Polycyclic compound containing benzene ring as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a polycyclic compound containing a benzene ring as well as a pharmaceutical composition and application thereof. Specifically provided is a compound represented by formula (I-E) or a pharmaceutically acceptable salt thereof. The compound provided by the invention has one or more of the following advantages: (1) the structure is novel; (2) the compound has a good retarding effect on the activity of a NaV1.8 channel; and (3) the method has good selectivity on other subtypes of Nav (such as Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and Nav1.9), and is high in safety. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a polycyclic compound containing a benzene ring, a pharmaceutical composition thereof, and an application thereof. Background Art

[0002] Pain originates from nociceptors in the peripheral nervous system. These are free nerve endings widely distributed in the skin, muscles, joints, and visceral tissues throughout the body. They can convert the sensed thermal, mechanical, or chemical stimuli into nerve impulses (action potentials) and transmit them via afferent nerve fibers to the cell body part located in the dorsal root ganglia (DRG), and ultimately to the higher nerve centers, causing the sensation of pain. The generation and conduction of action potentials in neurons also depend on voltage-gated sodium channels (VGSC / Nav) on the cell membrane. Nav is a key mediator for transmembrane information transmission along neurons.

[0003] According to statistics, about one-fifth of the people in the world suffer from moderate to severe chronic pain. The global analgesic market was approximately 36 billion US dollars in 2018 and is expected to reach 56 billion US dollars in 2023. Among them, acute moderate to severe pain will grow steadily at an annual compound growth rate of 2.5% in the future, and the chronic pain market will grow at an annual compound growth rate of about 18% in the future. Chronic pain is the main driving force for the continuous growth of the global pain market in the next decade.

[0004] The secondary structure of voltage-gated sodium channels (VGSC / Nav) contains four pore-forming domains (DI-DIV), each domain having six transmembrane α-helices (S1-S6), where the S4 segment is the voltage sensor containing positively charged ions; the intracellular loop between DIII and DIV is considered the fast inactivation gate; Nav exists in three different states, and the inactivated closed state has different kinetic manifestations of fast inactivation (within milliseconds) or slow inactivation (seconds). When the cell membrane depolarizes, the sodium channel is activated, the channel opens, causing sodium ions to flow inward, further depolarizing the cell membrane, and resulting in the generation of action potentials. Therefore, inhibiting abnormal sodium channel activity helps in the treatment and relief of pain, and Nav is a potential peripheral target for treating pain.

[0005] There are mainly 9 subtypes of human Nav, namely Nav1.1 - Nav1.9. According to 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, Nav1.3, Nav1.4, Nav1.6 and Nav1.7 are TTX-S types. Among them, Nav1.1, Nav1.2 and Nav1.3 are highly 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 central nervous system. Nav1.5, Nav1.8 and Nav1.9 are TTX-R types. Among them, Nav1.5 is mainly present in cardiomyocytes, and Nav1.8 and Nav1.9 are present in the dorsal root ganglion of the peripheral nervous system (PNS-DRG).

[0006] Studies have shown that ion channel alterations are the molecular basis for peripheral sensitization, central sensitization and disinhibition after inflammation or neuropathic injury, and are also important molecular mechanisms for pain occurrence. Currently, Nav inhibitors have been proven 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 existing Nav inhibitors used in clinical practice have a narrow therapeutic window and limited application scope due to the lack of subtype selectivity and the ability to inhibit sodium ion channels expressed in the heart and central nervous system. Nav1.8 is an important ion channel involved in chronic pain, atrial fibrillation, and Brugada syndrome. A highly selective Nav1.8 inhibitor is an ideal target for pain treatment, with relatively fewer side effects when producing analgesic effects.

[0007] In a model of neuropathic pain, nerve injury increases the expression level of Nav1.8 in axons and neuronal cell bodies. Using Nav1.8 antisense oligonucleotides can significantly relieve pain while reducing the expression of Nav1.8. Nav1.8 knockout mice cannot exhibit normal visceral inflammatory pain. After the human Nav1.8 gene generates a gain-of-function mutation, it will cause peripheral neuralgia. Based on a series of animal experiments and human gene evidence, selective inhibition of Nav1.8 has the potential to become a new type of analgesic therapy and can be used to treat various pain types such as inflammatory pain, neuropathic pain, postoperative pain, and cancer pain.

[0008] Highly selective Nav inhibitors are one of the key research directions for voltage-gated sodium channels. Since Nav1.8 is mainly distributed in the peripheral nervous system and is confined to pain-sensing neurons, it is a highly selective target for pain treatment. Therefore, selectively inhibiting Nav1.8 has good prospects for reducing potential toxic side effects. Nav inhibitors used in clinical practice have a narrow therapeutic window and limited application range because they lack subtype selectivity and can inhibit sodium channels expressed in the heart and central nervous system. 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

[0009] The technical problem to be solved by the present invention is to overcome the disadvantage of insufficient inhibition of the Nav1.8 channel in the prior art. To this end, the present invention provides a polycyclic compound containing a benzene ring, its pharmaceutical composition, and its application. The compounds of the present invention have one or more of the following advantageous effects: (1) novel structure; (2) good blocking effect (or inhibitory effect) on the activity of the NaV1.8 channel; (3) good selectivity for other Nav subtypes (such as Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.9) and high safety.

[0010] The technical problem of the present invention is solved by the following technical solutions:

[0011] The present invention provides a compound of formula (I-E) or a pharmaceutically acceptable salt thereof,

[0012]

[0013] wherein R 1 , R 2 and R 3 are each independently a halogen or -OR 1-1 ;

[0014] R 1-1 is a C1-C6 alkyl group, a 3-8 membered heterocyclic alkyl group, a C1-C6 alkyl group substituted with one or more R 1-1-1 or a 3-8 membered heterocyclic alkyl group substituted with one or more deuteriums; in the 3-8 membered heterocyclic alkyl group of the 3-8 membered heterocyclic alkyl group and the 3-8 membered heterocyclic alkyl group substituted with one or more deuteriums, the heteroatoms are selected from 1, 2, or 3 of N, O, and S, and the number of heteroatoms is 1, 2, or 3;

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

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

[0017] R 4 is hydrogen, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkyl-O-C1-C6 alkyl group, or an -O-C1-C6 alkyl group;

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

[0019] X 1 , X 3 , X 4 and X 5 are each independently N, N + -O - or CR X1 ;

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

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

[0022] Each R X2-1 is independently hydrogen, -CN, a C1-C6 alkyl group, an -O-C1-C6 alkyl group, -OH, or -NR’R”;

[0023] R’ and R” are each independently hydrogen or a C1-C6 alkyl group;

[0024] M is O or NH;

[0025] When M is O, one R X2-1 is -O-C1-C6 alkyl, and the other R X2-1 is hydrogen.

[0026] In some embodiments, the compound of formula (I-E) or a pharmaceutically acceptable salt thereof,

[0027]

[0028] wherein, R 1 , R 2 and R 3 are each independently a halogen or -OR 1-1 ;

[0029] R 1-1 is a C1-C6 alkyl group, a 3-8 membered heterocycloalkyl group, substituted with one or more R 1-1-1A substituted C1-C6 alkyl or a 3-8 membered heteroalkyl substituted with one or more deuteriums; in the 3-8 membered heteroalkyl of the 3-8 membered heteroalkyl and the 3-8 membered heteroalkyl substituted with one or more deuteriums, the heteroatoms are selected from 1, 2 or 3 of N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0030] Each R 1-1-1 is independently deuterium or -OR a ;

[0031] R a is a C1-C6 alkyl, a C3-C6 cycloalkyl or a C1-C6 alkyl substituted with one or more deuteriums;

[0032] R 4 is hydrogen, hydroxy, C1-C6 alkyl, C1-C6 alkyl-O-C1-C6 alkyl or -O-C1-C6 alkyl;

[0033] R 7 is hydrogen or C1-C6 alkyl;

[0034] X 1 , X 3 , X 4 and X 5 are each independently N, N + -O - or CR X1 ;

[0035] R X1 is hydrogen, halogen or -C(=O)N(R X1-1 )2;

[0036] Each R X1-1 is independently hydrogen or C1-C6 alkyl;

[0037] Each R X2-1 is independently hydrogen, C1-C6 alkyl, -O-C1-C6 alkyl, -OH or -NR’R”;

[0038] R’ and R” are each independently hydrogen or C1-C6 alkyl;

[0039] M is O or NH.

[0040] In some embodiments, in R 1 , R 2 and R 3 , the halogen is independently fluorine, chlorine, bromine or iodine, preferably fluorine.

[0041] In some embodiments, in R 1-1 , the C1-C6 alkyl and the C1-C6 alkyl substituted with one or more R 1-1-1Each of the C1-C6 alkyl groups in the substituted C1-C6 alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.

[0042] 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 deuteriums, 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 groups are each independently preferably 4-6 membered (e.g., 4-membered, 5-membered or 6-membered) heterocycloalkyl groups; more preferably For example

[0043] In some embodiments, R a In the C1-C6 alkyl group and the C1-C6 alkyl group substituted with one or more deuteriums, the C1-C6 alkyl groups are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0044] In some embodiments, R a In the C3-C6 cycloalkyl group, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclopropyl.

[0045] In some embodiments, R 4 In the C1-C6 alkyl group, the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0046] In some embodiments, R 7 In the C1-C6 alkyl group, the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0047] In some embodiments, R X1 In the halogen, the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine.

[0048] In some embodiments, R X2-1 In the C1-C6 alkyl group and the -O-C1-C6 alkyl group, the C1-C6 alkyl groups are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0049] In some embodiments, the compound represented by formula (I-E) is not

[0050]

[0051] In some embodiments, in formula (I-E), R 1 is -OR1-1 ;

[0052] R 1-1 is a C1-C6 alkyl group or a C1-C6 alkyl group substituted with one or more R 1-1-1 substituents;

[0053] Each R 1-1-1 is independently deuterium or -OR a ;

[0054] R a is a C1-C6 alkyl group, a C3-C6 cycloalkyl group or a C1-C6 alkyl group substituted with one or more deuterium atoms;

[0055] R 2 and R 3 are each independently a halogen;

[0056] R 4 is a C1-C6 alkyl group;

[0057] R 7 is a C1-C6 alkyl group;

[0058] X 1 、X 4 and X 5 are each independently CH;

[0059] X 3 is N or CR X1 ;

[0060] R X1 is a halogen;

[0061] M is O or NH;

[0062] Each R X2-1 is independently hydrogen, -OH, -CN or -O-C1-C6 alkyl;

[0063] When M is O, one R X2-1 is -O-C1-C6 alkyl and the other R X2-1 is hydrogen.

[0064] In some embodiments, in formula (I-E), R 1 is -OR 1-1 ;

[0065] R 1-1 is a C1-C6 alkyl group substituted with one or more R 1-1-1 substituents;

[0066] Each R 1-1-1 is independently deuterium or -OR a ;

[0067] R a is a C1-C6 alkyl group, a C3-C6 cycloalkyl group, or a C1-C6 alkyl group substituted with one or more deuteriums;

[0068] R 2 and R 3 are each independently a halogen;

[0069] R 4 is a C1-C6 alkyl group;

[0070] R 7 is a C1-C6 alkyl group;

[0071] X 1 、X 4 and X 5 are each independently CH;

[0072] X 3 is N or CR X1 ;

[0073] R X1 is a halogen;

[0074] M is O or NH;

[0075] When M is NH, each R X2-1 is independently hydrogen, -OH, or -CN;

[0076] When M is O, one R X2-1 is -O-C1-C6 alkyl, and the other R X2-1 is hydrogen.

