Morpholine derivatives, pharmaceutical compositions thereof and uses thereof

Abstract

The present application discloses a substituted morpholine derivative, the structure of which is shown as formula (I). In addition, the present application also discloses pharmaceutically acceptable salts, stereoisomers, pharmaceutical compositions of the derivative and uses thereof.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a morpholine derivative, its pharmaceutically acceptable salt, its stereoisomers, and pharmaceutical compositions and their uses. Background Technology

[0002] P2X purine receptors are a family of ion channels activated by extracellular adenosine triphosphate (ATP). Purine receptors are involved in a variety of biological functions, particularly in relation to pain sensitivity. The P2X3 receptor is one member of this family and was originally cloned from the rat dorsal root ganglion (Chen et al., Nature, Vol. 377, pp. 428-431 (1995)). The nucleotide and amino acid sequences of both rat P2X3 and human P2X3 are now known (Lewis et al., Nature, Vol. 377, pp. 432-435 (1995); and Garcia-Guzman et al., Brain Res. Mol. Brain Res., Vol. 47, pp. 59-66 (1997)).

[0003] P2X3 is reportedly involved in the afferent pathway that controls the bladder capacity reflex. Therefore, inhibiting P2X3 may treat conditions related to urine storage and voiding, such as overactive bladder (Cockayne et al., Nature, Vol. 407, pp. 1011-1015 (2000)).

[0004] P2X3 is also selectively expressed on nociceptive, small-diameter sensory neurons (i.e., neurons stimulated by pain or injury), which is consistent with its role in pain sensitivity. Furthermore, blocking P2X3 receptors has been reported to have analgesic effects in animal models of chronic inflammatory and neuropathic pain (Jarvis et al., PNAS, 99, 17179-17184 (2002)). Therefore, methods to reduce P2X3 levels or activity could be used to modulate pain perception in painful subjects.

[0005] P2X3 can also form the P2X2 / 3 heterodimer with P2X2, another member of the P2X purinergic ligand-gated ion channel family. P2X2 / 3 is highly expressed at the terminals (central and peripheral) of sensory neurons (Chen et al., Nature, Vol. 377, pp. 428-431 (1995)). Recent findings also indicate that P2X2 / 3 is primarily expressed (more than P2X3) in bladder sensory neurons and may play a role in the sensation of bladder fullness and nociceptive responses (Zhong et al., Neuroscience, Vol. 120, pp. 667-675 (2003)).

[0006] In view of the above, there is a need for new P2X3 and / or P2X2 / 3 receptor ligands, especially antagonists, that may be useful and safe for treating various conditions associated with P2X3 and / or P2X2 / 3. Summary of the Invention

[0007] The purpose of this invention is to provide novel morpholine derivatives, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, pharmaceutical compositions thereof, and their use as P2X3 antagonists. These compounds not only exhibit high inhibitory activity against P2X3 and low inhibitory activity against P2X2 / 3, demonstrating significant inhibitory selectivity, but more importantly, they also possess favorable pharmacokinetic parameters, such as low clearance and high in vivo exposure, making them more suitable for development into effective and safe drugs.

[0008] The first aspect of the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0009]

[0010] Where Q is -C(O)NHCH3,

[0011] R1 is hydrogen, C 1-6 Alkyl (preferably C) 1-3 Alkyl groups, more preferably methyl groups, or halogens (preferably fluorine or chlorine);

[0012] R2 is hydrogen or halogen (preferably fluorine);

[0013] R3 and R4 are as follows:

[0014] (i) R3 is hydrogen; R4 is -C(O)R 4a Or a 3- to 6-membered heterocyclic alkyl group, wherein the 3- to 6-membered heterocyclic alkyl group is unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the group consisting of: C 1-3 Alkyl (preferably methyl), hydroxyl, carboxyl, cyano, halogen (preferably fluorine), C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0 -SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6Cycloalkyloxy groups, 3- to 6-membered heterocyclic alkyl groups;

[0015] Where R 4a C 1-6 Alkyl (preferably C) 1-3 Alkyl), C 3-8 cycloalkyl (preferably C) 3-6 Cycloalkyl (more preferably cyclopropyl), 3- to 6-membered heterocyclic alkyl; the C 1-6 Alkyl (preferably C) 1-3 The alkyl group is substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: deuterium, hydroxyl, carboxyl, cyano, halogen (preferably fluorine), C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0 -SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyloxy or 3- to 6-membered heterocyclic alkyl; the C 3-8 cycloalkyl (preferably C) 3-6 The cycloalkyl group (more preferably cyclopropyl), and the 3- to 6-membered heterocycloalkyl group (preferably propylene oxide) are unsubstituted or substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: deuterium, C 1-3 Alkyl (preferably methyl), hydroxyl, carboxyl, cyano, halogen (preferably fluorine), C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0 -SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyloxy or 3 to 6-membered heterocyclic alkyl; or

[0016] (ii) R3 and R4 are connected together to form a 3- to 6-membered heterocyclic alkyl ring, a 5- to 6-membered heterocyclic alkenyl ring, or a 5- to 6-membered heteroaryl ring, together with the connected carbon and nitrogen atoms; wherein the 3- to 6-membered heterocyclic alkyl ring has 1, 2, or 3 nitrogen atoms and 0, 1, or 2 oxygen atoms as ring atoms; wherein the 5- to 6-membered heterocyclic alkenyl ring has 2, 3, or 4 nitrogen atoms and 0, 1, or 2 oxygen atoms as ring atoms; wherein the 5- to 6-membered heterocyclic alkyl ring, the 5- to 6-membered heterocyclic alkenyl ring, and the 5- to 6-membered heteroaryl ring are unsubstituted or substituted by 1, 2, or 3 substituents independently selected from the group consisting of: C 1-3 Alkyl, hydroxyl, carboxyl, cyano, halogen, C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0 -SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyloxy or 3- to 6-membered heterocyclic alkyl;

[0017] Z1, Z2, Z3, and Z4 represent ring atoms, each independently being C or N (preferably Z1 is C, Z2 is C or N, Z3 is C or N, and Z4 is N);

[0018] Z5 is CH2 or O;

[0019] Z6 and Z7 are each independently O, S, or NR. a0 ;

[0020] (R5) n Representing the ring The hydrogen atom is replaced by n R5 atoms, where n is 0, 1, 2, 3, or 4. Each R5 atom may be the same or different, and each is an independent C atom. 1-3 Alkyl, hydroxyl, carboxyl, cyano, halogen (preferably fluorine or chlorine), C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0 -SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyloxy or 3- to 6-membered heterocyclic alkyl; and

[0021] R a0 R b0 Each is independently hydrogen or C 1-3 alkyl.

[0022] In some embodiments, one of R1 and R2 is C 1-3 The alkyl group is one of the alkyl groups and the hydrogen group is the other; furthermore, one of R1 and R2 is methyl and the other is hydrogen; even further, R1 is methyl and R2 is hydrogen.

[0023] In some embodiments, one of R1 and R2 is a halogen and the other is hydrogen; further, one of R1 and R2 is chlorine and the other is hydrogen; even further, R1 is chlorine and R2 is hydrogen.

[0024] In some embodiments, one of R1 and R2 is a halogen and the other is C. 1-3 Alkyl; further, one of R1 and R2 is fluorine and the other is methyl; further, R1 is methyl and R2 is fluorine.

[0025] In some embodiments, for

[0026] In some embodiments, R 4a C 1-3 Alkyl, C 3-6 Cycloalkyl (preferably cyclopropyl), or 3- to 6-membered heterocyclic alkyl, wherein the C 1-3 The alkyl group is substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: deuterium, hydroxyl, halogen (preferably fluorine); the C 3-6 The cycloalkyl group (preferably cyclopropyl) and the 3- to 6-membered heterocycloalkyl group are unsubstituted or substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: deuterium, C 1-3 Alkyl (preferably methyl), hydroxyl, halogen (preferably fluorine).

[0027] In some embodiments, R 4a For deuterated C 1-6 Alkyl; further, R 4a For deuterated C 1-3 alkyl.

[0028] In some embodiments, R 4a For deuterated C1-3 Alkyl groups, further selected from: monodeuterated methyl, monodeuterated ethyl, dideuterated methyl, dideuterated ethyl, trideuterated methyl, and trideuterated ethyl.

[0029] In some embodiments, R 4a Hydroxyl-substituted C 1-6 Alkyl; further, R 4a Hydroxyl-substituted C 1-3 Alkyl; furthermore, R 4a It is a hydroxyl-substituted methyl group.

[0030] In some embodiments, R 4a C 3-6 cycloalkyl; further, R 4a It is cyclopropyl. In some embodiments, R 4a C replaced by halogen 3-6 Cycloalkyl or hydroxylated C 3-6 cycloalkyl; further, R 4a It is a fluorocyclopropyl or hydroxylated cyclopropyl group.

[0031] In some embodiments, R 4a C 1-3 Alkyl, C 3-6 Cycloalkyl (preferably cyclopropyl), or 3- to 6-membered heterocycloalkyl, wherein the 3- to 6-membered heterocycloalkyl contains 1, 2, or 3 oxygen atoms as ring atoms; the C 1-3 The alkyl group is substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: hydroxyl, halogen (preferably fluorine); the C 3-6 The cycloalkyl group and the 3- to 6-membered heterocycloalkyl group are unsubstituted or substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: hydroxyl, C 1-3 Alkyl groups, halogens (preferably fluorine).

[0032] In some embodiments, the 3- to 6-membered heterocyclic alkyl group is selected from: ethylene oxide, oxobutyric acid, tetrahydrofuranyl, tetrahydropyranyl, dioxolanecycloyl, and dioxanecycloyl.

[0033] In some embodiments, the 3- to 6-membered heterocyclic alkyl groups are selected from the following groups:

[0034]

[0035] In some embodiments, R3 and R4 are connected together to form a 3- to 6-membered heterocyclic alkyl ring, a 5- to 6-membered heterocyclic alkenyl ring, or a 5- to 6-membered heteroaryl ring together with the connected carbon and nitrogen atoms; wherein the 3- to 6-membered heterocyclic alkyl ring is The 5- to 6-membered heterocyclic alkenyl ring is The 5- to 6-membered heteroaryl ring is The 3- to 6-membered heterocyclic alkyl ring, 5- to 6-membered heterocyclic alkenyl ring, and 5- to 6-membered heteroaryl ring are unsubstituted or substituted by 1, 2, or 3 substituents independently selected from the group consisting of: C 1-3 Alkyl, hydroxyl, carboxyl, cyano, halogen, C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0 -SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyloxy or 3 to 6-membered heterocyclic alkyl groups.

[0036] In some embodiments, R3 and R4 are connected together to form the following structure together with the connected carbon and nitrogen atoms:

[0037]

[0038] In some embodiments, the compound represented by formula (I) has the structure represented by formula (II):

[0039]

[0040] The groups in the formula are as defined above.

[0041] In some embodiments, in formula (II), R1 is methyl and R2 is hydrogen.

[0042] In some embodiments, in formula (II), R3 and R4 are connected, and the 3- to 6-membered heterocyclic alkyl ring formed together with the connected carbon and nitrogen atoms is... 5- to 6-membered heterocyclic alkenyl rings are 5- to 6-membered heteroaryl rings Wherein the 3- to 6-membered heterocyclic alkyl ring, the 5- to 6-membered heterocyclic alkenyl ring, and the 5- to 6-membered heteroaryl ring are unsubstituted or substituted by 1, 2, or 3 independent substituents selected from the following group: C 1-3 Alkyl, hydroxyl, carboxyl, cyano, halogen, C 1-3 Alkoxy, halogenated C 1-3 Alkyl, Halogenated C 1-3 Alkoxy, -NR a0 R b0-SO2C 1-3 Alkyl, -C(O)NR a0 R b0 -C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyloxy or 3 to 6-membered heterocyclic alkyl groups.

[0043] In some embodiments, R3 and R4 are connected together to form the following structure together with the connected carbon and nitrogen atoms:

[0044]

[0045] In some embodiments, the compound represented by formula (I) has the structure represented by formula (III):

[0046]

[0047] The groups in the formula are as defined above.

[0048] In some embodiments, in the structure shown in equation (Ⅲ), R1 is C 1-6 Alkyl (preferably C) 1-3 R2 is an alkyl group, more preferably a methyl group; R2 is hydrogen or a halogen (preferably fluorine or chlorine).

[0049] In some embodiments, in the structure shown in formula (Ⅲ), R1 is methyl; R2 is hydrogen or fluorine.

[0050] In some embodiments, Z5 is CH2 in the structure shown in formula (Ⅲ).

[0051] In some embodiments, n is 0 in the structure shown in equation (Ⅲ).

[0052] In some embodiments, in the structure shown in formula (Ⅲ), R5 is a hydroxyl group, a carboxyl group, -COOCH3, or -CONH2.

[0053] In some embodiments, in the structure shown in equation (Ⅲ), R 4a For deuterated C 1-6 Alkyl groups, or deuterated C groups 1-3 Alkyl, or monodeuterated methyl, monodeuterated ethyl, dideuterated methyl, dideuterated ethyl, trideuterated methyl, trideuterated ethyl, or trideuterated methyl (CD3).

[0054] In some embodiments, in the structure shown in equation (Ⅲ), R 4a It is a trideuterated methyl group (CD3).

[0055] In some embodiments, in the structure shown in equation (Ⅲ), R 4a C 3-8 cycloalkyl (preferably C) 3-6 Cycloalkyl (more preferably cyclopropyl), or C substituted with 1, 2 or 3 hydroxyl groups 1-6 Alkyl groups (preferably C substituted with one hydroxyl group) 1-3 alkyl).

[0056] In some embodiments, in the structure shown in equation (Ⅲ), R 4a It consists of cyclopropyl, hydroxyl-substituted methyl, and hydroxyl-substituted isopropyl.

[0057] In some embodiments, the compound represented by formula (I) has the structure represented by formula (IV):

[0058]

[0059] The groups in the formula are as defined above.

[0060] In some embodiments, in the structure shown in formula (Ⅳ), R2 is H or fluorine.

[0061] In some embodiments, in the structure shown in formula (Ⅳ), Z6 is NH and Z7 is S; or Z6 is S and Z7 is NH.

[0062] In some embodiments, in the structure shown in formula (Ⅳ), Z6 is CH2 and Z7 is S; or Z6 is S and Z7 is CH2.

[0063] In some embodiments, in the structure shown in formula (Ⅳ), Z6 is CH2 and Z7 is S.

[0064] In some embodiments, in the structure shown in formula (Ⅳ), Z6 is S and Z7 is CH2.

[0065] In some embodiments, in the structure shown in equation (Ⅳ), R 4a C 1-3 Alkyl, C 3-6 Cycloalkyl (preferably cyclopropyl), or 3- to 6-membered heterocyclic alkyl, wherein the C 1-3 The alkyl group is substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: deuterium, hydroxyl, halogen (preferably fluorine); the C 3-6 The cycloalkyl group (preferably cyclopropyl) and the 3- to 6-membered heterocycloalkyl group are unsubstituted or substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of: deuterium, C 1-3 Alkyl (preferably methyl), hydroxyl, halogen (preferably fluorine).

