Benzene ring-containing polycyclic compound, and pharmaceutical composition comprising same and use thereof

Abstract

Disclosed in the present invention are a benzene ring-containing polycyclic compound, and a pharmaceutical composition comprising same and a use thereof. Specifically provided is a compound as represented by formula (I-E) or a pharmaceutically acceptable salt thereof. The compound of the present invention has one or more of the following effects / advantages: (1) a novel structure; (2) a good blocking effect on the activity of NaV1.8 channel; and (3) good selectivity on other Nav subtypes (such as Nav1.1, Nav1.2, Nav1.3 Nav1.4, Nav1.5, Nav1.6, Nav1.7 and Nav1.9), and high safety.

Description

A polycyclic compound containing a benzene ring, its pharmaceutical composition and its application

[0001] This application claims priority to Chinese Patent Application No. 2023117733761, filed on December 21, 2023, priority to Chinese Patent Application No. 2024101754103, filed on February 7, 2024, and priority to Chinese Patent Application No. 2024108296661, filed on June 25, 2024. The entire text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] The present invention relates to a polycyclic compound containing a benzene ring, a pharmaceutical composition thereof and application thereof. Background Art

[0003] Pain originates from nociceptors in the peripheral nervous system. These are free nerve endings widely distributed throughout the skin, muscles, joints, and internal organs. They convert thermal, mechanical, or chemical stimuli into nerve impulses (action potentials), which are transmitted via afferent nerve fibers to the cell bodies of the dorsal root ganglia (DRG), ultimately reaching higher neural centers, causing pain sensation. The generation and conduction of action potentials in neurons, in turn, rely on voltage-gated sodium channels (VGSC / Nav) on the cell membrane. Nav is a key mediator for transmembrane information transmission along neurons.

[0004] According to statistics, approximately one-fifth of the world's population suffers from moderate to severe chronic pain. The global analgesic market was approximately US$36 billion in 2018 and is projected to reach US$56 billion by 2023. The acute, moderate to severe pain market is expected to grow steadily at a compound annual growth rate of 2.5%, while the chronic pain market is projected to grow at a compound annual growth rate of approximately 18%. Chronic pain is expected to be the primary driver of continued growth in the global pain market over the next decade.

[0005] The secondary structure of voltage-gated sodium channels (VGSC / Nav) consists of four pore-forming domains (DI-DIV), each with six transmembrane α-helices (S1-S6). S4 is a voltage sensor containing positively charged ions. The intracellular loop between DIII and DIV is considered the rapid inactivation gate. Nav exists in three distinct states, with the inactivated closed state exhibiting either rapid inactivation (within milliseconds) or slow inactivation (seconds) kinetics. When the cell membrane depolarizes, the sodium channel activates and opens, causing an influx of sodium ions, further depolarizing the cell membrane and leading to the generation of an action potential. Therefore, inhibiting abnormal sodium channel activity may help treat and alleviate pain, making Nav a potential peripheral target for pain treatment.

[0006] There are nine major subtypes of human Nav, namely Nav1.1-Nav1.9. Based on whether they can be effectively inhibited by nanomolar tetrodotoxin (TTX), Nav is divided into TTX-sensitive (TTX-S) and TTX-insensitive (TTX-R), and the tissue expression of different subtypes is extremely different. Nav1.1, Nav1.2, Nav1.3Nav1.4, Nav1.6 and Nav1.7 are TTX-S subtypes. Among them, Nav1.1, Nav1.2 and Nav1.3 are abundantly expressed in the central nervous system (CNS), Nav1.4 is abundant in skeletal muscle, and Nav1.6 and Nav1.7 are mainly abundant in the CNS. Nav1.5, Nav1.8 and Nav1.9 are of TTX-R type, among which Nav1.5 is mainly present in cardiomyocytes, while Nav1.8 and Nav1.9 are present in the dorsal root ganglia of the peripheral nervous system (PNS-DRG).

[0007] Studies have shown that changes in ion channels are the molecular basis for peripheral sensitization, central sensitization, and disinhibition after inflammation or neuropathological damage, and are also an important molecular mechanism for the occurrence of pain. Currently, Nav inhibitors have been proven to be effective. For example, the local anesthetic lidocaine relieves pain by inhibiting Nav. Non-selective Nav inhibitors such as lamotrigine, lacosamide, and mexiletine have been successfully used to treat chronic pain. However, the Nav inhibitors currently used in clinical practice lack subtype selectivity and can inhibit sodium ion channels expressed in the heart and central nervous system. The therapeutic window is narrow and the scope of application is limited. Nav1.8 is an important ion channel involved in chronic pain, atrial fibrillation, and Budd-Chiari syndrome. Highly selective Nav1.8 inhibitors are ideal targets for pain treatment, and when producing analgesic effects, the side effects are relatively small.

[0008] In models of neuropathic pain, nerve damage increases the expression level of Nav1.8 in axons and neuronal cell bodies. The use of Nav1.8 antisense oligonucleotides can significantly relieve pain while reducing Nav1.8 expression. Nav1.8 knockout mice do not show normal visceral inflammatory pain. When the human Nav1.8 gene produces a gain-of-function mutation, it will cause peripheral neuropathic pain. Based on a series of animal experiments and human genetic evidence, selective inhibition of Nav1.8 has the potential to become a new analgesic therapy that can be used to treat various types of pain such as inflammatory pain, neuralgia, postoperative pain and cancer pain.

[0009] Highly selective Nav inhibitors are one of the key research and development directions for voltage-gated sodium channels. Nav1.8 is a highly selective target for pain treatment because it is mainly distributed in the peripheral nervous system and confined to neurons that sense pain. Therefore, selectively inhibiting Nav1.8 has good prospects for reducing potential toxic side effects. Nav inhibitors used in clinical practice lack subtype selectivity and can inhibit sodium ion channels expressed in the heart and central nervous system. Therefore, their therapeutic window is narrow and their scope of application is limited. Therefore, it is necessary to develop Nav1.8 inhibitors with higher activity, better selectivity, better pharmacokinetic properties, and fewer side effects to meet clinical needs. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art in that Nav1.8 channels are insufficiently inhibited. To this end, the present invention provides a polycyclic compound containing a benzene ring, a pharmaceutical composition thereof, and its application. The compound of the present invention has one or more of the following advantages: (1) novel structure; (2) good blocking effect (or inhibitory effect) on NaV1.8 channel activity; (3) good selectivity for other Nav subtypes (e.g., Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.9), and high safety.

[0011] The present invention solves the technical problems of the present invention through the following technical solutions:

[0012] The present invention provides a compound as shown in formula (IE) or a pharmaceutically acceptable salt thereof,

[0013] Among them, R 1 、R 2 and R 3 are each independently halogen or -OR 1-1 ;

[0014] R 1-1 is C1-C6 alkyl, 3-8 membered heterocycloalkyl,1-1-1 a substituted C1-C6 alkyl group or a 3-8 membered heterocycloalkyl group substituted with one or more deuterium groups; in the 3-8 membered heterocycloalkyl group and the 3-8 membered heterocycloalkyl group substituted with one or more deuterium groups, the heteroatoms are selected from one, two or three of N, O and S, and the number of heteroatoms is one, two or three;

[0015] Each R 1-1-1 Each independently is deuterium or -OR a ;

[0016] R a is C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C6 alkyl substituted by one or more deuteriums;

[0017] R 4 is hydrogen, hydroxy, C1-C6 alkyl, C1-C6 alkyl-O-C1-C6 alkyl or -O-C1-C6 alkyl;

[0018] R 7 is hydrogen or C1-C6 alkyl;

[0019] X 1 、X 3 、X 4 and X 5 Each independently is N, N + -O - or CR X1 ;

[0020] R X1 is hydrogen, halogen or -C(=O)N(R X1-1 )2;

[0021] Each R X1-1 Each is independently hydrogen or C1-C6 alkyl;

[0022] Each R X2-1 are each independently hydrogen, -CN, C1-C6 alkyl, -O-C1-C6 alkyl, -OH or -NR'R";

[0023] R' and R" are each independently hydrogen or C1-C6 alkyl;

[0024] M is O or NH;

[0025] When M is 0, an R X2-1 is -O-C1-C6 alkyl, another R X2-1 For hydrogen.

[0026] In some embodiments, the compound as shown in formula (IE) or a pharmaceutically acceptable salt thereof,

[0027] Among them, R 1 、R 2 and R 3 are each independently halogen or -OR 1-1 ;

[0028] R 1-1 is C1-C6 alkyl, 3-8 membered heterocycloalkyl, 1-1-1 a substituted C1-C6 alkyl group or a 3-8 membered heterocycloalkyl group substituted with one or more deuterium groups; in the 3-8 membered heterocycloalkyl group and the 3-8 membered heterocycloalkyl group substituted with one or more deuterium groups, the heteroatoms are selected from one, two or three of N, O and S, and the number of heteroatoms is one, two or three;

[0029] Each R 1-1-1 Each independently is deuterium or -OR a ;

[0030] R a is C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C6 alkyl substituted by one or more deuteriums;

[0031] R 4 is hydrogen, hydroxy, C1-C6 alkyl, C1-C6 alkyl-O-C1-C6 alkyl or -O-C1-C6 alkyl;

[0032] R 7 is hydrogen or C1-C6 alkyl;

[0033] X 1 、X 3 、X 4 and X 5 Each independently is N, N + -O - or CR X1 ;

[0034] R X1 is hydrogen, halogen or -C(=O)N(R X1-1 )2;

[0035] Each R X1-1 Each is independently hydrogen or C1-C6 alkyl;

[0036] Each R X2-1 are each independently hydrogen, C1-C6 alkyl, -O-C1-C6 alkyl, -OH or -NR'R";

[0037] R' and R" are each independently hydrogen or C1-C6 alkyl;

[0038] M is O or NH.

[0039] In some embodiments, R 1 、R 2 and R 3 wherein the halogen is independently fluorine, chlorine, bromine or iodine, preferably fluorine.

[0040] In some embodiments, R 1-1 wherein the C1-C6 alkyl group and the 1-1-1 The C1-C6 alkyl groups in the substituted C1-C6 alkyl groups are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.

[0041] In some embodiments, R 1-1 In the 3-8 membered heterocycloalkyl group and the 3-8 membered heterocycloalkyl group substituted with one or more deuterium groups, the heteroatoms are each independently N and / or O; the number of heteroatoms is each independently preferably 1 or 2. The 3-8 membered heterocycloalkyl group is each independently preferably a 4-6 membered (e.g., 4-membered, 5-membered, or 6-membered) heterocycloalkyl group; more preferably For example

[0042] In some embodiments, R a wherein the C1-C6 alkyl group and the C1-C6 alkyl group substituted by one or more deuteriums are each independently a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, preferably a methyl group.

[0043] In some embodiments, R a In the embodiment, the C3-C6 cycloalkyl group is a cyclopropyl group, a cyclobutyl group, a cyclopentyl group or a cyclohexyl group, and is preferably a cyclopropyl group.

[0044] In some embodiments, R 4 In the embodiment, the C1-C6 alkyl group is a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group or a tert-butyl group, and is preferably a methyl group.

[0045] In some embodiments, R 7 In the embodiment, the C1-C6 alkyl group is a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group or a tert-butyl group, and is preferably a methyl group.

[0046] In some embodiments, R X1 wherein the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine.

[0047] In some embodiments, R X2-1In the above-mentioned C1-C6 alkyl group and the C1-C6 alkyl group in the -O-C1-C6 alkyl group are each independently a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, preferably a methyl group.

[0048] In some embodiments, the compound shown in formula (IE) is not

[0049] In some embodiments, in Formula (IE), R 1 For-OR 1-1 ;

[0050] R 1-1 is C1-C6 alkyl or is replaced by one or more R 1-1-1 Substituted C1-C6 alkyl;

[0051] Each R 1-1-1 Each independently is deuterium or -OR a ;

[0052] R a is C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C6 alkyl substituted by one or more deuteriums;

[0053] R 2 and R 3 are each independently halogen;

[0054] R 4 is a C1-C6 alkyl group;

[0055] R 7 is a C1-C6 alkyl group;

[0056] X 1 、X 4 and X 5 Each independently is CH;

[0057] X 3 N or CR X1 ;

[0058] R X1 is a halogen;

[0059] M is O or NH;

[0060] Each R X2-1 Each is independently hydrogen, -OH, -CN or -O-C1-C6 alkyl;

[0061] When M is 0, an R X2-1 is -O-C1-C6 alkyl, another R X2-1 For hydrogen.