[0077] In some embodiments, in formula (I-E), when M is O, R 1-1 is a C1-C6 alkyl group substituted with one or more R 1-1-1 ; each R 1-1-1 is deuterium.

[0078] In some embodiments, in formula (I-E), when M is NH, at least one of the following conditions is satisfied:

[0079] (i) R 1 contains deuterium;

[0080] (ii) R a is a C3-C6 cycloalkyl group;

[0081] (iii) Two R X2-1 are simultaneously H.

[0082] In some embodiments, in formula (I-E), R 1-1 is a C1-C6 alkyl group substituted with one or more R 1-1-1 ; each R1-1-1 is deuterium.

[0083] In some embodiments, the compound represented by formula (I-E) is not

[0084]

[0085] In some embodiments, in formula (I-E), R 1 is

[0086] In some embodiments, in formula (I-E), R 1 is -OCH3 or

[0087] In some embodiments, in formula (I-E), R 2 is fluorine.

[0088] In some embodiments, in formula (I-E), R 3 is fluorine.

[0089] In some embodiments, in formula (I-E), R 4 is methyl.

[0090] In some embodiments, in formula (I-E), R 7 is methyl.

[0091] In some embodiments, in formula (I-E), is

[0092] In some embodiments, in formula (I-E), is

[0093] In some embodiments, the compound represented by formula (I-E) is any one of the following compounds:

[0094]

[0095]

[0096] In some embodiments, the compound represented by formula (I-E) is any one of the following compounds:

[0097]

[0098]

[0099] or its enantiomers, diastereomers or mixtures thereof.

[0100] The present invention provides a pharmaceutical composition, which comprises:

[0101] (1) The compound as shown in formula (I-E) above or its pharmaceutically acceptable salt, and

[0102] (2) Pharmaceutically acceptable excipients.

[0103] The present invention provides an application of the compound as shown in formula (I-E) above or its pharmaceutically acceptable salt, or the above pharmaceutical composition in the preparation of a drug for treating a disease; the disease may be pain, pain-related diseases, multiple sclerosis, incontinence or arrhythmia.

[0104] 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.

[0105] The present invention provides an application of the compound as shown in formula (I-E) above or its pharmaceutically acceptable salt, or the above 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.

[0106] The present invention provides an application of the compound as shown in formula (I-E) above or its pharmaceutically acceptable salt, or the above pharmaceutical composition in the preparation of a voltage-gated sodium channel inhibitor; the voltage-gated sodium channel is preferably Na V 1.8.

[0107] 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, such as: providing comparison as a standard sample or control sample, or being made into a kit according to conventional methods in the art to provide a rapid detection of the effect of inhibiting voltage-gated sodium channels.

[0108] The present invention provides an application of the compound as shown in formula (I-E) above or its pharmaceutically acceptable salt, or the above pharmaceutical composition in the preparation of a drug for a disease caused by abnormal activation of voltage-gated sodium channels; the voltage-gated sodium channel is preferably Na V 1.8; the disease may be pain, pain-related diseases, multiple sclerosis, incontinence or arrhythmia.

[0109] The present invention provides a method for rapidly detecting voltage-gated sodium channel Na VA kit for the inhibitory effect of 1.8, which comprises the compound shown in the above formula (I-E) or a pharmaceutically acceptable salt thereof.

[0110] Term Explanation

[0111] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a 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 a 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).

[0112] In the present invention, in the structural fragment it means that this structural fragment is connected to the rest of the molecule through this bond. For example, is cyclopropyl.

[0113] In the present invention, the "-" at the end of a group means that this group is connected to the rest of the molecule through this site. For example, -OH means hydroxyl.

[0114] In the present invention, the term "one or more" means 1, 2, 3, 4, 5, 6, 7, for example, 1, 2 or 3.

[0115] In the present invention, in the term "B substituted by one or more A", when B is substituted by "multiple" A, A is the same or different.

[0116] In the present invention, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0117] In the present invention, the term "alkyl" refers to a straight-chain or branched-chain, saturated monovalent hydrocarbon group having a specified number of carbon atoms (for example, C1-C6). Alkyl includes but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0118] In the present invention, the term "cycloalkyl" refers to a cyclic, saturated monovalent hydrocarbon group having a specified number of carbon atoms (for example, C3-C6). Cycloalkyl includes but is not limited to: etc.

[0119] In the present invention, the term "heterocycloalkyl" refers to a cyclic, saturated monovalent group having a specified number of ring atoms (e.g., 3 - 12 membered, 4 - 8 membered, 5 membered, 6 membered or 7 membered), a specified number of heteroatoms (e.g., 1, 2 or 3), and a specified type of heteroatoms (one or more of N, O and S). The heterocycloalkyl is linked to the rest of the molecule through a carbon atom or a heteroatom. Heterocycloalkyl includes, but is not limited to: etc.

[0120] In the present invention, the term "pharmaceutically acceptable excipients" refers to all substances contained in a pharmaceutical preparation other than the active pharmaceutical ingredient, and is generally divided into two categories: excipients and additives. For details, reference can be made to Pharmacopoeia of the People's Republic of China (2020 Edition), Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0121] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0122] The reagents and raw materials used in the present invention are all commercially available.

[0123] The positive and progressive effects of the present invention are as follows: The compounds of the present invention have one or more of the following advantageous effects: (1) novel structure; (2) good blocking effect (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. Detailed Description of the Invention

[0124] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0125] Preparation Example 1 Synthesis of Intermediate 43 - 3

[0126] First step: Synthesis of Intermediate A2

[0127] The synthesis route is shown in the following formula:

[0128]

[0129] Method: Dissolve A1 (18.00 g, 95.76 mmol) in toluene (300 mL), and successively add 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). Replace N2, and stir the reaction mixture at 100 °C for 12 h. After the reaction is completed, filter through diatomaceous earth, concentrate under reduced pressure, and purify by column chromatography to obtain white solid A2 (7.60 g, 40.4%). MS (ESI, m / z) 217 [M+H] + 。

[0130] Step 2: Synthesis of intermediate A3

[0131]

[0132] Method: Dissolve A2 (7.60 g, 35.18 mmol) in THF (80 mL), add an aqueous lithium hydroxide solution (3.39 g LiOH·H2O dissolved in 40 mL H2O, 80.87 mmol), and stir the reaction mixture at 50 °C for 3 h. After the reaction is completed, dilute with DCM and separate layers, extract with H2O three times, combine the aqueous phases, adjust the pH < 5 with 1N HCl, extract with DCM three times, combine the organic phases, dry the organic phase, and concentrate under reduced pressure to obtain white solid compound A3 (5.90 g, 83.0%). MS (ESI, m / z) 203 [M+H] + 。

[0133] Step 3: Synthesis of intermediate S2

[0134] The synthetic route is shown as follows:

[0135]

[0136] Method: Dissolve compound S1 (15.50 g, 98.05 mmol) in Et2O (155 mL), replace N2, cool down to about 0 °C, and add methyllithium lithium bromide complex (209 mL, 313.76 mmol, 1.5 M in Et2O) in batches while maintaining the temperature at 0 - 10 °C. After dropping, let it rise to room temperature naturally and stir for 12 hours. After the reaction is completed, quench with H2O (100 mL) and saturated sodium chloride solution (20 mL), add citric acid (25.04 g) and stir for 0.5 hour, extract with Et2O three times, combine the organic phases, dry the organic phase, and concentrate under reduced pressure to 30 g to obtain crude product S2 (35% in Et2O, 30 g).

[0137] Step 4: Synthesis of intermediate S3

[0138] The synthetic route is shown as follows:

[0139]

[0140] Method: Dissolve compound A3 (5.90 g, 29.20 mmol) in MeCN (210 mL), cool it in an ice-water bath, add CDI (7.00 g, 30.70 mmol), displace N2, stir at a temperature below 10 °C for 1.5 h, add S2 (about 35% in Et2O, 15 g, 30 mmol) and K2CO3 (5.00 g, 36.50 mmol), and stir the reaction mixture at 35 °C for 12 hours. After the reaction is completed, add water and dilute hydrochloric acid to the reaction solution and stir until it becomes clear, extract with EA, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain white solid S3 (7.40 g, 78.7%). MS (ESI, m / z) 323 [M+H] + 。

[0141] Step 5: Synthesis of S4

[0142] The synthetic route is shown as follows:

[0143]

[0144] Method: Dissolve compound S3 (9.00 g, 28.0 mmol) in anhydrous methanol (800 mL) and anhydrous tetrahydrofuran (160 mL), cool to -40 °C, add the first batch of NiCl2·6H2O (6.80 g, 28.6 mmol) and NaBH4 (5.60 g, 148.4 mmol), and the second batch of NiCl2·6H2O (6.80 g, 28.6 mmol) and NaBH4 (5.60 g, 148.4 mmol). The reaction is complete immediately after addition. After the reaction is completed, slowly add saturated ammonium chloride to quench the reaction while maintaining the temperature at -40 °C and stir for 30 min. Let it rise to room temperature naturally, filter through diatomaceous earth, extract with DCM / MeOH, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain colorless oily compound S4 (3.57 g, 39.3%). MS (ESI, m / z) 325 [M+H] + 。

[0145] The NMR spectrum is as follows:

[0146] 1 H NMR (400 MHz, CDCl3) δ=6.96 - 6.83 (m, 2H), 4.47 (d, J=9.6 Hz, 1H), 4.02 (d, J=2.8 Hz, 1H), 2.92 - 2.84 (m, 1H), 1.70 (s, 3H), 0.81 - 0.78 (m, 3H).

