CN116789644A - Amide compound and preparation method and application thereof - Google Patents

Abstract

The invention provides an amide compound described in formula I and its preparation method and use. The amide compound of the present invention has Nav1.8 selective inhibitory activity and can be used as a Nav1.8 selective inhibitor. It has better activity, higher selectivity and fewer side effects, and can be used for treatment, prevention or control of Nav1. 8 channel involvement or dysfunction-related diseases have important clinical application value.

Description

Amide compounds and preparation methods and uses thereof

Technical Field

The present invention relates to the technical field of inhibitor synthesis, and in particular to an amide compound and a preparation method thereof, and use thereof in treating diseases related to pain at a Nav1.8 target point.

Background Art

Pain, as a protective mechanism, can alert and protect tissues from further damage. Pain is mainly caused by nociceptors converting the stimulation into nerve impulses (action potentials) and transmitting them to the nerve center via afferent nerve fibers, causing pain. The generation and conduction of action potentials in neurons depends on voltage-gated sodium channels (Nav) on the cell membrane.

Voltage-gated sodium channels mediate the selective transmembrane flow of sodium ions and play a key role in mediating the initiation, conduction, and transmission of action potentials in excitable cells such as neurons (Catterall et al., Pharmacol Rev. 2005, 57(4): 397-409.). Nav channels are important drug targets, and Nav channel inhibitors are used to treat diseases such as pain, arrhythmia, epilepsy, anesthesia, and itching (Black et al., Neuron. 2013, 80(2): 280-91; Catterall et al., Annu Rev Pharmacol Toxicol. 2014, 54: 317-38; Bennett et al., Physiol Rev. 2019, 99(2): 1079-1151.). Currently, 9 channel subtypes Nav1.1 to Nav1.9 channels have been found in mammalian genomes. According to the homology of amino acid sequences, the similarity of Nav channel proteins is between 45% and 87%. According to their different sensitivities to tetrodotoxin (TTX), they can be divided into TTX-resistant channels (TTX-R) and TTX-sensitive channels (TTX-S). Among them, Nav1.5, Nav1.8 and Nav1.9 channels belong to TTX-R type sodium channels, and other subtypes belong to TTX-S subtypes (Catterall et al., Pharmacol Rev. 2005, 57(4):397-409.).

Voltage-gated Nav1.8 channel subtypes (TTX-R type) are mainly distributed in the peripheral nervous system, such as 75% of dorsal root neurons expressing Nav1.8 channels. Because Nav1.8 channels have higher activation and inactivation voltages, they become the main component of the action potential ascending branch (other Nav channel subtypes are already in a non-functional inactivated state) (Goodwin et al., Nat Rev Neurosci. 2021, 22 (5): 263-274.). Due to the characteristics of slow inactivation and fast resurrection of Nav1.8 channels, they are involved in the physiological and pathological processes of membrane potential depolarization and high-frequency discharge of neurons such as pain (Alsaloum et al., Nat Rev Neurol. 2020, 16 (12): 689-705.). Human genetic studies have shown that mutations in the Nav1.8 gene cause small fiber neuralgia and erythropoietic pain (Faber et al., Proc Natl Acad Sci U S A. 2012, 109(47): 19444-9; Kaluza et al., Pflugers Arch. 2018, 470(12): 1787-1801.). In rodents, gene knockout or knockdown of the Nav1.8 channel gene can relieve a variety of inflammatory pain and neuralgia; and administration of Nav1.8 channel inhibitors such as A-803467 can effectively relieve pain responses (Jarvis et al., Proc Natl Acad Sci U S A. 2007, 104(20): 8520-5.). Diabetic neuropathy is one of the most common neuropathic pain diseases. About 60% to 70% of diabetic patients suffer from this disease, and more than 70% of patients do not receive effective treatment (Jensen et al., Brain. 2021, 144(6): 1632-1645.). Methylglyoxal in patients with diabetic neuropathy directly enhances the function of Nav1.8 channels, and gene knockout or knockdown of Nav1.8 channels can effectively relieve neuropathy (Bierhaus et al., Nat Med. 2012, 18(6): 926-33.). In the STZ-induced diabetic neuropathy rat model, intraperitoneal or plantar administration of Nav1.8 channel inhibitor A-803467 can relieve the pain behavioral response of animals in a dose-dependent manner (Mert et al., J Am Assoc Lab Anim Sci. 2012, 51(5): 579-85.).

In addition to pain, Nav1.8 channels are also associated with multiple sclerosis, arrhythmias, cough, pruritus and epilepsy. Multiple sclerosis (MS) is an inflammatory demyelinating disease originating in the central nervous system, and its exact pathogenesis remains to be elucidated. Normal Purkinje fibers in the cerebellum do not express Nav1.8 channels, while the expression of Nav1.8 in the cerebellum of patients with multiple sclerosis is upregulated, and the expression of the channel increases in a dependent manner with the progression of the disease. The single nucleotide polymorphism (SNP) of the Nav1.8 encoding gene is also associated with the severity of MS (Craner et al., J Neuropathol Exp Neurol. 2003, 62(9): 968-75; Roostaei et al., Neurology. 2016, 86(5): 410-7.). The genetic mice (L7-1.8TG) with knock-in (overexpression) of Nav1.8 in the cerebellar Purkinje fibers showed multiple sclerosis behavior, and the administration of the Nav1.8 selective inhibitor PF-01247324 alleviated the MS behavior of L7-1.8TG transgenic mice (Shields et al., Ann Neurol. 2012, 71(2): 186-94; Shields et al., PLoS One. 2015, 10(3): e0119067.). Osteoarthritis is a degenerative bone and joint disease, and cartilage wear and pain are its main characteristics. Phosphorylated cAMP response element binding protein (CREB) directly binds to the promoter of the Nav1.8 encoding gene, promotes the transcription of Nav1.8 protein, and upregulates the expression level of Nav1.8 channel (Zhu et al., Elife. 2020, 9: e57656.). In the cardiovascular system, Nav1.8 channels have been shown to be expressed in cardiac nerves such as Purkinje fibers, and some studies have also shown that Nav1.8 is also expressed in cardiomyocytes (Verkerk et al., Circ Res. 2012, 111(3): 333-43.). Human genetic studies have found that Nav1.8 gene mutations are associated with Brugada syndrome (Hu et al., J Am Coll Cardiol. 2014, 64(1): 66-79.). Inhibition of Nav1.8 channels can improve cardiac remodeling, and Nav1.8 channels are considered to be potential therapeutic targets for cardiovascular diseases such as arrhythmias, atrial fibrillation, and heart failure (Dybkova et al., Cardiovasc Res. 2018, 114(13): 1728-1737.). Nav1.8 channels are expressed in the vagal plexus associated with coughing. The phosphorylation level and expression of Nav1.8 increase during pathological coughing, and participate in the cough reflex (Muroi et al., Lung. 2014, 192 (1): 15-20.). In mammalian itch perception, itch factors such as histamine released by lymphocytes and mast cells can activate Nav1.8 channels, and knocking out Nav1.8 in mice can effectively alleviate the itch behavior induced by histamine and endothelin (Riol-Blanco et al., 2014, 510 (7503): 157-61.). In addition, congenital mutations in human Nav1.8 have been reported to cause epilepsy and convulsions (Kambouris et al., Ann Clin Transl Neurol. 2016, 4 (1): 26-35.).

At present, the Nav1.8 selective inhibitor is VERTEX's VX-150, which has completed Phase II clinical trials in patients with osteoarthritis, acute pain, and pain caused by small fiber neuropathy, and has obtained positive results. The Nav1.8 selective inhibitor that has entered clinical trials in China is Hengrui's HRS-4800, which is currently undergoing Phase I clinical trials; many other selective inhibitors are in the preclinical research and development stage; therefore, the development of Nav1.8 inhibitors with better activity, higher selectivity, and fewer side effects has important clinical applications and innovative drug value.

In view of this, the present invention is proposed.

Summary of the invention

One of the purposes of the present invention is to provide an amide compound having Nav1.8 selective inhibitory activity.

A second object of the present invention is to provide a method for preparing amide compounds.

The third object of the present invention is to provide a pharmaceutical composition comprising the amide compound.

The fourth object of the present invention is to provide a use of the amide compound or pharmaceutical composition in the preparation of Nav1.8 inhibitors or drugs for treating, preventing or controlling diseases or symptoms associated with the Nav1.8 channel.

In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

In one aspect, the present invention provides a compound of formula I, its isomers, racemates, prodrugs or pharmaceutically acceptable salts thereof,

in:

X is selected from N or CH; Y is selected from N or CR ₃ ;

V and G are each independently selected from N or CH, Q and T are each independently selected from N or C;

A, W and Z are each independently selected from O, S, N, carbonyl, sulfoxide, sulfone, -NR _a -, -CR _b -, -NR _a -CO-, -CR _b =N-, -CR _b -NR _a -, and at least one of A, W and Z contains nitrogen;

R _a is independently selected at each occurrence from hydrogen, amino, hydroxy, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, C1-C6 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O, S, C6-C12 aryl or 5-10 membered heteroaryl containing 1 to 4 heteroatoms selected from N, O, S;

R _b is independently selected at each occurrence from hydrogen, halogen, nitro, amino, cyano, hydroxy, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, C1-C6 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O, S, C6-C12 aryl or 5-10 membered heteroaryl containing 1 to 4 heteroatoms selected from N, O, S;

n is selected from 0, 1 or 2; in particular 2;

R ₁ is selected from hydrogen, hydroxy, halogen, cyano, nitro or amino; preferably hydrogen, fluorine, chlorine, bromine, amino or hydroxy;

R ₂ is selected from halogen, hydroxy, cyano, nitro or halogenated C1-C6 alkyl; preferably chlorine, bromine, iodine, trifluoromethyl;

_R3 is selected from halogen, hydroxy, cyano, amino, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, halo-C2-C6 alkenyl, C2-C6 alkenyloxy, halo-C2-C6 alkenyloxy, C2-C6 alkynyl, halo-C1-C6 alkynyl, C2-C6 alkynyloxy, halo-C1-C6 alkynyloxy, C1-C6 alkoxy, halo-C1-C6 alkoxy, C1-C6 alkylamino, halo-C1-C6 alkylamino, C3-C6 cycloalkylamino, C1-C6 alkoxyamino, C3-C6 cycloalkyl, C3-C6 cycloalkyloxy or a 3-8 membered heterocyclic group containing 1 to 4 heteroatoms selected from N, O, and S; preferably fluorine, chlorine, bromine, amino, hydroxyl, methyl, cyclopropyl, methoxy, trifluoromethyl, and trifluoromethoxy;

R ₆ and R ₇ are each independently selected from hydrogen, fluorine, chlorine, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxy; preferably each independently hydrogen, fluorine, chlorine, methyl, ethyl or isopropyl;

R ₅ , R ₈ and R ₉ are each independently selected from hydrogen, fluorine, chlorine, halogenated C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxy, C3-C8 cycloalkyl; preferably each independently hydrogen, methyl, ethyl, isopropyl or cyclopropyl;

Indicates a single bond or a double bond.

