WO2024222677A1 - Wrn inhibitor - Google Patents

Abstract

The present invention provides a compound represented by formula (A) as a WRN inhibitor, a compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof. The present invention further provides a pharmaceutical composition containing the compound and a use of the pharmaceutical composition in treatment of cancers.

Description

WRN inhibitors Technical Field

The present invention belongs to the field of medicine, and in particular relates to a WRN inhibitor.

Background Art

Abnormal DNA mismatch repair (MMR) can lead to high mutations (deletions or insertions) in the DNA nucleotide repeat region, which is called microsatellite instability (MSI). High microsatellite instability (MSI-H) can induce the occurrence of tumors, including colorectal cancer, gastric cancer, endometrial cancer, and ovarian cancer (Nature, 2019, 568, 551-556), among which colorectal cancer (15%) and gastric cancer (22%) have the highest mutation rates. Although immunotherapy PD-1/PD-L1 currently has very good therapeutic effects in multiple tumors, such as pembrolizumab, which greatly improves the median progression-free survival (PFS) in patients with advanced colorectal cancer with MSI-H compared to chemotherapy, and has been approved by the FDA as a first-line therapy (N. Engl. J. Med. 2020, 383, 2207-2218), there are still many MSI-H tumor patients who cannot benefit from it. In addition, the 2022 ASCO conference reported that in the Phase 2 CheckMate 142 (NCT02060188) clinical trial: the dual immunotherapy PD-1+CTLA-4 (nivolumab+ipilimumab) was used to treat patients with metastatic colorectal cancer. Regardless of whether the patients were in first-line treatment or second-line treatment, more than half of the tumor patients would relapse after 4 years of follow-up, so new treatments are urgently needed.

Synthetic lethality means that in tumor cells, the inactivation of any single gene in the two genes has no obvious effect on the survival of tumor cells, but the simultaneous inactivation of the two genes can lead to the death of tumor cells (Nat. Rev. Drug Discov. 2020, 19, 23-38; Cancer Discov. 2021, 11, 1626-1635). Synthetic lethal targeted drugs generally produce a good therapeutic safety window, while also increasing the development accessibility of certain targets with high mutation rates but difficult to drug. Currently, the most successful cases of synthetic lethality are PARP1/2 inhibitors such as Olaparib, Rucaparib, Niraparib, etc. These drugs have achieved excellent therapeutic effects in the treatment of BRCA1/2 mutant ovarian cancer, breast cancer, etc. and have been approved for marketing one after another (Nat. Rev. Drug Discov. 2020, 19, 711-736; Nat. Rev. Clin. Oncol. 2020, 17, 136-137). In 2019, Adam J. Bass et al. published consecutive articles in Nature proving that Werner helicase (WRN) is a synthetic lethal target for MSI-H tumors (Nature, 2019, 568, 551-556; Nature, 2019, 586, 292-298). Knocking out WRN as a whole or mutating WRN's helicase to K557M (loss of helicase function) both induce cell cycle arrest and apoptosis in MSI-H tumor cells. Not only that, Mathew J. Garnett et al. also found that even if the tumor cells of MSI-H patients become resistant after chemotherapy and immunotherapy, they can still further inhibit tumor growth by inhibiting WRN (Cancer Discov. 2021, 11, 1923-1937).

Despite the progress made in WRN research, there is still no approved WRN inhibitor to treat MSI-H related cancers. The novel WRN inhibitor provided by the present invention is expected to meet clinical needs.

Summary of the invention

The present invention provides a novel WRN inhibitor, wherein the compound of the present invention has a good inhibitory effect on WRN DNA unwinding, has a good anti-proliferation effect on MSI-H tumor cells, and has no inhibitory effect on microsatellite stable (MSS) tumor cells.

In another aspect, the present invention provides a compound of formula (A), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

in,

Ring B or Ring C is a 5-6 membered heteroaryl or a 5-6 membered heterocyclic group, and at least one ring exists;

Y is selected from CH or N;

R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 R _x ;

_R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

Rx is selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ ) _p -OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a ;

R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

m is selected from 0, 1, 2 or 3;

n is selected from 0, 1 or 2;

p is selected from 0, 1 or 2.

In some embodiments, only one of Ring B and Ring C is present.

In some embodiments, Y is N.

In some embodiments, for Wherein M 1 and M ₂ are each independently selected from CH or N. When one of M ₁ or M ₂ is _{selected from N, M 1 and M 2 form a monomer; M 3 , M 4 and M 5 are each} independently selected from CH, O, S or N, and M ₃ , M ₄ and M ₅ contain at least one N atom; Y is selected from CH or N.

In some embodiments, for Wherein _M1 and _M2 are each independently selected from CH or N. In some embodiments, for Wherein R ₅ is H or C _1-6 alkyl.

In some embodiments, R ₁ is selected from a 5-6 membered monocyclic heteroaryl containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur, a 5-6 membered monocyclic heterocyclyl containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur, a 7-12 membered bicyclic heteroaryl containing 1 or 2 or 3 or 4 heteroatoms selected from oxygen, nitrogen and sulfur, or a 7-12 membered bicyclic heterocyclyl containing 1 or 2 or 3 or 4 heteroatoms selected from oxygen, nitrogen and sulfur, each of which may be substituted by 1, 2 or 3 R x selected from H, C _1-6 alkyl, C _{1-6 haloalkyl, C 1-6 alkoxy} , CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ ) _p -OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a . In some embodiments, R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 R x , wherein R x is selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ )p-OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a . In some embodiments, R ₁ is selected from In some embodiments, R is selected _from

In some embodiments, R ₂ is selected from C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 alkylthio, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In some embodiments, R ₂ is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

In some embodiments, R ₃ is selected from H or C _1-6 alkyl. In some embodiments, R ₃ is C _1-6 alkyl. In some embodiments, R ₃ is methyl, ethyl or propyl.

In some embodiments, Ring A is absent.

In some embodiments, for wherein R _4a , R _4b and R _4c are independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio or C _3-6 cycloalkyl. for wherein R _4a is halogen or C _1-6 haloalkyl, and R _4b is halogen or C _1-6 alkyl. In some embodiments, for wherein R _4a is C _1-6 haloalkyl, and R _4b is halogen. In some embodiments, for

In some embodiments, for wherein R _4a is selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio or C _3-6 cycloalkyl, wherein Ring A is a 5- or 6-membered heteroaryl or heterocyclic group, each of which contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, for wherein R _4a is selected from halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio or C _3-6 cycloalkyl, and ring A is In some embodiments, R _4a is C _1-4 haloalkyl. In some embodiments, R _4a is trifluoromethyl.

In another aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

in,

M ₁ and M ₂ are each independently selected from CH or N. When one of M ₁ or M ₂ is selected from N, M ₁ and M ₂ form a monomer;

M ₃ , M ₄ and M ₅ are each independently selected from CH, O, S or N, and M ₃ , M ₄ and M ₅ contain at least one N atom;

Y is selected from CH or N;

R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 R _x ;

_R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

R4 is independently selected from H, halogen, CN, _SF5 , _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkylthio, _C3-6 cycloalkyl or 4-10 membered heterocyclyl;

Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

Rx is selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ ) _p -OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a ;

R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

m is selected from 0, 1, 2 or 3;

n is selected from 0, 1 or 2.

In another aspect, the present invention provides a compound of formula (II), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

in,

R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 Rx;

_R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

R4 is independently selected from H, halogen, CN, _SF5 , _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkylthio, _C3-6 cycloalkyl or 4-10 membered heterocyclyl:

Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

Rx is selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ ) _p -OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a ;

R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

m is selected from 0, 1, 2 or 3;

n is selected from 0, 1 or 2.

In another aspect, the present invention provides a compound of formula (III) or formula (IV), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

in,

M ₁ and M ₂ are each independently selected from CH or N;

R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 Rx;

_R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

R4 is independently selected from H, halogen, CN, _SF5 , _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkylthio, _C3-6 cycloalkyl or 4-10 membered heterocyclyl;

Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

_R5 is selected from H, halogen, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

_Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

m is selected from 0, 1, 2 or 3;

n is selected from 0, 1 or 2.

In another more specific embodiment, the present invention relates to a compound of formula (II), (III) or (IV), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

Wherein, R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 Rx;

_R2 is selected from methyl, ethyl, trifluoroethyl, methylthio or cyclopropyl;

R ₃ is independently selected from H, F, CN, methyl, ethyl, trifluoromethyl, cyclopropyl; or two R ₃ are connected to the carbon atom where they are located to form a cyclopropyl or cyclobutyl group;

R ₄ is independently selected from H, F, Cl, Br, CN, SF ₅ , methyl, ethyl, trifluoromethyl, difluoromethyl, methylthio or cyclopropyl;

Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

R ₅ is selected from H, F, Cl, methyl, ethyl, trifluoromethyl, methoxy, methylthio or cyclopropyl;

_Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

m is selected from 0, 1, 2 or 3;

n is selected from 0, 1 or 2.

In another aspect, the present invention provides a compound of formula (III-1), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

in,

_M1 is selected from CH or N;

R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 Rx;

_R2 is selected from H, methyl, ethyl, trifluoromethyl, trifluoroethyl, methoxy, methylthio or cyclopropyl;

R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl:

Ring A is selected from 5-6 membered heteroaryl or Ring A is absent;

R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

_Rx is selected from H, methyl, trifluoromethyl, methoxy, CN, _NH2 , -C(O)Me, -C(O)OMe, -( _CH2 )-OH or -S(O) _2- Me;

m is selected from 0, 1, 2 or 3.

In some embodiments, _M1 is N.

In some embodiments, R ₁ is It may be substituted by 1, 2 or 3 Rx. In some embodiments, _R1 is

In some embodiments, R ₂ is methyl or ethyl. In some embodiments, R ₂ is ethyl.

In some embodiments, Ring A is absent.

In some embodiments, for wherein R _4a is halogen or C _1-6 haloalkyl, and R _4b is halogen or C _1-6 alkyl. In some embodiments, for wherein R _4a is C _1-6 haloalkyl, and R _4b is halogen. In some embodiments, for

In some embodiments, _R5 is H.

In another aspect, the present invention provides a compound of formula (IV-1), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

in,

R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 Rx;

_R2 is selected from H, methyl, ethyl, trifluoromethyl, trifluoroethyl, methoxy, methylthio or cyclopropyl;

R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

Ring A is selected from 5-6 membered heteroaryl or Ring A is absent;

R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

Rx is selected from H, methyl, trifluoromethyl, methoxy, CN, _NH2 , -C(O)Me, -C(O)OMe, -( _CH2 )-OH or -S(O) _2- Me;

m is selected from 0, 1, 2 or 3.

In some embodiments, R ₁ is It may be substituted by 1, 2 or 3 Rx. In some embodiments, _R1 is

In some embodiments, R ₂ is methyl or ethyl. In some embodiments, R ₂ is ethyl.

In some embodiments, R ₄ is independently selected from H, halogen, C _1-6 alkyl or C _1-6 haloalkyl, and in some embodiments, m is 2.

In some embodiments, Ring A is absent.

In some embodiments, for wherein R _4a is halogen or C _1-6 haloalkyl, and R _4b is halogen or C _1-6 alkyl. In some embodiments, for wherein R _4a is C _1-6 haloalkyl, and R _4b is halogen. In some embodiments, for

In another aspect, the present invention provides the following compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, and optionally a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient, which may also contain another therapeutic agent.

In another aspect, the present invention provides the use of a compound of the present invention in the preparation of a medicament for treating and/or preventing a WRN-mediated disease.

In another aspect, the present invention provides a method for treating and/or preventing a WRN-mediated disease in a subject, comprising administering to the subject a compound or composition of the present invention.

In another aspect, the present invention provides a compound of the present invention or a composition of the present invention for use in the treatment and/or prevention of a WRN-mediated disease.

In a specific embodiment, the disease treated by the present invention includes a cancer selected from the group consisting of colorectal cancer (e.g., colon cancer, rectal cancer, large intestine adenocarcinoma), gastric cancer (e.g., gastric adenocarcinoma), or lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma).

Other objects and advantages of the present invention will be apparent to those skilled in the art from the following detailed description, examples and claims.

Specific implementation plan

Related definitions

Definitions of specific functional groups and chemical terms are described in more detail below.

When a numerical range is listed, each value and subrange within the stated range is intended to be included. For example, "C _1-6 alkyl" includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C _1-6 , C _1-5 , C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-5 , C _{2-4 ,} C _2-3 , C 3-6, C _3-5 , C _3-4 , C _4-6 , C _4-5 , and C _5-6 alkyl.

