation, and neurodegeneration, leading to nerve cell damage and an increase in the infarct area. The effect of compound (I) on stroke was determined using a rat ischemic stroke (tMCAO) model. The efficacy of the test substance in rat ischemia-reperfusion stroke was evaluated. Male SD rats (6-8 weeks old male SD rats from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used as experimental animals. Animals in the sham-operated group underwent sham surgery for middle cerebral artery infarction (MCAO), while animals in other groups underwent MCAO infarction using the suture occlusion method. The suture was removed 48 hours later to create a rat model of MCAO ischemia-reperfusion infarction. The experiment continued until 168 hours after ischemia-reperfusion. The test substance group was administered the drug 1 hour before occlusion (PO, QD) for 5 consecutive days. Weigh the required mass of the test substance according to the experimental concentration. Add 30% PEG 400, 10% Kolliphor EL, and 60% pH 2.2 buffer containing 10% HP-β-CD sequentially. After each solvent addition, vortex and sonicate until completely dissolved before adding the next solvent. The control group received edaravone dexborneol (edaravone and dexborneol in a 4:1 ratio, purchased from Simcere Pharmaceutical) immediately after suture removal (IV, QD) for 5 consecutive days. Body weight was measured in all groups before surgery and on days 1, 3, 5, and 7. Behavioral observation was performed in all groups before surgery and on days 1, 3, 5, and 7. Animal behavior was observed using the mNSS scoring method with a total of 18 points, evaluating motor function, sensation, balance, and reflexes.

[0184] Table 1. mNSS Scoring Criteria

[0185] Measurement of cerebral infarction volume:

[0186] At the end of the experiment, all animals in each group were euthanized by CO2 inhalation. The brain was harvested, the cerebellum was dissected, and the cerebrum was placed in a brain mold, cut into 6 slices, and stained with TTC. A ruler was attached next to each slice, and images were taken using a digital camera. Finally, the infarct area and volume of each brain slice were calculated using image analysis software.

[0187] Figure 4 shows the effect of the test substance on the mNSS score of rats with ischemia-reperfusion neurobehavioral response. Figure 5 shows the effect of the test substance on the infarct volume in rats with ischemia-reperfusion. The experimental results show that the compound of formula (I) has a significant protective effect on the cranial nerves of rats with ischemic stroke (tMCAO) and has a dose-response effect.

[0188] Test Example 3: Effect of Compound (I) on Diabetic Macular Edema

[0189] The effect of compound (I) on diabetic macular edema was determined using a CA-1-induced rat DME model. Following administration of the test substance, IR+OCT scans were performed on CA-1-induced DME in rats (6-8 week old male SD rats from Beijing Vital River Laboratory Animal Technology Co., Ltd.), and the thickness of the entire retina was measured at specific points. The modeling point was recorded as 0h. Before modeling, animals were anesthetized with salbutamol (25-50 mg / kg, ip) and xylazine hydrochloride (5 mg / kg, ip). The modeling control group, control compound group, and test substance group were induced by bilateral IVT injection of CA-1. Animals in the blank control group received no treatment. Animals in the control compound group and test substance group were orally administered the drug once daily (-4h±0.5h, 20h±0.5h, 44h±0.5h, 68h±0.5h), for a total of four times. At 48h and 72h after modeling, IR+OCT scans were performed on the enrolled animals, and the thickness of the entire retina was measured at specific points. Animals were anesthetized with Supraxediol 50 (25-50 mg / kg, ip) and xylazine hydrochloride injection (5 mg / kg, ip) before the experiment. All animals were euthanized by exsanguination while under anesthesia after the experiment. Experimental data are expressed as mean ± standard error (Mean ± SEM). Data were analyzed using Graphpad Prism or SPSS with appropriate statistical methods. P < 0.05 was considered statistically significant.

[0190] Figure 6 shows the effect of the test substance on CA-1-induced retinal thickness in rats. The experimental results show that the compound of formula (I) has a significant alleviating effect on CA-1-induced DME in rat retina, and has a dose-dependent effect.