In some embodiments, the compound of formula I is selected from the following compounds of formula II:

wherein R ₃ , A, Z, W, Q, T, V and G are as defined above.

In some preferred embodiments, is one of the following groups:

In some preferred embodiments, the compound of formula I is selected from the following compounds:

The definitions of terms in this invention are as follows:

"Halogen" may be fluorine, chlorine, bromine or iodine.

"C1-C6" alkyl refers to a chain alkyl group having 1 to 6 carbon atoms; specific examples thereof may include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like; "haloalkyl" refers to a group in which at least one hydrogen of the alkyl group as described above is replaced by a halogen; specific examples thereof include trifluoromethyl and the like.

"C2-C6 alkenyl" refers to a straight or branched group containing 2 to 6 carbon atoms and at least one carbon-carbon double bond; specific examples thereof may include ethenyl, propenyl, 2-propenyl, (E)-2-butenyl, (Z)-2-butenyl, (E)-2-methyl-2-butenyl, (Z)-2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, (Z)-2-pentenyl, (E)-1-pentenyl, (E)-2-pentenyl, (Z)-2-hexenyl, (E)-1-hexenyl, (Z)-1-hexenyl, (E)-2-hexenyl, (Z)-3-hexenyl, (E)-3-hexenyl, (E)-1,3-hexadienyl, 4-methyl-3-pentenyl or norbornene.

"C2-C6 alkynyl" refers to a straight or branched group containing 2-6 carbon atoms and at least one carbon-carbon double bond; specific examples may include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, and 3-hexynyl.

"C1-C6 alkoxy" refers to a RO-group, wherein R is a C1-C6 alkyl group as described above; specific examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, isopentoxy, neopentoxy, n-hexoxy, isohexoxy, 3-methylpentoxy, 3,3-dimethylbutoxy, 2-ethylbutoxy, and the like. "Haloalkoxy" refers to a group in which at least one hydrogen of the alkoxy group as described above is replaced by a halogen; specific examples thereof include trifluoromethoxy, and the like.

"C2-C6 alkenyloxy" refers to a RO-group, wherein R is a C2-C6 alkenyl group as described above; specific examples of alkenyloxy include ethyleneoxy, propyleneoxy.

"C2-C6 alkynyloxy" refers to a RO- group, wherein R is a C2-C6 alkynyl group as described above; specific examples of alkynyloxy include ethynyloxy and propynyloxy.

"Amino" refers to _-NH2 .

"C1- _C6 alkylamino" refers to a group obtained by replacing one or two hydrogen atoms of _-NH2 with a C1-C6 alkyl group as described above, and can be represented by _R1R2N- , wherein _R1 and _R2 are each independently H or a C1-C6 alkyl group, and at most one of _R1 and _R2 is H. Specific examples of C1-C6 alkylamino include methylamino, dimethylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, tert-butylamino, sec-butylamino, n-pentylamino, isopentylamino, neopentylamino, n-hexylamino, isohexylamino, 3-methylpentylamino, 3,3-dimethylbutylamino, 2-ethylbutylamino, and the like.

“C1-C6 alkoxyamino” refers to a group obtained by replacing the two hydrogen atoms of -NH ₂ with a C1-C6 alkyl group and oxygen as described above, respectively; specific examples of the C1-C6 alkoxyamino group include methoxyamino, dimethoxyamino, ethoxyamino, n-propoxyamino and isopropoxyamino.

"C3-C8 cycloalkyl" refers to a fully saturated cyclic hydrocarbon compound group containing 3 to 8 carbon atoms, and specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

"C3-C6 cycloalkylamino" refers to a group obtained by replacing one or two hydrogen atoms of _-NH2 with a C3-C6 _{cycloalkyl group as described above, and can be represented by R1R2N- ,} wherein _R1 and _R2 are each independently H or a C3-C6 cycloalkyl group, and at most one of _R1 and _R2 is H. Specific examples of C3-C6 cycloalkylamino include cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, and the like.

The "3- to 8-membered heterocyclic group" refers to a 3- to 8-membered non-aromatic cyclic alkyl group containing at least one heteroatom selected from nitrogen, oxygen and sulfur in the ring; specific examples thereof include piperazine, piperidine, morpholine and the like.

The "C6-C12 aryl group" refers to a monocyclic or polycyclic aromatic group having 6 to 12 carbon atoms; specific examples thereof include phenyl and naphthyl.

"5-10 membered heteroaryl" refers to a 5-10 membered aromatic group containing at least one heteroatom selected from nitrogen, oxygen and sulfur in the ring; specific examples include pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyridazine-3-yl, pyridazine-4-yl, pyrimidine-2-yl, pyrimidine-4-yl, pyrimidine-5-yl, pyrazine-2-yl, pyrazine-3-yl, indolyl, isoindolyl and the like.

"Pharmaceutically acceptable salts" include salts formed between the compound of formula I and an acid or a base; the acid includes an inorganic acid and an organic acid; preferably, the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and carbonic acid; preferably, the organic acid includes formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, citric acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, glutamic acid, and pamoic acid; the base includes hydroxides, carbonates, bicarbonates, and the like of sodium, potassium, calcium, aluminum, lithium, and ammonium.

The compounds and pharmaceutically acceptable salts thereof involved in the present application may have isomers or racemates, such as optical isomers (including diastereomers and enantiomers), atropisomers, geometric isomers (cis-trans isomers), conformational isomers, tautomers and mixtures thereof, but are not limited thereto. These isomers are also included in the scope defined by the claims of the present invention.

Another aspect of the present invention provides a method for preparing the compound of formula I, which is achieved by the following reaction route:

Formula III and Formula IV are subjected to acylation reaction in the presence of a base to obtain Formula I,

Preferably, the base is selected from pyridine, sodium carbonate, sodium bicarbonate.

In another aspect, the present invention provides a pharmaceutical composition comprising one or more selected from the group consisting of a compound of formula I and its isomers, racemates, pharmaceutically acceptable salts and prodrugs, and optional pharmaceutically acceptable excipients.

In another aspect, the present invention provides the use of the compound of formula I or its isomers, racemates, pharmaceutically acceptable salts, prodrugs in the preparation of Nav1.8 inhibitors or drugs for treating, preventing or controlling diseases or symptoms associated with the Nav1.8 channel.

In another aspect, the present invention provides a method for treating, preventing or controlling diseases or symptoms associated with Nav1.8, the method comprising administering to a subject in need thereof one or more of a compound of formula I, its isomers, racemates, pharmaceutically acceptable salts and prodrugs, or the above-mentioned pharmaceutical composition.

The diseases or symptoms associated with the Nav1.8 channel include but are not limited to nociceptive pain, inflammatory pain, neuropathic pain, functional pain, pain associated with muscle or bone injury, pelvic pain, abdominal pain, chest pain, lumbosacral neuralgia, preoperative pain, intraoperative pain, postoperative pain, acute or chronic pain, migraine, trigeminal neuralgia, pancreatitis, renal colic, cancer pain, pain caused by chemotherapy or drug therapy, diabetic neuropathy, postherpetic neuralgia, back pain, phantom limb pain, sciatica, small fiber neuralgia, erythromelalgia, arthritis, pruritus, acute or chronic pruritus, asthma, multiple sclerosis, arrhythmia, atrial fibrillation, heart failure, Brugada syndrome, kidney stones, epilepsy, and convulsions.

The present invention has the following beneficial effects:

The amide compounds of the structure of the present invention have Nav1.8 selective inhibitory activity and can be used as Nav1.8 selective inhibitors. They have better activity, higher selectivity, and fewer side effects. They can be used to treat, prevent or control diseases related to the involvement or dysfunction of Nav1.8 channels and have important clinical application value.

The present invention has been described in detail above, but the above embodiments are only illustrative in nature and are not intended to limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed for the present invention.

Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, methods in the art.

The Chinese names of reagents represented by chemical formulas or English letter abbreviations are as follows: ℃ represents degrees Celsius; g represents gram; s represents singlet, d represents doublet, t represents triplet, m represents multiplet; min represents minute; ml represents milliliter; mmol represents millimole; h represents hour; TLC represents thin layer chromatography.

In the following examples, the H NMR spectra were recorded using a Bruker AMX-400 or AMX-600 NMR instrument, and the unit of chemical shift δ is ppm.

Unless otherwise specified, all reaction solvents were purified by conventional methods.

Thin layer chromatography used GF254 high-efficiency plates, produced by Yantai Chemical Research Institute.

Unless otherwise specified, all solvents were of analytical grade and all reagents used were purchased from Sinopharm Chemical Reagent Co., Ltd.

The color was developed by using 2,4-dinitrophenylhydrazine, iodine, and ultraviolet fluorescence.

The organic solvent was evaporated under reduced pressure in a rotary evaporator.

Example 1 Synthesis of Compound 1

[Reaction Scheme 1]

(1) Synthesis of intermediate 1-2

Compound 1-1 (12.2 g, 57.2 mmol) was dissolved in anhydrous dichloromethane (300 ml), placed in an ice bath, and bis(2-methoxyethyl)aminosulfur trifluoride (BAST) was slowly added dropwise under nitrogen protection. After the addition was completed, the mixture was moved to room temperature for reaction for 24 h. TLC detection showed that the reaction was basically complete. Saturated sodium bicarbonate solution was slowly added dropwise under ice bath to quench, and a large amount of gas was generated. The mixture was extracted with dichloromethane (DCM) and water, and the organic layer was dried over anhydrous sodium sulfate, and spin-dried for column chromatography separation (petroleum ether (PE)/ethyl acetate (EA) = 5/1) to obtain intermediate 1-2 (10 g, 74.3%). ¹ H NMR (400MHz, CDCl ₃ ) δ3.51–3.38(m,3H),3.37–3.30(m,1H),2.22–1.95(m,4H),1.88–1.75(m,2H),1.46(s,9H).