[0066] In some embodiments, in the structure shown in equation (Ⅳ), R 4a Selected from: C14 cells substituted with 1, 2, or 3 hydroxyl groups 3-8 Cycloalkyl groups (preferably C substituted with one hydroxyl group) 3-6 Cycloalkyl groups, more preferably cyclopropyl groups substituted with one hydroxyl group, and C groups substituted with one, two, or three halogens. 3-8 Cycloalkyl groups (preferably C14 substituted with one fluorine atom) 3-6 Cycloalkyl groups, more preferably cyclopropyl groups substituted with one fluorine atom, and C groups substituted with one, two, or three hydroxyl groups. 1-6 Alkyl groups (preferably C substituted with one hydroxyl group) 1-3 alkyl).

[0067] In some embodiments, in the structure shown in equation (Ⅳ), R 4a Selected from: hydroxy-substituted cyclopropyl, fluorocyclopropyl, hydroxy-substituted methyl, hydroxy-substituted ethyl.

[0068] In some embodiments, the compound of formula (I) is any one of the following compounds:

[0069]

[0070]

[0071] A second aspect of the present invention provides a pharmaceutical composition comprising a compound of formula (I) above, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0072] A third aspect of the present invention provides the use of the compound of formula (I) above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for treating diseases associated with P2X3 activity or P2X2 / 3 activity.

[0073] The fourth aspect of the present invention provides the use of the pharmaceutical composition described in the second aspect in the preparation of a medicament for treating diseases associated with P2X3 activity or P2X2 / 3 activity.

[0074] In some embodiments, the diseases associated with P2X3 activity or P2X2 / 3 activity are pain, urinary tract disorders, gastrointestinal disorders, cancer, immune-related diseases, cough, depression, anxiety, or stress-related conditions.

[0075] The fifth aspect of the present invention provides a method for treating diseases associated with P2X3 activity or P2X2 / 3 activity, comprising administering to a patient a therapeutically effective amount of the compound of the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a pharmaceutical composition of the second aspect of the present invention. Detailed Implementation

[0076] Through extensive and in-depth research, the inventors unexpectedly discovered that these morpholine derivatives not only possess significant P2X3 inhibitory activity and low P2X2 / 3 inhibitory activity, but also exhibit excellent in vivo pharmacokinetic parameters, particularly low clearance and high absorption. Therefore, this series of compounds holds promise for development into drugs for modulating P2X3 and / or P2X2 / 3 to treat various diseases mediated by (or otherwise associated with) P2X3 and / or P2X2 / 3. Based on this, the inventors completed this invention.

[0077] Terminology Definition

[0078] To better understand the technical content of this invention, the terminology of this invention will be further explained below.

[0079] "alkyl" refers to straight-chain and branched saturated aliphatic hydrocarbon groups. "C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms, preferably C14. 1-3 Alkyl; non-limiting examples of alkyl groups 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, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl. 2,3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers are preferred.

[0080] "Cycloalkyl" and "cycloalkyl ring" are used interchangeably, both referring to a saturated monocyclic, bicyclic, or polycyclic cyclic hydrocarbon group that may be fused with an aryl or heteroaryl group. The cycloalkyl ring may optionally be substituted. In some embodiments, the cycloalkyl ring contains one or more carbonyl groups, such as oxo groups. "C" 3-8"Cycloalkyl" refers to a monocyclic cycloalkyl group having 3 to 8 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclobutanone, cyclopentanone, cyclopentane-1,3-dione, etc. Preferably C 3-6 Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0081] "Heterocyclic alkyl" and "heterocyclic alkyl ring" are used interchangeably, both referring to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur, which may be fused with an aryl or heteroaryl group. The heterocyclic alkyl ring may optionally be substituted. In some embodiments, the heterocyclic alkyl ring contains one or more carbonyl or thiocarbonyl groups, such as groups containing oxo and thio groups. "3- to 6-membered heterocyclic alkyl" refers to a monocyclic cyclic hydrocarbon group having 3 to 6 ring atoms, wherein 1, 2, or 3 ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, preferably 1 or 2 ring atoms in a 3- to 6-membered heterocyclic alkyl group are heteroatoms selected from nitrogen, oxygen, and sulfur. Non-limiting examples of monocyclic heterocyclic alkyl groups include azircycloyl, ethylene oxide, azircyclobutyl, oxacyclobutyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyrrolyl, oxazolidinyl, dioxopentyl, piperidinyl, piperazinyl, morpholinyl, dioxohexyl, thiomorpholinyl, thiomorpholin-1,1-dioxide, tetrahydropyranyl, azircyclobutane-2-one, oxacyclobutane-2-one, dihydrofuran-2(3H)-one, pyrrolidine-2-one, pyrrolidine-2,5-diketone, dihydrofuran-2,5-diketone, piperidin-2-one, tetrahydro-2H-pyran-2-one, piperazin-2-one, morpholin-3-one, etc.

[0082] The terms “heterocyclic alkenyl” and “heterocyclic alkenyl ring” are used interchangeably and refer to a heterocyclic alkyl group containing one or more carbon-carbon or carbon-nitrogen double bonds within the ring, but are not intended to include a heteroaryl moiety as defined herein. The group may be fused with an aryl or heteroaryl group. The heterocyclic alkenyl ring may optionally be substituted. In some embodiments, the heterocyclic alkenyl ring contains one or more carbonyl or thiocarbonyl groups, such as groups containing oxo and thio groups. “5- to 6-membered heterocyclic alkenyl ring” refers to a heterocyclic alkenyl ring having 5 to 6 ring atoms, wherein 1, 2, or 3 ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur. Non-limiting examples of heterocyclic alkenyl rings include 4,5-dihydro-1H-imidazolium rings, 1,4,5,6-tetrahydropyrimidine rings, 3,4,7,8-tetrahydro-2H-1,4,6-oxadiazin rings, 1,6-dihydropyrimidine rings, 4,5,6,7-tetrahydro-1H-1,3-diazazoline rings, and 2,5,6,7-tetrahydro-1,3,5-oxadiazidine rings.

[0083] The terms "heteroaryl" and "heteroaryl ring" are used interchangeably, both referring to a group having a monocyclic, bicyclic, or polycyclic 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) with a ring carbon atom and a ring heteroatom, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In this invention, the heteroaryl also includes a ring system in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl rings, heterocycloalkyl rings, cycloalkenyl rings, heterocycloalkenyl rings, or aromatic rings. The heteroaryl ring may optionally be substituted. "5- to 6-membered heteroaryl" refers to a monocyclic heteroaryl having 5 to 6 ring atoms, wherein 1, 2, 3, or 4 ring atoms are heteroatoms. Non-limiting embodiments include thienyl, furanyl, thiazolyl, isothiazolyl, imidazole, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, thiadiazolyl, pyridinyl, pyridinyl, pyrazinyl, triazinyl, and tetraazinyl. "8 to 10-membered heteroaryl" refers to a bicyclic heteroaryl group having 8 to 10 ring atoms, of which 1, 2, 3, or 4 ring atoms are heteroatoms. Non-limiting examples include indole, isoindole, indazole, benzotriazolyl, benzothiophene, isobenzothiophene, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzooxazolyl, benzoisooxazolyl, benzooxadiazolyl, benzothiazolyl, and benzoisothiazolyl. Azolyl, benzothiadiazolyl, indazinyl, purine, pyrido[3,2-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, 1,8-naphthidyl, 1,7-naphthidyl, 1,6-naphthidyl, 1,5-naphthidyl, pteridyl, quinolinyl, isoquinolinyl, zolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. "Heteroatom" refers to nitrogen, oxygen, or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings.

[0084] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0085] "Halogenated" means that one or more (such as 1, 2, 3, 4 or 5) hydrogen atoms in a group are replaced by halogens.

[0086] "Halogenated alkyl" refers to an alkyl group that is substituted with one or more (e.g., 1, 2, 3, 4, or 5) halogens, wherein the definition of alkyl is as described above. Preferably, it is a halogenated C. 1-8 Alkyl groups, preferably halogenated C 1-6 Alkyl, more preferably halogenated C 1-3Alkyl groups. Examples of haloalkyl groups include (but are not limited to) monochloromethyl, dichloromethyl, trichloromethyl, monochloroethyl, 1,2-dichloroethyl, trichloroethyl, monobromoethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, etc.

[0087] "Deuterated alkyl" refers to an alkyl group that is substituted with one or more (e.g., 1, 2, 3, 4, or 5) deuterium atoms, as defined above. Preferably, it is a deuterated C-type alkyl group. 1-8 Alkyl groups, more preferably deuterated C4 groups 1-6 Alkyl groups, more preferably deuterated C4 groups 1-3 Alkyl groups. Examples of deuterated alkyl groups include (but are not limited to) monodeuterated methyl, monodeuterated ethyl, dideuterated methyl, dideuterated ethyl, trideuterated methyl, trideuterated ethyl, etc.

[0088] "Alkoxy" refers to -O-alkyl, where alkyl is defined as described above. Preferably C 1-8 Alkoxy, more preferably C 1-6 Alkoxy group, C is the most preferred. 1-3 Alkoxy groups. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, isobutoxy, pentoxy, etc.

[0089] "Cycloalkyloxy" refers to -O-cycloalkyl, where cycloalkyl is defined as described above. Preferably C 3-8 Cycloalkyloxy, more preferably C 3-6 Cycloalkyloxy groups. Non-limiting examples of cycloalkyloxy groups include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc.

[0090] "Haloalkoxy" refers to an alkoxy group that is substituted with one or more (e.g., 1, 2, 3, 4, or 5) halogens, as defined above. Preferably, it is a halogenated C. 1-8 Alkoxy, more preferably halogenated C 1-6 Alkoxy, more preferably halogenated C 1-3 Alkyl groups. Halogenated alkoxy groups include (but are not limited to) trifluoromethoxy, trifluoroethoxy, monofluoromethoxy, monofluoroethoxy, difluoromethoxy, difluoroethoxy, etc.

[0091] "Amino" refers to NH2, "cyano" refers to CN, "nitro" refers to NO2, "benzyl" refers to -CH2-phenyl, "oxo" refers to =O, "carboxyl" refers to -C(O)OH, "acetyl" refers to -C(O)CH3, "hydroxymethyl" refers to -CH2OH, "hydroxyethyl" refers to -CH2CH2OH or -CHOHCH3, "hydroxy" refers to -OH, and "thiol" refers to SH.

[0092] "Substituted" refers to one or more hydrogen atoms in a group, preferably 1 to 5 hydrogen atoms independently substituted by a corresponding number of substituents, more preferably 1 to 3 hydrogen atoms independently substituted by a 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 (through 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).

[0093] In this invention, when the number of substituents is not specified, it is stated that any number of substituents can be substituted.

[0094] Unless otherwise defined, the phrase "substituents selected independently of each other" in this invention means that when one or more hydrogen atoms on a group are replaced by substituents, the types of substituents may be the same or different, and the selected substituents are of independent types.

[0095] Unless otherwise defined, any group herein may be substituted or unsubstituted. When the above groups are substituted, the substituents are preferably 1 to 5 groups independently selected from: cyano, halogen (preferably fluorine or chlorine), C 1-8 Alkyl (preferably C) 1-6 Alkyl, more preferably C 1-3 Alkyl), C 1-8 Alkoxy (preferably C) 1-6 Alkoxy, more preferably C 1-3 alkoxy), halogenated C 1-8 Alkyl (preferably halogenated C) 1-6 Alkyl, more preferably halogenated C 1-3 Alkyl), C 3-8 cycloalkyl (preferably C) 3-6 cycloalkyl), halogenated C 1-8 Alkoxy (preferably halogenated C) 1-6 Alkoxy, more preferably halogenated C 1-3 alkoxy), C 1-8 Alkyl-substituted amino groups, halogenated C 1-8 Alkyl-substituted amino, acetyl, hydroxy, hydroxymethyl, hydroxyethyl, carboxyl, nitro, C 6-10 Aryl (preferably phenyl), C 3-8 Cycloalkyloxy (preferably C) 3-6 cycloalkyloxy), C 2-8 alkenyl (preferably C) 2-6 alkenyl, more preferably C 2-4 alkenyl), C 2-8 alkynyl group (preferably C) 2-6 Alkyne group, more preferably C 2-4 alkynyl group), -CONR a0 R b0-C(O)OC 1-10 Alkyl (preferably -C(O)OC) 1-6 Alkyl groups, more preferably -C(O)OC 1-3 Alkyl groups, -CHO, -OC(O)C 1-10 Alkyl groups (preferably -OC(O)C) 1-6 Alkyl groups, more preferably -OC(O)C 1-3 alkyl), -SO2C 1-10 Alkyl groups (preferably -SO2C) 1-6 Alkyl groups, more preferably -SO2C 1-3 alkyl), -SO2C 6-10 Aryl (preferably -SO2C6 aryl, such as -SO2-phenyl), -COC 6-10 aryl (preferably -COC6 aryl, such as -CO-phenyl), 4 to 6 membered saturated or unsaturated mono-heterocyclic rings, 4 to 6 membered saturated or unsaturated mono-cyclic rings, 5 to 6 membered monocyclic heteroaryl rings, 8 to 10 membered bicyclic heteroaryl rings, spirocyclic rings, spiroheterocyclic rings, bridged rings or bridged heterocyclic rings, wherein R a0 R b0 Each is independently hydrogen or C 1-3 alkyl.

[0096] The various substituents described above can themselves be replaced by the groups described in this article.

[0097] Pharmaceutical Composition

[0098] Typically, the compounds of this invention, or their pharmaceutically acceptable salts, solvates, stereoisomers, or prodrugs, can be formulated with one or more pharmaceutical carriers into suitable dosage forms for administration. These dosage forms are suitable for oral, rectal, topical, intraoral, and other non-gastrointestinal administration (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, suitable dosage forms for oral administration include capsules, tablets, granules, and syrups. The compounds of this invention contained in these formulations can be: solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, etc. The above dosage forms can be prepared from the active compound and one or more carriers or excipients using common pharmaceutical methods. The carriers mentioned above need to be compatible with the active compound or other excipients. For solid dosage forms, commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, and sucrose. Carriers used for liquid dosage forms include water, physiological saline, glucose aqueous solution, ethylene glycol, and polyethylene glycol, etc. The active compound can form solutions or suspensions with the above carriers.

[0099] "Pharmaceutically acceptable carrier" refers to a non-toxic, inert, solid, or semi-solid substance or liquid filling machine, diluent, encapsulation material, or excipient or any type of excipient that is compatible with patients, preferably mammalian, more preferably human, and suitable for delivering an active agent to a target site without terminating the agent's activity.

[0100] The compositions of the present invention are formulated, quantified, and administered in a manner consistent with medical practice. The "therapeutic effective amount" of the compound administered is determined by factors such as the specific condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the route of administration.