[0062] In some embodiments, in Formula (IE), R 1 For-OR 1-1 ;

[0063] R 1-1 For one or more R 1-1-1 Substituted C1-C6 alkyl;

[0064] Each R 1-1-1 Each independently is deuterium or -OR a ;

[0065] R a is C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C6 alkyl substituted by one or more deuteriums;

[0066] R 2 and R 3 are each independently halogen;

[0067] R 4 is a C1-C6 alkyl group;

[0068] R 7 is a C1-C6 alkyl group;

[0069] X 1 、X 4 and X 5 Each independently is CH;

[0070] X 3 N or CR X1 ;

[0071] R X1 is a halogen;

[0072] M is O or NH;

[0073] When M is NH, each R X2-1 are each independently hydrogen, -OH or -CN;

[0074] When M is 0, an R X2-1 is -O-C1-C6 alkyl, another R X2-1 For hydrogen.

[0075] In some embodiments, in Formula (IE), when M is O, R 1-1 For one or more R 1-1-1 Substituted C1-C6 alkyl; each R 1-1-1 For deuterium.

[0076] In some embodiments, in formula (IE), when M is NH, at least one of the following conditions is satisfied:

[0077] (i)R1 Contains deuterium;

[0078] (ii)R a is a C3-C6 cycloalkyl group;

[0079] (iii) Two Rs X2-1 At the same time, it is H.

[0080] In some embodiments, in Formula (IE), R 1-1 For one or more R 1-1-1 Substituted C1-C6 alkyl; each R 1-1-1 For deuterium.

[0081] In some embodiments, the compound shown in formula (IE) is not

[0082] In some embodiments, in Formula (IE), R 1 for

[0083] In some embodiments, in Formula (IE), R 1 for -OCH3

[0084] In some embodiments, in Formula (IE), R 2 For fluorine.

[0085] In some embodiments, in Formula (IE), R 3 For fluorine.

[0086] In some embodiments, in Formula (IE), R 4 It is a methyl group.

[0087] In some embodiments, in Formula (IE), R 7 It is a methyl group.

[0088] In some embodiments, in Formula (IE), for

[0089] In some embodiments, in Formula (IE), for

[0090] In some embodiments, the compound represented by formula (IE) is any of the following compounds:

[0091] In some embodiments, the compound represented by formula (IE) is any of the following compounds:

[0092] enantiomers thereof, diastereomers thereof or mixtures thereof.

[0093] The present invention provides a pharmaceutical composition comprising:

[0094] (1) the compound represented by formula (IE) or a pharmaceutically acceptable salt thereof, and

[0095] (2) Pharmaceutically acceptable excipients.

[0096] The present invention provides a use of the compound represented by formula (IE) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for treating a disease; the disease may be pain, a pain-related disease, multiple sclerosis, incontinence or arrhythmia.

[0097] In some embodiments, the pain is one or more of acute pain, chronic pain, inflammatory pain, cancer pain, neuropathic pain, musculoskeletal pain, primary pain, intestinal pain, and idiopathic pain.

[0098] The present invention provides a compound as shown in formula (IE) or a pharmaceutically acceptable salt thereof, or the use of the above-mentioned pharmaceutical composition in the preparation of a drug for inhibiting voltage-gated sodium channels; the voltage-gated sodium channel is preferably Na V 1.8.

[0099] The present invention provides a use of the compound represented by formula (IE) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a voltage-gated sodium channel inhibitor; the voltage-gated sodium channel is preferably Na V 1.8.

[0100] In some embodiments, the voltage-gated sodium channel inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the art to provide rapid detection of the effect of inhibiting voltage-gated sodium channels.

[0101] The present invention provides a use of the compound represented by formula (IE) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof in the preparation of a drug for a disease caused by abnormal activation of a voltage-gated sodium channel; the voltage-gated sodium channel is preferably Na V1.8; The disease may be pain, a pain-related disease, multiple sclerosis, incontinence or cardiac arrhythmia.

[0102] The present invention provides a method for rapidly detecting voltage-gated sodium channels Na V 1.8. A kit for inhibiting the effect of leukemia / aspartate amino acids, comprising the compound represented by formula (IE) or a pharmaceutically acceptable salt thereof.

[0103] Terminology

[0104] As used herein, the term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0105] In the present invention, the structural fragment This refers to the bond through which the structural fragment is connected to the rest of the molecule. For example, It refers to cyclopropyl.

[0106] In the present invention, the "-" at the end of a group means that the group is connected to the rest of the molecule through this site. For example, -OH refers to a hydroxyl group.

[0107] In the present invention, the term "one or more" refers to 1, 2, 3, 4, 5, 6, 7, such as 1, 2 or 3.

[0108] In the present invention, in the term "B substituted by one or more A", when B is substituted by "multiple" A, A may be the same or different.

[0109] In the present invention, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0110] As used herein, the term "alkyl" refers to a linear or branched, saturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0111] In the present invention, the term "cycloalkyl" refers to a cyclic, saturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C3-C6). Cycloalkyl groups include, but are not limited to: wait.

[0112] As used herein, the term "heterocycloalkyl" refers to a cyclic, saturated, monovalent group having a specified number of ring atoms (e.g., 3-12, 4-8, 5, 6, or 7 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S). Heterocycloalkyl groups are attached to the rest of the molecule through a carbon atom or a heteroatom. Heterocycloalkyl groups include, but are not limited to: wait.

[0113] As used herein, the term "pharmaceutically acceptable excipients" refers to all substances contained in a pharmaceutical formulation other than the active pharmaceutical ingredient, and is generally classified into two categories: excipients and additives. For details, see the Pharmacopoeia of the People's Republic of China (2020 Edition) and the Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0114] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0115] The reagents and raw materials used in the present invention are commercially available.

[0116] The positive progress of the present invention is that the compounds of the present invention have one or more of the following advantages: (1) novel structure; (2) good blocking effect (inhibitory effect) on Nav1.8 channel activity; (3) good selectivity for other Nav subtypes (such as Nav1.1, Nav1.2, Nav1.3Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.9) and high safety. DETAILED DESCRIPTION

[0117] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0118] Preparation Example 1 Synthesis of Intermediate 43-3

[0119] Step 1: Synthesis of intermediate A2

[0120] The synthetic route is shown below:

[0121] Method: A1 (18.00 g, 95.76 mmol) was dissolved in toluene (300 mL). Methyl bromoacetate (13.32 g, 87.07 mmol), Pd(PPh3)4 (3.19 g, 2.76 mmol), Cu2O (0.37 g, 2.62 mmol), and K2CO3 (41.19 g, 298.06 mmol) were added sequentially. The atmosphere was replaced with nitrogen. The reaction mixture was stirred at 100°C for 12 h. After completion of the reaction, the mixture was filtered through celite, concentrated under reduced pressure, and purified by column chromatography to yield A2 (7.60 g, 40.4%) as a white solid. MS (ESI, m / z) 217 ​​[M+H] + .

[0122] Step 2: Synthesis of Intermediate A3

[0123] Method: A2 (7.60 g, 35.18 mmol) was dissolved in THF (80 mL). A lithium hydroxide solution (3.39 g LiOH·H2O dissolved in 40 mL H2O, 80.87 mmol) was added. The reaction mixture was stirred at 50°C for 3 h. After completion of the reaction, the mixture was diluted with DCM and separated. The mixture was extracted three times with H2O. The aqueous phases were combined and the pH was adjusted to <5 with 1N HCl. The organic phases were then extracted three times with DCM. The organic phases were combined, dried, and concentrated under reduced pressure to yield compound A3 (5.90 g, 83.0%) as a white solid. MS (ESI, m / z) 203 [M+H] + .

[0124] Step 3: Synthesis of intermediate S2

[0125] The synthetic route is shown below:

[0126] Method: Compound S1 (15.50 g, 98.05 mmol) was dissolved in Et2O (155 mL). The atmosphere was replaced with N2, and the temperature was lowered to approximately 0°C. Methyllithium bromide complex (209 mL, 313.76 mmol, 1.5 M in Et2O) was added portionwise at 0-10°C. After completion of the addition, the mixture was naturally warmed to room temperature and stirred for 12 hours. After completion of the reaction, H2O (100 mL) and saturated sodium chloride solution (20 mL) were added dropwise to quench the reaction. Citric acid (25.04 g) was added, stirred for 0.5 hours, and extracted three times with Et2O. The organic phases were combined, dried, and concentrated under reduced pressure to 30 g to obtain crude S2 (35% in Et2O, 30 g).

[0127] Step 4: Synthesis of intermediate S3

[0128] The synthetic route is shown below:

[0129] Method: Compound A3 (5.90 g, 29.20 mmol) was dissolved in MeCN (210 mL) and cooled in an ice-water bath. CDI (7.00 g, 30.70 mmol) was added, the atmosphere was replaced with nitrogen, and the mixture was stirred below 10°C for 1.5 h. S2 (about 35% in Et2O, 15 g, 30 mmol) and K2CO3 (5.00 g, 36.50 mmol) were added, and the reaction mixture was stirred at 35°C for 12 h. After the reaction, water and dilute hydrochloric acid were added to the reaction mixture, and the mixture was stirred until clear. The mixture was extracted with EA, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to yield S3 (7.40 g, 78.7%) as a white solid. MS (ESI, m / z) 323 [M+H] + .

[0130] Step 5: Synthesis of S4

[0131] The synthetic route is shown below:

[0132] Methods: Compound S3 (9.00 g, 28.0 mmol) was dissolved in anhydrous methanol (800 mL) and anhydrous tetrahydrofuran (160 mL). The mixture was cooled to -40°C. The first batch of NiCl2·6H2O (6.80 g, 28.6 mmol) and NaBH4 (5.60 g, 148.4 mmol) were added, followed by the second batch of NiCl2·6H2O (6.80 g, 28.6 mmol) and NaBH4 (5.60 g, 148.4 mmol). The reaction was complete upon completion. After completion of the reaction, saturated ammonium chloride was slowly added dropwise to the reaction mixture while maintaining the temperature at -40°C. The mixture was stirred for 30 min and allowed to warm to room temperature. The mixture was filtered through celite, extracted with DCM / MeOH, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to afford compound S4 (3.57 g, 39.3%) as a colorless oil. MS (ESI, m / z) 325 [M+H] + .

[0133] The NMR spectrum is:

[0134] 1 H NMR (400MHz, CDCl3) δ = 6.96-6.83 (m, 2H), 4.47 (d, J = 9.6Hz, 1H), 4.02 (d, J = 2.8Hz, 1H), 2.92-2.84 (m, 1H), 1.70 (s, 3H), 0.81-0.78 (m, 3H).

[0135] Step 6: Synthesis of S5

[0136] The synthetic route is shown below:

[0137] Method: Compound S4 (3.57 g, 11.0 mmol) was dissolved in toluene (30 mL). The atmosphere was replaced with nitrogen and the temperature was lowered to -30°C. DiBAL-H (1.5 M, 7.7 mL, 11.6 mmol) was slowly added dropwise. The reaction mixture was stirred at -25 to -30°C for 2 h. After completion of the reaction, the reaction mixture was quenched by adding saturated ammonium chloride, filtered through celite, extracted with EA, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound S5 (3.53 g, crude) as a colorless oil. MS (ESI, m / z) 327 [M+H] + .

[0138] Step 7: Synthesis of S6

[0139] The synthetic route is shown below:

[0140] Method: Compound S5 (3.53 g, 10.8 mmol) was dissolved in anhydrous DCM (30 mL) and stirred until clear. Et3N (2.18 g, 21.6 mmol) was added, and the mixture was cooled to 0-10°C in an ice-water bath. AcCl (1.70 g, 21.6 mmol) was slowly added dropwise. After the mixture was warmed to room temperature and stirred for 1 h. After the reaction was complete, saturated ammonium chloride was added to the reaction mixture for quenching. The mixture was extracted with DCM, washed with concentrated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound S6 (4.36 g, crude) as a colorless oil. MS (ESI, m / z) 369 [M+H] + .

