Benzoheteroaromatic ring compound, composition comprising same, and use thereof

Abstract

Disclosed are a benzoheteroaromatic ring compound, a composition comprising same, and a use thereof. Provided are a compound of formula I, and a stereoisomer or pharmaceutically acceptable salt thereof. The benzoheteroaromatic ring compound has a novel structure and has good agonistic activity on KCNQ2 / 3.

Description

Benzoheterocyclic aromatic compounds, compositions containing them and their applications Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically to benzo[a]ary aromatic compounds, compositions containing them, and their applications. Background Technology

[0002] Ion channels are an important family of membrane proteins on the cell membrane, playing a crucial role in processes such as nerve and muscle excitation, hormone secretion, cell differentiation, sensory conduction, learning and memory, blood pressure control, and salt and water balance. Studies have found that mutations in more than 60 ion channels are closely related to diseases. Currently, ion channels have become the third largest drug target after GPCRs (G protein-coupled receptors) and protein kinases. There are more than 400 genes encoding ion channels in the human genome, among which the potassium ion channel superfamily has the most members. According to their function and structural characteristics, potassium ion channels can be mainly divided into four categories: inward rectifier potassium channels (Kir), two-pore potassium channels (K2p), calcium-activated potassium channels (KCa), and voltage-gated potassium channels (KV) (H. Wulff et al., Nature Reviews Drug Discovery, 2009, 8(12), 982-1001). Potassium channels play a crucial role in regulating neuronal excitability. Their ionic basis is that the intracellular potassium ion concentration is higher than the extracellular concentration. After the membrane potential depolarizes and activates the channel, positively charged potassium ions flow out, causing the membrane potential to become negative (negative polarization or even hyperpolarization), thus reducing cellular excitability. Recent studies on the genetics of epilepsy have shown that potassium channel abnormalities can directly lead to epilepsy (H. Wulff et al., Chemical Review, 2008, 108(5), 1744-1773), such as in benign familial neonatal seizures (BFNC).

[0003] Voltage-gated potassium channels (KV) are important members of the potassium channel superfamily, which consists of 12 members (KV1.X-KV12.X). The KCNQ channel is the 7th member of the voltage-gated potassium channel family (Kv7), and it includes five subtypes, named KCNQ1-KCNQ5. The distribution and function of different KCNQ subtypes vary. For example, KCNQ1 is mainly found in the heart and cochlea, and its mutations are closely related to congenital QT interval prolongation syndrome and congenital deafness; KCNQ2, 3, and 5 are mainly found in the brain and central nervous system, and are closely related to neural excitation; KCNQ4 is mainly found in the cochlea and vestibular system, and is closely related to hearing (DA Brown et al., British Journal of Pharmacology, 2009, 156, 1185-1195). Compared to other voltage-gated potassium channel members, KCNQ channels have a lower activation threshold, opening at an action potential of -60 mV. Furthermore, their activation is slow, and they do not become inactive under sustained depolarization. These characteristics place KCNQ channels at a basal level in regulating cellular excitability; their opening reduces neural excitability, while functional inhibition induces depolarization of the neuronal membrane potential, enhancing excitability and triggering more nerve impulses. Therefore, KCNQ channels are important pharmaceutical targets for the prevention and treatment of various neuroexcitability disorders.

[0004] Based on the aforementioned characteristics of the KCNQ target, KCNQ potassium channel agonists can reduce nerve cell excitability by activating potassium channels. They can be used not only to treat epilepsy, but also to treat other diseases caused by excessive nerve excitability, such as convulsions, neuropathic pain, acute ischemic stroke, and neurodegenerative diseases.

[0005] Currently, the most representative KCNQ potassium channel agonist in clinical practice is retigabine (RTG), an antiepileptic drug developed by GSK and launched in 2011. Retigabine was the first KCNQ potassium channel agonist to be systematically studied. It can activate KCNQ2-5 and is mainly used to treat some adult patients with seizure-type epilepsy.

[0006] Retigabine has a number of adverse reactions in clinical use, including dizziness, drowsiness, fatigue, confusion, tremor, poor coordination, diplopia, blurred vision, attention deficit, memory loss, motor incoordination, aphasia, dysarthria, balance disorders, increased appetite, hallucinations, myoclonus, peripheral edema, decreased motor function, dry mouth, and dysphagia. Urinary abnormalities are also common adverse reactions of retigabine, including bladder swelling, bladder wall thickening, and urinary retention. On April 26, 2013, the FDA's Drug Safety Committee disclosed that retigabine can also cause some pigmentary reactions in clinical use, including blue skin and retinal pigment changes, but the specific mechanism of action is unclear, and recommended that all patients taking this drug have regular eye examinations. Its original manufacturer, GlaxoSmithKline (GSK), voluntarily withdrew it from the market in 2017 for commercial reasons.

[0007] Therefore, it is necessary to develop novel KCNQ2&3 small molecule openers. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a benzo[a]heterocyclic aromatic compound, a composition containing the same, and the application thereof. The benzo[a]heterocyclic aromatic compound of the present invention has a novel structure.

[0009] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0010] This invention provides a compound of formula I, its stereoisomer, or a pharmaceutically acceptable salt thereof:

[0011] in:

[0012] Ring A is selected from benzo5-membered heterocyclic, benzo5-membered heteroaryl, 6-membered heteroaryl-5-membered heterocyclic, 6-membered heteroaryl-5-membered heteroaryl, or...

[0013] Ring B1 is selected from phenyl, 5-6 membered heterocyclic, or 5-6 membered heterocyclic alkenyl; ring B2 is selected from C 5-6 Cycloalkenyl, 5-6 membered heterocyclic alkenyl, phenyl or 5-6 membered heteroaryl; ring B3 is selected from 5-6 membered heteroaryl or 5-6 membered heterocyclic alkenyl;

[0014] L1 is selected from key, -NR a -、-NH-C(O)-、-C(O)-、-CR a =CR b -、-NH-CR a R b -、-CR a R b -C(O)-, -C(O)-NH-, -C(O)-CR a Rb -、

[0015] L2 is selected from the bond, -NR c -、-(CH2) n - Heterocyclic group, cycloalkyl group, -NH-CR d R e -、-O-、-S-、

[0016] R1 is selected from hydrogen, hydroxyl, cyano, nitro, amino, C 1-6 Hydroxyalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclic groups, -OR 1.0 or -NR a R b The amino group, C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, optionally surrounded by one or more R 1.1 replace;

[0017] R 1.0 Selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups;

[0018] R is independently selected from hydrogen, halogen, cyano, C 1-6 Alkyl, -OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Hydroxyalkyl, -C(O)R a C 3-6 Cycloalkyl, 3-6 membered heterocyclic groups or -P(=O)R a R b The C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally separated by one or more R 1.1 replace;

[0019] Alternatively, any two Rs together form = O;

[0020] Alternatively, any two Rs can be connected to form a C. 3-10 Cycloalkyl, 3-10 membered heterocyclic, 5-6 membered heteroaryl or C 6-10 Aryl; or, R connects to L1 to form C 3-10 Cycloalkyl, 3-10 membered heterocyclic, 5-6 membered heteroaryl or C 6-10 Aryl; the C 3-10Cycloalkyl, 3-10 membered heterocyclic, 5-6 membered heteroaryl and C 6-10 aryl, optionally constituting one or more R 1.1 replace;

[0021] R 1.1 Each is independently selected from halogens, hydroxyl groups, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups;

[0022] R a R b R c R d and R e Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -C(O)OR a.1 C 3-6 cycloalkyl or 3-6 membered heterocyclic groups; R a.1 Selected from halogens, C 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups;

[0023] n is selected from 1, 2, 3, or 4;

[0024] x is selected from 1, 2, 3, 4 or 5.

[0025] In a preferred embodiment of the present invention, the ring A mentioned above is selected from...

[0026] In a preferred embodiment of the present invention, the general formula (I) described above is further shown as general formulas (II-1) to (II-21):

[0027] in:

[0028] R2, R 2’ R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, halogen, cyano, cyano, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Hydroxyalkyl, -C(O)R a C 3-6 Cycloalkyl, 3-6 membered heterocyclic groups or -P(=O)R aR b The C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally separated by one or more R 1.1 replace;

[0029] Alternatively, R5 and R6 together with the atoms attached to them form 4-8 membered heterocyclic alkenyl groups;

[0030] Alternatively, R7, R2, and the atoms they are attached to form 4-8 membered cycloalkenyl or 4-8 membered heterocycloalkenyl;

[0031] The 4-8 membered cycloalkenyl and 4-8 membered heterocycloalkenyl groups are optionally replaced by one or more groups selected from halogen, cyano, hydroxyl, C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Haloalkyl, C 1-3 Halogenated alkoxy or C 1-3 The hydroxyalkyl group is substituted.

[0032] In a preferred embodiment of the present invention, the ring A mentioned above is... The Selected from

[0033] In a preferred embodiment of the present invention, the ring A mentioned above is... The Selected from Wherein, M is -CR7R8-, -O-, -S- or -NR6-, and R6, R7 and R8 are as defined above in this invention.

[0034] In a preferred embodiment of the present invention, the compound of general formula (I) described above is further shown as of general formulas (III-1) to (III-17):

[0035] Where M is -CR7R8-, -O-, -S-, or -NR6-; y is 0, 1, 2, 3, or 4; L1, L2, R1, R2, R 2’ R3, R4, R5, R6, R7 and R8 are as defined above in this invention.

[0036] In a preferred embodiment of the present invention, L1 mentioned above is selected from... -NR a -、-NH-C(O)-、-C(O)-、-CR a =CR b -、-NH-CRa R b -、-CR a R b -C(O)-, -C(O)-NH-, -C(O)-CR a R b -、

[0037] In a preferred embodiment of the present invention, L2 mentioned above is selected from bond, -NR c - Heterocyclic group, cycloalkyl group, -NH-CR d R e -、-O-、-S-、

[0038] In a preferred embodiment of the present invention, the R described above is... a Selected from hydrogen, halogens, C 1-6 Alkyl, C 1-3 Haloalkyl, C 1-3 Alkyl or -C(O)OR a.1 Preferred components include hydrogen, F, Cl, methyl, ethyl, -CF3, and -CH2CF3.

[0039] In a preferred embodiment of the present invention, the R described above is... b Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy group; preferably hydrogen, methyl, ethyl, -CF3 or -CH2CF3.

[0040] In a preferred embodiment of the present invention, the R described above is... c Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy; preferably hydrogen or methyl.

[0041] In a preferred embodiment of the present invention, the R described above is... d Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy; preferably hydrogen.

[0042] In a preferred embodiment of the present invention, the R described above is... e Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy; preferably hydrogen.

[0043] In a preferred embodiment of the present invention, R5 and R6, as described above, are formed together with the atoms connected to them.

[0044] In a preferred embodiment of the present invention, R7 and R2, as described above, together with the atoms connected to them, form...

[0045] In a preferred embodiment of the present invention, each of R1 mentioned above is independently selected from hydrogen, hydroxyl, cyano, amino, nitro, C 1-3 Hydroxyalkyl, C 1-6 Alkyl, C 1-3 Haloalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclic groups, -OR 1.0 or -NR a R b The amino group, C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, optionally surrounded by one or more R 1.1 replace.

[0046] In a preferred embodiment of the present invention, R2 and R mentioned above... 2’ Each is independently selected from hydrogen, halogen, or C. 1-3 alkyl.

[0047] In a preferred embodiment of the present invention, each of the R3 groups mentioned above is independently selected from hydrogen, halogen, cyano, and C. 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Hydroxyalkyl or C 3-6 Cycloalkyl.

[0048] In a preferred embodiment of the present invention, each of the R4 groups mentioned above is independently selected from cyano, halogen, and C. 3-6 cycloalkyl, 3-6 membered heterocyclic, C 1-3 Halogenated alkoxy or C 1-3 Halogenated alkyl; the C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally separated by one or more R 1.1 replace.

[0049] In a preferred embodiment of the present invention, each of the R5s mentioned above is independently selected from hydrogen or halogen.

[0050] In a preferred embodiment of the present invention, each of the R6 mentioned above is independently selected from hydrogen or C. 1-6 alkyl.

[0051] In a preferred embodiment of the present invention, R7 and R8 mentioned above are each independently selected from hydrogen and C.1-3 Alkyl or -C(O)R a .

[0052] In a preferred embodiment of the present invention, the R described above is... 1.1 Each is independently selected from hydrogen, hydroxyl, C 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups.

[0053] In a preferred embodiment of the present invention, the R described above is... 1.0 Selected from hydrogen, C 1-3 Alkoxy, C 3-6 cycloalkyl or C 1-3 alkyl.

[0054] In a preferred embodiment of the present invention, each of the R1s mentioned above is independently selected from hydrogen or C. 1-6 alkyl.

[0055] In a preferred embodiment of the present invention, R2 and R mentioned above... 2’ Each is independently selected from hydrogen, halogen, or C. 1-3 alkyl.

[0056] In a preferred embodiment of the present invention, each of the R3s mentioned above is independently selected from hydrogen, halogen, or C. 1-3 alkyl.

[0057] In a preferred embodiment of the present invention, each of the R4 groups mentioned above is independently selected from hydrogen, halogen, cyano, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Halogenated alkoxy groups.

[0058] In a preferred embodiment of the present invention, each of R5 mentioned above is independently selected from hydrogen, halogen, and C. 1-3 alkyl.

[0059] In a preferred embodiment of the present invention, each of the R6 mentioned above is independently selected from hydrogen and C. 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups.

[0060] In a preferred embodiment of the present invention, each of the R7s mentioned above is independently selected from hydrogen, halogens, and C. 1-3 Alkyl, C 3-6 Cycloalkyl groups, 3-6 membered heterocyclic groups.

[0061] In a preferred embodiment of the present invention, each of the R8s mentioned above is independently selected from hydrogen, halogens, and C. 1-3 alkyl.

[0062] In a preferred embodiment of the present invention, R5 and R8, as described above, together with the atoms they are attached to form a 4-6 membered heterocyclic group.