(2) Synthesis of intermediate 1-3

Intermediate 1-2 (10 g, 42.5 mmol) was placed in a 250 ml round-bottom flask, 4N hydrochloric acid dioxane solution (42.5 ml, 170 mmol) was added under ice bath condition, and the reaction was carried out at room temperature for about 4 hours. TLC detected that the reaction was complete, and the reaction liquid was directly spin-dried and dried to obtain intermediate 1-3 (7.3 g, 100%). ¹ H NMR (400 MHz, DMSO) δ9.47 (s, 2H), 3.15 (m, 4H), 2.49–2.37 (m, 2H), 2.30–2.13 (m, 2H), 1.83 (m, 2H).

[Reaction Scheme 2]

(3) Synthesis of Intermediates 1-5

Compound 1-4 (0.9 g, 6.76 mmol) was dissolved in 10 ml of concentrated sulfuric acid, moved to an ice bath, potassium nitrate (0.68 g, 6.76 mmol) was added, and the reaction was carried out for 3 h under ice bath conditions. The reaction was basically complete when detected by TLC. The reaction solution was slowly added to ice water, and a large amount of white solid precipitated. The filter cake was filtered and dried to obtain intermediate 1-5 (1.1 g, 91.3%). ¹ H NMR (400 MHz, DMSO) δ8.99 (s, 1H), 8.44 (dd, J = 8.3, 2.2 Hz, 1H), 8.33 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 4.54 (s, 2H).

(4) Synthesis of Intermediates 1-6

Dissolve the intermediate 1-5 (1.1 g, 6.17 mmol) in 100 ml methanol, add 60% palladium carbon (0.22 g), replace hydrogen three times, heat to 40 ° C and continue to react for 7 hours. After TLC detection, the reaction is complete, filter the reaction solution, and spin dry the filtrate to obtain the intermediate 1-6 (0.7 g, 76.6%). ¹ H NMR (400 MHz, DMSO) δ8.31 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.86-6.68 (m, 2H), 5.28 (s, 2H), 4.16 (s, 2H).

[Reaction Scheme 3]

(5) Synthesis of Intermediate 1-8

The intermediate 1-3 (2.38 g, 12.8 mmol) was dissolved in 40 ml of N,N-dimethylformamide (DMF), potassium carbonate (5.5 g, 38.5 mmol) was added, and then compound 1-7 (2.0 g, 11.7 mmol) was added, and the mixture was heated to 120°C and reacted overnight. The reaction was basically complete according to TLC detection. The mixture was extracted with EA and water, and the organic layer was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried for column chromatography (PE/EA=5/1) to obtain the intermediate 1-8 (2.0 g, 54.9%). ¹ H NMR(400MHz,CDCl ₃ )δ7.80(d,J＝7.8Hz,1H),6.51(d,J＝7.8Hz,1H),3.85(s,3H),3.75–3.68(m,2H),3.28(m,2H),2.44–2.31(m,5H),2.01–1.89(m,4H).

(6) Synthesis of Intermediate 1-9

The intermediate 1-8 (2.0 g, 7.04 mmol) was dissolved in DMAC (N, N-dimethylacetamide) (40 ml), and NCS (N-chlorosuccinimide) (1.8 g, 14.1 mmol) was added, and heated to 100°C. After 1 hour, the reaction was detected to be complete, and extracted with EA and water. The organic layer was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried for column chromatography (PE/EA=10/1) to obtain intermediate 1-9 (1.5 g, 66.8%). ¹ H NMR (400MHz, CDCl ₃ ) δ7.84 (s, 1H), 3.85 (s, 3H), 3.69 (dt, J = 11.9, 4.4Hz, 2H), 3.25 (t, J = 5.4Hz, 2H), 2.48 (s, 3H), 2.42–2.29 (m, 2H), 1.98–1.88 (m, 4H) .

(7) Synthesis of Intermediate 1-10

The intermediate 1-9 (1.5 g, 4.7 mmol) was dissolved in methanol (40 ml), lithium hydroxide (2.0 g, 47 mmol) was added, and 8 ml of water was added, and the mixture was heated to 50 °C for 4 h. The reaction was basically complete by TLC. The reaction solution was dried and the pH of the reaction solution was adjusted to about 3-4 with 2N hydrochloric acid. A large amount of white solid was precipitated and filtered to obtain the intermediate 1-10 (1.15 g, 80.3%). ¹ HNMR (400 MHz, DMSO) δ12.91 (s, 1H), 7.81 (s, 1H), 3.65–3.55 (m, 2H), 3.32 (m, 2H), 2.42 (s, 3H), 2.38–2.25 (m, 2H), 2.04–1.93 (m, 2H), 1.90–1.83 (m, 2H).

(8) Synthesis of Intermediate 1-11

The intermediate 1-10 (0.05 g, 0.16 mmol) was dissolved in anhydrous DCM (5 ml), the reaction solution was placed in an ice bath, 2-3 drops of DMF were added as a catalyst, oxalyl chloride (2M, 0.1 ml) was added under nitrogen protection, and the reaction was carried out in an ice bath for 2 h. The reaction solution was then dried to obtain the intermediate 1-11.

(9) Synthesis of Compound 1

Intermediate 1-6 (0.026 g, 0.18 mmol) was added to intermediate 1-11, and placed in an ice bath, 3 ml of anhydrous pyridine was added dropwise, and the reaction was completed after 2 h by TLC detection. The mixture was extracted with EA and water, and the organic layer was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried for column chromatography (DCM/MeOH=10/1) to obtain compound 1 (0.015 g, 21.2%). ¹ H NMR (400MHz, DMSO) δ10.63(s,1H),8.60(s,1H),8.11(d,J＝1.6Hz,1H),7.85–7.74(m,2H),7.54(d,J＝8.2Hz,1H),5.76(s,1H),4.34(s,2H),3.62(d,J＝ 3.1Hz,2H),3.43(t,J＝5.8Hz,2H),2.45(s,3H),2.31(m,2H),1.94(d,J＝13.5Hz,2H),1.84(d,J＝5.5Hz,2H).

Example 2 Synthesis of Compound 2

Referring to the method of step 9 of Example 1, intermediate 1-6 was replaced with 5-amino-2,3-dihydroisoindole-1-one (purchased from Bidler) to obtain compound 2 (0.021 g, 29.7%). ¹ H NMR (400 MHz, DMSO) δ 10.72 (s, 1H), 8.44 (s, 1H), 8.03 (s, 1H), 7.76 (s, 1H), 7.68-7.55 (m, 2H), 4.36 (s, 2H), 3.65-3.56 (m, 2H), 3.44-3.37 (m, 6H), 2.45 (s, 3H), 2.30 (s, 2H), 2.01-1.89 (m, 2H), 1.86-1.77 (m, 2H).

Example 3 Synthesis of Compound 3

Referring to the method of step 9 of Example 1, intermediate 1-6 was replaced with 6-aminoindolone (purchased from Shaoyuan) to obtain compound 3 (0.020 g, 29.7%). ¹ H NMR (400 MHz, DMSO) δ 10.45–10.38 (m, 2H), 7.70 (s, 1H), 7.43 (s, 1H), 7.17–7.07 (m, 2H), 3.64–3.58 (m, 2H), 3.46–3.42 (m, 4H), 2.44 (s, 2H), 2.37–2.24 (m, 2H), 2.03–1.90 (m, 2H), 1.87–1.78 (m, 2H).

Example 4 Synthesis of Compound 4

[Reaction Scheme 4]

(1) Synthesis of intermediate 4-2

Compound 4-1 (0.6 g, 3.4 mmol) was dissolved in 50 ml of methanol, and 60% water content palladium carbon (0.12 g) was added. After replacing hydrogen three times, the reaction was continued for 7 hours at room temperature. TLC detected that the reaction was complete, and the reaction solution was filtered and the filtrate was dried to obtain intermediate 4-2 (0.5 g, 100%). ¹ H NMR (400 MHz, DMSO) δ9.19 (s, 1H), 5.83–5.56 (m, 3H), 3.90 (s, 2H), 2.57 (d, J＝2.2 Hz, 2H).

(2) Synthesis of Compound 4

Referring to the method of step 9 of Example 1, intermediate 1-6 was replaced by intermediate 4-2 to obtain compound 4 (0.025 g, 32.5%). ¹ H NMR (400 MHz, DMSO) δ 10.33 (s, 1H), 10.28 (s, 1H), 7.67 (s, 1H), 7.58 (s, 1H), 7.45-7.38 (m, 1H), 6.77 (d, J = 8.3 Hz, 1H), 3.63-3.56 (m, 2H), 3.48 (s, 2H), 3.45-3.40 (m, 2H), 2.43 (s, 3H), 2.37-2.23 (m, 2H), 2.03-1.91 (m, 2H), 1.89-1.78 (m, 2H).

Example 5 Synthesis of Compound 5

[Reaction Scheme 5]

(1) Synthesis of intermediate 5-2

Compound 5-1 (1.9 g, 9.9 mmol) was dissolved in 10 ml of anhydrous ethanol, hydrazine hydrate (85%) (1.5 g, 47.7 mmol) was added, and the mixture was heated to reflux. After 2 h, the reaction was complete by TLC. The filter cake was filtered and dried to obtain the crude intermediate 5-2 (1.1 g, 59.2%). ¹ H NMR (400 MHz, DMSO) δ12.39 (s, 1H), 11.13 (s, 1H), 8.21 (d, J = 1.7 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.78 (dd, J = 8.8, 1.9 Hz, 1H).

(2) Synthesis of intermediate 5-3

The intermediate 5-2 (1.05 g, 5.7 mmol) was dissolved in 60 ml of DCM, triethylamine (0.7 g, 7.0 mmol) was added, and then di-tert-butyl dicarbonate ((Boc) ₂ O) (1.5 g, 7.0 mmol) was added. The mixture was reacted for 3 h at room temperature. The reaction was basically complete when detected by TLC. The mixture was extracted with DCM and water, and the organic layer was washed with saturated sodium chloride (NaCl), dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), and spin-dried by column chromatography (DCM/MeOH=20/1) to obtain the intermediate 5-3 (1.0 g, 68.5%). ¹ H NMR (400 MHz, DMSO) δ8.74 (s, 1H), 8.04 (d, J=8.3 Hz, 1H), 7.96 (d, J=8.6 Hz, 1H), 1.61 (s, 9H).