"C _1-6 alkyl" refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C _1-4 alkyl and C _1-2 alkyl are preferred. Examples of C _1-6 alkyl include: methyl (C ₁ ), ethyl (C ₂ ), n-propyl (C ₃ ), isopropyl (C 3), n-butyl (C ₄ ), tert-butyl (C ₄ ), sec-butyl (C ₄ ), isobutyl (C ₄ ), n-pentyl (C ₅ ), 3-pentyl (C ₅ ), pentyl (C ₅ ), neopentyl (C ₅ ), 3-methyl-2-butyl (C ₅ ), tert-pentyl (C ₅ ) and n-hexyl (C ₆ ). The term "C _1-6 alkyl" also includes heteroalkyl groups in which one or more (e.g., ₁ , 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, ₁ to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me _{( -CH3 ), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3 ) , or i - Bu} ( _{-CH2CH ( CH3} ) _{2 )} .

" _C2-6 alkenyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, _C2-4 alkenyl is preferred. Examples of _C2-6 alkenyl include: vinyl ( _C2 ), 1-propenyl ( _C3 ), 2-propenyl ( _C3 ), 1-butenyl ( _C4 ), 2-butenyl ( _C4 ), butadienyl ( _C4 ), pentenyl ( _C5 ), pentadienyl ( _C5 ), hexenyl ( _C6 ), and the like. The term " _C2-6 alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

"C _2-6 alkynyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C _2-4 alkynyl is preferred. Examples of C _2-6 alkynyl include, but are not limited to, ethynyl (C ₂ ), 1-propynyl (C ₃ ), 2-propynyl (C ₃ ), 1-butynyl (C ₄ ), 2-butynyl (C ₄ ), pentynyl (C ₅ ), hexynyl (C ₆ ), and the like. The term "C _2-6 alkynyl" also includes heteroalkynyl groups, in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"C _1-6 alkylene" refers to a divalent group formed by removing another hydrogen of a C _1-6 alkyl group, and may be substituted or unsubstituted. In some embodiments, C _1-4 alkylene, C _2-4 alkylene, and C _1-3 alkylene are preferred. The unsubstituted alkylene includes, but is not limited to, methylene (-CH ₂ -), ethylene (-CH ₂ CH ₂ -), propylene (-CH ₂ CH ₂ CH ₂ -), butylene (-CH _{2 CH 2 CH 2 CH 2 -), pentylene (-CH 2 CH 2 CH 2 CH 2 CH 2} -), hexylene (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -), and the like. Exemplary substituted alkylene groups, for example, substituted alkylene groups with one or more alkyl(methyl) groups, include, but are not limited to, substituted methylene groups (—CH(CH ₃ )—, —C(CH ₃ ) ₂ —), substituted ethylene groups (—CH(CH ₃ )CH ₂ —, —CH ₂ CH(CH ₃ )—, —C(CH ₃ ) ₂ CH ₂ —, —CH ₂ C(CH ₃ ) ₂ —), substituted propylene groups (—CH(CH ₃ )CH ₂ CH ₂ —, —CH ₂ CH(CH ₃ )CH ₂ —, —CH _{2 CH 2} CH ₍ CH ₃ )—, —C(CH ₃ ) ₂ CH ₂ CH ₂ —, —CH ₂ C(CH ₃ ) _{2 CH 2 — )} , and the like.

" _C2-6 alkenylene" refers to a divalent group formed by removing another hydrogen of a _C2-6 alkenyl group, and may be substituted or unsubstituted. In some embodiments, _C2-4 alkenylene is particularly preferred. Exemplary unsubstituted alkenylene groups include, but are not limited to, vinylene (-CH=CH-) and propenylene (e.g., -CH= _CHCH2- , _-CH2 -CH=CH-). Exemplary substituted alkenylene groups, for example, alkenylene groups substituted with one or more alkyl(methyl) groups, include, but are not limited to, substituted ethylene groups (—C(CH ₃ )═CH—, —CH═C(CH ₃ )—), substituted propenylene groups (—C(CH ₃ )═CHCH _{2 —, —CH═C(CH 3 )CH 2 — ,} —CH═CHCH ₍ CH ₃ )—, —CH═CHC(CH ₃ ) ₂ —, —CH(CH ₃ )—CH═CH—, —C(CH ₃ ) 2 —CH═CH—, —CH ₂ —C(CH ₃ )═CH—, —CH ₂ —CH═C(CH ₃ )—), and the like.

"C _2-6 alkynylene" refers to a divalent group formed by removing another hydrogen of a C _2-6 alkynyl group, and may be substituted or unsubstituted. In some embodiments, C _2-4 alkynylene is particularly preferred. Exemplary alkynylene groups include, but are not limited to, ethynylene (-C≡C-), substituted or unsubstituted propynylene (-C≡CCH ₂ -), and the like.

"Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

Therefore, "C _1-6 haloalkyl" refers to the above-mentioned "C _1-6 alkyl" substituted by one or more halogen groups. In some embodiments, C _1-4 haloalkyl is particularly preferred, more preferably C _1-2 haloalkyl. Exemplary haloalkyls include, but are not limited to: -CF ₃ , -CH ₂ F, -CHF ₂ , -CHFCH ₂ F, -CH ₂ CHF ₂ , -CF ₂ CF ₃ , -CCl ₃ , -CH ₂ Cl, -CHCl ₂ , 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group may be substituted at any available attachment point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"C _1-6 alkoxy" refers to a -OR group wherein R is a C _1-6 alkyl group as defined above. C _1-4 alkoxy is preferred.

"C _1-6 haloalkoxy" refers to a "C _1-6 alkoxy" group which is substituted with one or more halogen groups. In some embodiments, a C _1-4 haloalkoxyalkyl group is particularly preferred, and a C _1-2 haloalkoxyalkyl group is more preferred.

" _C3-10 cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, _C4-10 cycloalkyl, _C5-10 cycloalkyl, _C4-7 cycloalkyl, _C3-7 cycloalkyl, _C3-6 cycloalkyl, _C3-5 cycloalkyl and _C3-4 cycloalkyl are particularly preferred, more preferably _C5-6 cycloalkyl. Cycloalkyl also includes a ring system in which the above cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C ₃ ), cyclopropenyl (C ₃ ), cyclobutyl (C ₄ ), cyclobutenyl (C ₄ ), cyclopentyl (C ₅ ), cyclopentenyl (C ₅ ), cyclohexyl (C _{6 ), cyclohexenyl (C 6 )} , cyclohexadienyl (C ₆ ), cycloheptyl (C ₇ ), cycloheptenyl (C ₇ ), cycloheptadienyl (C ₇ ), cycloheptatrienyl (C ₇ ), etc. The cycloalkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"3-12 membered heterocyclyl" refers to a group of a 3- to 12-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. In some embodiments, a 5-12 membered heterocyclyl is preferably a 3-10 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, a 3-10 membered heterocyclyl is preferably a 3-10 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, a 4-10 membered heterocyclyl is preferably a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, a 5-10 membered heterocyclyl is preferably a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, a 5-8 membered heterocyclyl is preferably a 5-8 membered non-aromatic ring having ring carbon atoms and 1 to 5 ring heteroatoms. System; In some embodiments, 3-7 membered heterocyclyl is preferably a 3-7 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; 3-6 membered heterocyclyl is preferably a 3-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 4-7 membered heterocyclyl is preferably a 4-7 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 4-6 membered heterocyclyl is preferably a 4-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 5-6 membered heterocyclyl is more preferably a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl is more preferably a 3-5 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. In some embodiments, the heterocyclyl may contain an unsaturated double bond, but the heterocyclyl itself is not aromatic. Heterocyclyl also includes a ring system in which the above heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or a ring system in which the above heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidine, oxirane, and thiorenyl. Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, dihydropyranyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thianyl. Exemplary 5-membered heterocyclyl groups fused to a _C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a _C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydrobenzopyranyl, tetrahydropyranopyridinyl, and the like. The heterocyclyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"C _6-10 aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C ₆ aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C ₁₀ aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above aryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. The aryl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"5-14 membered heteroaryl" refers to a group of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above-mentioned heteroaryl rings are fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-12 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system with ring carbon atoms and 1-4 ring heteroatoms; in some embodiments, 5-10 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system with ring carbon atoms and 1-4 ring heteroatoms. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 6-10 membered monocyclic or bicyclic 4n+2 aromatic ring system with ring carbon atoms and 1-4 ring heteroatoms. In some embodiments, 5-9 membered heteroaryl is preferred, which is a 5-9 membered monocyclic or bicyclic 4n+2 aromatic ring system with ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryls are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5 membered heteroaryls containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5 membered heteroaryls containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5 membered heteroaryls containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5 membered heteroaryls containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6 membered heteroaryls containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6 membered heteroaryls containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryls containing three or four heteroatoms include, but are not limited to, triazine and tetrazine, respectively. Exemplary 7-membered heteroaryls containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxacycloheptatrienyl, and thiacycloheptatrienyl. Exemplary 5,6-bicyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryls include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"Cycloalkylene", "heterocyclylene", "arylene" or "heteroarylene" is a divalent group formed by removing another hydrogen from the above-defined "cycloalkyl", "heterocyclyl", "aryl" or "heteroaryl", and may be substituted or unsubstituted. For example, "C _5-7 cycloalkylene" refers to a divalent group formed by removing another hydrogen from C _5-7 cycloalkyl, "5-8 membered heterocyclylene" refers to a divalent group formed by removing another hydrogen from 5-8 membered heterocyclyl, "C _6-10 arylene" refers to a divalent group formed by removing another hydrogen from C _6-10 aryl, and "5-6 membered heteroarylene" refers to a divalent group formed by removing another hydrogen from 5-6 membered heteroaryl.

Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups and the like as defined herein are optionally substituted groups.

Exemplary substituents on carbon atoms _include , but are not limited to, halogen, -CN, _-NO2 , -N3, _-SO2H , _-SO3H , -OH, ^-ORaa , -ON( ^Rbb ) ₂ , -N( ^Rbb ) ₂ , -N( ^Rbb ) ₃ ^{+ X-} , -N( ^ORcc ) ^Rbb , -SH, ^-SRaa , ^-SSRcc , -C(=O) ^Raa , _-CO2H , -CHO, -C( ^ORcc ) ₂ , -CO2Raa _, -OC(=O) ^Raa , _-OCO2Raa , -C(=O ⁾ N( ^Rbb ) ₂ , -OC( ⁼ O)N( ^Rbb ) ₂ ^{, -NRbbC} (=O) ^Raa _, ^-NRbbCO2Raa , ^-NRbb C(=O)N(R ^bb ) ₂ , -C(=NR ^bb )R ^aa , -C(=NR ^bb )OR ^aa , -OC(=NR ^bb )R ^aa , - OC(=NR ^bb )OR ^aa , -C(=NR ^bb )N(R ^bb ) ₂ , -OC(=NR ^bb )N(R ^bb ) ₂ , -NR ^bb C(=NR ^bb )N(R ^bb ) ₂ , -C(=O)NR ^bb SO ₂ R ^aa , -NR ^bb SO ₂ R ^aa , -SO ₂ N(Rb ^b ) ₂ , -SO ₂ R ^aa , - SO ₂ OR ^aa , -OSO ₂ R ^aa , -S(=O)R ^aa , -OS(=O)R ^aa , -Si(R ^aa ) ₃ , -OSi(R ^aa ) ₃ , -C(=S)N(R ^bb ) ₂ , -C(=O)SR ^aa , -C(=S)SR ^aa , -SC(=S)SR ^aa , -SC(=O)SR ^aa , -OC(=O)SR ^aa , -SC(=O)OR ^aa , -SC(=O)R ^aa , -P(=O) ₂ R ^aa , -OP(=O) ₂ R ^aa , -P(=O)(R ^aa ) ₂ , -OP(=O)(R ^aa ) ₂ , -OP(=O)(OR ^cc ) ₂ , -P(=O) ₂ N(R ^bb ) ₂ , -OP(＝O) ₂ N(R ^bb ) ₂ , -P(＝O)(NR ^bb ) ₂ , -OP(＝O)(NR ^bb ) ₂ , -NR ^bb P(＝O)(OR ^cc ) ₂ , -NR ^bb P(＝O)(NR ^bb ) ₂ , -P(R ^cc ) ₂ , -P(R ^cc ) ₃ , -OP( ^Rcc ) ₂ , -OP( ^Rcc ) ₃ , -B( ^Raa ) ₂ , -B( ^ORcc ) ₂ , ^-BRaa ( ^ORcc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 ^Rdd groups;

or two geminal hydrogens on a carbon atom are replaced by groups =0, =S, =NN( ^Rbb ) ₂ , = ^NNRbbC (=O) ^Raa , =NNRbbC(=O) _ORaa ^, = ^NNRbbS (=O) ^2Raa , = ^{NRbb or} = ^NORcc ;

each of ^Raa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two ^Ra groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 ^Rdd groups;