[0141] Step 8: Synthesis of S7

[0142] The synthetic route is shown below:

[0143] Method: Compound S6 (4.36 g, 11.8 mmol) was dissolved in anhydrous DCM (40 mL). The atmosphere was replaced with N2, and the temperature was lowered to -60°C. TMSCN (3.52 g, 35.5 mmol) and BF3OEt2 (5.04 g, 35.5 mmol) were added dropwise sequentially. After the addition was complete, the mixture was stirred at -60°C for 1 hour, then naturally warmed to room temperature and stirred for 12 hours. After completion of the reaction, the reaction mixture was quenched with saturated sodium carbonate solution, filtered through celite, extracted with DCM, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to afford compound S7 (2.25 g, 56.9%) as a colorless oil. MS (ESI, m / z) 336 [M+H] + .

[0144] Step 9: Synthesis of Intermediate 39-1

[0145] The synthetic route is shown below:

[0146] Method: S7 (1.1 g, 3.28 mmol) was dissolved in DCM (15 mL), the atmosphere was replaced with N2, the temperature was lowered to -78°C, and BBr3 (2.1 mL, 21.32 mmol) was slowly added dropwise. After the addition was complete, the temperature was raised to 0°C and the reaction was stirred for 3 hours. After the reaction was complete, the reaction solution was slowly added dropwise to ice water, extracted with DCM, washed with saturated aqueous NaCl solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain compound 39-1 (1.0 g, crude) as a yellow oil. MS (ESI, m / z) 322 [M+H] + .

[0147] Step 10: Synthesis of Intermediate 43-1

[0148] The synthetic route is shown below:

[0149] Method: Compound 39-1 (300 mg, crude, approximately 0.93 mmol) was dissolved in DMF (5 mL). Cs2CO3 (609 mg, 1.87 mmol) and deuterated iodomethane (542 mg, 3.74 mmol) were added. The mixture was heated to 70°C and stirred for 16 hours. After completion of the reaction, the mixture was quenched with water, extracted with EA, washed with saturated aqueous NaCl solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford compound 43-1 (300 mg, crude) as a yellow oil. MS (ESI, m / z) 339 [M+H] + .

[0150] Step 11: Synthesis of Intermediate 43-2

[0151] The synthetic route is shown below:

[0152] Method: Compound 43-1 (300 mg, crude, approximately 0.93 mmol) was dissolved in MeOH (5 mL). A KOH solution (367 mg, 6.54 mmol, dissolved in 1 mL of water) was added. The reaction mixture was heated to 60°C and stirred for 16 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, diluted with water, and extracted once with MTBE. The aqueous phase was adjusted to pH <3 by dropwise addition of 1N HCl. The mixture was extracted with EA, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield compound 43-2 (260 mg, crude) as a yellow oil. MS (ESI, m / z) 358 [M+H] + .

[0153] Step 12: Synthesis of Intermediate 43-3

[0154] The synthetic route is shown below:

[0155] Methods: Dissolve 43-2 (260 mg, crude, 0.73 mmol) in DCM (4 mL), add 1 drop of DMF, cool to 0°C, and slowly add (COCl)2 (0.09 mL, 1.092 mmol) dropwise. Stir the reaction at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure, redissolve the mixture in DCM (4 mL), and slowly add dropwise to a solution of 3-aminopyridine (69 mg, 0.73 mmol) and TEA (294 mg, 2.91 mmol) in DCM / NMP (1:1, 2 mL). Stir at room temperature for 0.5 h. After completion of the reaction, concentrate the reaction mixture under reduced pressure, extract with EA, and combine the organic phases, wash once with water and once with saturated aqueous NaCl, dry over anhydrous Na2SO4, and concentrate under reduced pressure to yield compound 43-3 (280 mg, crude) as a yellow oil. MS (ESI, m / z) 434 [M+H] + .

[0156] Preparation Example 2 Synthesis of Intermediate 59-3

[0157] Step 1: Synthesis of Intermediate 39-1

[0158] Refer to Preparation Example 1 to synthesize intermediate 39-1.

[0159] Step 2: Synthesis of Intermediate 59-1

[0160] The synthetic route is shown below:

[0161] Method: 39-1 (140 mg, crude, approximately 0.44 mmol) was dissolved in DMF (5 mL), and Cs2CO3 (286 mg, 0.88 mmol) and 2-bromoethanol (220 mg, 6.2 mmol) were added. The mixture was heated to 70°C and stirred for 16 hours. After completion of the reaction, the mixture was quenched with water, extracted with EA, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 59-1 (240 mg, crude) as a yellow oil. MS (ESI, m / z) 366 [M+H] + .

[0162] Step 3: Synthesis of Intermediate 59-2

[0163] The synthetic route is shown below:

[0164] Method: Dissolve 59-1 (240 mg, crude, approximately 0.44 mmol) in DMF (5 mL). Add NaH (35 mg, 0.88 mmol) under ice-cooling. After stirring at room temperature for 0.5 hour, add CD3I (191 mg, 1.32 mmol) and continue stirring at room temperature for 1.5 hours. After completion of the reaction, quench with saturated aqueous NH4Cl solution and extract with EA. The combined organic phases are washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield compound 59-2 (220 mg, crude) as a brown oil. MS (ESI, m / z) 383 [M+H] + .

[0165] Step 4: Synthesis of Intermediate 59-3

[0166] The synthetic route is shown below:

[0167] Method: Dissolve 59-2 (220 mg, crude, approximately 0.44 mmol) in MeOH (5 mL), add an aqueous solution of KOH (172 mg, 3.08 mmol, dissolved in 1 mL of water), and heat to 60°C with stirring for 16 hours. After the reaction, concentrate under reduced pressure, dilute with water, and extract once with MTBE. The aqueous phase is adjusted to pH <3 by dropwise addition of 1N HCl. Extract with EA, and the combined organic phases are washed with saturated aqueous NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield compound 59-3 (110 mg, crude) as a yellow oil. MS (ESI, m / z) 402 [M+H] + .

[0168] Preparation Example 3 Synthesis of Intermediate 60-3

[0169] Step 1: Synthesis of Intermediate 39-1

[0170] Refer to Preparation Example 1 to synthesize intermediate 39-1.

[0171] Step 2: Synthesis of Intermediate 60-1

[0172] The synthetic route is shown below:

[0173] Method: 39-1 (140 mg, crude, approximately 0.44 mmol) was dissolved in DMF (5 mL), and Cs2CO3 (286 mg, 0.88 mmol) and 2-bromoethanol-D4 (227 mg, 1.76 mmol) were added. The mixture was heated to 70°C and stirred for 16 hours. After completion of the reaction, the mixture was quenched with water, extracted with EA, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 60-1 (300 mg, crude) as a yellow oil. MS (ESI, m / z) 370 [M+H] + .

[0174] Step 3: Synthesis of Intermediate 60-2

[0175] The synthetic route is shown below:

[0176] Method: 60-1 (300 mg, crude, approximately 0.44 mmol) was dissolved in DMF (5 mL). NaH (35 mg, 0.88 mmol) was added under ice-cooling. After stirring at room temperature for 0.5 hour, deuterated iodomethane (191 mg, 1.32 mmol) was added and stirring continued at room temperature for 1.5 hours. After completion of the reaction, the mixture was quenched with saturated aqueous NH4Cl solution and extracted with EA. The combined organic phases were washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford compound 60-2 (280 mg, crude) as a brown oil. MS (ESI, m / z) 387 [M+H] + .

[0177] Step 4: Synthesis of intermediate 60-3

[0178] The synthetic route is shown below:

[0179] Method: Dissolve 69-2 (280 mg, crude, approximately 0.44 mmol) in MeOH (5 mL), add an aqueous solution of KOH (172 mg, 3.08 mmol, dissolved in 1 mL of water), and heat to 60°C with stirring for 16 hours. After the reaction, concentrate under reduced pressure, dilute with water, and extract once with MTBE. The aqueous phase is adjusted to pH <3 by dropwise addition of 1N HCl, and extracted three times with EA. The combined organic phases are washed with saturated aqueous NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield compound 69-3 (100 mg, crude) as a yellow oil. MS (ESI, m / z) 406 [M+H] + .

[0180] Example 1 Synthesis of BX20-9-074

[0181] Step 1: Synthesis of Intermediate 43-2

[0182] Refer to Preparation Example 1 to synthesize intermediate 43-2.

[0183] Step 2: Synthesis of Compound 74-1

[0184] The synthetic route is shown below:

[0185] Method: Dissolve 43-2 (100 mg, crude) in DCM (2 mL), add one drop of DMF, and slowly add (COCl)2 (0.2 mL) dropwise. Stir at room temperature for half an hour. The reaction solution is directly concentrated under reduced pressure to obtain intermediate 43-3, which is then dissolved in DCM (2 mL) and slowly added dropwise to a solution of 4-amino-2-cyanopyridine (32 mg, 0.27 mmol) and TEA (62.5 mg, 0.62 mmol) in NMP (2 mL). Stir at room temperature for half an hour. After completion of the reaction, quench with water, extract three times with EA, combine the organic phases, wash once with water, then with saturated NaCl solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain 74-1 (100 mg, crude) as a yellow oil. MS (ESI, m / z) 459 [M+H] + .

[0186] Step 3: Synthesis of compound BX20-9-074

[0187] The synthetic route is shown below:

[0188] Method: Dissolve 74-1 (100 mg, crude) in EtOH (3 mL), add NH2OH (50% in water, 74 mg, 1.12 mmol), and reflux at 80°C with stirring for 1 hour. Dissolve the reaction mixture in MeOH, filter, and purify by preparative chromatography (neutral system). After lyophilization, obtain BX20-9-074 (15 mg, 13.9% yield) as a white solid powder. MS (ESI, m / z) 492 [M+H] + .

[0189] 1 H NMR (400MHz, DMSO-d6) δ = 8.40 (d, J = 5.6Hz, 1H), 8.12 (s, 1H), 7.62-7.60 (m, 1H), 7.16-7.11 (m, 2H), 5.7 6(s,2H),5.06(d,J=10.4Hz,1H),4.24-4.20(m,1H),2.77-2.73(m,1H),1.58(s,3H),0.71-0.69(m,3H).

[0190] Example 2 Synthesis of BX20-9-075

[0191] Step 1: Synthesis of Intermediate 43-2

[0192] Refer to Preparation Example 1 to synthesize intermediate 43-2.

[0193] Step 2: Synthesis of Intermediate 75-1

[0194] The synthetic route is shown below:

[0195] Method: Dissolve 43-2 (100 mg, 0.28 mmol) in DCM (4 mL), add 1 drop of DMF, and slowly add (COCl)2 (53 mg, 0.42 mmol) dropwise in an ice bath. Stir at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure, redissolve the mixture in DCM (4 mL), and slowly add dropwise to a solution of 5-amino-2-fluorobenzonitrile (34 mg, 0.25 mmol) and TEA (85 mg, 0.84 mmol) in DCM / NMP (1:1, 2 mL). Stir at room temperature for 0.5 h. After completion of the reaction, dilute with water and extract three times with EA. The organic phases are combined, washed once with water and once with saturated aqueous NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford compound 75-1 (118 mg, crude) as a brown oil. MS (ESI, m / z) 476 [M+H] + .

[0196] Step 3: Synthesis of BX20-9-075

[0197] The synthetic route is shown below:

[0198] Method: Compound 75-1 (118 mg, crude) was dissolved in EtOH (6 mL), and NH2OH (0.15 mL, 2.0 mmol, 50% in H2O) was added. The mixture was heated to 80°C and stirred for 1 hour. After the reaction, the mixture was filtered, purified by preparative chromatography, and lyophilized to obtain BX20-9-075 (29 mg, 23%) as a white solid. MS (ESI, m / z) 509 [M+H] + .

[0199] 1 H NMR (400MHz, DMSO-d6) δ = 10.32 (s, 1H), 9.62 (s, 1H), 7.75 (dd, J = 2.4, 6.4Hz, 1H), 7.67-7.62 (m, 1H), 7.21-7.11 (m, 3H), 5 .78(s,2H),5.04(d,J=10.4Hz,1H),4.23(dd,J=7.6,10.4Hz,1H),2.80-2.71(m,1H),1.59(s,3H),0.72(d,J=6.0Hz,3H).

[0200] Example 3 Synthesis of BX20-9-079

[0201] Step 1: Synthesis of Intermediate 39-1

[0202] Refer to Preparation Example 1 to synthesize intermediate 39-1.