[0063] In a preferred embodiment of the present invention, R7 and R8, as described above, together with the atoms connected to them, form C. 3-6 Cycloalkyl or 3-6 membered heterocyclic groups;

[0064] In a preferred embodiment of the present invention, R4 and R5 described above together form =O.

[0065] In a preferred embodiment of the present invention, R4 and R5, as described above, together with the atoms connected to them, form C. 3-6 cycloalkyl or 3-6 membered heterocyclic groups; the C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally surrounded by one or more groups selected from halogen, hydroxyl, C 1-3 Alkyl or C 1-3 Alkyl-substituted.

[0066] In a preferred embodiment of the present invention, the C described above... 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally surrounded by one or more groups selected from halogen, hydroxyl, C 1-3 Alkyl or C 1-3 Alkyl-substituted.

[0067] In a preferred embodiment of the present invention, each of R1 mentioned above is independently selected from hydrogen, hydroxyl, cyano, nitro, methyl, ethyl, methoxy

[0068] In a preferred embodiment of the present invention, each of R1 mentioned above is independently selected from hydrogen, methyl, ethyl, ...

[0069] In a preferred embodiment of the present invention, R2 and R mentioned above... 2’ Each is independently selected from hydrogen, F, or methyl.

[0070] In a preferred embodiment of the present invention, each of the R3 groups mentioned above is independently selected from hydrogen, cyano, -CF3, methyl, ... Or cyclopropyl.

[0071] In a preferred embodiment of the present invention, each of the R4 groups mentioned above is independently selected from hydrogen, F, cyano, methyl, cyclopropyl, ... Or -CF3.

[0072] In a preferred embodiment of the present invention, each of the R5 groups mentioned above is independently selected from hydrogen, F, cyano, ... Or methyl.

[0073] In a preferred embodiment of the present invention, each of the R6 molecules mentioned above is independently selected from hydrogen, methyl, and...

[0074] In a preferred embodiment of the present invention, each of the R7s mentioned above is independently selected from hydrogen, methyl, -CF3, CHF2, ...

[0075] In a preferred embodiment of the present invention, each of the R8s mentioned above is independently selected from hydrogen or methyl.

[0076] In a preferred embodiment of the present invention, R5 and R8, as described above, are formed together with the atoms connected to them. Optionally selected by one or more elements chosen from halogens, hydroxyl groups, C 1-3 Alkyl or C 1-3 The alkoxy group is replaced by a substituent.

[0077] In a preferred embodiment of the present invention, R7 and R8, as described above, are formed together with the atoms connected to them. The Optionally selected by one or more elements chosen from halogens, hydroxyl groups, C 1-3 Alkyl or C 1-3 The alkoxy group is replaced by a substituent.

[0078] In a preferred embodiment of the present invention, R4 and R5, as described above, are formed together with the atoms connected to them. The Optionally selected by one or more elements chosen from halogens, hydroxyl groups, C 1-3 Alkyl or C 1-3 The alkoxy group is replaced by a substituent.

[0079] In a preferred embodiment of the present invention, each of the L1 values ​​mentioned above is independently selected from a key, -NH-, -NH-C(O)-, -C(O)-, -C(O)-CH2- or

[0080] In a preferred embodiment of the present invention, each of the L2 values ​​mentioned above is independently selected from bonds, -C(O)-, -CH2-, -CH2CH2-, -NH-,

[0081] In a preferred embodiment of the present invention, the terms -L1-L2 mentioned above are each independently selected from the key, -NH-C(O)-、 -C(O)-、 Or -NH-.

[0082] In a preferred embodiment of the present invention, the compounds described above are selected from the compounds listed in Table 1 below:

[0083] The present invention also provides a pharmaceutical composition comprising:

[0084] (1) Any of the compounds described above in this invention or their pharmaceutically acceptable salts; and,

[0085] (2) Pharmaceutically acceptable excipients.

[0086] The present invention also provides the use of substance A in the preparation of a medicament for the prevention and / or treatment of diseases, symptoms or conditions;

[0087] The substance A is any compound described in this invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above in this invention; the disease, symptom or condition may be a neurological disease such as epilepsy, convulsions, neuropathic pain, acute ischemic stroke and neurodegenerative diseases.

[0088] Terminology Explanation

[0089] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0090] The term "oxo" refers to =O, where an oxygen atom replaces two hydrogen atoms on the same carbon atom; that is, a carbonyl group replaces a methylene group.

[0091] The term "alkyl" refers to a saturated aliphatic hydrocarbon group; for example, an alkyl group containing 1 to 8 carbon atoms; preferably an alkyl group containing 1 to 6 carbon atoms, more preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and their various branched isomers.

[0092] The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one double bond. Alkenes can contain 2-20 carbon atoms, preferably alkenyl groups containing 2 to 8 carbon atoms, more preferably alkenyl groups containing 2 to 6 carbon atoms, and most preferably alkenyl groups containing 2 to 3 carbon atoms. Non-limiting examples include: vinyl groups. 1-Propylene 2-Propylene, 1-Butenyl, Isobutenyl and 1,3-Butadienyl, etc.

[0093] The term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic hydrocarbon substituent; the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, further preferably 3 to 6 carbon atoms, and most preferably 3 to 4 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. Polycyclic cycloalkyl groups include spirocycloalkyl, bridged cycloalkyl, and fused cycloalkyl.

[0094] The term "cycloalkenyl" refers to a partially unsaturated cyclic hydrocarbon group; for example, a cycloalkenyl ring contains 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms. Non-limiting examples of cycloalkenyl groups include cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptanetrienyl, etc.

[0095] The term "heterocyclic group" refers to a saturated cyclic group having a specified number of ring atoms (e.g., 3-8, 5-6, 7-12), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (1, 2, or 3 of N, O, and S); for example, a heterocyclic group contains 3 to 8 ring atoms; most preferably, it contains 3 to 6 ring atoms. In this invention, "member" indicates the number of ring atoms, such as a 3-6 member heterocyclic group indicating that it contains 3-6 ring atoms. Heterocyclic groups include monocyclic heterocyclic groups and polycyclic heterocyclic groups. Non-limiting examples of monocyclic heterocyclic groups include oxetane, thiohepane, azirone, azironeheptane, pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolidinyl, dihydrofuranyl, morpholinyl, thiomorpholinyl, and piperazineyl, preferably oxetane, thiohepane, azirone, tetrahydrofuranyl, tetrahydropyranyl, 1-aminoethylene-1-oxothiran, and azironeheptane. Polycyclic heterocyclic groups include spirohepane, fused heterocyclic, and bridged heterocyclic groups. A spirohepane is a polycyclic heterocyclic group in which a single atom (called a spiro atom) is shared between monocyclic rings. Non-limiting examples include... etc.; Fused heterocyclic groups refer to polycyclic heterocyclic groups in which each ring in a system shares an adjacent pair of atoms with other rings in the system. Non-limiting examples include Etc.; Bridged heterocyclic groups refer to polycyclic heterocyclic groups in which any two rings share two atoms that are not directly connected. Non-limiting examples include wait.

[0096] The term "heterocyclic alkenyl" refers to a monocyclic cyclic group having a specified number of ring atoms (e.g., 5-6), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (1, 2, or 3 of N, O, and S), wherein at least one ring contains one or more double bonds. "Heterocyclic alkenyl" groups do not contain closed cyclic conjugated systems, but only isolated π bonds or discontinuous conjugated π bonds; for example... wait.

[0097] The term "alkoxy" refers to -O- (alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, and cyclopropoxy.

[0098] "Halogenated alkyl" refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above. Non-limiting examples include trifluoromethyl and difluoromethyl.

[0099] "Haloalkoxy" refers to an alkoxy group that has been substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0100] "Hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group, wherein the alkyl group is as defined above. Non-limiting examples include: -CH2OH, -C(CH3)2(OH).

[0101] The different terms "X is A, B, or C", "X is A, B, and C", "X is A, B, or C", and "X is A, B, and C" all express the same meaning, that is, X can be any one or more of A, B, and C.

[0102] Substituents of the present invention This indicates the position where the substituent is attached to the site of substitution.

[0103] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0104] "One or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more, preferably 1, 2, 3 or 4.

[0105] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0106] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0107] "Pharmaceutical acceptable" means relatively non-toxic, safe, and suitable for patient use.

[0108] "Pharmaceutically acceptable salts" refer to salts 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. See Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition) for details.

[0109] Unless otherwise specified, all technical and scientific terms used herein have the standard meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0110] Unless otherwise specified, the chiral carbon in the compounds of this invention is either in the R configuration or the S configuration.

[0111] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0112] The reagents and raw materials used in this invention are all commercially available. Detailed Implementation

[0113] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0114] Synthesis of intermediate A1

[0115] Step 1: Synthesis of intermediate A1-2

[0116] Under nitrogen protection and in an ice bath, N-bromosuccinimide (13.31 g, 74.78 mmol, 1.1 equiv) was added to a solution of 2,3,6-trifluoroaniline (10 g, 67.98 mmol, 1.0 equiv) in N,N-dimethylformamide (50 mL). The mixture was stirred at room temperature for 2 hours. The desired product was observed in LC-MS. The reaction mixture was diluted with ethyl acetate (150 mL), backwashed with saturated brine (3 x 150 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (0-20%) to give intermediate A1-2 (13.90 g, 90.47%) as a yellow liquid. LC-MS: (ESI, m / z): 226.0 [M+H] + .

[0117] Step 2: Synthesis of intermediate A1-3

[0118] Under nitrogen protection, sodium perborate (43.07 g, 526.56 mmol, 1.0 equiv) was added to a solution of compound A1-2 (11.90 g, 55.66 mmol, 1.0 equiv) in glacial acetic acid (120 mL) at room temperature. The mixture was stirred at 60 °C for 16 hours. The desired product was observed in LC-MS. The reaction mixture was filtered, diluted with ethyl acetate (300 mL), backwashed with saturated brine (3 x 300 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (0-10%) to give a yellow liquid intermediate A1-3 (1.67 g, 12.39%). LC-MS: (ESI, m / z): 256.0 [M+H] + .

[0119] 1 H NMR(400MHz,DMSO-d6)δ8.19-8.15(m,1H).

[0120] Step 3: Synthesis of intermediate A1-4

[0121] Under nitrogen protection, 2-methylpropyl-2-amine (249 mg, 3.40 mmol, 1 equiv) and N,N-diisopropylethylamine (1.32 g, 10.20 mmol, 3 equiv) were added to a 10 mL solution of intermediate A1-3 (870 mg, 3.40 mmol, 1 equiv) in dimethyl sulfoxide (DMSO) at room temperature, and the reaction was carried out for 2 hours at room temperature. The desired product was observed in liquid chromatography-mass spectrometry (LC-MS). The reaction mixture was diluted with ethyl acetate (50 mL), backwashed with saturated brine (3 x 50 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in ethyl acetate / petroleum ether (0-10%) to give intermediate A1-4 (800 mg, 76.15%) as a yellow liquid.

[0122] LCMS:(ESI,m / z):309.0[M+H] + .

[0123] 1 H NMR (400MHz, DMSO-d6) δ7.64-7.61 (m, 1H), 5.44 (s, 1H), 1.22 (d, J = 1.6Hz, 9H).

[0124] Step 4: Synthesis of intermediate A1-5

[0125] Under nitrogen protection, iron powder (1.45 g, 25.88 mmol, 10 equiv) and ammonium chloride (1.38 g, 25.88 mmol, 10 equiv) were added to a solution of intermediate A1-4 (800 mg, 2.59 mmol, 1.0 equiv) in ethanol (15 mL) and water (5 mL) at room temperature. The mixture was stirred at 80 °C for 1 hour. The desired product was observed in the liquid chromatography-mass spectrometry (LC-MS). The reaction mixture was filtered, diluted with ethyl acetate (50 mL), backwashed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography in ethyl acetate / petroleum ether (0-10%) to give intermediate A1-5 (700 mg, 96.90%) as a yellow solid.

[0126] LCMS:(ESI,m / z):279.0[M+H] + .

[0127] Step 5: Synthesis of intermediate A1

[0128] Under nitrogen protection, cyanogen bromide (672 mg, 6.34 mmol, 3.0 equiv) was added to a 10 mL ethanol solution of intermediate A1-5 (590 mg, 2.10 mmol, 1.0 equiv) at room temperature. The mixture was stirred for 16 hours at room temperature. The desired product was observed in LC-MS. The reaction mixture was diluted with ethyl acetate (30 mL), backwashed with saturated brine (3 x 30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0-10%) to give intermediate A1 (210 mg, 32.67%) as a yellow solid. LC-MS: (ESI, m / z): 304.0 [M+H] + .

[0129] Synthesis of intermediate A2

[0130] Step 1: Synthesis of intermediate A2-2

[0131] Under nitrogen protection, N,N-diisopropylethylamine (2103 mg, 16.304 mmol, 3.0 equiv) and 1-methylcyclobutane-1-amine hydrochloride (658 mg, 5.434 mmol, 1.0 equiv) were added to a solution of 3,5-difluoro-4-nitrobenzene (1000.0 mg, 5.434 mmol, 1.0 equiv) in dimethyl sulfoxide (10 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, water (20 mL) was added and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in dichloromethane / petroleum ether (0–50%) to give intermediate A2-2 (880.0 mg, 65.04% yield) as a yellow solid.

[0132] 1 H NMR (400MHz, DMSO-d6) δ7.21-7.15(m,2H),6.78(s,1H),2.21-2.16(m,4H),1.87-1.82(m,2H),1.47(s,3H).

[0133] Step 2: Synthesis of intermediate A2-3

[0134] Iron powder (1979.0 mg, 35.340 mmol, 10.0 equiv) and ammonium chloride (1891.0 mg, 35.340 mmol, 10.0 equiv) were added to a solution of intermediate A2-2 (880.0 mg, 35.340 mmol, 10.0 equiv) in ethanol (10 mL) and water (3 mL) at room temperature. The reaction mixture was heated to 80 °C under nitrogen protection and stirred for 2 hours. After the reaction was complete, the reaction mixture was concentrated and water (20 mL) was added, followed by extraction with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in dichloromethane / petroleum ether (0-50%) to give intermediate A2-3 (760.0 mg, 98.20% yield) as a yellow solid. LCMS: (ESI, m / z): 220.2 [M+H] + .