Each of R ^bb is independently selected from: hydrogen, -OH, -OR ^aa , -N(R ^cc ) ₂ , -CN, -C(=O)R ^aa , -C(=O)N(R ^cc ) _2. -CO ₂ R ^aa , -SO ₂ R ^aa , -C(=NR ^cc )OR ^aa , -C(=NR ^cc )N(R ^cc ) ₂ , -SO ₂ N(R ^cc ) ₂ , -SO ₂ R ^cc , -SO ₂ OR ^cc , -SOR ^aa , -C(=S)N(R ^cc ) ₂ , -C(=O)SR ^cc , -C(=S)SR ^cc , -P(=O ) ₂ R ^aa , -P(=O)(R ^aa ) ₂ , -P(=O) ₂ N(R ^cc ) ₂ , -P(=O)(NR ^cc ) ₂ , alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ^bb groups are combined to form A heterocyclic or heteroaryl ring in which each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently substituted by 0, 1, 2, 3, 4, or 5 R ^dd group substitution;

each of R ^cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ^cc groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

Each of R ^dd is independently selected from the group consisting of halogen, -CN, -NO ₂ , -N ₃ , -SO ₂ H, -SO ₃ H, -OH, -OR ^ee , -ON(R ^ff ) ₂ , -N(R ^ff ) ₂ , -N(R ^ff ) ₃ ⁺ X ^- , -N(OR ^ee )R ^ff , -SH, -SR ^ee , -SSR ^ee , -C(＝O)R ^ee , -CO ₂ H, -CO ₂ R ^ee , -OC(＝O)R ^ee , -OCO ₂ R ^ee , -C(＝O)N(R ^ff ) ₂ , -OC(＝O)N(R ^ff ) ₂ , -NR ^ff C(＝O)R ^ee , -NR ^ff CO ₂ R ^ee , -NR ^ff C(＝O)N(R ^ff ) ₂ , -C(=NR ^ff )OR ^ee , -OC(=NR ^ff )R ^ee , -OC(=NR ^ff )OR ^ee , -C(=NR ^ff )N(R ^ff ) ₂ , -OC(=NR ^ff )N(R ^ff ) ₂ , -NR ^ff C(=NR ^ff )N(R ^ff ) ₂ , -NR ^ff SO ₂ R ^ee , -SO ₂ N(R ^ff ) ₂ , -SO ₂ R ^ee , -SO ₂ OR ^ee , -OSO ₂ R ^ee , -S(=O)R ^ee , -Si(R ^ee ) ₃ , -OSi(R ^ee ) ₃ , -C(=S)N(R ^ff ) ₂ , -C(=O)SR ^ee , -C(=S)SR ^ee , -SC(=S)SR ^ee , -P(=O) _2Ree , -P(=O)( ^Ree ) ₂ , -OP(=O)( ^Ree ) ₂ , -OP( ⁼ O)( ^ORee ) ₂ , alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 ^Rgg groups, or two geminal ^Rdd substituents may combine to form =O or =S;

Each of R ^ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;

Each of ^Rff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two ^Rff groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 ^Rgg groups:

Each of Rgg is independently: halogen, -CN, _-NO2 , -N3, _-SO2H , _-SO3H , -OH, _-OC1-6alkyl , -ON( _C1-6alkyl ) ₂ , -N( _C1-6alkyl ) ₂ , _-N( C1-6alkyl) ₃ ^{+ X-} , -NH( _C1-6alkyl ) ₂ ^{+ X-} , _-NH2 (C1-6alkyl) ^{+ X-} , _-NH3 ^{+ X-} , -N( _OC1-6alkyl )(C1-6alkyl), _-N (OH)( _C1-6alkyl ), _-NH (OH), -SH, _-SC1-6alkyl , -SS( _C1-6alkyl ), _-C (＝O) ₍ C1-6alkyl), -CO2H, _{-CO2 (C1-6alkyl} ), -OC(＝O)( _C1-6alkyl ), _-OCO2 ( _C1-6alkyl ) _-C (＝NH) ₂ , -C(＝O)NH2, -C(＝O)N(C _1-6 alkyl) ₂ , -OC(＝O)NH(C _1-6 alkyl), -NHC(＝O)(C _1-6 alkyl), -N(C _1-6 alkyl)C(＝O)(C _1-6 alkyl), _-NHCO2 (C _1-6 alkyl), -NHC(＝O)N(C _1-6 alkyl) ₂ , -NHC(＝O)NH(C _1-6 alkyl), -NHC(＝O) _NH2 , -C(＝NH)O(C _1-6 alkyl), -OC(＝NH)(C _1-6 alkyl), -OC(＝NH)OC _1-6 alkyl, -C(＝NH)N(C _1-6 alkyl) ₂ , -C(＝NH)NH(C 1-6 alkyl), -C(＝NH) _NH2 , -OC(＝NH)N(C _1-6 alkyl) _1-6 alkyl) ₂ , -OC(NH)NH(C _1-6 alkyl), -OC(NH)NH ₂ , -NHC(NH)N(C _1-6 alkyl) _-NHC (＝NH) _NH2 , -NHSO2( _C1-6alkyl ), _-SO2N ( _C1-6alkyl ) ₂ , _-SO2NH ( _C1-6alkyl ), _-SO2NH2 , -SO2C1-6alkyl, -SO2OC1-6alkyl, _{-OSO2C1-6alkyl} , -SOC1-6alkyl, -Si( _C1-6alkyl ) ₃ , _-OSi ( _C1-6alkyl ) ₃ , _-C (＝S ₎ N( _{C1-6alkyl ) 2} , _C (＝S)NH( _C1-6alkyl ), _C (＝S) _NH2 , _-C (＝O) _S ( _C1-6alkyl ), -C(＝S) _SC1-6alkyl , -SC(＝S) _SC1-6alkyl , _-P (＝O) ₂ (C1-6alkyl) -P(＝O)(C _1-6 alkyl) ₂ , -OP(＝O)(C _1-6 alkyl) ₂ , _-OP (＝O)(OC _1-6 alkyl) ₂ , C _1-6 alkyl, C _1-6 haloalkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, C ₆ -C ₁₀ aryl, C ₃ -C ₇ heterocyclyl, C ₅ -C ₁₀ heteroaryl; or two geminal R ^gg substituents may combine to form ＝O or ＝S; wherein X ^- is a counter ion.

Exemplary substituents on the nitrogen atom include, but are not limited to, hydrogen, -OH, ^-ORaa , -N ^{( Rcc} ) ₂ , -CN, -C( ⁼ O) ^Raa , -C(=O)N( ^Rcc ) ₂ , -CO2Raa, -SO2Raa, -C( ₌ ^NRbb ) ^Raa , -C(= ^{NRcc ) ORaa} , -C(= ^NRcc )N ^{( Rcc} ) ₂ , -SO2N ₍ ^Rcc _{)2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2 ,} ^-C _{( =} ^O ₎ ^SRcc , -C(=S) ^SRcc , -P(=O) _2Raa , -P(=O)( ^Raa ₎ , -P( ⁼ O) ₂ N( ^Rcc ) ₂ , -P(=O)( ^NRcc ) ₂ , alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two ^Rcc groups attached to the nitrogen atom combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 ^Rdd groups, and wherein ^Raa , ^Rbb , ^Rcc and ^Rdd are as described above.

Other definitions

The term "pharmaceutically acceptable salt" as used herein refers to those carboxylates, amino acid addition salts of the compounds of the present invention which are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

"Subjects" for administration include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

"Disease," "disorder," and "condition" are used interchangeably herein.

Generally, the "effective amount" of a compound refers to an amount sufficient to cause a target biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of the compounds of the invention may vary depending on factors such as the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes a therapeutically effective amount and a prophylactically effective amount.

"Combination" and related terms refer to the simultaneous or sequential administration of the compounds of the invention and other therapeutic agents. For example, the compounds of the invention can be administered simultaneously or sequentially with the other therapeutic agents in separate unit dosage forms, or can be administered simultaneously with the other therapeutic agents in a single unit dosage form.

As used herein, the term "compound of the present invention" refers to a compound of the following formula (A) (including sub-general formulae, such as formula (I), (II), (III) or formula (IV), a pharmaceutically acceptable salt, enantiomer, diastereoisomer or isotopic variant thereof, and a mixture thereof.

The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and/or diastereoisomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

The compounds of the present invention may also exist as tautomers. For compounds that exist in different tautomeric forms, a compound is not limited to any specific tautomer, but is intended to encompass all tautomeric forms.

The present invention also includes isotopically labeled compounds (isotopic variants), which are equivalent to those described in formula (A), but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ^2H , ^3H , ^13C , ^11C , ^{14C, 15N , 18O} , ^17O , ^31P , ^32P , ^35S , ^18F and ^36Cl , respectively. Compounds of the present invention containing the above-mentioned isotopes and/or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are all within the scope of the present invention. Certain Isotopically labeled compounds of the invention, such as those into which radioactive isotopes (e.g., ³ H and ¹⁴ C) are introduced, can be used in drug and/or substrate tissue distribution assays. Tritium, i.e., ³ H, and carbon-14, i.e., ¹⁴ C isotopes are particularly preferred because they are easy to prepare and detect. Furthermore, substitution with heavier isotopes, such as deuterium, i.e., ² H, may be preferred in some cases because of the higher metabolic stability that can provide therapeutic benefits, such as extending the in vivo half-life or reducing the dosage requirements. Isotopically labeled compounds of the invention of formula (A) and their prodrugs can generally be prepared by replacing non-isotopically labeled reagents with readily available isotopically labeled reagents when performing the processes disclosed in the following flows and/or the Examples and Preparations.

The term "deuterium (D or 2H)" is a stable isotope of hydrogen that exists at a natural abundance of 0.015 mol%. The term "deuterated" refers to a group or compound in which one or more hydrogen atoms H are replaced by D.

Isotopic variants of the present invention include deuterated compounds. "Deuterated compound" refers to a compound in which one or more hydrogens bonded to carbon are replaced by one or more deuteriums. Similarly, "deuterated" refers to a chemical structure or organic group in which one or more hydrogens bonded to carbon are replaced by one or more deuteriums, for example, "deuterated alkyl", "deuterated cycloalkyl", "deuterated heterocycloalkyl", "deuterated aryl", etc. For example, "deuterated alkyl" refers to an alkyl group defined in the present application, in which at least one hydrogen atom bonded to carbon is replaced by deuterium. In a deuterated alkyl, at least one carbon atom is bonded to one deuterium; one carbon atom can be bonded to multiple deuteriums; and multiple carbon atoms in an alkyl group can also be bonded to deuterium. For example, deuterated methyl includes methyl-d3, in which three hydrogen atoms are replaced by deuterium; it also includes monodeuterated methyl and dideuterated methyl. For example, deuterated piperazinyl means that one or more hydrogen atoms on the piperazinyl group are replaced by deuterium, including all 8 hydrogen atoms replaced by deuterium. In some embodiments, the compounds of the present invention include deuterated compounds.

Pharmaceutical compositions and kits

On the other hand, the present invention provides a pharmaceutical composition comprising a compound of the present invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a compound of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of the present invention. In some embodiments, the pharmaceutical composition comprises a preventive effective amount of a compound of the present invention.

The pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the composition of the present invention include (but are not limited to) ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

The present invention also includes a kit (e.g., a pharmaceutical package). The kit provided may include a compound of the invention, other therapeutic agents, and a first and second container (e.g., a vial, an ampoule, a bottle, a syringe, and/or a dispersible package or other suitable container) containing the compound of the invention and other therapeutic agents. In some embodiments, the kit provided may also optionally include a third container containing a pharmaceutical excipient for diluting or suspending the compound of the invention and/or other therapeutic agents. In some embodiments, the compound of the invention and other therapeutic agents provided in the first container and the second container are combined to form a unit dosage form.

Drug administration

Pharmaceutical composition provided by the invention can be administered by many ways, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration by implant or other modes of administration. For example, parenteral administration used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial cavity administration, intrasternal administration, intrathecal administration, intralesional administration and intracranial injection or infusion technology.

Generally, an effective amount of the compounds provided herein is administered. The amount of the compound actually administered can be determined by a physician according to the relevant circumstances, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

When used to prevent a condition described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically on the advice of a physician and under the supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition typically include subjects with a family history of the condition, or subjects who have been identified by genetic testing or screening as being particularly susceptible to developing the condition. Those subjects who were affected.

The pharmaceutical compositions provided herein can also be administered over a long period of time ("chronic administration"). Chronic administration refers to administration of a compound or a pharmaceutical composition thereof over a long period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration may continue indefinitely, e.g., for the rest of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a long period of time, e.g., within a therapeutic window.

Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition can be administered by push injection, for example, in order to increase the concentration of the compound in the blood to an effective level. The push dose depends on the target systemic level of the active component through the body, for example, the intramuscular or subcutaneous push dose slowly releases the active component, and the push delivered directly to the vein (for example, by IV intravenous drip) can be delivered more quickly, so that the concentration of the active component in the blood is quickly increased to an effective level. In other embodiments, the pharmaceutical composition can be given in a continuous infusion form, for example, by IV intravenous drip, so as to provide a steady-state concentration of the active component in the subject's body. In addition, in other embodiments, the pharmaceutical composition of the push dose can be first given, and then continuous infusion.

