Aromatic ring derivative, preparation method therefor and use thereof

Abstract

The present invention relates to an aromatic ring derivative, a preparation method therefor, and the use of a pharmaceutical composition containing the derivative in medicine. Specifically, the present invention relates to an aromatic ring derivative represented by general formula (I), a preparation method therefor, and the use thereof as a therapeutic agent, especially as a WRN inhibitor. The definition of each substituent in general formula (I) is the same as that in the description.

Description

Aromatic ring derivatives, methods of making and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to the following patent applications: Patent application entitled “Aromatic ring derivatives, methods of making and uses thereof” filed with the China National Intellectual Property Office on December 11, 2024, application number CN202411817983.8; Patent application entitled “Aromatic ring derivatives, methods of making and uses thereof” filed with the China National Intellectual Property Office on April 9, 2025, application number CN202510442717.X; and Patent application entitled “Aromatic ring derivatives, methods of making and uses thereof” filed with the China National Intellectual Property Office on June 6, 2025, application number CN202510760775.7, the contents of each of the above patent applications are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to a kind of aromatic ring derivatives, its preparation method and the pharmaceutical composition containing the derivative and its purposes as therapeutic agent, especially as WRN inhibitor. BACKGROUND

[0004] WRN of human is composed of 1432 amino acid residues, and contains five important components from N terminal to C terminal—exonuclease domain, ATPase domain, RecQ carbon terminal domain, helicase / ribonuclease D carbon terminal domain and nuclear localization signal.Human body five kinds of RecQ helicases, WRN is the only one with 3'→5' exonuclease activity, which is realized by the specific activation of its N-terminal exonuclease domain by Ku70 / 80 complex bound to DNA ends.ATPase domain is the largest and most conserved component in RecQ helicase family, which acts as an ATP-dependent DNA translocation module by binding and hydrolyzing ATP. RecQ carbon terminal domain is the main site of DNA binding and catalyzes the unwinding of DNA double strand. Therefore, ATPase domain and RecQ carbon terminal domain jointly constitute the core of WRN helicase.WRN is a DNA helicase with multiple enzyme activities that can bind to DNA and other proteins.This makes the enzyme play an important role in maintaining genome integrity and stability, including participating in DNA damage repair, replication and transcription, and maintaining telomere and heterochromatin stability.

[0005] Synthetic lethality refers to the phenomenon that two non-lethal genes are simultaneously inhibited (inhibited forms include gene defects such as gene mutation, gene silencing, and / or molecular perturbations such as gene expression knock-out, drug inhibition) to cause cell death. Using this mechanism, a specific mutation in cancer is found, and its "synthetic lethal partner" is found and inhibited, thereby specifically killing cancer cells with the mutation.

[0006] Studies have shown that WRN is a "synthetic lethal partner" of high microsatellite instability (MSI-H), a type of genomic damage. High microsatellite instability is a hyper-variable state caused by frequent insertion and / or deletion mutations in nucleotide repeat regions due to defects in DNA mismatch repair (MMR), which is commonly seen in endometrial cancer (31%), colorectal cancer (25%), and gastric cancer (19%) and other cancers. In MSI-H cancer cells, thymine / adenine dinucleotide (TA) repeat sequences are highly unstable and undergo large-scale amplification, forming non-canonical right-handed double helix (non-B) DNA secondary structures (such as cruciform and G-quadruplex), which require WRN-specific unwinding to complete replication. In the absence of WRN, these DNA secondary structures are cut by the MUS81-EME1-SLX4 endonuclease complex, leading to extensive DNA end resection, depletion of replication protein A (RPA), chromosomal fragmentation, and cell death. In addition, in tumor models with MMR defects, the absence of WRN leads to the activation of multiple DNA damage signaling markers, inducing cell cycle arrest and apoptosis, thereby inhibiting the proliferation of tumor cells. Recent studies have shown that WRN small molecule inhibitors can specifically cause tumor regression in MSI-H tumor models, but have no effect in microsatellite stable (MSS) tumor models. Therefore, small molecule chemicals that can inhibit WRN helicase activity are expected to become a new method for effectively treating MSI-H cancer.

[0007] There is no effective marketed drug for WRN target inhibitors, and there is still a lack of satisfactory WRN inhibitors for treating tumor diseases, and there is a huge unmet clinical need in this regard. SUMMARY

[0008] To solve the above technical problems, the present application provides a compound represented by general formula (I) or a stereoisomer, tautomer, deuterated product or pharmaceutically acceptable salt thereof:

[0009] wherein:

[0010] a bond denotes may exist as (Z)- or (E)-stereoisomers;

[0011] X is selected from CH and N;

[0012] Y is selected from CR b and N;

[0013] Z is selected from CH and N;

[0014] R 1 is selected from C 2-6 alkenyl and C 2-6 alkynyl, wherein said C 2-6 alkenyl or C 2-6 alkynyl is optionally substituted with one or more R c ;

[0015] ring A is selected from phenyl, C 3-10 cycloalkyl, C 3-8 cycloalkenyl and 3-10 membered heterocyclyl, wherein said 3-10 membered heterocyclyl contains at least one double bond;

[0016] R c is each independently selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl and 5-6 membered heteroaryl, wherein said C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-6 membered heteroaryl is optionally substituted with one or more R j ;

[0017] Alternatively, two R c together with the same carbon atom to which they are attached form a -C(=0);

[0018] Alternatively, two R c together with the same carbon atom to which they are attached form a C 3-10 cycloalkyl or 3-10 membered heterocyclyl; wherein said C 3-10 cycloalkyl or 3-10 membered heterocyclyl is optionally substituted with one or more R j ;

[0019] R j is each independently selected from deuterium atom, hydroxy, halogen, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C1-6 Halogenated alkoxy groups, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 heteroaryl, =O, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 ;

[0020] R 2 Selected from C 1-6 Haloalkyl, C 2-6 Alkyne, -SF5 and -S(=O)2R e ; wherein C 2-6 The alkynyl group may be further selected by one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups;

[0021] R e Selected from C 1-6 Halogenated alkyl groups, preferably trifluoromethyl;

[0022] R b Selected from hydrogen atom, deuterium atom, halogen, nitro, cyano, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR7 , -NR 6 R 7 , -C(=O)NR 6 R 7 , -S(=O) r NR 6 R 7 , and -S(=O) r R 5 wherein said C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6- 10 aryl, 5-6 membered heteroaryl is optionally further substituted with one or more substituents independently selected from the group consisting of hydroxy, halogen, nitro, cyano, alkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR 8 , =O, -C(=O)R 8 , -C(=O)OR 8 , -OC(=O)R 8 , -NR 9 R 10 , -C(=O)NR 9 R 10 , -S(=O)2NR 9 R 10 , -N(R 9 )C(=O)R 10 , and -N(R 9 )C(=O)OR 10 ;

[0023] with the proviso that when is optionally substituted phenyl or optionally substituted C 3-10 cycloalkyl and R 2 is C 1-6 haloalkyl, Y is CR b , and R b is not selected from the group consisting of hydrogen atom, cyano, halogen and C 1-6 alkyl;

[0024] R 4 is selected from the group consisting of C 1-6 alkyl and C 3-10 cycloalkyl; wherein said C 1-6 alkyl or C 3-10 cycloalkyl is optionally further substituted with one or more substituents independently selected from the group consisting of deuterium atom, halogen, hydroxy, cyano, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-Substituents of 6-haloalkoxy groups;

[0025] R d Selected from hydrogen atoms, C 1-6 Alkyl and -C 0-6 Alkylene-R A ;

[0026] R A Selected from hydroxyl, alkenyl, alkynyl, cyano, C 3-12 cycloalkyl, C 6-10 aryl, 3-12 heterocyclic, 5-10 heteroaryl, and 8-10 fused rings, wherein the alkenyl, alkynyl, C 3-12 cycloalkyl, C 6-10 Aryl, 3-12 heterocyclic, 5-10 heteroaryl, or 8-10 fused ring optionally further modified by one or more R B Replaced;

[0027] R B Each is independently selected from deuterium, hydroxyl, halogen, nitro, cyano, and C atoms. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 aryl, or heteroaryl groups may be further divided by one or more R groups. C Replaced;

[0028] Or, two Rs BTogether with the same carbon atom it is attached to, it forms a -C (=O);

[0029] R C Each is independently selected from deuterium, hydroxyl, halogen, nitro, cyano, and C atoms. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 heteroaryl, optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Hydroxyalkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -OR 8 =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 -N(R) 9 )C(=O)R 10 and -N(R)9 )C(=O)OR 10 The substituents are replaced;

[0030] Or, two Rs C Together with the same carbon atom it is attached to, it forms a -C (=O);

[0031] R f Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 3-10 Cycloalkyl and 3-10 membered heterocyclic groups; wherein C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic groups optionally further selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups;

[0032] R g Each atom is independently selected from hydrogen atoms and deuterium atoms, preferably hydrogen atoms;

[0033] R 5 Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl and 5-6 heteroaryl, wherein the C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-6 heteroaryl groups may be further selected from one or more deuterium atoms, hydroxyl groups, halogens, nitro groups, cyano groups, C6 groups, etc. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 and -N(R) 9 )C(=O)R10 The substituents are replaced;

[0034] R 6 and R 7 Each is independently selected from hydrogen atoms, hydroxyl groups, and C atoms. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6- 10 Aryl and 5-6 membered heteroaryl, wherein the C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-6 heteroaryl groups may be further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-6 heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 and -N(R) 9 )C(=O)R 10 The substituents are replaced;

[0035] Or, R 6 and R 7 The atoms bonded to them together form a structure containing one or more N, O, or S atoms (=O). r The 4-8 membered heterocyclic group, wherein the 4-8 membered heterocyclic group is optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-6 heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 and -N(R) 9 )C(=O)R 10The substituents are replaced;

[0036] R 8 R 9 and R 10 Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl, amino, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl and 5-6-membered heteroaryl, wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is optionally further selected from one or more groups selected from hydroxyl, halogen, nitro, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Substituents include aryl, 5-6 heteroaryl, carboxyl, and carboxylic acid ester groups;

[0037] n is 0, 1, 2, 3, 4, or 5; and

[0038] r can be 0, 1, or 2 independently.

[0039] In some preferred embodiments of the present invention, a compound of general formula (I) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein: R 1 Selected from C 2-6 alkenyl and C 2-6 alkynyl group, wherein the C 2- 6-olefin or C 2-6 The alkynyl group is optionally surrounded by one or more R groups. c replace;

[0040] R c Each is independently selected from C 3-10 cycloalkyl, wherein the C 3-10 cycloalkyl groups are optionally surrounded by one or more R groups. j Replaced;

[0041] Or, two Rs c Together with the same carbon atom it is attached to, they form a C 3-10 Cycloalkyl or 3-10 membered heterocyclic group; wherein the C 3-10 Cycloalkyl or 3-10 membered heterocyclic groups are optionally surrounded by one or more R groups. j Replaced;

[0042] R j The definition is as stated in general formula (I).

[0043] In some preferred embodiments of the present invention, a compound of general formula (I) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein R 1 Selected from the following groups:

[0044] In some preferred embodiments of the present invention, a compound of general formula (I) or a stereoisomer, tautomer, deuterated product or a pharmaceutically acceptable salt thereof is provided, which is a compound of general formula (II) or a stereoisomer, tautomer, deuterated product or a pharmaceutically acceptable salt thereof:

[0045] Among them, rings A, X, Y, Z, and R d R g R f R j R 2 R 4 The definitions of n are as described in general formula (I).

[0046] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from C 3-8 Cycloalkenyl and 3-10 membered heterocyclic groups, wherein the 3-10 membered heterocyclic group contains at least one double bond.

[0047] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 Cycloalkyl.

[0048] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 cycloalkyl, R 2 Selected from -SF5, C 2-6 Alkyne group and -S(=O)2R e ; wherein C 2-6 The alkynyl group may be further selected by one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups;

[0049] R e Selected from C 1-6 Halogenated alkyl group, preferably trifluoromethyl.

[0050] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 cycloalkyl,

[0051] R 2 Selected from -SF5, C 2-6 Alkyne group and -S(=O)2R e ; wherein C 2-6 The alkynyl group may be further selected by one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups;

[0052] R e Selected from C 1-6 Halogenated alkyl groups, preferably trifluoromethyl;

[0053] Y is selected from N or CR b ,

[0054] R b Selected from hydrogen atom, deuterium atom, cyano group, halogen, -OR 5 and -NR 6 R 7 ;

[0055] R 5 R 6 R 7 Each is independently selected from hydrogen atoms or C atoms. 1-6 alkyl.

[0056] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 cycloalkyl,

[0057] R 2 Selected from -SF5, C 2-6 Alkyne group or -S(=O)2R e ; wherein C 2-6 The alkynyl group may be further selected by one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups;

[0058] R eSelected from C 1-6 Halogenated alkyl groups, preferably trifluoromethyl;

[0059] Y is selected from N or CR b ,

[0060] R b It consists of hydrogen atoms, deuterium atoms, fluorine, chlorine, bromine, cyano, amino, dimethylamino, hydroxyl, and methoxy groups.

[0061] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 cycloalkyl,

[0062] R 2 Selected from C 1-6 Halogenated alkyl groups;

[0063] Y is CR b ;

[0064] R b Selected from deuterium atoms, nitro groups, and C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 heteroaryl, optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, alkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -OR 8 =O, -C(=O)R8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 -N(R) 9 )C(=O)R 10 and -N(R) 9 )C(=O)OR 10 The substituents are replaced by the substituents.

[0065] r can be 0, 1, or 2 independently.

[0066] R 5 R 6 R 7 R 8 R 9 and R 10 The definition is as described in claim 1.

[0067] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 cycloalkyl,

[0068] R 2 Selected from C 1-6 Halogenated alkyl groups;

[0069] Y is CR b ;

[0070] R b Selected from deuterium atoms, nitro groups, and C 2-6 alkenyl, C 2-6 alkynyl group, -OR 5 -NR 6 R 7 -C(=O)R 5 -S (=O) r R 5 3-10 membered heterocyclic groups, C 3-10 cycloalkyl and 5-6-membered heteroaryl; wherein the C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 heterocyclic, 5-6 heteroaryl groups optionally further selected by one or more hydroxyl, halogen, cyano, C 1- 6-alkyl and C 1-6 Substituents of haloalkyl groups;

[0071] R 5 R 6 R 7 Each is independently selected from hydrogen atoms and C atoms. 1-6 alkyl;

[0072] r is 2.

[0073] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from phenyl and C. 3-10 cycloalkyl,

[0074] R 2 Selected from C 1-6 Halogenated alkyl groups;

[0075] Y is CR b ;

[0076] R b Selected from the following groups: deuterium, nitro, hydroxyl, methoxy, amino, dimethylamino, vinyl, ethynyl.

[0077] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein ring A is selected from the following groups:

[0078] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein R 4 Selected from C 1-6 Alkyl group, preferably methyl group.

[0079] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein R d Selected from hydrogen atoms.

[0080] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein R f Selected from C 3-10 Cycloalkyl, preferably cyclopropyl.

[0081] In some preferred embodiments of the present invention, a compound of general formula (I) or (II) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof is provided, wherein R 2 Selected from SF5

[0082] In a preferred embodiment of the present invention, the compound of the general formula is selected from the compounds in Table 1 below:

[0083] Table 1. Some compounds of the present invention

[0084] Or its stereoisomers, tautomers, or medicinal salts.