[0135] Step 3: Synthesis of intermediate A2

[0136] A solution of cyanogen bromide (552 mg, 5.205 mmol, 1.5 equiv) was added to 10 mL of ethanol containing intermediate A2-3 (760 mg, 3.470 mmol, 1 equiv) at room temperature. The reaction mixture was heated to 60 °C under nitrogen protection and stirred for 16 hours. After the reaction was complete, the reaction mixture was concentrated and water (20 mL) was added, followed by extraction with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0–10%) to give a yellow solid intermediate A2 (540.0 mg, yield 63.78%). LCMS: (ESI, m / z): 245.1 [M+H] + .

[0137] Synthesis of intermediate A3

[0138] Following the preparation method of intermediate A2, A2-1 was replaced with A3-1 to obtain intermediate A3 (500.0 mg, yield 65.75%). LCMS: (ESI, m / z): 318.0 [M+H] + .

[0139] Synthesis of intermediate A4

[0140] Following the preparation method of intermediate A2, A4-1 was replaced with A4-1, and 1-methylcyclobutylamine was replaced with cyclobutylamine to obtain intermediate A4 (1.53 g, 88.19%). LCMS:(ESI, m / z): 264.1 [M+H] + .

[0141] Synthesis of intermediate A5

[0142] Following the preparation method of intermediate A2, tert-butylamine was used instead of cyclobutylamine to obtain intermediate A5 (3.7 g, 43.5%). LCMS:(ESI, m / z): 233.1 [M+H] + .

[0143] Synthesis of intermediate A6

[0144] Step 1: Synthesis of intermediate A6-2

[0145] Under nitrogen protection, N,N-diisopropylethylamine (1320 mg, 10.235 mmol, 3.0 equiv) and 1-methylcyclobutane-1-amine hydrochloride (413 mg, 3.412 mmol, 1.0 equiv) were added to a solution of 1-bromo-2,3,5-trifluoro-4-nitrobenzene (870.0 mg, 3.412 mmol, 1.0 equiv) in dimethyl sulfoxide (10 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, water (20 mL) was added and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in dichloromethane / petroleum ether (0–50%) to give intermediate A6-2 (760.0 mg, 69.61% yield) as a yellow solid.

[0146] LCMS(ESI,m / z):338.4[M+H] + .

[0147] Step 2: Synthesis of intermediate A6-3

[0148] Iron powder (1330.0 mg, 23.750 mmol, 10.0 equiv) and ammonium chloride (1271.0 mg, 23.750 mmol, 10.0 equiv) were added to a solution of intermediate A6-2 (760.0 mg, 2.375 mmol, 1.0 equiv) in ethanol (10 mL) and water (3 mL) at room temperature. The reaction mixture was heated to 80 °C under nitrogen protection and stirred for 2 hours. After the reaction was complete, the reaction mixture was concentrated and water (20 mL) was added, followed by extraction with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in dichloromethane / petroleum ether (0-50%) to give intermediate A6-3 (700.0 mg, crude) as a yellow solid.

[0149] LCMS:(ESI,m / z):291.0[M+H] + .

[0150] Step 3: Synthesis of intermediate A6-4

[0151] A solution of cyanogen bromide (384 mg, 3.621 mmol, 1.5 equiv) was added to 10 mL of ethanol containing intermediate A6-3 (700 mg, 2.414 mmol, 1 equiv) at room temperature. The reaction mixture was heated to 60 °C under nitrogen protection and stirred for 16 hours. After the reaction was complete, the reaction mixture was concentrated and water (20 mL) was added, followed by extraction with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0–10%) to give intermediate A6-4 (500.0 mg, 65.75% yield) as a yellow solid.

[0152] LCMS:(ESI,m / z):318.0[M+H] + .

[0153] Step 4: Synthesis of intermediate A6

[0154] To a solution of intermediate A6-4 (200 mg, 0.634 mmol, 1.0 equiv) in N,N-dimethylformamide (3 mL), zinc powder (41 mg, 0.634 mmol, 1.0 equiv), zinc cyanide (148 mg, 1.28 mmol, 2.0 equiv), tris(dibenzylacetone)palladium (58 mg, 0.0634 mmol, 0.1 equiv), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (51.7 mg, 0.0634 mmol, 0.1 equiv) were added. The reaction mixture was protected with nitrogen at 120 °C and stirred for 2 hours. After the reaction was complete, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography with ethyl acetate / petroleum ether (0-10%) to give intermediate A6 (81 mg, yield 51.30%) as a yellow solid.

[0155] LCMS:(ESI,m / z):263.1[M+H] + .

[0156] Example 1: Synthesis of Compound 1

[0157] Under nitrogen protection, 2-(4,4-dimethyl-1,4-azasilane-1-yl)acetic acid (120 mg, 0.64 mmol, 1 equiv) was dissolved in N,N-dimethylformamide (5 mL), and compounds A5 (150 mg, 0.64 mmol, 1 equiv), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (366 mg, 0.96 mmol, 1.5 equiv), and N,N-diisopropylethylamine (330 mg, 2.56 mmol, 4.0 equiv) were added separately. The reaction was carried out at 50 °C for 16 hours. The desired product was observed in the liquid chromatography-mass spectrometry (LC-MS). The reaction solution was poured into water (20 mL). The system was extracted with ethyl acetate (2 x 30 mL), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10µm, 19mm x 250mm; Mobile phase A: water (0.1% formic acid); Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 19-29% B for 16 min; Detection wavelength: UV 254nm / 214nm; Retention time (min): 9.2-10.6), yielding a white solid compound 1 (8.97 mg, 3.5%). LCMS (ESI, m / z): 402.3 [M+H] + .

[0158] 1 H NMR (400MHz, DMSO-d) 6) δ8.30(s,1H),7.60(d,J=10.4Hz,1H),3.22(s,2H),2.85-2.76(m,4H),1.76(s,9H),0.83-0.73(m,4H),0.06(s,6H).

[0159] Example 2: Synthesis of Compound 2

[0160] Step 1: Synthesis of Compound 2-2

[0161] To a solution of 3,3-difluoropyrrolidine hydrochloride (500.0 mg, 3.484 mmol, 1.0 equiv) in dichloromethane (10 mL) at room temperature, p-nitrophenyl chloroformate (843.0 mg, 4.181 mmol, 1.2 equiv) and pyridine (826.0 mg, 10.453 mmol, 3.0 equiv) were added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was added to water (20 mL) and extracted with dichloromethane (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0–10%) to give a yellow oily compound 2–2 (450.0 mg, 47.49% yield). LCMS: (ESI, m / z): 273.1 [M+H] + .

[0162] Step 2: Synthesis of Compound 2

[0163] At 0 °C, sodium hydride (16.0 mg, 0.410 mmol, 2.0 equiv) was added to a tetrahydrofuran (0 mL) solution of compound 2-2 (50.0 mg, 0.205 mmol, 1.0 equiv), and the reaction mixture was stirred at 0 °C for 30 min. Then, 4-nitrophenyl 3-difluoropyrrolidine-1-carboxylic acid (67.0 mg, 0.246 mmol, 1.2 equiv) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction mixture was added to water (20 mL) and extracted with ethyl acetate (20 mL x 3). The organic phase was concentrated, and the residue was purified by preparative high-performance liquid chromatography (HPLC) of the crude product. Purification conditions: (Column specifications: Sunfire C18 10µm, 19mm*250mm; Mobile phase A: water (0.1% ammonium bicarbonate), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 58-68% B for 17 min; Detection wavelength: UV 254nm / 214nm; Retention time (min): 8.5-10.5), yielding compound 2 (20.20 mg, yield 26.13%). LCMS: (ESI, m / z): 378.2 [M+H] + .

[0164] 1H NMR (400MHz, CDCl3) δ12.06 (s, 1H), 7.19 (d, J = 9.2Hz, 1H), 7.15 (s, 1H), 3.90-3. 69(m,4H),2.84-2.77(m,2H),2.50-2.33(m,4H),2.08-1.93(m,2H),1.66(s,3H).

[0165] Example 3: Synthesis of Compound 3

[0166] Step 1: Synthesis of Compound 3-2

[0167] Under nitrogen protection, 3,3-dimethylbutyryl chloride (866 mg, 6.466 mmol, 1.5 equiv) was added to a pyridine (20 mL) solution of intermediate A5 (1000.0 mg, 4.310 mmol, 1.0 equiv). The reaction mixture was stirred at 85 °C for 2 hours. After the reaction was complete, the mixture was evaporated to dryness and then extracted with ethyl acetate (40 mL x 3) after adding water (40 mL). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0-10%) to give compound 3-2 (600.0 mg, yield 42.19%). LCMS: (ESI, m / z): 331.2 [M+H] + .

[0168] 1 H NMR (400MHz, DMSO-d6) δ10.30 (s, 1H), 8.21 (d, J = 88.0Hz, 1H), 7.63-7.51 (m, 1H), 2.29 (d, J = 22.8Hz, 2H), 1.84 (d, J = 76.0Hz, 9H), 1.03 (d, J = 26.4Hz, 9H).

[0169] Step 2: Synthesis of Compound 3

[0170] Under nitrogen protection, Lawson's reagent (245 mg, 0.606 mmol, 0.5 equiv) was added to a tetrahydrofuran (10 mL) solution of compound 3-2 (400.0 mg, 1.212 mmol, 1.0 equiv). The reaction mixture was stirred at 50 °C for 16 hours. After the reaction was complete, water (20 mL) was added and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with methanol / dichloromethane (0-10%) to give compound 3 (380.0 mg, 90.62% yield). LCMS: (ESI, m / z): 347.2 [M+H] + .

[0171] 1 H NMR (400MHz, CD3OD) δ8.20 (s, 1H), 7.41 (d, J = 9.6Hz, 1H), 2.79 (s, 2H), 1.85 (s, 9H), 1.19 (s, 9H).

[0172] Example 4: Synthesis of Compound 4

[0173] Step 1: Synthesis of Compound 4-1

[0174] Under nitrogen protection, iodomethane (123 mg, 0.867 mmol, 1.5 equiv) and potassium carbonate (239 mg, 1.734 mmol, 3.0 equiv) were added to a 5 mL solution of compound 3 (200.0 mg, 0.578 mmol, 1.0 equiv) in acetone (5 mL). The reaction mixture was stirred at 35 °C for 16 hours. After the reaction was complete, water (10 mL) was added and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0-10%) to give a yellow solid product 4-1 (150.0 mg, 72.09% yield). LCMS: (ESI, m / z): 361.2 [M+H] + .

[0175] Step 2: Synthesis of Compound 4

[0176] Under nitrogen protection, a 2M solution of methylamine in tetrahydrofuran (0.25 mL, 0.500 mmol, 3.0 equiv) was added to a 3 mL ethanol solution of compound 4-1 (60.0 mg, 0.167 mmol, 1.0 equiv) in a microwave tube. The reaction mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was evaporated to dryness and then extracted with ethyl acetate (10 mL x 3) after adding 10 mL of water. The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 70-80% B for 19 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 8.0-10.0), yielding compound 4 (38.86 mg, yield 67.98%). LCMS (ESI, m / z): 344.2 [M+H] + .

[0177] 1 H NMR (400MHz, CD3OD) δ7.88-7.84(m,1H),7.19-7.15(m,1H),3.04(d,J=75.2Hz ,3H),2.70(d,J=167.6Hz,2H),1.90(d,J=9.6Hz,9H),1.00(d,J=119.2Hz,9H).

[0178] Example 5: Synthesis of Compound 5

[0179] Under nitrogen protection, methoxyamine hydrochloride (42 mg, 0.500 mmol, 3.0 equiv) and N,N-diisopropylethylamine (108 mg, 0.833 mmol, 5.0 equiv) were added to a 3 mL ethanol solution of compound 4-1 (60.0 mg, 0.167 mmol, 1.0 equiv) in a microwave tube. The reaction mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was evaporated to dryness and then extracted with ethyl acetate (10 mL x 3) after adding 10 mL of water. The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 70-80% B for 16 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 9.8-11.0), yielding compound 5 (26.64 mg, yield 44.43%).

[0180] LCMS:(ESI,m / z):360.2[M+H] + .

[0181] 1 H NMR (400MHz, CDCl3) δ8.01 (d, J = 9.6Hz, 1H), 7.69 (s, 1H), 7.18-7.12 (m, 1H), 3. 87(d,J=32.0Hz,3H),2.77(d,J=198.4Hz,2H),1.88(s,9H),1.11-0.92(m,9H).

[0182] Example 6: Synthesis of Compound 6

[0183] Step 1: Synthesis of Compound 6-3

[0184] Triethylamine (1.15 g, 11.34 mmol, 2.0 equiv) and p-nitrophenyl chloroformate (1.14 g, 5.67 mmol, 1.0 equiv) were added to a solution of 2,2-dimethylpropane-1-ol (500.0 mg, 5.67 mmol, 1.0 equiv) in dichloromethane (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with dichloromethane (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (0-10%) to give compound 6-3 as a yellow solid (850.0 mg, yield 59.17%).

[0185] 1 HNMR: 1 ¹H NMR (400MHz, CDCl₃) δ 8.36–8.22 (m, 2H), 7.45–7.34 (m, 2H), 3.99 (s, 2H), 1.02 (s, 9H). Step 2: Synthesis of compound 6

[0186] At room temperature, sodium hydride (39.7 mg, 0.947 mmol, 2.0 equiv) was added to a tetrahydrofuran (5 mL) solution of compound A6 (110.0 mg, 0.473 mmol, 1.0 equiv) at 0 °C, and the mixture was stirred for 30 minutes. Then, compound 6-3 (120.0 mg, 0.473 mmol, 1.0 equiv) was added. The reaction mixture was protected with nitrogen at 0 °C and stirred for 2 hours. After the reaction was complete, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 60-70% B for 16 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 8.5), yielding compound 6 (76.54 mg, yield 46.63%).

[0187] LCMS:(ESI,m / z):347.1[M+H] + .