(3) Synthesis of intermediate 5-4

Dissolve the intermediate 5-3 (1.0 g, 4.0 mmol) in 50 ml of methanol, add 60% palladium carbon (0.20 g), replace hydrogen 3 times, continue to react at room temperature for 7 hours, TLC detection shows that the reaction is complete, filter the reaction solution, and spin dry the filtrate to obtain the intermediate 5-4 (0.6 g, 89.6%). ¹ H NMR (400 MHz, DMSO) δ7.30 (d, J＝8.5 Hz, 1H), 7.06 (s, 1H), 6.52 (dd, J＝8.5, 1.8 Hz, 1H), 5.86 (s, 2H), 1.56 (s, 9H).

(4) Synthesis of Compound 5

Referring to step 9 of Example 1, intermediate 1-6 was replaced with intermediate 5-4 to obtain intermediate 5-5. Intermediate 5-5 was dissolved in 5 ml of DCM, 2 ml of trifluoroacetic acid (TFA) was added under ice bath condition, and the reaction was carried out at room temperature for 2 h. The reaction was complete when detected by TLC, and the mixture was dried by spin drying, extracted with EA and water, and the organic layer was washed with saturated sodium bicarbonate and sodium chloride, dried over anhydrous sodium sulfate, and spin dried by column chromatography (DCM/MeOH=10/1) to obtain compound 5 (0.05 g, 33.1%). 1. 51(s,3H),2.35(td,J＝10.6,5.5Hz,2H),2.07–1.98(m ^, 2H),1.98–1.87(m,2H),1.78–1.68(m,2H),1.47(dt,J＝14.7,7.5Hz,2H).

Example 6 Synthesis of Compound 6

[Reaction Scheme 6]

(1) Synthesis of intermediate 6-2

Compound 6-1 (0.6 g, 2.6 mmol) was dissolved in 30 ml methanol, and 60% water content palladium carbon (0.12 g) was added. After replacing hydrogen three times, the reaction was continued for 7 hours at room temperature. TLC detected that the reaction was complete, and the reaction solution was filtered and the filtrate was dried to obtain intermediate 6-2 (0.4 g, 76.7%). ¹ H NMR (400 MHz, DMSO) δ7.59 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.89 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (s, 1H).

(2) Synthesis of intermediate 6-3

The intermediate 6-2 (0.4 g, 2.0 mmol) was dissolved in 5 ml of concentrated hydrochloric acid under ice bath conditions, and zinc powder (1.1 g, 16.2 mmol) was added in batches. After the addition, the reaction was naturally warmed to room temperature. After 2 h, TLC detected that the reaction was complete. The reaction solution was slowly poured into ice water to quench, and the pH was adjusted to about 7 with 2N sodium hydroxide. It was extracted with EA and water, and the organic layer was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried for column chromatography (DCM/MeOH=10/1) to obtain intermediate 6-3 (0.10 g, 27.1%). ¹ HNMR (400MHz, DMSO) δ7.55–7.52(m,1H),7.14(d,J＝8.3Hz,1H),6.83(dd,J＝8.3,2.1Hz,1H),6.79(d,J＝1.9Hz,1H),5.60(s,2H),4.18(d,J＝4.7Hz,2H).

(3) Synthesis of Compound 6

Referring to the reaction of step 9 of Example 1, intermediate 1-6 was replaced by intermediate 6-3 to obtain compound 6 (0.048 g, 37.3%). ¹ H NMR (600 MHz, DMSO) δ 10.79 (s, 1H), 8.19 (s, 1H), 7.86 (t, J = 4.6 Hz, 1H), 7.84-7.77 (m, 2H), 7.54 (d, J = 8.4 Hz, 1H), 4.37 (d, J = 4.5 Hz, 2H), 3.61 (dd, J = 6.5, 3.6 Hz, 2H), 3.40 (t, J = 5.9 Hz, 2H), 2.45 (s, 3H), 2.31 (m, 2H), 2.01-1.92 (m, 2H), 1.84 (d, J = 5.5 Hz, 2H).

Example 7 Synthesis of Compound 7

[Reaction Scheme 7]

(1) Synthesis of Intermediate 7-2

Compound 7-1 (1.0 g, 5.6 mmol) was dissolved in 40 ml methanol, and 60% water content palladium carbon (0.2 g) was added. After replacing hydrogen three times, the reaction was continued at room temperature for 7 hours. TLC detected that the reaction was complete, and the reaction solution was filtered and the filtrate was dried to obtain intermediate 7-2 (0.7 g, 83.2%). ¹ H NMR (400 MHz, DMSO) δ11.05 (s, 1H), 6.74 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 6.35 (dd, J = 8.3, 2.0 Hz, 1H), 5.00 (s, 2H).

(2) Synthesis of Compound 7

Referring to step 9 of Example 1, intermediate 1-6 was replaced with intermediate 7-2 to obtain compound 7 (0.018 g, 28.5%). ¹ H NMR (400 MHz, MeOD) δ7.74 (d, J = 1.9 Hz, 1H), 7.68 (s, 1H), 7.34 (dd, J = 8.4, 2.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 3.73-3.66 (m, 2H), 3.48 (t, J = 5.9 Hz, 2H), 2.48 (s, 3H), 2.39-2.26 (m, 2H), 1.99-1.86 (m, 4H).

Example 8 Synthesis of Compound 8

[Reaction Scheme 8]

(1) Synthesis of intermediate 8-2

Compound 8-1 (2.0 g, 9.9 mmol) was dissolved in 20 ml of anhydrous ethanol, hydrazine hydrate (85%) (2.92 g, 49.6 mmol) was added, and the mixture was heated to reflux. After 3 h, the reaction was complete as determined by TLC. The filter cake was filtered and dried to obtain the crude product 8-2 (2.0 g, 102.24%). ¹ H NMR (400 MHz, DMSO) δ12.45 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.11 (dd, J = 9.2, 2.2 Hz, 1H), 7.45 (d, J = 9.2 Hz, 1H).

(2) Synthesis of intermediate 8-3

Dissolve the crude product 8-2 (2.0 g, 10.1 mmol) in 30 ml of water, add 3 ml of concentrated hydrochloric acid, heat to 90 ° C, react overnight, TLC detection shows that the reaction is almost complete, filter to obtain a filter cake, and dry the filter cake to obtain a crude intermediate 8-3 (1.67 g, 91.9%). ¹ H NMR (400 MHz, DMSO) δ 12.43 (s, 1H), 8.67 (d, J = 1.9 Hz, 1H), 8.12 (dd, J = 9.2, 2.2 Hz, 1H), 7.45 (d, J = 9.2 Hz, 1H).

(3) Synthesis of intermediate 8-4

The intermediate 8-3 (1.5 g, 8.4 mmol) was dissolved in 50 ml of DCM, triethylamine (2.5 g, 25.1 mmol) was added, and then (Boc) ₂ O (2 g, 9.2 mmol) was added. The reaction was allowed to proceed for 3 h at room temperature. The reaction was basically complete when detected by TLC. The product was extracted with DCM and water, and the organic layer was washed with saturated NaCl, dried with anhydrous Na ₂ SO ₄ , and spin-dried by column chromatography (DCM/MeOH=20/1) to obtain the intermediate 8-4 (1.5 g, 64.1%). ¹ H NMR (400 MHz, DMSO) δ11.66 (s, 1H), 7.64 (s, 1H), 6.88 (d, J=8.7 Hz, 1H), 6.73 (s, 1H), 1.57 (s, 9H).

(4) Synthesis of Intermediate 8-5

Dissolve the intermediate 8-4 (1.5 g, 5.4 mmol) in 50 ml of methanol, add 60% palladium carbon (0.30 g), replace hydrogen 3 times, continue to react at room temperature for 7 hours, TLC detection shows that the reaction is complete, filter the reaction solution, and spin dry the filtrate to obtain the intermediate 8-5 (1.2 g, 89.6%). ¹ H NMR (400 MHz, DMSO) δ7.64 (d, J = 7.9 Hz, 1H), 6.88 (dd, J = 8.8, 2.2 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 5.18 (s, 2H), 1.56 (s, 9H).

(5) Synthesis of Compound 8

Referring to step 9 of Example 1, intermediate 1-6 was replaced with intermediate 8-5 to obtain intermediate 8-6 (0.091 g, 34.6%); intermediate 8-6 was dissolved in DCM (5 ml), 2 ml of TFA was added under ice bath conditions, and the reaction was carried out at room temperature for 2 h. The reaction was completed by TLC detection, and the mixture was spin-dried. The mixture was extracted with EA and water, and the organic layer was washed with saturated sodium bicarbonate and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The mixture was spin-dried and column chromatography (DCM/MeOH=10/1) was performed to obtain compound 8 (0.05 g, 25.5%). ¹ H NMR (400MHz, DMSO) δ11.28(s,1H),10.56(s,1H),10.37(s,1H),8.08(s,1H),7.72(s,1H),7.46(m,1H),7.26(d,J＝8.9Hz,1H),3.62(m,2H),3.46(t,J =5.8Hz,2H),2.44(s,3H),2.30(m,2H),1.96(m,2H),1.84(m,2H).

Example 9 Synthesis of Compound 9

Referring to step 9 of Example 1, intermediate 1-6 was replaced by intermediate 6-2 to obtain compound 9 (9 mg, 19.8%). ¹ H NMR (400 MHz, DMSO) δ 7.92 (s, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.20 (s, 1H), 7.08 (d, J = 1.9 Hz, 1H), 7.00 (dd, J = 8.7, 1.9 Hz, 1H), 3.60 (m, 2H), 3.20 (m, 2H), 2.46 (s, 3H), 2.29 (m, 2H), 1.90 (m, 4H).

Example 10 Synthesis of Compound 10

Referring to step 9 of Example 1, intermediate 1-6 was replaced with 4-aminophthalic acid hydrazide (purchased from Leyan) to obtain compound 10 (8.0 mg, 22.5%). ¹ H NMR (400 MHz, MeOD) δ8.60 (s, 1H), 8.17 (d, J = 14.0 Hz, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.76 (s, 1H), 3.73 (d, J = 5.0 Hz, 2H), 3.47 (t, J = 5.8 Hz, 2H), 2.50 (s, 3H), 2.27-2.40 (m, 2H), 2.03-1.86 (m, 4H).