[0085] Note: If there is a discrepancy between the drawn structure and the given name of the structure, the drawn structure shall prevail.

[0086] Furthermore, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or (II) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0087] The present invention provides the use of a compound of general formula (I) or (II) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a WRN inhibitor.

[0088] The present invention also provides the use of a compound of general formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of a medicament for treating or preventing WRN-mediated diseases, wherein the WRN-mediated diseases are preferably highly microsatellite unstable (MSI-H) cancers; wherein the WRN-mediated diseases are selected from colorectal cancer, gastric cancer, endometrial cancer, rectal adenocarcinoma, adrenocortical carcinoma, uterine sarcoma, cervical cancer, nephroblastoma, mesothelioma, esophageal cancer, breast cancer, clear cell renal cell carcinoma, ovarian serous cystadenocarcinoma, bile duct cancer, thymoma, liver cancer, head and neck squamous cell carcinoma, sarcoma, melanoma of the skin, squamous cell carcinoma of the lung, prostate cancer, lung adenocarcinoma, transitional cell carcinoma of the bladder, pediatric neuroblastoma, chronic lymphocytic leukemia or glioma, preferably colorectal cancer, gastric cancer or endometrial cancer.

[0089] The present invention further provides the use of a compound of formula (I) or (II) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for the treatment or prevention of highly microsatellite instability (MSI-H) cancer.

[0090] This invention provides the use of a compound of general formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of a medicament for the treatment or prevention of colorectal cancer, gastric cancer, endometrial cancer, rectal adenocarcinoma, adrenocortical carcinoma, uterine sarcoma, cervical cancer, nephroblastoma, mesothelioma, esophageal cancer, breast cancer, clear cell renal cell carcinoma, ovarian serous cystadenocarcinoma, bile duct carcinoma, thymoma, liver cancer, head and neck squamous cell carcinoma, sarcoma, melanoma of the skin, squamous cell carcinoma of the lung, prostate cancer, lung adenocarcinoma, transitional cell carcinoma of the bladder, neuroblastoma of children, chronic lymphocytic leukemia or glioma, preferably in the preparation of a medicament for the treatment or prevention of colorectal cancer, gastric cancer or endometrial cancer.

[0091] Accordingly, this application provides a method for treating or preventing WRN-mediated diseases, comprising administering to a subject in need a compound of formula (I) or (II) of this application, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in this application. The WRN-mediated diseases described herein are selected from colorectal cancer, gastric cancer, endometrial cancer, rectal adenocarcinoma, adrenocortical carcinoma, uterine sarcoma, cervical cancer, nephroblastoma, mesothelioma, esophageal cancer, breast cancer, clear cell renal cell carcinoma, ovarian serous cystadenocarcinoma, bile duct cancer, thymoma, liver cancer, head and neck squamous cell carcinoma, sarcoma, skin melanoma, lung squamous cell carcinoma, prostate cancer, lung adenocarcinoma, bladder transitional cell carcinoma, pediatric neuroblastoma, chronic lymphocytic leukemia, and glioma, preferably colorectal cancer, gastric cancer, or endometrial cancer. This application also provides a method for treating or preventing cancers with high microsatellite instability (MSI-H), comprising administering to a subject in need a compound of formula (I) or (II) of this application or a stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in this application.

[0092] The compounds of this invention have been shown to have good inhibitory effects on WRN helicase, SW48 cell proliferation, RL95-2 cell proliferation, and HCT116 cell proliferation; the compounds of this invention also have high permeability and no efflux; in addition, the compounds of this invention also have good plasma stability and efficacy.

[0093] Detailed description of the invention

[0094] Unless otherwise stated, some terms used in this specification and claims are defined as follows:

[0095] When "alkyl" is used as a group or part of a group, it refers to a group consisting of C1-C2. 20 Straight-chain or branched aliphatic hydrocarbon groups. Preferably C1-C. 10Alkyl groups, more preferably C1-C6 and C1-C4 alkyl groups. Examples of alkyl groups include, but are not limited to, 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, etc. The alkyl group may be substituted or unsubstituted.

[0096] "Alkenyl" refers to an alkyl group as defined above, consisting of at least two carbon atoms and at least one carbon-carbon double bond. Representative examples include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl. C2-C6 alkenyl groups are preferred, such as C2-C4 alkenyl groups. Alkenyl groups may be optionally substituted or unsubstituted.

[0097] "Alkyne group" refers to an aliphatic hydrocarbon group containing a single carbon-carbon triple bond, which can be straight-chain or branched. C2-C is preferred. 10 The alkynyl group is preferred, more preferably C2-C6 alkynyl, and most preferably C2-C4 alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl. The alkynyl group may be substituted or unsubstituted.

[0098] "Cycloalkyl" refers to a non-aromatic saturated cyclic alkyl group in which one or more cyclic atoms are carbon atoms, including monocyclic, polycyclic, fused, bridged, and spirocyclic rings, preferably having 3 to 10-membered rings, such as 3 to 7-membered monocyclic or 5 to 18-membered bicyclic or tricyclic rings.

[0099] Examples of "monocycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, and cyclopentyl.

[0100] Monocyclic alkyl groups can be substituted or unsubstituted.

[0101] "Spirocycloalkyl" refers to a 5- to 18-membered polycyclic aromatic hydrocarbon group with two or more ring structures, wherein the monocyclic rings share a carbon atom (called a spiro atom) with each other, preferably 6- to 14-membered, more preferably 7- to 10-membered. Based on the number of shared spiro atoms between the rings, spirocycloalkyl is classified into monospiro, bispiro, or polyspirocycloalkyl, preferably monospiro and bispirocycloalkyl, preferably 4 / 5, 4 / 4, 4 / 6, 3 / 6, 5 / 5, or 5 / 6. Non-limiting examples of "spirocycloalkyl" include, but are not limited to: spiro[4.5]decyl, spiro[4.4]nonyl, spiro[3.5]nonyl, and spiro[2.4]heptyl; spirocycloalkyl can be substituted or unsubstituted.

[0102] "Fused cycloalkyl" refers to a 5- to 18-membered saturated all-carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms, preferably 6- to 14-membered, more preferably 6- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or more cyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 3-membered / 5-membered, 5-membered / 5-membered, or 5-membered / 6-membered bicyclic fused cycloalkyl. Non-limiting examples of "fused cycloalkyl" include, but are not limited to: bicyclo[3.1.0]hexyl, bicyclo[3.2.0]hept-1-enyl, bicyclo[3.2.0]heptyl, decahydronaphthyl, tetradecylhydrophenanthrene; the fused cycloalkyl can be substituted or unsubstituted.

[0103] "Bridged cycloalkyl" refers to a 5- to 18-membered polycyclic aromatic hydrocarbon group containing two or more cyclic structures sharing two non-directly connected carbon atoms. It is preferably 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or more cyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to: (1s,4s)-bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, (1s,5s)-bicyclo[3.3.1]nonyl, bicyclo[2.2.2]octyl, (1r,5r)-bicyclo[3.3.2]decyl. The bridged cycloalkyl may be substituted or unsubstituted.

[0104] "Cycloalkenyl" refers to a non-aromatic unsaturated cyclic alkyl group in which all the cyclic atoms are carbon atoms, and at least two carbon atoms have carbon-carbon double bonds. Cycloalkenyl includes monocyclic cycloalkenyl and polycyclic cycloalkenyl. Polycyclic cycloalkenyl includes fused rings, bridged rings and spiro rings. Cycloalkenyl is preferably a 3-8 membered cycloalkenyl, such as a 3-7 membered monocyclic or a 5-18 membered bicyclic or tricyclic.

[0105] Examples of “monocyclic alkenyl” include, but are not limited to,

[0106] The monocyclic alkenyl group can be substituted or unsubstituted.

[0107] "Spirocycloalkenyl" refers to an unsaturated, fully carbon-based polycyclic group with 5 to 18 quintiles, consisting of two or more cyclic structures, where the monocyclic rings share a carbon atom (called a spiro atom) with each other. One or more rings contain at least one double bond, but none of the rings have fully conjugated π electrons. Preferably, it is a 6 to 14 quintile group, more preferably a 7 to 10 quintile group. Based on the number of shared spiro atoms between the rings, spirocycloalkenyl groups are classified as monospiro, dispiro, or polyspirocycloalkenyl groups, preferably monospiro and dispirocycloalkenyl groups, and preferably 4 / 5, 4 / 4, 4 / 6, 3 / 6, 5 / 5, or 5 / 6 quintile groups. Non-limiting embodiments of "spirocycloalkenyl" include, but are not limited to,

[0108] Spirocycloalkenes can be substituted or unsubstituted.

[0109] "Fused-ring alkenyl" refers to an 5- to 18-membered, unsaturated, fully carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms, with at least one double bond within one or more rings, but none of the rings having fully conjugated π electrons; preferably 6- to 14-membered, more preferably 6- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or more cyclic fused-ring alkenyl, preferably bicyclic or tricyclic, more preferably 3-membered / 5-membered, 5-membered / 5-membered, or 5-membered / 6-membered bicyclic fused-ring alkyl. Non-limiting examples of "fused-ring alkenyl" include, but are not limited to: The fused-cyclic alkenyl group can be substituted or unsubstituted.

[0110] "Bridged cyclic alkenyl" refers to an aromatic system with 5 to 18 cyclic members, containing two or more cyclic structures, sharing two unsaturated, fully carbon-based polycyclic groups that are not directly connected to each other, and where one or more rings contain at least one double bond, but none of the rings have fully conjugated π electrons. Preferably, it is a 6 to 14 cyclic alkenyl group, more preferably a 7 to 10 cyclic alkenyl group. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or more cyclic bridged cyclic alkenyl groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cyclic alkenyl" include, but are not limited to: Bridged cycloalkyl groups can be substituted or unsubstituted.

[0111] The terms “heterocyclic group,” “heterocyclic alkyl group,” “heterocyclic,” or “heterocyclic” are used interchangeably in this application and all refer to a non-aromatic heterocyclic group in which one or more cyclic atoms are selected from nitrogen, oxygen, or S(O). r(where r is selected from 0, 1 or 2) heteroatoms, containing 0, 1 or more double bonds in the ring, including monocyclic, polycyclic, fused ring, bridged ring and spirocyclic, preferably 3-10 membered heterocyclic groups, for example having 3 to 8 membered monocyclic or 5 to 18 membered bicyclic or tricyclic, which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and / or sulfur.

[0112] The heterocyclic group can be substituted or unsubstituted.

[0113] Examples of "monocyclic heterocyclic groups" include, but are not limited to, morpholino, oxetane, azabolane, thiomorpholino, tetrahydrofurano, tetrahydropyrano, 1,1-dioxo-thiomorpholino, piperidino, 2-oxo-piperidino, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazine-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl, piperazine, hexahydropyrimidine,

[0114] "Aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term "aryl" includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, and tetrahydronaphthyl aromatic groups. Preferably, the aryl group is C6-C. 10 Aryl, more preferably phenyl and naphthyl, most preferably naphthyl. The aryl group can be substituted or unsubstituted.

[0115] "Heteroaryl" refers to an aromatic 5- to 6-membered monocyclic or 8- to 10-membered bicyclic ring, which may contain 1 to 4, for example 1, 2 or 3 atoms selected from nitrogen, oxygen and sulfur. Preferred heteroaryl groups are 5- to 10-membered heteroaryl groups, for example 5- to 6-membered heteroaryl groups; the heteroaryl group may contain 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur. Examples of "heteroaryl" compounds include, but are not limited to, furanyl, pyridyl, 2-oxo-1,2-dihydropyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiopheneyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrroleyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzo[m]dioxacyclopentenyl, benzo[thiophene], benzimidazolyl, indoleyl, isoyindolyl, 1,3-dioxo-isoindolyl, quinolinyl, indoleyl, benzo[isothiazolyl], benzo[oxazolyl], benzo[isothiazolyl], isothiazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl, pyridyl, pyridine- 2(1H)-keto, pyrimidinyl, pyrazin-2(1H)-keto, pyrimidin-4(3H)-keto, pyrimidin-2(1H)-keto, pyridazin-3(2H)-keto, 1H-indolyl, 1H-benzo[d]imidazolyl, 1H-pyrrolo[2,3-c]pyridyl, 3H-imidazo[4,5-c]pyridyl, isoquinolinyl, quinazolinyl, 2H-isoindolyl, furan[3,2-b]pyridyl, furan[2,3-c]pyridyl, thieno[2,3-c]pyridyl, benzofuranyl, benzo[b]thienoyl, 1H-pyrrolo[3,2-b]pyridyl, 2H-pyrrolo[3,4-c]pyridyl

[0116] The heteroaryl group can be substituted or unsubstituted.

[0117] A "fused ring" refers to a polycyclic group in which two or more ring structures share a pair of atoms, wherein at least one ring has a fully conjugated π electron aromatic system, and one or more rings may contain 0, 1 or more double bonds, but at least one ring does not have a fully conjugated π electron aromatic system, wherein the ring atoms are selected from 0, 1 or more nitrogen, oxygen or S(O). r (where r is selected from 0, 1, or 2) heteroatoms, and the remaining ring atoms are carbon. The fused ring preferably comprises a bicyclic or tricyclic fused ring, wherein the bicyclic fused ring is preferably a fused ring of an aryl or heteroaryl group with a monocyclic heterocyclic group or a monocyclic cycloalkyl group. Preferably, it is 6 to 14 quinary, more preferably 8 to 10 quinary. Examples of "fused rings" include, but are not limited to:

[0118] "Alkoxy" refers to an (alkyl-O-) group. Alkyl groups are defined in the relevant section of this document. C1-C6 alkoxy groups are preferred. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, etc.

[0119] "Alkylthio" refers to a (alkyl-S-) group. Alkyl groups are defined in the relevant section of this document. C1-C6 alkylthio groups are preferred. Examples include, but are not limited to: methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, etc.

[0120] "Nitro" refers to the -NO2 group.

[0121] "Hydroxy" refers to the -OH group.

[0122] "Halogens" refers to fluorine, chlorine, bromine, and iodine.

[0123] "Amino" refers to -NH2.

[0124] "Hydroxyamino group" refers to -NHOH.

[0125] “Cyano” refers to -CN.

[0126] "Benzyl" refers to -CH2-phenyl.

[0127] "Carboxyl group" refers to -C(=O)OH.

[0128] "Carboxylic acid ester group" refers to -C(=O)O-alkyl or -C(=O)O-cycloalkyl, where the definitions of alkyl and cycloalkyl are as described above.

[0129] “Hydroxyalkyl” refers to an alkyl group substituted with a hydroxyl group, where the definition of alkyl is as described above.

[0130] "Aminoalkyl" refers to an amino-substituted alkyl group, where the definition of alkyl is as described above.

[0131] "Halogenated alkyl" refers to halogen-substituted alkyl groups, where the definition of alkyl is as described above.

[0132] "Haloalkoxy" refers to halogen-substituted alkoxy groups, where the definition of alkoxy groups is as described above.

[0133] "DMSO" refers to dimethyl sulfoxide.

[0134] “BOC” refers to tert-butoxycarbonyl.

[0135] “Bn” refers to benzyl.