Oral compositions can be in the form of bulk liquid solutions or suspensions or bulk powders. However, more generally, in order to facilitate accurate dosing, the composition is provided in unit dosage form. The term "unit dosage form" refers to a physical discrete unit suitable as a unit dose for human patients and other mammals, each unit containing a predetermined amount of active substances suitable for producing the desired therapeutic effect and a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions. In such compositions, the compound is usually a relatively small component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), and the remainder is various carriers or excipients and processing aids useful for forming the desired administration form.

For oral dosage, a representative regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses. Using these dosage modes, each dose provides about 0.01 to about 20 mg/kg of the compound of the invention, and preferred doses each provide about 0.1 to about 10 mg/kg, especially about 1 to about 5 mg/kg.

In order to provide blood levels similar to those using an injectable dose, or lower blood levels than using an injectable dose, a transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight.

From about 1 to about 120 hours, especially 24 to 96 hours, the injected dosage level is in the range of about 0.1 mg/kg/hour to at least 10 mg/kg/hour. In order to obtain sufficient steady state levels, a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given. For a 40 to 80 kg human patient, the maximum total dose cannot exceed about 2 g/day.

Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous carrier and buffers, suspending and dispersing agents, colorants, flavoring agents, etc. Solid forms may include, for example, any of the following components, or compounds of a similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, and the remainder is an injectable excipient, etc.

Typically, transdermal compositions are formulated as topical ointments or creams containing active ingredients. When formulated as an ointment, the active ingredient is typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated as an ointment together with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and typically include other components for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by the invention.

The compounds of the invention may also be administered by transdermal means. Thus, transdermal administration may be achieved using patches of the reservoir or porous membrane type, or a variety of solid matrices.

The above components for oral administration, injection or topical administration of the composition are representative only. Other materials and processing techniques are described in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, Part 8, which is incorporated herein by reference.

The compounds of the invention may also be administered in a sustained release form or from a sustained release delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

The present invention also relates to pharmaceutically acceptable formulations of the compounds of the present invention. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α-, β- and γ-cyclodextrins composed of 6, 7 and 8 α-1,4-linked glucose units, respectively, which optionally include one or more substituents on the linked sugar moiety, including but not limited to: methylated, hydroxyalkylated, acylated and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, for example, sulfobutyl ether β-cyclodextrin, also known as Captisol. See, for example, U.S.5,376,645. In some embodiments, the formulation includes hexapropyl-β-cyclodextrin (e.g., in water, 10-50%).

Example

The reagents used in the present invention are commercial reagents purchased directly or synthesized by common methods well known in the art.

Notes on commonly used abbreviations:

PE = petroleum ether; EA = ethyl acetate; MeOH = methanol; DCM = dichloromethane; DCE = dichloroethane; CH ₃ CN = acetonitrile; 1,4-dioxane = 1,4-dioxane; DMSO = dimethyl sulfoxide; HFIP = hexafluoroisopropanol; DMF = N,N-dimethylformamide; Hex = n-hexane; IPA = isopropanol; NMP = N-methylpyrrolidone; NMO = N-methylmorpholine-N-oxide; TEA = triethylamine; DIEA = diisopropylethylamine; CuI = cuprous iodide; CuCN = cuprous cyanide; triphosgene = triphosgene; p-TsOH = p-toluenesulfonic acid; T ₃ P = 1-propylphosphoric acid cyclic anhydride; TsN ₃ = p-toluenesulfonyl azide; PPA = polyphosphoric acid; SEM-Cl = 2-(trimethylsilyl)ethoxymethyl chloride; DMC = diethyl carbonate; DMEDA = N,N′-dimethylethylenediamine; AIBN = azobisisobutyronitrile; DMC = diethyl carbonate; NBS = N-bromosuccinimide; TBS-Cl = tert-butyldimethylchlorosilane; LDA = lithium diisopropylamide; HMPA = hexamethylphosphoric triamide; HATU = 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; DMA = N,N-dimethylacetamide; EDCI = carbodiimide hydrochloride; HOAT = 1-hydroxy-7-azabenzotriazole; EDA = ethylenediamine.

MSI-H = microsatellite instability-high; MSI-L = microsatellite instability-low; MSS = microsatellite stable.

Embodiment 1:

Preparation of key intermediates

Preparation of intermediate a1

Steps: Dissolve the raw material ethyl acetoacetate a1-1 (9.0 g, 69.2 mmol) and 5-bromo-1-H-3-amino 1,2,4-triazole a1-2 (11.3 g, 69.2 mmol) in 90 mL of ethanol, slowly add polyphosphoric acid PPA (8.0 g, 69.2 mmol), add dropwise, heat to 80°C and react for 12 hours, cool to room temperature, and evaporate the solvent under reduced pressure. Pour the reaction solution into 100 mL of ice water, adjust the pH to about 8 with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, dry with anhydrous sodium sulfate, and concentrate to obtain a white solid a1 (7.5 g), yield: 45%. LCMS ESI-MS m/z: 243 [M+H] ⁺ .

Preparation of intermediates a5, a7-a13

Step 1: Dissolve the raw material a5-1 (24.0 g, 134 mmol) and tert-butyl piperazine-1-carboxylate (25.0 g, 134 mmol) in 240 mL of acetonitrile, slowly add TEA (40.8 g, 403 mmol), and heat to 60°C for 16 hours, cool to room temperature, and evaporate the solvent under reduced pressure. Pour the reaction solution into 100 mL of ice water, extract with dichloromethane, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography (PE/EA, 9/1) to obtain a yellow oil a5-2 (24 g), yield: 54%. LCMS ESI-MS m/z: 329 [M+H] ⁺ .

Step 2: Dissolve the intermediate a5-2 (22.2 g, 67.6 mmol) and 5-bromo-1-H-3-amino 1,2,4-triazole a1-2 (11.0 g, 67.6 mmol) in 200 mL of ethanol, slowly add polyphosphoric acid PPA (7.8 g, 67.6 mmol), and heat to 80°C for 12 hours, cool to room temperature, and remove the solvent under reduced pressure. Pour the reaction solution into 100 mL of ice water, adjust the pH to about 8 with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography (PE/EA, 3/1) to obtain a yellow solid a5 (4.4 g), yield: 15%. LCMS ESI-MS m/z: 427 [M+H] ⁺ .

Referring to the synthetic route of intermediate a1 or a5, similar raw materials/analogues were used to synthesize the following target intermediates.

Preparation of intermediates a2, a6, a14-a32

Steps: Dissolve the intermediate a1 (7.5 g, 30.9 mmol) and the raw material a2-1 (10.1 g, 37.0 mmol) in 75 mL NMP, slowly add DIEA (12.0 g, 92.6 mmol), and heat to 50°C for 16 hours, cool to room temperature, and stop the reaction. Pour the reaction solution into 100 mL ice water, extract with dichloromethane, dry with anhydrous sodium sulfate, and concentrate. The crude product is separated by HPLC preparative chromatography (chromatographic column: XSelect Prep OBD C18 Column, 30*150 mm, 5 μm; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: acetonitrile; flow rate: 90 mL/min; retention time: 3 min) to obtain a yellow solid a2 (5.2 g), yield: 35%). LCMS ESI-MS m/z: 478 [M+H] ⁺ .

Steps: Dissolve the intermediate a5 (1.1 g, 2.57 mmol) and the raw material a2-1 (1.1 g, 3.86 mmol) in 11 mL NMP, slowly add DIEA (1.0 g, 7.72 mmol), and heat to 70°C for 12 hours, cool to room temperature, and stop the reaction. Pour the reaction solution into 50 mL ice water, extract with dichloromethane, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O=5/1) to obtain a yellow solid a6 (996 mg), yield: 58%. LCMS ESI-MS m/z: 662[M+H] ⁺ .

Referring to the synthetic route of intermediate a2 or a6, similar raw materials/analogues were used to synthesize the following target intermediates.

[Using similar heteroaryl NH ₂ as raw material, reacting with chloroacetyl chloride to synthesize a2-1 analogues]

Preparation of intermediate a3

Steps: Under nitrogen protection, intermediate a2 (5.0 g, 10.4 mmol), raw material a3-1 (2.9 g, 13.6 mmol) and sodium carbonate (3.3 g, 31.3 mmol) were dissolved in 50 mL of a mixed solution of 1,4-dioxane and water (v/v, 4/1), and catalyst Pd(dppf)Cl ₂ (900 mg, 1.0 mmol) was added. The mixture was heated to 100° C. and reacted for 2 hours, then the reaction was stopped and filtered. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by HPLC preparative chromatography (chromatographic column: WelFlash C18-I, 20-40 μm, 330 g; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: acetonitrile; flow rate: 80 mL/min; retention time: 12 min) to obtain a yellow solid a3 (2.6 g), yield: 52%. LCMS ESI-MS m/z: 482 [M+H] ⁺ .

Preparation of intermediate a4

Steps: Dissolve the intermediate a3 (2.4 g, 5.0 mmol) in 25 mL DMF, add NBS (1.8 g, 10.0 mmol), react the mixture at room temperature for 2 hours, stop the reaction, and filter. Add 100 mL of water to the reaction solution, extract with ethyl acetate, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by HPLC preparative chromatography (chromatographic column: C18-I, 20-40 μm, 330 g; mobile phase A water: (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: acetonitrile; flow rate: 90 mL/min; retention time: 11 min) to obtain a white solid a4 (1.6 g), yield: 57%. LCMS ESI-MS m/z: 560 [M+H] ⁺ .

Preparation of intermediate b1

Step 1: Under nitrogen protection, the raw material 4,6-dichloro-5-methoxypyrimidine b1-1 (20.0 g, 111 mmol), the raw material methylboric acid (7.0 g, 117 mmol) and potassium phosphate (59.2 g, 279 mmol) were dissolved in 120 mL DME, and the catalyst Pd(dppf)Cl ₂ (4.6 g, 5.6 mmol) was added. The mixture was heated to 85°C for 12 hours, the reaction was stopped, and the mixture was filtered. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 10/1) to obtain a white solid b1-2 (6.0 g). Rate: 34%. LCMS ESI-MS m/z: 159[M+H] ⁺ .

Step 2: Under carbon monoxide atmosphere, the intermediate b1-2 (6.0 g, 37.0 mmol) and TEA (7.66 g, 75.0 mmol) were dissolved in 90 mL of methanol, and the catalyst Pd(dppf)Cl ₂ (1.85 g, 2.3 mmol) was added. The mixture was heated to 100°C under CO (20 atm) for 12 hours, and the reaction was stopped and filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by HPLC preparative chromatography (chromatographic column: WelFlash C18-I, 20-40 um, 120 g; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ) mobile phase B: acetonitrile; flow rate: 60 mL/min) to obtain a white solid b1-3 (5.0 g) with a yield of 73%. LCMS ESI-MS m/z: 183 [M+H] ⁺ .

Step 3: Dissolve the intermediate b1-3 (3.0 g, 16.5 mmol) in 15 mL HBr aqueous solution (40%), heat to 40°C and react for 10 hours, then stop the reaction. Add HI (15 mL) to the reaction solution, continue to react at 40°C for 6 hours, and remove the solvent under reduced pressure. Adjust the pH of the crude product to about 8 with NaOH aqueous solution (1N), then adjust the pH to about 3 with concentrated hydrochloric acid, and remove the solvent under reduced pressure. The crude product is separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain a yellow solid b1 (1.5 g), yield: 59%. LCMS ESI-MS m/z: 155[M+H] ⁺ .

Preparation of intermediate b2

Step 1: In an ice bath, under nitrogen protection, the raw material b2-1 (500 mg, 3.81 mmol) was dissolved in 10 mL of tetrahydrofuran, and NBS (679 mg, 3.81 mmol) was added. The mixture was reacted at room temperature for 0.5 hours, the reaction was stopped, and filtered. Saturated sodium bicarbonate aqueous solution was added to the reaction solution to adjust the pH to about 8, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 10/1) to obtain a yellow oil b2-2 (540 mg), yield: 63%. LCMS ESI-MS m/z: 224 [M+H] ⁺ .

Step 2: Under nitrogen protection, the intermediate b2-2 (510 mg, 2.28 mmol) of the previous step was dissolved in 3 mL of diethyl carbonate DMC, and CH ₃ ONa (185 mg, 3.42 mmol) was added. The mixture was heated to 125°C for reaction for 0.5 hours and cooled to room temperature. 20 mL of ice water was added to the reaction solution, extracted with methyl tert-butyl ether, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 1/1) to obtain a yellow oil b2-3 (300 mg), with a yield of 64%. LCMS ESI-MS m/z: 205 [M+H] ⁺ .

Step 3: -78°C, under nitrogen protection, the intermediate b2-3 (300 mg, 1.46 mmol) of the previous step was dissolved in 6 mL of anhydrous tetrahydrofuran, and nBuLi (2.5 M, 1.47 mL) was added dropwise. After the addition was completed, stirring was continued for 1 hour. Isopropyl alcohol pinacol borate (327 mg, 1.75 mmol) was added to the reaction solution, and the reaction was continued at -78°C for 1 hour. The mixture was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a yellow oil b2 (300 mg), yield: 81%. LCMS ESI-MS m/z: 253 [M + H] ⁺ .