[0188] 1H NMR (400MHz, DMSO-d6) δ10.01(s,1H),8.32(s,1H),7.61(d,J=10.0Hz,1H),3.82(s,2H),1.76(s,9H),0.89(s,9H).

[0189] Example 7 Synthesis of Compound 7

[0190] Step 1: Synthesis of Compound 7-2

[0191] Triethylamine (703 mg, 6.95 mmol, 3.0 equiv) and p-nitrophenyl chloroformate (467 mg, 2.32 mmol, 1.0 equiv) were added to a solution of 3,3-difluoroazacyclobutane hydrochloride (300.0 mg, 2.32 mmol, 1.0 equiv) in dichloromethane (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with dichloromethane (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (0-10%) to give a yellow solid to compound 7-2 (410 mg, yield 68.57%).

[0192] 1 H NMR (400MHz, CDCl3) δ8.28-8.26(m,2H),7.35-7.32(m,2H),4.52(s,4H).

[0193] Step 2: Synthesis of Compound 7-3

[0194] To a solution of compound A6-4 (105 mg, 0.329 mmol, 1.0 equiv) in tetrahydrofuran (5 mL), sodium hydride (16 mg, 0.658 mmol, 2.0 equiv) was added at 0 °C and stirred for 30 min. Then, compound 7-2 (85 mg, 0.329 mmol, 1.0 equiv) was added. The reaction mixture was stirred at room temperature under nitrogen protection for 16 h. After the reaction was complete, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with ethyl acetate / petroleum ether (0-10%) to give a yellow solid product, yielding compound 7-3 (87 mg, 60.72% yield).

[0195] LCMS:(ESI,m / z):434.9[M+H]+ ,RT(min):2.051.

[0196] Step 3: Synthesis of Compound 7

[0197] To a solution of compound 7-3 (87.0 mg, 0.199 mmol, 1.0 equiv) in N,N-dimethylformamide (3 mL), zinc powder (13 mg, 0.199 mmol, 1.0 equiv), zinc cyanide (47 mg, 0.399 mmol, 2.0 equiv), tris(dibenzylacetone)dipalladium (18.3 mg, 0.0199 mmol, 0.1 equiv), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (16.2 mg, 0.0199 mmol, 0.1 equiv) were added. The reaction mixture was protected with nitrogen at 120 °C and stirred for 2 hours. After the reaction was complete, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 45-95% B for 16 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 9.3-10.0), yielding compound 7 (7.06 mg, yield 9.26%). LCMS (ESI, m / z): 382.1 [M+H] + .

[0198] 1 H NMR (400MHz, DMSO-d6) δ7.69-7.66(m,1H),4.45-4.39(m,4H),2.72-2.65(m,2H),2.32-2.31(m,2H),1.95-1.88(m,1H),1.80-1.75(m,4H)..

[0199] Example 8: Synthesis of Compound 8

[0200] Compound 8 was synthesized using the same method as compound 7, but intermediate 7-1 was replaced with intermediate 8-1 to obtain compound 8 (5.22 mg, yield 5.43%). LCMS (ESI, m / z): 374.1 [M+H] + .

[0201] 1H NMR(400MHz,DMSO-d6)δ8.25(s,1H),7.67-7.61(m,1H),4.07-3.96(m,1H),2.77-2.63(m,3H),2.34-2.21( m,3H),2.00-1.94(m,2H),1.92-1.83(m,2H),1.79-1.71(m,4H),1.59-1.55(m,1H),1.15(d,J=6.0Hz,3H)..

[0202] Example 9: Synthesis of Compound 9

[0203] Compound 9 was synthesized using the same method as compound 7, but intermediate 7-1 was replaced with intermediate 9-1 to obtain compound 9 (13.54 mg, yield 13.98%). LCMS (ESI, m / z): 397.1 [M+H] + .

[0204] 1 H NMR(400MHz,DMSO-d6)δ8.25(s,1H),7.48-7.43(m,1H),4.89-4.81(m,1H),3.06-2.99(m,2H),2.68-2.6 6(m,2H),2.63-2.55(m,2H),2.38-2.30(m,2H),1.95-1.85(m,1H),1.78-1.74(m,1H),1.68-1.63(m,3H).

[0205] Example 10: Synthesis of Compound 10

[0206] Compound 10 was synthesized using the same method as compound 7, but intermediate 7-1 was replaced with intermediate 10-1 to obtain compound 10 (10.44 mg, yield 12.96%).

[0207] LCMS:(ESI,m / z):349.1[M+H] + .

[0208] 1 H NMR(400MHz,DMSO-d6)δ10.25(s,1H),7.67-7.63(m,1H),4.93-4.86(m,1H),2.67-2.62 (m,2H),2.37-2.29(m,2H),1.98-1.91(m,1H),1.81-1.75(m,4H),1.26(d,J=6.4Hz,6H).

[0209] Example 11 Synthesis of Compound 11

[0210] Compound 11 was synthesized using the same method as compound 7, but intermediate 7-1 was replaced with intermediate 11-1 to obtain compound 11 (8.22 mg, yield 7.27%). LCMS (ESI, m / z): 361.1 [M+H] + .

[0211] 1 H NMR(400MHz,DMSO-d6)δ10.27(s,1H),7.69-7.66(m,1H),2.67-2.59(m,2H),2.35-2.30(m,2 H),1.98-1.91(m,1H),1.82-1.76(m,4H),1.54(s,3H),0.89-0.86(m,2H),0.72-0.69(m,2H).

[0212] Example 12 Synthesis of Compound 12

[0213] Compound 12 was synthesized using the same method as compound 7, but intermediate 7-1 was replaced with intermediate 12-1 to obtain compound 12 (13.96 mg, yield 23.39%).

[0214] LCMS:(ESI,m / z):374.1[M+H] + .

[0215] 1 H NMR(400MHz,DMSO-d6)δ8.90(s,1H),8.24(s,1H),7.65-7.62(m,1H),2.67-2.58(m,2H),2.46-2.4 3(m,2H),2.33-2.26(m,2H),2.00-1.89(m,3H),1.85-1.80(m,2H),1.78-1.70(m,4H),1.45(s,3H).

[0216] Example 13 Synthesis of Compound 13

[0217] Compound 13 was synthesized using the same method as compound 7, but intermediate 7-1 was replaced with intermediate 13-1 to obtain compound 13 (19.29 mg, yield 24.09%).

[0218] LCMS:(ESI,m / z):396.1[M+H] + .

[0219] 1H NMR(400MHz,DMSO-d6)δ9.30(s,1H),8.13(s,1H),7.66-7.63(m,1H),4.11-4.04(m,1H),3.01-2.90(m ,2H),2.70-2.64(m,2H),2.61-2.57(m,2H),2.43-2.38(m,2H),1.96-1.89(m,1H),1.77-1.70(m,4H).

[0220] Example 14 Synthesis of Compound 14

[0221] Compound 14-4 was prepared according to the method for intermediate A6. In a microwave-safe environment, 3,3-dimethylbutyryl chloride (18.0 mg, 0.132 mmol, 1.5 equiv) was added to a pyridine solution (2 mL) of compound 14-4 (20.0 mg, 0.088 mmol, 1.0 equiv). The reaction mixture was heated to 85 °C under nitrogen protection and stirred for 16 hours. After the reaction was complete, the mixture was concentrated, and water (20 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in methanol / dichloromethane (0-10%) to obtain a yellow crude solid. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 20 mL / min; elution gradient: 56-67% B for 16 min; detection wavelength: UV 254 nm / 214 nm; retention time (min): 9.8-11.0) to obtain compound 14 (1.95 mg, yield 6.80%).

[0222] LCMS:(ESI,m / z):327.1[M+H] + .

[0223] 1 H NMR (400MHz, DMSO-d6) δ10.63(s,1H),8.47(s,1H),7.41(d,J=8.4Hz,1H),7.26(d,J=8.4Hz,1H),4.87-4 .83(m,1H),4.77-4.72(m,1H),4.60-4.56(m,1H),2.36-2.31(m,4H),1.34(d,J=6.4Hz,3H),1.06(s,9H).

[0224] Example 15 Synthesis of Compound 15

[0225] Compound 15 was synthesized using the same method as compound 14, but intermediate 14-1 was replaced with intermediate 15-1 to obtain compound 15 (8.14 mg, yield 26.62%).

[0226] LCMS:(ESI,m / z):313.2[M+H] + ,RT(min):1.707.

[0227] 1 H NMR (400MHz, DMSO-d6) δ11.21(s,1H),7.35(d,J=8.0Hz,1H),7.20(d,J=8.4Hz,1H),4.70-4.66 (m,1H),4.50-4.46(m,1H),4.03-3.97(m,1H),2.33(s,2H),1.53(d,J=6.4Hz,3H),1.05(s,9H).

[0228] Example 16 Synthesis of Compound 16

[0229] Compound 16 was synthesized using the same method as compound 14, but intermediate 14-1 was replaced with intermediate 16-1 to obtain compound 16 (17.04 mg, yield 23.87%).

[0230] LCMS:(ESI,m / z):327.1[M+H] + .

[0231] 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),7.42(d,J=8.4Hz,1H),7.27-7.13(m,1H),4.37(s,2H),2.29(s,2H),1.57(s,6H),1.06(s,9H).

[0232] Example 17 Synthesis of Compound 17

[0233] Under nitrogen protection, Lawson's reagent (232.0 mg, 0.611 mmol, 2 equiv) was added to a solution of N-(6-cyano-4,7-difluoro-1-(1-methylcyclobutyl)-1H-benzo[d]imidazol-2-yl)-3,3-dimethylbutyramide (110.0 mg, 0.306 mmol, 1.0 equiv) in 1,2-dichloroethane (4 mL). The reaction mixture was heated to 85 °C and stirred for 3 hours. After the reaction was complete, the reaction mixture was added to water and extracted with dichloromethane (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with methanol / dichloromethane (0-10%) to give a yellow solid crude product. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 20 mL / min; elution gradient: 50-95%, time: 16 min; detection wavelength: UV 254 nm / 214 nm; retention time (min): 10.2-12) to give compound 17 (11.76 mg, yield 10.22%).

[0234] LCMS:(ESI,m / z):377.0[M+H]+.

[0235] 1 H NMR (400MHz, DMSO-d6) δ11.62 (s, 1H), 7.70 (d, J = 6.4Hz, 1H), 2.77-2.72 (m, 4H),2.34-2.32(m,2H),1.98-1.91(m,1H),1.83-1.76(m,4H),1.12(s,9H).

[0236] Example 18 Synthesis of Compound 18

[0237] Compound 18 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 18-1 to give compound 18 (4.16 mg, yield 7.03%). LCMS (ESI, m / z): 388.2 [M+H] + .

[0238] 1H NMR(400MHz,DMSO-d6)δ9.26(s,1H),7.71-7.61(m,1H),3.58-3.51(m,2H),2.77-2.70(m,2H),2.30-2.21(m,2H),1.95-1 .91(m,1H),1.86-1.73(m,6H),1.67-1.61(m,1H),1.55-1.48(m,1H),1.38(s,4H),1.29-1.23(m,1H),0.95-0.84(m,1H).

[0239] Example 19 Synthesis of Compound 19

[0240] Compound 19 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 19-1 to give compound 19 (4.62 mg, yield 7.84%). LCMS (ESI, m / z): 386.2 [M+H] + .

[0241] 1 H NMR (400MHz, DMSO-d6) δ7.65-7.64(m,1H),3.60-3.52(m,4H),2.73-2.69(m,2H),2.29-2. 25(m,2H),1.94-1.87(m,1H),1.83-1.79(m,2H),1.76-1.71(m,4H),0.59(d,J=4.4Hz,4H).

[0242] Example 20 Synthesis of Compound 20

[0243] Compound 20 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 20-1 to give compound 20 (6.12 mg, yield 9.78%). LCMS (ESI, m / z): 410.2 [M+H] + .

[0244] 1 H NMR (400MHz, DMSO-d6) δ7.69-7.66(m,1H),3.64(s,4H),2.72-2.67(m,2H),2.35-2.25(m,2H),2.03-1.87(m,5H),1.78-1.67(m,4H).

[0245] Example 21 Synthesis of Compound 21

[0246] Compound 21 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 21-1 to obtain compound 21 (10.94 mg, yield 9.45%). LCMS (ESI, m / z): 400.2 [M+H]+.

[0247] 1 H NMR (400MHz, DMSO-d6) δ7.68-7.59(m,1H),3.45-3.32(m,4H),2.73-2.66(m,2H),2.27-2.23(m,2H),1.96-1.87(m,9H),1.78-1.72(m,4H).

[0248] Example 22 Synthesis of Compound 22

[0249] Compound 22 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 22-1 to give compound 22 (9.39 mg, yield 7.85%). LCMS (ESI, m / z): 428.0 [M+H] + .

[0250] 1 H NMR(400MHz,DMSO-d6)δ7.71-7.67(m,1H),4.78-4.70(m,1H),3.64-3.57(m,2H),2.77-2 .63(m,2H),2.33-2.27(m,2H),2.18-2.13(m,1H),2.01-1.88(m,4H),1.77-1.63(m,4H).

[0251] Example 23 Synthesis of Compound 23

[0252] Compound 23 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 23-1 to give compound 23 (5.01 mg, yield 6.72%). LCMS (ESI, m / z): 374.2 [M+H] + .

[0253] 1 H NMR(400MHz,DMSO-d6)δ9.54(s,1H),7.66-7.63(m,1H),3.67(s,4H),2.71-2.6 4(m,2H),2.33-2.28(m,2H),1.96-1.89(m,1H),1.78-1.73(m,4H),1.24(s,6H).

[0254] Example 24 Synthesis of Compound 24

[0255] Compound 24 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 24-1 to give compound 24 (8.31 mg, yield 11.09%). LCMS (ESI, m / z): 372.2 [M+H] + .

[0256] 1 H NMR(400MHz,DMSO-d6)δ7.67-7.63(m,1H),4.53-4.48(m,1H),3.39(s,2H),2.90-2.88(m,1H), 2.73-2.66(m,2H),2.33-2.23(m,2H),1.97-1.87(m,3H),1.78-1.71(m,4H),1.36-1.30(m,2H).