[0101] "Therapeutic effective amount" refers to the amount of the compound of the present invention that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease.

[0102] The therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt, its solvate, its stereoisomer, or its prodrug contained in the pharmaceutical composition or the pharmaceutical composition of the present invention is preferably 0.1 mg to 5 g / kg (body weight).

[0103] "Patient" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, including animals such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, and humans.

[0104] "Treatment" refers to reducing, slowing the progression of, attenuating, preventing, or maintaining an existing disease or condition (such as cancer). Treatment also includes curing, preventing the development of, or reducing to some extent one or more symptoms of a disease or condition.

[0105] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0106] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, propionates, glycolates, gluconates, lactates, oxalates, maleates, succinates, fumarates, tartrates, citrates, glutamates, aspartates, benzoates, methanesulfonates, p-toluenesulfonates, and salicylates. These salts can be prepared using methods known in this field.

[0107] "Pharmaceutical-acceptable base addition salts" include, but are not limited to, salts of inorganic bases such as sodium, potassium, calcium, and magnesium salts, and, but are not limited to, salts of organic bases such as ammonium, triethylamine, lysine, and arginine salts. These salts can be prepared by methods known in this field.

[0108] The compounds of this invention may contain one or more chiral centers and exist in different optically active forms. When a compound contains one chiral center, the compound comprises enantiomers. This invention includes both isomers and mixtures of isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When a compound contains more than one chiral center, diastereomers may be present. This invention includes resolved optically pure specific isomers and mixtures of diastereomers. Diastereomers can be resolved by methods known in the art, such as crystallization and preparative chromatography.

[0109] Preparation method

[0110] This invention provides a method for preparing compounds of formula (I), which can be synthesized using standard synthetic techniques known to those skilled in the art or by combining methods known in the art with the methods described herein. The solvents, temperatures, and other reaction conditions given in this invention can be varied according to the art. The reactions can be used sequentially to provide the compounds of this invention, or they can be used to synthesize fragments subsequently added by the methods described herein and / or methods known in the art.

[0111] The compounds described in this invention can be synthesized using appropriate, selectable starting materials, similar to those described in the examples below or relevant publications used by those skilled in the art. The starting materials used to synthesize the compounds described in this invention can be synthetic or commercially available. The compounds described in this invention and other related compounds with different substituents can be synthesized using techniques and starting materials known to those skilled in the art. General methods for preparing the compounds disclosed in this invention can be derived from reactions known in the art, and these reactions can be modified by reagents and conditions deemed appropriate by those skilled in the art to introduce various parts of the molecules provided in this invention.

[0112] Compared with the prior art, the main advantage of the present invention is that it provides a series of novel morpholine derivatives with high inhibitory activity against P2X3 and low inhibitory activity against P2X2 / 3, which have the potential to treat diseases related to P2X3 activity or P2X2 / 3 activity.

[0113] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise defined, the terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the present invention.

[0114] Reagents and Instruments

[0115] 1 HNMR: Bruker AVANCE-400 NMR spectrometer, internal standard: tetramethylsilane (TMS).

[0116] LC-MS: Agilent 1290HPLC System / 6130 / 6150MS liquid chromatography-mass spectrometry (manufacturer: Agilent), Waters BEH / CHS column, 50×2.1mm, 1.7μm.

[0117] Preparative high performance liquid chromatography (pre-HPLC): GX-281 (manufacturer: Gilson).

[0118] An ISCO Combiflash-Rf75 or Rf200 automatic column chromatography system was used, with Agela disposable silicone columns in sizes of 4g, 12g, 20g, 40g, 80g, and 120g.

[0119] The known starting materials can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCRGmbH & Co.KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc., and Darui Chemicals.

[0120] In the examples, the reaction process can be monitored using thin-layer chromatography (TLC), and the compound can be purified using column chromatography. The developing solvent system used for column chromatography or TLC can be selected from: dichloromethane and methanol system, n-hexane and ethyl acetate system, petroleum ether and ethyl acetate system, and acetone system, etc., and the volume ratio of the solvent is adjusted according to the polarity of the compound.

[0121] As used in this article, DCM is dichloromethane, DCE is 1,2-dichloroethane, DMF is N,N-dimethylformamide, DMSO is dimethyl sulfoxide, THF is tetrahydrofuran, EA is ethyl acetate, PE is petroleum ether, n-BuLi is n-butyllithium, HATU is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, TEA is triethylamine, DIEA or DIPEA is N,N-diisopropylethylamine, NBS is N-bromosuccinimide, NCS is N-chlorosuccinimide, and TBAF is tetrabutylammonium fluoride.

[0122] As used in this article, room temperature refers to approximately 20-30°C.

[0123] Preparation of intermediate V1

[0124]

[0125] Step 1: Dissolve NaOH (14.42 g, 360.56 mmol) in water (300 mL), then lower the temperature to 0 °C. Slowly add Br2 (19.45 g, 121.69 mmol) dropwise to the reaction system and maintain the temperature at 0 °C for half an hour. Then add ((benzyloxy)carbonyl)-L-asparagine (30 g, 112.68 mmol) in portions to the reaction system and raise the temperature to 55 °C for 3 hours. Cool the reaction solution to room temperature, wash twice with EA, and then adjust the pH of the aqueous phase to 1 with 6 M HCl. A solid precipitates out; filter and evaporate the filter cake to obtain V1-1 (24.6 g). MS m / z (ESI): 265.0 [M+1] + .

[0126] Step 2: Dissolve V1-1 (6.3 g, 23.84 mmol) in MeOH (50 mL). Cool the reaction solution to -20 °C, then add SOCl2 (2.13 g, 17.88 mmol, 1.30 mL). Stir the reaction solution at -20 °C for 2 hours, then at 25 °C for 16 hours. After the reaction is complete, evaporate the solvent, add water and EtOAC, separate, extract twice with EtOAc, and dry with Na2SO4. EtOAC evaporate to obtain V1-2 (6 g). MS m / z (ESI): 279.0 [M+1] + .

[0127] Step 3: Dissolve V1-2 (4 g, 14.38 mmol) in DMF (40 mL), then add Cs₂CO₃ (7.03 g, 21.56 mmol) and methyl iodide (4.08 g, 28.75 mmol). Stir the reaction at room temperature for 16 hours. After the reaction is complete, add water, extract with ethyl acetate, and dry to anhydrous sodium sulfate. Filter and evaporate the solvent to obtain V1-3 (3.4 g). MS m / z (ESI): 293.0 [M+1] + .

[0128] Step 4: Dissolve V1-3 (3.4 g, 11.63 mmol) in methanol (40 mL), then add 10% Pd / C (1.23 g, 1.16 mmol, 10% purity). The reaction was stirred at room temperature in hydrogen atmosphere for 2 hours, and the reaction was monitored by LC-MS. After the reaction was complete, the mixture was filtered. V1-4 (1.5 g) was obtained after filtration. MS m / z (ESI): 159.1 [M+1] + .

[0129] Step 5: Dissolve V1-4 in DMF (20 mL), then add 3-bromoprop-1-ene (2.29 g, 18.97 mmol) and Cs₂CO₃ (4.64 g, 14.23 mmol). The reaction was stirred at room temperature for 16 hours, and the reaction was monitored by LC-MS. After the reaction was complete, water was added to obtain a solid, which was extracted with EtOAc and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain V1-5 (1.3 g). MS m / z (ESI): 199.1 [M+1] + .

[0130] Step 6: Dissolve V1-5 (1.3 g, 6.56 mmol) in ethanol (15 mL), then add NaBH4 (248.10 mg, 6.56 mmol). The reaction was stirred at room temperature for 2 hours, and the reaction was monitored by LC-MS. After the reaction was complete, acetic acid was added to quench the reaction, sodium carbonate was added for neutralization, the mixture was filtered, and the filtrate was evaporated to dryness to obtain V1-6 (1.1 g). MS m / z (ESI): 171.1 [M+1] + .

[0131] Step 7: Dissolve V1-6 (1.1 g, 6.46 mmol) in DCM (15 mL), then add p-toluenesulfonyl chloride (2.46 g, 12.93 mmol) and TEA (1.96 g, 19.39 mmol, 2.70 mL). The reaction was stirred at room temperature for 16 hours, and the reaction was monitored by LC-MS. After the reaction was complete, the solution was evaporated under reduced pressure and separated by column chromatography (20 g, 0–80% EA / PE) to obtain V1-7 (410 mg). MS m / z (ESI): 325.1 [M+1] + .

[0132] Step 8: AD-mix-beta (CAS NO: 148618-32-0) (9.85 g, 12.64 mmol) was added to a mixed solvent of water (150 mL) and tert-butanol (150 mL), and stirred at room temperature to obtain a clear yellow solution. After cooling to 0 °C, V1-7 (410 mg, 1.26 mmol) was added. The reaction solution was stirred at 0 °C for 24 hours, and the reaction was monitored by LC-MS until it was complete. After adding 35 g of sodium sulfite, the mixture was heated to room temperature and stirred for 30 minutes. Saturated sodium chloride solution (150 mL) was added, and the mixture was extracted with DCM (250 mL x 5). The organic phase was dried and concentrated under reduced pressure. V1-8 (170 mg) was purified by silica gel column chromatography with an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1). MS m / z (ESI): 359.0 [M+1] + .

[0133] Step 9: V1-8 (170 mg, 474.32 μmol) was dissolved in DMF (5 mL), followed by the addition of imidazole (129.16 mg, 1.90 mmol) and TBSCl (142.98 mg, 948.64 μmol). The reaction mixture was stirred at room temperature for 4 hours, and the reaction was monitored by LC-MS until completion. Saturated brine and ethyl acetate were added. The ethyl acetate was washed twice with brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness. V1-9 (190 mg) was purified by silica gel column chromatography using an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1). MS m / z (ESI): 473.2 [M+1] + .

[0134] Step 10: Dissolve V1-9 (190 mg, 401.97 μmol) in THF, cool to 0°C, and then add NaH (32.16 mg, 803.94 μmol, 60% purity). Stir the reaction mixture at 0°C for 4 hours, and monitor the reaction by LC-MS until complete. Quench with saturated ammonium chloride solution and extract with DCM. Dry the organic phase and concentrate under reduced pressure to obtain V1-10 (105 mg). MS m / z (ESI): 301.1 [M+1] + .

[0135] Step 11: Dissolve V1-10 (160 mg, 532.50 μmol) in THF (5 mL). Then add TBAF (1 M, 639.00 μL). Stir the reaction mixture at room temperature for 2 hours. Monitor the reaction by LC-MS. Once the reaction is complete, evaporate the solvent to dryness. Purify the residue by silica gel column chromatography using an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1) to obtain V1-11 (95 mg). MS m / z (ESI): 187.1 [M+1] + .

[0136] Step 12: V1-11 (95 mg, 510.18 μmol) was added to DCM (2 mL). After cooling to 0 °C, Dysmart oxidant (259.67 mg, 612.22 μmol) was added. The reaction mixture was stirred at room temperature for 2 hours, and the reaction was monitored by LC-MS until completion. Sodium sulfite was added, and the mixture was heated to room temperature and stirred for 5 minutes. Saturated sodium chloride solution was added, and the mixture was extracted with DCM. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1) to obtain V1 (90 mg). MS m / z (ESI): 203.1 [M+1+18] + .

[0137] Preparation of intermediate V2

[0138]

[0139] Step 1: A solution of triphosgene (19.07 g, 64.28 mmol) in THF (100 mL) was added to a solution of (2S)-2-amino-3-hydroxypropionate methyl ester (10 g, 64.28 mmol, HCl) in THF (200 mL). The mixture was heated to 80 °C and stirred for 4 hours. The solvent was evaporated under reduced pressure, and the solution was purified by column chromatography (using petroleum ether containing 80-90% ethyl acetate as the mobile phase) to obtain V2-1 (8 g). MS m / z (ESI): 146.1 [M+1] + .

[0140] Step 2: V2-1 (6 g, 41.35 mmol) and cesium carbonate (26.96 g, 82.69 mmol) were dissolved in DMF (25 mL), and then allyl bromo (10.00 g, 82.69 mmol) was added. The reaction was stirred at room temperature for 12 hours, and LC-MS showed that the reaction was complete. The mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The solution was purified by column chromatography (using petroleum ether containing 90% tetrahydrofuran as the mobile phase) to give V2-2 (5 g). MS m / z (ESI): 186.0 [M+1] + .

[0141] Step 3: Dissolve V2-2 (4 g, 21.60 mmol) in EtOH (5 mL), then add NaBH4 (4.09 g, 108.00 mmol). Stir the reaction at room temperature for 1 hour. Monitor the reaction completion by LC-MS. Quench the reaction with acetic acid, filter, evaporate the filtrate to dryness, dissolve in DCM, add potassium carbonate solid, filter, and evaporate the filtrate directly to dryness to obtain V2-3 (2.5 g). MS m / z (ESI): 158.1 [M+1] + .

[0142] Step 4: Dissolve V2-3 (2 g, 12.73 mmol) and p-toluenesulfonyl chloride (2.91 g, 15.27 mmol) in DCM (30 mL), then add TEA (2.57 g, 25.45 mmol, 3.54 mL). Stir the reaction mixture at room temperature for 6 hours. Monitor the reaction progress by LC-MS. Add saturated sodium bicarbonate to the reaction solution, extract with DCM, wash with saturated brine, dry under anhydrous sodium sulfate, evaporate to dryness under reduced pressure, and purify by column chromatography (using petroleum ether containing 50% tetrahydrofuran as the mobile phase) to obtain V2-4 (2.6 g), MS m / z (ESI): 312.0 [M+1]. + .

[0143] Step 5: Dissolve AD-MIX-BETA (55.29 g, 70.98 mmol) in water (100 mL) and cool to 0°C. Add V2-4 (1.7 g, 5.46 mmol) solution to the solution. Stir the reaction at 0°C for 24 hours. Monitor the reaction completion by LC-MS. Add 55 g of sodium sulfite and stir for half an hour. Extract with 500 mL of DCM, and extract the aqueous phase with 300 mL of DCM. Combine the organic phases, dry to anhydrous sodium sulfate, evaporate to dryness under reduced pressure, and purify by column chromatography (using dichloromethane with 20% methanol as the mobile phase) to obtain V2-5 (1.5 g, 4.34 mmol, 79.55% yield), MS m / z (ESI): 346.0 [M+1]. +

[0144] Step 6: Dissolve V2-5 (1.4 g, 4.05 mmol) and imidazole (1.10 g, 16.21 mmol) in DMF (15 mL), cool to 0 °C, and add TBSCl (1.22 g, 8.11 mmol). Stir the reaction at room temperature for 2 hours. Monitor the reaction completion by LC-MS. Quench the reaction with water and extract with ethyl acetate. Dry the organic phase, concentrate under reduced pressure, and purify by column chromatography (petroleum ether containing 80–100% ethyl acetate) to obtain product V2-6 (1.4 g). MS m / z (ESI): 460.1 [M+1] + .