[0136] "THP" refers to 2-tetrahydropyranyl.

[0137] "TFA" refers to trifluoroacetic acid.

[0138] “Ts” refers to p-toluenesulfonyl group.

[0139] “Bn” refers to benzyl.

[0140] “SEM” refers to (trimethylsilyl)ethoxymethyl.

[0141] "Formyl group" refers to

[0142] “FA” stands for nail acid.

[0143] A "leaving group," or simply a group, is an atom or functional group that breaks off from a larger molecule in a chemical reaction. It's a term used in nucleophilic substitution and elimination reactions. In a nucleophilic substitution reaction, the reactant attacked by the nucleophile is called the substrate, and the atom or group of atoms that breaks off with a pair of electrons from the substrate molecule is called the leaving group. Groups that readily accept electrons and have a strong ability to accept negative charges are desirable leaving groups. The smaller the pKa of the conjugate acid of the leaving group, the easier it is for the leaving group to break off from other molecules. This is because a smaller pKa means the leaving group doesn't need to bond with other atoms and has a stronger tendency to exist as an anion (or an electrically neutral leaving group). Common leaving groups include, but are not limited to, halogens, methanesulfonyl groups, -OTs, or -OH.

[0144] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0145] In this application, "one or more" means one or more, such as one, two, three, four or five or more.

[0146] Unless otherwise specified, the terms "substituted" or "substituted" in this specification refer to the substitution of a group by one or more (e.g., 1, 2, or 3) groups selected from the following: deuterium, alkyl, alkenyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio, amino, haloalkyl, haloalkoxy, hydroxyalkyl, carboxyl, carboxylic acid ester, SF5, =O, -OR 5 -C(=O)R 5 -C(=O)OR 5-N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -CH2NHC(=O)OR 5 -CH2NR 6 R 7 -S (=O) r NR 6 R 7 or -S(O) r R 5 The substituents are replaced;

[0147] R 5 Each is independently selected from alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups, wherein the alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl group is optionally further selected by one or more (e.g., 1, 2, or 3) from deuterium, hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 or -N(R) 9 )C(=O)R 10 The substituents are replaced;

[0148] R 6 and R 7 Each is independently selected from hydrogen atom, hydroxyl, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl, wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl may optionally be further selected by one or more (e.g., 1, 2 or 3) from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R10 or -N(R) 9 )C(=O)R 10 The substituents are replaced;

[0149] Or, R 6 and R 7 The atoms bonded to them together form a structure containing one or more N, O, or S (=O). r The 4-8 membered heterocyclic group, wherein the 4-8 membered heterocyclic group is optionally further selected by one or more (e.g., 1, 2 or 3) from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 or -N(R) 9 )C(=O)R 10 The substituents are replaced;

[0150] R 8 R 9 and R 10 Each is independently selected from hydrogen atoms, alkyl, amino, cycloalkyl, heterocyclic, aryl or heteroaryl, wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is optionally further substituted by one or more (e.g. 1, 2 or 3) substituents selected from hydroxyl, halogen, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, carboxyl or carboxylic acid ester group;

[0151] r can be 0, 1, or 2 independently.

[0152] In this article, the wavy line in a group usually indicates the position where the group is attached to the compound.

[0153] The compounds of this invention may contain asymmetric or chiral centers, and thus exist in different stereoisomer forms. It is contemplated that all stereoisomer forms of the compounds of this invention, including but not limited to diastereomers, enantiomers, atropisomers, and geometric (conformal) isomers, and mixtures thereof, such as racemic mixtures, are within the scope of this invention.

[0154] Unless otherwise stated, the structures described in this invention also include all isomers of this structure (e.g., diastereomers, enantiomers, and trans-isomers, and geometric (conformal) isomers; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers). Therefore, individual stereoisomers of the compounds of this invention, as well as mixtures of enantiomers, mixtures of diastereomers, and mixtures of geometric (conformal) isomers, are all within the scope of this invention.

[0155] "Medicinal salts" refer to certain salts of the above-mentioned compounds that retain their original biological activity and are suitable for medicinal use. Medicinal salts of compounds represented by general formula (I) can be metal salts or amine salts formed with suitable acids.

[0156] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as a physiologically pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity. Attached Figure Description

[0157] Figure 1 is a tumor formation curve after administration of the compound of the present invention or the control compound;

[0158] Figure 2 shows the changes in body weight of experimental animals after administration of the compound of the present invention or the control compound. Detailed Implementation

[0159] The following embodiments are used to further describe the present invention, but these embodiments are not intended to limit the scope of the present invention.

[0160] Example

[0161] The examples provide preparation and structural identification data for representative compounds represented by formula (I). It must be noted that the following examples are illustrative of the invention and not intended to limit it. 1 The 1H NMR spectra were obtained using a Bruker instrument (400 MHz), and chemical shifts are expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used. 1 H NMR representation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If the coupling constant is provided, the unit is Hz.

[0162] Mass spectrometry is performed using an LC / MS instrument, and the ionization method can be ESI or APCI.

[0163] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0164] Column chromatography typically uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0165] In the following examples, all temperatures are in Celsius unless otherwise specified. Unless otherwise specified, all starting materials and reagents are commercially available or synthesized according to known methods. Commercially available materials and reagents are used directly without further purification. Unless otherwise specified, they are purchased from manufacturers including but not limited to Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Guangzan Chemical Technology Co., Ltd., and Jingyan Chemical Technology Co., Ltd.

[0166] CD3OD: Deuterated methanol.

[0167] CDCl3: Deuterated chloroform.

[0168] DMSO-d6: Deuterated dimethyl sulfoxide.

[0169] Argon atmosphere refers to a reaction flask connected to an argon gas balloon with a volume of approximately 1L.

[0170] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0171] The compounds were purified using silica gel column chromatography and reversed-phase column chromatography. The eluent system was selected from: A: petroleum ether and ethyl acetate; B: dichloromethane and methanol; C: dichloromethane: ethyl acetate; D: trifluoroacetic acid aqueous solution and acetonitrile. The volume ratio of the solvent varied depending on the polarity of the compound and could be adjusted by adding small amounts of acidic or basic reagents, such as acetic acid or triethylamine.

[0172] Example 1

[0173] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-4-phenoxy-6-((trifluoromethyl)sulfonyl)nicotinamide

[0174] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-4-Phenyl-6-((Trifluoromethyl)sulfone)nicotinamide

[0175] first step

[0176] methyl 6-bromo-4-phenoxynicotinate

[0177] 6-Bromo-4-phenoxynicotinic acid methyl ester

[0178] 6-Bromo-4-chloronicotinic acid methyl ester 1a (6.8 g, 27.15 mmol, commercially available) and phenol (2.55 g, 27.15 mmol) were dissolved in N,N-dimethylformamide (70 mL), and cesium carbonate (22.11 g, 67.87 mmol) was added. The reaction mixture was reacted at room temperature for 12 hours. The reaction mixture was poured into water (200 mL), extracted with ethyl acetate (80 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated by silica gel column chromatography (eluent: system A) to give 6-bromo-4-phenoxynicotinic acid methyl ester 1b (7.7 g), yield 92%.

[0179] MS m / z (ESI): 307.9 / 309.9 [M+H] +

[0180] Step 2

[0181] methyl 4-phenoxy-6-((trifluoromethyl)thio)nicotinate

[0182] 4-Phenoxy-6-((trifluoromethyl)thio)nicotinic acid methyl ester

[0183] 6-Bromo-4-phenoxynicotinic acid methyl ester 1b (500 mg, 1.62 mmol), cuprous iodide (309.04 mg, 1.62 mmol), 2,2'-bipyridine (506.88 mg, 3.25 mmol), and silver trifluoromethyl thiocyanate (508.57 mg, 2.43 mmol) were added to acetonitrile (1 mL), and the reaction was carried out at 110 °C for 12 hours. After returning to room temperature, the reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (eluent: system A) to give 4-phenol-6-((trifluoromethyl)thio)nicotinic acid methyl ester 1c (476 mg), with a yield of 89%.

[0184] 1 H NMR (400MHz, CDCl3) δ8.97 (s, 1H), 7.50-7.43 (m, 2H), 7.31 (t, J = 7.4Hz, 1H), 7.13-7.09 (m, 2H), 6.81 (s, 1H), 3.94 (s, 3H) ppm.

[0185] Step 3

[0186] 4-phenoxy-6-((trifluoromethyl)thio)nicotinic acid

[0187] 4-Phenoxy-6-((trifluoromethyl)thio)nicotinic acid

[0188] Lithium hydroxide (61.09 mg, 2.55 mmol) was added to a mixed solution of methyl 4-phenoxy-6-((trifluoromethyl)thio)nicotinic acid 1c (420 mg, 1.28 mmol) in tetrahydrofuran (3 mL) and water (3 mL), and the reaction was carried out at room temperature for 2 hours. The pH was adjusted to 6 by slow dropwise addition of 0.5 M hydrochloric acid, and the mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 4-phenoxy-6-((trifluoromethyl)thio)nicotinic acid 1d (360 mg), yield 90%. MS m / z (ESI): 316.3 [M+H] +

[0189] Step 4

[0190] 4-phenoxy-6-((trifluoromethyl)sulfonyl)nicotinic acid

[0191] 4-Phenoxy-6-((trifluoromethyl)sulfone)nicotinic acid

[0192] 4-phenoxy-6-((trifluoromethyl)sulfonyl)nicotinic acid 1d (190 mg, 602.66 μmol), sodium periodate (386.71 mg, 1.81 mmol), and ruthenium trichloride hydrate (27.17 mg, 120.53 μmol) were added to a mixed solvent of water (2 mL), chloroform (1 mL), and acetonitrile (1 mL), and reacted at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (eluent: system A) to give 4-phenoxy-6-((trifluoromethyl)sulfonyl)nicotinic acid 1e (190 mg), yield 91%.

[0193] MS m / z(ESI): 348.0 [M+H] +

[0194] Step 5

[0195] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-4-phenoxy-6-((trifluoromethyl)sulfonyl)nicotinamide

[0196] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-4-phenoxy-6-((trifluoromethyl)sulfone)nicotinamide

[0197] 4-Phenoxy-6-((trifluoromethyl)sulfonyl)nicotinic acid 1e (190 mg, 547.13 μmol) and (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (228.12 mg, 656.56 μmol, commercially available) were dissolved in pyridine (3 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (104.89 mg, 547.13 μmol) was added. The reaction mixture was reacted at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C). 18 (19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, mobile phase B: acetonitrile; flow rate: 20mL / min), yielded (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-4-phenoxy-6-((trifluoromethyl)sulfone)nicotinamide 1 (109.0mg), yield 40%.

[0198] MS m / z(ESI): 505.0 [M+H] +

[0199] 1H NMR (400MHz, DMSO-d6) δ9.10 (d, J = 8.3Hz, 1H), 9.06 (s, 1H), 7.59 (t, J = 7.9Hz, 2H), 7.44-7.40 (m, 2H), 7.37 (d, J = 7.6Hz, 2H), 6.91-6. 81(m,2H),4.30(td,J=8.2,3.2Hz,1H),2.97(s,3H),1.13-1.02(m,1H),0.60-0.52(m,1H),0.51-0.44(m,2H),0.41-0.33(m,1H)ppm.

[0200] Example 2

[0201] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-4-(pentafluoro-λ 6 -sulfaneyl)-2-phenoxybenzamide

[0202] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-4-(Pentafluoro-λ) 6 (-thioalkyl)-2-phenoxybenzamide

[0203] first step

[0204] 2-bromo-4-(pentafluoro-λ 6 -sulfaneyl)aniline

[0205] 2-Bromo-4-(pentafluoro-λ) 6 -Thioalkyl)aniline

[0206] 4-(pentafluoro-λ) 6 2-Thioalkyl)aniline 2a (400 mg, 1.83 mmol, commercially available) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of N-bromosuccinimide (341 mg, 1.92 mmol), and the reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the reaction solution was diluted with water (30 mL), extracted with ethyl acetate (30 mL × 3), and the combined solutions were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to give 2-bromo-4-(pentafluoro-λ) 6 -Thioalkyl)aniline 2b (500 mg), yield 91.91%.

[0207] MS m / z(ESI): 297.9 [M+H] +

[0208] Step 2

[0209] (3-bromo-4-iodophenyl)pentafluoro-λ 6 -sulfane

[0210] (3-Bromo-4-iodophenyl)pentafluoro-λ 6 -Thiones

[0211] 2-bromo-4-(pentafluoro-λ) 6 (-Thioalkyl)aniline 2b (500 mg, 1.68 mmol) was dissolved in concentrated sulfuric acid (1 mL) and water (2.5 mL), followed by the addition of sodium nitrite (116 mg, 1.68 mmol), and reacted at 0 °C for 2 hours. Cuprous iodide (16 mg, 84.02 μmol) and potassium iodide (279 mg, 1.68 mmol) were added to the reaction solution, and the reaction was continued at 0 °C for 1 hour, followed by a final reaction at 25 °C for 12 hours. After the reaction, the reaction solution was diluted with water (30 mL), extracted three times with dichloromethane (30 mL), and the extracts were combined and washed with saturated sodium chloride solution (30 mL). The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification was achieved by silica gel column chromatography (eluent: system A) to obtain (3-bromo-4-iodophenyl)pentafluoro-λ.6 -Thioane 2c (430 mg), yield 62.68%.

[0212] 1 H NMR (400MHz, CDCl3) δ7.98 (m, 2H), 7.38 (dd, J = 8.8, 2.8Hz, 1H) ppm.

[0213] Step 3

[0214] methyl 2-bromo-4-(pentafluoro-λ 6 -sulfaneyl)benzoate

[0215] 2-Bromo-4-(pentafluoro-λ) 6 methyl benzoate (-thioalkyl)

[0216] (3-Bromo-4-iodophenyl)pentafluoro-λ 6 2-Thane 2C (470 mg, 1.15 mmol) and triethylamine (1.16 g, 11.49 mmol) were dissolved in methanol (10 mL). Palladium acetate (13 mg, 57.90 μmol) and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (67 mg, 115.79 μmol) were added to the reaction solution under a carbon monoxide atmosphere. The reaction solution was purged three times with carbon monoxide gas, and then reacted at 50 °C for 1 hour. After the reaction, the reaction solution was diluted with water (30 mL), extracted three times with dichloromethane (30 mL), and the combined extracts were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to give 2-bromo-4-(pentafluoro-λ) 6 Methyl benzoate (280 mg) 2d, yield 71.43%.

[0217] MS m / z (ESI): 340.8 [M+H] +

[0218] Step 4

[0219] methyl 4-(pentafluoro-λ 6 -sulfaneyl)-2-phenoxybenzoate

[0220] 4-(pentafluoro-λ) 6 Methyl 2-thioalkyl-2-phenoxybenzoate

[0221] 2-bromo-4-(pentafluoro-λ) 6280 mg (820.89 μmol) of methyl benzoate (2-thioalkyl) 2d, cesium carbonate (401 mg, 1.23 mmol), and phenol (93 mg, 988.15 μmol) were dissolved in toluene (10 mL), followed by the addition of cuprous iodide (156 mg, 819.10 μmol). The mixture was reacted at 120 °C for 2 hours. After the reaction, the reaction solution was diluted with water (30 mL), extracted three times with dichloromethane (30 mL), and the combined extracts were washed with saturated sodium chloride solution (30 mL). The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to obtain 4-(pentafluoro-λ) 6 Methyl 2e (245 mg) of 2-thioalkyl-2-phenoxybenzoate, yield 84.24%.