Preparation of intermediate b3

Step 1: In an ice bath, under nitrogen protection, the raw material 5-bromo-1H-pyrrolo[2,3-b]pyridine-6-carboxylic acid methyl ester b3-1 (600 mg, 2.35 mmol) and NaH (70 mg, 2.82 mmol) were dissolved in 12 mL DMF, SEM-Cl (570 mg, 3.52 mmol) was added, and the mixture was reacted at room temperature for 3 hours to stop the reaction. 100 mL of ice water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 7/10) to obtain a yellow solid b3-2 (500 mg), yield: 55%. LCMS ESI-MS m/z: 385[M+H] ⁺ .

Step 2: Under nitrogen protection, the intermediate b3-2 (500 mg, 1.29 mmol) and diboronic acid pinacol ester (490 mg, 1.95 mmol) were dissolved in 13 mL 1,4-dioxane, KOAc (380 mg, 3.89 mmol) and catalyst Pd(dppf)Cl ₂ .DCM (110 mg, 0.13 mmol) were added, and the mixture was heated to 120°C for 5 hours, cooled to room temperature, and filtered. The filter cake was washed and dried to obtain a brown solid b3 (400 mg), with a yield of 71%. LCMS ESI-MS m/z: 433 [M+H] ⁺ .

Preparation of intermediates b4 and b5

Step 1: In an ice bath, under nitrogen protection, the intermediate b3 (400 mg, 0.92 mmol) was dissolved in a mixed solution of 5 mL tetrahydrofuran and water (v/v, 1/1), sodium borate (210 mg, 1.38 mmol) was added, and the mixed solution was reacted at room temperature for 3 hours to stop the reaction. 20 mL of ice water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a brown oil b4-1 (100 mg), yield: 40%. LCMS ESI-MS m/z: 323 [M+H] ⁺ .

Step 2: Under nitrogen protection, the intermediate b4-1 (100 mg, 0.31 mmol) of the previous step was dissolved in a mixed solution of 3 mL of tetrahydrofuran and water (v/v, 1/1), and NaOH (25 mg, 0.62 mmol) was added. The mixture was reacted at room temperature for 2 hours, and the reaction was stopped. The solvent was evaporated under reduced pressure. 10 mL of water was added to the mixture, and the pH was adjusted to about 4 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain yellow solid b4 (70 mg), with a yield of 73%. LCMS ESI-MS m/z: 309 [M+H] ⁺ .

Referring to the synthetic route of intermediate b4, similar raw materials/analogues were used to synthesize the following target intermediate.

Preparation of intermediate b6

Step 1: Under nitrogen protection, the raw material 4,6-dichloro-5-methoxypyrimidine b1-1 (5.0 g, 27.9 mmol), the raw material b6-1 (9.2 g, 27.9 mmol) and CuI (50 mg, 0.28 mmol) were dissolved in 5 mL DME, and the catalyst Pd(PPh ₃ ) ₄ (320 mg, 0.28 mmol) was added. The mixed solution was heated to 85° C. for 1 hour, the reaction was stopped, and filtered. 30 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 9/1) to obtain a yellow solid b6-2 (3.0 g), with a yield of 59%. LCMS ESI-MS m/z: 183 [M+H] ⁺ .

Step 2: Under carbon monoxide atmosphere, the intermediate b6-2 (3.0 g, 16.4 mmol) and TEA (3.3 g, 32.9 mmol) were dissolved in 60 mL of methanol, and the catalyst Pd(dppf)Cl ₂ (1.2 g, 1.64 mmol) was added. The mixture was heated to 100°C under CO (30 atm) for 4 hours, the reaction was stopped, and the mixture was filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography (PE/EA, 4/1) to obtain a yellow solid b6-3 (1.45 g), with a yield of 43%. LCMS ESI-MS m/z: 207 [M+H] ⁺ .

Step 3: Under nitrogen protection, the intermediate b6-3 (1.4 g, 6.79 mmol) and TEA (7.0 mL) were dissolved in 7 mL DMA, and the catalyst CuI (130 mg, 0.68 mmol) was added. The mixture was heated to 105 ° C for 2 hours, the reaction was stopped, and filtered. 30 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 1/1) to obtain yellow solid b6-4 (150 mg), yield: 11%. LCMS ESI-MS m/z: 207 [M+H] ⁺ .

Step 4: Dissolve the intermediate b6-4 (150 mg, 0.73 mmol) in a mixed solution of 3 mL of methanol and water (v/v, 1/1), add NaOH (58 mg, 1.5 mmol), react at room temperature for 2 hours, and stop the reaction. Add 2 M dilute hydrochloric acid to the reaction solution to adjust the pH to about 6, and evaporate the solvent under reduced pressure. The crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 3/2) to obtain a white solid b6 (110 mg), yield: 79%. LCMS ESI-MS m/z: 193 [M+H] ⁺ .

Preparation of intermediate b7

Step 1: In an ice bath, under nitrogen protection, the raw material b7-1 (11.0 g, 59.3 mmol) was dissolved in 115 mL of acetonitrile, and NBS (10.5 g, 59.3 mmol) was added. The mixed solution was reacted for 1 hour in an ice bath, the reaction was stopped, and filtered. 300 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 1/1) to obtain yellow solid b7-2 (9.0 g), yield: 91%. LCMS ESI-MS m/z: 245 [M+H] ⁺ .

Step 2: Under nitrogen protection, the intermediate b7-2 (10.0 g, 40.8 mmol) of the previous step was dissolved in a mixed solution of 150 mL of acetic acid and tetrahydrofuran (v/v, 2/1), and NIS (13.8 g, 61.2 mmol) was added. The mixed solution was reacted at 35°C for 12 hours, the reaction was stopped, and filtered. 300 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 1/2) to obtain yellow solid b7-3 (8.0 g), yield: 53%. LCMS ESI-MS m/z: 371 [M+H] ⁺ .

Step 3: Under nitrogen protection, the intermediate b7-3 (8.0 g, 21.6 mmol), K ₃ PO ₄ (13.7 g, 64.70 mmol) and the raw material b7-4 (8.5 g, 43.1 mmol) were dissolved in 100 mL of isopropanol, and the catalyst Pd(OAc) ₂ (500 mg, 2.16 mmol) and the ligand RuPhos (1.0 g, 2.16 mmol) were added. The mixed solution was reacted at room temperature for 14 hours, the reaction was stopped, and the mixture was filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography (PE/EA, 1/1) to obtain a yellow solid b7-5 (5.3 g), with a yield of 72%. LCMS ESI-MS m/z: 343 [M+H] ⁺ .

Step 4: Dissolve the intermediate b7-5 (5.3 g, 15.4 mmol) in 53 mL of methanol, add concentrated hydrochloric acid (11 mL), and react the mixture at 40 ° C for 12 hours to stop the reaction. The solvent was evaporated under reduced pressure, and a saturated sodium bicarbonate aqueous solution was added to the reaction solution to adjust the pH to about 7, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography (PE/EA, 1/2) to obtain a yellow solid b7-6 (2.0 g), with a yield of 48%. LCMS ESI-MS m/z: 269 [M+H] ⁺ .

Step 5: In an ice bath, under nitrogen protection, the intermediate b7-6 (2.0 g, 7.43 mmol) and NaH (200 mg, 8.92 mmol) of the previous step were dissolved in 32 mL of DMF, and SEM-Cl (1.9 g, 11.2 mmol) was added. The mixture was reacted at room temperature for 3 hours to stop the reaction. 100 mL of ice water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain yellow oil b7-7 (1.2 g), yield: 40%. LCMS ESI-MS m/z: 399 [M+H] ⁺ .

Step 6: Under nitrogen protection, the intermediate b7-7 (1.2 g, 3.0 mmol) and biboronic acid pinacol ester (1.1 g, 4.5 mmol) were dissolved in 24 mL 1,4-dioxane, KOAc (900 mg, 9.02 mmol) and catalyst Pd(dppf)Cl ₂ .DCM (210 mg, 0.30 mmol) were added, and the mixture was heated to 100°C for 2 hours, cooled to room temperature, and filtered. The solvent was evaporated under reduced pressure to obtain a brown oil b7-8 (1.0 g). LCMS ESI-MS m/z: 447 [M+H] ⁺ .

Step 7: In an ice bath, under nitrogen protection, the intermediate b7-8 (1.0 g, 2.24 mmol) of the previous step was dissolved in a mixed solution of 12 mL of tetrahydrofuran and water (v/v, 1/1), sodium borate (310 mg, 3.38 mmol) was added, and the mixed solution was reacted at 50°C for 3 hours to stop the reaction. 50 mL of ice water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by column chromatography (PE/EA, 1/1) to obtain a yellow solid b7-9 (100 mg), yield: 13%. LCMS ESI-MS m/z: 337 [M+H] ⁺ .

Step 8: Under nitrogen protection, the intermediate b7-9 (100 mg, 0.30 mmol) of the previous step was dissolved in a mixed solution of 3 mL of tetrahydrofuran and water (v/v, 2/1), and NaOH (25 mg, 0.62 mmol) was added. The mixture was reacted for 2 hours at room temperature, and the reaction was stopped. The solvent was evaporated under reduced pressure. 10 mL of water was added to the mixture, and the pH was adjusted to about 4 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain yellow solid b7 (37 mg), with a yield of 39%. LCMS ESI-MS m/z: 323 [M+H] ⁺ .

Preparation of intermediates c1-c4, c16-c22

Step 1: Under nitrogen protection, intermediate a4 (210 mg, 0.3 mmol) and TEA (114 mg, 1.1 mmol) were dissolved in 2 mL DMSO, and raw material c1-1 (371 mg, 1.9 mmol) was added. The mixture was heated to 85°C for 12 hours to stop the reaction. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by HPLC chromatography (chromatographic column: WelFlash C18-I, 20-40 μm, 180 g; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: acetonitrile; flow rate: 70 mL/min; retention time: 12 min) to obtain white solid c1-2 (120 mg), yield: 47%. LCMS ESI-MS m/z: 678 [M+H] ⁺ .

Step 2: Dissolve the intermediate c1-1 (120 mg, 0.2 mmol) and trifluoroacetic acid (0.5 mL) in 2 mL of dichloromethane, react at room temperature for 2 hours, and stop the reaction. Evaporate the solvent under reduced pressure, and separate the crude product by HPLC preparative chromatography (chromatographic column: WelFlash C18-I, 20-40 um, 40 g; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ) mobile phase B: acetonitrile; flow rate: 50 mL/min; retention time: 9 min) to obtain white solid c1 (80 mg), yield: 78%. LCMS ESI-MS m/z: 578 [M+H] ⁺ .

Referring to the synthetic route of intermediate c1, similar raw materials/analogues were used to synthesize the following target intermediate.

Referring to the synthetic route of intermediate c1, similar raw materials/analogues were used to synthesize the following target intermediate.

Preparation of intermediates C5-C6, C12-C15, C23

Step 1: Dissolve the intermediate a6 (996 mg, 1.5 mmol) in 10 mL of dichloromethane, add 0.5 mL of trifluoroacetic acid, and react the mixture at room temperature for 1 hour to stop the reaction. Add 20 mL of water to the reaction solution, adjust the pH to about 9 with a saturated sodium bicarbonate aqueous solution, extract with dichloromethane, dry with anhydrous sodium sulfate, and concentrate to obtain a yellow solid c5-2 (694 mg), yield: 82%. LCMS ESI-MS m/z: 562 [M+H] ⁺ .

Step 2: Dissolve the intermediate c5-2 (494 mg, 0.88 mmol) and DIEA (567 mg, 4.4 mmol) in 5 mL of dichloromethane, add the raw material 3-hydroxy-2-pyridinecarbonyl chloride c5-1 (277 mg, 1.76 mmol), react at room temperature for 2 hours, and stop the reaction. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography (chromatographic column: WelFlash C18-I, 20-40um, 130g; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 60 mL/min; retention time: 14 min) to obtain a yellow solid c5 (360 mg), yield: 60%. LCMS ESI-MS m/z: 683 [M+H] ⁺ .

Steps: In an ice bath, under nitrogen protection, the intermediate b1 (49 mg, 0.32 mmol) and 1-chloro-N, N, 2-trimethylpropyl-1-enyl-1-amine (43 mg, 0.32 mmol) were dissolved in 2 mL of dichloromethane and stirred for 1 hour in an ice bath. DIEA (138 mg, 1.06 mmol) and intermediate c5-2 (120 mg, 0.21 mmol) were added to the reaction solution, reacted at room temperature for 1 hour, and the reaction was stopped. 15 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 4/5) to obtain a light yellow solid c6 (45 mg), yield: 30%. LCMS ESI-MS m/z: 698 [M+H] ⁺ .

Referring to the synthetic route of intermediate C5 or C6, similar raw materials/analogues were used to synthesize the following target intermediates.