[0145] Step 7: Dissolve V2-6 in THF (5 mL), cool to 0 °C, and add NaH (365.48 mg, 9.14 mmol, 60% purity). Stir the reaction at room temperature for 2 hours. Monitor the reaction completion by LC-MS. After the reaction is complete, quench the reaction with saturated brine, extract with ethyl acetate, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure. Purify by column chromatography (using dichloromethane with 20% methanol as the mobile phase) to obtain V2-7 (0.6 g), MS m / z (ESI): 288.1 [M+1] + .

[0146] Step 8: Dissolve V2-7 (0.6 g, 2.09 mmol) in THF (6 mL), then add TBAF (654.96 mg, 2.50 mmol). Stir the reaction at room temperature for 2 hours. Monitor the reaction completion by LC-MS. Quench the reaction with water, and evaporate the solvent under reduced pressure. Purify by column chromatography (using petroleum ether containing 90% tetrahydrofuran as the mobile phase) to obtain V2-8 (320 mg), MS m / z (ESI): 174.1 [M+1]. + .

[0147] Step 9: Dissolve V2-8 (300 mg, 1.73 mmol) in acetonitrile (10 mL), then add IBX (727.68 mg, 2.60 mmol). Stir the reaction at 100 °C for 2 hours. Monitor the reaction completion by LC-MS. Cool to room temperature, filter, and wash the filter cake with DCM. Dry the filtrate under reduced pressure to obtain V2 (230 mg). MS m / z (ESI): 172.0 [M+1] + .

[0148] Preparation of intermediate V3

[0149]

[0150] Step 1: Dissolve the raw material (R)-3-aminopropane-1,2-diol (9.1 g, 0.1 mol) in DCM (500 ml), cool to -20 °C, add TEA (12.1 g, 0.12 mol), and then slowly add a DCM solution of chloroacetyl chloride (12.43 g, 0.12 mol). Then, allow the mixture to naturally warm to room temperature, stir overnight, filter, wash the filtrate with water, dry, concentrate, wash the remaining yellow solid with methyl tert-butyl ether, and dry the remaining solid under vacuum to obtain V3-1 (16 g). MS m / z (ESI): 168.0 [M+1] + .

[0151] Step 2: A solution of potassium tert-butoxide (27 g, 0.241 mol, 2.5 eq) in tert-amyl alcohol (200 ml) was slowly added dropwise to a solution of V3-1 (16 g, 0.0952 mol) in tert-amyl alcohol (300 ml). The mixture was stirred for 1 hour at room temperature. The pH of the reaction solution was adjusted to 3-4 with acetic acid, resulting in the precipitation of a large amount of yellow solid. The solid was filtered, washed with water until neutral, and dried under vacuum to obtain V3-2 (7.7 g). MS m / z (ESI): 132.1 [M+1] + .

[0152] Step 3: V3-2 (7.7 g, 58.8 mmol) was suspended in DCM (300 ml), TEA (7.12 g, 70.6 mmol) was added, and then benzoyl chloride (9.06 g, 64.7 mmol) was slowly added dropwise. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction mixture was washed with water, dried, concentrated, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 3 / 1) to obtain V3-3 (9.12 g) MSm / z (ESI): 236.1 [M+1] + .

[0153] Step 4: Dissolve V3-3 (2.35 g, 10 mmol) in DCM (80 ml), add trimethyloxonium tetrafluoroborate (1.78 g, 12 mmol), stir at room temperature for 6 hours, then add methyl hydrazine formate (1.08 g, 12 mmol), stir overnight at room temperature, and concentrate the reaction solution to obtain V3-4 (5 g). MS m / z (ESI): 308.1 [M+1] + .

[0154] Step 5: Dissolve V3-4 (2g) in DMF (20ml), heat to 170℃ under microwave conditions, react for 1 hour, cool to room temperature, and concentrate the reaction solution to obtain a red oily substance. The product was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20) to obtain V3-5 (0.46g). MS m / z (ESI): 276.1 [M+1] + .

[0155] Step 6: Dissolve V3-5 (0.46 g, 1.67 mmol) in DMF (15 ml), add potassium carbonate (0.69 g, 5 mmol) and methyl iodide (0.71 g, 5 mmol), stir at room temperature for 20 hours, filter the reaction solution, concentrate, and purify the product by silica gel column chromatography (methanol / dichloromethane = 1 / 20) to obtain V3 (0.26 g). MS m / z (ESI): 290.2 [M+1] + .

[0156] Preparation of intermediate V4

[0157]

[0158] Step 1: Following the method in step 4 of intermediate V3, the difference is that acetylhydrazine is used instead of methyl hydrazine to prepare V4-1.

[0159] Step 2: Following the method described in step 5 for intermediate V3, V4 can be prepared. MS m / z (ESI): 274.1 [M+1] + .

[0160] Preparation of intermediate V5

[0161]

[0162] Following the method in step 6 of intermediate V3, except that iodopropane isopropane is used instead of iodomethane, V5 can be prepared. MS m / z (ESI): 318.2 [M+1] + .

[0163] Preparation of intermediate V6

[0164]

[0165] Following the method in step 6 of intermediate V3, except that iodomethane is replaced with 2,2,2-trifluoroethyl methanesulfonate, V6 can be prepared. MS m / z (ESI): 358.1 [M+1] + .

[0166] Preparation of intermediate V7

[0167]

[0168] Following the method in step 6 of intermediate V3, except that iodomethane is replaced with 2,2'-bipyridine, V7 can be prepared. MS m / z (ESI): 316.1 [M+1] + .

[0169] Preparation of intermediate V8

[0170]

[0171] Step 1: N-chloroacetamide (20 g, 145.39 mmol) and triphenylphosphine (38.13 g, 145.39 mmol) were dissolved in acetonitrile (200 mL), and the mixture was reacted at 90 °C for 12 hours. The reaction solution was evaporated to dryness, dissolved in DCM, washed with 2N potassium hydroxide aqueous solution, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to give compound V8-1 (45 g). MS m / z (ESI): 364.1 [M+1] + .

[0172] Step 2: Compound V8-1 (18 g, 83.63 mmol) and N-methoxy-N-methyl-2-(triphenylphosphine)acetamide (30.39 g, 83.63 mmol) were added to DCM (200 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using an eluent system (petroleum ether / ethyl acetate: 1 / 0 to 1 / 1) to obtain compound V8-2 (27 g), MS m / z (ESI): 245.1 [M+1]. + .

[0173] Step 3: Compound V8-2 (30 g, 99.88 mmol) was dissolved in methanol (300 mL), and Pd / C (10%) (30 g, 99.88 mmol) was added. The reaction mixture was stirred for 12 hours under hydrogen protection. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound V8-3 (30 g). MS m / z (ESI): 247.1 [M-55] - .

[0174] Step 4: Dissolve 1-bromo-3,5-difluorobenzene (15.32 g, 79.37 mmol) in THF (100 mL), cool to -78 °C under nitrogen protection, and add LDA (2 M, 49.61 mL) dropwise. Stir the reaction mixture at -78 °C for 1 hour. Then add a tetrahydrofuran (20 mL) solution of compound V8-3 (20 g, 66.15 mmol). Stir the reaction mixture at -78 °C for 1 hour, then raise the temperature to 25 °C and continue stirring for 3 hours. Quench the reaction with saturated ammonium chloride solution (50 mL), and extract with dichloromethane (50 mL x 3). Wash the organic phase with saturated brine, dry to anhydrous sodium sulfate, and evaporate to dryness under reduced pressure. Purify the residue by silica gel column chromatography with an eluent system (petroleum ether / ethyl acetate: 1 / 0 to 3 / 1) to give compound V8-4 (20 g). MS m / z (ESI): 336.0 [M+1] + .

[0175] Step 5: Compound V8-4 (20 g, 46.05 mmol) was dissolved in THF (200 mL), cooled to 0 °C, and then NaH (3.53 g, 88.24 mmol, 60% purity) was added. The reaction mixture was stirred at room temperature for half an hour. Then tert-butyldimethylchlorosilane (13.88 g, 92.11 mmol) was added, and the reaction mixture was stirred for another hour. The reaction was quenched with saturated ammonium chloride solution (100 mL), extracted with ethyl acetate (100 mL x 3), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound V8-5 (25 g). MS m / z (ESI): 492.0 [M+1]+ .

[0176] Step 6: Compound V8-5 (25 g, 45.58 mmol) was added to water (100 mL) and THF (100 mL), cooled to 0 °C, and then N-bromosuccinimide (8.11 g, 45.58 mmol) was added. The reaction mixture was stirred at 0 °C for 1 hour. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The filtrate was then subjected to silica gel column chromatography with an eluent system of petroleum ether / ethyl acetate: 1 / 0 to 4 / 1 to give compound V8 (19 g). MS m / z (ESI): 413.9 [M+1] + .

[0177] Preparation of intermediate V9

[0178]

[0179] Step 1: Compound V10 (2.4 g, 4.79 mmol) was dissolved in water (2 mL) and methanol (20 mL), and lithium hydroxide (343.81 mg, 14.36 mmol) was added. The mixture was stirred at room temperature for 2 hours. The pH was adjusted to 1 with 6N hydrochloric acid solution, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound V9-1 (2.2 g). MS m / z (ESI): 488.1 [M+1] + .

[0180] Step 2: Compound V9-1 (2.2 g, 4.51 mmol) was dissolved in DMF (21.85 mL), and methylamine hydrochloride (609.40 mg, 9.03 mmol), HATU (2.55 g, 6.77 mmol), and triethylamine (2.28 g, 22.56 mmol, 3.15 mL) were added. The mixture was stirred overnight at room temperature and concentrated under reduced pressure. V9-2 (2.1 g) was obtained by column chromatography (0-30%, DCM / MeOH), MS m / z (ESI): 501.1 [M+1] + .

[0181] Step 3: Dissolve V9-2 (2.1 g, 4.20 mmol) in methanol (20 mL), add HCl (4.0 M, 3.15 mL), and stir at room temperature for 2 hours. Concentrate under reduced pressure, adjust pH to 8 with saturated sodium bicarbonate solution, and extract with dichloromethane / methanol (10 / 1). Dry the organic phase with anhydrous sodium sulfate and concentrate under reduced pressure to obtain V9 (2 g), MS m / z (ESI): 401.1 [M+1] + .

[0182] Preparation of intermediate V10

[0183]

[0184] V10 can be prepared by referring to the preparation method of intermediate compound V25-3, except that 2-chloro-4-methylpyridine is used instead of 2-chloro-5-fluoro-4-methylpyridine. MS m / z (ESI): 502.1 [M+1] + .

[0185] Preparation of intermediate V11

[0186]

[0187] Step 1: 4-Methyl-2-nitroaniline (1 g, 6.57 mmol) was dissolved in THF (20 mL). Sodium hydroxide (788.61 mg, 19.72 mmol, 60% purity) was added at 0 °C, and the mixture was stirred at 0 °C for half an hour. Boc₂O (2.15 g, 9.86 mmol) was added, and the mixture was stirred at room temperature for half an hour. The reaction was quenched with water, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to give compound V11-1 (1.6 g). MS m / z (ESI): 153.1 (M+H-100).

[0188] Step 2: Compound V11-1 (1.6 g, 6.34 mmol) was dissolved in methanol (20 mL), and 10% Pd / C (770.33 mg, 6.34 mmol) was added. The mixture was reacted at room temperature under hydrogen atmosphere for 2 hours. After filtration and concentration under reduced pressure, compound V11 (1.4 g) was obtained. MS m / z (ESI): 167.1 (M+H-56).

[0189] Preparation of intermediate V12

[0190]

[0191] Step 1: 3,5-Difluoro-4-formylbenzoic acid (1 g, 5.37 mmol) and methylamine hydrochloride (362.78 mg, 5.37 mmol) were dissolved in DMF (12.75 mL), followed by the addition of HATU (3.04 g, 8.06 mmol) and TEA (1.63 g, 16.12 mmol, 2.25 mL). The reaction was stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated under reduced pressure, and the solution was separated by column chromatography (40 g, 0–60% EA / PE) to give compound V12-1 (650 mg). MS m / z (ESI): 218.0 (M+H+18).

[0192] Step 2: Compound V12-1 (5.5 g, 27.62 mmol) was dissolved in DMF (6 mL), and potassium peroxide (25.44 g, 41.43 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow oil. The oil was separated by column chromatography (80 g, 0–30% MeOH / DCM) to obtain compound V12-2 (2.1 g). MS m / z (ESI): 216.1 [M+1].

[0193] Step 3: Compound V11 (300 mg, 1.35 mmol) was dissolved in acetonitrile (20 mL), and compound V12-2 (290.38 mg, 1.35 mmol) and N,N,N',N'-tetramethylchloromethylaminohexafluorophosphate (1.14 g, 4.05 mmol) were added. The mixture was stirred at room temperature for half an hour. N-methylimidazole (224.34 mg, 2.70 mmol) was added, and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the oil was separated by column chromatography (20 g, 0-90% EA / PE) to give compound V12-3 (420 mg). MS m / z (ESI): 320.1 (M+H-100).

[0194] Step 4: Compound V12-3 (410 mg, 977.54 μmol) was dissolved in methanol (10 mL), and methanol hydrochloric acid (10 mL) was added. The reaction mixture was stirred for 4 hours, concentrated under reduced pressure, and saturated sodium bicarbonate solution was added. A solid precipitated out, and the mixture was filtered to obtain compound V12 (180 mg). Yield: 57.67%. MS m / z (ESI): 320.1 [M+1].

[0195] Preparation of intermediate V13

[0196]

[0197] Step 1: 4-Bromo-2,6-difluorobenzaldehyde (9.38 g, 42.43 mmol), 5-fluoro-4-methylpyridin-2-amine (4.46 g, 35.36 mmol), (2S)-2-ethynylmorpholine-4-carboxylic acid tert-butyl ester (8.96 g, 42.43 mmol), copper(II) trifluoromethanesulfonate (3.84 g, 10.61 mmol), N,N-dimethylacetamide (924.17 mg, 10.61 mmol), and cuprous chloride (1.05 g, 10.61 mmol) were dissolved in Xylene (100 mL). The reaction mixture was stirred at 85 °C for 16 hours under nitrogen protection. The reaction was monitored by LC-MS. After the reaction was complete, the solvent was evaporated to dryness. After evaporation under reduced pressure, the solution was separated by column chromatography (120 g, 0–80% EA / PE) to obtain compound V13-1 (7.1 g). MS m / z(ESI): 540.1 [M+1].

[0198] Step 2: Compound V13-1 (2.3 g, 4.26 mmol) was dissolved in methanol (10 mL) and HCl / dioxane (10 mL). The reaction was stirred at room temperature for 4 hours, and the reaction was monitored by LC-MS. After the reaction was complete, the solvent was evaporated to give compound V13-2 (1.7 g). MS m / z (ESI): 440.0 [M+1].