Example 11 Synthesis of Compound 11

[Reaction Scheme 9]

(1) Synthesis of Intermediate 11-2

Referring to step 9 of Example 1, intermediate 1-6 was replaced with compound 11-1 to obtain intermediate 11-2 (0.48 g, 69.1%). ¹ H NMR (400 MHz, DMSO) δ 10.77 (s, 1H), 8.20–8.16 (m, 1H), 7.94 (s, 1H), 7.79 (s, 1H), 7.55 (t, J=9.1 Hz, 1H), 3.60 (m, 2H), 3.40–3.35 (m, 2H), 2.44 (s, 3H), 2.25-2.35 (m, 2H), 1.91-1.98 (m, 2H), 1.80-1.87 (m, 2H).

(2) Synthesis of Compound 11

The intermediate 11-2 (0.12 g, 0.28 mmol) was dissolved in super dry DMF (3 ml), potassium tert-butoxide (0.048 g, 0.426 mmol) and acetohydroxamic acid (0.032 g, 0.43 mmol) were added, and the reaction was carried out at 50°C for 4 hours. The reaction was completed by TLC detection, and the mixture was extracted with EA and water. The organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. The mixture was spin-dried and subjected to column chromatography (DCM/MeOH=20/1) to obtain compound 11 (0.035 g, 28.3%). ¹ H NMR (600MHz, DMSO) δ10.55(s,1H),8.27(d,J＝1.7Hz,1H),7.73(s,1H),7.58(dd,J＝8.9,2.0Hz,1H),7.43(d,J＝8.9Hz,1H),6.40(s,2H),3.64–3.61(m,2H ),3.46(t,J=6.1Hz,2H),2.45(s,3H),2.31(2.26-2.35,2H),1.91-97(m,2H),1.86–1.82(m,2H).

Example 12 Synthesis of Compound 12

[Reaction Scheme 10]

The intermediate 11-2 (0.10 g, 0.28 mmol) was dissolved in ethanol (10 ml), 0.3 ml of hydrazine hydrate solution was added, and the mixture was heated to 80°C and refluxed overnight. The next day, TLC detected that the reaction was complete. The mixture was extracted with EA and water, and the organic layer was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried for column chromatography (DCM/MeOH=20/1) to obtain compound 12 (0.067 g, 65.1%). ¹ HNMR (400MHz, DMSO) δ11.34(s,1H),10.31(s,1H),8.05(s,1H),7.69(s,1H),7.35(d,J＝8.6Hz,1H),7.20(d,J＝8.7Hz,1H),5.29(s,2H),3.61-3.66(m,2 H),3.50–3.46(m,2H),2.44(s,3H),2.25-2.37(m,2H),1.93-2.03(m,2H),1.82-1.87(m,2H).

Example 13 Synthesis of Compound 13

[Reaction Scheme 11]

(1) Synthesis of Intermediate 13-1

Compound 2,6-dichloronicotinic acid (2.0 g, 10.4 mmol) and intermediate 1-3 (2.1 g, 12.5 mmol) were dissolved in DMF (100 ml), potassium carbonate (7.0 g, 52 mmol) was added, and the reaction solution was heated to 80°C for 4 hours. After TLC detection, the reaction was basically complete, and water was added to quench the reaction. The pH was adjusted to about 4 with 2M hydrochloric acid, and extracted with EA and water. The organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate, and then spin-dried for column chromatography separation (DCM/MeOH=20/1) to obtain intermediate 13-1 (2.57 g, 85%). ¹ H NMR (400MHz, Chloroform-d) δ8.25(t,J＝8.3Hz,1H),6.35(dd,J＝8.3Hz,1H),3.69–3.65(m,2H),3.37(m,2H),2.40(m,2H),2.07–2.01(m,2H),1.98(m,2H).

(2) Synthesis of Intermediate 13-2

Compound 13-1 (0.5 g, 1.72 mmol) was dissolved in anhydrous dichloromethane (40 ml) and placed in an ice bath. Oxalyl chloride (0.18 ml, 2.1 mmol) was added dropwise under nitrogen protection, and a catalytic amount of DMF was added. The reaction was allowed to react for about 2 h under ice bath conditions. The reaction liquid was directly dried to obtain a crude product and placed in a reaction bottle. 5-amino-2-fluorobenzonitrile was added to the reaction bottle, and anhydrous pyridine (5 ml) was added dropwise under nitrogen protection and ice bath conditions. After 2 h, TLC detected that the reaction was basically complete, and the mixture was extracted with EA and water. The organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. The mixture was dried by spin drying and subjected to column chromatography (DCM/MeOH=50/1) to obtain intermediate 13-2 (0.4 g, 56%). ¹ H NMR (400MHz, DMSO) δ10.77(s,1H),8.19(d,J＝5.8,1H),7.95(m,1H),7.90(m,1H),7.55(t,J＝9.2Hz,1H),6.45(dd,J＝8.0,3.1Hz,1H),3.57(s,2H),3.42 –3.37(m,2H),2.31(s,2H),1.96(s,2H),1.86(d,J＝5.0Hz,2H).

(3) Synthesis of Intermediate 13-3

Intermediate 13-2 (0.4 g, 0.98 mmol), potassium tetrafluorocyclopropane (0.36 g, 2.5 mmol) and potassium carbonate (0.41 g, 2.9 mmol) were dissolved in dioxane/water (10 ml/2 ml). After purging with nitrogen for 3-4 times, Pd(dppf) ₂ Cl ₂ (0.07 g, 0.098 mmol) was added. The reaction solution was heated to 100°C and reacted overnight. TLC detected that the reaction was basically complete. The product was extracted with EA and water, and the organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. The spin-dried agent was subjected to column chromatography (DCM/MeOH=50/1) to obtain intermediate 13-3 (0.1 g, 25%). ¹ H NMR (400MHz, DMSO) δ10.22(s,1H),8.19(m,1H),7.95(m,1H),7.90(m,1H),7.55(t,J＝9.2Hz,1H),6.64(d,J＝8.0,1H),3.57(s,2H),3.42–3.37(m,2H) ,2.35–2.26(m,2H),2.05–1.91(m,2H),1.88–1.81(m,2H).

(4) Synthesis of Intermediate 13-4

The intermediate 13-3 (0.1 g, 0.24 mmol) was dissolved in 2 ml of DMAC solution, and NCS (35 mg, 0.26 mmol) was added. The reaction was carried out at 100 ° C for 0.5 h. The reaction was basically completed after TLC monitoring. The mixture was extracted with EA and water, and the organic phase was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. After spin drying, it was separated by column chromatography (DCM/MeOH=20:1) to obtain intermediate 13-4 (85 mg, 78%). ¹ H NMR (400MHz, DMSO) δ10.22(s,1H),8.17(s,1H),7.88(m,2H),7.48(t,J＝9.2Hz,1H),3.57(m,2H),3.45–3.39(m,2H),2.38–2.29(m,2H),2.15–2.03(m ,2H),1.88–1.85(m,2H),1.24(m,2H),0.99(m,2H).

(5) Synthesis of Compound 13

Referring to step (2) of Example 11, intermediate 11-2 was replaced with intermediate 13-4 to obtain compound 13 (8 mg, 18.3%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.73 (s, 1H), 7.97 (s, 1H), 7.77 (s, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.40-7.35 (d, J = 8.5 Hz, 1H), 6.35 (s, 2H), 3.61 (m, 2H), 3.40 (t, 2H), 2.45 (s, 3H), 2.30 (m, 2H), 1.93 (m, 2H), 1.83 (m, 2H) 1.23 (m, 2H), 0.98 (m, 2H).

Example 14 Synthesis of Compound 14

[Reaction Scheme 12]

(1) Synthesis of Intermediate 14-1

The intermediate 13-2 (0.2 g, 0.49 mmol) was dissolved in 1 ml of DMSO solution, and 2 ml of methanol and K ₂ CO ₃ (0.06 g, 0.49 mmol) were added and reacted at 80°C for 1 h in a microwave oven. The reaction was basically completed after TLC monitoring. The mixture was extracted with EA and water, and the organic phase was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. After spin drying, it was separated by column chromatography (DCM/MeOH=20:1) to obtain the intermediate 14-1 (0.09 g, 45%). ¹ H NMR (600MHz, DMSO) δ10.56(s,1H),8.19(dd,J＝5.7,2.8Hz,1H),7.94(m,1H),7.68(d,J＝8.2Hz,1H),7.53(d,J＝9.1Hz,1H),6.16(d,J＝8.2Hz,1H),3.84(s, 3H),3.63–3.60(m,2H),3.42–3.39(m,2H),2.39–2.33(m,2H),1.98(m,2H),1.85(m,2H).

(2) Synthesis of Intermediate 14-2

Referring to step (4) of Example 13, intermediate 13-3 was replaced with 14-1 to obtain intermediate 14-2 (0.06 g, 64%). ¹ H NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 8.18 (dd, J = 5.7, 2.8 Hz, 1H), 7.93 (m, 1H), 7.85 (s, 1H), 7.55 (d, J = 9.1 Hz, 1H), 3.94 (s, 3H), 3.61 (m, 2H), 3.37 (m, 2H), 2.37-2.31 (m, 2H), 1.96 (m, 2H), 1.86 (m, 2H).

(3) Synthesis of Compound 14

Referring to step (2) of Example 11, intermediate 11-2 was replaced by intermediate 14-2 to obtain product 14 (11 mg, 20.8%). ¹ H NMR (400 MHz, DMSO) δ 10.45 (s, 1H), 8.27 (m, 1H), 7.79 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 6.41 (s, 2H), 3.93 (s, 3H), 3.65 (m, 2H), 3.46 (m, 2H), 2.33 (m, 2H), 1.99 (m, 2H), 1.87 (m, 2H).

Example 15 Synthesis of Compound 15

Referring to step (1) of Example 11, intermediate 11-1 was replaced with 4-amino-2-fluorobenzonitrile (purchased from Bissac) to obtain intermediate 15-1. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.05 (s, 1H), 7.91 (s, 1H), 7.82 (s, 1H), 7.65 (t, J=8.2 Hz, 1H), 7.57 (dd, J=8.6, 1.9 Hz, 1H), 3.60 (m, 2H), 3.08 (m, 2H), 2.45 (s, 3H), 2.31 (m, 2H), 1.90-1.96 (d, 2H), 1.80-1.85 (m, 2H).