[0257] Example 25 Synthesis of Compound 25

[0258] Compound 25 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 25-1 to give compound 25 (4.79 mg, yield 6.42%). LCMS (ESI, m / z): 374.2 [M+H] + .

[0259] 1 H NMR(400MHz,DMSO-d6)δ9.41(s,1H),7.66-7.64(m,1H),3.61-3.51(m,2H),2.96-2.90(m,1H),2.77-2.65(m,2H),2. 32-2.18(m,4H),2.04-1.97(m,1H),1.93-1.88(m,1H),1.78-1.69(m,4H),1.55-1.46(m,1H),1.03(d,J=6.4Hz,3H).

[0260] Example 26 Synthesis of Compound 26

[0261] Compound 26 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 26-1 to give compound 26 (3.20 mg, yield 4.29%). LCMS (ESI, m / z): 374.2 [M+H] + .

[0262] 1H NMR (400MHz, DMSO-d6) δ7.66-7.63(m,1H),3.95(s,2H),2.71-2.67(m,2H),2.33-2.28(m,2H),2.06-1.87(m,3H),1.80-1.75(m,4H),1.46(s,6H).

[0263] Example 27 Synthesis of Compound 27

[0264] Compound 27 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 27-1 to give compound 27 (10.16 mg, yield 11.49%). LCMS (ESI, m / z): 384.1 [M+H] + .

[0265] 1 H NMR (400MHz, DMSO-d6) δ8.15 (s, 1H), 7.69-7.64 (m, 1H), 6.16 (t, J = 56.0Hz, 1H), 3.88-3.69 (m, 2H ),3.09-3.03(m,3H),2.75-2.66(m,2H),2.35-2.25(m,2H),1.94-1.87(m,1H),1.79-1.68(m,4H).

[0266] Example 28 Synthesis of Compound 28

[0267] Compound 28 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 28-1 to give compound 28 (1.13 mg, yield 0.77%). LCMS (ESI, m / z): 388.2 [M+H] + .

[0268] 1 H NMR (400MHz, DMSO-d6) δ7.68-7.65(m,1H),4.03-3.97(m,1H),3.69-3.63(m,1H),2.72-2.68(m,2H),2.29-2. 26(m,2H),2.02-1.97(m,2H),1.93-1.88(m,1H),1.79-1.74(m,4H),1.64-1.63(m,2H),1.24(d,J=6.0Hz,6H).

[0269] Example 29 Synthesis of Compound 29

[0270] Compound 29 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 29-1 to give compound 29 (16.95 mg, yield 20.04%). LCMS (ESI, m / z): 372.3 [M+H] + .

[0271] 1 H NMR(400MHz,DMSO-d6)δ9.63(s,1H),7.67-7.65(m,1H),3.78-3.61(m,1H),3.59-3.46(m,1H),3.09-3.06(m,1H),2.81-2.63(m,2H), 2.35-2.21(m,2H),2.16-2.03(m,1H),2.00-1.89(m,2H),1.79-1.72(m,4H),1.68-1.63(m,1H),0.76-0.71(m,1H),0.66-0.55(m,1H).

[0272] Example 30 Synthesis of Compound 30

[0273] Compound 30 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 30-1 to give compound 30 (8.52 mg, yield 9.61%). LCMS (ESI, m / z): 388.1 [M+H] + .

[0274] 1 H NMR(400MHz,DMSO-d6)δ9.36(s,1H),7.66-7.63(m,1H),3.91-3.72(m,1H),3.49-3.44(m,2H),2.77-2.6 7(m,2H),2.33-2.20(m,2H),1.92-1.86(m,4H),1.82-1.65(m,6H),1.38-1.30(m,1H),0.86-0.82(m,3H).

[0275] Example 31 Synthesis of Compound 31

[0276] Compound 31 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 31-1 to obtain compound 31 (3.23 mg, yield 3.52%). LCMS: (ESI, m / z): 402.1 [M+H] + .

[0277] 1H NMR(400MHz,DMSO-d6)δ9.42(s,1H),7.66-7.63(m,1H),3.87-3.82(m,1H),3.57-2.51(m,1H),3.45-3.36(m,1H),2.90-2.56(m ,2H),2.24-2.12(m,2H),1.94-1.87(m,2H),1.82-1.74(m,7H),1.23-1.13(m,1H),0.85(d,J=6.8Hz,3H),0.80(d,J=6.8Hz,3H).

[0278] Example 32 Synthesis of Compound 32

[0279] Compound 32 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 32-1 to give compound 32 (2.21 mg, yield 2.41%). LCMS (ESI, m / z): 402.2 [M+H] + .

[0280] 1 H NMR (400MHz, DMSO-d6) δ9.42(s,1H),7.66-7.63(m,1H),3.87-3.82(m,1H),3.57-3.51(m,1H),3.46-3.37(m,1H),2.77(dd,J=38.2,2 3.6Hz,2H),2.22-2.10(m,2H),1.94-1.87(m,2H),1.83-1.71(m,7H),1.29-1.27(m,1H),0.85(d,J=6.8Hz,3H),0.80(d,J=6.8Hz,3H).

[0281] Example 33 Synthesis of Compound 33

[0282] Compound 33 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 33-1 to give compound 33 (14.97 mg, yield 17.06%). LCMS (ESI, m / z): 386.1 [M+H] + .

[0283] 1H NMR(400MHz,DMSO-d6)δ9.36(s,1H),7.66-7.62(m,1H),3.66-3.63(m,2H),2.73-2.65(m,2H), 2.33-2.22(m,2H),1.96-1.85(m,5H),1.80-1.69(m,4H),1.67-1.60(m,2H),0.48-0.45(m,2H).

[0284] Example 34 Synthesis of Compound 34

[0285] Compound 34 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 34-1 to obtain compound 34 (28.34 mg, yield 34.91%).

[0286] LCMS:(ESI,m / z):357.1[M+H] + .

[0287] 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.47(s,1H),7.86(s,1H),7.74-7.71(m,1H),6. 98(s,1H),2.74-1.67(m,2H),2.33-2.19(m,2H),1.94-1.87(m,1H),1.82-1.70(m,4H).

[0288] Example 35 Synthesis of Compound 35

[0289] Compound 22 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 22-1 to give compound 22 (27.28 mg, yield 27.61%). LCMS (ESI, m / z): 425.0 [M+H] + .

[0290] 1 H NMR(400MHz,DMSO-d6)δ10.35(s,1H),7.70-7.66(m,1H),4.93-4.86(m,1H), 2.67-2.61(m,2H),2.35-2.32(m,2H),2.07-1.92(m,7H),1.81-1.77(m,6H).

[0291] Example 36 Synthesis of Compound 36

[0292] Step 1: Synthesis of Compound 36-1

[0293] At -10 °C, 5-fluoro-2,3-dihydro-1H-inden-1-one (6 g, 39.96 mmol, 1.0 equiv) was added in portions to fuming nitric acid (60 mL). The reaction mixture was stirred at -10 °C for 1 hour. After the reaction was complete, the reaction mixture was slowly poured into ice water (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (30%) to give a yellow oily product, compound 36-1 (4.54 g, 58.22%). 1 H NMR (400MHz, CDCl3) δ8.44(d,J=7.2Hz,1H),7.41(d,J=10.0Hz,1H),3.33-3.19(m,2H),2.87-2.76(m,2H).

[0294] Step 2: Synthesis of compound 36-2

[0295] At 0 °C, sodium borohydride (1.76 g, 46.53 mmol, 2.0 equiv) was added in portions to a methanol (100 mL) solution of compound 36-1 (4.54 g, 23.26 mmol, 1.0 equiv). The reaction mixture was stirred at 0 °C for 2 hours. After the reaction was complete, the reaction mixture was slowly poured into ice water (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (50%) to give a yellow oily product, compound 36-2 (4.55 g, 99.20%).

[0296] 1 H NMR (400MHz, CDCl3) δ8.09 (d, J = 7.2Hz, 1H), 7.15 (d, J = 10.8Hz, 1H), 5.29 (t, J = 6.4Hz, 1H) ,3.17-3.09(m,1H),2.96-2.82(m,1H),2.65-2.54(m,1H),2.12-2.00(m,1H),1.81(s,1H).

[0297] Step 3: Synthesis of compound 36-3

[0298] At 0 °C, triethylsilane (2.36 g, 20.39 mmol, 2.0 equiv) was slowly added to a trifluoroacetic acid (20 mL) solution of compound 36-2 (2 g, 10.14 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was slowly poured into ice water (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (30%) to give a yellow oily compound 36-3 (1.5 g, 81.62%).

[0299] 1 H NMR (400MHz, CDCl3) δ7.88 (d, J = 7.0Hz, 1H), 7.10 (d, J = 10.8Hz, 1H), 3.03-2.90 (m, 4H), 2.22-2.14 (m, 2H).

[0300] Step 4: Synthesis of compound 36-4

[0301] At room temperature, iron powder (4.62 g, 82.8 mmol, 10.0 equiv) and ammonium chloride (4.43 g, 82.8 mmol, 10.0 equiv) were added to a solution of compound 36-3 (1.5 g, 8.28 mmol, 1.0 equiv) in ethanol (20 mL) and water (4 mL). The reaction mixture was stirred at 80 °C for 3 hours. After the reaction was complete, the mixture was filtered, and the filtrate was slowly poured into ice water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (50%) to give compound 36-4 (1.2 g, 95.87%) as a yellow oil.

[0302] LCMS:(ESI,m / z):152.1[M+H] + .

[0303] Step 5: Synthesis of compound 36-5

[0304] At room temperature, N-bromosuccinimide (1.41 g, 7.94 mmol, 1.0 equiv) was added to a solution of compound 36-4 (1.2 g, 7.94 mmol, 1.0 equiv) in N,N-dimethylformamide (20 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was slowly poured into ice water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (30%) to give compound 36-5 (910 mg, 49.83%) as a yellow oil.

[0305] LCMS:(ESI,m / z):230.1[M+H] + .

[0306] Step 6: Synthesis of compound 36-6

[0307] At room temperature, triethylamine (845 mg, 8.34 mmol, 3.0 equiv) and 4-dimethylaminopyridine (34 mg, 0.278 mmol, 0.1 equiv) were added to a solution of compound 36-5 (640 mg, 2.78 mmol, 1.0 equiv) in di-tert-butyl dicarbonate (10 mL). The reaction mixture was stirred at 60 °C for 16 hours. After the reaction was complete, the reaction mixture was slowly poured into ice water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (10%) to give compound 36-6 (900 mg, 75.19%) as a yellow oil.

[0308] LCMS:(ESI,m / z):318.0[M-56-56] + .

[0309] Step 7: Synthesis of compound 36-7

[0310] At room temperature, sodium hydroxide (251 mg, 6.27 mmol, 3.0 equiv) was added to a tetrahydrofuran (10 mL) and water (2 mL) solution of compound 36-6 (900 mg, 2.09 mmol, 1.0 equiv). The reaction mixture was stirred at 100 °C for 16 hours. After the reaction was complete, the reaction mixture was slowly poured into ice water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (20%) to give a yellow oily compound 36-7 (620 mg, 89.78%).

[0311] LCMS:(ESI,m / z):274.1[M-56] + .

[0312] Step 8: Synthesis of compound 36-8

[0313] At room temperature, 1-methylcyclobutane-1-amine hydrochloride (230 mg, 1.89 mmol, 1.2 equiv), L-proline (91 mg, 0.787 mmol, 0.5 equiv), cuprous iodide (150 mg, 0.787 mmol, 0.5 equiv), and cesium carbonate (1.54 g, 4.72 mmol, 3.0 equiv) were added to a 10 mL solution of dimethyl sulfoxide (DMSO) of compound 36-7 (520 mg, 1.57 mmol, 1.0 equiv). The reaction mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the reaction mixture was slowly poured into ice water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography in ethyl acetate / petroleum ether (50%) to give a yellow solid compound 36-8 (27 mg, 5.13%).

[0314] LCMS:(ESI,m / z):335.2[M+H] + .

[0315] Step 9: Synthesis of compound 36-9

[0316] At room temperature, a solution of 1,4-dioxane hydrochloride (1 mL, 4 M) was added to a 2 mL solution of dichloromethane containing compound 36-8 (27 mg, 0.08 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was evaporated to dryness, added to ice water (10 mL), and the pH was adjusted to neutral with an aqueous sodium bicarbonate solution. The mixture was then extracted with ethyl acetate (5 mL x 3). The combined organic layers were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (50%) to give a yellow solid compound 36-9 (14 mg, 74.01%).

[0317] LCMS:(ESI,m / z):235.2[M+H] + .

[0318] Step 10: Synthesis of compound 36-10

[0319] At room temperature, cyanogen bromide (32 mg, 0.3 mmol, 5.0 equiv) was added to a 2 mL ethanol solution of compound 36-9 (14 mg, 0.06 mmol, 1.0 equiv). The reaction mixture was stirred at 60 °C for 16 hours. After the reaction was complete, the reaction mixture was evaporated to dryness, added to ice water (10 mL), and extracted with ethyl acetate (5 mL x 3). The combined organic layers were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to methanol / dichloromethane (5%) to give a yellow solid compound 36-10 (10 mg, 64.54%). LCMS: (ESI, m / z): 260.2 [M+H] + .

[0320] Step 11: Synthesis of Compound 36

[0321] Under nitrogen protection, 3,3-dimethylbutyryl chloride (11 mg, 0.077 mmol, 2.0 equiv) was added to a pyridine (2 mL) solution of compound 36-10 (10 mg, 0.038 mmol, 1.0 equiv) in a microwave tube, and the reaction mixture was stirred at 85 °C for 3 hours. After the reaction was completed, the reaction mixture was evaporated to dryness, and the crude product was purified by preparative high performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 66-71% B for 18 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 12-13), yielding compound 36 (1.73 mg, yield 12.55%).

[0322] LCMS:(ESI,m / z):358.2[M+H] + .

[0323] 1 H NMR (400MHz, CDCl3) δ6.87 (d, J = 9.6Hz, 1H), 3.03-2.99 (m, 2H), 2.97-2.93 (m, 2H), 2.72-2.5 8(m,4H),2.38(s,2H),2.14-2.11(m,2H),1.97-1.87(m,1H),1.84-1.81(m,4H),1.06(s,9H).

