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-C) 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 thermal, mechanical, or chemical stimuli they sense 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 a compound annual growth rate of 2.5% in the future, and the chronic pain market will grow at a compound annual 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 domains (DI-DIV) that form the pore. Each domain has 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 is depolarized, the sodium channel is activated, the channel opens, causing sodium ions to flow in, further depolarizing the cell membrane and resulting in the generation of an action potential. 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 present 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 currently used Nav inhibitors in clinical practice have a narrow therapeutic window and limited application range 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 produces 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 for the treatment of 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 limited 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-C) 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, a 3-8 membered heterocyclic alkyl, a C1-C6 alkyl substituted with one or more R 1-1-1 or a 3-8 membered heterocyclic alkyl substituted with one or more deuteriums; in the 3-8 membered heterocyclic alkyl of the 3-8 membered heterocyclic alkyl and the 3-8 membered heterocyclic alkyl 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] R 5 and R 6 are both hydrogen or both deuterium at the same time;

[0020] X 1 、X 3 、X 4 and X 5 are each independently N, N + -O - or CR X1 ;

[0021] R X1 is hydrogen, a halogen, or -C(=O)N(R X1-1 )2;

[0022] Each R X1-1 is independently hydrogen or a C1-C6 alkyl group;

[0023] And at least one of the following conditions is satisfied:

[0024] 1) R 5 and R 6 are both deuterium at the same time;

[0025] 2) R 1-1 is a C1-C6 alkyl group substituted with one or more R 1-1-1 or a 3-8 membered heteroalkyl group substituted with one or more deuteriums; Each R 1-1-1 is independently deuterium or -OR a ; Wherein R a is a C1-C6 alkyl group substituted with one or more deuteriums.

[0026] In some embodiments, the compound of formula (I-C) 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- to 8-membered heteroalkyl group, a C1-C6 alkyl group substituted by one or more R 1-1-1 groups, or a 3- to 8-membered heteroalkyl group substituted by one or more deuteriums; in the 3- to 8-membered heteroalkyl group of the 3- to 8-membered heteroalkyl group and the 3- to 8-membered heteroalkyl group substituted by 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 group, a C3-C6 cycloalkyl group, or a C1-C6 alkyl group substituted by one or more deuteriums;

[0032] 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;

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

[0034] R 5 and R 6 are both hydrogen or both deuterium at the same time;

[0035] X 1 、X 3 、X 4 and X 5 are independently N, N + -O - or CR X1 ;

[0036] R X1 is hydrogen, a halogen, or -C(=O)N(R X1-1 )2;

[0037] Each R X1-1 is independently hydrogen or a C1-C6 alkyl group.

[0038] In some embodiments, in R 1 , R 2 and R 3 , the halogen is independently fluorine, chlorine, bromine, or iodine, preferably fluorine.

[0039] In some embodiments, in R 1-1 , the C1-C6 alkyl group and the C1-C6 alkyl group substituted by 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.

[0040] 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 group is each independently preferably a 4-6 membered (e.g., 4-membered, 5-membered or 6-membered) heterocycloalkyl group; more preferably For example

[0041] 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 group is each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0042] In some embodiments, R a In the C3-C6 cycloalkyl group, it is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclopropyl.

[0043] In some embodiments, R 4 In the C1-C6 alkyl group, it is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0044] In some embodiments, R 7 In the C1-C6 alkyl group, it is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0045] In some embodiments, R X1 In the halogen, it is fluorine, chlorine, bromine or iodine, preferably fluorine.

[0046] In some embodiments, in formula (I-C), R 1 is -OR 1-1 ;

[0047] R 1-1 is C1-C6 alkyl, 3-8 membered heterocycloalkyl, C1-C6 alkyl substituted with one or more R 1-1-1 or 3-8 membered heterocycloalkyl substituted with one or more deuteriums; in the 3-8 membered heterocycloalkyl group and the 3-8 membered heterocycloalkyl 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;

[0048] Each R 1-1-1 is independently deuterium or -OR a ;

[0049] R a is a C1-C6 alkyl group, a C3-C6 cycloalkyl group or a C1-C6 alkyl group substituted by one or more deuteriums;

[0050] R 2 and R 3 are each independently a halogen;

[0051] R 4 is a C1-C6 alkyl group;

[0052] R 5 and R 6 are both hydrogen or both deuterium at the same time;

[0053] R 7 is a C1-C6 alkyl group;

[0054] X 1 , X 3 , X 4 and X 5 are each independently CR X1 ;

[0055] R X1 is hydrogen or a halogen, preferably hydrogen.

[0056] In some embodiments, in formula (I-C), R 1-1 is a C1-C6 alkyl group or a C1-C6 alkyl group substituted by one or more R 1-1-1 ;

[0057] In some embodiments, in formula (I-C), each R 1-1-1 is independently deuterium.

[0058] In some embodiments, in formula (I-C), R 5 and R 6 are both deuterium at the same time.

[0059] In some embodiments, in formula (I-C), R 1 is -OCH3,

[0060] In some embodiments, in formula (I-C), R 2 is fluorine.

[0061] In some embodiments, in formula (I-C), R 3 is fluorine.

[0062] In some embodiments, in formula (I-C), R 4 is methyl.

[0063] In some embodiments, in formula (I-C), R 5 and R 6 are both hydrogen or both deuterium.

[0064] In some embodiments, in formula (I-C), R 7 is methyl. In some embodiments, in formula (I-C), is In some embodiments, the compound represented by formula (I-C) is any one of the following compounds:

[0065]

[0066] In some embodiments, the compound represented by formula (I-C) is any one of the following compounds:

[0067]

[0068]

[0069] or its enantiomer, its diastereomer or a mixture thereof.

[0070] The present invention provides a compound represented by formula (II-C):

[0071]

[0072] wherein the definitions of R 1 , R 2 , R 3 , R 4 and R 7 are as described in any one of the above;

[0073] R 8 is -CN, -COOH or

[0074] In some embodiments, the compound represented by formula (II-C) is any one of the following compounds:

[0075]

[0076] In some embodiments, the compound represented by formula (II-C) is any one of the following compounds:

[0077] or its enantiomer, its diastereomer or a mixture thereof.

[0078] The present invention provides a pharmaceutical composition, which comprises:

[0079] (1) The compound represented by the following formula (I-C) or a pharmaceutically acceptable salt thereof, and

[0080] (2) Pharmaceutically acceptable excipients.

[0081] The present invention provides an application of the compound represented by the following formula (I-C) or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating diseases; the diseases may be pain, pain-related diseases, multiple sclerosis, incontinence or arrhythmia.

[0082] 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.

[0083] The present invention provides an application of the compound represented by the following formula (I-C) or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for inhibiting voltage-gated sodium channels; the voltage-gated sodium channel is preferably Na V 1.8.

[0084] The present invention provides an application of the compound represented by the following formula (I-C) or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a voltage-gated sodium channel inhibitor; the voltage-gated sodium channel is preferably Na V 1.8.