Preparation of intermediates C7-C11

Step 1: Under nitrogen protection, intermediate a5 (2.5 g, 5.85 mmol), raw material a3-1 (1.2 g, 5.85 mmol) and sodium carbonate (1.9 g, 17.5 mmol) were dissolved in 50 mL of a mixed solution of 1,4-dioxane and water (v/v, 1/1), and catalyst Pd(dppf)Cl ₂ (400 mg, 0.59 mmol) was added. The mixture was heated to 100° C. and reacted for 12 hours. The reaction was stopped and filtered. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by HPLC preparative chromatography (chromatographic column: WelFlash C18-I, 20-40 μm, 330 g; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: acetonitrile; flow rate: 40 mL/min; retention time: 16 min) to obtain yellow oil c7-1 (1.5 g), yield: 58%. LCMS ESI-MS m/z: 431 [M+H] ⁺ .

Step 2: Dissolve the intermediate c7-1 (1.5 g, 3.48 mmol) and the raw material ethyl bromoacetate c7-2 (0.8 g, 4.88 mmol) in 25 mL 1,4-dioxane, slowly add DIEA (1.4 g, 10.5 mmol), and heat to 80°C for 4 hours, cool to room temperature, and stop the reaction. Pour the reaction solution into 100 mL ice water, extract with dichloromethane, dry with anhydrous sodium sulfate, and concentrate. The crude product is separated by HPLC preparative chromatography (chromatographic column: XSelect Prep OBD C18 Column, 30*150 mm, 5 μm; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: acetonitrile; flow rate: 40 mL/min; retention time: 16 min) to obtain yellow solid c7-3 (1.4 g), yield: 78%. LCMS ESI-MS m/z: 517[M+H] ⁺ .

Step 3: Dissolve the intermediate c7-3 (1.2 g, 2.32 mmol) in a mixed solution of 18 mL tetrahydrofuran and water (v/v, 2/1), slowly add NAOH aqueous solution (3.5 mL, 1 M), dropwise, react at room temperature for 1 hour, and stop the reaction. Adjust the pH of the reaction solution to about 4 with dilute hydrochloric acid, extract with dichloromethane, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by HPLC preparative chromatography (chromatographic column: XSelect Prep OBD C18, 30*150 mm, 5 μm; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 40 mL/min; retention time: 16 min) to obtain yellow solid c7 (900 mg), yield: 92%. LCMS ESI-MS m/z: 489 [M+H] ⁺ .

Referring to the synthetic route of intermediate c7, similar raw materials/analogues were used to synthesize the following target intermediate.

Preparation of intermediates d1-d7

Step 1: Under nitrogen protection, the raw material d1-1 (2.0 g, 7.40 mmol) and methylhydrazine sulfate (1.1 g, 7.40 mmol) were dissolved in 40 mL of ethanol, acetic acid (100 mg, 0.14 mmol) was added, and the mixture was heated to 80 ° C for 2 hours, the reaction was stopped, and the mixture was evaporated under reduced pressure. Solvent. Saturated sodium bicarbonate aqueous solution was added to the mixed solution to adjust the pH to about 8, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a yellow oil d1-2 (2.5 g), yield: 89%. LCMS ESI-MS m/z: 297 [M+H] ⁺ .

Step 2: Under nitrogen protection, the intermediate d1-2 (2.5 g, 8.38 mmol) and potassium phosphate (1.78 g, 8.39 mmol) of the previous step were dissolved in 38 mL DMSO, and the catalyst CuI (160 mg, 0.83 mmol) was added. The mixture was heated to 100 ° C for 3 hours, the reaction was stopped, and filtered. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by HPLC preparative chromatography (chromatographic column: WelFlash C18-I, 20-40 μm, 330 g; mobile phase A water: (0.1% TFA), mobile phase B: acetonitrile; flow rate: 40 mL/min; retention time: 12 min) to obtain a white solid d1-3 (130 mg), yield: 7%. LCMS ESI-MS m/z: 217 [M+H] ⁺ .

Step 3: -78°C, under nitrogen protection, the intermediate d1-3 (130 mg, 0.59 mmol) of the previous step was dissolved in 2.5 mL of anhydrous tetrahydrofuran, and n-BuLi (0.28 mL, 2.5 M) was slowly added. After the addition was complete, the mixture was stirred at -78°C for 1 hour. 2-isopropoxyboronic acid pinacol ester (134 mg, 1.23 mmol) was added to the reaction solution, and stirring was continued for 30 minutes to stop the reaction. 10 mL of saturated aqueous ammonium chloride solution and ethyl acetate were added to the reaction solution, dried over anhydrous sodium sulfate, and concentrated to obtain a yellow oil d1 (110 mg), with a yield of 70%. LCMS ESI-MS m/z: 265 [M+H] ⁺ .

Referring to the synthetic route of intermediate d1, similar raw materials/analogues were used to synthesize the following target intermediate.

Preparation of intermediates d8 and d15

The first step: under nitrogen protection, the raw material 1-bromo-2-fluoro-4-iodobenzene d8-1 (1.95 g, 6.5 mmol) and the raw material d8-2 (2.32 g, 7.8 mmol) were dissolved in 40 mL of acetonitrile, potassium tert-butoxide (2.2 g, 19.4 mmol) was added, and the mixture was heated to 50 ° C for 6 hours, the reaction was stopped, and filtered. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 20/1) to obtain a yellow oil d8-3 (2.22 g), with a yield of 79%.

Step 2: Dissolve the intermediate d8-3 (2.22 g, 5.15 mmol) in 133 mL of chlorobenzene, add polyphosphoric acid PPA (2.37 g, 20.6 mmol), heat the mixture to 130°C for 5 hours, stop the reaction, and cool to room temperature. Add 100 mL of ice water to the reaction solution, extract with ethyl acetate, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 9/10) to obtain yellow oil d8-4 (1.05 g), yield: 60%.

Step 3: Under nitrogen protection, the intermediate d8-4 (1.05 g, 3.1 mmol) and the raw material d8-5 (1.95 g, 9.3 mmol) of the previous step were dissolved in 21 mL DMF, and the catalyst CuI (290 mg, 1.55 mmol) and HMPA (2.39 g, 13.3 mmol) were added. The mixture was heated to 100 ° C for 2 hours, the reaction was stopped, and filtered. 60 mL of water was added to the reaction solution, extracted with methyl tert-butyl ether, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 100/1) to obtain a yellow solid d8-6 (0.8 g), with a yield of 92%.

Step 4: Under nitrogen protection, the intermediate d8-6 (0.8 g, 2.85 mmol) and the raw material diphenyl ketone imine (1.03 g, 5.69 mmol) were dissolved in 16 mL of toluene, and catalyst Pd(OAc) ₂ (60 mg, 0.28 mmol), ligand BINAP (0.35 g, 0.57 mmol) and cesium carbonate (1.85 g, 5.69 mmol) were added. The mixed solution was heated to 110°C for 2 hours, the reaction was stopped, and filtered. 60 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a crude mixture. The mixture was dissolved in 4 mL of tetrahydrofuran, and a 2M concentration of hydrogen chloride in tetrahydrofuran solution (16 mL) was added, and the reaction was carried out at room temperature for 1 hour, and the reaction was stopped. Saturated sodium bicarbonate aqueous solution was added to the reaction solution to adjust the pH to about 8, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 10/7) to obtain yellow solid d8 (0.5 g), yield: 81%. LCMS ESI-MS m/z: 218 [M+H] ⁺ .

Referring to the synthetic route of intermediate d8, similar raw materials/analogues were used to synthesize the following target intermediate.

Preparation of intermediate d9

Step 1: Under CO ₂ (20 atm), the raw material d9-1 (2.5 g, 15.7 mmol) and TEA (21.8 mL, 157 mmol) were dissolved in 50 mL of methanol, and the catalyst Pd(dppf)Cl ₂ (1.2 g, 1.57 mmol) was added. The mixture was heated to 80°C for 12 hours, the reaction was stopped, and the mixture was filtered. The solvent was evaporated under reduced pressure, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 3/10) to obtain a yellow solid d9-2 (900 mg), with a yield of 31%. LCMS ESI-MS m/z: 184 [M+H] ⁺ .

Step 2: Dissolve the intermediate d9-2 (900 mg, 4.91 mmol) and chloroacetaldehyde (771 mg, 9.8 mmol) in 18 mL of ethanol, heat the mixture to 80°C for 5 hours, stop the reaction, and cool to room temperature. Add 100 mL of ice water to the reaction solution, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by ASFC chromatography (chromatographic column: DAICEL DCpak P4VP 3*25 cm, 5 μm; mobile phase: CO ₂ , mobile phase B: MeOH (0.1% formic acid); flow rate: 60 mL/min; retention time (min) of d9-3: 3.41; retention time (min) of d9-4: 5.94). d9-3 (300 mg), yield: 30%; d9-4 (150 mg), yield: 15%.

Step 3: Under nitrogen protection, the intermediate d9-3 (200 mg, 0.97 mmol) of the previous step was dissolved in 4 mL of dichloromethane, and BBr ₃ (1.93 mL, 1.93 mmol) was added. The mixture was reacted at room temperature for 12 hours, the reaction was stopped, and the mixture was filtered. 20 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated to obtain a crude mixture. The mixture was dissolved in 3 mL of tetrahydrofuran, 2N aqueous NAOH solution (1 mL) was added, reacted at room temperature for 1 hour, and the reaction was stopped. Dilute hydrochloric acid was added to the reaction solution to adjust the pH to about 6, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a yellow solid d9 (40 mg), with a yield of 23%. LCMS ESI-MS m/z: 180 [M+H] ⁺ .

Preparation of intermediate d10

Step 1: Under nitrogen protection, the raw materials NBS (3.81 g, 21.4 mmol) and AIBN (200 mg, 1.22 mmol) were dissolved in 40 mL of carbon tetrachloride, the mixed solution was heated to 80 ° C, and the raw material d10-1 (3.0 g, 21.4 mmol) was added dropwise to the reaction solution. After the addition was completed, the reaction was continued for 5 hours, the reaction was stopped, and the reaction was filtered. The solvent was evaporated under reduced pressure, 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 1/10) to obtain a yellow oil d10-2 (4.09 g), with a yield of 87%.

Step 2: The intermediate d10-2 (2.5 g, 11.4 mmol) and the raw material d10-3 (3.05 g, 12.5 mmol) of the previous step were dissolved in 18 mL of DMF, and KI (189 mg, 1.1 mmol) and K ₂ CO ₃ (2.36 g, 17.1 mmol) were added. The mixture was heated to 50°C for 3 hours, the reaction was stopped, and the mixture was cooled to room temperature. 100 mL of ice water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by chromatography (PE/EA, 1/4) to obtain a yellow oil d10-4 (3.77 g), with a yield of 87%. LCMS ESI-MS m/z: 382 [M+H] ⁺ .

Step 3: -70°C, under nitrogen protection, the intermediate d10-4 (2.11 g, 5.54 mmol) of the previous step was dissolved in 40 mL of anhydrous tetrahydrofuran, and LiHMDS (15.51 mL, 15.51 mmol) was added. The mixture continued to react for 1 hour, the reaction was stopped, and the temperature was raised to room temperature. 20 mL of water was added to the reaction solution, and the pH was adjusted to about 6 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by chromatography (PE/EA, 1/5) to obtain a yellow oil d10-5 (951 mg), with a yield of 89%. LCMS ESI-MS m/z: 194 [M+H] ⁺ .

Step 4: The intermediate d10-5 (903 mg, 4.68 mmol) of the previous step was dissolved in a mixed solution of 12 mL of tetrahydrofuran and water (v/v, 2/1), and LiOH (280 mg, 11.7 mmol) was added. The mixed solution was heated to 50°C for 4 hours to stop the reaction. 20 mL of water was added to the reaction solution, and the pH was adjusted to about 3 with dilute hydrochloric acid. The solid was precipitated, filtered, and the filter cake was dried to obtain a white solid d10 (265 mg), with a yield of 32%. LCMS ESI-MS m/z: 180 [M+H] ⁺ .

Preparation of intermediate d11

Step 1: Under hydrogen protection (1 atm), the intermediate d10-5 (800 mg, 4.14 mmol) and Pd/C (320 mg, 40% wt) were dissolved in 16 mL of ethanol, mixed and reacted at room temperature for 2 hours, the reaction was stopped, and filtered. The solvent was evaporated under reduced pressure to obtain a white solid d11-1 (707 mg), yield: 88%. LCMS ESI-MS m/z: 196 [M+H] ⁺ .

Step 2: Dissolve the intermediate d11-1 (700 mg, 3.58 mmol) in a mixed solution of 10 mL tetrahydrofuran and water (v/v, 3/1), add LiOH (215 mg, 9.0 mmol), heat the mixed solution to 50°C for 4 hours, and stop the reaction. Add 20 mL of water to the reaction solution, adjust the pH to about 3 with dilute hydrochloric acid, precipitate the solid, filter, and dry the filter cake to obtain a white solid d11 (485 mg), yield: 75%. LCMS ESI-MS m/z: 182 [M+H] ⁺ .