[0199] Step 3: Compound V13-2 (1.7 g, 3.86 mmol) was dissolved in DCM (19.33 mL), followed by DIEA (998.09 mg, 7.72 mmol, 1.35 mL). The reaction mixture was cooled to 0 °C, and then 2,2,2-trideuterated acetyl chloride (472.15 mg, 5.79 mmol) was added. The reaction was stirred at room temperature for 0.5 h. After the reaction was complete, water and DCM were added, and the aqueous phase was extracted twice with DCM and dried over anhydrous sodium sulfate. The solution was evaporated to dryness under reduced pressure to give compound V13 (1.8 g). MS m / z (ESI): 485.0 [M+1].

[0200] Preparation of intermediate V15

[0201]

[0202] Following the preparation method of intermediate V18, except that 5-fluoro-4-methylpyridin-2-amine is used instead of 4-methylpyridin-2-amine, V15 can be prepared. MS m / z (ESI): 427.1 [M+1] + .

[0203] Preparation of intermediate V18

[0204]

[0205] Step 1: Dissolve intermediate V8 (147.47 mg) in ethanol (1 mL). The reaction was carried out at 120 °C with stirring in air. After approximately half an hour, the solvent evaporated completely. Ethanol was repeatedly added (1 mL x 8), and the mixture was stirred for 8 hours. LC-MS showed the reaction was complete. The solvent was evaporated under reduced pressure, and the solution was purified by column chromatography (using petroleum ether containing 50-60% ethyl acetate as the mobile phase) to obtain V18-1 (60 mg). MS m / z (ESI): 540.0 [M+1] + .

[0206] Step 2: V18-1 (59.73 mg) and pyrrolidone-2-one (18.81 mg) were dissolved in dioxane (10 mL), followed by the addition of Pd2dba3 (10.12 mg), Xantphos (12.79 mg), and cesium carbonate (72.07 mg). The reaction was stirred at 100 °C for 8 hours under argon protection. LC-MS showed that the reaction was complete. After the reaction was complete, the solvent was evaporated under reduced pressure, and the solution was purified by column chromatography (using dichloromethane containing 10-15% methanol as the mobile phase) to obtain V18-2 (40 mg). MS m / z (ESI): 545.2 [M+1] + .

[0207] Step 3: Dissolve V18-2 (40 mg, 73.45 μmol) in dioxane (2.85 mL), then add dioxane hydrochloric acid gas (4 M, 146.91 μL). Stir the reaction at room temperature for 1 hour. Monitor the reaction completion by LC-MS. Recycle the solvent under reduced pressure to obtain V18 (35 mg), MS m / z (ESI): 445.1 [M+1] + .

[0208] Preparation of intermediate V21

[0209]

[0210] Following the preparation method of intermediate V25, except that 2-chloro-4-methylpyridine is used instead of 2-chloro-5-fluoro-4-methylpyridine, V21 can be prepared. MS m / z (ESI): 444.0 [M+1] + .

[0211] Preparation of intermediate V25

[0212]

[0213] Step 1: 2-Chloro-5-fluoro-4-methylpyridine (4 g, 27.48 mmol) and tert-butyl carbamate (4.83 g, 41.22 mmol) were dissolved in toluene (40 mL). Cesium carbonate (13.43 g, 41.22 mmol), tris(dibenzylacetone)dipalladium (2.52 g, 2.75 mmol), and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (1.59 g, 2.75 mmol) were added. The reaction mixture was stirred at 100 °C for 12 hours under nitrogen protection. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an eluent system (petroleum ether / ethyl acetate: 5 / 0~3 / 1) to give V25-1 (4 g). MS m / z (ESI): 171.0 [M-55] - .

[0214] Step 2: Add V25-1 (5 g, 22.10 mmol) to DCM (50 mL), then add trifluoroacetic acid (8.53 g, 74.79 mmol, 5.56 mL) dropwise to the reaction mixture. Stir at room temperature for 12 hours. Quench the reaction mixture with 1 N sodium hydroxide aqueous solution (50 mL), then extract three times with DCM (50 mL). Combine the organic phases, wash with saturated brine, dry to anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness to obtain V25-2 (2.5 g). MS m / z (ESI): 127.0 [M+1] + .

[0215] Step 3: V25-2 (630.22 mg, 5.00 mmol) and methyl 3,5-difluoro-4-carboxymethyl benzoate (1 g, 5.00 mmol) were added to ethanol (20 mL), and the mixture was stirred at 80 °C for 12 hours. The reaction solution was then evaporated to dryness and dissolved in toluene (20 mL). Tert-butyl(S)-2-ethynylmorpholine-4-carboxylate (1.06 g, 5.00 mmol), copper(II) trifluoromethanesulfonate (542.14 mg, 1.50 mmol), cuprous chloride (148.40 mg, 1.50 mmol), and N,N-dimethylacetamide (130.59 mg, 1.50 mmol) were added, and the reaction solution was stirred at 85 °C for 12 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography using an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1) to obtain V25-3 (1 g). MS m / z (ESI): 520.1 [M+1] + .

[0216] Step 4: Dissolve V25-3 (0.3 g, 577.47 μmol) in DCM (30 mL), and add 1.44 mL of 4 M hydrochloric acid in 1,4-dioxane. Stir the reaction mixture at room temperature for 2 hours. Directly evaporate the reaction mixture to dryness to obtain V25-4 (220 mg). MS m / z (ESI): 420.1 [M+1] + .

[0217] Step 5: Add V25-4 (0.5 g, 1.19 mmol) to DCM (20 mL), then add TEA (241.28 mg, 2.38 mmol, 332.56 μL) and methyl chloroformate (168.99 mg, 1.79 mmol). React at room temperature for 2 hours. Add water (20 mL) and extract with DCM (20 mL x 3). Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography with an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1) to obtain V25-5 (400 mg). MS m / z (ESI): 478.1 [M+1]+ .

[0218] Step 6: Dissolve V25-5 (0.4 g, 837.82 μmol) in ethanol (20 mL), add hydrazine hydrate (524.27 mg, 8.38 mmol), and stir the reaction solution at 90 °C for 4 hours. The reaction solution is then evaporated to dryness to obtain V25-6 (400 mg). MS m / z (ESI): 478.1 [M+1] + .

[0219] Step 7: Dissolve V25-6 (0.4 g, 837.81 μmol) in DCM (20 mL), add TEA (169.56 mg, 1.68 mmol, 233.71 μL) and di-tert-butyl dicarbonate (274.27 mg, 1.26 mmol), and stir overnight at room temperature. Concentrate the reaction solution under reduced pressure and elute using silica gel column chromatography with a petroleum ether / ethyl acetate eluent system of 2 / 1 to 1 / 1 to obtain V25-7 (400 mg). MS m / z (ESI): 578.2 [M+1] + .

[0220] Step 8: Add V25-7 (200 mg, 346.29 μmol) and Lawson's reagent (210.10 mg, 519.43 μmol) to 1,4-dioxane (4 mL) and stir at 140 °C for 40 minutes in a microwave reactor. Cool to room temperature and concentrate the reaction solution under reduced pressure. Purify the residue by silica gel column chromatography with an eluent system (dichloromethane / methanol: 10 / 1 to 5 / 1) to obtain V25-8 (150 mg). MS m / z (ESI): 594.2 [M+1] + .

[0221] Step 9: Dissolve V25-8 (150 mg, 252.69 μmol) in DCM (6 mL), and add trifluoroacetic acid (2 mL) dropwise. Stir the reaction mixture at room temperature for 1 hour. After concentrating the reaction mixture under reduced pressure, dilute with DCM (20 mL), wash with saturated sodium bicarbonate aqueous solution (20 mL), wash the organic phase with saturated brine, dry to anhydrous sodium sulfate, and concentrate the organic phase under reduced pressure to obtain V25-9 (100 mg). MS m / z (ESI): 494.1 [M+1] + .

[0222] Step 10: Dissolve V25-9 (100 mg, 243.16 μmol) and N,N'-carbonyldiimidazole (39.43 mg, 243.16 μmol) in tetrahydrofuran (20 mL). Stir the reaction mixture at room temperature for 1 hour. Concentrate under reduced pressure and purify the residue by silica gel column chromatography using an eluent system (dichloromethane / methanol: 10 / 1 to 5 / 1) to obtain V25-10 (57 mg). MS m / z (ESI): 520.1 [M+1] + .

[0223] Step 11: Add V25-10 (57 mg, 109.72 μmol) to a 1,4-dioxane solution (2 mL) in 4N hydrochloric acid and concentrated hydrochloric acid (2 mL). Heat the reaction solution to 110 °C and stir overnight. Concentrate the reaction solution under reduced pressure to obtain V25 (50 mg). MS m / z (ESI): 462.1 [M+1] + .

[0224] Preparation of intermediate V26

[0225]

[0226] Following the preparation method of intermediate V28, except that 4-methylpyridin-2-amine is used instead of 4-chloropyridin-2-amine, V26 (100 mg) can be prepared. MS m / z (ESI): 444.0 [M+1] + .

[0227] Preparation of intermediate V28

[0228]

[0229] Step 1: Intermediate V8 (450 mg, 876.91 μmol) and 4-chloropyridin-2-amine (112.73 mg, 876.91 μmol) were placed in a microwave-safe tube, and 0.5 mL of ethanol was added. The reaction was stirred at 120 °C for 4 hours (open reaction, maintaining a very small amount of solvent). The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography with an eluent system (petroleum ether / ethyl acetate: 1 / 0 to 2 / 1) to give V28-1 (142 mg). LC-MS m / z (ESI): 542.1 [M+1] + .

[0230] Step 2: V28-1 (120 mg, 0.221 mmol) and zinc cyanide (18.17 mg, 154.75 μmol) were placed in a microwave-safe tube, DMF (5 mL) was added, and then bis(tri-tert-butylphosphine)palladium(0) (11.30 mg, 22.11 μmol) was added. The reaction was stirred at 90 °C for 30 minutes in a microwave reactor. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure to obtain V28-2 (108 mg). MS m / z (ESI): 489.2 [M+1] + .

[0231] Step 3: V28-2 (108 mg, 220.90 μmol) and hydroxylamine hydrochloride (23.03 mg, 331.35 μmol) were added to ethanol (10 mL), followed by DIPEA (57.10 mg, 441.80 μmol). The reaction mixture was stirred at 80 °C for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was purified by preparative thin-layer chromatography using a chromatographic system (dichloromethane / methanol: 100 / 7) to obtain V28-3 (60 mg). MS m / z (ESI): 522.2 [M+1] + .

[0232] Step 4: Dissolve V28-3 (60 mg, 114.96 μmol) and N,N'-thiocarbonyldiimidazole (30.73 mg, 172.43 μmol) in tetrahydrofuran (10 mL). Stir the reaction mixture at room temperature for 2 hours. Add 20 mL of water to the reaction mixture and extract with ethyl acetate (30 mL x 3). After drying the organic phase, filter and concentrate under reduced pressure to obtain V28-4 (72 mg). MS m / z (ESI): 632.2 [M+1] + .

[0233] Step 5: Dissolve V28-4 (72 mg, 126.41 μmol) in tetrahydrofuran (20 mL) and cool to 0 °C. Add boron trifluoride diethyl ether (89.71 mg, 632.04 μmol). Stir the reaction mixture at room temperature for 16 hours. Concentrate under reduced pressure to obtain V28-5 (63 mg). MS m / z (ESI): 564.1 [M+1] + .

[0234] Step 6: Dissolve V28-5 (63 mg, 111.70 μmol) in methanol (2.86 mL), and add 1,4-dioxane hydrochloric acid solution (4 M, 1 mL). Stir the reaction mixture at room temperature for 1 hour. Concentrate under reduced pressure, and adjust the pH to neutral by adding 7N ammonia in methanol. Purify the residue by preparative thin-layer chromatography using a chromatographic system (dichloromethane / methanol / ammonia in methanol: 100 / 10 / 2) to obtain V28 (50 mg). MS m / z (ESI): 464.1 [M+1] + .

[0235] Example 1: Preparation of compound Z-1

[0236]

[0237] Intermediate V9 (0.1 g, 249.74 μmol) and 2-hydroxy-2-methylpropionic acid (31.20 mg, 299.68 μmol) were dissolved in DMF (10 mL), followed by the sequential addition of DIPEA (32.28 mg, 249.74 μmol) and HATU (94.22 mg, 249.74 μmol), and the reaction was carried out at 25 °C for 2 hours. The reaction solution was quenched with 10 mL of water, and then extracted with 20 mL of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%-60% acetonitrile) to obtain compound Z-1 (15 mg). MS m / z (ESI): 487.2 [M+1] + ; 1 H NMR (400MHz, DMSO-d6) δ8.66(d,J=4.7Hz,1H),8.36(d,J=7.1Hz,1H),7.61(d,J= 8.0Hz,2H),6.80(dd,J=7.3,1.7Hz,1H),5.30(s,1H),4.56(s,1H),4.07(s,1H),3 .64(d,J=11.5Hz,1H),3.44(d,J=19.5Hz,2H),3.19(d,J=2.9Hz,2H),3.01(d,J= 6.9Hz, 3H), 2.78 (d, J = 4.5Hz, 3H), 2.34 (d, J = 1.1Hz, 3H), 1.18 (d, J = 10.1Hz, 6H).

[0238] Example 2: Preparation of compound Z-2

[0239]

[0240] Example 2 can be prepared by referring to the method of Example 1, except that 2-hydroxyacetic acid is used instead of 2-hydroxy-2-methylpropionic acid to obtain compound Z-2 (12 mg). MS m / z (ESI): 459.2 [M+1] + ;1H NMR (400MHz, CDCl3)8.17(brs,1H),7.45-7.41(m,3H),7.03-6.89(m,1H),6.73-6.69(m,1H),4.30-4.27(m,1H),4.15-3.79(m,3H),3.57(br s,1H),3.45-3.42(m,1H),3.38-2.83(m,7H),2.79-2.72(m,1H),2.55-2.42(m,4H).

[0241] Example 3: Preparation of compound Z-3

[0242]

[0243] Example 3 can be prepared by referring to the method of Example 1, except that 2-hydroxypropionic acid is used instead of 2-hydroxy-2-methylpropionic acid to obtain compound Z-3 (12 mg). MS m / z (ESI): 473.2 [M+1] + ;1H NMR (400MHz, CDCl3)8.17(brs,1H),7.43(br s,3H),7.13(br s,1H),6.72(br s,1H),5.32(d,J=8Hz,1H),4.39-4.19(m,2H),3.91-3.77(m,1H),3.57(br s,1H),3.44-3.36(m,2H),3.16-3.00(m,5H),2.81-2.74(m,1H),2.48-2.42(m,4H),1.27-1.23(m,3H).