[0085] In some embodiments, the voltage-gated sodium channel inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, for example: provided as a standard sample or a control sample for comparison, or made into a kit according to the conventional methods in the art to provide a rapid detection for the effect of inhibiting voltage-gated sodium channels.

[0086] The present invention provides an application of the compound represented by the following formula (I-C) or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for diseases caused by abnormal activation of voltage-gated sodium channels; the voltage-gated sodium channel is preferably Na V 1.8; the diseases may be pain, pain-related diseases, multiple sclerosis, incontinence or arrhythmia.

[0087] The present invention provides a kit for rapidly detecting the inhibitory effect of voltage-gated sodium channel Na V 1.8, which comprises the compound represented by the following formula (I-C) or a pharmaceutically acceptable salt thereof.

[0088] Explanation of Terms

[0089] 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, the 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, the 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).

[0090] 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.

[0091] 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 group.

[0092] In the present invention, the term "one or more" means 1, 2, 3, 4, 5, 6, 7, for example, 1, 2 or 3.

[0093] In the present invention, in the term "B substituted by one or more A", when B is substituted by "multiple" A, A can be the same or different.

[0094] In the present invention, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] In the present invention, the term "pharmaceutically acceptable excipients" refers to all substances contained in a pharmaceutical preparation except 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).

[0099] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain the preferred examples of the present invention.

[0100] The reagents and raw materials used in the present invention are all commercially available.

[0101] 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

[0102] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples described herein. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0103] Example 1 Synthesis of BX20 - 9 - 043

[0104] First step: Synthesis of intermediate A2

[0105] The synthesis route is shown as follows:

[0106]

[0107] 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 completion of the reaction, 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] + 。

[0108] Step 2: Synthesis of intermediate A3

[0109]

[0110] Method: Dissolve A2 (7.60 g, 35.18 mmol) in THF (80 mL), add 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 completion of the reaction, 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] + 。

[0111] Step 3: Synthesis of intermediate S2

[0112] The synthetic route is shown as follows:

[0113]

[0114] Method: Dissolve compound S1 (15.50 g, 98.05 mmol) in Et2O (155 mL), replace N2, cool 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 completion of the reaction, 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).

[0115] Step 4: Synthesis of intermediate S3

[0116] The synthetic route is shown as follows:

[0117]

[0118] 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] + 。

[0119] Step 5: Synthesis of S4

[0120] The synthetic route is shown as follows:

[0121]

[0122] 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, keep the temperature at -40 °C and slowly add saturated ammonium chloride dropwise to the reaction solution to quench 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] + 。

[0123] The NMR spectrum is as follows:

[0124] 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).

[0125] Step 6: Synthesis of S5

[0126] The synthesis route is shown as follows:

[0127]

[0128] 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 diatomaceous earth, 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] + 。

[0129] Step 7: Synthesis of S6

[0130] The synthesis route is shown as follows:

[0131]

[0132] 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 dropping, 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] + 。

[0133] Step 8: Synthesis of S7

[0134] The synthesis route is shown as follows:

[0135]

[0136] 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, stir and react at -60 °C for 1 h, then naturally warm up to room temperature and stir and react for 12 h. After the reaction is completed, quench the reaction solution with 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] + 。

[0137] Step 9: Synthesis of Intermediate 39-1

[0138] The synthetic route is shown as follows:

[0139]

[0140] 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, warm up to 0 °C and stir and react for 3 hours. After the reaction is completed, slowly add the reaction solution 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] + 。

[0141] Step 10: Synthesis of Intermediate 43-1

[0142] The synthetic route is shown as follows:

[0143]

[0144] Method: Dissolve 39-1 (300 mg, crude product, about 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 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 yellow oily compound 43-1 (300 mg, crude product). MS (ESI, m / z) 339 [M+H] + 。

[0145] Step 11: Synthesis of Intermediate 43-2

[0146] The synthesis route is shown as follows:

[0147]

[0148] 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 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 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 43-2 (260 mg, crude product). MS (ESI, m / z) 358 [M+H] + .

[0149] The twelfth step: Synthesis of intermediate 43-3

[0150] The synthesis route is shown as follows:

[0151]

[0152] Method: Dissolve 43-2 (260 mg, crude product, 0.73 mmol) in DCM (4 mL), add 1 drop of DMF, cool to 0 °C, and slowly add dropwise (COCl)2 (0.09 mL, 1.092 mmol). React with stirring at room temperature for 0.5 hour. After the reaction is completed, concentrate under reduced pressure, redissolve in DCM (4 mL), and slowly add dropwise to a solution of 3-aminopyridine (69 mg, 0.73 mmol) and TEA (294 mg, 2.91 mmol) in DCM / NMP (1:1, 2 mL). Stir at room temperature for 0.5 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] + .

[0153] The thirteenth step: Synthesis of BX20-9-043

[0154] The synthesis route is shown as follows:

[0155]

[0156] Method: Dissolve compound 43-3 (280 mg, crude, about 0.65 mmol) in DCM (5 mL), add m-CPBA (307 mg, 1.61 mmol, 85% purity) under ice bath, and stir at room temperature for 16 h. After the reaction is completed, drop the reaction solution into saturated aqueous NaHCO3 (20 mL), extract once with DCM, extract three times with DCM / MeOH (10:1), combine the organic phases, wash once with saturated aqueous NaCl, dry over anhydrous Na2SO4, concentrate under reduced pressure, purify by preparative chromatography (neutral system), and lyophilize to obtain white solid BX20-9-043 (163 mg, 55%). MS (ESI, m / z) 450 [M+H] + .

[0157] 1 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.66 (s, 1H), 7.99 (d, J = 6.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.39 - 7.33 (m, 1H), 7.29 - 7.11 (m, 2H), 5.08 (d, J = 10.0 Hz, 1H), 4.24 (dd, J = 8.0, 10.4 Hz, 1H), 2.81 - 2.72 (m, 1H), 1.60 (s, 3H), 0.72 (d, J = 6.0 Hz, 3H).