Preparation of intermediate d12

Step 1: Under carbon monoxide protection, the raw material d12-1 (1.0 g, 6.26 mmol) and methanol (3.0 mL) were dissolved in 10 mL DMF, and catalyst Pd(dppf)Cl ₂ (0.9 g, 1.25 mmol) and TEA (3.0 mL) were added. The mixed solution was heated to 120°C under carbon monoxide (30 atm) for 12 hours, the reaction was stopped, and the mixture was filtered. The solvent was evaporated under reduced pressure, 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain a yellow solid d12-2 (500 mg), yield: 44%. LCMS ESI-MS m/z: 184 [M+H] ⁺ .

Step 2: Dissolve the intermediate d12-2 (500 mg, 2.73 mmol) and the raw material chloroacetaldehyde (642 mg, 8.2 mmol) in 10 mL of tert-butyl alcohol, heat the mixture to 90 °C and react for 12 hours, stop the reaction, and cool to room temperature. Add 50 mL of ice water to the reaction solution. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain a yellow solid d12-3 (200 mg), yield: 35%. LCMS ESI-MS m/z: 208 [M+H] ⁺ .

Step 3: Dissolve the intermediate d12-3 (190 mg, 0.91 mmol) in a mixed solution of 4 mL tetrahydrofuran and water (v/v, 3/1), add NaOH (73 mg, 1.8 mmol), and react the mixed solution at room temperature for 1 hour to stop the reaction. Add 10 mL of water to the reaction solution, adjust the pH to about 3 with dilute hydrochloric acid, extract with ethyl acetate, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain a white solid d12 (100 mg), yield: 56%. LCMS ESI-MS m/z: 194 [M+H] ⁺ .

Preparation of intermediate d13

Step 1: Dissolve the raw material d13-1 (5.0 g, 17.7 mmol) and ethanol (50 mL) in 100 mL of concentrated HBr, add KSCN (5.2 g, 53.2 mmol), heat the mixture to 100°C for 2 hours, and stop the reaction. Evaporate the solvent under reduced pressure, add 100 mL of water to the reaction solution, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain a brown solid d13-2 (1.1 g), yield: 24%. LCMS ESI-MS m/z: 260 [M+H] ⁺ .

Step 2: Under nitrogen protection, the intermediate d13-2 (1.1 g, 4.22 mmol) and the raw material iPrONO (1.49 g, 12.7 mmol) were dissolved in 17 mL of anhydrous tetrahydrofuran, and DMSO (0.3 g, 3.38 mmol) was added. The mixture was heated to 50°C for 12 hours, the reaction was stopped, and the mixture was cooled to room temperature. 50 mL of ice water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain a yellow solid d13-3 (460 mg), with a yield of 44%. LCMS ESI-MS m/z: 245 [M+H] ⁺ .

Step 3: Under carbon monoxide protection, the intermediate d13-3 (460 mg, 1.87 mmol) and TEA (380 mg, 3.75 mmol) of the previous step were dissolved in 10 mL of methanol, and the catalyst Pd(dppf)Cl ₂ (137 mg, 0.18 mmol) was added. The mixed solution was heated to 100°C under carbon monoxide (30 atm) for 12 hours, the reaction was stopped, and the mixture was filtered. The solvent was evaporated under reduced pressure, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a brown solid d13-4 (525 mg). LCMS ESI-MS m/z: 225 [M+H] ⁺ .

Step 4: The intermediate d13-4 (525 mg, 2.34 mmol) of the previous step was dissolved in a mixed solution of 10 mL of tetrahydrofuran and water (v/v, 7/3), and NaOH (187 mg, 4.8 mmol) was added. The mixed solution was reacted at room temperature for 1 hour to stop the reaction. 10 mL of water was added to the reaction solution, and the pH was adjusted to about 6 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain a yellow solid d13 (230 mg), with a yield of 50%. LCMS ESI-MS m/z: 211 [M+H] ⁺ .

Preparation of intermediates d18-d19

Step 1: Under nitrogen protection, the raw material 2-bromo-5-trifluoromethylphenol d18-1 (5.0 g, 20.75 mmol) and potassium carbonate (8.6 g, 62.2 mmol) were dissolved in 100 mL DMF, and the raw material d18-2 (12.3 g, 62.2 mmol) was added dropwise. The mixture was heated to 80 ° C for 12 hours, the reaction was stopped, and filtered. 250 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (PE/EA, 20/1) to obtain a yellow oil d18-3 (6.0 g), with a yield of 81%.

Step 2: Dissolve the intermediate d18-3 (6.0 g, 16.8 mmol) and polyphosphoric acid (30 g, 261 mmol) in 72 mL of toluene, heat the mixture to 120°C and react for 5 hours to stop the reaction. Add 100 mL of water to the reaction solution, adjust the pH to about 8 with ammonia water, extract with methyl tert-butyl ether, dry with anhydrous sodium sulfate, concentrate, and separate by column chromatography (PE/EA, 20/1) to obtain a yellow oil d18-4 (1.0 g), with a yield of 23%.

Step 3: Under nitrogen protection, the intermediate d18-4 (1.0 g, 3.77 mmol) and the raw material diphenyl ketone imine (1.4 g, 7.55 mmol) were dissolved in 20 mL of toluene, and the catalyst Pd(OAc) ₂ (80 mg, 0.38 mmol), the ligand BINAP (0.5 g, 0.8 mmol) and Cesium carbonate (2.5 g, 7.55 mmol), the mixture was heated to 110 ° C and reacted for 2 hours, the reaction was stopped and filtered. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a crude mixture. The mixture was dissolved in 5 mL of tetrahydrofuran, and a 2M concentration of hydrogen chloride in tetrahydrofuran solution (20 mL) was added, and the reaction was carried out at room temperature for 1 hour, and the reaction was stopped. Saturated sodium bicarbonate aqueous solution was added to the reaction solution to adjust the pH to about 8, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain a yellow solid d18 (150 mg), yield: 20%. LCMS ESI-MS m/z: 202[M+H] ⁺ .

Referring to the synthetic route of intermediate d18, similar raw materials/analogues were used to synthesize the following target intermediate.

Preparation of intermediate d20

Steps: -78°C, under nitrogen protection, the raw material 7-aminobenzofuran d20-1 (600 mg, 4.13 mmol) was dissolved in 14 mL of anhydrous dichloromethane, and a pre-prepared liquid Br ₂ solution (0.7 g, 4.13 mmol, DCM 11 mL) was added, and the mixture was reacted at -78°C for 1 hour to stop the reaction. 20 mL of saturated sodium thiosulfate aqueous solution was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain a yellow oil d20 (200 mg), with a yield of 23%. LCMS ESI-MS m/z: 212 [M+H] ⁺ .

Embodiment 2:

Preparation of target molecules P1-P2, P5-P9, P22-P23

Step 1: In an ice bath, under nitrogen protection, the intermediate b4 (70 mg, 0.22 mmol) and 1-chloro-N, N, 2-trimethylpropyl-1-en-1-amine (61 mg, 0.45 mmol) were dissolved in 2 mL of dichloromethane and stirred for 1 hour in an ice bath. The intermediate c2 (129 mg, 0.23 mmol) and DIEA (88 mg, 0.68 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 2 hours to stop the reaction. 10 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 7/10) to obtain a yellow solid P1-1 (40 mg), yield: 21%. LCMS ESI-MS m/z: 856 [M+H] ⁺ .

Step 2: In an ice bath, under nitrogen protection, the compound P1-1 (40 mg, 0.05 mmol) and trifluoroacetic acid (0.4 mL) were dissolved in 1 mL of dichloromethane, stirred at room temperature for 2 hours, and the solvent was evaporated under reduced pressure. The mixture was dissolved in 1 mL of dichloromethane again, and DMEDA (0.08 mL) was added. The reaction solution was reacted at room temperature for 8 hours, and the reaction was stopped. The solvent was evaporated under reduced pressure, and the crude product was separated by HPLC chromatography (chromatographic column: XBridge Prep Shield RP18 OBD Column, 19*250 mm, 5 μm; mobile phase A: water (10 mmol/L NH ₄ HCO ₃ ), mobile phase B: CH ₃ CN; flow rate: 25 mL/min mL/min; G retention time (min): 9.6) to obtain a white solid P1 (12 mg), yield: 35%. LCMS ESI-MS m/z: 726 [M+H] ⁺ .

¹H NMR (300 MHz, DMSO-d₆) δ11.46 (s, 1H), 10.25 (s, 1H), 9.62 (s, 1H), 8.06 (d, J=8.6Hz, 1H), 7.96 (d, J=2.1Hz, 1H), 7.71 (dd , J=8.7, 2.1Hz, 1H), 7.46 (t, J=2.9Hz, 1H), 7.41 (s, 1H), 6.83 (dd, J=3.2, 1.7Hz, 1H), 6.33 (dd, J= 3.3, 1.7Hz, 1H), 5.31 (s, 2H), 4.57 (s, 1H), 4.25 (d, J=2.9Hz, 2H), 3.84-3.76 (m,2H), 3.55 (d, J=39.2Hz, 4H), 3.25 (d, J=13.1Hz, 2H), 2.98 (d, J=9.8Hz, 3H), 2.81 (d, J=11.1Hz, 1H) , 2.61 (d, J=10.9Hz, 1H), 1.19 (t, J=7.5Hz, 3H).

Referring to the synthetic route of compound P1, similar raw materials/intermediates (such as intermediates b5, b7, c16-c22, d10, etc.) were used to synthesize the following target molecules.

Preparation of target molecules P3-P4, P24

Step 1: In an ice bath, under nitrogen protection, the intermediate b4 (602 mg, 1.95 mmol) and 1-chloro-N, N, 2-trimethylpropyl-1-en-1-amine (261 mg, 1.95 mmol) were dissolved in 12 mL of dichloromethane and stirred for 1 hour in an ice bath. The intermediate c5-2 (550 mg, 0.98 mmol) and DIEA (379 mg, 2.9 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 2 hours to stop the reaction. 50 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 4/5) to obtain a yellow oil P3-1 (400 mg), with a yield of 48%. LCMS ESI-MS m/z: 852 [M+H] ⁺ .

Step 2: In an ice bath, under nitrogen protection, the compound P3-1 (300 mg, 0.35 mmol) and trifluoroacetic acid (3.0 mL) were dissolved in 3 mL of dichloromethane, stirred at room temperature for 2 hours, and the solvent was evaporated under reduced pressure. The mixture was dissolved in 3 mL of dichloromethane again, and DMEDA (0.08 mL) was added. The reaction solution was reacted at room temperature for 8 hours, and the reaction was stopped. The solvent was evaporated under reduced pressure, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 4/5) to obtain a yellow oil P3-2 (150 mg), with a yield of 59%. LCMS ESI-MS m/z: 722 [M+H] ⁺ .

Step 3: Under nitrogen protection, the intermediate P3-2 (34 mg, 0.05 mmol) and the intermediate d7 (23 mg, 0.09 mmol) of the previous step were dissolved in a mixed solution of 1 mL DMF and water (3/1), and catalyst Pd(dppf)Cl ₂ (60 mg, 0.28 mmol) and potassium phosphate (30 mg, 0.14 mmol) were added. The mixed solution was heated to 70°C for 2 hours, the reaction was stopped, and filtered. 5 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a crude mixture. The crude product was separated by HPLC chromatography (chromatographic column: Xselect CSH Prep C18 OBD Colum, 19*250 nm, 5 μm; mobile phase A: water (0.1% trifluoroacetic acid), mobile phase B: MeOH; flow rate: 25 mL/min; retention time (min): 12.4) to obtain a white solid P3 (4.4 mg), yield: 3%. LCMS ESI-MS m/z: 757[M+H] ⁺ . ¹ H NMR (300MHz, DMSO-d ₆ ) δ 11.47 (s, 1H), 10.37 (s, 1H), 9.63 (s, 1H), 8.29 (s, 1H), 8.10-7.88 (m, 2H), 7.71 (dd, J=8.8, 2.1Hz, 1H), 7.51-7.44 (m, 1H), 7 .41 (s, 1H), 6.33 (dd, J=3.4, 1.8Hz, 1H), 6.28 (t, J=5.9 Hz, 1H), 5.36 (s, 2H), 4.78 (d, J=5.5Hz, 2H), 4.60 (d, J=12.2Hz, 1H), 3.51 (t, J=12.3Hz, 3H), 3.24 (s, 1H), 3.01 (s, 3H), 2.83 (d, J=11.1Hz, 1H), 2.63 (d, J =10.3Hz, 1H), 1.23(s, 3H).

Referring to the synthetic route of compound P3, similar raw materials/intermediates (such as intermediates b2, b7) were used to synthesize the following target molecules.