[0147] Step 6: Synthesis of S5

[0148] The synthesis route is shown as follows:

[0149]

[0150] Method: Dissolve compound S4 (3.57 g, 11.0 mmol) in toluene (30 mL), displace N2, cool down to -30 °C, slowly add dropwise DiBAL-H (1.5 M, 7.7 mL, 11.6 mmol), and stir the reaction mixture at -25 to -30 °C for 2 h. After the reaction is completed, add the reaction solution to saturated ammonium chloride for quenching, filter through diatomite, extract with EA, wash with concentrated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain colorless oily compound S5 (3.53 g, crude product). MS (ESI, m / z) 327 [M+H] + 。

[0151] Step 7: Synthesis of S6

[0152] The synthesis route is shown as follows:

[0153]

[0154] Method: Dissolve compound S5 (3.53 g, 10.8 mmol) in anhydrous DCM (30 mL) and stir until clear, add Et3N (2.18 g, 21.6 mmol), cool down to 0 - 10 °C in an ice-water bath and slowly add dropwise AcCl (1.70 g, 21.6 mmol). After adding, raise the temperature to room temperature and stir for 1 h. After the reaction is completed, add saturated ammonium chloride to the reaction solution for quenching, extract with DCM, wash with concentrated brine, dry over anhydrous sodium sulfate, and concentrate to obtain colorless oily compound S6 (4.36 g, crude product). MS (ESI, m / z) 369 [M+H] + 。

[0155] Step 8: Synthesis of S7

[0156] The synthesis route is shown as follows:

[0157]

[0158] Method: Dissolve compound S6 (4.36 g, 11.8 mmol) in anhydrous DCM (40 mL), displace N2, cool down to -60 °C, successively add dropwise TMSCN (3.52 g, 35.5 mmol) and BF3OEt2 (5.04 g, 35.5 mmol). After the addition is complete, stir the reaction at -60 °C for 1 h, and then naturally warm up to room temperature and stir the reaction for 12 h. After the reaction is completed, quench the reaction mixture by adding saturated sodium carbonate solution, filter through diatomaceous earth, extract with DCM, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain colorless oily compound S7 (2.25 g, 56.9%). MS (ESI, m / z) 336 [M+H] + 。

[0159] Step 9: Synthesis of Intermediate 39-1

[0160] The synthetic route is shown as follows:

[0161]

[0162] Method: Dissolve S7 (1.1 g, 3.28 mmol) in DCM (15 mL), displace with N2, cool down to -78 °C, slowly add dropwise BBr3 (2.1 mL, 21.32 mmol). After the addition is complete, warm up to 0 °C and stir the reaction for 3 h. After the reaction is completed, slowly add the reaction mixture dropwise to ice water, then extract with DCM, wash with saturated NaCl aqueous solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain yellow oily compound 39-1 (1.0 g, crude product). MS (ESI, m / z) 322 [M+H] + 。

[0163] Step 10: Synthesis of Intermediate 43-1

[0164] The synthetic route is shown as follows:

[0165]

[0166] Method: Dissolve 39-1 (300 mg, crude product, approximately 0.93 mmol) in DMF (5 mL), add Cs2CO3 (609 mg, 1.87 mmol) and deuterated iodomethane (542 mg, 3.74 mmol), warm up to 70 °C and stir for 16 h. After the reaction is completed, quench with water, extract with EA, wash with saturated NaCl aqueous solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain yellow oily compound 43-1 (300 mg, crude product). MS (ESI, m / z) 339 [M+H] + 。

[0167] Step 11: Synthesis of Intermediate 43-2

[0168] The synthesis route is shown as follows:

[0169]

[0170] Method: Dissolve 43-1 (300 mg, crude product, about 0.93 mmol) in MeOH (5 mL), add an aqueous solution of KOH (367 mg, 6.54 mmol, dissolved in 1 mL of water), heat up to 60 °C and react with stirring for 16 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure, dilute with water, extract once with MTBE, adjust the pH of the aqueous phase to < 3 by dropwise adding 1N HCl, extract with EA, wash with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow oily compound 43-2 (260 mg, crude product). MS (ESI, m / z) 358 [M+H] + 。

[0171] The twelfth step: Synthesis of intermediate 43-3

[0172] The synthesis route is shown as follows:

[0173]

[0174] Method: Dissolve 43-2 (260 mg, crude product, 0.73 mmol) in DCM (4 mL), add 1 drop of DMF, cool down to 0 °C, slowly dropwise add (COCl)2 (0.09 mL, 1.092 mmol), and react with stirring at room temperature for 0.5 hour. After the reaction is completed, concentrate under reduced pressure, then dissolve in DCM (4 mL) and slowly dropwise add it 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), and stir at room temperature for 0.5 hour. After the reaction is completed, concentrate the reaction solution under reduced pressure, extract with EA, combine the organic phases, wash once with water and saturated aqueous NaCl solution respectively, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow oily compound 43-3 (280 mg, crude product). MS (ESI, m / z) 434 [M+H] + 。

[0175] Synthesis of intermediate 59-3 in Preparation Example 2

[0176] The first step: Synthesis of intermediate 39-1

[0177] Refer to Preparation Example 1 to synthesize intermediate 39-1.

[0178] The second step: Synthesis of intermediate 59-1

[0179] The synthesis route is shown as follows:

[0180]

[0181] Method: Dissolve 39-1 (140 mg, crude product, about 0.44 mmol) in DMF (5 mL), add Cs2CO3 (286 mg, 0.88 mmol) and 2-bromoethanol (220 mg, 6.2 mmol), and heat to 70 °C and stir for 16 hours. After the reaction is completed, quench with water, extract with EA, wash with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain yellow oily compound 59-1 (240 mg, crude product). MS (ESI, m / z) 366 [M+H] + .

[0182] Step 3: Synthesis of intermediate 59-2

[0183] The synthesis route is shown as follows:

[0184]

[0185] Method: Dissolve 59-1 (240 mg, crude product, about 0.44 mmol) in DMF (5 mL), add NaH (35 mg, 0.88 mmol) under ice bath, stir at room temperature for 0.5 hour, then add CD3I (191 mg, 1.32 mmol), and continue to stir at room temperature for 1.5 hours. After the reaction is completed, quench with saturated aqueous NH4Cl solution, extract with EA, combine the organic phases, wash with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain brown oily compound 59-2 (220 mg, crude product). MS (ESI, m / z) 383 [M+H] + .

[0186] Step 4: Synthesis of intermediate 59-3

[0187] The synthesis route is shown as follows:

[0188]

[0189] Method: Dissolve 59-2 (220 mg, crude product, about 0.44 mmol) in MeOH (5 mL), add an aqueous solution of KOH (172 mg, 3.08 mmol, dissolved in 1 mL of water), heat to 60 °C and stir for 16 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure, dilute with water, extract once with MTBE, adjust the pH of the aqueous phase to <3 by dropwise addition of 1N HCl, extract with EA, combine the organic phases, wash with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain yellow oily compound 59-3 (110 mg, crude product). MS (ESI, m / z) 402 [M+H] + .

[0190] Synthesis of intermediate 60-3 in Preparation Example 3

[0191] Step 1: Synthesis of Intermediate 39-1

[0192] Synthesize Intermediate 39-1 with reference to Preparation Example 1.

[0193] Step 2: Synthesis of Intermediate 60-1

[0194] The synthesis route is shown as follows:

[0195]

[0196] Method: Dissolve 39-1 (140 mg, crude product, about 0.44 mmol) in DMF (5 mL), add Cs2CO3 (286 mg, 0.88 mmol) and 2-bromoethanol-D4 (227 mg, 1.76 mmol), and heat to 70 °C and stir for 16 hours. After the reaction is completed, quench with water, extract with EA, wash with saturated NaCl aqueous solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow oily compound 60-1 (300 mg, crude product). MS (ESI, m / z) 370 [M+H] + 。

[0197] Step 3: Synthesis of Intermediate 60-2

[0198] The synthesis route is shown as follows:

[0199]

[0200] Method: Dissolve 60-1 (300 mg, crude product, about 0.44 mmol) in DMF (5 mL), add NaH (35 mg, 0.88 mmol) under ice bath, stir at room temperature for 0.5 hour, then add deuterated iodomethane (191 mg, 1.32 mmol), and continue to stir at room temperature for 1.5 hours. After the reaction is completed, quench with saturated NH4Cl aqueous solution, extract with EA, combine the organic phases, wash with saturated NaCl aqueous solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown oily compound 60-2 (280 mg, crude product). MS (ESI, m / z) 387 [M+H] + 。

[0201] Step 4: Synthesis of Intermediate 60-3

[0202] The synthesis route is shown as follows:

[0203]

[0204] Method: Dissolve 69-2 (280 mg, crude product, about 0.44 mmol) in MeOH (5 mL), add an aqueous solution of KOH (172 mg, 3.08 mmol, dissolved in 1 mL of water), heat to 60 °C and stir for 16 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure, dilute with water, extract once with MTBE, adjust the pH of the aqueous phase to <3 by dropwise addition of 1N HCl, extract three times with EA, combine the organic phases, wash with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the yellow oily compound 69-3 (100 mg, crude product). MS (ESI, m / z) 406 [M+H] + 。

[0205] Synthesis of Example 1 BX20-9-074

[0206] Step 1: Synthesis of Intermediate 43-2

[0207] Refer to Preparation Example 1 to synthesize Intermediate 43-2.

[0208] Step 2: Synthesis of Compound 74-1

[0209] The synthetic route is shown as follows:

[0210]

[0211] Method: Dissolve 43-2 (100 mg, crude product) in DCM (2 mL), add one drop of DMF, slowly add dropwise (COCl)2 (0.2 mL), stir at room temperature for half an hour, directly concentrate the reaction solution under reduced pressure to obtain Intermediate 43-3, then dissolve it in DCM (2 mL), and slowly add it 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 the reaction is completed, quench with water, extract three times with EA, combine the organic phases, wash once with water, then wash with saturated NaCl solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain the yellow oily liquid 74-1 (100 mg, crude product). MS (ESI, m / z) 459 [M+H] + 。

[0212] Step 3: Synthesis of Compound BX20-9-074

[0213] The synthetic route is shown as follows:

[0214]

[0215] Method: Dissolve 74-1 (100 mg, crude product) in EtOH (3 mL), add NH2OH (50% in water, 74 mg, 1.12 mmol), reflux and stir at 80 °C for 1 hour. Dissolve the reaction solution in MeOH, filter, purify by preparative chromatography (neutral system), and obtain a white solid powder BX20-9-074 (15 mg, yield 13.9%) after lyophilization. MS (ESI, m / z) 492 [M+H] + 。

[0216] 1 1H NMR (400 MHz, DMSO-d6) δ = 8.40 (d, J = 5.6 Hz, 1H), 8.12 (s, 1H), 7.62 - 7.60 (m, 1H), 7.16 - 7.11 (m, 2H), 5.76 (s, 2H), 5.06 (d, J = 10.4 Hz, 1H), 4.24 - 4.20 (m, 1H), 2.77 - 2.73 (m, 1H), 1.58 (s, 3H), 0.71 - 0.69 (m, 3H).

[0217] Example 2 Synthesis of BX20-9-075

[0218] Step 1: Synthesis of Intermediate 43-2

[0219] Synthesize Intermediate 43-2 with reference to Preparation Example 1.

[0220] Step 2: Synthesis of Intermediate 75-1

[0221] The synthesis route is shown as follows:

[0222]

[0223] Method: Dissolve 43-2 (100 mg, 0.28 mmol) in DCM (4 mL), add 1 drop of DMF, slowly dropwise add (COCl)2 (53 mg, 0.42 mmol) under ice bath, and stir at room temperature for 0.5 hour. After completion of the reaction, concentrate under reduced pressure, then dissolve in DCM (4 mL), and slowly dropwise add it 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 hour. After completion of the reaction, dilute with water, extract three times with EA, combine the organic phases, wash once with water and once with saturated NaCl aqueous solution respectively, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown oily compound 75-1 (118 mg, crude product). MS (ESI, m / z) 476 [M+H] + 。

[0224] Step 3: Synthesis of BX20-9-075

[0225] The synthesis route is shown as follows:

[0226]

[0227] Method: Dissolve compound 75-1 (118 mg, crude product) in EtOH (6 mL), add NH2OH (0.15 mL, 2.0 mmol, 50% in H2O), and heat to 80 °C with stirring for 1 hour. After completion of the reaction, filter and purify by preparative chromatography, and lyophilize to obtain a white solid BX20-9-075 (29 mg, 23%). MS (ESI, m / z) 509 [M+H] + .