[0158] Example 2 Synthesis of BX20-9-044

[0159] First step: Synthesis of intermediate S3

[0160] Refer to Example 1 to synthesize intermediate S3

[0161] Second step: Synthesis of 44-1

[0162] The synthetic route is shown as follows:

[0163]

[0164] Method: Dissolve compound S3 (2.00 g, 5.9 mmol) in CD3OD (100 mL) and anhydrous tetrahydrofuran (20 mL), cool down to -20 °C, add NiCl2 (782 mg, 6.0 mmol), and slowly add NaBD4 (1.3 g, 31.27 mmol) in batches; after addition, continue to add the second batch of NiCl2 (782 mg, 6.0 mmol), and slowly add NaBD4 (1.3 g, 31.27 mmol) in batches, and stir at -20 °C for half an hour. After the reaction is completed, keep the temperature at -20 °C and slowly add heavy water (25 mL) and deuterated hydrochloric acid (10 mL, 20% in D2O) to the reaction solution to quench the reaction. Let it rise to room temperature naturally, filter through diatomaceous earth, extract with DCM, wash with concentrated brine, dry with anhydrous sodium sulfate, and after concentration, purify by column chromatography to obtain the product with relatively high polarity, colorless oily liquid 44-1 (840 mg, 43.7%). MS (ESI, m / z) 327 [M+H] + 。

[0165] Step 3: Synthesis of 44-2

[0166] The synthesis route is shown as follows:

[0167]

[0168] Method: Dissolve compound 44-1 (0.84 g, 2.6 mmol) in toluene (15 mL), displace N2, cool down to -30 °C, slowly add dropwise DiBAL-H (1.5 M in toluene, 2.6 mL, 3.9 mmol), and stir the reaction mixture at -25 to -30 °C for 1 h. After the reaction is completed, add the reaction solution to saturated ammonium chloride to quench, filter through diatomaceous earth, extract with EA, wash with concentrated brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain brown oily substance 44-2 (0.79 g, 92.6%). MS (ESI, m / z) 329 [M+H] + 。

[0169] Step 4: Synthesis of 44-3

[0170] The synthesis route is shown as follows:

[0171]

[0172] Method: Dissolve compound 44-2 (0.79 g, 2.4 mmol) in anhydrous DCM (20 mL), add Et3N (0.48 g, 4.8 mmol), cool the mixture to 0 - 10 °C in an ice-water bath, and slowly add AcCl (0.38 g, 4.8 mmol) dropwise. After the addition, raise the temperature to room temperature and stir for 1 h. After the reaction is completed, quench the reaction mixture with saturated ammonium chloride, extract with DCM, wash with brine, dry over anhydrous sodium sulfate, and concentrate to obtain a brown oil 44-3 (0.84 g, 94.6%). MS (ESI, m / z) 371 [M+H] + 。

[0173] Step 5: Synthesis of 44-4

[0174] The synthesis route is shown as follows:

[0175]

[0176] Method: Dissolve compound 44-3 (0.84 g, 2.3 mmol) in anhydrous DCM (15 mL), displace N2, cool to -78 °C, and sequentially add TMSCN (0.68 g, 6.9 mmol) and BF3OEt2 (0.50 g, 3.5 mmol) dropwise. After the addition, stir the reaction mixture at -60 °C for 1 h, and then naturally raise the temperature to room temperature and stir for 2 h. After the reaction is completed, quench the reaction mixture with saturated sodium carbonate solution, filter through diatomaceous earth, extract with DCM, wash with brine, dry over anhydrous sodium sulfate, and concentrate to obtain a brown oil 44-4 (0.76 g, 98.3%). MS (ESI, m / z) 338 [M+H] + 。

[0177] Step 6: Synthesis of 44-5

[0178] The synthesis route is shown as follows:

[0179]

[0180] Method: Dissolve compound 44-4 (0.76 g, 2.3 mmol) in MeOH (15 mL), add an aqueous KOH solution (0.90 g KOH dissolved in 3 mL H2O, 16.1 mmol), and stir the reaction mixture at 60 °C for 12 h. After the reaction is completed, extract with MTBE. Adjust the pH of the aqueous phase to <5 with hydrochloric acid, extract with MTBE again. Wash the organic phase with brine, dry over anhydrous sodium sulfate, and concentrate to obtain a brown oil 44-5 (0.61 g, 74.5%). MS (ESI, m / z) 357 [M+H] +

[0181] Step 7: Synthesis of 44-7

[0182] The synthetic route is shown as follows:

[0183]

[0184] Method: Dissolve 44-5 (150 mg, 0.42 mmol) in DCM (3 mL), add 1 drop of DMF, and dropwise add (COCl)2 (160 mg, 1.26 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 44-6 (180 mg, crude product), which is directly used for the next step; dissolve 3-aminopyridine (47 mg, 0.50 mmol) in NMP (2 mL), add TEA (85 mg, 0.84 mmol), and then slowly add 44-6 (crude product, diluted with 5 mL of DCM, about 0.42 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 brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 44-7 (0.25 g, crude product). MS (ESI, m / z) 433 [M+H] + 。

[0185] Step 8: Synthesis of BX20-9-044

[0186] The synthetic route is shown as follows:

[0187]

[0188] Method: Dissolve compound 44-7 (crude, 0.25 g, about 0.42 mmol) in DCM (10 mL), cool to 0 °C, add m-CPBA (0.17 g, 0.84 mmol), and stir the reaction mixture at room temperature for 12 h. After completion of the reaction, quench the reaction mixture by adding it to saturated sodium bicarbonate solution, extract with DCM / MeOH, combine the organic phases, wash with brine, dry the organic phase, and purify by preparative chromatography (neutral system) after concentration under reduced pressure to obtain white solid BX20-9-044 (50 mg, 26.6%). MS (ESI, m / z) 449 [M+H] + 。

[0189] 1 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.66 - 8.63 (m, 1H), 8.00 - 7.96 (m, 1H), 7.53 - 7.46 (m, 1H), 7.39 - 7.32 (m, 1H), 7.21 - 7.10 (m, 2H), 5.01 (s, 1H), 3.94 (d, J = 2.0 Hz, 3H), 1.59 (s, 3H), 0.74 - 0.69 (m, 3H).

[0190] Synthesis of Example 3 BX20-9-059

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

[0192] Refer to the first to ninth steps of Example 1 to synthesize Intermediate 39-1

[0193] Step 2: Synthesis of Intermediate 59-1

[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 (220 mg, 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 sodium sulfate, and concentrate under reduced pressure to obtain a yellow oily compound 59-1 (240 mg, crude product). MS (ESI, m / z) 366 [M+H] + 。

[0197] Step 3: Synthesis of Intermediate 59-2

[0198] The synthesis route is shown as follows:

[0199]

[0200] 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 hours, 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 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 59-2 (220 mg, crude product). MS (ESI, m / z) 383 [M+H] + 。

[0201] Step 4: Synthesis of Intermediate 59-3

[0202] The synthesis route is shown as follows:

[0203]

[0204] 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), and 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 a yellow oily compound 59-3 (110 mg, crude product). MS (ESI, m / z) 402 [M+H] + 。

[0205] Step 5: Synthesis of Intermediate 59-4

[0206] The synthesis route is shown as follows:

[0207]

[0208] Method: Dissolve 59-3 (110 mg, crude product, about 0.27 mmol) in DCM (4 mL), add 1 drop of DMF, cool to 0 °C, and slowly add dropwise (COCl)2 (52 mg, 0.41 mmol), and stir at room temperature for 0.5 hour. After the reaction is completed, concentrate under reduced pressure, then dissolve in DCM (4 mL), and slowly add dropwise to a solution of 3-aminopyridine (23 mg, 0.25 mmol) and TEA (83 mg, 0.82 mmol) in DCM / NMP (1:1, 2 mL), and stir at room temperature for 0.5 hour. After the reaction is completed, quench with water, extract with EA, combine the organic phases, wash once with water and saturated aqueous NaCl solution respectively, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a brown oily compound 59-4 (170 mg, crude product). MS (ESI, m / z) 478 [M+H] + 。