[0222] MS m / z(ESI): 355.0 [M+H] +

[0223] Step 5

[0224] 4-(pentafluoro-λ 6 -sulfaneyl)-2-phenoxybenzoic acid

[0225] 4-(pentafluoro-λ) 6 -Thioalkyl)-2-phenoxybenzoic acid

[0226] 4-(pentafluoro-λ) 6 Methyl 2e of (-thioalkyl)-2-phenoxybenzoate (100 mg, 282.25 μmol) was dissolved in tetrahydrofuran (5 mL) and water (2 mL), followed by the addition of lithium hydroxide monohydrate (13 mg, 310.48 μmol), and then reacted at 25 °C for 1 hour. The reaction solution was concentrated under reduced pressure to give 4-(pentafluoro-λ) 6 2f (95mg) of 2-thioalkyl-2-phenoxybenzoic acid, yield 98.92%.

[0227] MS m / z (ESI): 341.2 [M+H] +

[0228] Step 6

[0229] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-4-(pentafluoro-λ 6 -sulfaneyl)-2-phenoxybenzamide

[0230] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-4-(Pentafluoro-λ)6 (-thioalkyl)-2-phenoxybenzamide

[0231] 4-(pentafluoro-λ) 6 (-thioalkyl)-2-phenoxybenzoic acid 2f (95 mg, 279.19 μmol), (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (107 mg, 307.94 μmol) and N,N-diisopropylethylamine (108 mg, 837.58 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (138 mg, 362.95 μmol) was added. The mixture was then reacted at 25 °C for 1 hour. After the reaction was complete, the reaction solution was diluted with water (30 mL), extracted with ethyl acetate (30 mL × 3), and the combined solution was washed with saturated sodium chloride solution (30 mL). The solution was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C). 18 19*250mm, 10μm; Mobile phase A: 0.1% FA / H2O, Mobile phase B: Acetonitrile; Flow rate: 20mL / min), yielding (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-4-(pentafluoro-λ 6 (-thioalkyl)-2-phenoxybenzamide 2 (70 mg), yield 50.40%.

[0232] MS m / z(ESI): 498.0 [M+H] +

[0233] 1 H NMR (400MHz, DMSO-d6) δ8.83(d,J=8.0Hz,1H),7.87–7.74(m,2H),7.45(m,2H),7.34(d,J=2.0Hz,1H),7.22(t,J=7 .6Hz,1H),7.10(d,J=7.6Hz,2H),6.85–6.68(m,2H),4.16(m,1H),2.92(s,3H),1.00(m,1H),0.54–0.22(m,4H)ppm.

[0234] Example 3

[0235] (S,E)-5-acetyl-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide

[0236] (S,E)-5-acetyl-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide

[0237] first step

[0238] ethyl 6-acetyl-2-chloronicotinate

[0239] Ethyl 6-acetyl-2-chloronicotinate

[0240] Ethyl 2,6-dichloronicotinate 3a (500 mg, 2.27 mmol, commercially available) and tributyl(1-ethoxyvinyl)tin (820 mg, 2.27 mmol, 774 μL) were dissolved in dioxane (10 mL), followed by the addition of triethylamine (690 mg, 6.82 mmol, 947 μL) and tetrakis(triphenylphosphine)palladium (262 mg, 227 μmol). The reaction was carried out at 100 °C for 2 hours under a nitrogen atmosphere. After cooling, 4 M hydrochloric acid aqueous solution was added, and the reaction was continued at room temperature for 1 hour. The reaction solution was quenched with potassium fluoride aqueous solution (30 mL), extracted with dichloromethane (30 mL × 3), and the combined solutions were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (eluent: system A) to give ethyl 6-acetyl-2-chloronicotinic acid 3b (350 mg), with a yield of 67.67%.

[0241] 1 H NMR (400MHz, CDCl3) δ8.23(d,J=7.6Hz,1H),7.99(d,J=8.0Hz,1H),4.44(q,J=7.2Hz,2H),2.72(s,3H),1.42(t,J=7.2Hz,3H)ppm.

[0242] Step 2

[0243] ethyl 2-chloro-6-(1,1-difluoroethyl)nicotinate

[0244] 2-Chloro-6-(1,1-difluoroethyl)nicotinic acid ethyl ester

[0245] Ethyl 6-acetyl-2-chloronicotinic acid 3b (350 mg, 1.54 mmol) was dissolved in diethylaminosulfur trifluoride (2 mL) and reacted at 25 °C for 12 hours. The reaction mixture was quenched by slowly pouring it into water (20 mL), extracted with dichloromethane (20 mL × 3), and the combined extracts were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (eluent: system A) to give ethyl 2-chloro-6-(1,1-difluoroethyl)nicotinic acid 3c (220 mg), yield 57.32%. MS m / z (ESI): 250.0 [M+H] +

[0246] Step 3

[0247] 6-(1,1-difluoroethyl)-2-hydroxynicotinic acid

[0248] 6-(1,1-Difluoroethyl)-2-hydroxynicotinic acid

[0249] Ethyl 2-chloro-6-(1,1-difluoroethyl)nicotinic acid 3c (500 mg, 2.00 mmol) was dissolved in concentrated hydrochloric acid (3 mL) and water (3 mL) and reacted at 100 °C for 24 hours. The reaction solution was diluted with water (20 mL), extracted with dichloromethane (20 mL × 3), and the combined extracts were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 6-(1,1-difluoroethyl)-2-hydroxynicotinic acid 3d (400 mg), yield 98.31%.

[0250] MS m / z(ESI): 204.0 [M+H] +

[0251] Step 4

[0252] ethyl 6-(1,1-difluoroethyl)-2-hydroxynicotinate

[0253] 6-(1,1-Difluoroethyl)-2-hydroxynicotinic acid ethyl ester

[0254] 6-(1,1-difluoroethyl)-2-hydroxynicotinic acid 3d (300 mg, 1.48 mmol) was dissolved in ethanol (5 mL), and thionyl chloride (527 mg, 4.43 mmol, 320 μL) was added at 0 °C. The reaction was carried out at 60 °C for 1 hour. After the reaction was completed, the reaction solution was quenched by slowly pouring it into water (20 mL), extracted with dichloromethane (20 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to give ethyl 6-(1,1-difluoroethyl)-2-hydroxynicotinic acid 3e (270 mg), with a yield of 79.08%.

[0255] MS m / z(ESI): 232.0 [M+H] +

[0256] Step 5

[0257] ethyl 5-bromo-6-(1,1-difluoroethyl)-2-hydroxynicotinate

[0258] 5-Bromo-6-(1,1-difluoroethyl)-2-hydroxynicotinic acid ethyl ester

[0259] Ethyl 6-(1,1-difluoroethyl)-2-hydroxynicotinic acid ester 3e (220 mg, 951 μmol) was dissolved in dichloromethane (5 mL), and N-bromosuccinimide (220 mg, 1.24 mmol) was added. The reaction mixture was reacted at 40 °C for 1 hour. The reaction solution was slowly poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to give ethyl 5-bromo-6-(1,1-difluoroethyl)-2-hydroxynicotinic acid ester 3f (290 mg), yield 98.28%.

[0260] MS m / z(ESI): 310.0 [M+H] +

[0261] Step 6

[0262] ethyl 5-bromo-2-chloro-6-(1,1-difluoroethyl)nicotinate

[0263] 5-Bromo-2-chloro-6-(1,1-difluoroethyl)nicotinic acid ethyl ester

[0264] Ethyl 5-bromo-6-(1,1-difluoroethyl)-2-hydroxynicotinic acid ester 3g (800 mg, 2.58 mmol) was dissolved in N,N-dimethylformamide (1 mL) and thionyl chloride (8 mL) and reacted at 75 °C for 12 h. After the reaction was complete, the reaction solution was diluted with water (30 mL), extracted with dichloromethane (30 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to give ethyl 5-bromo-2-chloro-6-(1,1-difluoroethyl)nicotinic acid ester 3g (450 mg), yield 53.09%.

[0265] MS m / z(ESI): 328.0 [M+H] +

[0266] Step 7

[0267] ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinate

[0268] 5-Bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid ethyl ester

[0269] 3 g (200 mg, 608 μmol) of ethyl 5-bromo-2-chloro-6-(1,1-difluoroethyl)nicotinate and phenol (114.58 mg, 1.22 mmol) were dissolved in N,N-dimethylformamide (5 mL), followed by the addition of cesium carbonate (396.69 mg, 1.22 mmol). The reaction mixture was reacted at 80 °C for 1 hour. After the reaction was complete, the reaction solution was diluted with water (30 mL), extracted with ethyl acetate (30 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: system A) to give ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinate 3 h (130 mg), yield 55.30%.

[0270] MS m / z (ESI): 387.9 [M+H] +

[0271] Step 8

[0272] 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid

[0273] 5-Bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid

[0274] Ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid 3h (80 mg, 207 μmol) was dissolved in tetrahydrofuran (2 mL) and water (1 mL), followed by the addition of lithium hydroxide monohydrate (9.56 mg, 228 μmol), and the reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to obtain 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid 3i (80 mg), which was directly used in the next step of the reaction.

[0275] MS m / z (ESI): 357.9 [M+H] +

[0276] Step 9

[0277] (S,E)-5-bromo-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide

[0278] (S,E)-5-bromo-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide

[0279] 5-Bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid 3i (80 mg, 223 μmol), (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (80.0 mg, 230 μmol), and N,N-diisopropylethylamine (86.61 mg, 670 μmol) were dissolved in N,N-dimethylformamide (1 mL), followed by the addition of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (127 mg, 335.07 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. After the reaction was complete, the reaction solution was diluted with water (30 mL), extracted with ethyl acetate (20 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography (eluent: system A) yielded (S,E)-5-bromo-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide 3j (85 mg), with a yield of 73.8%.

[0280] MS m / z(ESI): 515.0 [M+H]

[0281] Step 10

[0282] (S,E)-5-acetyl-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide

[0283] (S,E)-5-acetyl-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide

[0284] (S,E)-5-bromo-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide 3j (55 mg, 106.72 μmol) and tributyl(1-ethoxyvinyl)tin (58 mg, 160.61 μmol) were dissolved in dioxane (1 mL), followed by the addition of triethylamine (33 mg, 326.09 mmol) and tetrakis(triphenylphosphine)palladium (13 mg, 11.25 μmol). The reaction was carried out at 100 °C for 2 hours under a nitrogen atmosphere. After cooling, 4 M hydrochloric acid aqueous solution was added, and the reaction was continued at room temperature for 1 hour. The reaction solution was quenched with potassium fluoride aqueous solution (20 mL), extracted with dichloromethane (30 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C18, 19*250 mm, 10 μm; mobile phase A: 0.1% FA / H2O, mobile phase B: acetonitrile; flow rate: 20 mL / min) to obtain (S,E)-5-acetyl-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide 3 (13 mg), yield 25.46%.

[0285] MS m / z (ESI): 479.2 [M+H] +

[0286] 1 H NMR(400MHz,DMSO-d6)δ8.92(d,J=8.0Hz,1H),8.30(s,1H),7.51–7.45(m,2H),7.29(m,3H),6.88(m,2 H),4.30(m,1H),2.98(s,3H),2.57(s,3H),1.79(t,J=19.2Hz,3H),1.11(m,1H),0.61–0.37(m,4H)ppm.

[0287] Example 4

[0288] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinamide

[0289] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-6-(1,1-Difluoroethyl)-2-phenoxy-5-vinylnicotinamide

[0290] first step

[0291] ethyl 6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinate

[0292] 6-(1,1-Difluoroethyl)-2-phenoxy-5-vinylnicotinic acid ethyl ester

[0293] Ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinate (200 mg, 517 μmol) and potassium vinyltrifluoroborate (104 mg, 776 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL). Then, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (38 mg, 51.8 μmol) and potassium carbonate (179 mg, 1.29 mmol) were added. The mixture was purged with nitrogen three times and stirred at 90 °C for 18 hours. The mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography (eluent: system A) yielded ethyl 6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinate 4a (150 mg), with a yield of 86.89%.

[0294] MS m / z(ESI): 334.1 [M+H] +

[0295] Step 2

[0296] 6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinic acid

[0297] 6-(1,1-Difluoroethyl)-2-phenoxy-5-vinylnicotinic acid

[0298] Ethyl 6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinic acid 4a (140 mg, 420 μmol) was dissolved in tetrahydrofuran (4 mL) and water (0.5 mL), followed by the addition of lithium hydroxide monohydrate (21 mg, 504 μmol). The resulting mixture was stirred at 25 °C for 1 hour. The mixture was concentrated to dryness under reduced pressure to give 6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinic acid 4b (120 mg), with a yield of 93.59%.

[0299] MS m / z (ESI): 306.2 [M+H] +

[0300] Step 3

[0301] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinamide

[0302] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-6-(1,1-Difluoroethyl)-2-phenoxy-5-vinylnicotinamide

[0303] To a solution of 6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinic acid 4b (130 mg, 425 μmol) and (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (138 mg, 511 μmol) in N,N-dimethylformamide (4 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (323 mg, 851 μmol) and N,N-diisopropylethylamine (220 mg, 1.70 mmol) were added, the reaction was stirred at 25 °C for 2 hours. The mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C). 18 (19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, mobile phase B: acetonitrile; flow rate: 20mL / min), yielded (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxy-5-vinylnicotinamide 4 (100mg), yield 50.77%.

[0304] MS m / z (ESI): 463.2 [M+H] +

[0305] 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=8.4Hz,1H),8.40(s,1H),7.48–7.42(m,2H),7.29–7.22(m,3H),7.18–7.08(m,1H),6.92–6.82(m,2H),5.97(d,J =17.2Hz,1H),5.48(d,J=11.6Hz,1H),4.28(td,J=8.4,2.8Hz,1H),2.97( s,3H),1.72(t,J=19.6Hz,3H),1.15–1.06(m,1H),0.58–0.37(m,4H)ppm.

[0306] Example 5

[0307] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinamide

[0308] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-6-(1,1-Difluoroethyl)-5-ethynyl-2-phenoxynicotinamide

[0309] first step

[0310] ethyl 6-(1,1-difluoroethyl)-2-phenoxy-5-((trimethylsilyl)ethynyl)nicotinate

[0311] 6-(1,1-Difluoroethyl)-2-phenoxy-5-((trimethylsilyl)ethynyl)nicotinic acid ethyl ester

[0312] Ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid ester (120 mg, 311 μmol), trimethylsilylacetylene (305 mg, 3.11 mmol, 439 μL), cuprous iodide (5.92 mg, 31.07 μmol), tetrabutylammonium iodide (688.64 mg, 1.86 mmol), and triethylamine (189 mg, 1.86 mmol) were dissolved in N,N-dimethylformamide (1 mL), and then palladium dichloride dichloride (21.8 mg, 31.1 μmol) was added. The reaction was carried out in a microwave environment at 80 °C for 6 hours. After the reaction was completed, the reaction solution was diluted with water (20 mL), extracted with ethyl acetate (20 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography (eluent: system A) yielded ethyl 6-(1,1-difluoroethyl)-2-phenoxy-5-((trimethylsilyl)ethynyl)nicotinic acid 5a (87 mg), 69.4% yield. MS m / z (ESI): 404.2 [M+H] +

[0313] Step 2

[0314] ethyl 6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinate

[0315] 6-(1,1-Difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid ethyl ester

[0316] Ethyl 6-(1,1-difluoroethyl)-2-phenoxy-5-((trimethylsilyl)ethynyl)nicotinic acid ester 5a (67 mg, 166 μmol) was dissolved in tetrahydrofuran (3 mL), and then tetrabutylammonium fluoride (332 μmol, 1 M, 332.10 μL) was added. The reaction mixture was reacted at 25 °C for 1 hour. The reaction mixture was poured into water (10 mL), extracted with ethyl acetate (10 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (eluent: system A) to give ethyl 6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid ester 5b (45 mg), yield 81.80%.