[0228] 1 1H NMR (400 MHz, DMSO-d6) δ = 10.32 (s, 1H), 9.62 (s, 1H), 7.75 (dd, J = 2.4, 6.4 Hz, 1H), 7.67 - 7.62 (m, 1H), 7.21 - 7.11 (m, 3H), 5.78 (s, 2H), 5.04 (d, J = 10.4 Hz, 1H), 4.23 (dd, J = 7.6, 10.4 Hz, 1H), 2.80 - 2.71 (m, 1H), 1.59 (s, 3H), 0.72 (d, J = 6.0 Hz, 3H).

[0229] Synthesis of Example 3 BX20-9-079

[0230] Step 1: Synthesis of Intermediate 39-1

[0231] Synthesize Intermediate 39-1 with reference to Preparation Example 1.

[0232] Step 2: Synthesis of 79-1

[0233] The synthesis route is shown as follows:

[0234]

[0235] Method: Dissolve 39-1 (0.30 g, 0.93 mmol) in DMF (10 mL), add CsCO3 (0.63 g, 1.92 mmol), 2-cyclopropoxyethyl 4-methylbenzenesulfonate (0.38 g, 1.92 mmol), and heat to 70 °C with stirring for 12 hours. After completion of the reaction, add water and EA for extraction, combine the organic phases, wash with saturated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 79-1 (0.31 g, 82.3%). MS (ESI, m / z) 406 [M+H] + .

[0236] Step 3: Synthesis of 79-2

[0237] The synthesis route is shown as follows:

[0238]

[0239] Method: Dissolve compound 79-1 (0.31 g, 0.76 mmol) in MeOH (5 mL), add an aqueous KOH solution (0.30 g KOH dissolved in 1 mL H2O, 5.33 mmol), and stir the reaction mixture at 60 °C for 5 hours. After the reaction is completed, extract with MTBE, adjust the pH of the aqueous phase to <5 with hydrochloric acid, extract with MTBE, wash the organic phase with concentrated brine, dry over anhydrous sodium sulfate, and concentrate to obtain a brown oil 79-2 (0.21 g, 65.2%). MS (ESI, m / z) 425 [M+H] +

[0240] Step 4: Synthesis of 79-4

[0241] The synthesis route is shown as follows:

[0242]

[0243] Method: Dissolve 79-2 (0.21 g, 0.50 mmol) in DCM (6 mL), add a drop of DMF, and dropwise add (COCl)2 (0.13 g, 1.00 mmol) at 0 °C. Stir the reaction solution at room temperature for 0.5 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure to obtain the crude product 79-3 (0.23 g, crude product), which is directly used for the next step; dissolve compound 4-fluoro-3-cyanoaniline (82 mg, 0.60 mmol) in NMP (2 mL), add TEA (101 mg, 1.00 mmol), and then slowly add 79-3 (crude product, diluted with 4 mL DCM, about 0.50 mmol). Stir at room temperature for 0.5 hours. After the reaction is completed, quench with saturated ammonium chloride, extract with EA, combine the organic phases, wash with concentrated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 79-4 (0.31 g, crude product). MS (ESI, m / z) 543 [M+H] + 。

[0244] Step 5: Synthesis of BX20-9-079

[0245] The synthesis route is shown as follows:

[0246]

[0247] Method: Dissolve compound 79-4 (0.31 g, crude product, about 0.50 mmol) in EtOH (5 mL), add NH2OH (151 mg, 2.29 mmol), and raise the temperature to 80 °C for stirring reaction for 1 hour. After the reaction is completed, directly concentrate under reduced pressure. After concentration, purify by preparative chromatography under neutral conditions to obtain white solid BX20-9-079 (80 mg, 27.8%). MS (ESI, m / z) 576 [M+H] + .

[0248] 1 1H NMR (400 MHz, 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 = 7.2 Hz, 3H), 0.49 - 0.37 (m, 4H).

[0249] Synthesis of Example 4 BX20-9-054

[0250] Step 1: Synthesis of Intermediate 43-2

[0251] Synthesize Intermediate 43-2 with reference to Preparation Example 1.

[0252] Step 2: Synthesis of Intermediate 54-1

[0253] The synthesis route is shown in the following formula:

[0254]

[0255] Method: Dissolve 43-2 (200 mg, crude product, 0.56 mmol) in DCM (4 mL), add 1 drop of DMF, cool down to 0 °C, and slowly add dropwise (COCl)2 (107 mg, 0.84 mmol). Stir at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure, then dissolve 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, extract with EA, combine the organic phases, wash once with water and once with saturated NaCl aqueous solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a black oily compound 53-1 (310 mg, crude product). MS (ESI, m / z) 509 [M+H] + 。

[0256] Step 3: Synthesis of Intermediate 54-2

[0257] The synthetic route is shown as follows:

[0258]

[0259] Method: Dissolve 54-1 (310 mg, crude product, approximately 0.56 mmol) in THF (5 mL), add an aqueous solution of LiOH·H2O (282 mg, 6.72 mmol, dissolved in 3 mL of water), and stir at room temperature for 3 h. After completion of the reaction, concentrate the reaction solution under reduced pressure, dilute with water, adjust the pH to <3 by dropwise addition of 1 N HCl, extract with EA, wash with saturated NaCl aqueous solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a black oily compound 53-2 (280 mg, crude product). MS (ESI, m / z) 495 [M+H] + 。

[0260] Step 4: Synthesis of BX20-9-054

[0261] The synthetic route is shown as follows:

[0262]

[0263] Method: Dissolve compound 54-2 (280 mg, crude product, about 0.56 mmol) in DCM (4 mL), add 1 drop of DMF, cool down to 0 °C, and slowly add dropwise (COCl)2 (107 mg, 0.84 mmol). Stir at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure to obtain the crude acyl chloride. Separately, add methoxylamine hydrochloride (94 mg, 1.12 mmol) and K2CO3 (255 mg, 1.85 mmol) to the EA / H2O (2:1, 9 mL) solution, stir at room temperature for 20 min, then add dropwise the EA solution of the crude acyl chloride, and continue to stir at room temperature for 0.5 h. After completion of the reaction, dilute with water, extract with EA, combine the organic phases, wash once with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, concentrate under reduced pressure, purify by preparative chromatography, and lyophilize to obtain the white solid BX20-9-054 (112 mg, 38%). MS (ESI, m / z) 524 [M+H] + 。

[0264] 1 H NMR (400 MHz, DMSO-d6) δ=11.39 (s, 1H), 10.34 (s, 1H), 7.82 (d, J=6.0 Hz, 1H), 7.72 - 7.65 (m, 1H), 7.21 (t, J=9.2 Hz, 1H), 7.15 - 7.06 (m, 2H), 5.00 (d, J=10.0 Hz, 1H), 4.18 (dd, J=8.0, 10.8 Hz, 1H), 3.64 (s, 3H), 2.75 - 2.66 (m, 1H), 1.55 (s, 3H), 0.67 (d, J=6.0 Hz, 3H).

[0265] Example 5 Synthesis of BX20-9-080

[0266] First step: Synthesis of intermediate 43-2

[0267] Refer to Preparation Example 1 to synthesize intermediate 43-2.

[0268] Second step: Synthesis of intermediate 80-2

[0269] The synthesis route is shown as follows:

[0270]

[0271] Method: Dissolve 43-2 (250 mg, 0.70 mmol) in DCM (4 mL), add a drop of DMF, and dropwise add (COCl)2 (178 mg, 1.40 mmol) at 0 °C. Stir the reaction mixture at room temperature for 0.5 h. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain crude product 80-1 (0.27 g, crude product), which is directly used for the next step; dissolve 4-fluoro-3-cyanoaniline (114 mg, 0.84 mmol) in NMP (2 mL), add TEA (141 mg, 1.40 mmol), and then slowly add 80-1 (crude product, diluted with 4 mL of DCM, about 0.70 mmol). Stir at room temperature for 0.5 h. After completion of the reaction, quench with saturated ammonium chloride, extract with EA, combine the organic phases, wash with concentrated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 80-2 (350 mg, crude product). MS (ESI, m / z) 476 [M+H] + 。

[0272] Step 3: Synthesis of Intermediate 80-3

[0273] The synthetic route is shown as follows:

[0274]

[0275] Method: Dissolve compound 80-2 (350 mg, crude product, about 0.70 mmol) in EtOH (5 mL), cool in an ice bath, add NH2OH (185 mg, 2.80 mmol), and stir at 80 °C for 1 h. After completion of the reaction, directly concentrate under reduced pressure to obtain a brown oil 80-3 (380 mg, crude product). MS (ESI, m / z) 509 [M+H] + 。

[0276] Step 4: Synthesis of Intermediate 80-4

[0277] The synthetic route is shown as follows:

[0278]

[0279] Method: Dissolve compound 80-3 (380 mg, crude product, about 0.70 mmol) in AcOH (6 mL), add Pd / C (500 mg) and ammonium formate (800 mg, 12.7 mmol), displace N2, and stir at 120 °C for 4 h. After completion of the reaction, filter through diatomaceous earth, concentrate under reduced pressure, extract with EA, dry, and concentrate under reduced pressure to obtain a brown solid 80-4 (180 mg, 52.3%). MS (ESI, m / z) 493 [M+H] + 。

[0280] Step 5: Synthesis of Intermediate BX20-9-080

[0281] The synthetic route is shown as follows:

[0282]

[0283] Method: Dissolve compound 80-4 (180 mg, 0.36 mmol) in EtOH (6 mL), add TEA (91 mg, 0.90 mmol), cool down in an ice bath, slowly dropwise add BrCN (69 mg, 0.65 mmol, dissolved in 2 mL ACN), and continue stirring for 10 minutes after dropping. After the reaction is completed, quench with water, extract with EA, dry, and purify by preparative chromatography in a formic acid system to obtain white solid BX20-9-080 (100 mg, 49.3%). MS (ESI, m / z) 518 [M+H] + 。

[0284] 1 H NMR (400 MHz, 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=10.4 Hz, 1H), 4.28 - 4.20 (m, 1H), 2.81 - 2.71 (m, 1H), 1.60 (s, 3H), 0.76 - 0.69 (m, 3H).

[0285] Synthesis of Example 6 BX20-9-082

[0286] First step: Synthesis of intermediate 59-3

[0287] Refer to Preparation Example 2 to synthesize intermediate 59-3

[0288] Second step: Synthesis of intermediate 82-1

[0289] The synthetic route is shown as follows:

[0290]

[0291] Method: Dissolve 59-3 (180 mg, 0.45 mmol) in DCM (5 mL), add 1 drop of DMF, cool down to 0 °C, and slowly add dropwise (COCl)2 (0.2 mL). Stir at room temperature for 0.5 h. After completion of the reaction, concentrate under reduced pressure, then dissolve 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 completion of the reaction, quench with water, extract with EA. Combine the organic phases, wash once with water and once with saturated NaCl aqueous solution respectively. Dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow oily compound 82-1 (200 mg, crude product). MS (ESI, m / z) 520 [M+H] + 。

[0292] Step 3: Synthesis of compound BX20-9-082

[0293] The synthesis route is shown as follows:

[0294]

[0295] Method: Dissolve 82-1 (200 mg, crude product) in EtOH (5 mL), add NH2OH aqueous solution (50%, 153 mg, 2.32 mmol), stir at 80 °C for 1 h. After completion of the reaction, concentrate the reaction solution under reduced pressure to remove EtOH, dissolve in MeOH, filter, purify by preparative chromatography (FA system), and lyophilize to obtain a white powdery solid BX20-9-082 (33 mg, yield 15.7%). MS (ESI, m / z) 553 [M+H] + 。

[0296] 1 H NMR (400 MHz, DMSO-d6) δ = 10.28 (s, 1H), 9.59 (s, 1H), 7.74 - 7.72 (dd, J = 6.4 Hz, J = 2.8 Hz, 1H), 7.64 - 7.60 (m, 1H), 7.17 - 7.12 (m, 3H), 5.75 (s, 2H), 5.03 (d, J = 10.8 Hz, 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).