[0209] Step 6: Synthesis of BX20-9-059

[0210] The synthesis route is shown as follows:

[0211]

[0212] Method: Dissolve 59-4 (170 mg, crude product, about 0.27 mmol) in DCM (4 mL), slowly add m-CPBA (220 mg, 1.08 mmol, 85% purity) under ice bath, and stir at room temperature for 16 hours. After the reaction is completed, drop the reaction solution into saturated aqueous NaHCO3 solution, extract it once with DCM first, then extract it four times with DCM / MeOH (10:1), combine the organic phases, wash it once with saturated aqueous NaCl solution, dry it over anhydrous sodium sulfate, concentrate it under reduced pressure, purify it by preparative chromatography, and lyophilize to obtain white solid BX20-9-059 (11 mg, 8%). MS (ESI, m / z) 494 [M+H] + 。

[0213] 1 1H NMR (400 MHz, DMSO-d6) δ = 10.51 (s, 1H), 8.65 (s, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 6.0, 8.4 Hz, 1H), 7.19 - 7.14 (m, 2H), 5.11 (d, J = 10.8 Hz, 1H), 4.37 - 4.25 (m, 2H), 4.22 - 4.15 (m, 1H), 3.65 - 3.56 (m, 1H), 3.64 (s, 3H), 2.86 - 2.78 (m, 1H), 1.60 (s, 3H), 0.70 (d, J = 5.2 Hz, 3H).

[0214] Example 4 Synthesis of BX20-9-060

[0215] First step: Synthesis of intermediate 39-1

[0216] Refer to the first to ninth steps of Example 1 to synthesize intermediate 39-1

[0217] Second step: Synthesis of intermediate 60-1

[0218] The synthesis route is shown in the following formula:

[0219]

[0220] 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), heat to 70 °C and stir for 16 hours. After the reaction is completed, quench it with water, extract it with EA, wash it with saturated aqueous NaCl solution, dry it over anhydrous sodium sulfate, and concentrate it under reduced pressure to obtain yellow oily compound 60-1 (300 mg, crude product). MS (ESI, m / z) 370 [M+H]+ 。

[0221] Step 3: Synthesis of Intermediate 60-2

[0222] The synthesis route is shown as follows:

[0223]

[0224] 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. After stirring at room temperature for 0.5 h, add iodomethane-d3 (191 mg, 1.32 mmol), and continue to stir at room temperature for 1.5 h. 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 a brown oily compound 60-2 (280 mg, crude product). MS (ESI, m / z) 387 [M+H] + 。

[0225] Step 4: Synthesis of Intermediate 60-3

[0226] The synthesis route is shown as follows:

[0227]

[0228] 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), and heat to 60 °C and stir for 16 h. 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 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 a yellow oily compound 69-3 (100 mg, crude product). MS (ESI, m / z) 406 [M+H] + 。

[0229] Step 5: Synthesis of Intermediate 60-4

[0230] The synthesis route is shown as follows:

[0231]

[0232] Method: Dissolve 60-3 (100 mg, crude product, about 0.25 mmol) in DCM (4 mL), add 1 drop of DMF, cool down to 0 °C, and slowly add dropwise (COCl)2 (47 mg, 0.37 mmol). Stir 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 3-aminopyridine (21 mg, 0.22 mmol) and TEA (74 mg, 0.74 mmol) in DCM / NMP (1:1, 2 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 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 60-4 (160 mg, crude product). MS (ESI, m / z) 482 [M+H] + 。

[0233] Step 6: Synthesis of BX20-9-060

[0234] The synthetic route is shown as follows:

[0235]

[0236] Method: Dissolve 60-4 (160 mg, crude product, about 0.22 mmol) in DCM (4 mL), slowly add m-CPBA (135 mg, 0.66 mmol, 85% purity) under ice bath, and stir at room temperature for 16 h. After the reaction is completed, directly concentrate under reduced pressure and then purify by preparative chromatography, and lyophilize to obtain a white solid BX20-9-060 (25 mg, 22%). MS (ESI, m / z) 498 [M+H] + 。

[0237] 1 1H NMR (400 MHz, DMSO-d6) δ = 10.49 (s, 1H), 8.55 (s, 1H), 8.00 - 7.96 (m, 1H), 7.54 - 7.50 (m, 1H), 7.35 (dd, J = 6.4, 8.4 Hz, 1H), 7.18 - 7.13 (m, 2H), 5.10 (d, J = 10.8 Hz, 1H), 4.34 (dd, J = 7.2, 10.8 Hz, 1H), 2.86 - 2.78 (m, 1H), 1.60 (s, 3H), 0.70 (d, J = 6.8 Hz, 3H).

[0238] Example 5 Synthesis of BX20-9-062

[0239] Step 1: Synthesis of Intermediate 44-4

[0240] Refer to the first to fifth steps of Example 2 to synthesize Intermediate 44-4

[0241] Step 2: Synthesis of Intermediate 62-1

[0242] The synthesis route is shown as follows:

[0243]

[0244] Method: Dissolve 44-4 (4.9 g, 14.54 mmol) in DCM (60 mL), displace with N2, cool down to -78 °C, slowly add dropwise BBr3 (6.3 mL, 63.96 mmol), after the addition, warm up to room temperature and stir for 2 hours. After the reaction is completed, slowly add the reaction solution dropwise to ice water, extract with DCM three times, combine the organic phases, wash once with saturated NaCl aqueous solution, dry with anhydrous Na2SO4, and concentrate under reduced pressure to obtain white solid 62-1 (5.0 g, crude product). MS (ESI, m / z) 324 [M+H] + 。

[0245] Step 3: Synthesis of Intermediate 62-2

[0246] The synthesis route is shown as follows:

[0247]

[0248] Method: Dissolve compound 62-1 (450 mg, 1.39 mmol) in DMF (5 mL), add 1-iodo-2-methoxyethane (1.03 g, 5.56 mmol) and Cs2CO3 (585 mg, 4.2 mmol), stir at 70 °C for 16 hours. After the reaction is completed, quench with water, extract with EA (30 mL × 3), combine the organic phases, wash the organic phases with saturated brine, dry with anhydrous Na2SO4, and concentrate under reduced pressure to obtain yellow oily liquid 62-2 (565 mg, crude product). MS (ESI, m / z) 382 [M+H] + 。

[0249] Step 4: Synthesis of Intermediate 62-3

[0250] The synthesis route is shown as follows:

[0251]

[0252] Method: Dissolve compound 62-2 (565 mg, crude) in MeOH (5 mL), add a solution of KOH (581 mg, 10.38 mmol) in H2O (1 mL), and stir at 60 °C for 12 hours. After completion of the reaction, concentrate under reduced pressure to remove methanol. Add water and EA to the residue for extraction. Adjust the aqueous phase to pH < 4, extract with EA, combine the organic phases, wash with saturated brine, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow oily liquid 62-3 (450 mg, crude). MS (ESI, m / z) 401 [M+H] + 。