Referring to step (2) of Example 11, intermediate 11-2 was replaced by intermediate 15-1 to obtain compound 15. ¹ H ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.73 (s, 1H), 7.97 (s, 1H), 7.77 (s, 1H), 7.73 (d, J=8.5 Hz, 1H), 7.40-7.35 (d, J=8.5 Hz, 1H), 6.35 (s, 2H), 3.61 (m, 2H), 3.40 (t, 2H), 2.45 (s, 3H), 2.30 (m, 2H), 1.93 (m, 2H), 1.83 (m, 2H).

Example 16 Synthesis of Compound 16

Referring to the method of Example 12, compound 16 was obtained by replacing intermediate 11-2 with intermediate 15-1.

¹ H NMR (400MHz, DMSO-d ₆ ) δ11.28 (s, 1H), 10.45 (s, 1H), 7.89 (s, 1H), 7.72 (s, 1H), 7.58 (d, J = 8.6Hz, 1H),7.02–6.97(dd,J＝8.6Hz,1H),5.29(s,2H),3.61(m,2H),3.44(t,2H),2.44(s,3H),2.30(m,2H ),1.95(m,2H),1.83(m,2H).

Example 17 Synthesis of Compound 17

[Reaction Scheme 13]

(1) Synthesis of Intermediate 17-2

Dissolve the intermediate 17-1 (0.5 g, 2.7 mmol) in 20 ml of methanol, add 60% water content palladium carbon (0.1 g), replace hydrogen three times, continue to react at room temperature for 7 hours, TLC detection reaction is complete, filter the reaction solution, spin dry the filtrate to obtain intermediate 17-2 (0.093 g, 22.4%). ¹ H NMR (400 MHz, DMSO) δ7.96 (d, J = 2.7 Hz, 1H), 7.39 (d, J = 2.7 Hz, 1H), 6.03 (s, 2H).

(2) Synthesis of Intermediate 17-3

Referring to step (1) of Example 11, intermediate 11-1 was replaced with intermediate 17-2 to obtain intermediate 17-3 (0.11 g, 29.6%). ¹ H NMR (600 MHz, MeOD) δ 8.81 (d, J = 1.7 Hz, 1H), 8.67 (d, J = 1.9 Hz, 1H), 7.78 (s, 1H), 3.72-3.68 (m, 2H), 3.41 (q, J = 5.1 Hz, 2H), 2.50 (s, 3H), 2.33 (td, J = 10.5, 5.3 Hz, 2H), 1.99-1.90 (m, 4H).

(3) Synthesis of Compound 17

Referring to step (2) of Example 11, intermediate 11-2 was replaced by intermediate 17-3 to obtain compound 17 (9 mg, 19.8%). ¹ H NMR (600 MHz, CDCl ₃ ) δ 10.48 (s, 1H), 8.83 (d, J = 2.3 Hz, 1H), 8.41 (d, J = 2.4 Hz, 1H), 8.14 (s, 1H), 4.61 (s, 2H), 3.59-3.56 (m, 2H), 3.39 (t, J = 5.7 Hz, 2H), 2.57 (s, 3H), 2.41 (ddd, J = 15.3, 9.9, 5.5 Hz, 2H), 2.21-2.15 (m, 2H), 1.94 (dd, J = 11.3, 5.6 Hz, 2H).

Example 18 Synthesis of Compound 18

Referring to Example 12, intermediate 11-2 was replaced with intermediate 17-3 to obtain compound 18 (25 mg, 53%). ¹ HNMR (400 MHz, DMSO) δ 11.92 (s, 1H), 10.51 (s, 1H), 8.49 (d, J = 2.2 Hz, 1H), 8.43 (d, J = 2.3 Hz, 1H), 7.77 (s, 1H), 5.57 (s, 2H), 3.66-3.60 (m, 2H), 3.49-3.44 (m, 2H), 2.45 (s, 3H), 2.31 (d, J = 15.8 Hz, 2H), 1.98 (d, J = 7.5 Hz, 2H), 1.86 (d, J = 5.3 Hz, 2H).

Example 19 Synthesis of Compound 19

Referring to step (1) of Example 11, intermediate 11-1 was replaced with intermediate 6-amino-2-chlorocyanopyridine to obtain intermediate 19-1. ¹ H NMR (400 MHz, DMSO) δ 11.92 (s, 1H), 8.72 (d, J = 14.9 Hz, 1H), 8.23 (d, J = 15.1 Hz, 1H), 7.79 (s, 1H), 3.72-3.68 (m, 2H), 3.41 (3.49-3.46, m, 2H), 2.51 (s, 3H), 2.01-1.98 (m, 2H), 1.99-1.90 (m, 4H).

Referring to step (2) of Example 11, intermediate 11-2 was replaced by intermediate 19-1 to obtain compound 19. ¹ H NMR (400 MHz, CDCl ₃ ) δ 11.67 (s, 1H), 8.24 (s, 1H), 7.86 (d, J = 15.1 Hz, 1H), 7.71 (d, J = 15.3 Hz, 1H), 6.18 (s, 2H), 3.58-3.55 (m, 2H), 3.39 (m, 2H), 2.45 (s, 3H), 2.39-2.37 (m, 2H), 2.21-2.15 (m, 2H), 1.92-1.89 (m, 2H).

Example 20 Synthesis of Compound 20

Referring to Example 12, intermediate 11-2 was replaced with intermediate 19-1 to obtain compound 20. ¹ H NMR (400 MHz, DMSO) δ 12.12 (s, 1H), 11.01 (s, 1H), 8.26 (s, 1H), 7.87 (d, J = 15.3 Hz, 1H), 7.43 (d, J = 14.9 Hz, 1H), 5.28 (s, 2H), 3.69-3.64 (m, 2H), 3.49-3.44 (m, 2H), 2.51 (s, 3H), 2.31-2.29 (m, 2H), 1.98-1.95 (m, 2H), 1.86-1.82 (m, 2H).

Example 21 Synthesis of Compound 21

[Reaction Scheme 14]

(1) Synthesis of Intermediate 21-1

5-Bromo-2-hydrazinopyridine (2 g, 10.6 mmol) was dissolved in tetrahydrofuran, N,N'-carbonyldiimidazole (2.6 g, 15.9 mmol) was added at room temperature, and the reaction solution was heated to reflux for overnight reaction; TLC detected that the reaction was basically complete, and the mixture was extracted with ethyl acetate and water. The organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate, and then dried to give the crude product intermediate 21-1 (1.3 g, 58%).

(2) Synthesis of Intermediate 21-2

Dissolve the intermediate 21-1 (1.3 g, 6.07 mmol) in 15 ml of phosphorus oxychloride, heat to 110°C and react overnight. The reaction is basically complete according to TLC. Cool the reaction solution and slowly drop it into ice water, extract it with ethyl acetate and water, wash the organic layer with saturated sodium bicarbonate, wash it with saturated sodium chloride, and dry it with anhydrous sodium sulfate, spin dry, and perform column chromatography (DCM/MeOH＝20/1) to obtain the intermediate 21-2 (0.86 g, 61%). ¹ H NMR (400 MHz, CDCl ₃ )δ8.17(s,1H),7.67(d,J＝9.7Hz,1H),7.38(dd,J＝9.7,1.6Hz,1H).

(3) Synthesis of Intermediate 21-3

Compound 21-2 (0.86 g, 3.69 mmol) was dissolved in benzylamine (5 ml), and the mixture was reacted at 110° C. overnight. The reaction solution was extracted with ethyl acetate and water, and the organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. The mixture was spin-dried and subjected to column chromatography (DCM/MeOH=20/1) to obtain intermediate 21-3.

(4) Synthesis of Intermediate 21-4

The intermediate 21-3 was dissolved in DMSO, potassium carbonate, L-proline and cuprous iodide were added, and the reaction solution was stirred at room temperature for 5 minutes under nitrogen protection, and then ammonia water was added and the reaction solution was heated to 90°C overnight. TLC detection showed that the reaction was basically complete, and the mixture was extracted with ethyl acetate and water. The organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate, spin-dried and separated by column chromatography to obtain the intermediate 21-4.

(5) Synthesis of Intermediate 21-5

Compound 21-4 was dissolved in 50 ml of methanol, and 60% palladium carbon was added. After replacing hydrogen three times, the reaction was continued for 7 hours at room temperature. TLC detected that the reaction was complete, and the reaction solution was filtered and the filtrate was dried to obtain intermediate 21-5.

(6) Synthesis of Compound 21

Referring to step (9) of Example 1, intermediate 1-6 was replaced with intermediate 21-5 to obtain compound 21.

Example 22 Synthesis of Compound 22

Referring to step (9) of Example 1, intermediate 1-6 was replaced with 4-aminophthalimide to obtain compound 22. ¹ H NMR (400 MHz, DMSO) δ 11.26 (s, 1H), 10.99 (s, 1H), 8.23 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.84-7.79 (m, 2H), 3.61 (m, 2H), 3.38 (m, 2H), 2.45 (s, 3H), 2.32 (m, 2H), 2.02-1.97 (m, 2H), 1.84 (d, J = 5.6 Hz, 2H).

Example 23 Synthesis of Compound 23

[Reaction Scheme 15]

(1) Synthesis of Intermediate 23-1

Dissolve 4-chloro-7-nitroquinazoline (0.5 g, 2.39 mmol) in 20 ml of ammonia methanol solution (7 N), react at room temperature for 2 h, TLC detection shows that the reaction is complete, and the reaction solution is dried to obtain a crude product 23-1 (0.3 g). ¹ H NMR (400 MHz, DMSO) δ8.52 (m, 2H), 8.38 (d, J = 2.1 Hz, 1H), 8.20 (dd, J = 9.0, 2.2 Hz, 1H), 7.30 (brs, 2H).

(2) Synthesis of Intermediate 23-2

The intermediate 23-1 (0.3 g, 0.9 mmol) was dissolved in 30 ml of methanol, and 60% palladium carbon (0.06 g) was added. After replacing hydrogen three times, the reaction was continued at room temperature for 7 hours. TLC detected that the reaction was complete, and the reaction solution was filtered and the filtrate was dried to obtain the intermediate 23-2. ¹ H NMR (400 MHz, DMSO) δ8.21 (s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.61 (s, 2H), 6.78 (dd, J = 8.9, 2.2 Hz, 1H), 6.59 (d, J = 2.2 Hz, 1H), 6.11 (s, 2H).