[0244] Example 4: Preparation of compound Z-4

[0245]

[0246] Compound V9 (60 mg, 149.84 μmol) was dissolved in acetonitrile (5 mL), and triethylamine (45.49 mg, 449.53 μmol, 62.70 μL) and 3-methoxyethane-3-trifluoromethanesulfonate (CAS NO: 1379585-89-3) (65.98 mg, 299.68 μmol) were added. The mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure, and compound Z-4 (4.89 mg) was obtained by pre-HPLC. MS m / z (ESI): 471.1 [M+1] + ; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.37-8.35 (d, J = 8Hz, 1H), 7.62 (m, 2H) ,7.31(s,1H),6.80-6.78(d,J=8Hz,1H),3.54(m,3H),2.95-2.91(m,2H),2. 78(s,3H),2.60-2.58(m,2H),2.56-2.55(m,1H),2.26(s,3H),2.18-2.15(m ,2H),2.10-2.07(m,1H),1.88-1.85(m,1H),1.68-1.62(m,1H),1.15(s,3H).

[0247] Example 5: Preparation of compound Z-5

[0248]

[0249] Example 5 can be prepared by referring to the method of Example 1, except that oxetane-2-carboxylic acid is used instead of 2-hydroxy-2-methylpropionic acid to obtain compound Z-5 (25 mg). MS m / z (ESI): 485.1 [M+1] + ; 1 H NMR (400MHz, CDCl3) δ8.27(d,J=22.6Hz,1H),7.52(dd,J=23.4,12.3Hz,3H),7.06(d,J=86.6Hz,1H),6.81(s,1H),5. 35–5.01(m,1H),4.69–4.19(m,3H),3.92–3.11(m,5H),2.71(q,J=16.9,11.5Hz,2H),2.50–2.40(m,3H),1.23(s,6H).

[0250] Example 6: Preparation of compound Z-6

[0251]

[0252] Example 6 can be prepared by referring to the method of Example 1, except that tetrahydrofuran-2-carboxylic acid is used instead of 2-hydroxy-2-methylpropionic acid to obtain compound Z-6 (25 mg). MS m / z (ESI): 499.1 [M+1] + ; 1 H NMR(400MHz, CDCl3)8.24(s,1H),7.54-7.45(m,3H),6.79(s,1H),4.45-4.32(m,2H) ,3.85-3.39(m,6H),3.02-3.01(m,5H),2.45(s,3H),2.20-1.93(m,4H),1.23(s,3H).

[0253] Example 7: Preparation of compound Z-7

[0254]

[0255] Example 6 can be prepared by referring to the method of Example 1, except that tetrahydro-2H-pyran-2-carboxylic acid is used instead of 2-hydroxy-2-methylpropionic acid to obtain compound Z-7 (20 mg). MS m / z (ESI): 513.2 [M+1] + ;1H NMR (400MHz, CDCl3) δ8.34–7.94(m,1H),7.44(t,J=9.9Hz,3H),6.95–6.42(m,2H),4. 30(d,J=13.4Hz,1H),4.08–3.31(m,7H),3.22–2.79(m,6H),2.43(s,4H),1.23(s,6H).

[0256] Example 8: Preparation of compound Z-8

[0257]

[0258] Step 1: Compound V12 (156.02 mg, 488.62 μmol) and compound V1 (90 mg, 488.62 μmol) were added to methanol (10 mL) and stirred at room temperature for 0.5 hours. Then, NaBH3CN (92.11 mg, 1.47 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours, and the reaction was monitored by LC-MS. After the reaction was complete, the solution was concentrated under reduced pressure to obtain a yellow oil. The residue was purified by silica gel column chromatography using an eluent system (dichloromethane / methanol: 1 / 0 to 10 / 1) to obtain 8-1 (120 mg). MS m / z (ESI): 488.2 [M+1] + .

[0259] Step 2: Compound 8-1 (120 mg, 246.15 μmol) was added to 1,2-dichloroethane (5 mL). Then, anhydrous magnesium sulfate (100 mg) and TFA (28.07 mg, 246.15 μmol) were added. The reaction mixture was microwaved at 120 °C for 1 hour, and the reaction was monitored by LC-MS. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%-60% acetonitrile) to obtain Z-8 (5.04 mg). MS m / z (ESI): 470.1 [M+1] + ; 1HNMR (400MHz, DMSO-d6) δ: 8.74 (s, 1H), 7.68 (d, J=8Hz, 2H), 7.62 (d, J= 8Hz,1H),7.47(s,1H),7.18-7.14(m,1H),4.32-4.28(m,1H),4.07-4.01(m ,1H),3.55-3.50(m,2H),3.37-3.30(m,3H),3.20-3.18(m,1H),2.81-2.80 (m,1H),2.80(d,J=4Hz,3H),2.75-2.74(m,1H),2.55(s,3H),2.41(s,3H).

[0260] Example 9: Preparation of compound Z-9

[0261]

[0262] Example 9 can be prepared by referring to the method of Example 8, except that compound V1 is replaced by compound V2, to obtain compound Z-9 (20.86 mg). LCMS: MS m / z (ESI): 457.1 [M+1] + ; 1 H NMR (400MHz, CDCl3) δ7.87 (s, 1H), 7.64 (s, 1H), 7.49 (d, J = 8.5Hz, 2H), 7.35 (d, J=8.4Hz,1H),7.25(s,1H),4.30(t,J=8.5Hz,1H),4.25–4.12(m,1H),4.03(dd,J =15.2,7.6Hz,1H),3.83–3.64(m,4H),3.58(dd,J=10.6,7.8Hz,1H),3.09(t,J= 10.9Hz, 1H), 3.01 (d, J = 4.5Hz, 3H), 2.68 (dd, J = 12.9, 11.3Hz, 1H), 2.52 (s, 3H).

[0263] Example 10: Preparation of compound Z-10

[0264]

[0265] Step 1: Intermediate V3 (0.26 g, 0.9 mmol) was dissolved in a tetrahydrofuran / methanol / water (10 / 1 / 1) mixed solvent (24 ml), and lithium hydroxide (0.19 g, 4.5 mmol) was added. The mixture was stirred at room temperature for 2 hours, and the reaction solution was concentrated to obtain 10⁻¹ (0.4 g). MS m / z (ESI): 186.1 [M+1] +

[0266] Step 2: Dissolve compound 10⁻¹ (0.4 g) in DCM (20 ml), add TEA (0.33 g, 3.3 mmol), and slowly add a solution of methanesulfonyl chloride (0.23 g, 2.0 mmol) in DCM (10 ml). Stir at room temperature for 2 hours. Wash the reaction solution directly with water, dry, and concentrate. Purify the product by silica gel column chromatography (methanol / dichloromethane = 1 / 25) to give 10⁻² (0.25 g). MS m / z (ESI): 248.1 [M+1] +

[0267] Step 3: Compound 10⁻² (0.25 g, 1 mmol) was dissolved in DMF (15 ml), sodium azide (0.14 g, 2 mmol) was added, and the mixture was heated to 80 °C and stirred overnight. After cooling to room temperature, the reaction solution was diluted with ethyl acetate, washed with water and saturated sodium chloride solution, dried, and concentrated to give 10⁻³ (0.2 g). MS m / z (ESI): 211.1 [M+1]⁺

[0268] Step 4: Compound 10⁻³ (0.2 g) was dissolved in ethanol (20 ml), and 10% wet palladium on carbon (80 mg) was added. The mixture was purged with hydrogen three times, stirred at room temperature for 20 hours, filtered, and the filtrate was concentrated to obtain 10⁻⁴ (0.17 g). MS m / z (ESI): 185.2 [M+1]

[0269] Step 5: Add compound 10⁻⁴ (0.17 g), 1-fluoro-4-methylnitrobenzene (0.2 g, 1.2 mmol), potassium carbonate (0.25 g, 1.8 mmol), and DMF (15 ml) to a round-bottom flask. Heat to 80 °C, stir overnight, cool to room temperature, filter, and concentrate the filtrate. Purify the product by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give 10⁻⁵ (0.11 g). MS m / z (ESI): 320.2 [M+1] +

[0270] Step 6: Compound 10⁻⁵ (0.11 g, 0.34 mmol) was suspended in anhydrous ethanol (20 ml), 10% wet palladium on carbon (60 mg) was added, the mixture was purged with hydrogen three times, stirred at room temperature for 1 hour, filtered, and concentrated to obtain 10⁻⁶ (0.07 g). MS m / z (ESI): 290.1 ​​[M+1] +

[0271] Step 7: 10⁻⁶ (70 mg, 0.24 mmol), 2,6-difluoro-4-(methylcarbamoyl)benzoic acid (63 mg, 0.29 mmol), HATU (110 mg, 0.29 mmol), and TEA (88 mg, 0.87 mmol) were dissolved in DMF (3 mL). The mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the product was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 25) to give 10⁻⁷ (76 mg). MS m / z (ESI): 487.1 [M+1] +

[0272] Step 8: Compound 10-7 (76 mg, 0.156 mmol) was dissolved in 1,2-dichloroethane (8 mL), trifluoroacetic acid (1 mL) and anhydrous magnesium sulfate solid (76 mg) were added, and the mixture was heated to 130 °C under microwave conditions for 40 minutes. After cooling to room temperature, the mixture was filtered and concentrated. The product was purified by preparative HPLC (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to obtain Z-10 (19 mg). MS m / z (ESI): 469.2 [M+1] + ; 1 H NMR (400MHz, DMSO-d6) δ8.74-8.71(m,1H),7.69-7.66(m,3H),7.51-7.49(m,1H),7.18-7.15(m,1H),4.55-4.52(m,1H),4.47-4.45(m,1 H),4.28-4.25(m,1H),4.17-4.13(m,1H),4.06–3.93(m,1H),3.78(dd,J=12.1,3.5Hz,1H),3.23–3.09(m,4H),2.80(s,3H),2.42(s,3H).

[0273] Example 11: Preparation of compound Z-11

[0274]

[0275] Example 11 can be prepared by referring to the method of Example 10, except that compound V3 is replaced by compound V4, to obtain compound Z-11 (24 mg). MS m / z (ESI): 453.2 [M+1] + ; 1 H NMR (400MHz, DMSO-d6) δ8.75-8.72(m,1H),7.75–7.62(m,3H),7.53-7.50(m,1H),7.20-7.16(m,1H),4.80-4.76(m,1H),4. 63–4.47(m,2H),4.25–4.14(m,2H),4.10–4.00(m,1H),3.66-3.62(m,1H),2.80(d,J=4.5Hz,3H),2.43(s,3H),2.30(s,3H).

[0276] Example 12: Preparation of compound Z-12

[0277]

[0278] Example 12 can be prepared by referring to the method of Example 10, except that compound V5 is used instead of compound V3, to obtain compound Z-12 (32 mg). MS m / z (ESI): 497.2 [M+1] + ; 1 H NMR(400MHz, DMSO-d6)δ8.74-8.72(m,1H),7.73–7.64(m,3H),7.50(s,1H),7.17(dd,J=8.3,1.5Hz,1H),4.58–4.43(m,2H),4.32–4 .10(m,3H),4.02-4.00(m,1H),3.79-3.76(m,1H),3.16-3.14(m,1H),2.81-2.79(m,3H),2.43(s,3H),1.15(dd,J=6.7,3.3Hz,6H).

[0279] Example 13: Preparation of compound Z-13

[0280]

[0281] Example 13 can be prepared by referring to the method of Example 10, except that compound V3 is replaced by compound V6, to obtain compound Z-13 (34 mg). MS m / z (ESI): 537.2 [M+1] + ; 1H NMR (400MHz, DMSO-d6) δ8.74-8.72(m,1H),7.70-7.68(m,3H),7.50(s,1H),7.21–7.16(m,1H),4.59–4.42(m,4H),4.32-4. 30(m,1H),4.17-4.14(m,1H),4.07-4.02(m,1H),3.84-3.81(m,1H),3.23-3.20(m,1H),2.80(d,J=4.5Hz,3H),2.43(s,3H).

[0282] Example 14: Preparation of compound Z-14

[0283]

[0284] Example 14 can be prepared by referring to the method of Example 10, except that compound V7 is used instead of compound V3, to obtain compound Z-14 (27.19 mg). MS m / z (ESI): 495.2 [M+1] + , 1 H NMR (400MHz, CD3OD) δ7.75–7.58(m,3H),7.54(s,1H),7.27(d,J=8.4Hz,1H),4.57(dd,J=25.9,15.4Hz,2H),4.28(dd,J=15.5,6.1Hz,2 H),4.08(s,1H),3.82(dd,J=12.2,3.6Hz,1H),3.26–3.18(m,1H),3.09(d,J=5.9Hz,1H),2.95(s,3H),2.50(s,3H),1.00–0.79(m,4H).

[0285] Example 15: Preparation of compound Z-15

[0286]

[0287] Intermediate V15 (35 mg) was dissolved in DCM (5 mL) and cooled to 0 °C. TEA (0.726 M, 226.09 μL) was added, followed by dropwise addition of cyclopropanecarboxylic acid chloride (8.58 mg, 82.07 μmol). The reaction was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS until complete. The solvent was evaporated under reduced pressure. The solution was purified by preparative chromatography (21.2 x 250 mm C18 column, system: 10 mM NH4HCO3 H2O, wavelength: 254 / 214 nm, gradient: 30%-60% acetonitrile) to obtain compound Z-15 (6.0 mg). MS m / z (ESI): 495.2 [M+1] +;1H NMR (400MHz, CDCl3) δ8.19 (d, J=7.0Hz, 1H), 7.45 (dd, J=25.7, 10.7Hz, 3H), 6 .71(s,1H),4.33(s,1H),3.85(t,J=7.1Hz,4H),3.57(s,1H),3.40(td,J=11. 8,2.6Hz,1H),3.27–2.93(m,3H),2.83(s,1H),2.65(t,J=8.1Hz,2H),2.41(s ,3H),2.28–2.14(m,2H),1.52(s,1H),0.98–0.87(m,2H),0.77–0.67(m,2H).

[0288] Example 16: Preparation of compound Z-16

[0289]

[0290] Intermediate V15 (crude product 30 mg), glycolic acid (8 mg), HATU (38 mg), and TEA (40 mg) were dissolved in DMF (6 mL) and stirred overnight at room temperature. The product was purified by preparative HPLC (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-16 (4 mg). MS m / z (ESI): 485.1 [M+1] + ; 1 H NMR(400MHz,DMSO-d6)δ8.37-8.35(m,1H),7.57-7.55(m,2H),7.30(s,1H),6.79-6.77(m,1H),4.53-4.52(m,1H),4.08–3.81(m,5H),3.6 6-3.64(m,1H),3.58–3.37(m,2H),3.17-3.15(m,1H),2.99-2.97(m,2H),2.59(s,1H),2.54-2.52(m,2H),2.34(s,4H),2.09–2.01(m,2H).

[0291] Example 17: Preparation of compound Z-17

[0292]

[0293] Example 17 can be prepared by referring to the method of Example 16, except that 2-hydroxy-2-methylpropionic acid is used instead of glycolic acid, to obtain compound Z-17 (16 mg). MS m / z (ESI): 513.2 [M+1]+ ; 1 H NMR(400MHz,DMSO-d6)δ8.35-8.33(m,1H),7.57-7.55(m,2H),7.30(s,1H),6.79-6.77(m,1H),5.30(s,1H),4.57(s,1H),4.07(s,1H),3.85-3.8 3(m,2H),3.67-3.64(m,1H),3.44(s,1H),3.23-3.20(m,2H),3.00-2.98 (m,3H),2.54-2.52(m,2H),2.34(s,3H),2.09–2.01(m,2H),1.17(s,6H).