[0253] Step 5: Synthesis of Intermediate 62-4

[0254] The synthetic route is shown as follows:

[0255]

[0256] Method: Dissolve 62-3 (150 mg, crude, about 0.35 mmol) in DCM (5 mL), add one drop of DMF, slowly add (COCl)2 (0.2 mL, 2.13 mmol) dropwise under ice bath. After addition, stir at room temperature for 0.5 hour. Directly concentrate the reaction solution under reduced pressure to obtain Intermediate 62-4, then dissolve it in DCM (5 mL) and slowly add it dropwise to a solution of 3-aminopyridine (36 mg, 0.38 mmol) and TEA (100 mg, 1 mmol) in NMP (3 mL). Stir at room temperature for 0.5 hour. After completion of the reaction, 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 a yellow oily liquid 62-5 (230 mg, crude). MS (ESI, m / z) 477 [M+H] + 。

[0257] Step 6: Synthesis of Compound BX20-9-062

[0258] The synthetic route is shown as follows:

[0259]

[0260] Method: Dissolve compound 62-5 (230 mg, crude, about 0.3 mmol) in DCM (5 mL), add m-CPBA (152 mg, 85%, 0.75 mmol) at 0 °C, and stir at room temperature for 16 hours. After completion of the reaction, concentrate the reaction solution at 40 °C under reduced pressure, purify by preparative chromatography (neutral system), and lyophilize to obtain a white solid powder BX20-9-062 (67 mg, yield 36.8%). MS (ESI, m / z) 493 [M+H] + 。

[0261] 1 1H NMR (400 MHz, DMSO-d6) δ = 10.44 (s, 1H), 8.63 - 8.62 (m, 1H), 7.97 - 7.94 (m, 1H), 7.51 - 7.48 (m, 1H), 7.34 - 7.31 (m, 1H), 7.16 - 7.12 (m, 2H), 5.07 (s, 1H), 4.28 - 4.14 (m, 2H), 3.60 - 3.57 (m, 2H), 3.25 (s, 3H), 1.58 (s, 3H), 0.67 - 0.66 (m, 3H).

[0262] Synthesis of Example 6 BX20 - 9 - 063

[0263] Step 1: Synthesis of Intermediate 62 - 1

[0264] Refer to Example 5 for the synthesis of Intermediate 62 - 1.

[0265] Step 2: Synthesis of 63 - 1

[0266] The synthesis route is shown as follows:

[0267]

[0268] Method: Dissolve 62 - 1 (190 mg, 0.45 mmol) in DMF (5 mL), add CsCO3 (290 mg, 0.90 mmol) and 2 - cyclopropoxyethyl 4 - methylbenzenesulfonate (230 mg, 0.90 mmol), and heat to 70 °C and stir for 12 hours. After the reaction is completed, add water and EA for extraction, combine the organic phases, wash with concentrated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 63 - 1 (150 mg, crude product). MS (ESI, m / z) 408 [M + H] + .

[0269] Step 3: Synthesis of 63 - 2

[0270] The synthesis route is shown as follows:

[0271]

[0272] Method: Dissolve Compound 63 - 1 (150 mg, crude product) in MeOH (5 mL), add KOH (260 mg, 4.64 mmol) and water (1 mL), and stir the reaction mixture at 60 °C for 12 h. After the reaction is completed, extract with MTBE, adjust the pH of the aqueous phase to < 5 with hydrochloric acid, extract three times with EA, combine the organic phases, wash with concentrated brine, dry with anhydrous sodium sulfate, and concentrate to obtain a brown oil 63 - 2 (120 mg, crude product). MS (ESI, m / z) 427 [M + H] +

[0273] Step 4: Synthesis of 63-4

[0274] The synthesis route is shown as follows:

[0275]

[0276] Method: Dissolve 63-2 (60 mg, crude product) in DCM (4 mL), add one drop of DMF, and dropwise add (COCl)2 (70 mg, 0.56 mmol) at 0 °C. Stir the reaction solution at room temperature for 0.5 h. After the reaction is completed, concentrate the reaction solution under reduced pressure to obtain crude 63-3 (80 mg, crude product), which is directly used for the next step; dissolve compound 3-aminopyridine (32 mg, 0.34 mmol) in NMP (2 mL), add TEA (56 mg, 0.56 mmol), and then slowly add the DCM solution (2 mL) of 63-3. Stir at room temperature for 0.5 h. After the reaction is completed, quench with saturated ammonium chloride, extract three times with EA, combine the organic phases, wash with concentrated brine, dry the organic phase, and concentrate under reduced pressure to obtain a brown oil 63-4 (100 mg, crude product). MS (ESI, m / z) 503 [M+H] + 。

[0277] Step 5: Synthesis of BX20-9-063

[0278] The synthesis route is shown as follows:

[0279]

[0280] Method: Dissolve compound 63-4 (100 mg, crude product) in DCM (5 mL), cool to 0 °C in an ice bath, add m-CPBA (99 mg, 0.48 mmol), and stir the reaction at room temperature for 12 h. After the reaction is completed, directly concentrate under reduced pressure and purify by preparative chromatography under neutral conditions to obtain a light yellow solid BX20-9-063 (2 mg, 1.6%). MS (ESI, m / z) 519 [M+H] + 。

[0281] 11H NMR (400 MHz, DMSO-d6) δ = 10.48 (s, 1H), 8.67 - 8.64 (m, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.38 - 7.32 (m, 1H), 7.22 - 7.11 (m, 2H), 5.10 (s, 1H), 4.33 - 4.16 (m, 2H), 3.74 - 3.68 (m, 2H), 3.33 - 3.29 (m, 1H), 1.61 (s, 3H), 0.69 (s, 3H), 0.48 - 0.41 (m, 2H), 0.41 - 0.35 (m, 2H).

[0282] Synthesis of Example 7 BX20 - 9 - 064

[0283] Step 1: Synthesis of Intermediate 62 - 1

[0284] Intermediate 62 - 1 was synthesized with reference to Example 5.