[0317] MS m / z(ESI): 332.2 [M+H] +

[0318] Step 3

[0319] 6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid

[0320] 6-(1,1-Difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid

[0321] Ethyl 6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid ester 5b (35 mg, 106 μmol) was dissolved in tetrahydrofuran (2 mL) and water (1 mL), followed by the addition of lithium hydroxide monohydrate (4.88 mg, 116 μmol), and the reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to obtain 6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid 5c (35 mg), which was directly used in the next step of the reaction.

[0322] MS m / z(ESI): 304.0 [M+H] +

[0323] Step 4

[0324] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinamide

[0325] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-6-(1,1-Difluoroethyl)-5-ethynyl-2-phenoxynicotinamide

[0326] 6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinic acid 5c (35 mg, 115 μmol), (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (24.3 mg, 69.9 μmol), and N,N-diisopropylethylamine (44.7 mg, 346 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (57.05 mg, 150 μmol) was added. The mixture was then reacted at 25 °C for 1 hour. After the reaction was complete, the mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C). 18(19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, mobile phase B: acetonitrile; flow rate: 20mL / min), yielded (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-5-ethynyl-2-phenoxynicotinamide 5 (13mg), yield 40.3%.

[0327] MS m / z(ESI): 461.1 [M+H] +

[0328] 1 H NMR(400MHz, DMSO-d6)δ8.87(d,J=8.4Hz,1H),8.28(s,1H),7.49–7.43(m,2H),7.26(m,3H),6.91–6.81(m,2H),4 .63(s,1H),4.26(td,J=8.4,3.2Hz,1H),2.98(s,3H),1.76(t,J=19.2Hz,3H),1.09(m,1H),0.58–0.33(m,4H)ppm.

[0329] Example 6

[0330] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinamide

[0331] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-6-(1,1-Difluoroethyl)-5-(1-Methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinamide

[0332] first step

[0333] ethyl 6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinate

[0334] 6-(1,1-Difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid ethyl ester

[0335] Ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinic acid ester 3h (300 mg, 776.82 μmol), 4-bromo-1-methyl-1H-1,2,3-triazole 6a (378 mg, 2.33 mmol, commercially available), pinacol diboronate (592 mg, 2.33 mmol), bis(1-adamantyl)-butylphosphine (112 mg, 312.38 μmol), and potassium carbonate (537 mg, 3.88 mmol) were dissolved in ethylene glycol dimethyl ether (10 mL) and water (2 mL). The reaction solution was purged three times with nitrogen. Under a nitrogen atmosphere, palladium acetate (35 mg, 155.89 μmol) was added, and the reaction solution was reacted at 70 °C for 12 hours. The reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (20 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (eluent: system A) to give 6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid ethyl ester 6b (160 mg), yield 53.03%.

[0336] MS m / z (ESI): 389.2 [M+H] +

[0337] Step 2

[0338] 6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid

[0339] 6-(1,1-Difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid

[0340] Ethyl 6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid 6b (150 mg, 386.23 μmol) was dissolved in tetrahydrofuran (2 mL) and water (1 mL), followed by the addition of lithium hydroxide monohydrate (18 mg, 428.91 μmol), and the reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to give 6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid 6c (130 mg), with a yield of 93.41%.

[0341] MS m / z(ESI): 361.0 [M+H] +

[0342] Step 3

[0343] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinamide

[0344] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-6-(1,1-Difluoroethyl)-5-(1-Methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinamide

[0345] 6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinic acid 6c (130 mg, 229.65 μmol), (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (80 mg, 230.25 μmol) and N,N-diisopropylethylamine (89 mg, 688.94 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (114 mg, 298.83 μmol) was added. The mixture was then reacted at 25 °C for 1 hour. The mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C). 18 (19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, mobile phase B: acetonitrile; flow rate: 20mL / min) yielded (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-5-(1-methyl-1H-1,2,3-triazol-4-yl)-2-phenoxynicotinamide 6 (35mg), yield 29.45%.

[0346] MS m / z (ESI): 518.1 [M+H] +

[0347] 1H NMR (400MHz, DMSO-d6) δ8.91(d,J=8.0Hz,1H),8.44(s,1H),8.28(s,1H),7.55–7.43(m,2H),7.37–7.25(m,3H),6.89 (s,2H),4.30(t,J=8.0Hz,1H),4.14(s,3H),2.99(s,3H),1.77(t,J=19.2Hz,3H),1.13(m,1H),0.63–0.38(m,4H)ppm.

[0348] Example 7

[0349] 4-(cyclohex-3-en-1-yloxy)-N-((S,E)-1-cyclopropyl-3-(methylsulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide

[0350] 4-(cyclohexyl-3-en-1-yloxy)-N-((S,E)-1-cyclopropyl-3-(methanesulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidin-5-carboxamide

[0351] first step

[0352] ethyl 4-(benzyloxy)-2-(methylthio)pyrimidine-5-carboxylate

[0353] 4-(benzyloxy)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester

[0354] Benzyl alcohol (2.09 g, 19.3 mmol) and potassium tert-butoxide (1.45 g, 12.9 mmol) were dissolved in tetrahydrofuran (30 mL). The resulting mixture was stirred at 80 °C for 0.5 h. The reaction mixture was cooled to -78 °C and slowly added dropwise to a solution of ethyl 4-chloro-2-methylthio-pyrimidin-5-carboxylic acid 7a (3 g, 12.89 mmol) in N,N-dimethylformamide (30 mL). The reaction mixture was heated to 25 °C and stirred for 1 h. The mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (eluent: system A) to give ethyl 4-(benzyloxy)-2-(methylthio)pyrimidine-5-carboxylic acid 7b (3.5 g), yield 89.19%.

[0355] MS m / z (ESI): 305.1 [M+H] +

[0356] Step 2

[0357] ethyl 4-(benzyloxy)-2-chloropyrimidine-5-carboxylate

[0358] 4-(benzyloxy)-2-chloropyrimidine-5-carboxylic acid ethyl ester

[0359] Ethyl 4-(benzyloxy)-2-(methylthio)pyrimidine-5-carboxylic acid ester 7b (3.1 g, 10.19 mmol) was dissolved in acetonitrile (60 mL), and thionyl chloride (13.75 g, 101.9 mmol) in dichloromethane (60 mL) was added dropwise at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour. The mixture was poured into water (50 mL), extracted with ethyl acetate (50 mL × 3), and the combined organic phases were washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (eluent: system A) to give ethyl 4-(benzyloxy)-2-chloropyrimidine-5-carboxylic acid ester 7c (2.8 g), yield 93.92%.

[0360] MS m / z(ESI): 293.0 [M+H] +

[0361] Step 3

[0362] ethyl 4-(benzyloxy)-2-(1-ethoxyvinyl)pyrimidine-5-carboxylate

[0363] 4-(benzyloxy)-2-(1-ethoxyvinyl)pyrimidine-5-carboxylic acid ethyl ester

[0364] To a solution of ethyl 4-(benzyloxy)-2-chloropyrimidine-5-carboxylate 7c (501 mg, 1.71 mmol) and tributyl(1-ethoxyvinyl)stanane (741.75 mg, 2.05 mmol) in toluene (2 mL), tetrakis(triphenylphosphine)palladium (197.78 mg, 171.16 μmol) was added. The mixture was degassed and purged three times with nitrogen, then heated to 100 °C and stirred for 18 hours. The mixture was quenched with saturated potassium fluoride solution (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (eluent: system A) to give ethyl 4-(benzyloxy)-2-(1-ethoxyvinyl)pyrimidine-5-carboxylate 7d (450 mg), yield 87.55%.

[0365] MS m / z(ESI): 329.1 [M+H] +

[0366] Step 4

[0367] ethyl 2-acetyl-4-(benzyloxy)pyrimidine-5-carboxylate

[0368] 2-Acetyl-4-(benzyloxy)pyrimidine-5-carboxylic acid ethyl ester

[0369] Ethyl 4-(benzyloxy)-2-(1-methoxyvinyl)pyrimidine-5-carboxylic acid ester 7d (1.1 g, 3.35 mmol) was dissolved in a dioxane solution of hydrochloric acid (10 mL, 4 N), and the resulting mixture was stirred at 25 °C for 6 hours. The reaction mixture was concentrated to dryness under reduced pressure to give ethyl 2-acetyl-4-(benzyloxy)pyrimidine-5-carboxylic acid ester 7e (1 g), in 99.40% yield.

[0370] MS m / z (ESI): 301.1 [M+H] +

[0371] Step 5

[0372] ethyl 4-(benzyloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate

[0373] 4-(benzyloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ethyl ester

[0374] To a solution of ethyl 2-acetyl-4-(benzyloxy)pyrimidine-5-carboxylic acid 7e (3 g, 9.99 mmol) in dichloromethane (5 mL), 10 mL of diethylaminosulfur trifluoride was added, and the mixture was stirred at 25 °C under a nitrogen atmosphere for 18 hours. The mixture was poured into ice water (100 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (eluent: system A) to give ethyl 4-(benzyloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid 7f (1.9 g), yield 59.01%. MS m / z (ESI): 323.2 [M+H] +

[0375] Step 6

[0376] ethyl 2-(1,1-difluoroethyl)-4-hydroxypyrimidine-5-carboxylate

[0377] 2-(1,1-Difluoroethyl)-4-hydroxypyrimidine-5-carboxylic acid ethyl ester

[0378] Palladium on carbon 10% (564.61 mg, 5.31 mmol) was added to a suspension of 7 f (1.71 g, 5.31 mmol) of 4-(benzyloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ethyl ester in ethanol (20 mL). The resulting mixture was degassed and purged three times with hydrogen, and then stirred at 25 °C under hydrogen (15 psi) for 18 h. The mixture was filtered and the filter cake was washed with ethyl acetate (20 mL × 3). The filtrate was concentrated to dryness under reduced pressure to give 7 g (1 g) of 2-(1,1-difluoroethyl)-4-hydroxypyrimidine-5-carboxylic acid ethyl ester, yield 81.18%.

[0379] MS m / z(ESI): 233.0 [M+H] +

[0380] Step 7

[0381] ethyl 4-(cyclohex-3-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate

[0382] 4-(cyclohexyl-3-en-1-oxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ethyl ester

[0383] Ethyl 2-(1,1-difluoroethyl)-4-hydroxypyrimidine-5-carboxylate 7 g (100 mg, 430 μmol), cyclohexyl-3-en-1-ol 7 h (43 mg, 430 μmol, commercially available), and triphenylphosphine (226 mg, 862 μmol) were dissolved in tetrahydrofuran (2 mL). Diisopropyl azodicarbonate (174 mg, 861 μmol) was added dropwise at 0 °C, and the resulting mixture was stirred at 25 °C for 2 h. The mixture was then poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (eluent: system A) to give 7i (130 mg) of ethyl 4-(cyclohexyl-3-en-1-oxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid, with a yield of 96.6%.

[0384] MS m / z(ESI): 313.1 [M+H] +

[0385] Step 8

[0386] 4-(cyclohex-3-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid

[0387] 4-(cyclohexyl-3-en-1-oxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid

[0388] Lithium hydroxide monohydrate (6 mg, 142 μmol) was added to a solution of ethyl 4-(cyclohexyl-3-en-1-oxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid 7i (37 mg, 118 μmol) in tetrahydrofuran (1 mL) and water (0.3 mL). The resulting mixture was stirred at 25 °C for 1 hour. The mixture was concentrated to dryness under reduced pressure to give 4-(cyclohexyl-3-en-1-oxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid 7j (33 mg), yield 97.9%.

[0389] MS m / z(ESI): 285.1 [M+H] +

[0390] Step 9

[0391] 4-(cyclohex-3-en-1-yloxy)-N-((S,E)-1-cyclopropyl-3-(methylsulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide

[0392] 4-(cyclohexyl-3-en-1-yloxy)-N-((S,E)-1-cyclopropyl-3-(methanesulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidin-5-carboxamide

[0393] To a solution of 4-(cyclohexyl-3-en-1-oxy)-2-(1,1-difluoroethyl)pyrimidin-5-carboxylic acid 7j (33 mg, 116 μmol) and (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (31 mg, 116 μmol) in N,N-dimethylformamide (2 mL), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (88 mg, 232 μmol) and N,N-diisopropylethylamine (75 mg, 580 μmol) were added, and the resulting mixture was stirred at 25 °C for 2 hours. The mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by preparative liquid chromatography (Waters 3767 / QDA column: SunFire Sunfire C). 18 (19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, mobile phase B: acetonitrile; flow rate: 20mL / min), yielded 4-(cyclohexyl-3-en-1-yloxy)-N-((S,E)-1-cyclopropyl-3-(methanesulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide 7 (5.4mg), yield 10.54%.

[0394] MS m / z(ESI): 442.1 [M+H] +

[0395] 1H NMR(400MHz, DMSO-d6)δ8.92(s,1H),8.45(dd,J=8.4,3.3Hz,1H),6.89–6.80(m,2H),5.79–5.73(m,1H),5.69–5.61(m,1H),5.58–5.50(m,1H),4.2 9–4.10(m,1H),3.04(s,3H),2.67–2.54(m,2H),2.32–2.17(m,2H),2.10– 1.96(m,5H),1.12–1.02(m,1H),0.63–0.46(m,3H),0.41–0.35(m,1H)ppm.

[0396] Example 8

[0397] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-2-phenoxy-4-((trifluoromethyl)sulfonyl)benzamide

[0398] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-2-phenoxy-4-((trifluoromethyl)sulfone)benzamide

[0399] first step

[0400] methyl 4-bromo-2-phenoxybenzoate

[0401] methyl 4-bromo-2-phenoxybenzoate

[0402] 4-Bromo-2-fluorobenzoate methyl ester 8a (2 g, 8.58 mmol) and phenol (1.21 g, 12.87 mmol) were dissolved in N,N-dimethylformamide (20 mL), and potassium carbonate (2.37 g, 17.16 mmol) was added. The mixture was reacted at 80 °C for 8 hours, then cooled to room temperature. The reaction solution was poured into water (200 mL), and extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated by silica gel column chromatography (eluent: system A) to give 4-bromo-2-phenoxybenzoate methyl ester 8b (1.8 g), with a yield of 68%.