[0203] Step 2: Synthesis of 79-1

[0204] The synthetic route is shown below:

[0205] Method: Dissolve 39-1 (0.30 g, 0.93 mmol) in DMF (10 mL), add CsCO3 (0.63 g, 1.92 mmol) and 2-cyclopropyloxyethyl 4-methylbenzenesulfonate (0.38 g, 1.92 mmol), and stir at 70°C for 12 hours. After the reaction, extract with water and EA. The combined organic phases are washed with brine, dried, and concentrated under reduced pressure to yield 79-1 (0.31 g, 82.3%) as a brown oil. MS (ESI, m / z) 406 [M+H] + .

[0206] Step 3: Synthesis of 79-2

[0207] The synthetic route is shown below:

[0208] Method: Compound 79-1 (0.31 g, 0.76 mmol) was dissolved in MeOH (5 mL), and a KOH aqueous solution (0.30 g KOH dissolved in 1 mL H2O, 5.33 mmol) was added. The reaction mixture was stirred at 60°C for 5 hours. After completion of the reaction, MTBE was added for extraction. The aqueous phase was adjusted to pH <5 with hydrochloric acid and extracted with MTBE. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated to yield a brown oil, 79-2 (0.21 g, 65.2%). MS (ESI, m / z) 425 [M+H] +

[0209] Step 4: Synthesis of 79-4

[0210] The synthetic route is shown below:

[0211] Method: 79-2 (0.21 g, 0.50 mmol) was dissolved in DCM (6 mL), and one drop of DMF was added. (COCl)2 (0.13 g, 1.00 mmol) was added dropwise at 0°C, and the reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford crude product 79-3 (0.23 g, crude), which was used directly in the next step. 4-Fluoro-3-cyanoaniline (82 mg, 0.60 mmol) was dissolved in NMP (2 mL), and TEA (101 mg, 1.00 mmol) was added. Then, 79-3 (crude, diluted with 4 mL of DCM, approximately 0.50 mmol) was slowly added to the mixture, and stirred at room temperature for 0.5 h. After completion of the reaction, the mixture was quenched with saturated ammonium chloride and extracted with EA. The organic phases were combined, washed with brine, dried, and concentrated under reduced pressure to afford 79-4 (0.31 g, crude) as a brown oil. MS (ESI, m / z) 543 [M+H] + .

[0212] Step 5: Synthesis of BX20-9-079

[0213] The synthetic route is shown below:

[0214] Method: Compound 79-4 (0.31 g, crude product, approximately 0.50 mmol) was dissolved in EtOH (5 mL), and NH2OH (151 mg, 2.29 mmol) was added. The mixture was heated to 80°C and stirred for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by preparative chromatography under neutral conditions to yield BX20-9-079 (80 mg, 27.8%) as a white solid. MS (ESI, m / z) 576 [M+H] + .

[0215] 1 H NMR (400MHz, DMSO-d6) δ=10.28(s,1H),9.62(s,1H),7.77-7.73(m,1H),7.68-7.62(m,1H),7.22-7.11(m,3H),5.77(s,2H),5.06(d,J=10.8 Hz,1H),4.33-4.18(m,3H),3.75-3.70(m,2H),3.37-3.34(m,1H),2.88-2.79(m,1H),1.61(s,3H),0.70(d,J=7.2Hz,3H),0.49-0.37(m,4H).

[0216] Example 4 Synthesis of BX20-9-054

[0217] Step 1: Synthesis of Intermediate 43-2

[0218] Refer to Preparation Example 1 to synthesize intermediate 43-2.

[0219] Step 2: Synthesis of Intermediate 54-1

[0220] The synthetic route is shown below:

[0221] Methods: Dissolve 43-2 (200 mg, crude, 0.56 mmol) in DCM (4 mL), add 1 drop of DMF, cool to 0°C, and slowly add (COCl)2 (107 mg, 0.84 mmol) dropwise. Stir at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure, redissolve the mixture in DCM (4 mL), and slowly add dropwise to a solution of methyl 2-fluoro-5-aminobenzoate (95 mg, 0.56 mmol) and TEA (170 mg, 1.68 mmol) in DCM / NMP (1:1, 2 mL). Stir at room temperature for 0.5 h. After completion of the reaction, quench with water and extract with EA. The combined organic phases are washed once with water and once with saturated aqueous NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford compound 53-1 (310 mg, crude) as a black oil. MS (ESI, m / z) 509 [M+H] + .

[0222] Step 3: Synthesis of Intermediate 54-2

[0223] The synthetic route is shown below:

[0224] Method: Dissolve 54-1 (310 mg, crude product, approximately 0.56 mmol) in THF (5 mL) and add a solution of LiOH·H2O (282 mg, 6.72 mmol, dissolved in 3 mL of water). Stir at room temperature for 3 hours. After completion of the reaction, concentrate under reduced pressure, dilute with water, and adjust the pH to <3 with the addition of 1N HCl. Extract with EA, wash with saturated aqueous NaCl, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound 53-2 (280 mg, crude product) as a black oil. MS (ESI, m / z) 495 [M+H] + .

[0225] Step 4: Synthesis of BX20-9-054

[0226] The synthetic route is shown below:

[0227] Methods: Compound 54-2 (280 mg, crude, approximately 0.56 mmol) was dissolved in DCM (4 mL). One drop of DMF was added, the temperature was lowered to 0°C, and (COCl)₂ (107 mg, 0.84 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 0.5 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to yield the crude acid chloride. Separately, methoxyamine hydrochloride (94 mg, 1.12 mmol) and K₂CO₃ (255 mg, 1.85 mmol) were added to an EA / H₂O (2:1, 9 mL) solution. After stirring at room temperature for 20 minutes, the crude acid chloride in EA was added dropwise, and stirring was continued at room temperature for 0.5 hours. After completion of the reaction, the mixture was diluted with water and extracted with EA. The combined organic phases were washed once with saturated aqueous NaCl, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by preparative chromatography, and lyophilized to yield BX20-9-054 (112 mg, 38%) as a white solid. MS (ESI, m / z) 524 [M+H] + .

[0228] 1 H NMR (400MHz, DMSO-d6) δ = 11.39 (s, 1H), 10.34 (s, 1H), 7.82 (d, J = 6.0Hz, 1H), 7.72-7.65 (m, 1H), 7.21 (t, J = 9.2Hz, 1H), 7.15-7.06 (m,2H),5.00(d,J=10.0Hz,1H),4.18(dd,J=8.0,10.8Hz,1H),3.64(s,3H),2.75-2.66(m,1H),1.55(s,3H),0.67(d,J=6.0Hz,3H).

[0229] Example 5 Synthesis of BX20-9-080

[0230] Step 1: Synthesis of Intermediate 43-2

[0231] Refer to Preparation Example 1 to synthesize intermediate 43-2.

[0232] Step 2: Synthesis of Intermediate 80-2

[0233] The synthetic route is shown below:

[0234] Method: 43-2 (250 mg, 0.70 mmol) was dissolved in DCM (4 mL), and one drop of DMF was added. (COCl)2 (178 mg, 1.40 mmol) was added dropwise at 0°C, and the reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford crude product 80-1 (0.27 g, crude), which was used directly in the next step. 4-Fluoro-3-cyanoaniline (114 mg, 0.84 mmol) was dissolved in NMP (2 mL), and TEA (141 mg, 1.40 mmol) was added. 80-1 (crude, diluted with 4 mL of DCM, approximately 0.70 mmol) was slowly added to the mixture, and stirred at room temperature for 0.5 h. After completion of the reaction, the mixture was quenched with saturated ammonium chloride and extracted with EA. The organic phases were combined, washed with brine, dried, and concentrated under reduced pressure to afford 80-2 (350 mg, crude) as a brown oil. MS (ESI, m / z) 476 [M+H] + .

[0235] Step 3: Synthesis of Intermediate 80-3

[0236] The synthetic route is shown below:

[0237] Method: Dissolve compound 80-2 (350 mg, crude, approximately 0.70 mmol) in EtOH (5 mL), cool in an ice bath, add NH2OH (185 mg, 2.80 mmol), and heat to 80°C with stirring for 1 hour. After the reaction, concentrate under reduced pressure to obtain a brown oil 80-3 (380 mg, crude). MS (ESI, m / z) 509 [M+H] + .

[0238] Step 4: Synthesis of intermediate 80-4

[0239] The synthetic route is shown below:

[0240] Method: Compound 80-3 (380 mg, crude product, approximately 0.70 mmol) was dissolved in AcOH (6 mL). Pd / C (500 mg) and ammonium formate (800 mg, 12.7 mmol) were added. The atmosphere was replaced with N2 and the temperature was raised to 120°C with stirring for 4 hours. After completion of the reaction, the mixture was filtered through Celite, concentrated under reduced pressure, extracted with EA, dried, and concentrated under reduced pressure to afford 80-4 (180 mg, 52.3%) as a brown solid. MS (ESI, m / z) 493 [M+H] + .

[0241] Step 5: Synthesis of intermediate BX20-9-080

[0242] The synthetic route is shown below:

[0243] Method: Compound 80-4 (180 mg, 0.36 mmol) was dissolved in EtOH (6 mL), and TEA (91 mg, 0.90 mmol) was added. The mixture was cooled in an ice bath, and BrCN (69 mg, 0.65 mmol, dissolved in 2 mL ACN) was slowly added dropwise. Stirring was continued for 10 minutes. After completion of the reaction, the mixture was quenched with water, extracted with EA, dried, and concentrated under reduced pressure. Purification by preparative formic acid chromatography afforded BX20-9-080 (100 mg, 49.3%) as a white solid. MS (ESI, m / z) 518 [M+H] + .

[0244] 1 H NMR (400MHz, DMSO-d6) δ = 10.48 (s, 1H), 8.99 (s, 1H), 8.77 (s, 1H), 8.00-7.82 (m, 1H), 7.81-7.76 (m, 1H), 7.35 (s, 1H) ),7.21-7.11(m,2H),5.07(d,J=10.4Hz,1H),4.28-4.20(m,1H),2.81-2.71(m,1H),1.60(s,3H),0.76-0.69(m,3H).

[0245] Example 6 Synthesis of BX20-9-082

[0246] Step 1: Synthesis of Intermediate 59-3

[0247] Reference Preparation Example 2 Synthesis of Intermediate 59-3

[0248] Step 2: Synthesis of Intermediate 82-1

[0249] The synthetic route is shown below:

[0250] Method: Dissolve 59-3 (180 mg, 0.45 mmol) in DCM (5 mL), add 1 drop of DMF, cool to 0°C, and slowly add (COCl)2 (0.2 mL) dropwise. Stir at room temperature for 0.5 h. After the reaction, concentrate under reduced pressure, redissolve in DCM (5 mL), and slowly add dropwise to a solution of 5-amino-2-fluorobenzonitrile (55 mg, 0.40 mmol) and TEA (91 mg, 0.90 mmol) in NMP (3 mL). Stir at room temperature for 0.5 h. After the reaction, quench with water, extract with EA, and combine the organic phases, wash once with water and once with saturated aqueous NaCl solution. Dry the organic phase over anhydrous sodium sulfate and concentrate under reduced pressure to obtain compound 82-1 (200 mg, crude) as a yellow oil. MS (ESI, m / z) 520 [M+H] + .

[0251] Step 3: Synthesis of compound BX20-9-082

[0252] The synthetic route is shown below:

[0253] Method: 82-1 (200 mg, crude product) was dissolved in EtOH (5 mL), and an aqueous solution of NH2OH (50%, 153 mg, 2.32 mmol) was added. The mixture was stirred at 80°C for 1 hour. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove EtOH, dissolved in MeOH, filtered, and purified by preparative chromatography (FA system). After lyophilization, a white powder solid BX20-9-082 (33 mg, yield 15.7%) was obtained. MS (ESI, m / z) 553 [M+H] + .

[0254] 1 H NMR (400MHz, DMSO-d6) δ=10.28(s,1H),9.59(s,1H),7.74-7.72(dd,J=6.4Hz,J=2.8Hz,1H),7.64-7.60(m,1H),7.17-7.12(m,3H ),5.75(s,2H),5.03(d,J=10.8Hz,1H),4.32-4.14(m,3H),3.60-3.58(m,2H),2.82-2.78(m,1H),1.57(s,3H),0.68-0.66(m,3H).

[0255] Example 7 Synthesis of BX20-9-083

[0256] Step 1: Synthesis of Intermediate 60-3

[0257] Refer to Preparation Example 3 Intermediate 60-3.