[0285] Step 2: Synthesis of Intermediate 64 - 1

[0286] The synthesis route is shown as follows:

[0287]

[0288] Method: Dissolve 62 - 1 (400 mg, crude product, about 1.2 mmol) in DMF (5 mL), add Cs2CO3 (808 mg, 2.48 mmol) and 3 - iodooxetane (912 mg, 4.96 mmol), and heat to 70 °C and stir for 16 hours. After the reaction was completed, it was diluted with water, extracted three times with EA, the organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a yellow oily compound 64 - 1 (300 mg, crude product). MS (ESI, m / z) 380 [M + H] + 。

[0289] Step 3: Synthesis of Intermediate 64 - 2

[0290] The synthesis route is shown as follows:

[0291]

[0292] Method: Dissolve 64-1 (300 mg, crude product, about 0.6 mmol) in MeOH (7.5 mL), add an aqueous solution of KOH (486 mg, 8.68 mmol, dissolved in 1.5 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 three times with EA, combine the organic phases, wash with saturated aqueous NaCl solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow oily compound 64-2 (200 mg, crude product). MS (ESI, m / z) 399 [M+H] + 。

[0293] Step 4: Synthesis of Intermediate 64-3

[0294] The synthesis route is shown as follows:

[0295]

[0296] Method: Dissolve 64-2 (200 mg, crude product) in DMF (6 mL), add EDCI (288 mg, 1.5 mmol), HOBT (122 mg, 0.9 mmol) and DIEA (290 mg, 2.25 mmol), stir at room temperature for 10 minutes, then add 3-aminopyridine (56 mg, 0.6 mmol), and continue to stir at room temperature for 4 hours. After the reaction is completed, quench with water, extract three times with EA, combine the organic phases, wash once with water and once with saturated aqueous NaCl solution, dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow oily compound 64-3 (160 mg, crude product). MS (ESI, m / z) 475 [M+H] + 。

[0297] Step 5: Synthesis of BX20-9-064

[0298] The synthesis route is shown as follows:

[0299]

[0300] Method: Dissolve 64-3 (160 mg, crude product, about 0.3 mmol) in DCM (5 mL), slowly add m-CPBA (160 mg, 0.8 mmol, 85% purity) under ice bath, and stir at room temperature for 16 hours. After the reaction is completed, directly concentrate under reduced pressure and purify by preparative chromatography, and lyophilize to obtain a white solid BX20-9-064 (7 mg, 5%). MS (ESI, m / z) 491 [M+H] + 。

[0301] 11H NMR (400 MHz, DMSO-d6) δ = 10.54 (s, 1H), 8.65 (s, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.54 - 7.50 (m, 8.4 Hz, 1H), 7.36 (dd, J = 6.4, 8.4 Hz, 1H), 7.19 - 7.14 (m, 2H), 5.33 - 5.26 (m, 1H), 5.11 (s, 1H), 4.90 - 4.82 (m, 2H), 4.73 - 4.66 (m, 2H), 1.60 (s, 3H), 0.72 (s, 3H).

[0302] Reference compound:

[0303] Synthesize the reference compound (Compound 7) according to the synthesis method of Example 3 of Patent CN114945566A

[0304]

[0305] 1 1H 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)

[0306] Biological test evaluation

[0307] The present invention will be further described and explained in combination with test examples below

[0308] Test Example 1 Blocking activity of the compound of the present invention on sodium channel 1.8 (Nav1.8)

[0309] 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

[0310] 2. Experimental materials and equipment

[0311] 2.1 Cell line: A CHO cell line stably expressing the Nav1.8 sodium channel. The Nav1.8 cells were self-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, 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: Sodium channel, voltage-gated, type 8, alpha (SCN10A), cDNA strictly similar to GenBank accession number: NM_006514

[0312] 2.2 Compound: Dissolved in DMSO

[0313] 3. Experimental methods

[0314] 3.1 Cell culture

[0315] (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%.

[0316] (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 amplification 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).

[0317] (3) To maintain the electrophysiological activity of the cells, the cell density must not exceed 80%.

[0318] (4) For patch-clamp detection, the cells are separated with 0.25%-Trypsin-EDTA before the experiment. Seed 6.5×103 cells onto a coverslip and culture in a 24-well plate (final volume: 500 μL). After 18 hours, perform the experimental detection.

[0319] 3.2. Patch-Clamp Detection

[0320] (1) When a whole-cell seal is formed, the cell voltage is clamped at -120 mV. First, step the voltage from -130 mV in 10-mV increments to -10 mV and maintain for 5 s, then apply a 0-mV depolarizing pulse to obtain the half-inactivation voltage (Vhalf). The resting state and half-inactivated state of the sodium current are detected using a double-pulse protocol. First, apply the first depolarizing pulse (TP1) to 0 mV for 50 ms to detect the sodium current in the resting state. Then adjust the voltage to Vhalf and maintain for 5 s, then restore the voltage to -120 mV and maintain for 20 ms, and then apply the second depolarizing pulse (TP2) to 0 mV for 50 ms to detect the sodium current in the half-inactivated state. Finally, restore to the clamped voltage of -120 mV. Collect data every 20 ms and observe the effect of the drug on the peak sodium current in the two different states. The experimental data are collected by an EPC 10 amplifier (HEKA) and stored in the PatchMaster (HEKA) software.

[0321] (2) During patch clamp operation, first use a microelectrode puller to pull the capillary glass tube into a recording electrode, then load the electrode filled with intracellular fluid into the microelectrode holder, and under an inverted microscope, use 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 break the cell membrane to form a whole-cell recording mode. Finally, perform slow capacitance compensation and record relevant parameters. Do not apply leakage compensation.

[0322] (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.

[0323] 3.3. Data analysis

[0324] 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.

[0325] 4. Experimental results

[0326] Table 1 Blocking rate of the compound of the present invention on NaV1.8 at 10 nM

[0327] Compound Number Inhibition Rate (%) Reference Compound 95.49 Example 1 85.74 Example 2 87.77 Example 3 84.62 Example 4 86.09 Example 5 80.63

[0328] It can be seen that the compound of the present invention has an obvious blocking effect on the activity of the NaV1.8 channel.

[0329] Test Example 2 Blocking activity of the compound of the present invention on sodium channel 1.8 (Nav1.8) (IC 50 )

[0330] 1. Research purpose

[0331] Use 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 or 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 IC 50 . This experiment includes the determination of 2 parallel samples.

[0332] 2. Test Methods

[0333] 2.1. Test Materials

[0334] 1) Cells: The HEK293 cell line stably expressing hNav1.8 / β3 ion channels was prepared by the Biology department of Catalent (Beijing) Biopharma 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 the 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 according to a total number of 5×10 5 and seeded on glass slides for subsequent manual patch clamp experiments.

[0335] 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 test compound gradient intermediate solutions 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 .

[0336] 2.2. Test Procedures

[0337] 1) Place the glass slide with HEK293 cells in the perfusion chamber on the micromanipulation stage.

[0338] 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.

[0339] 3) When approaching the cell closely, switch to a ×40 objective lens for observation, and gradually approach the cell surface with the electrode through the fine adjustment knob of the micromanipulator.

[0340] 4) Apply negative pressure to form a seal with a resistance higher than 1 GΩ between the electrode tip and the cell membrane.

[0341] 5) Compensate for the instantaneous capacitive current Cfast in the voltage clamp mode. Then repeatedly apply short negative pressures to rupture the membrane, and finally form the whole-cell recording mode.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 10) Test for the inhibitory effect of the test compound on hNav1.8 current: First, take 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 simultaneously 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 this 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).