[0297] Example 7 Synthesis of BX20-9-083

[0298] Step 1: Synthesis of intermediate 60-3

[0299] Refer to Preparation Example 3 for intermediate 60-3.

[0300] Step 2: Synthesis of Intermediate 83-1

[0301] The synthesis route is shown as follows:

[0302]

[0303] Method: Dissolve 60-3 (260 mg, crude product, about 0.64 mmol) in DCM (5 mL), add 1 drop of DMF, cool down to 0 °C, and slowly add dropwise (COCl)2 (163 mg, 1.28 mmol). Stir at room temperature for 0.5 h. After the reaction is completed, concentrate under reduced pressure, then dissolve it in DCM (5 mL) and slowly add it 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 the reaction is completed, quench with water, extract with EA, combine the organic phases, wash with water and saturated NaCl aqueous solution respectively once, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown oily compound 83-1 (330 mg, crude product). MS (ESI, m / z) 524 [M+H] + 。

[0304] Step 3: Synthesis of BX20-9-083

[0305] The synthesis route is shown as follows:

[0306]

[0307] Method: Dissolve 83-1 (330 mg, crude product, about 0.64 mmol) in EtOH (5 mL), add an aqueous solution of NH2OH (169 mg, 2.56 mmol, 50 wt% aqueous solution), heat up to 80 °C and stir for 1 h. After the reaction is completed, concentrate under reduced pressure and purify by preparative chromatography, then lyophilize to obtain a white solid BX20-9-83 (22 mg, 6.2%). MS (ESI, m / z) 557 [M+H] + 。

[0308] 1 1H NMR (400 MHz, DMSO-d6) δ = 10.89 (s, 1H), 10.52 (d, J = 3.2 Hz, 1H), 7.95 - 7.90 (m, 1H), 7.81 - 7.75 (m, 1H), 7.41 - 7.33 (m, 1H), 7.18 - 7.11 (m, 2H), 5.09 (d, J = 10.8 Hz, 1H), 4.37 - 4.30 (m, 1H), 2.90 - 2.79 (m, 1H), 1.59 (s, 3H), 0.75 - 0.67 (m, 3H).

[0309] Synthesis of Example 8 BX20-9-084

[0310] First Step: Synthesis of Intermediate 39-1

[0311] Refer to Preparation Example 1 to synthesize Intermediate 39-1.

[0312] Second Step: Synthesis of Intermediate 39-2

[0313] The synthesis route is shown in the following formula:

[0314]

[0315] Method: Dissolve 39-1 (500 mg, crude product, about 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), heat to 70 °C and stir for 16 hours. After the reaction is completed, quench with water, extract with EA, wash with saturated NaCl aqueous solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow oily compound 39-2 (450 mg, crude product). MS (ESI, m / z) 380 [M+H] + .

[0316] Third Step: Synthesis of Intermediate 39-3

[0317] The synthesis route is shown in the following formula:

[0318]

[0319] Method: Dissolve 39-2 (450 mg, crude product, 1.18 mmol) in MeOH (7.5 mL), add an aqueous solution of KOH (462 mg, 8.26 mmol, dissolved in 1.5 mL of water), heat to 60 °C and react with stirring for 16 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure, dilute with water, extract with MTBE, adjust the pH of the aqueous phase to <3 by dropwise adding 1N HCl, extract with EA, wash with saturated NaCl aqueous solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow oily compound 39-3 (430 mg, crude product). MS (ESI, m / z) 399 [M+H] + .

[0320] Fourth Step: Synthesis of Intermediate 39-4

[0321] The synthesis route is shown in the following formula:

[0322]

[0323] Method: Dissolve 39-3 (160 mg, crude product, 0.402 mmol) in DCM (4 mL), add 1 drop of DMF, cool down to 0 °C, slowly add dropwise (COCl)2 (0.1 mL, 1.206 mmol), and stir the reaction at room temperature for 0.5 h. After the reaction is completed, concentrate under reduced pressure, then dissolve in DCM (4 mL), and slowly add 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), and stir at room temperature for 0.5 h. After the reaction is completed, concentrate the reaction solution under reduced pressure, dissolve with EA, quench with water, extract with EA, combine the organic phases, wash once with water and saturated NaCl aqueous solution respectively, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain the yellow oily compound 39-4 (207 mg, crude product). MS (ESI, m / z) 517 [M+H] + 。

[0324] Step 5: Synthesis of Intermediate 39-5

[0325] The synthetic route is shown as follows:

[0326]

[0327] Method: Dissolve compound 39-4 (207 mg, crude product, about 0.4 mmol) in EtOH (5 mL), add NH2OH (0.1 mL, 1.608 mmol, 50% in H2O), heat up to 80 °C and stir the reaction for 1 h. After the reaction is completed, concentrate under reduced pressure and purify by preparative chromatography (formic acid system), and lyophilize to obtain the white solid 39-5 (112 mg, 51%). MS (ESI, m / z) 550 [M+H] + 。

[0328] Step 6: Synthesis of Intermediate BX20-9-084

[0329] The synthetic route is shown as follows:

[0330]

[0331] Method: Dissolve compound 39-5 (90 mg, 0.16 mmol) in AcOH (5 mL), add Pd / C (100 mg, 10% purity) and HCOONH4 (202 mg, 3.2 mmol), replace nitrogen and heat up to 120 °C and stir for 2.5 h. After the reaction is completed, concentrate under reduced pressure and purify by preparative chromatography (formic acid system), and lyophilize to obtain the white solid BX20-9-084 (48 mg, 56%). MS (ESI, m / z) 534 [M+H] + 。

[0332] 1 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.43 (s, 1H), 7.97 (dd, J = 2.4, 6.0 Hz, 1H), 7.90 - 7.83 (m, 1H), 7.41 (t, J = 9.6 Hz, 1H), 7.22 - 7.11 (m, 2H), 5.13 (d, J = 10.8 Hz, 1H), 4.34 (dd, J = 7.2, 10.8 Hz, 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 = 5.6 Hz, 3H).

[0333] Synthesis of Example 9 BX20 - 9 - 090

[0334] Step 1: Synthesis of Intermediate 39 - 3

[0335] Synthesize Intermediate 39 - 3 with reference to Example 8.

[0336] Step 2: Synthesis of Intermediate 90 - 1

[0337] The synthesis route is shown as follows:[[]]

[0338]

[0339] Method: Dissolve 39 - 3 (160 mg, 0.40 mmol) in DCM (4 mL), add one drop of DMF, and dropwise add (COCl)2 (102 mg, 0.80 mmol) at 0 °C. Stir the reaction mixture at room temperature for 0.5 h. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain crude 39 - 4 (200 mg, crude product), which is directly used for the next step; dissolve 4 - fluoro - 3 - cyanobenzenamine (66 mg, 0.48 mmol) in NMP (2 mL), add TEA (81 mg, 0.80 mmol), and then slowly add 39 - 4 (crude product, diluted with 4 mL of DCM, about 0.40 mmol). Stir at room temperature for 0.5 h. After completion of the reaction, quench with saturated ammonium chloride, extract with EA, combine the organic phases, wash with concentrated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 90 - 1 (250 mg, crude product). MS (ESI, m / z) 517 [M + H] + 。

[0340] Step 3: Synthesis of 90 - 2

[0341] The synthesis route is shown as follows:[[]]

[0342]

[0343] Method: Dissolve compound 90-1 (250 mg, crude, approximately 0.40 mmol) in EtOH (5 mL), cool in an ice bath, add NH2OH (50% aqueous solution, 106 mg, 1.60 mmol), and raise the temperature to 80 °C and stir for 1 hour. After completion of the reaction, directly concentrate under reduced pressure to obtain a brown oil 90-2 (280 mg, crude). MS (ESI, m / z) 550 [M+H] + .

[0344] Step 4: Synthesis of 90-3

[0345] The synthesis route is shown as follows:

[0346]

[0347] Method: Dissolve compound 90-2 (280 mg, crude, approximately 0.40 mmol) in AcOH (6 mL), add Pd / C (10%, 500 mg) and ammonium formate (800 mg, 12.7 mmol), displace N2, raise the temperature to 120 °C and stir for 4 hours. After completion of the reaction, filter through diatomaceous earth, concentrate under reduced pressure, extract with EA, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown solid 90-3 (170 mg, 79.7%). MS (ESI, m / z) 534 [M+H] + .

[0348] Step 5: Synthesis of BX20-9-090

[0349] The synthesis route is shown as follows:

[0350]

[0351] Method: Dissolve compound 90-3 (170 mg, 0.31 mmol) in EtOH (6 mL), add TEA (79 mg, 0.78 mmol), cool in an ice bath, slowly add dropwise a solution of BrCN (59 mg, 0.56 mmol) in acetonitrile (2 mL). After addition, continue stirring for 10 minutes. After completion of the reaction, quench with water, extract with EA, dry over anhydrous sodium sulfate, and concentrate under reduced pressure, then purify by preparative chromatography in a neutral system to obtain a white solid BX20-9-090 (36 mg, 20.8%). MS (ESI, m / z) 559 [M+H] + .

[0352] 11H NMR (400 MHz, 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 = 10.8 Hz, 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).

[0353] Synthesis of Example 10 BX20 - 9 - 095

[0354] Step 1: Synthesis of Intermediate 39 - 1

[0355] Synthesize Intermediate 39 - 1 with reference to Preparation Example 1.

[0356] Step 2: Synthesis of Intermediate 40 - 1

[0357] The synthesis route is shown as follows:

[0358]

[0359] Method: Dissolve 39 - 1 (500 mg, crude product, 1.55 mmol) in DMF (5 mL), add Cs2CO3 (2.1 mL, 21.32 mmol) and 3 - iodooxetane (1.14 g, 6.2 mmol), heat to 70 °C and react with stirring for 16 hours. After the reaction is completed, quench with water, extract with EA, wash with saturated NaCl aqueous solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow oily compound 40 - 1 (565 mg, crude product). MS (ESI, m / z) 378 [M + H] + .