[0324] Example 37 Synthesis of Compound 37

[0325] Step 1: Synthesis of Compound 37-2

[0326] At 0°C, sodium hydroxide solution (1.52 g, 37.95 mmol, 1.2 equiv) and 30% hydrogen peroxide (28 mL) were added to a tetrahydrofuran (130 mL) solution of 3,5-difluoro-2-hydroxybenzaldehyde (5 g, 31.63 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was cooled to 0°C, and sodium hydroxide solution (2 N) was added until the pH was 10–11. The reaction mixture was stirred at 0°C for 30 minutes, and the reaction was quenched with hydrochloric acid until the pH was 2–3. The mixture was extracted with dichloromethane, and the combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to give a colorless, oily crude product, 3,5-difluorobenzene-1,2-diol (6 g, crude product), which was used directly in the next reaction without further purification.

[0327] LCMS:(ESI,m / z):145.0[MH] + .

[0328] Step 2: Synthesis of Compound 37-3

[0329] Under nitrogen protection, 1,2-dibromoethane (9.64 g, 51.37 mmol, 1.5 equiv) and potassium carbonate (10.41 g, 75.35 mmol, 2.2 equiv) were added to a solution of 3,5-difluorobenzene-1,2-diol (6.0 g, crude) in N,N-dimethylformamide (50 mL) in a single-necked flask. The reaction mixture was heated to 105 °C and stirred for 3 hours. After the reaction was complete, the reaction mixture was quenched in ice water (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to petroleum ether / ethyl acetate (5%) to give compound 37-3 (3.2 g, 45.38% yield) as a white solid.

[0330] 1 H NMR (400MHz, CDCl3) δ6.42-6.34(m,2H),4.24-4.20(m,4H).

[0331] Step 3: Synthesis of compound 37-4

[0332] At 0 °C, nitric acid (540 mg, 5.80 mmol, 1.0 equiv) was added to a sulfuric acid (10 mL) solution of compound 37-3 (1 g, 5.80 mmol, 1.0 equiv) in a single-necked flask. The reaction mixture was stirred at 0 °C for 2 hours. After the reaction was complete, the reaction mixture was quenched with ice water (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to petroleum ether / ethyl acetate (20%) to give a yellow oily compound 37-4 (100 mg, yield 6.22%).

[0333] 1 H NMR (400MHz, CDCl3) δ6.62-6.59(m,1H),4.39-4.38(m,2H),4.35-4.33(m,2H).

[0334] Step 4: Synthesis of Compound 37-5

[0335] At room temperature, 1-methyl-1-cyclobutylamine hydrochloride (56.04 mg, 0.46 mmol, 1.0 equiv) and N,N-diisopropylethylamine (178.37 mg, 1.38 mmol, 3.0 equiv) were added to a solution of compound 37-4 (100 mg, 0.46 mmol, 1.0 equiv) in N,N-dimethylformamide (5 mL) in a single-necked flask. The reaction mixture was stirred at 60 °C for 16 hours. After the reaction was complete, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to petroleum ether / ethyl acetate (50%) to give a yellow oily compound 37-5 (70 mg, yield 53.96%).

[0336] LCMS:(ESI,m / z):283.0[M+H] + .

[0337] Step 5: Synthesis of Compound 37-6

[0338] At room temperature, palladium on carbon (70 mg) was added to a 2 mL solution of tetrahydrofuran containing compound 37-5 (70 mg, 0.25 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 3 hours. After the reaction was complete, the reaction mixture was filtered and concentrated to give a yellow oily compound 37-6 (70 mg, 99% yield), which was used directly in the next reaction without further purification.

[0339] LCMS:(ESI,m / z):252.9[M+H] + .

[0340] Step 6: Synthesis of compound 37-7

[0341] At room temperature, cyanogen bromide (44.08 mg, 0.42 mmol, 1.5 equiv) was added to a 5 mL ethanol solution of compound 37-6 (70 mg, 0.28 mmol, 1.0 equiv) in a single-necked flask. The reaction mixture was stirred at 60 °C for 16 hours. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography to methanol / dichloromethane (5%) to give a yellow solid compound 37-7 (50 mg, yield 64.46%). LCMS: (ESI, m / z): 277.9 [M+H] + .

[0342] Step 7: Synthesis of Compound 37

[0343] At room temperature, 3,3-dimethylbutyryl chloride (32.6 mg, 0.22 mmol, 1.2 equiv) was added to a pyridine (1 mL) solution of compound 37-7 (50 mg, 0.18 mmol, 1.0 equiv) in a microwave-safe tube. The reaction mixture was sealed and stirred at 85 °C for 16 hours. After the reaction was complete, the reaction mixture was concentrated, and the crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Waters 3767 / Qda Column; Pursuit C18 10 μm, 21.2 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 58-68% B for 16 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 9), yielding compound 37 (1.22 mg, yield 1.8%).

[0344] LCMS:(ESI,m / z):376.3[M+H] + .

[0345] 1 H NMR (400MHz, DMSO-d6) δ9.99 (s, 1H), 6.66 (d, J = 10.8Hz, 1H), 4.28 (s, 4H), 2.60-2.56 (m, 2H),2.37-2.33(m,2H),2.22(s,2H),1.88-1.81(m,1H),1.74-1.70(m,4H),1.05(s,9H).

[0346] Example 38 Synthesis of Compound 38

[0347] Step 1: Synthesis of Compound 38-2

[0348] In a single-necked flask, 2-methyl-5-nitroaniline (5.0 g, 32.895 mmol, 1.0 equiv) was dissolved in acetonitrile (50 mL), and N-chlorosuccinimide (5.3 g, 39.474 mmol, 1.2 equiv) was added. The reaction mixture was heated to 80 °C and stirred for 16 hours. After the reaction was complete, the reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (70 mL x 3). The combined organic layers were washed with saturated brine (70 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in dichloromethane / petroleum ether (40%) to give a yellow solid product, compound 38-2 (2.7 g, 44.26%).

[0349] LCMS:(ESI,m / z):186.9[M+H] +.

[0350] Step 2: Synthesis of compound 38-3

[0351] Compound 38-2 (700.0 mg, 3.763 mmol, 1.0 equiv) was added to a 20 mL acetic acid solution of amyl nitrite (581.0 mg, 5.645 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (25%) to give the yellow solid product compound 38-3 (624.0 mg, 84.21%).

[0352] 1 H-NMR: 1 H NMR (400MHz, DMSO) δ14.28(s,1H),8.41(s,1H),8.00-7.98(m,1H),7.79-7.77(m,1H).

[0353] Step 3: Synthesis of compound 38-4

[0354] In a single-necked flask, trimethoxynil tetrafluoroborate (586, 3.959 mmol, 1.3 equiv) was added to a 10 mL solution of 7-chloro-6-nitro-2H-indazole (600.0 mg, 3.046 mmol, 1.0 equiv) in ethyl acetate (10 mL). The reaction mixture was stirred at room temperature for 5 hours. After the reaction was complete, the reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography in ethyl acetate / petroleum ether (33%) to give a yellow solid product, compound 38-4 (333.0 mg, 51.79%).

[0355] LCMS:(ESI,m / z):212.1[M+H] + .

[0356] Step 4: Synthesis of compound 38-5

[0357] In a single-necked flask, 1-methylcyclobutane-1-amine (260.0 mg, 2.133 mmol, 1.5 equiv) and N,N-diisopropylethylamine (550.0 mg, 4.3265 mmol, 3.0 equiv) were added to a solution of compound 38-4 (300.0 mg, 1.422 mmol, 1.0 equiv) in N,N-dimethylformamide (5 mL). The reaction mixture was heated to 100 °C and stirred for 16 hours. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (17%) to give compound 38-5 (194.0 mg, 52.43%) as a yellow solid.

[0358] LCMS:(ESI,m / z):261.2[M+H] + .

[0359] Step 5: Synthesis of compound 38-6

[0360] In a single-necked flask, 15.0 mg of palladium catalyst on carbon was added to a 5 mL solution of compound 38-5 (130.0 mg, 0.500 mmol, 1.0 equiv), and hydrogen gas was introduced. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated to give a brown oily product, compound 38-6 (74.0 mg, 64.35%).

[0361] LCMS:(ESI,m / z):231.3[M+H] + .

[0362] Step 6: Synthesis of Compound 38-7

[0363] In a single-necked flask, cyanogen bromide (171.0 mg, 1.609 mmol, 5.0 equiv) was added to a 5 mL ethanol solution of compound 38-6 (74.0 mg, 0.322 mmol, 1.0 equiv). The reaction mixture was heated to 60 °C under nitrogen protection and stirred for 16 hours. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography to methanol / dichloromethane (10%) to give a brown oily product, compound 38-7 (63.0 mg, 38.41%).

[0364] LCMS:(ESI,m / z):256.3[M+H] + .

[0365] Step 7: Synthesis of Compound 38

[0366] Under nitrogen protection, 3,3-dimethylbutyryl chloride (50.0 mg, 0.371 mmol, 1.2 equiv) was added to a pyridine (3 mL) solution of compound 38-7 (63.0 mg, 0.247 mmol, 1.0 equiv) in a microwave tube. The reaction mixture was heated to 85 °C and stirred for 2 hours. After the reaction was complete, the reaction solution was concentrated, the residue was quenched with water (20 mL), and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 25 mL / min; Elution gradient: 30-35% B for 18 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 9.7-10.3), yielding compound 38 (10.61 mg, yield 12.20%).

[0367] LCMS:(ESI,m / z):354.3[M+H] + .

[0368] 1 H NMR(400MHz,DMSO-d6)δ9.90(s,1H),8.41(s,1H),8.35(s,1H),7.43-7.41(m,1H),7.24-7.22(m,1H),4.16(s,3H ),2.72(s,2H),2.61-2.58(m,2H),2.23(s,2H),1.97-1.91(m,1H),1.81(s,3H),1.76-1.74(m,1H),1.06(s,9H).

[0369] Example 39 Synthesis of Compounds 39 and 40

[0370] Step 1: Synthesis of Compound 39-1

[0371] 5-Bromo-7-fluoro-2,3-dihydro-1H-indene-1-one (5.0 g, 21.930 mmol, 1.0 equiv) was dissolved in fuming nitric acid (20 mL) in a single-necked flask in an ice-salt bath at -10 °C. The reaction mixture was stirred at -10 °C for 1 hour. After the reaction was complete, the reaction mixture was quenched in ice water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (26%) to give compound 39-1 (400.0 mg, 6.68%) as a yellow solid.

[0372] 1 H NMR (400MHz, CDCl3) δ7.62 (d, J = 0.8Hz, 1H), 3.25-3.22 (m, 2H), 2.82-2.79 (m, 2H).

[0373] Step 2: Synthesis of compound 39-2

[0374] In a single-necked flask, 1-methylcyclobutane-1-amine hydrochloride (266.0 mg, 2.198 mmol, 1.5 equiv) and N,N-diisopropylethylamine (567 mg, 4.396 mmol, 3.0 equiv) were added to a solution of compound 39-1 (400.0 mg, 1.465 mmol, 1.0 equiv) in N,N-dimethylformamide (5 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (10%) to give compound 39-2 (440.0 mg, 88.89%) as a yellow solid.

[0375] LCMS:(ESI,m / z):341.1[M+H+2] + .

[0376] Step 3: Synthesis of compound 39-3

[0377] Sodium borohydride (98.0 mg, 2.604 mmol, 2.0 equiv) was added to a methanol (5 mL) solution of compound 39-2 (440.0 mg, 1.302 mmol, 1.0 equiv) in a single-necked flask in an ice-water bath at 0 °C. The reaction mixture was heated to room temperature and stirred for 1 hour. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (12%) to give a yellow oily product, compound 39-3 (422.0 mg, 95.26%).

[0378] LCMS:(ESI,m / z):341.2[M+H] + .

[0379] Step 4: Synthesis of compound 39-4

[0380] In a single-necked flask, triethylsilane (288.0 mg, 2.482 mmol, 2.0 equiv) was added to a trifluoroacetic acid (5 mL) solution of compound 39-3 (422.0 mg, 1.241 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (4%) to give a yellow oily compound 39-4 (387.0 mg, 96.27%).

[0381] 1 H NMR(400MHz, CDCl3)δ7.08(s,1H),4.30(s,1H),2.92-2.88(m,2H),2.77-2.73 (m,2H),2.14-2.04(m,4H),2.00-1.95(m,2H),1.72-1.64(m,2H),1.27(s,3H).

[0382] Step 5: Synthesis of Compound 39-5

[0383] In a single-necked flask, iron powder (268.0 mg, 4.778 mmol, 4.0 equiv), ammonium chloride (253.0 mg, 4.778 mmol, 4.0 equiv), and water (1 mL) were added to a 5 mL ethanol solution of compound 39-4 (387.0 mg, 1.194 mmol, 1.0 equiv). The reaction mixture was heated to 70 °C under nitrogen protection and stirred for 16 hours. After the reaction was complete, the reaction mixture was filtered, the filtrate was quenched with ice water (20 mL), and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (5%) to give the yellow solid product compound 39-5 (308.0 mg, 87.75%). LCMS: (ESI, m / z): 297.2 [M+H] + .

[0384] Step 6: Synthesis of compound 39-6

[0385] In a single-necked flask, cyanogen bromide (555.0 mg, 5.238 mmol, 5.0 equiv) was added to a 5 mL ethanol solution of compound 39-5 (308.0 mg, 1.048 mmol, 1.0 equiv). The reaction mixture was heated to 60 °C under nitrogen protection and stirred for 16 hours. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography to methanol / dichloromethane (10%) to give compound 39-6 (257.0 mg, 76.95%) as a yellow solid.

[0386] LCMS:(ESI,m / z):320.1[M+H] + .