[0258] Step 2: Synthesis of Intermediate 83-1

[0259] The synthetic route is shown below:

[0260] Methods: Dissolve 60-3 (260 mg, crude, approximately 0.64 mmol) in DCM (5 mL), add 1 drop of DMF, cool to 0°C, and slowly add (COCl)2 (163 mg, 1.28 mmol) dropwise. Stir at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure, redissolve in DCM (5 mL), and slowly add dropwise to a solution of 3-cyano-4-fluoroaniline (105 mg, 0.77 mmol) and TEA (194 mg, 1.92 mmol) in NMP (3 mL). Stir at room temperature for 0.5 h. After completion of the reaction, quench with water, extract with EA, and combine the organic phases, wash once with water and once with saturated aqueous NaCl, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to afford compound 83-1 (330 mg, crude) as a brown oil. MS (ESI, m / z) 524 [M+H] + .

[0261] Step 3: Synthesis of BX20-9-083

[0262] The synthetic route is shown below:

[0263] Method: Dissolve 83-1 (330 mg, crude product, approximately 0.64 mmol) in EtOH (5 mL), add aqueous NH2OH (169 mg, 2.56 mmol, 50 wt% aqueous solution), and heat to 80°C with stirring for 1 hour. After completion of the reaction, concentrate under reduced pressure, purify by preparative chromatography, and lyophilize to obtain BX20-9-83 (22 mg, 6.2%) as a white solid. MS (ESI, m / z) 557 [M+H] + .

[0264] 1 H NMR (400MHz, DMSO-d6) δ = 10.89 (s, 1H), 10.52 (d, J = 3.2Hz, 1H), 7.95-7.90 (m, 1H), 7.81-7.75 (m, 1H), 7.41-7.33 (m, 1 H),7.18-7.11(m,2H),5.09(d,J=10.8Hz,1H),4.37-4.30(m,1H),2.90-2.79(m,1H),1.59(s,3H),0.75-0.67(m,3H).

[0265] Example 8 Synthesis of BX20-9-084

[0266] Step 1: Synthesis of Intermediate 39-1

[0267] Refer to Preparation Example 1 to synthesize intermediate 39-1.

[0268] Step 2: Synthesis of Intermediate 39-2

[0269] The synthetic route is shown below:

[0270] Method: Dissolve 39-1 (500 mg, crude, approximately 1.5 mmol) in DMF (5 mL), add Cs2CO3 (2.1 mL, 21.32 mmol) and 1-iodo-2-methoxyethane (1.15 g, 6.2 mmol), and heat to 70°C with stirring for 16 hours. After completion of the reaction, quench with water, extract with EA, wash with saturated aqueous NaCl, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain compound 39-2 (450 mg, crude) as a yellow oil. MS (ESI, m / z) 380 [M+H] + .

[0271] Step 3: Synthesis of Intermediate 39-3

[0272] The synthetic route is shown below:

[0273] Method: Dissolve 39-2 (450 mg, crude, 1.18 mmol) in MeOH (7.5 mL) and add an aqueous solution of KOH (462 mg, 8.26 mmol, dissolved in 1.5 mL of water). Heat to 60°C and stir for 16 hours. After completion of the reaction, concentrate under reduced pressure, dilute with water, extract with MTBE, and adjust the aqueous phase to pH <3 by dropwise addition of 1N HCl. Extract with EA, wash with saturated aqueous NaCl, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to yield compound 39-3 (430 mg, crude) as a yellow oil. MS (ESI, m / z) 399 [M+H] + .

[0274] Step 4: Synthesis of Intermediate 39-4

[0275] The synthetic route is shown below:

[0276] Methods: 39-3 (160 mg, crude, 0.402 mmol) was dissolved in DCM (4 mL), 1 drop of DMF was added, the temperature was lowered to 0°C, and (COCl)2 (0.1 mL, 1.206 mmol) was slowly added dropwise. The reaction was stirred at room temperature for 0.5 h. After completion of the reaction, the mixture was concentrated under reduced pressure, redissolved in DCM (4 mL), and slowly added dropwise to a solution of 5-amino-2-fluorobenzonitrile (55 mg, 0.402 mmol) and TEA (122 mg, 1.206 mmol) in DCM / NMP (4:1, 5 mL). The mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, dissolved in EA, quenched with water, and extracted with EA. The organic phases were combined, washed once with water and once with saturated aqueous NaCl, dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford compound 39-4 (207 mg, crude) as a yellow oil. MS (ESI, m / z) 517 [M+H] + .

[0277] Step 5: Synthesis of Intermediate 39-5

[0278] The synthetic route is shown below:

[0279] Methods: Compound 39-4 (207 mg, crude product, approximately 0.4 mmol) was dissolved in EtOH (5 mL), and NH2OH (0.1 mL, 1.608 mmol, 50% in H2O) was added. The reaction was heated to 80°C and stirred for 1 hour. After completion of the reaction, the product was concentrated under reduced pressure and purified by preparative chromatography (formic acid system). The product was lyophilized to afford 39-5 (112 mg, 51%) as a white solid. MS (ESI, m / z) 550 [M+H] + .

[0280] Step 6: Synthesis of intermediate BX20-9-084

[0281] The synthetic route is shown below:

[0282] Method: Compound 39-5 (90 mg, 0.16 mmol) was dissolved in AcOH (5 mL), and Pd / C (100 mg, 10% purity) and HCOONH4 (202 mg, 3.2 mmol) were added. After replacing the nitrogen atmosphere, the temperature was raised to 120°C and stirred for 2.5 hours. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by preparative chromatography (formic acid system). The mixture was lyophilized to obtain BX20-9-084 (48 mg, 56%) as a white solid. MS (ESI, m / z) 534 [M+H] + .

[0283] 1H NMR(400MHz,DMSO-d6)δ10.72(s,1H),8.43(s,1H),7.97(dd,J=2.4,6.0Hz, 1H),7.90-7.83(m,1H),7.41(t,J=9.6Hz,1H),7.22-7.11(m,2H),5.13(d,J =10.8Hz,1H),4.34(dd,J=7.2,10.8Hz,1H),4.30-4.17(m,2H),3.65-3.61( m,2H),3.30(s,3H),2.88-2.80(m,1H),1.60(s,3H),0.70(d,J=5.6Hz,3H).

[0284] Example 9 Synthesis of BX20-9-090

[0285] Step 1: Synthesis of intermediate 39-3

[0286] Refer to Example 8 to synthesize intermediate 39-3.

[0287] Step 2: Synthesis of Intermediate 90-1

[0288] The synthetic route is shown below:

[0289] Method: 39-3 (160 mg, 0.40 mmol) was dissolved in DCM (4 mL), and one drop of DMF was added. (COCl)2 (102 mg, 0.80 mmol) was added dropwise at 0°C, and the reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford crude product 39-4 (200 mg, crude), which was used directly in the next step. 4-Fluoro-3-cyanoaniline (66 mg, 0.48 mmol) was dissolved in NMP (2 mL), and TEA (81 mg, 0.80 mmol) was added. 39-4 (crude product, diluted with 4 mL of DCM, approximately 0.40 mmol) was then slowly added and stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was quenched with saturated ammonium chloride and extracted with EA. The organic phases were combined, washed with brine, dried, and concentrated under reduced pressure to afford 90-1 (250 mg, crude) as a brown oil. MS (ESI, m / z) 517 [M+H] + .

[0290] Step 3: 90-2 Synthesis

[0291] The synthetic route is shown below:

[0292] Method: Compound 90-1 (250 mg, crude, approximately 0.40 mmol) was dissolved in EtOH (5 mL), cooled in an ice bath, and NH2OH (50% aqueous solution, 106 mg, 1.60 mmol) was added. The temperature was raised to 80°C and stirred for 1 hour. After the reaction, the mixture was concentrated under reduced pressure to obtain a brown oil, 90-2 (280 mg, crude). MS (ESI, m / z) 550 [M+H] + .

[0293] Step 4: Synthesis of 90-3

[0294] The synthetic route is shown below:

[0295] Method: Compound 90-2 (280 mg, crude product, approximately 0.40 mmol) was dissolved in AcOH (6 mL). Pd / C (10%, 500 mg) and ammonium formate (800 mg, 12.7 mmol) were added. The atmosphere was replaced with N2 and the mixture was heated to 120°C with stirring for 4 hours. After completion of the reaction, the mixture was filtered through celite, concentrated under reduced pressure, extracted with EA, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford 90-3 (170 mg, 79.7%) as a brown solid. MS (ESI, m / z) 534 [M+H] + .

[0296] Step 5: Synthesis of BX20-9-090

[0297] The synthetic route is shown below:

[0298] Method: Compound 90-3 (170 mg, 0.31 mmol) was dissolved in EtOH (6 mL), and TEA (79 mg, 0.78 mmol) was added. The mixture was cooled in an ice bath, and a solution of BrCN (59 mg, 0.56 mmol) in acetonitrile (2 mL) was slowly added dropwise. After the addition was complete, stirring was continued for 10 minutes. After completion of the reaction, the mixture was quenched with water, extracted with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative neutral chromatography to afford BX20-9-090 (36 mg, 20.8%) as a white solid. MS (ESI, m / z) 559 [M+H] + .

[0299] 1H NMR (400MHz, DMSO-d6)δ=10.41(s,1H),8.97(s,1H),8.79(s,1H),7.85(s,1H),7.81-7.75(m,1H),7.35(s,1H),7.21-7.12(m,2H),5.08(d ,J=10.8Hz,1H),4.37-4.24(m,2H),4.23-4.16(m,1H),3.68-3.57(m,2H),3.28(s,3H),2.88-2.80(m,1H),1.60(s,3H),0.73-0.67(m,3H).

[0300] Example 10 Synthesis of BX20-9-095

[0301] Step 1: Synthesis of Intermediate 39-1

[0302] Refer to Preparation Example 1 to synthesize intermediate 39-1.

[0303] Step 2: Synthesis of Intermediate 40-1

[0304] The synthetic route is shown below:

[0305] Method: Dissolve 39-1 (500 mg, crude, 1.55 mmol) in DMF (5 mL), add Cs2CO3 (2.1 mL, 21.32 mmol) and 3-iodooxetane (1.14 g, 6.2 mmol), and heat to 70°C with stirring for 16 hours. After completion of the reaction, quench with water, extract with EA, wash with saturated aqueous NaCl, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain compound 40-1 (565 mg, crude) as a yellow oil. MS (ESI, m / z) 378 [M+H] + .

[0306] Step 3: Synthesis of Intermediate 40-2

[0307] The synthetic route is shown below:

[0308] Method: Dissolve 40-1 (565 mg, crude product, 1.5 mmol) in MeOH (7.5 mL) and add an aqueous solution of KOH (587 mg, 10.5 mmol, dissolved in 1.5 mL of water). Heat to 60°C and stir for 16 hours. After completion of the reaction, concentrate under reduced pressure, dilute with water, extract with MTBE, and adjust the aqueous phase to pH <3 by dropwise addition of 1N HCl. Extract with EA, wash with saturated aqueous NaCl, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to yield compound 40-2 (330 mg, crude product) as a yellow oil. MS (ESI, m / z) 397 [M+H]+ .

[0309] Step 4: Synthesis of Intermediate 95-1

[0310] The synthetic route is shown below:

[0311] Method: Dissolve 40-2 (440 mg, 1.11 mmol) in MeCN (8 mL), add 2-cyano-4-aminopyridine (146 mg, 1.22 mmol), TCFH (374 mg, 1.33 mmol), and DIPEA (574 mg, 4.44 mmol), and stir at room temperature for 3 hours. After completion of the reaction, extract with EA. The organic phase is washed once with water and once with saturated aqueous NaCl, dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford compound 95-1 (620 mg, crude) as a brown oil. MS (ESI, m / z) 498 [M+H] + .

[0312] Step 5: Synthesis of 95-2

[0313] The synthetic route is shown below:

[0314] Method: Compound 95-1 (200 mg, crude, approximately 0.4 mmol) was dissolved in DCE (5 mL), and m-CPBA (85%, 244 mg, 1.2 mmol) was added. The mixture was heated to 80°C and stirred for 4 hours. After completion of the reaction, the mixture was cooled to room temperature, quenched with NaOH solution, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford 95-2 (220 mg, crude) as a brown oil. MS (ESI, m / z) 514 [M+H] + .