[0360] Step 3: Synthesis of Intermediate 40 - 2

[0361] The synthesis route is shown as follows:

[0362]

[0363] Method: Dissolve 40-1 (565 mg, crude product, 1.5 mmol) in MeOH (7.5 mL), add an aqueous solution of KOH (587 mg, 10.5 mmol, dissolved in 1.5 mL of water), heat to 60 °C and react with stirring for 16 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure, dilute with water, extract with MTBE, adjust the pH of the aqueous phase to <3 by dropwise addition of 1N HCl, extract with EA, wash with saturated aqueous NaCl solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow oily compound 40-2 (330 mg, crude product). MS (ESI, m / z) 397 [M+H] + 。

[0364] Step 4: Synthesis of Intermediate 95-1

[0365] The synthetic route is shown as follows:

[0366]

[0367] 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 the reaction at room temperature for 3 hours. After the reaction is completed, extract with EA, wash the organic phase with water and saturated aqueous NaCl solution once respectively, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a brown oily compound 95-1 (620 mg, crude product). MS (ESI, m / z) 498 [M+H] + 。

[0368] Step 5: Synthesis of 95-2

[0369] The synthetic route is shown as follows:

[0370]

[0371] Method: Dissolve compound 95-1 (200 mg, crude product, about 0.4 mmol) in DCE (5 mL), add m-CPBA (85%, 244 mg, 1.2 mmol), heat to 80 °C and stir the reaction for 4 hours. After the reaction is completed, cool to room temperature, quench with NaOH solution, extract with EA, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown oily substance 95-2 (220 mg, crude product). MS (ESI, m / z) 514 [M+H] + 。

[0372] Step 6: Synthesis of BX20-9-095

[0373] The synthetic route is shown as follows:

[0374]

[0375] Method: Dissolve compound 95-2 (220 mg, crude product, about 0.4 mmol) in EtOH (8 mL), add dropwise NH2OH (50% aqueous solution, 106 mg, 1.6 mmol), and raise the temperature to 80 °C, then stir and react for 1 hour. After the reaction is completed, directly concentrate under reduced pressure and then purify by preparative chromatography in a formic acid system to obtain white solid BX20-9-095 (54 mg, 24.7%). MS (ESI, m / z) 547 [M+H] + 。

[0376] 1 H NMR (400 MHz, 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 = 10.0 Hz, 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).

[0377] Example 11 Synthesis of BX20-9-096

[0378] First step: Synthesis of intermediate 39-3

[0379] Refer to Example 8 to synthesize intermediate 39-3.

[0380] Second step: Synthesis of 96-1

[0381] The synthesis route is shown in the following formula:

[0382]

[0383] Method: Dissolve 39-3 (140 mg, 0.35 mmol) in DCM (4 mL), add a drop of DMF, and dropwise add (COCl)2 (89 mg, 0.70 mmol) at 0 °C. Stir the reaction mixture at room temperature for 0.5 h. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain crude 39-4 (180 mg, crude product), which is directly used for the next step; dissolve 2-cyano-4-aminopyridine (50 mg, 0.42 mmol) in NMP (2 mL), add TEA (71 mg, 0.80 mmol), and then slowly add 39-4 (180 mg of crude product, diluted with 4 mL of DCM, about 0.40 mmol). Stir at room temperature for 0.5 h. After completion of the reaction, quench with water, extract with EA, combine the organic phases, wash with saturated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 96-1 (210 mg, crude product). MS (ESI, m / z) 500 [M+H] + 。

[0384] Step 3: Synthesis of 96-2

[0385] The synthetic route is shown as follows:

[0386]

[0387] Method: Dissolve compound 96-1 (500 mg, crude product, about 1.0 mmol) in DCE (7 mL), add m-CPBA (85%, 406 mg, 2.0 mmol), and stir the reaction at 80 °C for 4 h. After completion of the reaction, cool to room temperature, quench with NaOH solution, extract with EA, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown oil 96-1 (560 mg, crude product). MS (ESI, m / z) 516 [M+H] + 。

[0388] Step 4: Synthesis of BX20-9-096

[0389] The synthetic route is shown as follows:

[0390]

[0391] Method: Dissolve compound 96-2 (560 mg, crude product, about 1.09 mmol) in EtOH (5 mL), add NH2OH (50% aqueous solution, 287 mg, 4.36 mmol), and stir the reaction at 80 °C for 1 h. After completion of the reaction, directly concentrate under reduced pressure and purify by preparative chromatography in a neutral system to obtain a white solid BX20-9-096 (196 mg, 32.8%). MS (ESI, m / z) 549 [M+H] + 。

[0392] 11H NMR (400 MHz, 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 = 10.8 Hz, 1H), 4.37 - 4.30 (m, 1H), 4.30 - 4.14 (m, 2H), 3.66 - 3.55 (m, 2H), 3.26 (d, J = 0.8 Hz, 3H), 2.87 - 2.76 (m, 1H), 1.60 (s, 3H), 0.73 - 0.66 (m, 3H).

[0393] Synthesis of Example 12 BX20 - 9 - 098

[0394] Step 1: Synthesis of Intermediate 96 - 1

[0395] Refer to Example 10 to synthesize Intermediate 95 - 1.

[0396] Step 2: Synthesis of BX20 - 9 - 098

[0397] The synthesis route is shown as follows:

[0398]

[0399] Method: Dissolve Compound 95 - 1 (250 mg, 0.5 mmol) in EtOH (5 mL), add NH2OH (50% aqueous solution, 132 mg, 2.0 mmol), raise the temperature to 80 °C and stir for 1 hour. After the reaction is completed, directly concentrate under reduced pressure and then purify through preparative chromatography in a neutral system to obtain white solid BX20 - 9 - 098 (85 mg, 32.1%). MS (ESI, m / z) 531 [M + H] + .

[0400] 1 1H NMR (400 MHz, 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 = 10.4 Hz, 1H), 4.91 - 4.81 (m, 2H), 4.73 - 4.65 (m, 2H), 4.31 - 4.23 (m, 1H), 2.85 - 2.74 (m, 1H), 1.61 (s, 3H), 0.77 - 0.68 (m, 3H).

[0401] Synthesis of Example 13 BX20 - 9 - 099

[0402] Step 1: Synthesis of Intermediate 96-1

[0403] Refer to the first and second steps of Reference Example 11 to synthesize Intermediate 96-1

[0404] Step 2: Synthesis of BX20-9-099

[0405] The synthesis route is shown as follows:

[0406]

[0407] Method: Dissolve Compound 96-1 (210 mg, crude product, about 0.42 mmol) in EtOH (5 mL), cool it in an ice bath, add NH2OH (50% aqueous solution, 111 mg, 1.68 mmol), and stir at 80 °C for 1 hour. After the reaction is completed, directly concentrate under reduced pressure, and purify by preparative chromatography to obtain white solid BX20-9-099 (61 mg, 27.3%). MS (ESI, m / z) 533 [M+H] + 。

[0408] 1 1H NMR (400 MHz, 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).

[0409] Reference compound:

[0410] Refer to the synthesis method of Example 3 of Patent CN114945566A to synthesize the reference compound (Compound 7)

[0411]

[0412] 11H NMR (400 MHz, CD3OD) δ = 8.48 (d, J = 5.6 Hz, 1H), 8.25 (d, J = 2.0 Hz, 1H), 7.89 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.13 - 7.09 (m, 1H), 7.00 - 6.94 (m, 1H), 5.08 (d, J = 10.4 Hz, 1H), 4.34 - 4.30 (m, 1H), 3.99 (d, J = 2.4 Hz, 3H), 2.83 - 2.75 (m, 1H), 1.65 (s, 3H), 0.82 - 0.80 (m, 3H)

[0413] Biological test evaluation

[0414] The present invention will be further described and explained below in combination with test examples

[0415] Test Example 1 Blocking activity of the compound of the present invention against sodium channel 1.8 (Nav1.8)

[0416] 1. Experimental purpose: To detect the effect of the compound on the current of voltage-gated sodium channel (NaV) subtype 1.8 by patch clamp technique. 2. Experimental materials and equipment

[0417] 2.1 Cell line: A CHO cell line stably expressing the Nav1.8 sodium channel. The Nav1.8 cells were constructed by the laboratory of Beijing Ace Medicine Co., Ltd. (Reference: Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, KrambisM, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, GauvinD, 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, KrafteDS. A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuatesneuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A. 2007May15;104(20):8520-5. doi:10.1073 / pnas.0611364104. Epub 2007May 2. PMID:17483457; PMCID:PMC1895982.), Gene information: Sodium channel, voltage-gated, type 8, alpha (SCN10A), cDNA strictly similar to GenBank accession number: NM_006514

[0418] 2.2 Compound: Dissolved in DMSO

[0419] 3. Experimental methods

[0420] 3.1 Cell culture

[0421] (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 a culture temperature of 37 °C and a carbon dioxide concentration of 5%.

[0422] (2) Cell passage: Remove the old 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 min. When the cells detach from the bottom of the dish, add about 5 mL of pre-warmed complete medium at 37°C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min to collect the cells. For expansion or maintenance culture, inoculate the cells into a 6-cm cell culture dish, with the cell seeding density of 2.5×105 cells per cell culture dish (final volume: 5 mL).

[0423] (3) To maintain the electrophysiological activity of the cells, the cell density must not exceed 80%.

[0424] (4) Patch-clamp recording: Before the experiment, the cells were dissociated with 0.25%-Trypsin-EDTA. 6.5×103 cells were plated onto coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 hours, the experiment was performed for recording.

[0425] 3.2. Patch-clamp recording

[0426] (1) When the whole-cell patch-clamp configuration was established, the cell was voltage-clamped at -120 mV. First, the voltage was stepped from -130 mV to -10 mV in 10-mV increments and maintained for 5 s, then a 0-mV depolarizing pulse was applied to obtain the half-inactivation voltage (Vhalf). The resting state and half-inactivated state of the sodium current were detected using a double-pulse protocol. First, a depolarizing pulse (TP1) to 0 mV for 50 ms was applied to detect the sodium current in the resting state. Then the voltage was adjusted to Vhalf and maintained for 5 s, followed by restoring the voltage to -120 mV and maintaining for 20 ms. Then a second depolarizing pulse (TP2) to 0 mV for 50 ms was applied to detect the sodium current in the half-inactivated state. Finally, the voltage was restored to the clamped voltage of -120 mV. Data were acquired every 20 ms to observe the effect of the drug on the peak sodium current in the two different states. The experimental data were acquired by an EPC 10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0427] (2) During patch clamp operation, first use a microelectrode puller to pull the capillary glass tube into a recording electrode, then insert the electrode filled with intracellular fluid into the microelectrode holder. Under an inverted microscope, operate the microelectrode manipulator to bring the recording electrode into contact with the cell, and apply negative pressure suction to form a GΩ seal. At this time, perform fast capacitance compensation, then continue to apply negative pressure to rupture the cell membrane to form the whole-cell recording mode. Finally, perform slow capacitance compensation and record relevant parameters. Do not apply leakage compensation.

[0428] (3) When the sodium current in the whole-cell recording is stable, start drug administration. After each drug concentration acts for 5 min (or until the current is stable), detect the next concentration. Place the cover glass with cells in the recording bath under the inverted microscope. The blank control external solution and the working solution of the test compound flow through the recording bath from low concentration to high concentration in sequence by gravity perfusion to act on the cells, and a peristaltic pump is used for liquid exchange during recording. The current detected for each cell in the external solution without the compound serves as its own control group. Each concentration is independently detected 2 times. All electrophysiological experiments are carried out at room temperature.