(3) Synthesis of Compound 23

Referring to step (9) of Example 1, intermediate 1-6 was replaced by intermediate 23-2 to obtain compound 23 (12 mg, 11%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.14 (s, 1H), 8.60 (s, 1H), 8.15 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.90 (s, 1H), 7.78 (d, J = 9.0 Hz, 1H), 5.71 (s, 2H), 3.61-3.57 (m, 2H), 3.45-3.40 (m, 2H), 2.59 (s, 3H), 2.46-2.36 (m, 2H), 2.23-2.12 (m, 2H), 1.96-1.90 (m, 2H).

Example 24 Synthesis of Compound 24

Referring to the synthesis method of Example 23, 4-chloro-7-nitroquinazoline was replaced with 4-chloro-6-nitroquinazoline to obtain Compound 24 (61 mg, 56%). ¹ H NMR (400MHz, CDCl ₃ ) δ10.41(s,1H),8.64(d,J＝2.1Hz,1H),8.60(s,1H),8.19(s,1H),7.88(d,J＝9.0Hz,1H),7.64–7.60(m,1H),5.78(s,2H),3.62–3. 58(m,2H),3.41–3.37(m,2H),2.58(s,1H),2.45(ddd,J＝14.7,10.3,5.1Hz,2H),2.26–2.16(m,2H),1.97–1.91(m,2H).

Example 25 Synthesis of Compound 25

[Reaction Scheme 16]

(1) Synthesis of Intermediate 25-1

Dissolve 4-chloro-7-nitroquinazoline (0.5 g, 2.39 mmol) in methanol (20 ml), add sodium hydride (60% in kerosene) (0.14 g, 3.6 mmol) under ice bath, react overnight at room temperature, TLC shows that the reaction is almost complete, add ice water to quench, extract with EA and water, wash the organic layer with saturated sodium bicarbonate and sodium chloride, dry with anhydrous sodium sulfate, spin dry column chromatography (DCM/MeOH=20/1) to obtain intermediate 25-1 (0.4 g, 81.6%). ¹ H NMR (400 MHz, DMSO) δ9.00 (s, 1H), 8.67 (s, 1H), 8.43–8.34 (m, 2H), 4.20 (s, 3H).

(2) Synthesis of Intermediate 25-2

Dissolve the intermediate 25-1 (0.4 g, 1.94 mmol) in 50 ml of methanol, add 60% palladium carbon (0.08 g), replace hydrogen three times, and continue to react for 7 hours at room temperature. TLC detection shows that the reaction is complete. Filter the reaction solution and spin dry the filtrate to obtain the intermediate 25-2 (0.3 g, 91.2%). ¹ H NMR (400 MHz, DMSO) δ8.47 (s, 1H), 7.78 (d, J = 8.8 Hz, 1H), 6.93 (dd, J = 8.9, 2.2 Hz, 1H), 6.76 (d, J = 2.1 Hz, 1H), 6.21 (s, 2H), 4.01 (s, 3H).

(3) Synthesis of Intermediate 25-3

Referring to step (9) of Example 1, intermediate 1-6 was replaced with intermediate 25-2 to obtain intermediate 25-3 (0.09 g, 61%).

(4) Synthesis of Compound 25

The intermediate 25-3 (0.09 g, 0.2 mmol) was dissolved in DMF (5 ml), lithium chloride (0.04 g, 1.0 mmol) and p-toluenesulfonic acid (0.17 g, 1.0 mmol) were added, and the reaction solution was heated to 80 ° C for about 3 h. TLC showed that the reaction was basically complete. The mixture was cooled and extracted with EA and water. The organic layer was washed with saturated sodium bicarbonate and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The mixture was spin-dried and column chromatography (DCM/MeOH=10/1) was performed to obtain compound 25 (33 mg, 39%). ¹ H NMR (400MHz, DMSO) δ12.15(s,1H),10.84(s,1H),8.13–8.04(m,3H),7.81(s,1H),7.75–7.70(m,1H),3.62(s,2H),3.40(t,J＝5.8Hz,2H),2.45(s,3H) ,2.32(s,2H),1.99–1.90(m,2H),1.84(d,J＝5.2Hz,2H).

Example 26 Synthesis of Compound 26

[Reaction Scheme 17]

(1) Synthesis of Intermediate 26-1

Dissolve 3-hydroxy-1,2-benzisoxazole (purchased from Bidler) (1.0 g, 7.4 mmol) in 10 ml of concentrated sulfuric acid, transfer to an ice bath, add potassium nitrate (0.74 g, 7.4 mmol), react for 3 h under ice bath conditions, TLC detection shows that the reaction is almost complete, slowly add the reaction solution to ice water, a large amount of white solid precipitates, filter to obtain a filter cake, and dry to obtain intermediate 26-1 (1.3 g, 97.5%). ¹ H NMR (400 MHz, DMSO) δ13.05 (s, 1H), 8.68 (d, J = 2.1 Hz, 1H), 8.46 (dd, J = 9.2, 2.3 Hz, 1H), 7.82 (dd, J = 9.2, 4.6 Hz, 1H).

(2) Synthesis of Intermediate 26-2

The intermediate 26-1 (0.4 g, 2.2 mmol) was dissolved in 30 ml of dichloromethane, and the reaction solution was placed in an ice-salt bath. 2-(Trimethylsilyl)ethoxymethyl chloride (0.74 g, 4.4 mmol) was added under nitrogen protection. After stirring for 5 min, triethylamine (0.67 g, 6.7 mmol) was slowly added dropwise. After reacting in an ice-salt bath for 2 h, TLC detected that the reaction was complete. The mixture was extracted with DCM and water, and the organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. The mixture was spin-dried and separated by column chromatography (PE/EA=10/1) to obtain the intermediate 26-2 (0.28 g, 41%). ¹ H NMR (400MHz, CDCl ₃ ) δ8.79(d,J＝2.0Hz,1H),8.55(dd,J＝9.2,2.3Hz,1H),7.41(d,J＝9.2Hz,1H),5.40(s,2H),3.75–3.64(m,2H),1.00–0.91(m,2H),-0.01 (s,9H).

(3) Synthesis of Intermediate 26-3

The intermediate 26-2 (0.28 g, 0.9 mmol) was dissolved in 100 ml methanol, and 60% palladium carbon (0.05 g) was added. After replacing hydrogen three times, the reaction was continued for 1 h at room temperature. The reaction was complete after TLC detection. The reaction solution was filtered, the filtrate was dried, and column chromatography (DCM/MeOH=20/1) was used to obtain the intermediate 26-3 (0.07 g, 28%). ¹ H NMR (400 MHz, CDCl ₃ ) δ7.05 (dd, J=9.1, 5.4 Hz, 2H), 7.00 (dd, J=8.7, 2.4 Hz, 1H), 5.32 (s, 2H), 3.71–3.64 (m, 2H), 0.98–0.92 (m, 2H), -0.01 (s, 9H).

(4) Synthesis of Intermediate 26-4

Referring to step (9) of Example 1, intermediate 1-6 was replaced by intermediate 26-3 to obtain intermediate 26-4. ¹ H NMR (400 MHz, CDCl ₃ ) δ9.78 (s, 1H), 8.02 (d, J=3.2 Hz, 2H), 7.23 (d, J=3.9 Hz, 1H), 5.31 (s, 2H), 3.67-3.61 (m, 2H), 3.59-3.54 (m, 2H), 3.41-3.36 (m, 2H), 2.51 (s, 3H), 2.34 (td, J=10.3, 5.8 Hz, 2H), 2.14-2.05 (m, 2H), 1.93-1.86 (m, 2H), 0.96-0.89 (m, 2H), -0.04 (s, 9H).

(5) Synthesis of Compound 26

The intermediate 26-4 (50 mg, 0.09 mmol) was dissolved in dichloromethane, and 1 ml of trifluoroacetic acid was added under ice bath conditions. After stirring at room temperature for 1 h, TLC detected that the reaction was complete. The reaction solution was spin-dried and separated by column chromatography (DCM/MeOH=20/1) to obtain compound 26 (10 mg, 25.4%). ¹ H NMR (600MHz, DMSO) δ12.36(s,1H),10.62(s,1H),8.23(d,J＝1.6Hz,1H),7.77(s,1H),7.71(d,J＝9.1Hz,1H),7.54(d,J＝9.0Hz,1H),3.62(s,2H),3.44(t ,J＝6.1Hz,2H),3.17(d,J＝4.5Hz,1H),2.45(s,3H),2.31(s,2H),1.96(s,2H),1.84(d,J＝5.5Hz,2H).

Example 27 Synthesis of Compound 27

[Reaction Scheme 18]

(1) Synthesis of Intermediate 27-1

3,6-dichloropyridazine-4-carboxylic acid (0.5 g, 2.59 mmol), compound 1-3 (0.534 g, 3.11 mmol), potassium carbonate (1.79 g, 12.95 mmol) were dissolved in 30 ml of DMF and reacted at 80°C overnight. After TLC detection, the reaction solution was acidified with 3N HCl solution, extracted with EA and water, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product 27-1 (0.3 g, 39.7%). ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 12.0 (s, 1H) 7.72 (s, 1H), 3.73 (m, 2H), 3.38 (t, 2H), 2.37–2.31 (m, 2H), 2.04 (m, 2H), 1.92 (m, 2H).

(2) Synthesis of Compound 27

Intermediate 27-1 (0.10 g, 0.34 mmol) was dissolved in anhydrous DCM (5 ml), the reaction solution was placed in an ice bath, 2-3 drops of DMF were added, oxalyl chloride (2M, 0.2 ml) was added under nitrogen protection, and the reaction was carried out in an ice bath for 2 h. The reaction solution was dried to obtain intermediate 27-2.

1-6 (0.061 g, 0.41 mmol) was added to the reaction bottle of intermediate 27-2, and placed in an ice bath. 5 ml of anhydrous pyridine was added. After 2 h, TLC detected that the reaction was complete. The mixture was extracted with EA and water, and the organic layer was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. The mixture was spin-dried and subjected to column chromatography (DCM/MeOH=20/1) to obtain product 27 (0.03 g, 17.3%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ10.96(s,1H),8.63(s,1H),8.07(s,1H),7.84(s,1H),7.76(dd,1H),7.58(d,1H),4.35(m,2H),3.73(m,2H),3.53(t,2H),2. 33(m,2H),2.02-2.10(m,2H),1.85-1.90(m,2H).