[0347] 2.3. Data Analysis

[0348] 1) The data shall meet the following criteria: the initial sealing resistance is greater than 1 GΩ; the breakdown 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;

[0349] 2) The data is analyzed according to the following steps (the data is output by PatchMaster software):

[0350] 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.

[0351]

[0352] ② The dose-effect curve is fitted by Graphpad Prism 8.0 software and the IC 50 value is calculated.

[0353] The standard deviation range of the two groups of data is less than 15 (SD < 15)

[0354] 3. Test Results

[0355] Table 2 Blocking Activity of the Compounds of the Present Invention against NaV18

[0356] Compound Number <![CDATA[Inhibitory activity (IC 50 )]]> Reference Compound 0.297 Example 1 0.917 Example 2 1.448

[0357] It can be seen that the compounds of the present invention have strong inhibitory activity against the NaV1.8 channel.

[0358] Test Example 3 Selectivity Test of the Compounds of the Present Invention for Sodium Ion Channels

[0359] 1. Experimental Purpose: To detect the effect of the compound on the currents of voltage-gated sodium channel (NaV) subtypes 1.1 - 1.9 by patch clamp technique

[0360] 2. Experimental Materials and Instrumentation

[0361] 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.

[0362] 2.2. Compound: Dissolved in DMSO to prepare a sample concentration of 30 μM.

[0363] 3. Experimental methods

[0364] 3.1. Cell culture

[0365] (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%.

[0366] (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 seeding amount in each cell culture dish is 2.5×10 5 cells (final volume: 5 mL).

[0367] (3) To maintain the electrophysiological activity of the cells, the cell density must not exceed 80%.

[0368] (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 performed for detection.

[0369] 3.2. Patch clamp detection

[0370] Same as 3.2 in Test Example 1

[0371] 3.3. Data analysis

[0372] Same as 3.3 in Test Example 1

[0373] 4. Experimental results

[0374] Table 3 Blocking rates of the compounds of the present invention on NaV1.1-1.9 at 30 μM

[0375]

[0376] “ / ” indicates not done.

[0377] Conclusion: It can be seen that the compounds of the present invention have no obvious activity on the NaV1.1-1.9 channels. At the same concentration, the inhibitory activity on the NaV1.1-1.9 channels is lower than that of the reference compounds, with strong target selectivity, indicating that the compounds of the present invention have better safety.

[0378] Test Example 4 Pharmacokinetic Determination of the Compounds of the Present Invention in SD Rats

[0379] 1. Test Purpose

[0380] 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.

[0381] 2. Test Method

[0382] 2.1. Test Drugs

[0383] The examples of the present invention and the reference compound, self-made.

[0384] 2.2. Test Animals

[0385] Male SPF-grade SD rats, weighing (200±20) g, supplied by Beijing SPF Biotechnology Co., Ltd., animal production license number: SCXK(Beijing)2022-0030.

[0386] 2.3. Preparation of Test Drugs

[0387] 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.

[0388] 2.4. Administration

[0389] Male SPF-grade SD rats, after 3-4 days of adaptive feeding, were respectively given intragastric administration at a dose of 10 mg / kg and a volume of 10 mL / kg.

[0390] Before (0 h) and after administration, blood samples were collected from rats 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, and the blood collection volume was about 0.5 mL. The blood samples were 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 then 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 promptly aliquoted into EP tubes with corresponding labels attached, and stored in a -80°C refrigerator.

[0391] 2.6. Sample Detection

[0392] 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.

[0393] 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.

[0394] 2.6.2. LC-MS analysis:

[0395] 1) Liquid phase conditions

[0396] Chromatographic column: Welch CB-C8, 2.1 * 50 mm, 5 μm; Serial number: 217

[0397] Pressure: 10 Mpa

[0398] Pre-column: Phenomenex guard column

[0399] Column temperature: 40 °C

[0400] Injection volume: 5 μL

[0401] Injection temperature: 4 °C

[0402] Running time: 3.5 min

[0403] Flow rate: 0.6 mL / min

[0404] Column pressure: 10 Mpa

[0405] Needle wash solution: 50% methanol in water

[0406] Needle wash mode: Before and after aspiration

[0407] Mobile phase A: 0.1% formic acid in 10 mM ammonium acetate aqueous solution

[0408] Mobile phase B: 0.1% formic acid in 10 mM ammonium acetate methanol solution

[0409] Elution program and mobile phase ratio: Isocratic elution, A:B = 40:60.

[0410] 2) Mass spectrometry conditions:

[0411] Ion detection mode: Multiple reaction monitoring (MRM)

[0412] Ion polarity: Positive ion

[0413] Ionization method: Pneumatically assisted electrospray ionization (ESI)

[0414] CAD: 8

[0415] CUR: 40 psi

[0416] GS1: 50 psi

[0417] GS2: 50 psi

[0418] TEM: 600 °C

[0419] IS: 4500 v

[0420] 3. Test Results and Analysis

[0421] The main pharmacokinetic parameters were calculated using WinNonlin 7.0. The results of the pharmacokinetic experiments in rats are shown in Table 3 below.

[0422] Table 4 Pharmacokinetic Test Results in Rats

[0423]

[0424] 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, indicating that the compound of the present invention exhibits better absorption characteristics.

[0425] Test Example 5 Pharmacokinetic Determination in KM Mice

[0426] 1. Test Purpose

[0427] Using KM mice as the test animals, the pharmacokinetic behavior of the compound of the example in plasma in mice after intragastric administration at a dose of 10 mg / kg / intravenous injection at a dose of 1 mg / kg was studied.

[0428] 2. Test Method

[0429] 2.1. Test Drugs

[0430] The examples and reference compounds of the present invention were prepared in-house.

[0431] 2.2. Test Animals

[0432] Male SPF-grade KM mice, weighing (20 ± 2) g, Hubei Center for Experimental Animals, Production License No.: SCXK(E)2020-0018.

[0433] 2.3. Preparation of Test Drugs

[0434] 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).

[0435] Drug preparation for the intravenous administration group: The drug concentration was prepared at 0.2 mg / mL, and the preparation solvent was 5% DMA + 5% Solutol HS-15 + 90% normal saline.

[0436] 2.4. Administration:

[0437] Male SPF-grade KM mice were adaptively fed for 3 - 4 days.

[0438] Gavage administration group: The administration dose was 10 mg / kg, and the administration volume was 10 mL / kg.

[0439] Intravenous administration group: The administration dose was 1 mg / kg, and the administration volume was 5 mL / kg.

[0440] 2.5. Sample collection

[0441] Gavage administration: Blood was collected from mice before (0 h) and after administration at 0, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h. For intravenous injection, blood was collected before (0 h) and after administration at 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h.

[0442] Blood was collected by puncturing the fundus of the eye, with a blood collection volume of approximately 0.1 mL. It was 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 then immediately placed in an ice-water bath. Centrifugation was performed at 4500 rpm for 10 min at 4 °C. After the centrifugation operation was completed, the plasma was taken and promptly aliquoted into EP tubes with corresponding labels attached, and stored in a -80 °C refrigerator.