[0403] Step 2

[0404] methyl 2-phenoxy-4-((trifluoromethyl)thio)benzoate

[0405] 2-Phenoxy-4-((trifluoromethyl)thio)methyl benzoate

[0406] Under nitrogen protection, methyl 4-bromo-2-phenoxybenzoate 8b (1.8 g, 5.86 mmol), 2,2-bipyridine (1.83 g, 11.72 mmol), silver trifluoromethyl thiocyanate (1.83 g, 8.79 mmol), and cuprous iodide (1.11 g, 5.86 mmol) were sequentially added to acetonitrile (15 mL), and the reaction was carried out at 110 °C for 24 hours. After cooling to room temperature, the mixture was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate (60 mL), the organic phase was washed with water (30 mL × 2), the organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was separated by silica gel column chromatography (eluent: system A) to give methyl 2-phenoxy-4-((trifluoromethyl)thio)benzoate 8c (1.4 g), yield 73%.

[0407] MS m / z(ESI): 329.3 [M+H] +

[0408] Step 3

[0409] 2-phenoxy-4-((trifluoromethyl)thio)benzoic acid

[0410] 2-Phenoxy-4-((trifluoromethyl)thio)benzoic acid

[0411] Methyl 2-phenoxy-4-((trifluoromethyl)thio)benzoate 8c (500 mg, 1.52 mmol) and lithium hydroxide monohydrate (191.73 mg, 4.57 mmol) were added to a mixed solvent of tetrahydrofuran (3 mL) and water (3 mL). The reaction was carried out at 20 °C for 2 hours. Hydrochloric acid aqueous solution (0.5 M) was added dropwise to adjust the pH to 6. The mixture was extracted with ethyl acetate (15 mL × 2). The organic phases were combined, washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain 2-phenoxy-4-((trifluoromethyl)thio)benzoic acid 8d (500 mg), which was directly used in the next reaction.

[0412] MS m / z(ESI): 315.0 [M+H] +

[0413] Step 4

[0414] 2-phenoxy-4-((trifluoromethyl)sulfonyl)benzoic acid

[0415] 2-Phenoxy-4-((trifluoromethyl)sulfonyl)benzoic acid

[0416] 2-phenoxy-4-((trifluoromethyl)thio)benzoic acid 8d (500 mg, 1.59 mmol) and sodium periodate (1.02 g, 4.77 mmol) were added to a mixed solvent of water (10 mL), chloroform (5 mL), and acetonitrile (5 mL), followed by the addition of ruthenium trichloride hydrate (71.73 mg, 318.19 μmol). The reaction mixture was reacted at 20 °C for 12 hours. The reaction solution was washed with ethyl acetate (50 mL) and saturated ammonium chloride solution (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 2-phenoxy-4-((trifluoromethyl)sulfonyl)benzoic acid 8e (550 mg), which was directly used in the next reaction.

[0417] MS m / z (ESI): 345.0 [MH] -

[0418] Step 5

[0419] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-2-phenoxy-4-((trifluoromethyl)sulfonyl)benzamide

[0420] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)allyl)-2-phenoxy-4-((trifluoromethyl)sulfone)benzamide

[0421] 2-Phenoxy-4-((trifluoromethyl)sulfonyl)benzoic acid 8e (150 mg, 433.18 μmol) was dissolved in acetonitrile (2.5 mL), and (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (180.61 mg, 519.81 μmol), N-methylimidazolium (126.00 mg, 1.52 mmol), and tetramethylchlorourea hexafluorophosphate (145.85 mg, 519.81 μmol) were added. The mixture was reacted at 20 °C for 2 hours. The reaction solution was concentrated under reduced pressure and then separated by preparative liquid chromatography (Waters 3767 / Qda column: SunFire Sunfire C18, 19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, B: acetonitrile; flow rate: 20mL / min) to obtain (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-2-phenoxy-4-((trifluoromethyl)sulfone)benzamide (148.4mg), yield 66%.

[0422] MS m / z (ESI): 504.1 [M+H] +

[0423] 1 H NMR(400MHz,CD3OD)δ8.00(d,J=8.0Hz,1H),7.96–7.89(m,1H),7.50(dd,J=8.5,7 .6Hz,2H),7.44(s,1H),7.31(t,J=7.4Hz,1H),7.21–7.14(m,2H),6.94(dd,J=15.2 ,4.6Hz,1H),6.76(dd,J=15.2,1.6Hz,1H),4.15(dd,J=7.7,4.6Hz,1H),2.86(s,3H ),1.13–1.02(m,1H),0.71–0.64(m,1H),0.61–0.53(m,1H),0.52–0.40(m,2H)ppm.

[0424] Implementing Column 9

[0425] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide-5-d

[0426] (S,E)-N-(1-Cyclopropyl-3-(Methylsulfonyl)allyl)-6-(1,1-Difluoroethyl)-2-phenoxynicotinamide-5-deuterated

[0427] first step

[0428] ethyl 6-(1,1-difluoroethyl)-2-phenoxynicotinate-5-d

[0429] 6-(1,1-Difluoroethyl)-2-phenoxynicotinic acid ethyl ester-5-deuterated

[0430] To a toluene solution (1 mL) of ethyl 5-bromo-6-(1,1-difluoroethyl)-2-phenoxynicotinate (3h) (30 mg, 77.68 μmol) and deuterated methanol (30 mg, 831.7 μmol, commercially available), potassium phosphate (41 mg, 193.40 μmol), n-butyldi(1-adamantyl)phosphine (6 mg, 16.73 μmol, commercially available) and palladium acetate (2 mg, 8.93 μmol) were added. The mixture was purged with nitrogen three times and stirred at 80 °C for 18 hours. The reaction solution was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The solution was purified by silica gel chromatography (eluent: system A) to give 6-(1,1-difluoroethyl)-2-phenoxynicotinic acid ethyl ester-5-deuterated 9a (25 mg), yield 83.51%.

[0431] MS(ESI,pos.ion)m / z:309.2[M+H] +

[0432] Step 2

[0433] 6-(1,1-difluoroethyl)-2-phenoxynicotinic-5-d acid

[0434] 6-(1,1-Difluoroethyl)-2-phenoxy-5-deuterated-nicotinic acid

[0435] 6-(1,1-difluoroethyl)-2-phenoxynicotinic acid ethyl ester-5-deuterated 9a (25 mg, 81.09 μmol) was dissolved in a mixed solution of tetrahydrofuran (1 mL) and water (0.3 mL), and lithium hydroxide monohydrate (4.08 mg, 97.31 μmol) was added. The reaction solution was stirred at 25 °C for 1 hour, and then concentrated to dryness under reduced pressure to give 6-(1,1-difluoroethyl)-2-phenoxy-5-deuterated-nicotinic acid 9b (22 mg), with a yield of 96.81%.

[0436] MS(ESI,pos.ion)m / z:281.0[M+H] +

[0437] Step 3

[0438] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide-5-d

[0439] (S,E)-N-(1-Cyclopropyl-3-(Methylsulfonyl)allyl)-6-(1,1-Difluoroethyl)-2-phenoxynicotinamide-5-deuterated

[0440] To a solution of 6-(1,1-difluoroethyl)-2-phenoxy-5-d-nicotinic acid-9b (22 mg, 78.50 μmol) and (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (21 mg, 60.44 μmol) in N,N-dimethylformamide (2 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (60 mg, 157 μmol) and N,N-diisopropylethylamine (51 mg, 392 μmol) were added, and the mixture was reacted at 25 °C for 2 hours. The reaction solution was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3), and the combined organic phases were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to dryness, and purified by preparative high performance liquid chromatography (Nouryon-Kromasil-C18-10 μm-25*250 mm eluent: 10%-90% (v / v) acetonitrile and water (containing 0.025% ammonium bicarbonate) to obtain (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-6-(1,1-difluoroethyl)-2-phenoxynicotinamide-5-deuterated 9 (9 mg), with a yield of 26.41%.

[0441] MS(ESI,pos.ion)m / z:438.1[M+H] +

[0442] 1 H NMR(400MHz, DMSO-d6)δ8.84(d,J=8.4Hz,1H),8.24(s,1H),7.50–7.45(m,2H),7.30–7.25(m,3H),6.91–6.85(m,2H),4 .30(t,J=8.4Hz,1H),2.97(s,3H),1.79(t,J=20.0Hz,3H),1.15–1.07(m,1H),0.58–0.46(m,3H),0.44–0.36(m,1H)ppm.

[0443] Example 10

[0444] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-4-(pentafluoro-λ 6 -sulfaneyl)-2-(phenoxy-d5)benzamide

[0445] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)propenyl)-4-(Pentafluoro-λ) 6 -Thio)-2-(pentadeuterated phenoxy)benzamide

[0446] first step

[0447] methyl 4-(pentafluoro-λ 6 -sulfaneyl)-2-(phenoxy-d5)benzoate

[0448] Methyl 4-(pentafluoro-λ) 6 -Thio)-2-(pentadeuteroxy)benzoate

[0449] 2-bromo-4-(pentafluoro-λ) 6 Methyl 4-(pentafluoro-λ)benzoate 2d (100 mg, 293.18 μmol), deuterated phenol (34.88 mg, 351.81 μmol), cuprous iodide (55.70 mg, 293.18 μmol), and cesium carbonate (143.28 mg, 439.76 μmol) were dissolved in toluene (2 mL) and reacted at 120 °C for 1 hour. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain methyl 4-(pentafluoro-λ)benzoate. 6 10a (70 mg) of 2-(pentadeuterated phenoxy)benzoate 10a was prepared with a yield of 96.81%.

[0450] MS(ESI,pos.ion)m / z:360.1[M+H] +

[0451] Step 2

[0452] 4-(pentafluoro-λ 6 -sulfaneyl)-2-(phenoxy-d5)benzoic acid

[0453] 4-(pentafluoro-λ) 6 -Thio)-2-(pentadeuteroxy)benzoic acid

[0454] Methyl 4-(pentafluoro-λ) 610a of 4-(pentadeuteroxy)benzoate 10a (70 mg, 194.81 μmol) was dissolved in a mixed solution of tetrahydrofuran (2 mL) and water (0.4 mL). Lithium hydroxide monohydrate (24.52 mg, 584.43 μmol) was added, and the mixture was stirred for 1 hour. The reaction solution was diluted with dichloromethane (30 mL) and water (20 mL). The pH of the system was adjusted to weakly acidic using 2N HCl. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4-(pentafluoro-λ)benzoate. 6 10b (60 mg) of 2-(pentadeuterated phenoxy)benzoic acid (-thio)-2-(pentadeuterated phenoxy)benzoic acid was prepared in a yield of 89.20%.

[0455] MS(ESI,pos.ion)m / z:346.1[M+H] +

[0456] Step 3

[0457] (S,E)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-4-(pentafluoro-λ 6 -sulfaneyl)-2-(phenoxy-d5)benzamide

[0458] (S,E)-N-(1-Cyclopropyl-3-(Methanesulfonyl)propenyl)-4-(Pentafluoro-λ) 6 -Thio)-2-(pentadeuterated phenoxy)benzamide

[0459] 4-(pentafluoro-λ) 6 The following solutions were added sequentially: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (66.62 mg, 347.53 μmol), 1-hydroxybenzotriazole (46.96 mg, 347.53 μmol), and N,N-diisopropylethylamine (89.83 mg, 695.06 μmol) in a solution of (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (72.45 mg, 208.52 μmol) in N,N-dimethylformamide (2 mL). The reaction mixture was allowed to react overnight at room temperature. The reaction solution was concentrated under reduced pressure and then separated by preparative liquid chromatography (Waters 3767 / Qda column: SunFire Sunfire). C18, 19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, B: acetonitrile; flow rate: 20mL / min), yielded (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-4-(pentafluoro-λ) 610 (4 mg) of 2-thio-2-(pentadeuterated phenoxy)benzamide was obtained, with a yield of 4.12%.

[0460] MS(ESI,pos.ion)m / z:503.1[M+H] +

[0461] 1 H NMR (400MHz, Methanol-d4) δ8.93(d,J=7.8Hz,1H),7.85(d,J=8.5Hz,1H),7.70(dd,J=8.5 ,2.1Hz,1H),7.32(d,J=2.1Hz,1H),6.90(dd,J=15.2,4.7Hz,1H),6.70(dd,J=15.3,1.7Hz ,1H),4.10(dp,J=9.1,4.0Hz,1H),2.85(s,3H),1.06(dtt,J=13.0,8.4,4.8Hz,1H),0.65( td,J=8.5,4.1Hz,1H),0.53(tq,J=7.8,4.5Hz,1H),0.41(ddt,J=20.6,9.4,4.6Hz,2H)ppm.

[0462] Example 11

[0463] (S,E)-4-(cyclohex-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide

[0464] (S,E)-4-(cyclohexyl-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-2-(1,1-difluoroethyl)pyrimidin-5-carboxamide

[0465] first step

[0466] ethyl 4-(cyclohex-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate

[0467] Ethyl 4-(cyclohexyl-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ester

[0468] 7 g (40 mg, 172.28 μmol) of ethyl 2-(1,1-difluoroethyl)-4-hydroxypyrimidine-5-carboxylate, 11a (26.04 mg, 206.73 μmol, commercially available), and 27.75 mg (346.88 μmol) of copper oxide were sequentially added to 1 mL of dimethyl sulfoxide and reacted at 100 °C for 4 hours under air. The reaction solution was extracted with ethyl acetate (30 mL × 2), the organic phase was washed with saturated sodium chloride aqueous solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: system A) to obtain ethyl 4-(cyclohexyl-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate 11b (40 mg), with a yield of 74.34%.

[0469] MS(ESI,pos.ion)m / z:313.2[M+H] +

[0470] Step 2

[0471] 4-(cyclohex-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid

[0472] 4-(cyclohexyl-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid

[0473] Ethyl 4-(cyclohexyl-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ester 11b (40 mg, 128.08 μmol) was dissolved in a mixed solution of tetrahydrofuran (1 mL) and water (0.25 mL). Lithium hydroxide monohydrate (26.87 mg, 640.39 μmol) was added to the solution, and the mixture was stirred at room temperature for 1 hour. The pH of the reaction mixture was adjusted to weakly acidic using 1N HCl. The mixture was extracted with dichloromethane (30 mL × 2), and the organic phase was washed with saturated sodium chloride aqueous solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: system B) to obtain 4-(cyclohexyl-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ester 11c (20 mg), with a yield of 54.93%.

[0474] MS(ESI,pos.ion)m / z:285.1[M+H] +

[0475] Step 3

[0476] (S,E)-4-(cyclohex-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide

[0477] (S,E)-4-(cyclohexyl-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-2-(1,1-difluoroethyl)pyrimidin-5-carboxamide

[0478] 4-(cyclohexyl-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidin-5-carboxylic acid 11c (20 mg, 70.36 μmol), (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (29.34 mg, 84.43 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (26.98 mg, 140.72 μmol), 1-hydroxybenzotriazole (19.01 mg, 140.72 μmol), and N,N-diisopropylethylamine (45.47 mg, 351.79 μmol) were sequentially added to a solution of N,N-dimethylformamide (1 mL). The reaction was carried out at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure and then separated by preparative liquid chromatography (Waters 3767 / Qda column: SunFire). Sunfire C18, 19*250mm, 10μm; mobile phase A: 0.1% FA / H2O, B: acetonitrile; flow rate: 20mL / min), yielded (S,E)-4-(cyclohexyl-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide 11 (2.14 mg), in a yield of 6.75%.