[0429] 3.3. Data analysis

[0430] First, normalize the current after the action of each drug concentration and the blank control current, then calculate the inhibition rate corresponding to each drug concentration, that is, (1 - current after compound action / blank control current), and calculate the mean (Mean), standard deviation (SD), and standard error (SE) for the inhibition rate of each concentration. The data is expressed as mean ± SE.

[0431] 4. Experimental results

[0432] Table 1 Blocking rate of the compound of the present invention on NaV1.8 at 10 nM

[0433]

[0434]

[0435] It can be seen that the compound of the present invention has an obvious blocking effect on the activity of the NaV1.8 channel.

[0436] Test Example 2 Blocking activity of the compound of the present invention on sodium channel 1.8 (Nav1.8) (IC 50 )

[0437] 1. Research purpose

[0438] Apply the manual patch clamp technique to evaluate whether the test compound has a potential inhibitory effect on the voltage-gated sodium channel hNav1.8. This experiment detects the effects of 5 concentrations, single point, or two concentration points of the compound on the hNav1.8 channel current, obtains the dose-effect curve of the compound, and calculates the IC50 This experiment included the determination of two parallel samples.

[0439] 2. Test method

[0440] 2.1. Test materials

[0441] 1) Cells: The HEK293 cell line stably expressing hNav1.8 / β3 ion channels was prepared by the Biology Department of Catalent (Beijing) New Drug Technology Co., Ltd. (by transducing lentiviral vectors expressing human Nav1.8 and β3 into HEK293 respectively and preparing them after screening. For specific reference: Proc Natl Acad Sci U S A. 2022 Jul 26; 119(30):e2208211119.doi:10.1073 / pnas.2208211119. and Cell Lines / BSYS CHO NaV1.8 / β3 Cell Line instruction manual). This 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 increased to 40% - 80% of the bottom area of the culture dish, it was digested with trypsin and passaged three times a week. Before the experiment, the cells were cultured in a 6 cm culture dish with a total number of 5×105 and seeded on glass slides for subsequent manual patch clamp experiments.

[0442] 2) Compounds: The test compounds were dissolved in DMSO and prepared into stock solutions with a final concentration of 10 or 30 mM. The stock solutions were diluted with DMSO as the solvent into the required intermediate solutions. Before the start of the experiment, the gradient intermediate solutions of the test compounds were diluted again with extracellular fluid at a ratio of 1:1000 into a series of concentration working solutions. The content of DMSO in the working solutions was 0.1% (volume ratio). The working solutions with different concentration gradients were used to determine the potential inhibitory effect of the compounds on the hNav1.8 channel and to fit the dose-effect curve and calculate the IC 50 .

[0443] 2.2. Test procedures

[0444] 1) Place the small glass slide with HEK293 cells in the perfusion chamber of the micromanipulation stage.

[0445] 2) Under an Olympus IX71 or IX73 inverted microscope, place the appropriate cells in the center of the field of view. Use a ×10 objective lens to find the tip of the glass electrode and place it in the center of the field of view. Then use the micromanipulator to lower the electrode while adjusting the coarse focus screw to slowly approach the cells.

[0446] 3) When approaching the cell closely, switch to the ×40 objective lens for observation, and gradually approach the cell surface with the electrode through the fine adjustment knob of the micromanipulator.

[0447] 4) Apply negative pressure to form a seal with a resistance higher than 1 GΩ between the electrode tip and the cell membrane.

[0448] 5) Compensate for the instantaneous capacitive current Cfast in the voltage-clamp mode. Then repeatedly apply short negative pressure to rupture the membrane, and finally form the whole-cell recording mode.

[0449] 6) Under the condition that the membrane potential is clamped at -60 mV, compensate for the slow capacitive current Cslow, the cell membrane capacitance (Cm), and the input membrane resistance (Ra) respectively.

[0450] 7) After the cell is stable, change the clamping voltage to -80 mV, set the sampling frequency to 20 kHz, and the filtering frequency to 10 kHz. The detection condition for the leakage current is that the clamping voltage changes to -80 mV with a duration of 200 ms.

[0451] 8) The method for testing hNav1.8 current is as follows: Apply a 20-ms depolarizing command voltage to depolarize the membrane potential from -80 mV to -10 mV, and then repolarize the membrane potential to -80 mV to close the channel. Stimulate once every 15 seconds. The peak value of the instantaneous current under the depolarizing voltage is the magnitude of the Nav1.8 sodium channel current.

[0452] 9) The hNav1.8 current used to detect the test compound is continuously recorded for 120 seconds before drug administration to evaluate the stability of the test cells in generating hNav1.8 current. Only stable cells within the acceptable range of the evaluation criteria can enter the subsequent compound detection.

[0453] 10) Test for the inhibitory effect of the test compound on hNav1.8 current: First, use the hNav1.8 current measured in the extracellular fluid containing 0.1% DMSO as the detection baseline. After the hNav1.8 current remains stable for at least 5 minutes, perfuse the solutions containing the test compound around the cell in ascending order of concentration from low to high. Wait about 5 minutes after each perfusion to allow the compound to fully act on the cell and synchronously record the hNav1.8 current. After the recorded current tends to be stable, record the last 5 hNav1.8 current values and take their average as the final current value at a specific concentration. After testing the compound, add 5 nM reference compound to the same cell to completely inhibit its current as the positive control for the cell. At the same time, the positive compound reference compound is synchronously detected with the same patch-clamp system before and after the test of the test drug 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).

[0454] 2.3. Data Analysis

[0455] 1) The data shall meet the following criteria: the initial sealing resistance is greater than 1 GΩ; the membrane-breaking resistance Ra is less than 15 MΩ; the leakage current under the detection voltage is less than 50% of the current value under this condition; the Nav1.8 peak current magnitude is at least greater than 200 pA;

[0456] 2) The data shall be analyzed according to the following steps (the data is output by PatchMaster software):

[0457] ① After perfusing the blank solvent or the compound gradient solution, stabilize the 5 consecutive current values obtained and calculate the average value, which are respectively used as "current magnitude 空白 " and "current magnitude 化合物 ". The percentage of current inhibition is calculated by the following formula.

[0458]

[0459] ② The dose-effect curve is fitted by Graphpad Prism 8.0 software and the IC50 value is calculated.

[0460] The standard deviation range of the two groups of data is less than 15 (SD < 15)

[0461] 3. Test Results

[0462] Table 2 Blocking Activity of the Compounds of the Present Invention against NaV1.8

[0463]

[0464]

[0465] It can be seen that the compounds of the present invention have strong inhibitory activity against the NaV1.8 channel, which is better than the reference compound.

[0466] Test Example 3 Selectivity Test of the Compounds of the Present Invention against Sodium Ion Channels

[0467] 1. Experimental Purpose: Use the patch clamp technique to detect the effects of compounds on the currents of voltage-gated sodium channel (NaV) subtypes 1.1 - 1.7

[0468] 2. Experimental Materials and Instrument Equipment

[0469] 2.1. Cell line: CHO / HEK293 cell lines stably expressing Nav1.1 - 1.9 sodium channels were constructed by the laboratories of Beijing Ace Medicine Discovery Co., Ltd. / the Biology department of Pharmaron (Beijing) Co., Ltd. (Reference: 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, MarshK, 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 U S A. 2007 May 15;104(20):8520 - 5. doi:10.1073 / pnas.0611364104. Epub 2007 May 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.

[0470] 2.2. Compound: Dissolved in DMSO

[0471] 3. Experimental methods

[0472] 3.1. Cell culture

[0473] (1) Maintenance medium: CHO cells were cultured in HAM’S / F-12 medium containing 10% fetal bovine serum, 100 μg / mL Zeocin and 10 μg / mL Blasticidin at a culture temperature of 37°C and a carbon dioxide concentration of 5%. HEK-293 cells were cultured in DMEM medium containing 10% fetal bovine serum and 800 μg / mL G418 at a culture temperature of 37°C and a carbon dioxide concentration of 5%.

[0474] (2) Cell passage: Remove the old 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 min. When the cells detached from the bottom of the dish, add about 5 mL of pre-warmed complete medium at 37°C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min to collect the cells. For amplification or maintenance culture, inoculate the cells into a 6 cm cell culture dish, and the cell inoculation amount for each cell culture dish is 2.5×10 5 cells (final volume: 5 mL).

[0475] (3) To maintain the electrophysiological activity of the cells, the cell density must not exceed 80%.

[0476] (4) Patch clamp detection: Before the experiment, the cells were separated with 0.25%-Trypsin-EDTA, and 6.5×10 3 cells were seeded onto cover slips and cultured in a 24-well plate (final volume: 500 μL). After 18 hours, the experiment was carried out for detection.

[0477] 3.2. Patch clamp detection

[0478] Same as 3.2 of Test Example 1

[0479] 3.3. Data analysis

[0480] Same as 3.3 of Test Example 1

[0481] 4. Experimental results

[0482] Table 3 Blocking rate of the compounds of the present invention on NaV1.1-1.7 at 30 μM

[0483]

[0484] “ / ” indicates not measured.

[0485] Conclusion: It can be seen that the compounds of the present invention have no obvious activity on the NaV1.1-1.7 channels. At the same concentration, the inhibitory activity on the NaV1.1-1.7 channels is lower than that of the reference compounds, with strong target selectivity, indicating that the compounds of the present invention have better safety.

[0486] Test Example 4 Pharmacokinetic Determination of the Compounds of the Present Invention in SD Rats

[0487] 1. Test Purpose

[0488] Using SD rats as test animals, study the pharmacokinetic behavior of the following compound examples in rat plasma after oral administration at a dose of 10 mg / kg.

[0489] 2. Test Method

[0490] 2.1. Test Drugs

[0491] The examples of the present invention and the reference compound, self-made.

[0492] 2.2. Test Animals

[0493] Male SPF-grade SD rats, weighing (200±20) g, supplied by Beijing Spevogene Biotechnology Co., Ltd., animal production license number SCXK (Beijing) 2022-0030.

[0494] 2.3. Preparation of Test Drugs

[0495] Drug preparation: Prepare a drug concentration of 1 mg / mL, and the preparation solution is Tween 80 + 0.5% methylcellulose (MC, CAS: 9004-67-5) (1:99, v / v) solution.

[0496] 2.4. Administration

[0497] Male SPF-grade SD rats, after 3-4 days of adaptive feeding, were administered by gavage respectively, with a dose of 10 mg / kg and a volume of 10 mL / kg.

[0498] Before (0 h) and after administration of the rats, blood was collected at 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h. Blood was collected by puncturing the fundus of the eye, with a blood collection volume of about 0.5 mL, and placed in an EDTA-K2 anticoagulant test tube with a label attached. After blood collection, the blood collection tube was gently inverted completely 3 times immediately to mix with the anticoagulant, and immediately placed in an ice-water bath. Centrifuged at 4500 rpm for 10 min at 4 °C. After the centrifugation operation was completed, the plasma was taken and timely aliquoted into EP tubes with corresponding labels attached, and stored in a -80 °C refrigerator.

[0499] 2.6. Sample Detection

[0500] In this experiment, a briefly validated LC-MS / MS method was used to determine the concentrations of Example X and Example Y in plasma after administration.