[0315] Step 6: Synthesis of BX20-9-095

[0316] The synthetic route is shown below:

[0317] Method: Compound 95-2 (220 mg, crude product, approximately 0.4 mmol) was dissolved in EtOH (8 mL), and NH2OH (50% aqueous solution, 106 mg, 1.6 mmol) was added dropwise. The temperature was raised to 80°C and stirred for 1 hour. After the reaction, the mixture was concentrated under reduced pressure and purified by preparative formic acid chromatography to afford BX20-9-095 (54 mg, 24.7%) as a white solid. MS (ESI, m / z) 547 [M+H] + .

[0318] 1H NMR (400MHz, DMSO-d6) δ = 10.73 (s, 1H), 10.15 (s, 1H), 8.22-8.17 (m, 2H), 7.72-7.67 (m, 1H), 7.19-7.13 (m, 2H), 6.78 (s, 2H), 5.33-5.25 (m ,1H),5.10(d,J=10.0Hz,1H),4.91-4.80(m,2H),4.73-4.64(m,2H),4.30-4.22(m,1H),2.82-2.72(m,1H),1.61(s,3H),0.76-0.67(m,3H).

[0319] Example 11 Synthesis of BX20-9-096

[0320] Step 1: Synthesis of intermediate 39-3

[0321] Refer to Example 8 to synthesize intermediate 39-3.

[0322] Step 2: Synthesis of 96-1

[0323] The synthetic route is shown below:

[0324] Method: 39-3 (140 mg, 0.35 mmol) was dissolved in DCM (4 mL), and one drop of DMF was added. (COCl)2 (89 mg, 0.70 mmol) was added dropwise at 0°C, and the reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford crude product 39-4 (180 mg, crude), which was used directly in the next step. 2-Cyano-4-aminopyridine (50 mg, 0.42 mmol) was dissolved in NMP (2 mL), and TEA (71 mg, 0.80 mmol) was added. 39-4 (180 mg crude, diluted with 4 mL of DCM, approximately 0.40 mmol) was slowly added to the mixture, and stirred at room temperature for 0.5 h. After completion of the reaction, the mixture was quenched with water and extracted with EA. The organic phases were combined, washed with brine, dried, and concentrated under reduced pressure to afford 96-1 (210 mg, crude) as a brown oil. MS (ESI, m / z) 500 [M+H] + .

[0325] Step 3: Synthesis of 96-2

[0326] The synthetic route is shown below:

[0327] Method: Compound 96-1 (500 mg, crude, approximately 1.0 mmol) was dissolved in DCE (7 mL), and m-CPBA (85%, 406 mg, 2.0 mmol) was added. The mixture was heated to 80°C and stirred for 4 hours. After completion of the reaction, the mixture was cooled to room temperature, quenched with NaOH solution, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 96-1 (560 mg, crude) as a brown oil. MS (ESI, m / z) 516 [M+H] + .

[0328] Step 4: Synthesis of BX20-9-096

[0329] The synthetic route is shown below:

[0330] Method: Compound 96-2 (560 mg, crude product, approximately 1.09 mmol) was dissolved in EtOH (5 mL). NH2OH (50% aqueous solution, 287 mg, 4.36 mmol) was added and the mixture was heated to 80°C with stirring for 1 hour. After the reaction, the mixture was concentrated under reduced pressure and purified by preparative neutral chromatography to afford BX20-9-096 (196 mg, 32.8%) as a white solid. MS (ESI, m / z) 549 [M+H] + .

[0331] 1 H NMR (400MHz, DMSO-d6) δ=8.23-8.15(m,2H),7.73-7.67(m,1H),7.21-7.12(m,2H),6.77(s,2H),5.08(d,J=10.8Hz,1H),4.37- 4.30(m,1H),4.30-4.14(m,2H),3.66-3.55(m,2H),3.26(d,J=0.8Hz,3H),2.87-2.76(m,1H),1.60(s,3H),0.73-0.66(m,3H).

[0332] Example 12 Synthesis of BX20-9-098

[0333] Step 1: Synthesis of Intermediate 96-1

[0334] Refer to Example 10 to synthesize intermediate 95-1.

[0335] Step 2: Synthesis of BX20-9-098

[0336] The synthetic route is shown below:

[0337] Method: Compound 95-1 (250 mg, 0.5 mmol) was dissolved in EtOH (5 mL), and NH2OH (50% aqueous solution, 132 mg, 2.0 mmol) was added. The mixture was heated to 80°C and stirred for 1 hour. After the reaction, the mixture was concentrated under reduced pressure and purified by preparative neutral chromatography to afford BX20-9-098 (85 mg, 32.1%) as a white solid. MS (ESI, m / z) 531 [M+H] + .

[0338] 1 H NMR (400MHz, DMSO-d6) δ = 10.65 (s, 1H), 9.84 (s, 1H), 8.45-8.40 (m, 1H), 8 .18-8.12(m,1H),7.68-7.61(m,1H),7.22-7.10(m,2H),5.79(s,2H),5.34 -5.25(m,1H),5.11(d,J=10.4Hz,1H),4.91-4.81(m,2H),4.73-4.65(m,2 H),4.31-4.23(m,1H),2.85-2.74(m,1H),1.61(s,3H),0.77-0.68(m,3H).

[0339] Example 13 Synthesis of BX20-9-099

[0340] Step 1: Synthesis of Intermediate 96-1

[0341] Reference Example 11 Step 1 to Step 2 Synthesis of Intermediate 96-1

[0342] Step 2: BX20-9-099 synthesis

[0343] The synthetic route is shown below:

[0344] Method: Compound 96-1 (210 mg, crude product, approximately 0.42 mmol) was dissolved in EtOH (5 mL), cooled in an ice bath, and NH2OH (50% aqueous solution, 111 mg, 1.68 mmol) was added. The temperature was raised to 80°C and stirred for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by preparative chromatography to afford BX20-9-099 (61 mg, 27.3%) as a white solid. MS (ESI, m / z) 533 [M+H] + .

[0345] 1H NMR (400MHz, DMSO-d6) δ=8.43-8.40(m,1H),8.17-8.13(m,1H),7.67-7.62(m,1H),7.22-7.11(m,2H),5.78(s,2H),5.09(d,J=10.8 Hz,1H),4.38-4.24(m,2H),4.22-4.15(m,1H),3.67-3.56(m,2H),3.27(s,3H),2.87-2.78(m,1H),1.61(s,3H),0.73-0.67(m,3H).

[0346] Reference compound:

[0347] Reference patent CN114945566A Example 3 synthesis method, the reference compound (Compound 7)

[0348] 1 H NMR (400MHz, CD3OD) δ = 8.48 (d, J = 5.6Hz, 1H), 8.25 (d, J = 2.0Hz, 1H), 7.89 (dd, J = 2.0Hz, 5.2Hz, 1H), 7.13-7.09 (m, 1H), 7.00-6. 94(m,1H),5.08(d,J=10.4Hz,1H),4.34-4.30(m,1H),3.99(d,J=2.4Hz,3H),2.83-2.75(m,1H),1.65(s,3H),0.82-0.80(m,3H)

[0349] Biological test evaluation

[0350] The present invention is further described below in conjunction with test examples.

[0351] Test Example 1 Blocking activity of the compound of the present invention on sodium ion channel 1.8 (Nav1.8)

[0352] 1. Experimental purpose: To detect the effect of compounds on the current of voltage-gated sodium channel (NaV) 1.8 subtype using patch clamp technique

[0353] 2. Experimental materials and equipment

[0354] 2.1 Cell Line: A CHO cell line stably expressing the Nav1.8 sodium channel was constructed in-house by the Beijing Aisiyipu Biotechnology Co., Ltd. (referring to Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A, Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR, Krafte DS. A-803467, a potent and selective Nav1.8sodium channel blocker,attenuates neuropathic and inflammatory pain in the rat.Proc Natl Acad Sci US A.2007May 15;104(20):8520-5.doi:10.1073 / pnas.0611364104.Epub 2007 May 2. PMID: 17483457; PMCID: PMC1895982.), gene information: Sodium channel, voltage-gated, type 8, alpha (SCN10A), cDNA strictly similar to GenBank accession number: NM_006514

[0355] 2.2 Compound: Dissolved in DMSO

[0356] 3. Experimental Methods

[0357] Cell culture

[0358] (1) Maintenance medium: The cells were cultured in HAM'S / F-12 medium containing 10% fetal bovine serum, 10 μg / mL blasticidin, 200 μg / mL hygromycin B, and 100 μg / mL zeocin at 37°C and 5% carbon dioxide.

[0359] (2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of 0.25%-Trypsin-EDTA solution and incubate at 37°C for about 1.5 minutes. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37°C. Gently blow the cell suspension with a pipette to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes to collect the cells. For expansion or maintenance culture, inoculate the cells into 6 cm cell culture dishes, with 2.5×105 cells in each cell culture dish (final volume: 5 mL).

[0360] (3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0361] (4) Patch clamp assay: Before the test, cells were detached with 0.25%-Trypsin-EDTA, 6.5×10 3 cells were plated on a coverslip, and cultured in a 24-well plate (final volume: 500 μL). After 18 hours, the test was performed.

[0362] 3.2. Patch clamp assay

[0363] (1) After the whole-cell seal is formed, the cell voltage is clamped at -120mV. First, the voltage is stepped from -130mV to -10mV in 10mV steps and maintained for 5s, and then a 0mV depolarizing pulse is given to obtain the half-inactivated voltage (Vhalf). The resting state (Resting state) and half-inactivated state (Half-inactivated state) of the sodium current are detected using a double pulse mode. First, the first depolarizing pulse (TP1) is given to 0mV for 50ms to detect the resting state sodium current. Then the voltage is adjusted to Vhalf and maintained for 5s, and then the voltage is restored to -120mV and maintained for 20ms. Then a second depolarizing pulse (TP2) is given to 0mV for 50ms to detect the half-inactivated state sodium current. Finally, it is restored to the clamping voltage of -120mV. Data are repeatedly collected every 20ms to observe the effect of the drug on the peak sodium current in the two different states. The experimental data were collected by EPC 10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0364] (2) During patch clamping, a microelectrode puller is first used to pull a capillary glass tube into a recording electrode. The electrode, filled with intracellular fluid, is then placed in a microelectrode holder. Under an inverted microscope, the microelectrode manipulator is used to bring the recording electrode into contact with the cell. Negative pressure is applied to form a GΩ seal. Fast capacitance compensation is then performed, and negative pressure is continued to be applied to rupture the cell membrane, forming a whole-cell recording mode. Finally, slow capacitance compensation is performed and the relevant parameters are recorded. No leakage compensation is performed.

[0365] (3) When the sodium current recorded in the whole cell is stable, the drug is administered. Each drug concentration is applied for 5 minutes (or the current is stable) before the next concentration is detected. The coverslip with cells is placed in a recording bath under an inverted microscope. The blank control external solution and the working solution of the compound to be tested are flowed through the recording bath in sequence from low concentration to high concentration by gravity perfusion to act on the cells. A peristaltic pump is used for liquid exchange during the recording. The current detected for each cell in the external solution without the compound serves as its own control group. Each concentration is tested twice independently. All electrophysiological experiments are performed at room temperature.

[0366] Data analysis

[0367] First, the current after each drug concentration and the blank control current were normalized, and then the inhibition rate corresponding to each drug concentration was calculated, that is, (1-compound action current / blank control current), and the mean (Mean), standard deviation (SD) and standard error (SE) of the inhibition rate at each concentration were calculated, and the data were expressed as mean ± SE.

[0368] 4. Experimental Results

[0369] Table 1 Blockade rate of the compounds of the present invention on NaV1.8 at 10 nM

[0370] It can be seen that the compound of the present invention has a significant blocking effect on the activity of NaV1.8 channels.

[0371] Test Example 2 Blocking activity (IC) of the compounds of the present invention on sodium ion channel 1.8 (Nav1.8) 50 )

[0372] 1. Research Objectives

[0373] Manual patch clamp technique is used to evaluate whether the test compound has a potential inhibitory effect on the voltage-gated sodium channel hNav1.8. This experiment measures the effect of the compound at 5 concentrations or at a single or dual concentration point on the hNav1.8 channel current to obtain a dose-effect curve and calculate the IC 50 This experiment included 2 parallel sample measurements.