[0479] MS(ESI,pos.ion)m / z:442.2[M+H] +

[0480] 1H NMR (400MHz, DMSO) δ8.92(s,1H),8.77(d,J=8.2Hz,1H),6.85(d,J=2.8Hz,2H),5.57(s,1H),4.24(t,J=8.4Hz,1H),3.03(s,3H),2.27(s,2H),2. 14(s,2H),2.00(t,J=19.0Hz,3H),1.74(d,J=6.4Hz,2H),1.60(d,J=7.2 Hz,2H),1.13–1.03(m,1H),0.60–0.43(m,3H),0.37(d,J=6.2Hz,1H)ppm.

[0481] Example 12

[0482] (S,E)-4-(cyclopent-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide

[0483] (S,E)-4-(cyclopent-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-2-(1,1-difluoroethyl)pyrimidin-5-carboxamide

[0484] first step

[0485] ethyl 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate

[0486] Ethyl 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ester

[0487] 7 g (40 mg, 172.28 μmol) of ethyl 2-(1,1-difluoroethyl)-4-hydroxypyrimidine-5-carboxylate, 12a (19.28 mg, 172.28 μmol, commercially available), and copper oxide (13.88 mg, 173.50 μmol) were sequentially added to dimethyl sulfoxide (2 mL), and the mixture was reacted at 100 °C for 4 hours under air. The reaction solution was extracted with ethyl acetate (30 mL × 2), the organic phase was washed with saturated sodium chloride aqueous solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: system A) to obtain ethyl 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate 12b (20 mg), with a yield of 38.92%.

[0488] MS(ESI,pos.ion)m / z:299.1[M+H] +

[0489] Step 2

[0490] 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid

[0491] 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid

[0492] Ethyl 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ester 12b (20 mg, 67.05 μmol) was dissolved in a mixed solution of tetrahydrofuran (1 mL) and water (0.25 mL). Lithium hydroxide monohydrate (2.81 mg, 67.05 μmol) was added to the solution, and the mixture was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to weakly acidic using 1N HCl. The mixture was extracted with dichloromethane (30 mL × 2), and the organic phase was washed with saturated sodium chloride aqueous solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: system B) to obtain 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidine-5-carboxylic acid ester 12c (15 mg), with a yield of 82.79%.

[0493] MS(ESI,pos.ion)m / z:271.1[M+H] +

[0494] Step 3

[0495] (S,E)-4-(cyclopent-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methylsulfonyl)allyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide

[0496] (S,E)-4-(cyclopent-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-2-(1,1-difluoroethyl)pyrimidin-5-carboxamide

[0497] The following substances were added: 4-(cyclopent-1-en-1-yloxy)-2-(1,1-difluoroethyl)pyrimidin-5-carboxylic acid 12c (19.01 mg, 70.36 μmol), (S,E)-1-cyclopropyl-3-(methanesulfonyl)propyl-2-en-1-amine p-toluenesulfonate 1f (29.34 mg, 84.43 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (26.9 μmol). 8 mg (140.72 μmol), 1-hydroxybenzotriazole (19.01 mg, 140.72 μmol), and N,N-diisopropylethylamine (45.47 mg, 351.79 μmol) were sequentially added to a solution of N,N-dimethylformamide (1 mL). The reaction was carried out at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure and then separated by preparative liquid chromatography (Waters 3767 / Qda column: SunFire Sunfire C18, 19*250 mm, 10 μm; mobile phase A: 0.1% FA / H2O, B: acetonitrile; flow rate: 20 mL / min) to obtain (S,E)-4-(cyclohexyl-1-en-1-yloxy)-N-(1-cyclopropyl-3-(methanesulfonyl)propenyl)-2-(1,1-difluoroethyl)pyrimidine-5-carboxamide 12 (1.46 mg), with a yield of 4.81%.

[0498] MS(ESI,pos.ion)m / z:428.2[M+H] +

[0499] 1H NMR(400MHz,DMSO)δ8.95(s,1H),8.84(d,J=8.2Hz,1H),6.85(d,J=2.7Hz,2H) ,5.64(s,1H),4.28–4.20(m,1H),3.03(s,3H),2.58(d,J=7.8Hz,2H),2.39(d, J=8.4Hz,2H),2.06(s,1H),2.01(d,J=6.0Hz,2H),1.97(d,J=6.2Hz,2H),1.12 –1.04(m,1H),0.52(ddd,J=25.6,12.9,5.4Hz,3H),0.38(d,J=5.8Hz,1H)ppm.

[0500] Biological evaluation

[0501] Test Example 1: Test on the inhibitory effect of the compound of the present invention on WRN helicase activity

[0502] The following method was used to determine the degree of inhibition of recombinant human WRN helicase activity by the compounds of the present invention under in vitro conditions.

[0503] The experimental procedure is briefly described as follows: The test compound was first dissolved in DMSO to prepare a stock solution, and then serially diluted using reaction buffer (25mM Tris-HCl Ph8.0, 5mM NaCl, 2mM MgCl2, 1mM TCEP, 0.1mg / mL BSA, 0.01% Triton X-100, 1mM DTT, 200uM ADP, 200uM ATP). The final concentration range of the test compound in the reaction system was 1000nM to 0.004nM. WRN protein (purchased from Sinobiological, catalog number 17475-HNCB) and fluorescent substrate (synthesized by Suzhou Genewiz Biotechnology Co., Ltd., sequences 5'-TGTGTGTGGTTCGCTGGG-(BHQ)-3' and 5'-(TAMRA)-CCCAGCGAACTGGTGTGTGT-3', and internally annealed to obtain double-stranded fluorescent substrate) were prepared using reaction buffer. The reaction was carried out in 384-well microplates. First, the compound and recombinant human WRN protein (final concentration 75 nM) were added to the wells and incubated at room temperature for 30 minutes. Then, a fluorescent substrate (final concentration 100 nM) and Trap-10bp solution (synthesized by Suzhou Genewiz Biotechnology Co., Ltd., sequence 5'-GTTCGCTGGG-3', final concentration 1 μM) were added to the reaction solution and incubated at room temperature for 30 minutes. After incubation, the fluorescence intensity of each well was measured using a microplate reader in Fluorescence mode, with excitation and emission wavelengths set to 535 nm and 585 nm, respectively. The percentage inhibition rate of the compound at each concentration was calculated by comparing the fluorescence intensity ratio with that of the control group (0.1% DMSO). The IC50 of the compound was obtained by nonlinear regression analysis using GraphPad Prism9 software with the compound concentration as the logarithm of the inhibition rate. 50 Values. See Table 1. The results show that the compounds of this invention have good inhibitory effects on WRN helicase.

[0504] IC of the compound of the present invention 50 The values ​​are represented by A and B, respectively:

[0505] A:IC 50 <1000nM

[0506] B:IC 50 ≥1000nM

[0507] The exemplary inhibitory effects of the compounds of the present invention on WRN helicase are shown in Table 1 below:

[0508] Table 1. IC50 values ​​of the compounds of this invention for inhibiting WRN helicase activity. 50

[0509] Conclusion: The compounds of this invention have a good inhibitory effect on WRN helicase.

[0510] Test Example 2: Determination of the inhibitory effect of the compound of the present invention on the proliferation of SW48 cells.

[0511] The following methods were used to determine the effect of the compounds of this invention on the proliferation of SW48 cells. SW48 cells (MSI-H cells) were purchased from the ATCC cell bank in the United States and cultured in Leibovitz's L-15 (Gibco, catalog number 11415064) medium containing 10% fetal bovine serum, 100 U penicillin, and 100 μg / mL streptomycin. Cell viability was determined by... The Luminescent Cell Viability Assay kit (Promega, catalog number G7573) was used for the determination.

[0512] The experimental method was performed according to the kit instructions, and is briefly described below: The test compound was first dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with the above-mentioned culture medium to prepare the test sample. The final concentration range of the compound was 10000 nM-1.52 nM. Cells in the logarithmic growth phase were seeded at a density of 500 cells per well into 96-well cell culture plates and cultured overnight at 37°C in an air incubator. The test compound was then added, and the cells were cultured for another 120 hours. After the culture, 40 μL of CellTiter-Glo assay solution was added to each well, shaken for 5 minutes, and allowed to stand for 10 minutes. The fluorescence values ​​of each well were then read using the Luminescence mode on a microplate reader. The percentage inhibition rate of the compound at each concentration was calculated by comparing the values ​​with the control group (0.1% DMSO). Then, a nonlinear regression analysis was performed in GraphPad Prism 9 software using the logarithm of the compound concentration versus the inhibition rate to obtain the IC50 of the compound inhibiting cell proliferation. 50 The results showed that the compounds of this invention had a good inhibitory effect on the proliferation of SW48 cells.

[0513] IC of the compound of the present invention 50 The values ​​are represented by A and B, respectively:

[0514] A:IC 50 ≤10nM

[0515] B:10 <IC 50 ≤100nM

[0516] The exemplary inhibitory effects of the compounds of the present invention on the proliferation of SW48 cells are shown in Table 2 below:

[0517] Table 2. Inhibitory effect of exemplary compounds of the present invention on SW48 cell proliferation

[0518] Conclusion: The compound of this invention has a good inhibitory effect on the proliferation of SW48 cells.

[0519] Test Example 3: Determination of the inhibitory effect of the compound of the present invention on the proliferation of RL95-2 cells.

[0520] The following methods were used to determine the effect of the compounds of this invention on the proliferation of RL95-2 cells. RL95-2 cells (MSI-H cells) were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in DMEM / F12 medium (Gibco, catalog number A4192001) containing 10% fetal bovine serum, 100 U penicillin, and 100 μg / mL streptomycin. Cell viability was determined by... The Luminescent Cell Viability Assay kit (Promega, catalog number G7573) was used for the determination.

[0521] The experimental method was performed according to the kit instructions, and is briefly described below: The test compound was first dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with the above-mentioned culture medium to prepare the test sample. The final concentration range of the compound was 10000 nM-1.52 nM. Cells in the logarithmic growth phase were seeded at a density of 500 cells per well into 96-well cell culture plates and cultured overnight at 37°C in a 5% CO2 incubator. The test compound was then added, and the cells were cultured for another 120 hours. After the culture, 50 μL of CellTiter-Glo assay solution was added to each well, shaken for 5 minutes, and allowed to stand for 10 minutes. The fluorescence values ​​of each well were then read using the Luminescence mode on a microplate reader. The percentage inhibition rate of the compound at each concentration was calculated by comparing the values ​​with the control group (0.1% DMSO). Then, a nonlinear regression analysis was performed in GraphPad Prism 9 software using the logarithm of the compound concentration versus the inhibition rate to obtain the IC50 of the compound inhibiting cell proliferation. 50 Values. The results showed that the compounds of this invention had a good inhibitory effect on the proliferation of RL95-2 cells.

[0522] IC of the compound of the present invention 50 The values ​​are represented by A and B, respectively:

[0523] A:IC 50 ≤10nM

[0524] B:10 <IC 50 ≤100nM

[0525] The exemplary inhibitory effects of the compounds of the present invention on the proliferation of RL95-2 cells are shown in Table 3 below:

[0526] Table 3. Inhibitory effect of exemplary compounds of the present invention on RL95-2 cell proliferation

[0527] Conclusion: The compound of this invention has a good inhibitory effect on the proliferation of RL95-2 cells.

[0528] Test Example 4: Determination of the inhibitory effect of the compound of the present invention on the proliferation of HCT116 cells.

[0529] The following methods were used to determine the effect of the compounds of this invention on the proliferation of HCT116 cells. HCT116 cells were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in McCoy's 5a medium containing 10% fetal bovine serum, 100 U penicillin, and 100 μg / mL streptomycin. Cell viability was determined by... The Luminescent Cell Viability Assay kit (Promega, catalog number G7573) was used for the determination.

[0530] The experimental method was performed according to the kit instructions, and is briefly described below: The test compound was first dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with the above-mentioned culture medium to prepare the test sample. The final concentration range of the compound was 10000 nM-1.52 nM. Cells in the logarithmic growth phase were seeded at a density of 300 cells per well into 96-well cell culture plates and cultured overnight at 37°C in a 5% CO2 incubator. The test compound was then added, and the cells were cultured for another 120 hours. After the culture, 50 μL of CellTiter-Glo assay solution was added to each well, shaken for 5 minutes, and allowed to stand for 10 minutes. The fluorescence values ​​of each well were then read using the Luminescence mode on a microplate reader. The percentage inhibition rate of the compound at each concentration was calculated by comparing the values ​​with the control group (0.1% DMSO). Then, a nonlinear regression analysis was performed in GraphPad Prism 9 software using the logarithm of the compound concentration versus the inhibition rate to obtain the IC50 of the compound inhibiting cell proliferation. 50 The results showed that the compounds of this invention had a good inhibitory effect on the proliferation of HCT116 cells.

[0531] IC of the compound of the present invention 50 The values ​​are represented by A and B, respectively:

[0532] A:IC 50 ≤10nM

[0533] B:10 <IC50 ≤100nM

[0534] The exemplary inhibitory effects of the compounds of the present invention on the proliferation of HCT116 cells are shown in Table 4 below:

[0535] Table 4. Inhibitory effect of exemplary compounds of the present invention on HCT116 cell proliferation

[0536] Conclusion: The compound of this invention has a good inhibitory effect on the proliferation of HCT116 cells.

[0537] Test Example 5: Determination of the membrane permeability of the compound Caco-2 of the present invention

[0538] A monolayer of Caco-2 cells grown on a polycarbonate membrane (monolayer cell integrity verified) was used. The culture medium was removed, and the cells were washed once with preheated HBSS buffer. Working solution containing the compound of the present invention (final compound concentration 10 μM, solvent: buffer containing appropriate amount of DMSO) was added to the donor side (A side or B side). An equal volume of blank buffer was added to the recipient side. The culture plate was incubated with shaking at 37°C and appropriate CO2 concentration. Samples were taken from both the recipient and donor sides at predetermined time points (e.g., 120 minutes after the start of incubation). Two parallel samples were prepared for each transport direction. The samples were treated with acetonitrile solution containing an internal standard (e.g., glibenclamide) for protein precipitation, followed by centrifugation, and the supernatant was transferred to a sample vial. The concentration of the analyte in each sample was quantitatively analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Based on the measured concentrations, the apparent permeability coefficients of the compound from the top to the base (AB) and from the base to the top (BA) were calculated. The efflux ratio was calculated based on the ratio of the permeability coefficients along the BA and AB directions. The efflux ratio was evaluated by comparing the permeability coefficients of the compound along the AB direction with those of known highly permeable standards. The results are shown in Table 5 below, demonstrating that the compound of this invention exhibits excellent membrane permeability.

[0539] Table 5. Membrane permeability properties of exemplary compounds of the present invention

[0540] Conclusion: The compound of this invention exhibits excellent membrane permeability and, compared to the reference compound RO7589831, does not show a tendency for efflux. The structure of the reference compound RO7589831 is as follows: Formula A:

[0541] Test Example 6: Determination of plasma stability of the compounds of the present invention in various genera.