[0501] 2.6.1. Sample preparation: Take 20 μL of blank plasma and add it to a 96-well deep plate pre-added with 20 μL of internal standard working solution. Then add 400 μL of precipitant methanol, vortex for 10 min, centrifuge at 4000 rpm for 15 min. Take 200 μL of the supernatant and add it to another 96-well deep 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.

[0502] 2.6.2. LC-MS analysis:

[0503] 1) Liquid phase conditions

[0504] Chromatographic column: Welch CB-C8, 2.1 * 50 mm, 5 μm; No.: 217

[0505] Pressure: 10 Mpa

[0506] Pre-column: Phenomenex guard column

[0507] Column temperature: 40 °C

[0508] Injection volume: 5 μL

[0509] Injection temperature: 4 °C

[0510] Running time: 3.5 min

[0511] Flow rate: 0.6 mL / min

[0512] Column pressure: 10 Mpa

[0513] Needle wash solution: 50% methanol in water

[0514] Needle wash mode: Before and after aspiration

[0515] Mobile phase A: 0.1% formic acid in 10 mM ammonium acetate aqueous solution

[0516] Mobile phase B: 0.1% formic acid in 10 mM ammonium acetate methanol solution

[0517] Elution program and mobile phase ratio: Isocratic elution A:B = 40:60.

[0518] 2) Mass spectrometry conditions:

[0519] Ion detection mode: Multiple reaction monitoring (MRM)

[0520] Ion polarity: Positive ion

[0521] Ionization method: Pneumatically assisted electrospray ionization (ESI)

[0522] CAD: 8

[0523] CUR: 40 psi

[0524] GS1: 50 psi

[0525] GS2: 50 psi

[0526] TEM: 600 °C

[0527] IS: 4500 v

[0528] 3. Test Results and Analysis

[0529] The main pharmacokinetic parameters were calculated using WinNonlin 7.0. The results of the pharmacokinetic experiment in rats are shown in Table 3 below.

[0530] Table 4 Results of the Pharmacokinetic Experiment in Rats

[0531]

[0532]

[0533] 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.

[0534] Test Example 5 Pharmacokinetic Determination in KM Mice

[0535] 1. Test Purpose

[0536] Using KM mice as the test animals, study the pharmacokinetic behavior of the compound of the example in the plasma of mice after intragastric administration at a dose of 10 mg / kg / intravenous injection at a dose of 1 mg / kg.

[0537] 2. Test Method

[0538] 2.1. Test Drugs

[0539] The examples and reference compounds of the present invention were prepared in-house.

[0540] 2.2. Test Animals

[0541] Male SPF-grade KM mice, weighing (20 ± 2) g, Hubei Experimental Animal Research Center, production license number: SCXK(E)2020-0018.

[0542] 2.3. Preparation of Test Drugs

[0543] 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).

[0544] Drug preparation for the intravenous administration group: Prepare the drug concentration at 0.2 mg / mL, and the preparation solvent is 5% DMA + 5% Solutol HS-15 + 90% normal saline.

[0545] 2.4. Administration:

[0546] Male SPF-grade KM mice were adaptively fed for 3 - 4 days.

[0547] Gavage administration group: The administration dose was 10 mg / kg, and the administration volume was 10 mL / kg.

[0548] Intravenous administration group: The administration dose was 1 mg / kg, and the administration volume was 5 mL / kg.

[0549] 2.5. Sample collection

[0550] Gavage administration: Before (0 h) and after administration to the mice, blood was collected at 0, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h. For intravenous injection, before (0 h) and after administration, blood was collected at 5, 15, 30 min, 1, 2, 4, 6, 8, 24 h.

[0551] Blood was collected by puncturing the fundus of the eye, with the blood collection volume of about 0.1 mL, and placed in an EDTA-K2 anticoagulant test tube with a label. After blood collection, the blood collection tube was gently inverted completely 3 times immediately to mix with the anticoagulant, and immediately placed in an ice-water bath. Centrifuged at 4500 rpm for 10 min at 4 °C. After the centrifugation operation was completed, the plasma was taken and timely aliquoted into an EP tube with a corresponding label, and stored in a -80 °C refrigerator.

[0552] 2.6. Sample detection

[0553] Take 20 μL of mouse plasma after administration, add 400 μL of precipitating agent methanol to a 96-well deep plate pre-added with 20 μL of internal standard working solution, vortex for 10 min, centrifuge at 4000 rpm for 15 min, take 200 μL of the supernatant and add it to another 96-well deep plate pre-added with 200 μL of ultrapure water, vortex for 5 min, centrifuge at 4000 rpm for 3 min, and take 5 μL of the supernatant for LC-MS / MS analysis of plasma drug concentration.

[0554] 3. Experimental results and analysis

[0555] The main pharmacokinetic parameters were calculated using WinNonlin 7.0, and the results of the mouse pharmacokinetic experiment are shown in Table 5 below.

[0556] Table 5 Results of mouse pharmacokinetic experiment

[0557]

[0558] 4. Experimental conclusion

[0559] As can be seen from the above data, at the same dosing dose, the exposure of the compound of the present invention is higher than that of the reference compound, and the bioavailability is also higher. It can be seen that the compound of the present invention exhibits better absorption characteristics.

[0560] Pharmacodynamic test in the acetic acid writhing model of mice in Test Example 6

[0561] 1. Purpose: To evaluate the analgesic pharmacodynamic effect of the examples in the acetic acid writhing model of KM mice.

[0562] 2. Test method:

[0563] 2.1. Test drugs: The examples of the present invention, reference compounds, self-made. Naproxen, Shanghai Yuanye Bio-Technology Co., Ltd., product number S63435. Prepared using a solvent (Tween 80 + 0.5% MC (v:v = 1:99 pH = 3)).

[0564] 2.2. Test animals: Male KM mice, purchased from Hubei Center for Disease Control and Prevention (Hubei Academy of Preventive Medicine), with a body weight of 20 - 25 g at the time of purchase.

[0565] 2.3. Test grouping:

[0566] Table 6 Grouping of pharmacodynamic tests of compounds in the acetic acid writhing model

[0567]

[0568]

[0569] 2.4. Drug administration and model establishment

[0570] One hour after the administration of the corresponding drugs in each group, 0.6% acetic acid (0.15 mL / 10 g (0.1 mL / 10 g is also acceptable)) was intraperitoneally injected, and the writhing latency (the time when the mouse first showed a writhing reaction after the injection of glacial acetic acid) was recorded. The number of writhing times of the mice within 20 minutes was observed and recorded.

[0571] · Writhing index: When the mouse showed typical abdominal concavity, accompanied by trunk torsion, hip elevation and other characteristic reactions, it was considered that writhing occurred.

[0572] 2.5. Data collection and analysis

[0573] Use Excel software to collect data.

[0574] Use Prism (Graph pad software, Inc.) software to analyze data.

[0575] 3. Results

[0576] Table 7 Analgesic Pharmacodynamic Results of Compounds in the Mouse Acetic Acid Writhing Pain Model

[0577] Grouping Administration Route Dose Writhing Times p (vs Solvent Control) Number of Animals Solvent Control Group Gavage, single dose 0.1 mL / 10 g 13.50±8.34 <0.05 10 Positive Drug Group (Naproxen) Gavage, single dose 100 mg / kg 4.80±3.52 <0.05 10 Reference Compound Gavage, single dose 30 mg / kg 10.14±6.18 <0.05 10 Example 2 Gavage, single dose 30 mg / kg 3.44±3.21 <0.05 10 Solvent Control Group Gavage, single dose 0.1 mL / 10 g 22.38±7.07 / 10 Positive Drug Group (Naproxen) Gavage, single dose 100 mg / kg 10.33±4.06 <0.05 10 Reference Compound Gavage, single dose 10 mg / kg 16.44±4.42 <0.05 10 Example 2 Gavage, single dose 10 mg / kg 15.40±5.44 <0.05 10 Example 2 Gavage, single dose 30 mg / kg 12.43±3.74 <0.05 10 Example 2 Gavage, single dose 100 mg / kg 9.00±3.12 <0.05 10

[0578] 4. Conclusion

[0579] As can be seen from the above data, at the same dosing dose, Compound Example 2 of the present invention can inhibit the pain in mice caused by acetic acid and reduce the number of writhing in mice. And the analgesic effect of Example 2 is stronger than that of the reference compound.

[0580] Test Example 7 Pharmacodynamic Determination of Mouse Foot Incision

[0581] 1. Purpose: To evaluate the analgesic pharmacodynamic effect of the example in the ICR mouse foot incision model.

[0582] 2. Test Method:

[0583] 2.1. Test Drugs: The examples of the present invention are self-made and prepared using a solvent (Tween 80 + 0.5% MC (v:v = 1:99, PH = 3)). Tramadol hydrochloride, Shijiazhuang Pharmaceutical Group Ouyi Pharmaceutical Co., Ltd., batch number 012230682, is prepared using normal saline.

[0584] 2.2. Test Animals: Male ICR mice, purchased from Sichuan Vital River Laboratory Animal Technology Co., Ltd., with a body weight of 28 - 35 g at the time of purchase.

[0585] 2.3. Test Grouping:

[0586] Table 8 The pharmacodynamic test grouping of the compounds in the mouse foot incision model is as follows:

[0587]

[0588] 2.4. Model Establishment and Drug Administration

[0589] After the animals are adapted for 3 - 5 days, the baseline before model establishment is measured before model establishment. One day before the drug administration test, a mouse plantar incision pain model is established through surgery. The pain sensitivity baseline test after model establishment is carried out on the second day after model establishment. The 50% paw withdrawal threshold (50% PWT) of the hind paw on the surgical side of the mouse is measured using a Von Frey test filament. Animals meeting the baseline requirements are selected for grouping, randomly divided into 6 groups, with 10 animals in each group, and the drugs are administered orally once according to the above grouping. The pain thresholds of the animals are measured before drug administration (baseline), 1 h, 2 h, 4 h, and 6 h after drug administration respectively.

[0590] The specific surgical modeling process is as follows: Place the mice in an anesthesia induction box and administer 3%-4% isoflurane for induction anesthesia. After induction anesthesia, place the mice in a supine position. Disinfect the surgical site with alcohol and povidone-iodine. Under sterile conditions, make a 0.5-cm longitudinal incision from the heel of the left hind foot surface, 0.2 cm away from the root of the foot, cut through the skin and fascia, separate the plantar muscle, slightly lift it, and make a longitudinal cut. Then suture the skin with 5-0 suture and disinfect it.

[0591] 2.5. Data collection and analysis

[0592] All statistical analyses were performed using two-tailed analysis. The significance level was set at 0.05 or P < 0.05. The means (Mean) and standard deviations (SD) of all results of the solvent group and the drug-administered group animals were statistically analyzed using the SPSS 23.0 statistical software. One-way analysis of variance (One Way ANOVA) was used for testing and LSD multiple comparisons were performed.

[0593] 3. Results

[0594] Table 9 Analgesic efficacy results of the compound in the mouse foot incision pain model

[0595]

[0596]

[0597] *: P < 0.05, vs solvent group

[0598] 4. Conclusions

[0599] Under the conditions of this experiment, intragastric administration of Test Example 2 of the test article at a dose of 60 mg / kg could significantly increase the pain threshold of mice in the incision pain model, had an obvious analgesic effect, and the analgesic effect was 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 enantiomers thereof, diastereomers thereof or mixtures 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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