[0443] 2.6. Sample detection

[0444] Take 20 μL of mouse plasma after administration and add it to a 96-well deep well 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 well plate pre-added with 200 μL of ultrapure water, vortex for 5 min, centrifuge at 4000 rpm for 3 min. Take 5 μL of the supernatant for LC-MS / MS analysis of plasma drug concentration.

[0445] 3. Experimental results and analysis

[0446] The main pharmacokinetic parameters were calculated using WinNonlin 7.0. The results of the mouse pharmacokinetic experiment are shown in Table 5 below.

[0447] Table 5 Results of mouse pharmacokinetic experiment

[0448]

[0449] 4. Experimental conclusion

[0450] 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.

[0451] Pharmacodynamic test in the acetic acid writhing model of mice in Test Example 6

[0452] 1. Purpose: To evaluate the analgesic pharmacodynamic effect of the examples in the acetic acid writhing model of KM mice.

[0453] 2. Test method:

[0454] 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% methylcellulose (MC, CAS: 9004-67-5) (v:v = 1:99, PH = 3)).

[0455] 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.

[0456] 2.3. Test grouping:

[0457] Table 6 Grouping of pharmacodynamic tests of compounds in the acetic acid writhing model

[0458]

[0459] 2.4. Drug administration and model establishment

[0460] 1 hour after the corresponding drugs of each group were administered as described above, 0.6% acetic acid (0.15 mL / 10 g (0.1 mL / 10 g is also acceptable)) was injected intraperitoneally. Record the writhing latency (the time when the mouse first showed a writhing response after injecting glacial acetic acid), and observe and record the number of writhing times of the mice within 20 minutes.

[0461] · Writhing index: When the mouse showed typical abdominal concavity, accompanied by trunk twisting, hip elevation and other characteristic responses, it was considered that writhing occurred.

[0462] 2.5. Data collection and analysis

[0463] Use Excel software to collect data.

[0464] Use Prism (Graph pad software, Inc.) software to analyze data.

[0465] 3. Results

[0466] Table 7 Analgesic Pharmacodynamic Results of Compounds in the Mouse Acetic Acid Writhing Pain Model

[0467]

[0468]

[0469] 4. Conclusion

[0470] As can be seen from the above data, at the same dosing dose, Compound Examples 1 and 2 of the present invention can inhibit the pain in mice caused by acetic acid and reduce the number of writhing in mice. Among them, the analgesic effect of Example 1 is stronger than that of the reference compound.

[0471] Test Example 7 Pharmacodynamic Determination of Mouse Foot Incision

[0472] 1. Purpose: To evaluate the analgesic pharmacodynamics of the examples in the ICR mouse foot incision model.

[0473] 2. Test Method:

[0474] 2.1. Test Drugs: The examples of the present invention are self-made and formulated 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 formulated using normal saline.

[0475] 2.2. Test Animals: Male ICR mice were purchased from Sichuan Vital River Laboratory Animal Technology Co., Ltd. and weighed 28 - 35 g at the time of purchase.

[0476] 2.3. Test Grouping:

[0477] Table 8 The pharmacodynamic test grouping of compounds in the mouse foot incision model is as follows:

[0478]

[0479] 2.4. Model Establishment and Drug Administration

[0480] After the animals were acclimatized for 3 - 5 days, the baseline before model establishment was measured before model establishment. One day before the drug administration test, a mouse plantar incision pain model was established by surgery. The pain sensitivity baseline test after model establishment was 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 was measured using Von Frey test filaments. Animals meeting the baseline requirements were selected for grouping and randomly divided into 6 groups with 10 animals in each group. The drugs were administered orally once according to the above grouping. The pain thresholds of the animals were measured before drug administration (baseline), 1 h, 2 h, 4 h, and 6 h after drug administration.

[0481] 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 root of the left hind foot surface 0.2 cm away from the heel towards the fingertip, cut through the skin and fascia, separate the plantar muscle, slightly lift it, and make a longitudinal incision. Then suture the skin with 5-0 suture thread and disinfect it.

[0482] 2.5. Data collection and analysis

[0483] 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 dosing 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.

[0484] 3. Results

[0485] Table 9 Analgesic efficacy results of compounds in the mouse foot incision pain model

[0486]

[0487] *: P < 0.05, vs solvent group

[0488] 4. Conclusions

[0489] Under the conditions of this experiment, intragastric administration of Test Example 1 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 represented by formula (IC) or a pharmaceutically acceptable salt thereof, in, R 1 is - OR 1-1 ; R 1-1 is C1-C6 alkyl, 3-8 membered heterocycloalkyl, 1-1-1 Substituted C1-C6 alkyl or 3-8 membered heterocycloalkyl substituted by one or more deuteriums; in the 3-8 membered heterocycloalkyl and the 3-8 membered heterocycloalkyl in the 3-8 membered heterocycloalkyl substituted by 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; 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 5 and R 6 Both hydrogen and both deuterium; R 7 is a C1-C6 alkyl group; X 1 , X 3 , X 4 and X 5 Each independently is CR X1 ; R X1 is hydrogen or halogen.

2. The compound of formula (IC) 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 1-1 In the 3-8 membered heterocycloalkyl and the 3-8 membered heterocycloalkyl 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 is each independently preferably a 4-6 membered heterocycloalkyl; more preferably For example (4)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; (5)R a In the above, the C3-C6 cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclopropyl; (6)R 4 wherein the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; (7)R 7 wherein the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; and (8)R X1 In the above, the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine.

3. The compound of formula (IC) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)R 1-1 is C1-C6 alkyl or is replaced by one or more R 1-1-1 Substituted C1-C6 alkyl; (2) Each R 1-1-1 Each independently is deuterium; and (3) R 5 and R 6 At the same time, it is deuterium.

4. The compound of formula (IC) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1) In formula (IC), R 1 -OCH3, (2) In formula (IC), R 2 For fluorine; (3) In formula (IC), R 3 For fluorine; (4) In formula (IC), R 4 is methyl; (5) In formula (IC), R 7 is methyl; and (6) in formula (IC), for 5. The compound of formula (IC) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (IC) is any of the following compounds: Preferably or an enantiomer thereof, a diastereomer thereof, or a mixture thereof.

6. A compound represented by formula (II-C): in, The R 1 , R 2 , R 3 , R 4 and R 7 The definition as in any one of claims 1 to 5; R 8 -CN, -COOH or Preferably, the compound represented by formula (II-C) is any of the following compounds: More preferably, the compound represented by formula (II-C) is any of the following compounds: or an enantiomer thereof, a diastereomer thereof, or a mixture thereof.

7. A pharmaceutical composition, comprising: (1) a compound of formula (IC) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, and (2) Pharmaceutically acceptable excipients.

8. Use of a compound of formula (IC) or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, 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 is 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 (IC) as described in any one of claims 1 to 5 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 (IC) as described in any one of claims 1 to 5 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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