Test Case Biological Activity Test

1. Detection method: Whole-cell manual patch clamp technique to detect the effect of compounds on voltage-gated Nav1.8 channel current 2. Preparation and analysis of detection compounds

Negative control: electrophysiological extracellular solution containing 0.5% DMSO

Positive control: VX-150 is a positive control drug

Test compound: Weigh a certain mass of compound and dissolve it in DMSO to prepare a 20mM DMSO stock solution. On the day of the test, dilute the 20mM compound stock solution with extracellular fluid to the final concentration to be tested, ensuring that the DMSO content in the test drug solution does not exceed 0.5%. This concentration of DMSO has no effect on the detected Nav1.8 channel current. For example, to prepare 100nM and 1μM compound solutions, the gradient dilution method is as follows: first draw 5μL DMSO stock solution and add it to 10mL of extracellular fluid, dissolve evenly, and obtain a 10μM compound solution; then draw 1mL of 10μM compound, add it to 9mL of extracellular fluid, dissolve evenly, and obtain a 1μM compound solution; then draw 1mL of 1μM compound, add it to 9mL of extracellular fluid, dissolve evenly, and obtain a 100nM compound solution.

3. Cell Culture

(1) Nav1.8 cell line: HEK293 (Flp-In T-Rex-293) cells stably expressing human Nav1.8 sodium channel, the encoding gene information is as follows: NM_001293306.2.

(2) Culture and subculture conditions and methods: Cell lines were cultured in a constant temperature incubator at 37°C and 5% _CO2 . Nav1.8 stable transfectants were cultured in a complete medium containing 10% tetracycline-free fetal bovine serum (HyClone) and 100 μg/mL Hygromycin B in DMEM (Gibco). The day before the experiment, cells were digested and subcultured when they grew to about 90% density. First, the medium was aspirated and the cells were washed with phosphate buffered saline (PBS) preheated at 37°C. After discarding the PBS buffer, trypsin was added for digestion and the cells were transferred to a centrifuge tube. Centrifuged at 800 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in a complete medium containing 1 μg/mL Doxcycline and subcultured to a 6-well plate. After induction culture for 20 hours, the cells were separated and subcultured to a coverslip coated with poly-lysine and cultured for 1-2 hours before being used for electrophysiological recording experiments.

4. Electrophysiological Experiments

(1) The Nav1.8 sodium channel currents were recorded using the whole-cell voltage clamp technique at room temperature (23-25°C).

(2) The whole-cell voltage clamp recording experiment used an Axon patch 700B patch clamp amplifier (Molecular Devices), a digital-to-analog converter Digidata 1440A (Molecular Devices), and a glass microelectrode was pulled from a glass electrode blank (World Precision Instruments) using a puller (P97, Sutter). The tip resistance after perfusion of the electrode solution was about 1.5-2.5 MΩ. The glass microelectrode was connected to the patch clamp amplifier by inserting it into the amplifier probe. The clamp voltage and data recording were controlled and recorded by a computer using pClamp 10 software (Molecular Devices), with a sampling frequency of 20 kHz and a filter frequency of 2 kHz.

(3) Extracellular and intracellular fluids for electrophysiological experiments:

Formula of extracellular solution: 140 mM NaCl, 3 mM KCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM HEPES and 20 mM glucose, pH adjusted to 7.3 with NaOH.

Intracellular solution formula: 140 mM CsF, 10 mM NaCl, 10 mM HEPES, 1.1 mM EGTA and 20 mM glucose, pH adjusted to 7.3 with CsOH.

Abbreviations: HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); EGTA: ethylene glycol bis(2-aminoethyl ether) tetraacetic acid; all chemicals were purchased from Sigma.

(4) Electrophysiological stimulation protocol: After obtaining the whole-cell recording, wait for 4-5 minutes at a clamp voltage of -80mV until the electrode fluid and the intracellular fluid are balanced, and then start electrophysiological recording (to achieve high-impedance GΩ sealing conditions). Current stimulation and compound activity detection protocol: The cells are clamped at -80mV, and a depolarizing voltage stimulus of +10mV is given for 20ms, and then repolarized to -80mV, with a stimulation frequency of 0.5Hz. After confirming that the Nav1.8 sodium channel current is stable (about 1 minute), the drug administration process is started until the cell current no longer changes (the compound inhibition reaches a steady state). At least 3 cells (n≥3) are tested for each concentration of the compound. A single concentration of 100nM VX-150 is given as a positive control after all tested compounds.

5. Data Analysis

Data acquisition and analysis were performed using pClamp10 (Molecular Devices), GraphPad Prism 5 (GraphPad Software) and Excel (Microsoft). All data were expressed as mean ± standard error (Mean ± SEM). The effect of the compound on the current was calculated using the following formula:

Inhibition rate (%) = [1-the magnitude of the current after drug addition (I _Drug )/the magnitude of the current before drug addition (I _Control )]×100.

The dose-effect curve was fitted using the Hill equation: Y = Bottom + (Top - Bottom) / (1 + 10^(LogIC ₅₀ -X) × k), where Bottom and Top represent the minimum and maximum values of inhibition, respectively, X represents the logarithmic value of the compound concentration, Y represents the I _Drug /I _Control value, IC ₅₀ represents the drug dose that produces a half-maximal inhibitory effect, and k represents the Hill coefficient.

The results are shown in Tables 1 and 2.

Table 1 _IC50 values of the compounds in the examples for the Nav1.8 channel

Table 2 Percentage blocking activity of the compounds in the examples on Nav1.8 channels

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents, without departing from the spirit and essence of the claims of the present invention; and these modifications or replacements are still within the scope defined by the claims of the present invention.

Claims (10)

1. A compound of formula I, its isomer, racemate, prodrug or a pharmaceutically acceptable salt thereof, in: X is selected from N or CH; Y is selected from N or CR ₃ ; V and G are each independently selected from N or CH, Q and T are each independently selected from N or C; A, W and Z are each independently selected from O, S, N, carbonyl, sulfoxide, sulfone, -NR _a -, -CR _b -, -NR _a -CO-, -CR _b =N-, - CR _b -NR _a -, and at least one of A, W and Z contains nitrogen; R _a is independently selected at each occurrence from hydrogen, amino, hydroxyl, C1-C6 alkyl, haloC1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkoxy, C1-C6 Alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O, S, C6-C12 aryl or 1 selected from N, O, S 5-10 membered heteroaryl groups with 4 heteroatoms; R _b at each occurrence is independently selected from hydrogen, halogen, nitro, amino, cyano, hydroxyl, C1-C6 alkyl, haloC1-C6 alkyl, C1-C6 alkoxy, haloC1- C6 alkoxy, C1-C6 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O, S, C6-C12 aryl or containing selected 5-10 membered heteroaryl group with 1 to 4 heteroatoms from N, O, and S; n is selected from 0, 1 or 2; especially 2; R ₁ is selected from hydrogen, hydroxyl, halogen, cyano, nitro or amino; R ₂ is selected from halogen, hydroxyl, cyano, nitro or halogenated C1-C6 alkyl; R ₃ is selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, halogenated C2-C6 alkenyl, C2-C6 alkenyloxy, halogenated C2-C6 alkenyloxy, C2-C6 alkynyl, halogenated C1-C6 alkynyl, C2-C6 alkynyloxy, halogenated C1-C6 alkynyloxy, C1-C6 alkoxy, halogenated C1-C6 alkane Oxygen, C1-C6 alkylamino, halogenated C1-C6 alkylamino, C3-C6 cycloalkylamino, C1-C6 alkoxyamino, C3-C6 cycloalkyl, C3-C6 cycloalkoxy or containing selected from N , 3-8 membered heterocyclic group with 1 to 4 heteroatoms of O and S; R ₆ and R ₇ are each independently selected from hydrogen, fluorine, chlorine, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxy; R ₅ , R ₈ and R ₉ are each independently selected from hydrogen, fluorine, chlorine, halogenated C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxy, C3-C8 cycloalkyl; Represents a single or double bond. 2. The compound according to claim 1, its isomer, racemate, prodrug or pharmaceutically acceptable salt thereof, wherein, R ₁ is selected from hydrogen, fluorine, chlorine, bromine, amino or hydroxyl; R ₂ is selected from chlorine, bromine, iodine or trifluoromethyl; R ₃ is selected from fluorine, chlorine, bromine, amino, hydroxyl, methyl, cyclopropyl, methoxy, trifluoromethyl, and trifluoromethoxy; R ₆ and R ₇ are each independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or isopropyl; R ₅ , R ₈ and R ₉ are each independently selected from hydrogen, methyl, ethyl, isopropyl or cyclopropyl. 3. The compound according to claim 1, its isomer, racemate, prodrug or pharmaceutically acceptable salt thereof, wherein, The compound of formula I is selected from the following compounds of formula II: Among them, the definitions of R ₃ , A, Z, W, Q, T, V and G are the same as those in the corresponding claims. 4. The compound according to any one of claims 1 to 3, its isomer, racemate, prodrug or pharmaceutically acceptable salt thereof, wherein, Be one of the following groups: 5. The compound according to claim 1, its isomer, racemate, prodrug or pharmaceutically acceptable salt thereof, wherein, The compound of formula I is selected from the following compounds: 6. A method for preparing the compound according to any one of claims 1 to 5, comprising the following steps: The compound of formula I is obtained by acylation reaction of formula III and formula IV in the presence of a base; Preferably, the base is selected from pyridine, sodium carbonate, sodium bicarbonate. 7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5, its isomer, racemate, prodrug or pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable salt thereof. of excipients. 8. A compound according to any one of claims 1 to 5, its isomer, racemate, prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 7 in the preparation of Nav1. 8 Uses in inhibitors. 9. A compound according to any one of claims 1 to 5, its isomer, racemate, prodrug or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 7 in the preparation of Use in drugs to treat, prevent or control diseases or symptoms related to Nav1.8 channels. 10. Use according to claim 9, wherein, The diseases or symptoms related to the Nav1.8 channel include nociceptive pain, inflammatory pain, neuropathic pain, functional pain, muscle or bone injury-related pain, pelvic pain, abdominal pain, chest pain, lumbosacral neuralgia, Preoperative pain, interoperative pain, postoperative pain, acute or chronic pain, migraine, trigeminal neuralgia, pancreatitis, renal colic, cancer pain, pain due to chemical or drug therapy, diabetic neuralgia, post-herpetic neuralgia Neuralgia, back pain, phantom limb pain, sciatica, small fiber neuralgia, erythromelalgia, arthritis, pruritus, acute or chronic pruritus, asthma, multiple sclerosis, cardiac arrhythmias, atrial fibrillation, heart failure , Brugada syndrome, kidney stones, epilepsy, convulsions.