[0387] Step 7: Synthesis of Compound 39

[0388] In a microwave-safe container, 3,3-dimethylbutyryl chloride (109 mg, 0.81 mmol, 1.3 equiv) was added to a pyridine (2 mL) solution of compound 39-6 (200.0 mg, 0.62 mmol, 1.0 equiv). The reaction mixture was stirred at 85 °C for 2 hours. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (20%) to give the crude product compound 39 (204 mg, 78.07%). 20 mg of the crude product was purified by preparative high performance liquid chromatography (HPLC). The purification conditions were as follows: (Column specifications: Waters Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 68-78% B for 17 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 9.1-10.5), yielding compound 39 (8.65 mg).

[0389] LCMS:(ESI,m / z):419.9[M+2] + .

[0390] 1 H NMR(400MHz,DMSO-d6)δ10.00(s,1H),7.36(s,1H),3.03-3.00(m,2H),2.96-2.92(m,2H),2.62-2.57(m,2 H),2.44-2.37(m,2H),2.25(s,2H),2.09-2.05(m,2H),1.96-1.89(m,1H),1.80-1.73(m,4H),1.06(s,9H).

[0391] Step 8: Synthesis of Compound 40

[0392] Under nitrogen protection, in a microwave tube, cuprous iodide (110 mg, 0.574 mmol, 2 equiv) and (1,10-diazaphenanthroline)(trifluoromethyl)copper(I) (180 mg, 0.574 mmol, 2 equiv) were added to a solution of compound 39 (120 mg, 0.287 mmol, 1.0 equiv) in N,N-dimethylformamide (2 mL). The reaction mixture was heated to 80 °C and stirred for 16 hours. After the reaction was complete, the reaction mixture was quenched in ice water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried with anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Waters Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 80-90% B for 17 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 8.5-9.3), yielding compound 40 (28.87 mg, yield 24.70%).

[0393] LCMS:(ESI,m / z):408.2[M+H] + .

[0394] 1 H NMR(400MHz,DMSO-d6)δ10.10(s,1H),7.45(s,1H),3.14-3.11(m,2H),3.01-2.98(m,2H),2.63-2.59(m,2 H),2.45-2.41(m,2H),2.28(s,2H),2.15-2.07(m,2H),1.97-1.90(m,1H),1.80-1.73(m,4H),1.06(s,9H).

[0395] Example 40 Synthesis of Compound 41

[0396] Compound 41 was synthesized using the same method as compound 12, but intermediate 12-1 was replaced with intermediate 41-1 to obtain compound 41 (12.00 mg, yield 15.69%). LCMS (ESI, m / z): 402.3 [M+H] + .

[0397] 1H NMR (400MHz, DMSO-d6) δ10.98(s,1H),7.70-7.66(m,1H),2.72-2.69(m,4H),2.33(s,2H),1.98-1.91(m,3H),1.80-1.74(m,4H),1.51(s,6H).

[0398] Example 41 Synthesis of Compound 42

[0399] Step 1: Synthesis of Compound 42-1

[0400] Under nitrogen protection, N,N'-dicyclohexylcarbodiimide (910 mg, 4.4 mmol, 1.2 equiv) and 2,3,4,5,6-pentafluorophenol (676.0 mg, 3.67 mmol, 1.0 equiv) were added to a solution of 3,3-difluorocyclobutane-1-carboxylic acid (500.0 mg, 3.67 mmol, 1.0 equiv) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was added to water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography to ethyl acetate / petroleum ether (10%) to give a yellow oily product, compound 42-1 (800.0 mg, 72.4% yield). 1 H NMR (400MHz, CDCl3) δ3.41-3.27(m,1H),3.14-2.92(m,4H).

[0401] Step 2: Synthesis of Compound 42

[0402] Under nitrogen protection, sodium hydride (9.0 mg, 0.232 mmol, 2.0 equiv) was added to an ultradry tetrahydrofuran (4 mL) solution of compound 36-10 (30.0 mg, 0.116 mmol, 1.0 equiv), and the reaction mixture was stirred at 0 °C for 30 min. Then, compound 42-1 (35.0 mg, 0.116 mmol, 1.0 equiv) was added, and the reaction mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction mixture was added to water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: (Column specifications: Sunfire C18 10 μm, 19 mm * 250 mm; Mobile phase A: water (0.1% formic acid), Mobile phase B: acetonitrile; Flow rate: 20 mL / min; Elution gradient: 70-80% B for 18 min; Detection wavelength: UV 254 nm / 214 nm; Retention time (min): 9.6-10.0). The result was a white solid compound 42 (8.46 mg, yield 12.90%).

[0403] LCMS:(ESI,m / z):378.0[M+H] + .

[0404] 1 H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 6.97 (d, J = 10.0Hz, 1H), 3.20-3.15 (m, 1H), 3.02 -2.99(m,2H),2.94-2.91(m,2H),2.85-2.76(m,4H),2.59-2.54(m,2H),2.43-2. 33(m,2H),2.19-2.06(m,2H),1.95-1.88(m,1H),1.82-1.75(m,1H),1.71(s,3H).

[0405] Referring to the preparation method of the embodiments, the following final product was synthesized.

[0406] Biological testing evaluation

[0407] Example 1: Detection of the effect of the compound of the present invention on stably overexpressed KCNQ2 / 3 channels using FLIPR potassium flow technology.

[0408] 1. Test compound: Homemade compound

[0409] 2. Information on experimental reagents and instruments

[0410] 2.1 Reagent Information

[0411] 2.2 Instrument Information

[0412] 3. Test methods

[0413] Cell culture

[0414] The HEK-293 cell line, which stably expresses the KCNQ2 / 3 receptor, was constructed in-house by the laboratory of Beijing Aisiyipu Biotechnology Co., Ltd. Its gene information is as follows:

[0415] KCNQ2: NM_172107; KCNQ3: NM_004519;

[0416] 1) Digest and collect cells, count them, and then seed the cells in black-bottomed 384-well plates and culture them overnight.

[0417] 2) Prepare 1×Assay buffer and 1×Chloride-free assay buffer according to the kit instructions. At the same time, prepare 2×dye using 1×Assay buffer.

[0418] 3) Invert the plate and centrifuge to remove the culture medium from the 384-well plate, then immediately add 20 μL of 1×Assay buffer.

[0419] 4) Take 20 μL of the dye prepared in step 2 and add it to the corresponding experimental well. Incubate at 37°C in the dark for 1 hour.

[0420] 5) Use 3mM TI + Prepare 5× positive compounds using 1× Chloride-free assay buffer and transfer 30 μL to the corresponding 384 source plate.

[0421] After the test substance is incubated, 10 μL of the test substance prepared in step 5 is added to the test well using a FLIPR instrument. Data is collected and recorded for 5 minutes. The excitation light for potassium flow detection is 470-495 nm, and the emission light is 515-575 nm.

[0422] 4. Data Analysis

[0423] 1) Z'factor = 1 - 3 * (SD) High +SD Low ) / (AVEHigh -AVE Low )

[0424] 2)CV High =(SD) High / AVE High )*100%

[0425] 3)CV Low =(SD) Low / AVE Low )*100%

[0426] 4) S / B = AVE High / AVE Low

[0427] 5) Calculate compound EC using GraphPad nonlinear fitting formula. 50 :

[0428] Y=Bottom+(Top-Bottom) / (1+10^((LogEC 50 -X)×HillSlope))

[0429] 6) % activation rate formula:

[0430] The average value of the positive control

[0431] Mean of negative control (DMSO)

[0432] Table 2: Note: The relative half-maximal effective concentration (EC50) of the compound tested is shown in this paper. 50 Scope definition: A:EC 50 =<500nM; B:EC 50 >500nM and <1000nM; C: EC 50 >1000nM and <2000Nm; D:EC 50 >2000nM.

[0433] Other compounds of the present invention were tested according to the above test plan to obtain the EC values ​​of the compounds of the present invention. 50 The value is in the range of less than 10 μM; the preferred compound of this invention is EC. 50 Value less than 500 nM; the preferred compound of the present invention is EC. 50 The value is less than 100 nM.

[0434] Experimental results show that the compound of this invention has good KCNQ2 / 3 agonist activity.

[0435] Example 2 uses manual patch-clamp technique to study the effect of the compound of the present invention on KCNQ2 / 3 potassium channels.

[0436] 1. Test substance: The compound prepared in this invention;

[0437] 2. Reagent and Instrument Information

[0438] 2.1 Reagent and Instrument Information

[0439] 2.2 Reagent Information

[0440] 3. Compound preparation

[0441] Weigh an appropriate amount of the test sample. Calculate the required DMSO volume using the formula: DMSO volume = actual compound mass × content (purity) / (molecular weight × theoretical concentration). Take the corresponding volume of DMSO, dissolve the test sample, and weigh the DMSO simultaneously. Calculate the volume using the solvent density to ensure accurate dosage. Calculate the actual stock solution concentration based on the final DMSO usage. The calculated stock solution concentration will generally differ slightly from the planned concentration. This process is automatically completed by the Aisiyipu compound preparation table. If solubility issues arise, the stock solution concentration will be adjusted. The entire preparation process should be conducted in the dark.

[0442] For the highest detectable concentration of the test substance, the stock solution is directly diluted with extracellular fluid or further diluted with DMSO. Other concentrations are first diluted sequentially with DMSO from high to low concentration to create intermediate dilutions, which are then further diluted with extracellular fluid to the working solution concentration. The concentration of DMSO in each working solution does not exceed 0.1%. Before patch-clamp testing, the working solution of the test substance is sonicated for 20 minutes in the dark.

[0443] 4. Cell Culture

[0444] This study used the HEK-293 cell line, which stably expresses the KCNQ2 / 3 receptor, and was constructed by the laboratory of Beijing Aisiyipu Biotechnology Co., Ltd. The gene information is as follows:

[0445] KCNQ2: NM_172107; KCNQ3: NM_004519;

[0446] 4.1. HEK-293 cell lines stably expressing KCNQ2 / 3 receptors were cultured in DMEM medium containing 10% fetal bovine serum and 800 μg / mL G418 at 37°C and 5% carbon dioxide.

[0447] 4.2. Cell Passage: Remove the old culture medium and wash once with PBS, then add 1 mL of 0.25% Trypsin-EDTA solution and incubate at 37°C for about 1 min. When the cells detach from the bottom of the dish, add about 5 mL of preheated (37°C) complete culture medium. Gently pipette the cell suspension to separate aggregated cells. Transfer the cell suspension to sterile centrifuge tubes and centrifuge at 1000 rpm for 5 min to collect the cells. For expansion or maintenance culture, seed the cells in 6 cm cell culture dishes at a density of 2.5 × 10⁶ cells per dish. 5 100 cells (final volume: 5 mL).

[0448] 4.3 To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0449] 4.4 Before manual patch-clamp detection, cells were separated using 0.25% Trypsin-EDTA, and 8 × 10⁸ cells were used. 3 The cells were seeded onto a coverslip and cultured in a 24-well plate (final volume: 500 μL). After 18 hours, the cells were tested.

[0450] 4.5 Before automated patch-clamp detection, cells were subjected to TrypLE... TM After separating the cells using Express culture medium to stop digestion, centrifuge, resuspend the cells, count them, and adjust the cell density to 2-3 × 10⁶ cells / year. 6 Cells / mL were collected, and then the cells were gently mixed on a balanced shaker for 15-20 minutes at room temperature before being analyzed.

[0451] All operations follow the standard operating procedures for cell culture of Beijing Aisiyipu Biotechnology Co., Ltd.

[0452] 5 Electrophysiological Recording

[0453] 5.1 Record the liquids used

[0454] 1) Extracellular fluid: KCNQ-001-1

[0455] 140mM NaCl, 5mM KCl, 1mM MgCl2·6H2O, 2mM CaCl2·2H2O, 10mM HEPES, 5mM D-Glucose, and NaOH to adjust pH to 7.4.

[0456] 2) Intracellular fluid: KCNQ-001-2

[0457] 120mM K-Aspartic, 20mM KCl, 1mM MgCl2·6H2O, 5mM HEPES, 10mM EGTA, 5mM Mg-ATP, KOH adjust pH=7.2.

[0458] Extracellular fluid was stored at 4°C for 2 weeks. Intracellular fluid was prepared, aliquoted into 1 mL tubes, and stored at -20°C. Freshly thawed intracellular fluid was used daily for experiments. All intracellular fluid was used within three months; otherwise, it was discarded and reconstituted.

[0459] 5.2 Patch clamp testing

[0460] The voltage stimulation protocol for whole-cell patch-clamp recording of KCNQ2 / 3 potassium current is as follows: After whole-cell sealing, the cell membrane voltage is clamped at -80 mV. The clamping voltage is depolarized from -110 mV to +40 mV in 10 mV steps and maintained for 3 s. The mean steady-state current is used for IV curve analysis. Then, recording is continued for 500 ms at -30 mV (Protocol-1) and 0 mV (Protocol-2), and the tail current peak is used for activation curve analysis.

[0461] 5.3 Manual Patch Clamp

[0462] The manual patch-clamp procedure begins by using a microelectrode puller to draw a glass capillary into a recording electrode. The electrode, filled with intracellular fluid, is then placed into a microelectrode holder. A coverslip containing cells is placed in a recording bath under an inverted microscope. Under the microscope, the microelectrode manipulator is used to immerse the electrode in the extracellular fluid, and the electrode resistance (Rpip) is recorded. The electrode is then slowly brought into contact with the cell surface, and negative pressure is applied to create a GΩ high-resistance seal. Fast capacitance compensation is then performed, and negative pressure is continued to rupture the cell membrane, establishing a whole-cell recording mode. Finally, slow capacitance compensation is performed, and experimental parameters such as series resistance (Rs) are recorded. No leakage compensation is applied. Experimental data are acquired by an EPC 10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0463] Once the current amplitude stabilized in the control extracellular solution, drug administration began. Each drug concentration was recorded repeatedly until the current stabilized before measuring the next concentration. Blank control extracellular solution and the working solution of the test compound were administered to the cells sequentially from low to high concentration via gravity perfusion in the recording bath, while a peristaltic pump was used for fluid replacement during recording. The current detected in each cell in extracellular solution without the compound served as its control group. All electrophysiological experiments were performed at room temperature.