[0542] The metabolic stability of the compounds of the present invention was assessed using an in vitro incubation method. Briefly, the test compound was mixed with plasma from different species (including but not limited to human, rat, mouse, and dog) and incubated at 37°C. Samples were taken at predetermined time points (e.g., 0 to 120 minutes), and the reaction was terminated by adding acetonitrile solution containing an internal standard, resulting in protein precipitation. The treated samples were analyzed by liquid chromatography-tandem mass spectrometry to determine the residual concentration of the original compound at each time point. The in vitro half-life of the compounds demonstrated that the compounds of the present invention exhibited excellent metabolic stability in the plasma of the aforementioned species, and showed superior plasma stability compared to the reference compound RO7589831, as shown in Table 6 below.

[0543] Table 6. Plasma half-life of exemplary compounds of the present invention

[0544] Conclusion: The compound of this invention has better plasma stability than the reference compound RO7589831.

[0545] Test Example 7: Pharmacological efficacy test of the compound of the present invention on RL95-2.

[0546] A subcutaneous xenograft tumor model was established in BALB / c nude mice by subcutaneously inoculating RL95-2 human endometrial cancer cells. The experiment was divided into three groups: a treatment group (RO7589831), a treatment group (compound 8), and a solvent control group, with six mice in each group. Oral administration was administered for 21 days. Efficacy was evaluated based on tumor growth, and safety was evaluated based on changes in animal weight and mortality.

[0547] BALB / c nude mice, female, 6-7 weeks old, were purchased from Hangzhou Qizhen Laboratory Animal Technology Co., Ltd. Animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee. Animals were housed in the experimental environment for at least 7 days after arrival before experiments could begin. All animals were housed in intelligent, individually ventilated cages (IVCs) with constant temperature and humidity. The temperature in the housing was 20-26℃, and the humidity was 40-70%. Animals had free access to food and water. Animals were marked using ear tags.

[0548] RL95-2 human endometrial cancer cells were cultured in DMEM and F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphoterase B solution. RL95-2 cells in the exponential growth phase were collected and resuspended in PBS and Matrigel (1:1). Approximately 7.5 × 10⁶ RL95-2 cells were subcutaneously seeded on the right back of BALB / c nude mice. The cells were cultured until the average tumor volume reached approximately 100-150 mm². 3Animals were randomly divided into three groups based on tumor size: a treatment group for test substance RO7589831, a treatment group for test substance compound 8, and a solvent control group, with six animals in each group. Administered orally once daily by gavage for 21 days. Tumor volume and animal weight were measured twice weekly. The tumor volume (TV) was calculated using the following formula: TV (tumor volume) = 1 / 2 × a × b², where a and b represent the length and width of the tumor, respectively.

[0549] The tumor growth curve is shown in Figure 1, and the changes in body weight of the experimental animals are shown in Figure 2. The experimental results show that compound 8 of this invention has a significant inhibitory effect on tumor proliferation in the RL95-2 human endometrial cancer cell subcutaneous xenograft model, and its inhibitory effect on tumor growth is superior to that of the reference compound RO7589831. No decrease in body weight was observed in the test animals during the experiment, indicating that the compound of this invention has good safety after administration.

[0550] The compounds of the present invention, including compound 8, have been shown to have good inhibitory effects on the proliferation of SW48 cells, RL95-2 cells, and HCT116 cells; the compounds of the present invention also have high permeability and no efflux; in addition, the compounds of the present invention also have good plasma stability and efficacy.

Claims

1. A compound of general formula (I) or a stereoisomer, tautomer, deuterated derivative or pharmaceutically acceptable salt thereof: in: key express It can exist as a (Z)- or (E)- stereoisomer; X is selected from CH and N; Y is selected from CR b and N; Z is selected from CH and N; R 1 Selected from C 2-6 alkenyl and C 2-6 alkynyl group, wherein the C 2-6 alkenyl or C 2-6 The alkynyl group is optionally surrounded by one or more R groups. c replace; Ring A is selected from phenyl, C 3-10 cycloalkyl, C 3-8 Cycloalkenyl and 3-10 membered heterocyclic groups, wherein the 3-10 membered heterocyclic group contains at least one double bond; R c Each is independently selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl and 5-6 membered heteroaryl, wherein the C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl and 5-6 heteroaryl groups are optionally enclosed by one or more R groups. j Replaced; Or, two Rs c Together with the same carbon atom it is attached to, it forms a -C (=O); Or, two Rs c Together with the same carbon atom it is attached to, they form a C 3-10 Cycloalkyl or 3-10 membered heterocyclic group; wherein the C 3-10 Cycloalkyl or 3-10 membered heterocyclic groups are optionally surrounded by one or more R groups. j Replaced; R j Each is independently selected from deuterium, hydroxyl, halogen, nitro, cyano, and C atoms. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 heteroaryl, =O, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 ; R 2 Selected from C 1-6 Haloalkyl, C 2-6 Alkyne, -SF5 and -S(=O)2R e ; wherein C 2-6 The alkynyl group may be further selected by one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups; R e Selected from C 1-6 Halogenated alkyl groups, preferably trifluoromethyl; R b Selected from hydrogen atom, deuterium atom, halogen, nitro, cyano, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6- 10 aryl, 5-6 heteroaryl, optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, alkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -OR 8 =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 -N(R) 9 )C(=O)R 10 and -N(R) 9 )C(=O)OR 10 The substituents are replaced; The condition is that when Optionally substituted phenyl or optionally substituted C 3-10 cycloalkyl and R 2 C 1-6 When alkyl haloside is used, Y is CR. b And R b Not selected from hydrogen atom, cyano group, halogen and C 1-6 alkyl; R 4 Selected from C 1-6 Alkyl and C 3-10 cycloalkyl; wherein the C 1-6 Alkyl or C 3-10 The cycloalkyl group may optionally be further selected from one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1- Substituents of 6-haloalkoxy groups; R d Selected from hydrogen atoms, C 1-6 Alkyl and -C 0-6 Alkylene-R A ; R A Selected from hydroxyl, alkenyl, alkynyl, cyano, C 3-12 cycloalkyl, C 6-10 aryl, 3-12 heterocyclic, 5-10 heteroaryl, and 8-10 fused rings, wherein the alkenyl, alkynyl, C 3-12 cycloalkyl, C 6-10 Aryl, 3-12 heterocyclic, 5-10 heteroaryl, or 8-10 fused ring optionally further modified by one or more R B Replaced; R B Each is independently selected from deuterium, hydroxyl, halogen, nitro, cyano, and C atoms. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 aryl, or heteroaryl groups may be further divided by one or more R groups. C Replaced; Or, two Rs B Together with the same carbon atom it is attached to, it forms a -C (=O); R C Each is independently selected from deuterium, hydroxyl, halogen, nitro, cyano, and C atoms. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 heteroaryl, optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Hydroxyalkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -OR 8 =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 -N(R) 9 )C(=O)R 10 and -N(R) 9 )C(=O)OR 10 The substituents are replaced; Or, two Rs C Together with the same carbon atom it is attached to, it forms a -C (=O); R f Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 3-10 Cycloalkyl and 3-10 membered heterocyclic groups; wherein C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic groups optionally further selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups; R g Each atom is independently selected from hydrogen atoms and deuterium atoms, preferably hydrogen atoms; R 5 Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl and 5-6 heteroaryl, wherein the C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-6 heteroaryl groups may be further selected from one or more deuterium atoms, hydroxyl groups, halogens, nitro groups, cyano groups, C6 groups, etc. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 and -N(R) 9 )C(=O)R 10 The substituents are replaced; R 6 and R 7 Each is independently selected from hydrogen atoms, hydroxyl groups, and C atoms. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6- 10 Aryl and 5-6 membered heteroaryl, wherein the C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-6 heteroaryl groups may be further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-6 heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 and -N(R) 9 )C(=O)R 10 The substituents are replaced; Or, R 6 and R 7 The atoms bonded to them together form a structure containing one or more N, O, or S atoms (=O). r The 4-8 membered heterocyclic group, wherein the 4-8 membered heterocyclic group is optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-6 heteroaryl, =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 and -N(R) 9 )C(=O)R 10 The substituents are replaced; R 8 R 9 and R 10 Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl, amino, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl and 5-6-membered heteroaryl, wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is optionally further selected from one or more groups selected from hydroxyl, halogen, nitro, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Substituents include aryl, 5-6 heteroaryl, carboxyl, and carboxylic acid ester groups; n is 0, 1, 2, 3, 4, or 5; and r can be 0, 1, or 2 independently.

2. The compound according to claim 1, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein: R 1 Selected from C 2-6 alkenyl and C 2-6 alkynyl group, wherein the C 2-6 alkenyl or C 2-6 The alkynyl group is optionally surrounded by one or more R groups. c replace; R c Each is independently selected from C 3-10 cycloalkyl, wherein the C 3-10 cycloalkyl groups are optionally surrounded by one or more R groups. j Replaced; Or, two Rs c Together with the same carbon atom it is attached to, they form a C 3-10 Cycloalkyl or 3-10 membered heterocyclic group; wherein the C 3-10 Cycloalkyl or 3-10 membered heterocyclic groups are optionally surrounded by one or more R groups. j Replaced; R j The definition is as described in claim 1.

3. The compound according to claim 1 or 2, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein R 1 Selected from the following groups:

4. The compound according to claim 1, or its stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof, wherein it is the compound according to general formula (II), or its stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof: in, Rings A, X, Y, Z, R d R g R f R j R 2 R 4 The definitions of n are as described in claim 1.

5. The compound according to claim 1 or 4, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein, Ring A is selected from C 3-8 Cycloalkenyl and 3-10 membered heterocyclic groups, wherein the 3-10 membered heterocyclic group contains at least one double bond.

6. The compound according to claim 1 or 4, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein, Ring A is selected from phenyl and C 3-10 Cycloalkyl.

7. The compound according to claim 6, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein, R 2 Selected from -SF5, C 2-6 Alkyne group and -S(=O)2R e ; wherein C 2-6 The alkynyl group may be further selected by one or more atoms selected from deuterium, halogen, hydroxyl, cyano, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups; R e Selected from C 1-6 Halogenated alkyl group, preferably trifluoromethyl.

8. The compound according to any one of claims 1-7, or a stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof, wherein Y is selected from N and C. b R b Selected from hydrogen atom, deuterium atom, cyano group, halogen, -OR 5 and -NR 6 R 7 ; R 5 R 6 R 7 Each is independently selected from hydrogen atoms and C atoms. 1-6 alkyl.

9. The compound according to claim 8, or a stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof, wherein R b It can be a hydrogen atom, a deuterium atom, fluorine, chlorine, bromine, cyano, amino, dimethylamino, hydroxyl, or methoxy.

10. The compound according to claim 6, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein, R 2 Selected from C 1-6 Halogenated alkyl groups; Y is CR b ; R b Selected from deuterium atoms, nitro groups, and C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-6 quinone heteroaryl, -SF5, -OR 5 -OC(=O)R 5 -C(=O)R 5 -C(=O)OR 5 -N(R) 6 )C(=O)R 7 -N(R) 6 )C(=O)OR 7 -NR 6 R 7 -C(=O)NR 6 R 7 -S (=O) r NR 6 R 7 and -S (=O) r R 5 The C mentioned therein 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl, 5-6 heteroaryl, optionally further selected by one or more groups selected from hydroxyl, halogen, nitro, cyano, alkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -OR 8 =O, -C(=O)R 8 -C(=O)OR 8 -OC(=O)R 8 -NR 9 R 10 -C(=O)NR 9 R 10 -S(=O)2NR 9 R 10 -N(R) 9 )C(=O)R 10 and -N(R) 9 )C(=O)OR 10 The substituents are replaced by the substituents. r can be 0, 1, or 2 independently; R 5 R 6 R 7 R 8 R 9 and R 10 The definition is as described in claim 1.

11. The compound according to claim 10, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein, R b Selected from deuterium atoms, nitro groups, and C 2-6 alkenyl, C 2-6 alkynyl group, -OR 5 -NR 6 R 7 -C(=O)R 5 -S (=O) r R 5 3-10 membered heterocyclic groups, C 3-10 cycloalkyl and 5-6-membered heteroaryl; wherein the C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 heterocyclic, 5-6 heteroaryl groups optionally further selected by one or more hydroxyl, halogen, cyano, C 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups; R 5 R 6 R 7 Each is independently selected from hydrogen atoms and C atoms. 1-6 alkyl; r is 2.

12. The compound according to claim 11, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein R b Selected from the following groups: deuterium, nitro, hydroxyl, methoxy, amino, dimethylamino, vinyl, ethynyl.

13. The compound according to any one of claims 1, 4-12, or a stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof, wherein ring A is selected from the following groups:

14. The compound according to claims 1-13, or its stereoisomers, tautomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein, R 4 Selected from C 1-6 Alkyl group, preferably methyl group.

15. The compound according to any one of claims 1-14, or a stereoisomer, tautomer, deuterated product, or pharmaceutically acceptable salt thereof, wherein R d Selected from hydrogen atoms.

16. The compound according to any one of claims 1-15, or a stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof, wherein R f Selected from C 3-10 Cycloalkyl, preferably cyclopropyl.

17. The compound according to any one of claims 1-16, or a stereoisomer, tautomer, deuterated product, or pharmaceutically acceptable salt thereof, wherein R 2 Selected from SF5 18. The compound according to any one of claims 1-17, or a stereoisomer, tautomer, deuterated derivative, or pharmaceutically acceptable salt thereof, wherein the compound is selected from:

19. A pharmaceutical composition comprising an effective dose of the compound or its stereoisomer, tautomer or pharmaceutically acceptable salt according to any one of claims 1-17, and a pharmaceutically acceptable carrier.

20. Use of the compound or its stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 1-18, in the preparation of a WRN inhibitor.

21. Use of the compound or stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 1-18, in the preparation of a medicament for treating or preventing WRN-mediated diseases; preferably, wherein the WRN-mediated disease is a highly microsatellite unstable cancer; more preferably, the highly microsatellite unstable cancer is selected from colorectal cancer, gastric cancer, endometrial cancer, rectal adenocarcinoma, adrenocortical carcinoma, uterine sarcoma, cervical cancer, nephroblastoma, mesothelioma, esophageal cancer, breast cancer, clear cell renal cell carcinoma, ovarian serous cystadenocarcinoma, bile duct carcinoma, thymoma, liver cancer, head and neck squamous cell carcinoma, sarcoma, melanoma of the skin, squamous cell carcinoma of the lung, prostate cancer, lung adenocarcinoma, transitional cell carcinoma of the bladder, pediatric neuroblastoma, chronic lymphocytic leukemia, and glioma, and even more preferably colorectal cancer, gastric cancer, or endometrial cancer.

22. Use of the compound or its stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 1-18, in the preparation of a medicament for the treatment or prevention of highly microsatellite unstable cancers; preferably, wherein the highly microsatellite unstable cancers are selected from colorectal cancer, gastric cancer, endometrial cancer, rectal adenocarcinoma, adrenocortical carcinoma, uterine sarcoma, cervical cancer, nephroblastoma, mesothelioma, esophageal cancer, breast cancer, clear cell renal cell carcinoma, ovarian serous cystadenocarcinoma, bile duct cancer, thymoma, liver cancer, head and neck squamous cell carcinoma, sarcoma, skin melanoma, lung squamous cell carcinoma, prostate cancer, lung adenocarcinoma, bladder transitional cell carcinoma, pediatric neuroblastoma, chronic lymphocytic leukemia, and glioma, more preferably colorectal cancer, gastric cancer, or endometrial cancer.

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