Claims

1. A method for preparing a compound represented by formula (I), It includes step a): the compound of formula (III) and the hydroxylamine salt are reacted in a solvent in the presence of a base to prepare the compound of formula (I).

2. The preparation method according to claim 1, wherein, The solvent is selected from ethanol, methanol, or isopropanol; preferably, the solvent is selected from ethanol. and / or The hydroxylamine salt is selected from hydroxylamine hydrochloride and hydroxylamine sulfate; preferably, the hydroxylamine salt is selected from hydroxylamine hydrochloride. and / or The base is selected from triethylamine and N,N-diisopropylethylamine; preferably, the base is selected from triethylamine. and / or The molar ratio of the hydroxylamine salt to the compound of formula (III) is 3:1 to 5:1; preferably, the molar ratio of the hydroxylamine salt to the compound of formula (III) is 3:

1. and / or The reaction temperature is selected from 50℃ to 70℃; preferably, the reaction temperature is selected from 60℃ to 65℃.

3. The preparation method according to claim 1 or 2, wherein, Step b) precedes step a): Compound (IV) and compound (V) are reacted in a solvent in the presence of a condensing agent and a base to prepare compound (III).

4. The preparation method according to claim 3, wherein, The solvent is selected from DMF, DME, and dichloromethane; preferably, the solvent is selected from DMF. and / or The base is selected from triethylamine and N,N-diisopropylethylamine; preferably, the base is selected from N,N-diisopropylethylamine. and / or The condensing agent is selected from EDCI / HOBT, HATU, and CDI; preferably, the condensing agent is selected from EDCI / HOBT. and / or The reaction temperature is room temperature, preferably 20-30℃.

5. The preparation method according to any one of claims 1 to 4, wherein, Steps c1) and c2) precede step b): Step c1): Compound (VI) and compound (VII) react in a solvent in the presence of an acid to give the imine intermediate compound (VIII); Step c2): Compound (V) is prepared by Wolff-Kishner reduction reaction in the presence of a base; 6. The preparation method according to claim 5, wherein, The solvent is selected from ethanol, ethylene glycol, and diethylene glycol; preferably, the solvent is selected from ethylene glycol. and / or The acid is selected from acetic acid, sulfuric acid, and p-toluenesulfonic acid; preferably, the acid is selected from acetic acid. and / or The molar ratio of the acid to the compound of formula (VI) is 1:10; and / or The molar ratio of compound (VI) to compound (VII) is 1:1 to 1:3; preferably, the molar ratio of compound (VI) to compound (VII) is 1:

2. and / or The reaction temperature in step c1) is selected from 100℃ to 120℃; preferably, the reaction temperature is selected from 100℃ to 110℃. and / or The alkali mentioned in step c2) is selected from sodium hydroxide and potassium hydroxide; preferably, the alkali is selected from potassium hydroxide. The reaction temperature in step c2) is selected from 130–140 °C; and / or Steps c1 and c2) are completed by a one-pot method, wherein the molar ratio of compound (VI) to base is 1:5 to 1:8, preferably 1:

6.

7. The preparation method according to any one of claims 1 to 6, wherein, Step c1) precedes step d): Compound (IX) is prepared by hydrolysis in a solvent in the presence of a base to obtain compound (VI).

8. The preparation method according to claim 7, wherein, The solvent is selected from ethanol and methanol; preferably, the solvent is selected from methanol. and / or The alkali is selected from sodium hydroxide and potassium hydroxide; preferably, the alkali is selected from sodium hydroxide. and / or The molar ratio of the compound of formula (IX) to the base is 1:2 to 1:3, preferably 1:2; and / or The reaction temperature is 60℃.

9. The preparation method according to any one of claims 1 to 8, wherein, Step e) precedes step d): Compound (X) and compound (XI) are reacted in a solvent in the presence of a base to prepare compound (IX) via hydrolysis.