[0374] 2. Test methods

[0375] Test materials

[0376] 1) Cells: The HEK293 cell line stably expressing the hNav1.8 / β3 ion channel was prepared by the Biology Department of Kanglong Chemical (Beijing) Pharmaceutical Technology Co., Ltd. (the lentiviral vectors expressing human Nav1.8 and β3 were transduced into HEK293 cells and screened. For details, see: Proc Natl Acad Sci US A. 2022 Jul 26; 119(30): e2208211119. doi: 10.1073 / pnas.2208211119. and Cell Lines / BSYS CHO NaV1.8 / β3 Cell Line instructions). The cell line was cultured in a medium containing 90% DMEM, 10% fetal bovine serum, 100 U / mL penicillin-streptomycin solution, 0.75 μg / mL puromycin, and 100 μg / mL hygromycin. When the cell density reached 40% to 80% of the bottom area of ​​the culture dish, the cells were digested with trypsin and passaged three times a week. Before the experiment, the cells were cultured in a 6 cm culture dish at a total number of 5 × 105 and seeded on glass slides for subsequent manual patch clamp experiments.

[0377] 2) Compounds: The test compound is dissolved in DMSO and prepared into a stock solution with a final concentration of 10 or 30 mM. The stock solution is diluted with DMSO as a solvent to the required intermediate solution. Before the experiment begins, the gradient intermediate solution of the test compound is diluted again with extracellular fluid at a ratio of 1:1000 to form a series of working solutions at the concentration. The content of DMSO in the working solution is 0.1% (volume ratio). Working solutions with different concentration gradients are used to determine the potential inhibitory effect of the compound on the hNav1.8 channel and to fit the dose-effect curve and calculate the IC 50 .

[0378] 2.2. Experimental procedures

[0379] 1) Place the small glass slide containing HEK293 cells in the culture dish into the perfusion tank on the microscope stage.

[0380] 2) Using an Olympus IX71 or IX73 inverted microscope, center the field of view of an appropriate cell. Using a ×10 objective lens, locate the tip of the glass electrode and position it in the center of the field of view. Then, use the micromanipulator to lower the electrode while adjusting the coarse focus knob to gradually bring the electrode closer to the cell.

[0381] 3) When approaching the cell, switch to a ×40 objective lens for observation and use the micromanipulator to fine-tune the gear to gradually bring the electrode closer to the cell surface.

[0382] 4) Apply negative pressure to form a seal with a resistance higher than 1 GΩ between the electrode tip and the cell membrane.

[0383] 5) Compensate for the transient capacitive current Cfast in voltage clamp mode. Repeated short negative pressures are then applied to rupture the membrane, ultimately establishing whole-cell recording mode.

[0384] 6) Under the condition that the membrane potential is clamped at -60 mV, the slow capacitive current Cslow, cell membrane capacitance (Cm) and input membrane resistance (Ra) are compensated separately.

[0385] 7) After the cells stabilize, change the clamping voltage to -80 mV, set the sampling frequency to 20 kHz, and the filtering frequency to 10 kHz. Leakage current is detected with a clamping voltage of -80 mV and a duration of 200 ms.

[0386] 8) hNav1.8 current measurement method is as follows: Depolarize the membrane potential from -80 mV to -10 mV by applying a 20 ms depolarizing command voltage, followed by repolarization to -80 mV to close the channel. Stimulation is repeated every 15 seconds. The instantaneous peak current at the depolarizing voltage is the magnitude of the Nav1.8 sodium channel current.

[0387] 9) The hNav1.8 current used to detect the test compound was recorded continuously for 120 seconds before administration to assess the stability of the hNav1.8 current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria were used for subsequent compound testing.

[0388] 10) Testing the inhibitory effect of the test compound on hNav1.8 current: First, the hNav1.8 current measured in extracellular solution containing 0.1% DMSO is used as the detection baseline. After the hNav1.8 current remains stable for at least 5 minutes, solutions containing the test compound are sequentially perfused around the cells from low to high concentrations. After each perfusion, wait approximately 5 minutes to allow the compound to fully act on the cells and simultaneously record the hNav1.8 current. After the recorded current stabilizes, record the last 5 hNav1.8 current values ​​and take the average as the final current value at the specific concentration. After the compound is tested, add 5nM of the reference compound to the same cell to completely inhibit its current and serve as a positive control for the cell. Simultaneously, the positive compound and the reference compound are simultaneously detected using the same patch clamp system before and after the test drug experiment to ensure the reliability and sensitivity of the entire detection system. The above test steps will be repeated on two separate test cells (n=2).

[0389] Data Analysis

[0390] 1) Data must meet the following criteria: initial sealing resistance greater than 1 GΩ; membrane rupture resistance Ra less than 15 MΩ; leakage current at the test voltage less than 50% of the current value under that condition; Nav1.8 peak current greater than 200 pA at least;

[0391] 2) Data were analyzed according to the following steps (data were exported by PatchMaster software):

[0392] ① After perfusing the blank solvent or compound gradient solution, the average value of the 5 consecutive current values ​​obtained was calculated and used as the "current magnitude" 空白 ” and “current magnitude 化合物 The current suppression percentage is calculated using the following formula.

[0393] ②The dose-effect curve was fitted using Graphpad Prism 8.0 software and the IC50 value was calculated.

[0394] The standard deviation of the two sets of data is less than 15 (SD<15)

[0395] 3. Test results

[0396] Table 2 The inhibitory activity of the compounds of the present invention on NaV1.8

[0397] It can be seen that the compound of the present invention has a strong inhibitory activity on NaV1.8 channels, which is better than the reference compound.

[0398] Test Example 3 Selectivity test of the compound of the present invention for sodium ion channels

[0399] 1. Experimental purpose: To detect the effect of compounds on the current of voltage-gated sodium ion channel (NaV) 1.1-1.7 subtypes using patch clamp technique

[0400] 2. Experimental materials and equipment

[0401] 2.1. Cell Line: CHO / HEK293 cell lines stably expressing Nav1.1-1.9 sodium channels were constructed in-house by the laboratory of Beijing Aisiyipu Biotechnology Co., Ltd. / Biology Department of Pharmaron Pharmaceuticals (Beijing) Co., Ltd. (referring to Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A, Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR,Krafte DS.A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.Proc Natl Acad Sci US A.2007May 15;104(20):8520-5.doi:10.1073 / pnas.0611364104.Epub 2007May 2.PMID:17483457;PMCID:PMC1895982.), gene information Nav1.1:NM_006920; Nav1.2:NM_001040142; Nav1.3:NM_006922; Nav1.4:NM_000334; Nav1.5:NM198056; Nav1.6:NM014191; Nav1.7:NM006922; Nav1.9:NM_001349253.2.

[0402] 2.2. Compound: Dissolved in DMSO

[0403] 3. Experimental Methods

[0404] Cell culture

[0405] (1) Maintenance medium: CHO cells were cultured in HAM'S / F-12 medium supplemented with 10% fetal bovine serum, 100 μg / mL Zeocin, and 10 μg / mL Blasticidin at 37°C and 5% CO2. HEK-293 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 800 μg / mL G418 at 37°C and 5% CO2.

[0406] (2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of 0.25%-Trypsin-EDTA solution and incubate at 37°C for about 1.5 minutes. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37°C. Gently blow the cell suspension with a pipette to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes to collect the cells. For expansion or maintenance culture, inoculate the cells in a 6 cm cell culture dish with 2.5×10 cells in each cell culture dish. 5 cells (final volume: 5 mL).

[0407] (3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0408] (4) Patch clamp assay: Before the test, cells were separated with 0.25%-Trypsin-EDTA and 6.5×10 3 The cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 hours, the assay was performed.

[0409] 3.2. Patch clamp assay

[0410] Same as test case 1 3.2

[0411] Data analysis

[0412] Same as test case 1 3.3

[0413] 4. Experimental Results

[0414] Table 3 Blockade rate of the compounds of the present invention on NaV1.1-1.7 at 30 μM “ / ” means not tested.

[0415] Conclusion: It can be seen that the compounds of the present invention have no obvious activity on NaV1.1-1.7 channels. At the same concentration, the inhibitory activity on NaV1.1-1.7 channels is lower than that of the reference compound, and the target selectivity is strong, suggesting that the compounds of the present invention have better safety.

[0416] Test Example 4 Pharmacokinetics of the compounds of the present invention in SD rats

[0417] 1. Purpose of the study

[0418] SD rats were used as test animals to study the pharmacokinetic behavior of the following compound examples in rat plasma after oral administration at a dose of 10 mg / kg.

[0419] 2. Test methods

[0420] 2.1. Investigational Drugs

[0421] The examples and reference compounds of the present invention were prepared in-house.

[0422] 2.2. Experimental animals

[0423] Male SPF-grade SD rats, weighing (200±20) g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., with animal production license number SCXK (Beijing) 2022-0030.

[0424] 2.3. Preparation of experimental drugs

[0425] Drug preparation: The drug concentration was prepared to 1 mg / mL in a Tween 80 + 0.5% methylcellulose (MC, CAS: 9004-67-5) solution (1:99, v / v).

[0426] 2.4. Administration:

[0427] After 3-4 days of adaptive feeding, male SPF SD rats were gavaged and administered with a dose of 10 mg / kg and a volume of 10 mL / kg.

[0428] Before administration (0h) and after administration, blood was collected from rats at 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, and 24h. Blood was collected by fundus puncture, and the blood volume was about 0.5mL. The blood was placed in a labeled EDTA-K2 anticoagulant tube. Immediately after blood collection, the blood collection tube was gently and completely inverted 3 times to mix with the anticoagulant, and immediately centrifuged at 4500rpm in an ice water bath at 4°C for 10min. After the centrifugation operation, the plasma was promptly divided into EP tubes with corresponding labels and stored in a -80°C refrigerator.

[0429] 2.6. Sample testing

[0430] This experiment used a simple validated LC-MS / MS method to determine the concentrations of Example X and Example Y in plasma after administration.

[0431] 2.6.1. Sample Processing: Place 20 μL of blank plasma in a 96-deep-well plate pre-added with 20 μL of internal standard working solution. Add 400 μL of precipitant methanol, vortex for 10 min, and centrifuge at 4000 rpm for 15 min. Place 200 μL of the supernatant in another 96-deep-well plate pre-added with 200 μL of ultrapure water. Vortex for 5 min, centrifuge at 4000 rpm for 3 min, and perform LC-MS / MS analysis with an injection volume of 5 μL.

[0432] 2.6.2. Liquid chromatography-mass spectrometry analysis:

[0433] 1) Liquid phase conditions

[0434] Chromatographic column: Welch CB-C8, 2.1*50mm, 5μm; No.: 217

[0435] Pressure: 10Mpa

[0436] Pre-column: Pheromone guard column

[0437] Column temperature: 40°C

[0438] Injection volume: 5 μL

[0439] Injector temperature: 4°C

[0440] Run time: 3.5 minutes

[0441] Flow rate: 0.6 mL / min

[0442] Column pressure: 10Mpa

[0443] Needle washing solution: 50% methanol in water

[0444] Needle wash mode: Before and after aspiration

[0445] Mobile phase A: 0.1% formic acid in 10 mM ammonium acetate in water

[0446] Mobile phase B: 0.1% formic acid 10 mM ammonium acetate in methanol

[0447] Elution procedure and mobile phase distribution ratio: isocratic elution A:B = 40:60.

[0448] 2) Mass spectrometry conditions:

[0449] Ion detection method: Multiple reaction ion monitoring (MRM)

[0450] Ionic polarity: positive ion

[0451] Ionization method: pneumatically assisted electrospray ionization (ESI)

[0452] CAD: 8

[0453] CUR: 40 psi

[0454] GS1: 50 psi

[0455] GS2: 50 psi

[0456] TEM: 600 °C

[0457] IS: 4500 v

[0458] 3. Test Results and Analysis

[0459] The main pharmacokinetic parameters were calculated using WinNonlin 7.0. The results of the pharmacokinetic experiment in rats are shown in Table 3 below.

[0460] Table 4 Results of the Pharmacokinetic Experiment in Rats

[0461] Conclusion: From the above data, it can be seen that at the same dosing dose, the exposure of the compound of the present invention is higher than that of the reference compound. It can be seen that the compound of the present invention exhibits better absorption characteristics.