[0294] Example 18: Preparation of compound Z-18

[0295]

[0296] Intermediate V18 (35 mg) and 2-hydroxyacetic acid (11.98 mg) were dissolved in DMF (4 mL), followed by the addition of HATU (29.71 mg) and DIPEA (20.36 mg). The reaction was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS until completion. Preparative chromatography (21.2 x 250 mm C18 column, system: 10 mM NH4HCO3H2O, wavelength: 254 / 214 nm, gradient: 30%–60% acetonitrile) was used to purify compound Z-18 (4.44 mg). MS m / z (ESI): 503.1 [M+1] + ;1H NMR (400MHz, DMSO) δ8.64(d,J=5.6Hz,1H),7.56(d,J=10.3Hz,2H),7.46(d,J=7.3Hz,1H),4.52(d,J=5.2Hz,1H),4.11–3.80(m,5H),3.69–3.59(m, 1H),3.57–3.38(m,2H),3.22–3.09(m,1H),2.97(dd,J=21.3,7.4Hz,2H), 2.77–2.59(m,1H),2.53(t,J=8.1Hz,2H),2.31(s,3H),2.12–1.97(m,2H).

[0297] Example 19: Preparation of compound Z-19

[0298]

[0299] Intermediate V15 (50 mg, 117.24 μmol) was added to DCM (10 mL), followed by TEA (35.59 mg, 351.73 μmol, 49.06 μL), and then deuterated acetyl chloride (14.34 mg, 175.87 μmol). The reaction mixture was stirred at room temperature for 1 hour. The solution was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-19 (24.61 mg). MS m / z (ESI): 472.2 [M+1] + ; 1 H NMR(400MHz,DMSO-d6)δ8.36(dd,J=11.0,7.1Hz,1H),7.55(d,J=10.1Hz,2H),7.29(s,1H) ,6.77(t,J=5.9Hz,1H),4.03(dd,J=26.7,12.8Hz,1H),3.84(t,J=7.1Hz,2H),3.65(d,J=8. 9Hz,1H),3.55–3.31(m,2H),3.17(dd,J=39.6,9.2Hz,1H),2.97(dd,J=12.6,7.4Hz,2H),2. 76–2.62(m,1H),2.53(t,J=8.1Hz,2H),2.33(s,3H),2.26–2.14(m,1H),2.09–1.99(m,2H).

[0300] Example 20-1: Preparation of compound Z-20

[0301]

[0302] Intermediate V18 (42 mg, 94.50 μmol) was added to DCM (10.01 mL), followed by TEA (28.69 mg, 283.50 μmol, 39.54 μL), and then deuterated acetyl chloride (11.55 mg, 141.75 μmol). The reaction mixture was stirred at room temperature for 1 hour. The solution was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-20 (19.63 mg). MS m / z (ESI): 490.2 [M+1] +;1H NMR (400MHz, DMSO-d6) δ8.71–8.62(m,1H),7.58(d,J=12.0Hz,2H),7.49(d,J=8. 0Hz,1H),4.14-4.00(m,1H),3.87(t,J=6.9Hz,2H),3.69-3.63(m,2H),3.57–3.44 (m,1H),3.37(s,0.5H),3.29–3.20(m,0.5H),3.17–2.92(m,3H),2.80–2.71(m,0. 5H),2.55(t,J=8.0Hz,2H),2.33(s,3H),2.29–2.21(m,0.5H),2.13–2.00(m,2H).

[0303] Example 20-2: Preparation of compound Z-20

[0304]

[0305] Intermediate compound V13 (1 g, 2.06 mmol) and pyrrolidone-2-one (350.72 mg, 4.12 mmol) were dissolved in toluene (50 mL), followed by the addition of Pd2dba3 (188.69 mg, 206.05 μmol), Xantphos (238.44 mg, 412.11 μmol), and cesium carbonate (1.34 g, 4.12 mmol). The reaction was stirred at 100 °C for 16 hours under Ar protection. After filtration and solvent drying, the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-20 (492 mg). MS m / z (ESI): 490.2 [M+1] + ; 1 HNMR (400MHz, DMSO-d6) δ8.71–8.62(m,1H),7.58(d,J=12.0Hz,2H),7.49(d,J=8. 0Hz,1H),4.14-4.00(m,1H),3.87(t,J=6.9Hz,2H),3.69-3.63(m,2H),3.57–3.44 (m,1H),3.37(s,0.5H),3.29–3.20(m,0.5H),3.17–2.92(m,3H),2.80–2.71(m,0. 5H), 2.55 (t, J = 8.0Hz, 2H), 2.33 (s, 3H), 2.29–2.21 (m, 0.5H), 2.13–2.00 (m, 2H).

[0306] Example 21: Preparation of compound Z-21

[0307]

[0308] Intermediate V21 (40 mg) and 1-hydroxycyclopropane-1-carboxylic acid (32.50 mg) were dissolved in DMF (10 mL), followed by the sequential addition of DIPEA (23.31 mg) and HATU (51.04 mg). The mixture was then reacted at 25 °C for 2 hours. The reaction solution was quenched with 10 mL of water, and then extracted with 20 mL of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3H2O; wavelength: 254 / 214 nm; gradient: 30%-60% acetonitrile) to obtain compound Z-21 (20 mg). MS m / z (ESI): 528.2 [M+1] + ; 1H NMR (400MHz, CDCl3) 8.37 (d, J = 8Hz, 1H), 7.55-7.51 (m, 2H), 7.49 (s, 1H), 7.32-6.81 (m, 1H), 6.80 (br s,1H),4.15-3.55(m,3H),3.12-2.52(m,7H),2.34(s,3H),0.81-0.80(m,2H),0.78-0.62(m,2H).

[0309] Example 22: Preparation of compound Z-22

[0310]

[0311] Intermediate V21 (90 mg) was dissolved in DMF (4 mL), and 1-fluorocyclopropane-1-carboxylic acid (18.78 mg), HATU (68.06 mg), and TEA (27.38 mg) were added. The mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure, and compound Z-22 (15.87 mg) was obtained by pre-HPLC. MS m / z (ESI): 530.1 [M+1] + ; 1H NMR (400MHz, DMSO-d6) δ13.34(s,1H),8.40-8.38(d,J=8Hz,1H),7.55-7.53(d,J=8Hz,2H),7.33(s,1H),6.82-6.80(m,1H),3. 92-3.89(m,2H),3.72-3.69(m,1H),3.54-3.52(m,1H),3.26-3.25(m,2H),3.07-3.05(m,3H),2.29(s,3H),1.06-1.04(m,4H).

[0312] Example 23: Preparation of compound Z-23

[0313]

[0314] Intermediate V21 (31 mg), 2-hydroxyacetic acid (10.63 mg), and HATU (52.74 mg) were added to DMF (5 mL), followed by TEA (21.22 mg). The reaction mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-23 (13.99 mg). MS m / z (ESI): 502.2 [M+1] + ; 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=7.1Hz,1H),7.52(d,J=7.9Hz,2H),7.32(s,1H),6.80(d,J=7.2Hz,1H),4.55-4.52 (m,2H),4.09-3.89(m,3H),3.64-3.54(m,3H),3.25-3.20(m,2H),3.03-3.00(m,1H),2.84–2.60(m,2H),2.34(s,3H).

[0315] Example 24: Preparation of compound Z-24

[0316]

[0317] Intermediate V21 (31 mg), 2-hydroxy-2-methylpropionic acid (14.55 mg), and HATU (52.74 mg) were added to DMF (971.58 μL), followed by TEA (21.22 mg). The reaction mixture was stirred at room temperature for 16 hours. The mixture was then concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-24 (20.12 mg). MS m / z (ESI): 530.2 [M+1] + ; 1 HNMR (400MHz, DMSO-d6) δ8.37(d,J=6.7Hz,1H),7.53(d,J=7.8Hz,2H),7.32(s,1H),6.80(dd,J=7.2,1.6Hz,1H),3. 72–3.56(m,2H),3.45(s,1H),3.21(d,J=26.8Hz,3H),3.02(d,J=6.1Hz,3H),2.63(s,1H),2.34(s,3H),1.17(s,6H).

[0318] Example 25: Preparation of compound Z-25

[0319]

[0320] Intermediate V25 (50 mg) and 2-hydroxyacetic acid (8.24 mg) were dissolved in DMF (10 mL), followed by the sequential addition of DIPEA (28.01 mg) and HATU (61.32 mg). The mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to obtain compound Z-25 (5 mg). MS m / z (ESI): 520.1 [M+1] + ;1H NMR (400MHz, CDCl3) δ8.27(d,J=4.9Hz,1H),7.55(s,1H),7.31(t,J=8.4Hz,2H),4.34(t,J=12.8Hz,1H),4.23–3.78(m,3H) ,3.53(d,J=67.3Hz,3H),3.37–3.08(m,2H),2.99(d,J=7.3Hz,2H),2.85(d,J=12.9Hz,1H),2.66–2.49(m,1H),2.40(s,3H).

[0321] Example 26: Preparation of compound Z-26

[0322]

[0323] Intermediate V26 and 1-fluorocyclopropanecarboxylic acid (3.52 mg) were dissolved in DMF (5 mL), followed by the addition of HATU (19.14 mg) and TEA (6.83 mg). The reaction was stirred at room temperature for 2 hours, and LC-MS showed complete reaction. The crude product was purified by preparative chromatography (21.2 x 250 mm C18 column, system: 10 mM NH4HCO3H2O, wavelength: 254 / 214 nm, gradient: 30%–60% acetonitrile) to prepare compound Z-26 (1.64 mg). MS m / z (ESI): 530.1 [M+1] + ;1HNMR(400MHz,CD3OD)δ8.41(d,J=6.9Hz,1H),7.69(d,J=8.1Hz,2H),7.33(s,1H),6.86(d,J=6.9Hz,1H),4.10(d, J=12.9Hz,2H),3.82(d,J=10.4Hz,1H),3.64(s,1H),3.41(t,J=11.7Hz,2H),3.12(s,3H),2.43(s,3H),1.13(s,4H).

[0324] Example 27: Preparation of compound Z-27

[0325]

[0326] Intermediate V26 (100 mg) and 1-hydroxycyclopropane-1-carboxylic acid (23.02 mg) were dissolved in DMF (10 mL), followed by the sequential addition of DIPEA (58.29 mg) and HATU (127.61 mg). The mixture was stirred at room temperature for 12 hours. The reaction solution was quenched with 10 mL of water, and then extracted with 20 mL of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-27 (25 mg). MS m / z (ESI): 528.1 [M+1] +; 1H NMR (400MHz, DMSO-d6) 8.38 (d, J = 4Hz, 1H), 7.72 (d, J = 8Hz, 2H), 7.32 (s, 1H), 6.81 (d, J = 8Hz, 1H), 6.21 (s, 1H), 4.1 (br s,1H),3.68(d,J=12Hz,1H),3.51-3.35(m,6H),3.04-3.01(m,2H),2.34(s,3H),0.83-0.63(m,4H).

[0327] Example 28: Preparation of compound Z-28

[0328]

[0329] Intermediate V28 (50 mg, 107.78 μmol), 1-hydroxycyclopropanecarboxylic acid (13.20 mg, 129.34 μmol), HATU (61.00 mg, 161.68 μmol), and DIPEA (41.79 mg, 323.35 μmol) were dissolved in DMF (10 mL) and stirred at room temperature for 16 hours. The solution was concentrated under reduced pressure to obtain a brown crude product. The residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile). The purified product was lyophilized to give compound Z-28 (18.39 mg). MS m / z (ESI): 548.0 [M+1] + ; 1 H NMR (400MHz, CD3OD) δ8.54(d,J=7.2Hz,1H),7.69(d,J=8.0Hz,2H),7.60(s,1H),7.01(d,J=7.3Hz,1H),4.57(s,2H),4.29(s,2H ),3.80(d,J=8.7Hz,1H),3.66(d,J=19.4Hz,1H),3.42–3.33(m,1H),3.14(d,J=5.2Hz,2H),0.95(d,J=9.4Hz,2H),0.78(s,2H).

[0330] Example 29: Preparation of compound Z-29

[0331]

[0332] Intermediate compound V13 (120 mg, 247.26 μmol) and (2R)-5-oxopyrrolidine-2-carboxylic acid methyl ester (70.79 mg, 494.53 μmol) were dissolved in dioxane (10 mL), followed by the addition of Pd2dba3 (22.64 mg, 24.73 μmol), Xantphos (28.61 mg, 49.45 μmol), and cesium carbonate (161.22 mg, 494.53 μmol). The reaction was stirred at 100 °C for 16 hours under Ar protection. After filtration and solvent drying, the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-29 (64.61 mg). MS m / z (ESI): 548.2 [M+1] + ; 1 HNMR(400MHz,DMSO-d6)δ8.70(t,J=6.0Hz,1H),7.61–7.35(m,3H),5.21(d,J=9.0Hz,1H),4.10(dd ,J=51.1,13.2Hz,1H),3.77–3.46(m,6H),3.26–2.96(m,4H),2.84–2.54(m,3H),2.41–2.11(m,5H).

[0333] Example 30: Preparation of compound Z-30

[0334]

[0335] Example 30 can be prepared by referring to the method of Example 29, except that (2S)-5-oxopyrrolidine-2-carboxylic acid methyl ester is used instead of (2R)-5-oxopyrrolidine-2-carboxylic acid methyl ester, to obtain compound Z-30 (18 mg). MS m / z (ESI): 548.3 [M+1] + ;1H NMR (400MHz, DMSO) δ8.71(t,J=6.3Hz,1H),7.48(dd,J=22.1,8.5Hz,3H),5.21(dd,J=9.1,2.6Hz,1H),4.09(dd,J= 52.8,13.1Hz,1H),3.85–3.49(m,4H),3.46–3.22(m,3H),3.14–2.92(m,2H),2.81–2.50(m,5H),2.45–1.99(m,4H).

[0336] Example 31: Preparation of compound Z-31

[0337]

[0338] Intermediate compound V13 (50 mg, 103.03 μmol) and (4R)-4-hydroxypyrrolidone-2-one (20.83 mg, 206.05 μmol) were dissolved in dioxane (10 mL), followed by the addition of Pd2dba3 (9.43 mg, 10.30 μmol), Xantphos (11.92 mg, 20.61 μmol), and cesium carbonate (67.17 mg, 206.05 μmol). The reaction was stirred at 100 °C for 16 hours under Ar protection. After filtration and solvent drying, the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-31 (8.59 mg). MS m / z (ESI): 506.2 [M+1] + ; 1 HNMR (400MHz, DMSO-d6) δ8.74–8.62(m,1H),7.61(d,J=10.0Hz,2H),7.51(d,J=8.0Hz,1H),5.46(d,J=4Hz,1H),4.42(s,1 H),4.19–3.99(m,2H),3.80–3.45(m,5H),3.27–3.11(m,1H),3.02(t,J=8.3Hz,2H),2.94–2.76(m,1H),2.41–2.21(m,5H).