10. The preparation method according to claim 9, wherein, The solvent is selected from DMF, DMSO, and DMA; preferably, the solvent is selected from DMF. and / or The molar ratio of the compound of formula (X) to the compound of formula (XI) is 1:1 to 1:1.5; preferably, the molar ratio of the compound of formula (X) to the compound of formula (XI) is 1:1.

3. and / or The alkali is selected from sodium carbonate, potassium carbonate, and cesium carbonate; preferably, the alkali is selected from potassium carbonate. and / or The molar ratio of the compound of formula (X) to the base is 1:2 to 1:4, preferably 1:3; and / or The reaction temperature is room temperature, preferably 20-30℃.

11. The preparation method according to any one of claims 1 to 10, wherein, Step f) precedes step e): Compound (XII) is prepared in a solvent in the presence of NBS or AIBN via a free radical bromination reaction to obtain compound (X).

12. The preparation method according to claim 11, wherein, The solvent is selected from carbon tetrachloride, 1,2-dichloroethane, and chlorobenzene; preferably, the solvent is selected from chlorobenzene. and / or The molar ratio of the compound of formula (XII) to NBS is 1:1 to 1:1.5; preferably, the molar ratio of the compound of formula (XII) to NBS is 1:1.

4. and / or The molar ratio of the compound of formula (XII) to AIBN is 1:0.05 to 1:0.2; preferably, the molar ratio of the compound of formula (XII) to AIBN is 1:0.

1. and / or The reaction temperature is selected from 70℃ to 90℃; preferably, the reaction temperature is selected from 70℃ to 75℃. and / or In the post-reaction treatment, dibromo byproducts are removed by adding DIEA and diethyl phosphite. The molar ratio of the compound of formula (XII) to DIEA and diethyl phosphite is 1:1.2:1.1 to 1:1.6:1.

4. Preferably, the molar ratio of the compound of formula (XII) to DIEA and diethyl phosphite is 1:1.5:1.

3.

13. The preparation method according to any one of claims 1 to 12, wherein, Step g) precedes step f): Compound (XIII) is prepared in toluene in the presence of aluminum trichloride via a Friedel-Crafts reaction to give compound (XII).

14. The preparation method according to claim 13, wherein, The molar ratio of the compound of formula (XIII) to aluminum trichloride is 1:1 to 1:1.2; preferably, the molar ratio of the compound of formula (XIII) to aluminum trichloride is 1:

1. and / or The reaction temperature for adding aluminum trichloride is selected from -10℃ to 10℃, preferably 0℃ to 5℃; after the addition is completed, the reaction temperature is controlled at 20℃ to 30℃.

15. The preparation method according to any one of claims 1 to 14, comprising the following steps: Step g): Compound (XII) was prepared by Friedel-Crafts reaction in toluene in the presence of aluminum trichloride, yielding compound (XIII); Step f): Compound (X) is prepared by radical bromination in a solvent in the presence of NBS or AIBN to obtain compound (X); Step e): Compound (X) and compound (XI) are prepared by hydrolysis in a solvent in the presence of a base to obtain compound (IX); Step d): Compound (IX) is prepared by hydrolysis in a solvent in the presence of a base to obtain compound (VI); Step c): Compound (VI) and compound (VII) are reacted in a solvent in the presence of an acid to give imine intermediate compound (VIII). Compound (VIII) is not separated and is prepared by Wolff-Kishner reduction reaction in the presence of a base to give compound (V). Step b): Compound (V) and compound (IV) are reacted in a solvent in the presence of a condensing agent and a base to prepare compound (III); Step a): Compound (I) is prepared by addition reaction of compound (III) with hydroxylamine salt in a solvent in the presence of a base.

16. Use of the compound of formula (I) prepared by any one of claims 1 to 15 in the preparation of a medicament for the prevention and / or treatment of post-stroke cerebral edema, hereditary angioedema (HAE) or diabetic macular edema (DME).

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