[0462] Test Example 5 Pharmacokinetic Determination in KM Mice

[0463] 1. Test Purpose

[0464] Using KM mice as test animals, study the pharmacokinetic behavior of the compound of the example in mouse plasma after intragastric administration at a dose of 10 mg / kg / intravenous injection at a dose of 1 mg / kg.

[0465] 2. Test Method

[0466] 2.1. Test Drugs

[0467] The examples and reference compounds of the present invention, self-made.

[0468] 2.2. Test Animals

[0469] Male SPF-grade KM mice, body weight (20 ± 2) g, Hubei Center for Experimental Animals, production license number: SCXK(E)2020-0018.

[0470] 2.3. Preparation of Test Drugs

[0471] Drug preparation for the intragastric administration group: Prepare a drug concentration of 1 mg / mL, and the preparation solvent is Tween 80 + 0.5% MC (v / v 1:99).

[0472] Drug preparation for the intravenous administration group: Prepare a drug concentration of 0.2 mg / mL, and the preparation solvent is 5% DMA + 5% Solutol HS-15 + 90% normal saline.

[0473] 2.4. Administration:

[0474] Male SPF KM mice were fed adaptively for 3-4 days.

[0475] Oral gavage group: the dosage was 10 mg / kg, and the administration volume was 10 mL / kg.

[0476] Intravenous administration group: the dosage was 1 mg / kg, and the administration volume was 5 mL / kg.

[0477] 2.5. Sample collection

[0478] Oral administration: Blood samples were collected from mice before (0 h) and at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 h after administration. Blood samples were collected before (0 h) and at 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h after intravenous administration.

[0479] Blood was collected by fundus puncture, with a volume of about 0.1 mL, and placed in a labeled EDTA-K2 anticoagulant tube. Immediately after blood collection, the tube was gently and completely inverted three times to mix with the anticoagulant, and immediately centrifuged at 4500 rpm for 10 min at 4°C in an ice water bath. After the centrifugation operation was completed, the plasma was promptly divided into EP tubes with corresponding labels and stored in a -80°C refrigerator.

[0480] 2.6. Sample testing

[0481] After administration, 20 μL of mouse plasma was taken and placed in a 96-deep-well plate pre-added with 20 μL of internal standard working solution. 400 μL of precipitant methanol was added, vortexed for 10 minutes, and centrifuged at 4000 rpm for 15 minutes. 200 μL of the supernatant was taken and placed in another 96-deep-well plate pre-added with 200 μL of ultrapure water. The supernatant was vortexed for 5 minutes and centrifuged at 4000 rpm for 3 minutes. 5 μL of the supernatant was taken for LC-MS / MS analysis of plasma drug concentration.

[0482] 3. Test results and analysis

[0483] The main pharmacokinetic parameters were calculated using WinNonlin 7.0. The results of the mouse pharmacokinetic experiment are shown in Table 5 below.

[0484] Table 5 Results of mouse pharmacokinetic test

[0485] 4. Experimental Conclusion

[0486] It can be seen from the above data that, at the same dosage, the exposure amount of the compound of the present invention is higher than that of the reference compound, and the bioavailability is also higher, which shows that the compound of the present invention exhibits better absorption characteristics.

[0487] Test Example 6: Drug Efficacy Test in the Mouse Acetic Acid Writhing Model

[0488] 1. Objective: To evaluate the analgesic efficacy of the examples in the acetic acid writhing model in KM mice.

[0489] 2. Test method:

[0490] 2.1. Test drugs: Examples of the present invention and reference compounds, homemade. Naproxen, Shanghai Yuanye Biotechnology Co., Ltd., product number S63435, prepared using a solvent (Tween 80 + 0.5% MC (v:v = 1:99, pH = 3)).

[0491] 2.2. Experimental animals: Male KM mice were purchased from Hubei Provincial Center for Disease Control and Prevention (Hubei Academy of Preventive Medicine), weighing 20-25 g at the time of purchase.

[0492] 2.3. Experimental Grouping:

[0493] Table 6: Grouping of compounds in the acetic acid writhing model

[0494] 2.4. Drug administration and modeling

[0495] One hour after each group completed the administration of the corresponding drugs, 0.6% acetic acid (0.15 mL / 10 g (0.1 mL / 10 g is also acceptable)) was injected intraperitoneally, and the writhing latency (the time when the mouse first writhes after the injection of glacial acetic acid) was recorded. The number of writhing reactions of the mice within 20 minutes was observed and recorded.

[0496] Writhing index: The mouse shows typical abdominal concavity, accompanied by characteristic reactions such as trunk twisting and hip elevation, which is considered to be writhing.

[0497] Data Collection and Analysis

[0498] Data were collected using Excel software.

[0499] Data were analyzed using Prism (Graph pad software, Inc.) software.

[0500] 3. Results

[0501] Table 7 Analgesic efficacy of compounds in the acetic acid writhing pain model in mice

[0502] 4. Conclusion

[0503] The above data show that, at the same dosage, Example 2 of the present invention can inhibit the pain induced by acetic acid in mice and reduce the number of writhings in mice. Moreover, Example 2 has a stronger analgesic effect than the reference compound.

[0504] Test Example 7: Mouse Foot Incision Drug Efficacy Determination

[0505] 1. Objective: To evaluate the analgesic efficacy of the Example in the ICR mouse foot incision model.

[0506] 2. Test method:

[0507] 2.1. Test drugs: Examples of the present invention, homemade, prepared using a solvent (Tween 80 + 0.5% MC (v:v = 1:99 pH = 3)). Tramadol hydrochloride, CSPC Ouyi Pharmaceutical Co., Ltd., batch number 012230682, prepared using normal saline.

[0508] 2.2. Experimental Animals: Male ICR mice were purchased from Sichuan Weitonglihua Experimental Animal Technology Co., Ltd., weighing 28-35 g at the time of purchase.

[0509] 2.3. Experimental Grouping:

[0510] The groups of the compounds in Table 8 for efficacy testing in the mouse foot incision model are as follows:

[0511] 2.4. Modeling and drug administration

[0512] After the animals have adapted for 3-5 days, the baseline before modeling is measured before modeling. A plantar incision pain model of mice is established by surgery one day before the drug administration test. A post-modeling pain sensitivity baseline test is performed on the second day after modeling. The 50% paw withdrawal threshold (50% PWT) of the hind foot on the surgical side of the mouse is measured using Von Frey test wire. Animals that meet the baseline requirements are selected for inclusion in the group and randomly divided into 6 groups of 10 animals each. The drug is orally administered once according to the above grouping. The pain threshold of the animals is tested before drug administration (baseline), 1h, 2h, 4h and 6h after drug administration.

[0513] The specific surgical modeling process is as follows: the mouse is placed in an anesthesia induction box and anesthesia is induced with 3%-4% isoflurane. After anesthesia induction, the mouse is placed in a supine position and the surgical site is disinfected with alcohol and iodine. Under sterile conditions, a 0.5 cm longitudinal incision is made from a blade toward the fingertip at 0.2 cm from the heel of the left hind foot. The skin and fascia are cut open, the plantar muscles are separated, slightly elevated, and longitudinally cut. The skin is then sutured with 5-0 sutures and disinfected.

[0514] Data Collection and Analysis

[0515] All statistical analyses were performed using two-tailed analysis. The significance level was set at 0.05 or P < 0.05. All results for the vehicle and drug-treated groups were calculated using SPSS 23.0 statistical software. One-way ANOVA with LSD multiple comparisons was performed.

[0516] 3. Results

[0517] Table 9 Analgesic efficacy of compounds in mouse foot incision pain model *: P<0.05, vs solvent group

[0518] 4. Conclusion

[0519] Under the conditions of this experiment, oral administration of the test sample Example 2 at a dose of 60 mg / kg can significantly increase the pain threshold of the incisional pain model mice and has a significant analgesic effect, which is comparable to that of the reference compound.

Claims

1. A compound as shown in formula (IE) or a pharmaceutically acceptable salt thereof, in, R 1 is - OR 1-1 ; R 1-1 is C1-C6 alkyl or is replaced by one or more R 1-1-1 Substituted C1-C6 alkyl; Each R 1-1-1 Each independently is deuterium or -OR a ; R a is C1-C6 alkyl, C3-C6 cycloalkyl or C1-C6 alkyl substituted by one or more deuteriums; R 2 and R 3 are each independently a halogen; R 4 is a C1-C6 alkyl group; R 7 is a C1-C6 alkyl group; X 1 , X 4 and X 5 Each independently is CH; X 3 N or CR X1 ; R X1 is a halogen; M is O or NH; Each R X2-1 Each is independently hydrogen, -OH, -CN or -O-C1-C6 alkyl; When M is 0, an R X2-1 is -O-C1-C6 alkyl, another R X2-1 For hydrogen.

2. The compound of formula (IE) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)R 2 and R 3 wherein the halogen is independently fluorine, chlorine, bromine or iodine, preferably fluorine; (2)R 1-1 wherein the C1-C6 alkyl group and the 1-1-1 The C1-C6 alkyl in the substituted C1-C6 alkyl is each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl; (3)R a wherein the C1-C6 alkyl group and the C1-C6 alkyl group substituted by one or more deuteriums are each independently a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, preferably a methyl group; (4)R a In the above, the C3-C6 cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclopropyl; (5)R 4 wherein the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; (6)R 7 wherein the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; (7)R X1 wherein the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine; and (8)R X2-1 In the -O-C1-C6 alkyl group, the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

3. The compound of formula (IE) or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: In formula (I-E), R 1 is -OR 1-1 ; R 1-1 For one or more R 1-1-1 Substituted C1-C6 alkyl; Each R 1-1-1 Each independently is deuterium or -OR a ; R a is C1-C6 alkyl, C3-C6 cycloalkyl or C1-C6 alkyl substituted by one or more deuteriums; R 2 and R 3 are each independently a halogen; R 4 is a C1-C6 alkyl group; R 7 is a C1-C6 alkyl group; X 1 , X 4 and X 5 Each independently is CH; X 3 N or CR X1 ; R X1 is a halogen; M is O or NH; When M is NH, each R X2-1 are each independently hydrogen, -OH or -CN; When M is 0, an R X2-1 is -O-C1-C6 alkyl, another R X2-1 is hydrogen; Preferably, R 1-1 For one or more R 1-1-1 Substituted C1-C6 alkyl; each R 1-1-1 For deuterium.

4. The compound of formula (IE) or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: When M is 0, R 1-1 For one or more R 1-1-1 Substituted C1-C6 alkyl; each R 1-1-1 for deuterium; and / or, when M is NH, at least one of the following conditions is satisfied: (i) R 1 Contains deuterium; (ii) R a is a C3-C6 cycloalkyl group; (iii) two R X2-1 At the same time, H.

5. The compound of formula (IE) or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: It meets one or more of the following conditions: (1) In formula (IE), R 1 for (2) In formula (IE), R 2 For fluorine; (3) In formula (IE), R 3 For fluorine; (4) In formula (IE), R 4 is methyl; (5) In formula (IE), R 7 is methyl; and (6) in formula (IE), for 6. A compound as represented by formula (IE) or a pharmaceutically acceptable salt thereof, characterized in that: The compound represented by formula (IE) is any of the following compounds: Preferably or an enantiomer thereof, a diastereomer thereof, or a mixture thereof.

7. A pharmaceutical composition, comprising: (1) a compound of formula (IE) according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, and (2) Pharmaceutically acceptable excipients.

8. A use of a compound of formula (IE) or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 6, or a pharmaceutical composition as described in claim 7, in the preparation of a medicament for treating a disease; the disease is pain, a pain-related disease, multiple sclerosis, incontinence or arrhythmia; the pain is preferably one or more of acute pain, chronic pain, inflammatory pain, cancer pain, neuropathic pain, musculoskeletal pain, primary pain, intestinal pain and idiopathic pain; Alternatively, the disease may be a disease for treatment by inhibiting voltage-gated sodium channels; the voltage-gated sodium channels are preferably Na V 1.

8.

9. Use of a compound as shown in formula (IE) as described in any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 7 in the preparation of a voltage-gated sodium channel inhibitor; the voltage-gated sodium channel is preferably Na V 1.

8.

10. Use of a compound as shown in formula (IE) as described in any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 7 in the preparation of a drug for a disease caused by abnormal activation of a voltage-gated sodium channel; the voltage-gated sodium channel is preferably Na V 1.8; The disease may be pain, a pain-related disease, multiple sclerosis, incontinence or cardiac arrhythmia.

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