[0464] 5.4 Automatic Patch Clamp

[0465] When performing electrophysiological assays using the fully automated patch-clamp QPatch 48X (Sophion) device, the prepared cells are first placed on the centrifuge of the Qpatch workbench and washed using multiple centrifugation / resuspension methods to replace the cell culture medium with extracellular fluid. An MTP-96 plate is removed and placed in the MTP source position. The QPlate chip is removed and placed in the QPlate source position. The robotic arm scans the barcodes on the MTP-96 plate and the QPlate chip and picks it up to the measurement station. Intracellular fluid and extracellular fluid are aspirated from the liquid pools and added to the intracellular fluid pool and cell and compound pool of the QPlate chip, respectively. At the measurement station, all measurement sites on the QPlate undergo initial quality control. The quality control process includes aspirating the cell suspension from the centrifuge's cell container and positioning the cells onto the chip wells using a pressure controller to establish a high-resistance seal, forming a whole-cell recording mode. Once a stable control current baseline is obtained, the test substances are sequentially aspirated from the MTP-96 plate according to their concentration and applied to the cells.

[0466] Each drug concentration was designed for multiple administration. Blank control solution and working solution of the test compound were applied to the cells sequentially from low to high concentrations. The current detected in each cell in the solution without the compound served as its control group. All electrophysiological experiments were performed at room temperature.

[0467] 6 Data Analysis

[0468] First, a current-voltage (IV) curve was plotted for each concentration, with voltage on the horizontal axis and normalized steady-state current on the vertical axis. The relative current magnitudes of each compound concentration at -30 mV were compared, and the increase factor of the current amplitude (Icompound) after each concentration was calculated relative to the control current amplitude (Icontrol), i.e., Potency% = (Icompound / Icontrol) * 100%. Data are expressed as Mean ± SE. Then, the EC50 value of the compound's agonist effect was calculated using the Hill equation, and a nonlinear fit was performed on the concentration-effect curve, i.e., Y = 1 / (1 + 10^((LogEC50 - X) * HillSlope)), where X is the logarithm of the concentration, Y is the Potency%, and EC50 is the half-maximal effective concentration.

[0469] The activation curve is fitted using the Boltzmann equation, i.e., I / Imax = 1 / (1 + exp((V1 / 2 - Vm) / к)), where I / Imax is the normalized tail current, V1 / 2 is the half-maximal activation voltage, Vm is the test voltage, and к is the slope factor affecting the slope of the activation curve. The maximum shift in V1 / 2 for each compound concentration is calculated using ΔV1 / 2 = V1 / 2test - V1 / 2control. The shift EC is then calculated by fitting a concentration-effect curve based on the change in shift amplitude with compound concentration. 50 value.

[0470] Curve fitting and graphical analysis were both performed using GraphPad Prism software.

[0471] Testing the compounds of this invention, EC 50 The value is in the range of less than 10 μM; the preferred compound of this invention is EC. 50 Value less than 500 nM;

[0472] The preferred compound of the present invention is EC 50 The value is less than 100 nM.

[0473] Experimental results show that the compound of this invention has good KCNQ2 / 3 agonist activity.

[0474] Test Example 3 investigated the pharmacokinetic behavior of the compound of the present invention in mice.

[0475] Experimental protocol:

[0476] Three healthy male ICR mice (SPF grade, source: Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 18-25g, were administered the compound at a dose of 1mg / kg via intravenous injection, with an administration volume of 5ml / kg, prepared with 5% DMSO + 10% Solutol + 85% Saline (w / v). The animals were fasted prior to the experiment. Three healthy male ICR mice (SPF grade, source: Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 18-25g, were administered the compound at a dose of 5mg / kg via gavage, with an administration volume of 10ml / kg, prepared with 5% DMSO + 10% Solutol + 85% Saline (w / v). The animals were fasted prior to the experiment. Blood samples were collected from the cheek at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after intravenous administration and at the same time after gavage administration. Approximately 0.05 mL of blood was collected per sample. Heparin sodium was used for anticoagulation. Blood samples were placed on ice after collection and centrifuged within one hour to separate the plasma (centrifugation conditions: 6000g, 3 minutes, 2-8℃). The concentration of compounds in the plasma was determined by liquid chromatography-tandem mass spectrometry. Plasma samples were stored at -80℃ before analysis. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.0 based on the blood drug concentration data at different time points. The experimental results are as follows:

[0477] Reference compound A0 of this invention: The compound 517 was synthesized according to the preparation method described in WO2021055538A1. Experimental results showed that the compound exhibited high plasma exposure in ICR rodent mice and possessed favorable pharmacokinetic properties.

[0478] Test Example 4 investigated the pharmacokinetic behavior of the compound of the present invention in rats.

[0479] Experimental reagents: The compounds of this invention, prepared in-house.

[0480] Experimental plan:

[0481] Three healthy male SD rats (SPF grade, source: Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 180-250g, were administered the compound at a dose of 1mg / kg via intravenous injection at a volume of 2ml / kg. The compound was prepared using 5% DMSO + 10% Solutol + 85% Saline (w / v). The animals were not fasted before the experiment.

[0482] Three healthy male SD rats (SPF grade, source: Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 180-250g, were administered the compound at a dose of 5mg / kg by gavage at a volume of 10ml / kg. The compound was prepared using 5% DMSO + 10% Solutol + 85% Saline (w / v). The animals were not fasted before the experiment.

[0483] Blood samples were collected intravenously at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after intravenous administration and at the same time after gavage administration. Approximately 0.25 mL of blood was collected per sample. Heparin sodium was used for anticoagulation. Blood samples were placed on ice after collection and centrifuged within one hour to separate the plasma (centrifugation conditions: 6000 g, 3 min, 2-8℃). The concentration of compounds in the plasma was determined by liquid chromatography-tandem mass spectrometry. Plasma samples were stored at -80℃ before analysis. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.0 based on the blood drug concentration data at different time points. The experimental results are as follows:

[0484] Experimental results show that the compound of this invention has high plasma exposure in rodent SD rats and exhibits good pharmacokinetic properties.

Claims

1. A compound of general formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof: in: Ring A is selected from benzo5-membered heterocyclic, benzo5-membered heteroaryl, 6-membered heteroaryl-5-membered heterocyclic, 6-membered heteroaryl-5-membered heteroaryl, or... Ring B1 is selected from phenyl, 5-6 membered heterocyclic, or 5-6 membered heterocyclic alkenyl; ring B2 is selected from C 5-6 Cycloalkenyl, 5-6 membered heterocyclic alkenyl, phenyl or 5-6 membered heteroaryl; ring B3 is selected from 5-6 membered heteroaryl or 5-6 membered heterocyclic alkenyl; L1 is selected from key, -NR a -、-NH-C(O)-、-C(O)-、-CR a =CR b -、-NH-CR a R b -、-CR a R b -C(O)-、-C(O)-NH-、-C(O)-CR a R b -、 L2 is selected from bond, -C(O)-, -NR c -、-(CH2) n - Heterocyclic group, cycloalkyl group, -NH-CR d R e -、-O-、-S-、 R1 is selected from hydrogen, hydroxyl, cyano, nitro, C 1-6 Hydroxyalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclic groups, -OR 1.0 or -NR a R b The C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, optionally surrounded by one or more R 1.1 replace; R 1.0 Selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; R is independently selected from hydrogen, halogen, cyano, C 1-6 Alkyl, -OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Hydroxyalkyl, -C(O)R a C 3-6 Cycloalkyl, 3-6 membered heterocyclic groups or -P(=O)R a R b The C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally separated by one or more R 1.1 replace; Alternatively, any two Rs together form = O; Alternatively, any two Rs can be connected to form a C. 3-10 Cycloalkyl, 3-10 membered heterocyclic, 5-6 membered heteroaryl or C 6-10 Aryl; or, R connects to L1 to form C 3-10 Cycloalkyl, 3-10 membered heterocyclic, 5-6 membered heteroaryl or C 6-10 Aryl; the C 3-10 Cycloalkyl, 3-10 membered heterocyclic, 5-6 membered heteroaryl and C 6-10 aryl, optionally constituting one or more R 1.1 replace; R 1.1 Each is independently selected from halogens, hydroxyl groups, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; R a R b R c R d and R e Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -C(O)OR a.1 C 3-6 cycloalkyl or 3-6 membered heterocyclic groups; R a.1 Selected from halogens, C 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; n is selected from 1, 2, 3, or 4; x is selected from 1, 2, 3, 4 or 5.

2. The compound according to claim 1, its stereoisomers, or pharmaceutically acceptable salts thereof, characterized in that, The ring A is selected from 3. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, The general formula (I) is further shown in general formulas (II-1) to (II-22): in: R2, R 2’ R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, halogen, cyano, cyano, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Hydroxyalkyl, -C(O)R a C 3-6 Cycloalkyl, 3-6 membered heterocyclic groups or -P(=O)R a R b The C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally separated by one or more R 1.1 replace; Alternatively, R5 and R6 together with the atoms attached to them form 4-8 membered heterocyclic alkenyl groups; Alternatively, R7, R2, and the atoms they are attached to form 4-8 membered cycloalkenyl or 4-8 membered heterocycloalkenyl; The 4-8 membered cycloalkenyl and 4-8 membered heterocycloalkenyl groups are optionally replaced by one or more groups selected from halogen, cyano, hydroxyl, C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Haloalkyl, C 1-3 Halogenated alkoxy or C 1-3 The hydroxyalkyl group is substituted.

4. The compound according to claim 1, its stereoisomers, or pharmaceutically acceptable salts thereof, characterized in that, The ring A is The Selected from 5. The compound according to claim 1 or 4, its stereoisomer, or a pharmaceutically acceptable salt thereof, characterized in that, The ring A is The Selected from Wherein, M is -CR7R8-, -O-, -S- or -NR6-, and R6, R7 and R8 are as defined in claim 3.

6. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1, 4, or 5, characterized in that, The compounds of general formula (I) are further shown as those of general formulas (III-1) to (III-17): Where M is -CR7R8-, -O-, -S-, or -NR6-; y is 0, 1, 2, 3, or 4; L1, L2, R1, R2, R 2’ R3, R4, R5, R6, R7 and R8 are as defined in claim 3.

7. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, characterized in that, It satisfies at least one of the following conditions: (1) The L1 is selected from -NR a -、-NH-C(O)-、-C(O)-、-CR a =CR b -、-NH-CR a R b -、-CR a R b -C(O)-、-C(O)-NH-、-C(O)-CR a R b -、 (2) The L2 is selected from bond, -NR c - Heterocyclic group, cycloalkyl group, -NH-CR d R e -、-O-、-S-、 (3) The R a Selected from hydrogen, halogens, C 1-6 Alkyl, C 1-3 Haloalkyl, C 1-3 Alkyl or -C(O)OR a.1 Preferred components include hydrogen, F, Cl, methyl, ethyl, -CF3, and -CH2CF3. (4) The R b Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy group; preferably hydrogen, methyl, ethyl, -CF3 or -CH2CF3; (5) The R c Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy; preferably hydrogen or methyl; (6) The R d Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy; preferably hydrogen; (7) The R e Selected from hydrogen, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkoxy; preferably hydrogen.

8. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 or 7, characterized in that, It satisfies one or more of the following conditions: (1) R1 is independently selected from hydrogen, hydroxyl, cyano, nitro, C 1-3 Hydroxyalkyl, C 1-6 Alkyl, C 1-3 Haloalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclic groups, -OR 1.0 or -NR a R b The C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, optionally surrounded by one or more R 1.1 replace; (2) R2 and R 2’ Each is independently selected from hydrogen, halogen, or C. 1-3 alkyl; (3) R3 is independently selected from hydrogen, halogen, cyano, C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Hydroxyalkyl or C 3-6 cycloalkyl; (4) R4 is independently selected from cyano, halogen, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 1-3 Halogenated alkoxy or C 1-3 Halogenated alkyl; the C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally separated by one or more R 1.1 replace; (5) Each of the R5 elements is independently selected from hydrogen or halogen; (6) R6 is independently selected from hydrogen or C. 1-6 alkyl; (7) R7 and R8 are each independently selected from hydrogen and C. 1-3 Alkyl or -C(O)R a ; (8)R 1.1 Each is independently selected from hydrogen, hydroxyl, C 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; (9)R 1.0 Selected from hydrogen, C 1-3 Alkoxy, C 3-6 cycloalkyl or C 1-3 alkyl.

9. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 4-7, characterized in that, It satisfies one or more of the following conditions: (1) R1 is independently selected from hydrogen or C. 1-6 alkyl; (2) R2 and R 2’ Each is independently selected from hydrogen, halogen, or C. 1-3 alkyl; (3) R3 is independently selected from hydrogen, halogen, or C. 1-3 alkyl; (4) R4 is independently selected from hydrogen, halogen, cyano, C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Halogenated alkoxy groups, (5) R5 is independently selected from hydrogen, halogen, or C. 1-3 alkyl; (6) R6 is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; (7) R7 is independently selected from hydrogen, halogens, and C. 1-3 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; (8) R8 is independently selected from hydrogen, halogen, or C. 1-3 alkyl; (9) R5 and R8 together with the atoms attached to them form 4-6 membered heterocyclic groups; (10) R7 and R8 together with the atoms they are attached to form C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups; (11) R4 and R5 together form = O; (12) R4 and R5 together with the atoms they are attached to form C 3-6 cycloalkyl or 3-6 membered heterocyclic groups; the C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally surrounded by one or more groups selected from halogen, hydroxyl, C 1-3 Alkyl or C 1-3 Alkyl substitution; (13) The C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, optionally surrounded by one or more groups selected from halogen, hydroxyl, C 1-3 Alkyl or C 1-3 Alkyl-substituted.

10. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, characterized in that, The compounds are selected from those in Table 1.

11. A pharmaceutical composition comprising: (1) The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-10; and, (2) Pharmaceutically acceptable excipients.

12. The use of substance A in the preparation of a medicament for the prevention and / or treatment of diseases, symptoms or conditions; The substance A is a compound as described in any one of claims 1-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 11; The diseases, symptoms, or conditions mentioned may be neurological diseases such as epilepsy, convulsions, neuropathic pain, acute ischemic stroke, and neurodegenerative diseases.

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