[0339] Example 32: Preparation of compound Z-32

[0340]

[0341] Compound Z-29 (50 mg, 91.32 μmol) was dissolved in MeOH (3 mL) and H₂O (3 mL), and then LiOH (10.94 mg, 456.59 μmol) was added. The reaction was stirred at room temperature for 5 hours. The solvent was evaporated and the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM formic acid / H₂O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to give compound Z-32 (17.80 mg). MS m / z (ESI): 534.2 [M+1] + ; 1HNMR(400MHz,DMSO-d6)δ8.70(t,J=6.0Hz,1H),7.58–7.37(m,3H),4.96(s,1H),4.18-4 .01(m,1H),3.73-3.53(m,2H),3.13-3.02(m,4H),2.66-2.50(m,3H),2.44–2.04(m,6H).

[0342] Example 33: Preparation of compound Z-33

[0343]

[0344] Compound Z-30 (50 mg, 91.32 μmol) was dissolved in tetrahydrofuran (6 mL), water (2 mL), and methanol (2 mL), then lithium hydroxide (21.87 mg, 913.18 μmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was adjusted to pH 6 with 2N hydrochloric acid, concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography (preparative column: 21.2 x 250 mm C18 column; system: 10 mM formic acid / H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to obtain compound Z-33 (20 mg). MS m / z (ESI): 534.2 [M+1] + ;1H NMR (400MHz, DMSO) δ13.37(s,1H),8.71(dd,J=8.0,5.5Hz,1H),7.49(dd,J=23.3,8.6Hz,3H),5.04(dd,J=9.2,2.6Hz,1H),4.10(dd,J=56.9 ,13.2Hz,1H),3.76–3.45(m,3H),3.25(td,J=11.6,2.6Hz,1H),3.19–2.92(m,3H),2.83–2.54(m,3H),2.38–2.24(m,4H),2.21–2.06(m,1H).

[0345] Example 34: Preparation of compound Z-34

[0346]

[0347] Example 34 can be prepared by referring to the method of Example 31, except that (4S)-4-hydroxypyrrolidone-2-one is used instead of (4R)-4-hydroxypyrrolidone-2-one, to obtain compound Z-34 (23 mg). MS m / z (ESI): 506.2 [M+1] +;1HNMR (400MHz, DMSO) δ8.70(t,J=6.4Hz,1H),7.56(dd,J=42.4,8.7Hz,3H),5.45(d,J=3.9Hz,1H),4.41(d,J=5.1Hz,1H),4 .20–3.93(m,2H),3.75–3.61(m,2H),3.59–3.44(m,1H),3.32–2.69(m,5H),2.45–2.12(m,4H),1.19(dd,J=55.6,6.7Hz,2H).

[0348] Example 35: Preparation of compound Z-35

[0349]

[0350] Compound Z-33 (20 mg, 37.49 μmol) and ammonium chloride (4.01 mg, 74.97 μmol) were dissolved in DMF (5 mL). HATU (14.14 mg, 37.49 μmol) and N,N-diisopropylethylamine (4.84 mg, 37.49 μmol, 6.53 μL) were added, and the mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography (21.2 x 250 mm C18 column; system: 10 mM NH4HCO3 H2O; wavelength: 254 / 214 nm; gradient: 30%–60% acetonitrile) to obtain compound Z-35 (8 mg). MS m / z (ESI): 533.2 [M+1] + ;1H NMR (400MHz, DMSO) δ8.68(dd,J=9.4,5.5Hz,1H),7.86(s,1H),7.60–7.24(m,4H),4.76(d,J=8.5Hz,1H),4.08(dd,J=56.6 ,13.2Hz,1H),3.74–3.35(m,3H),3.28–2.91(m,4H),2.84–2.53(m,2H),2.46–2.16(m,5H),1.97(dd,J=12.5,9.5Hz,1H).

[0351] Example 36: Preparation of compound Z-36

[0352]

[0353] Example 36 can be prepared by referring to the method of Example 29, except that methyl (2R)-5-oxopyrrolidine-2-carboxylic acid is replaced with oxazolidinyl but-2-one to obtain compound Z-36 (13.32 mg). MS m / z (ESI): 492.2 [M+1]+ ; 1 HNMR(400MHz,DMSO-d6)δ8.70(dd,J=9.0,6.0Hz,1H),7.49(dd,J=16.0,8.0Hz,3H),4.49(t,J=8.0Hz ,2H),4.21–3.96(m,3H),3.76–3.38(m,3H),3.31–3.11(m,1H),3.07–2.75(m,3H),2.42–2.22(m,4H).

[0354] Example 37: Preparation of compound Z-37

[0355]

[0356] Example 37 can be prepared by referring to the method of Example 19, except that acetyl chloride is used instead of deuterated acetyl chloride, to obtain compound Z-37. MS m / z (ESI): 469.2 [M+H] + ; 1 H NMR(400MHz, CDCl3)δ8.14(d,J=7.0Hz,1H),7.47–7.33(m,3H),6.66(dd,J=17.9,6.7H z,1H),4.33(t,J=13.0Hz,1H),3.92–3.75(m,3H),3.49(dd,J=21.4,7.4Hz,2H),3.36(d d,J=15.8,6.8Hz,1H),3.21–2.92(m,3H),2.77(dd,J=12.9,10.8Hz,1H),2.65(td,J=8. 0,4.8Hz,2H),2.43–2.36(m,3H),2.21(dt,J=14.2,7.2Hz,2H),1.98(d,J=35.8Hz,3H).

[0357] Example 38: Preparation of compound Z-38

[0358]

[0359] Example 38 can be prepared by referring to the method of Example 29, except that methyl chloroformate is used instead of deuterated acetyl chloride, to obtain compound Z-38. MS m / z (ESI): 485.2 [M+H] + .

[0360] Test Example 1: Screening of compounds for hP2X3 / hP2X using FLIPR assay 2 / 3 antagonistic activity of receptors

[0361] Material:

[0362]

[0363] Cell preparation: Cells 1321N1 / hP2X3 and 1321N1 / hP2X2 / 3 (supplier: Chempartner) were stably transfected using Versene digestion solution. After centrifugation, the cells were resuspended in plating medium (DMEM + 10% DFBS) and counted. The cell volume was adjusted to 3*10⁻⁶ cells / year. 5 Cells / mL, 50 μL of cells were seeded into each well of a 384-well test plate and incubated in a 5% CO2, 37°C incubator for 16-24 h.

[0364] Cell culture medium formulation:

[0365] hP2X3 working fluid concentration Stock solution concentration Dilution factor Required volume (ml) DMEM 1* 1* 1 447.006 FBS 10％ 100％ 10 50 G418 disulfate 300μg / ml 50mg / ml 167 2.994012 Sample volume 500 hP2X2 / 3 working fluid concentration Stock solution concentration Dilution factor Required volume (ml) DMEM 1* 1* 1 447.7489 FBS 10％ 100％ 10 50 G418 disulfate 150μg / ml 50mg / ml 333 1.501502 Hygromycin B 75μg / ml 50mg / ml 667 0.749625 Sample volume 500

[0366] Experimental Dye Recipe:

[0367] hP2X3 working fluid concentration Stock solution concentration Dilution factor Required volume (ml) 10*dye stock 0.5* 10* 20 0.7 Probenecid 1.25 0.25 200 0.07 Test buffer 1* 1* 1 13.23 ATP hydrolytic enzyme 0.5U / ml 10μl = 1U 200 0.07 Sample volume 14 hP2X2 / 3 working fluid concentration Stock solution concentration Dilution factor Required volume (ml) 10*dye stock 0.5* 10* 20 0.7 Probenecid 1.25 0.25 200 0.07 Test buffer 1* 1* 1 13.23 Sample volume 14

[0368] Compound preparation: 1. Test sample: Prepare the required concentration of the test compound (54 mM DMSO stock solution) at 180 times the concentration using DMSO in a 384-well polypropylene microplate conforming to Echo standards. Add 500 nmol to each well of the 384-well plate and supplement with 30 μL of test buffer (containing 1.26 mM Ca). 2+ Mix 1*HBSS+2mM CaCl2+20mM HEPES) and shake for 20-40 minutes to ensure homogeneity.

[0369] 2. Agonists: Prepare 3 times the required concentration of agonists (α,β-meATP) (hP2X3 and hP2X) using test buffer. 2 / 3 All cells require a final concentration of 3000 nM. Add 45 μL of agonist to each well of a 384-well compound plate.

[0370] Dye incubation: Remove the cell plate, aspirate the cell supernatant, and add 30 μL of dye to each well. Calcium 4 Assay Kit (diluted with test buffer), incubate for 1 hour.

[0371] FLIPR assay: Add 15 μL of the compound to each well of the cell plate (FLIPR instrument loading). After 15 minutes, add 22.5 μL of the agonist to each well and detect the fluorescence signal (excitation wavelength 470 nm-495 nm, emission wavelength 515 nm-575 nm).

[0372] Data processing: The difference between the signal peak and trough was used as the basic data. The highest concentration of the positive control drug was taken as the 100% inhibition rate, and the DMSO data was taken as the 0% inhibition rate. The inhibition effect curve of the compound was fitted in Graphpad Prism 6 software using (log(inhibitor) vs. response -- variable slope) and the IC50 was calculated. 50 value.

[0373] Experimental uniformity standard: For each plate, ≥12 max values ​​(results from DMSO action) and ≥12 min values ​​(results from the highest concentration of positive control agent) are recorded. The Z-value is calculated. If Z ≥ 0.5, the parallel wells are considered uniform and the data are reliable. The formula for calculating the Z-value is: Z = 1 - 3 * (SDmax + SDmin) / (MEANmax - MEANmin).

[0374] Table 1

[0375]

[0376] As shown in Table 1, the compounds in the embodiments of the present invention have high inhibitory activity against P2X3 and low inhibitory activity against P2X2 / 3, exhibiting significant inhibitory selectivity.

[0377] Test Example 2: In vivo pharmacokinetic test in rats

[0378] The drug concentration in the plasma of rats after intravenous injection and gavage administration of the compound of the present invention was determined by LC / MS / MS at different time points to study the pharmacokinetic behavior of the compound of the present invention in rats and evaluate its pharmacokinetic characteristics.

[0379] Experimental plan:

[0380] Experimental animals: Healthy adult male SD rats (6 rats, weighing 200-300g each; the intravenous injection group had free access to water and food, while the gavage group was fasted overnight and then had free access to water and food 4 hours after administration), provided by Vital River Laboratory Animal Co.LTD.

[0381] Administration route and dosage: SD rats were administered via tail vein (1 mg / kg, 5% DMSO in 0.9% saline) and by gavage (5 mg / kg, 5% DMSO in 0.9% saline).

[0382] Blood Sampling: Animals meeting experimental requirements were selected, weighed, and marked before drug administration. Before blood sampling, rats were tethered, and blood was collected from each administered rat at predetermined time points (for intravenous administration: blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 hours post-administration, for a total of 8 time points; for gavage administration: blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 hours post-administration, for a total of 8 time points), approximately 200 μL of blood was collected via the orbital rim. The blood was transferred to 1.5 mL tubes pre-filled with K2EDTA, centrifuged for 6 min (8000 rpm, 4℃), and the plasma was collected. The entire process was completed within 15 min of blood collection. All samples were stored at -20℃ until analysis. Drug concentration was determined using LC / MS / MS.

[0383] Table 2 shows the pharmacokinetic properties of some of the compounds in this invention administered intravenously at the same dose in rats.

[0384] Table 2

[0385]

[0386] The pharmacokinetic properties of some of the compounds in this invention, administered via gavage at the same dose in rats, are shown in Table 3.

[0387] Table 3

[0388]

[0389] Positive compound D1

[0390]

[0391] As can be seen from the table above, the compound structure has a significant impact on the pharmacokinetic parameters. The compound protected by this invention has a higher in vivo exposure and a lower clearance rate.

[0392] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: （I） in, R1 is hydrogen, C 1-6 Alkyl or halogen; R2 is hydrogen or halogen; (A)Q is hour; R3 is hydrogen; R4 is -C(O)R 4a ; R 4a To replace C 1-6 Alkyl, wherein the substituted C 1-6 The alkyl group is substituted by one or more substituents independently selected from the group consisting of: deuterium, hydroxyl; Z5 is CH2; for ; (R5) n Representing the ring The hydrogen atoms on the surface are replaced by n R5 atoms, where n is 0; (B) When Q is -C(O)NHCH3; R3 and R4 are connected, forming a 5- to 6-membered heterocyclic alkenyl ring or a 5- to 6-membered heteroaryl ring together with the connected carbon and nitrogen atoms, wherein the 5- to 6-membered heterocyclic alkenyl ring is... The 5- to 6-membered heteroaryl ring is The 5- to 6-membered heterocyclic alkenyl ring and the 5- to 6-membered heteroaryl ring are unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the group consisting of: C 1-3 Alkyl, C 3-6 cycloalkyl; for .

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, R1 is methyl; R2 is hydrogen.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, R1 is methyl; R2 is fluorine.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound of formula (I) has the structure shown in formula (II): （Ⅱ）， In this structure, R3 and R4 are connected, and together with the connected carbon and nitrogen atoms, they form the following structure: a 5- to 6-membered heterocyclic alkenyl ring. ; 5 to 6-membered heteroaryl rings are The 5- to 6-membered heterocyclic alkenyl ring and the 5- to 6-membered heteroaryl ring are unsubstituted or substituted by 1, 2 or 3 substituents independently selected from the following group: C 1-3 Alkyl, C 3-6 Cycloalkyl.

5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, characterized in that, When R3 and R4 are connected, together with the attached carbon and nitrogen atoms, they form the following structure: , or .

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound of formula (I) has the structure shown in formula (III): （Ⅲ）。 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, characterized in that, R 4a C replaced by one or more deuterium 1-6 Alkyl groups, or C groups substituted with 1, 2, or 3 hydroxyl groups 1-6 alkyl.

8. The compound of claim 6, or a pharmaceutically acceptable salt thereof, characterized in that, R 4a It is a trideuterated methyl (CD3), a hydroxylated methyl, or a hydroxylated isopropyl.

9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound of formula (I) is any one of the following compounds: 。 10. A pharmaceutical composition, characterized in that, include: 1) The compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof; and 2) Pharmaceutically acceptable carrier.

11. The use of any compound of claims 1-9, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 10, in the preparation of a medicament for treating diseases associated with P2X3 activity or P2X2 / 3 activity.

12. The application according to claim 11, characterized in that, The diseases associated with P2X3 activity or P2X2 / 3 activity are pain, urinary tract disorders, gastrointestinal disorders, cancer, immune-related diseases, cough, depression, anxiety, or stress-related conditions.

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