Preparation of target molecule P10

Step 1: Under nitrogen protection, intermediate c23 (100 mg, 0.12 mmol) and morpholine (51 mg, 0.59 mmol) were dissolved in 2 mL DMSO, KOAc (69 mg, 0.70 mmol) was added, and the temperature was raised to 120°C for reaction for 5 hours to stop the reaction. 20 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 4/5) to obtain yellow solid P10-1 (60 mg), yield: 60%. LCMS ESI-MS m/z: 859 [M+H] ⁺ .

Step 2: In an ice bath, under nitrogen protection, the compound P10-1 (60 mg, 0.07 mmol) and trifluoroacetic acid (1.0 mL) were dissolved in 1 mL of dichloromethane, stirred at room temperature for 2 hours, and the solvent was evaporated under reduced pressure. The mixture was dissolved in 1 mL of dichloromethane again, and DMEDA (0.12 mL) was added. The reaction solution was reacted at room temperature for 8 hours, and the reaction was stopped. The solvent was evaporated under reduced pressure, and the crude product was separated by HPLC chromatography (chromatographic column: XSelectPrep OBD C18 Column, 30*150 mm, 5 μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL/min; retention time (min): 9.9) to obtain a white solid P10 (6.1 mg), yield: 11%. LCMS ESI-MS m/z: 729 [M+H] ⁺ . ¹ H NMR (300 MHz, DMSO-d ₆ )δ11.46 (s, 1H), 10.35 (s, 1H), 9.60 (s, 1H), 8.04 (d, J=8.6Hz, 1H), 7.97 (d, J=2.1Hz, 1H), 7.72 (dd, J=8.9, 2.2Hz, 1H), 7.49-7.43 (m, 1H), 7.40 (s, 1H), 6 .33(dd, J=3.4, 1.8Hz, 1H), 5.22(s, 2 H), 4.57 (d, J = 12.2Hz, 1H), 3.66 (t, J = 4.8Hz, 4H), 3.50 (t, J = 13.3Hz, 3H), 3.42-3.37 (m, 4H), 3.23 (d, J = 12.9Hz, 1H), 2.95 (s, 3H), 2.77 (d, J = 13.4Hz, 1H) ), 2.58 (s, 1H), 1.16 (t, J=7.4Hz, 3H).

Preparation of target molecules P11-P15, P17

Step 1: Under nitrogen protection, intermediate c2 (687 mg, 1.21 mmol) and 1-chloro-N, N, 2-trimethylpropyl-1-en-1-amine (161 mg, 1.2 mmol) were dissolved in 3 mL of dichloromethane and stirred for 1 hour under ice bath. Intermediate d11 (110 mg, 0.60 mmol) and DIEA (392 mg, 3.0 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 2 hours to stop the reaction. 10 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 1/1) to obtain white solid P13 (19 mg), yield: 4%. LCMS ESI-MS m/z: 729 [M+H] ⁺ .

¹ H NMR (300MHz, DMSO-d ₆ ) δ 11.01 (s, 1H), 10.39 (s, 1H), 8.07 (d, J = 8.6Hz, 1H), 7.98 (d, J = 2.1Hz, 1H) , 7.78-7.66 (m, 2H), 6.84 (s, 1H), 5.33 (s, 2H), 4.66 (t, J=8.8Hz, 3H), 4.26 (d, J=3.0Hz, 3H), 3.81 (t, J=5.5Hz, 2H), 3.51 (t, J=11.0Hz, 2H), 3.33 (s, 3H), 3.20 (t, J=8.9Hz, 2H), 3.00 (d, J=7.8Hz , 3H), 2.74 (s, 2H), 1.20 (t, J=7.4Hz, 3H).

Referring to the synthetic route of compound P13, similar raw materials/intermediates (such as intermediates b6, d9-d12, c20) were used to synthesize the following target molecules.

Preparation of target molecule P16

Step 1: Under nitrogen protection, intermediate c2 (563 mg, 1.0 mmol), DIEA (428 mg, 3.32 mmol) and intermediate d13 (131 mg, 0.66 mmol) were dissolved in 3 mL of acetonitrile. EDCI (254 mg, 1.32 mmol) and HOAt (180 mg, 1.33 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 3 hours to stop the reaction. 10 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain yellow solid P16-1 (96 mg), yield: 19%. LCMS ESI-MS m/z: 758 [M+H] ⁺ .

Step 2: Under nitrogen protection, the compound P16-1 (88 mg, 0.45 mmol) and LiCl (76 mg, 1.8 mmol) were dissolved in 2 mL DMF. The mixture was heated to 150°C for 13 hours and the reaction was stopped. 10 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by HPLC preparative chromatography (chromatographic column: Xselect CSH F-Phenyl OBD Column 30*150mm 5μmn; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL/min; retention time: 9.55 min) to obtain a light yellow solid P16 (11 mg), yield: 3%. LCMS ESI-MS m/z: 744 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ )δ9.51 (s, 1H), 8.06 (d, J = 8.6Hz, 1H), 7.92 (d, J = 36.3Hz, 2H), 7.71 (d, J = 8.6Hz, 1H), 6.83 (s, 1H), 5.32 (s, 2H), 4.57 (d, J = 12.4Hz, 1H), 4.25 (s, 2H), 3.8 0 (t, J=5.6Hz, 2H), 3.54 (s, 2H), 3.30 (dd, J=21.8, 12.7Hz, 3H), 2.99 (d, J=11.0Hz, 3H), 2.82 (d, J=11.3Hz, 1H), 1.20 (q, J=9.5, 7.7Hz, 3H).

Preparation of target molecules P18-P21, P25-P27

Step 1: Under nitrogen protection, intermediate c7 (150 mg, 0.31 mmol), DIEA (238 mg, 1.84 mmol) and intermediate d18 (74 mg, 0.37 mmol) were dissolved in 5 mL of dichloromethane and stirred for 5 minutes under ice bath. T ₃ P (110 mg, 0.60 mmol) and DMAP (113 mg, 0.92 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 12 hours to stop the reaction. 30 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain yellow solid P18-1 (162 mg), yield: 79%. LCMS ESI-MS m/z: 672 [M+H] ⁺ .

Step 2: Dissolve the compound P18-1 (162 mg, 0.24 mmol) in 1 mL of 1,4-dioxane and stir for 5 minutes under ice bath. Add 1,4-dioxane solution of HCl (2 M, 3.3 mL) to the reaction solution, and react the mixture at room temperature for 1 hour to stop the reaction. Evaporate the solvent under reduced pressure to obtain a brown solid P18-2 (162 mg). LCMS ESI-MS m/z: 572 [M+H] ⁺ .

Step 3: Under nitrogen protection, the compound P18-2 (162 mg, 0.28 mmol), DIEA (238 mg, 1.84 mmol) and intermediate b4 (175 mg, 0.57 mmol) were dissolved in 4 mL of dichloromethane and stirred for 5 minutes under ice bath. 1-Chloro-N, N, 2-trimethylpropyl-1-en-1-amine (76 mg, 0.56 mmol) was added to the reaction solution, and the mixture was reacted at room temperature for 2 hours to stop the reaction. 30 mL of water was added to the reaction solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography (chromatographic column: C18; CH ₃ CN/H ₂ O, 2/1) to obtain yellow solid P18-3 (94 mg), two-step yield: 38%. LCMS ESI-MS m/z: 862 [M+H] ⁺ .

Step 4: Dissolve the compound P18-3 (93 mg, 0.11 mmol) in a mixed solution of 2 mL trifluoroacetic acid and dichloromethane (v/v, 1/1), react for 2 hours under ice bath, and remove the solvent under reduced pressure. Dissolve the mixture in 1 mL dichloromethane, add ethylenediamine EDA (1.0 mL) to the reaction solution, and continue to react at room temperature for 1 hour to stop the reaction. Add 10 mL of water to the reaction solution, extract with dichloromethane, dry with anhydrous sodium sulfate, concentrate, and separate the crude product by HPLC preparative chromatography (chromatographic column: Xbridge Prep phenyl OBD Colum, 19*250 nm, 5 μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL/min; retention time: 11.2 min) to obtain white solid P18 (13.5 mg), yield: 16%. LCMS ESI-MS m/z: 732 [M+H] ⁺ .

¹ H NMR (300MHz, DMSO-d ₆ ) δ 11.47 (s, 1H), 9.67 (s, 1H), 8.36 (d, J = 2.2Hz, 1H), 8.06 (d, J = 8.3Hz, 1H), 7.61 (d, J=8.5Hz, 1H), 7.49-7.38 (m, 2H), 7.14 (q, J=1.8Hz, 1H), 6.81 (t, J=2.3Hz, 1H), 6.33 (dd, J =3.4, 1.6Hz, 1H), 5.37 (s, 2H), 4.59 (d, J = 12.4Hz, 1H), 4.24 (q, J=2.7Hz, 2H), 3.80 (t, J=5.5Hz, 2H), 3.57 (t, J=10.6Hz, 2H), 3.51 (s, 1H), 3.47 (s, 1H), 3.36 (s, 1H), 3.24 (s, 1H), 3.09-2.89 (m, 3H), 2.82 (d, J = 11.0Hz, 1H), 2.62 (d, J = 10.5Hz, 1H), 1.20 (q, J=7.4, 6.4Hz, 3H).

Referring to the synthetic route of compound P18, similar raw materials/intermediates (such as intermediates d10, d20, c8) were used to synthesize the following target molecules.

Embodiment 3:

The activity of the molecules of the present invention on the hydrolysis of double-stranded DNA by WRN helicase was tested (Table 1).

Prepare the working solution and buffer to be tested. The test compound was dissolved in DMSO and diluted 4 times (10 μM final concentration as the starting concentration). 0.2 μL of the test compound solution was added to a 384-well plate, and 10 μL (2X) WRN enzyme solution was added. After incubation in the dark for 30 minutes, 10 μL of substrate detection solution containing double-stranded DNA was added to initiate the reaction (DNA length 19 bp, 3′ and 5′ labeled TAMRA and BHQ2, respectively), and incubated at room temperature for 60 minutes. The inhibitory activity (IC ₅₀ ) for WRN enzyme was calculated based on the changes in fluorescence Ex530/Em590 of the blank group (DMSO) and the compound group.

Calculation formula: Y = lower platform signal + (upper platform signal - lower platform signal) / (1 + 10^((LogIC ₅₀ -X) × Hill slope))

X: log value of compound concentration

Y: Inhibition rate (%)

Table 1. Inhibitory effects of compounds on the unwinding of WRN enzyme

Unwinding

The above results show that the molecule of the present invention has a good inhibitory effect on WRN DNA unwinding, and is expected to achieve a better tumor inhibition effect by inhibiting the WRN helicase activity.

Embodiment 4:

The molecules of the present invention were tested for their anti-proliferative activity against MSI-H tumor cells.

The proliferation of tumor cells with microsatellite instability MSI-H is sensitive to WRN inhibitors, while the proliferation of tumor cells with microsatellite stability MSS is insensitive to WRN inhibitors. By testing the activity of both, it is shown that the molecules of the present invention have an inhibitory and synthetic lethal effect on WRN at the cellular level.

MSI unstable SW48 colorectal cancer cells were cultured in RMI1640 medium containing 10% FBS and 1% penicillin-streptomycin, placed in a 37°C, 5% CO ₂ constant temperature incubator, and 40 μL of cell suspension was added to each well of a 384-well microplate. 40 nL of compounds of different concentrations were added to each well using Echo, and placed in a 37°C, 5% CO ₂ constant temperature incubator for 5 days. 40 μL of CTG solution (Promega, CatNo. G7573) was added to each well and placed in a 37°C, 5% CO ₂ constant temperature incubator incubated in the dark for 30 minutes. The luminescence value was read using an Envision multifunctional microplate reader (Perkin Elmer, catalog number Envision 2104). The light signal was proportional to the amount of ATP in the system, and the ATP content directly characterized the number of viable cells in the system.

_IC50 value calculation:

Y = lower platform signal + (upper platform signal - lower platform signal) / (1 + 10^((LogIC ₅₀ -X) × Hill slope))

X: log value of compound concentration

Y: Inhibition rate (%)

Table 2.1: 2D antiproliferative effects of compounds on MSI-H colorectal cancer SW48 cell line

N.D. = Not Tested

Referring to WO2022249060, the following control molecule A1 was synthesized

The above results show that the molecule of the present invention has a good anti-proliferation effect on MSI-H tumor cells and is expected to achieve a better tumor inhibition effect by inhibiting the WRN helicase activity.

The above results also show that the control molecule P11b has no inhibitory effect on SW48 cells, while the molecules of the present invention such as P13, P14, P15, P16, P26 and the like have significant inhibitory effects, indicating that different ring structures significantly affect the anti-proliferation effect.

Embodiment 5:

The molecules of the present invention were tested for their antiproliferative activity against MSS tumor cells.

The HT-29 colorectal cancer cells of MSS were cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin-streptomycin, placed in a 37°C, 5% CO ₂ constant temperature incubator, and 40 μL of cell suspension was added to each well of a 384-well microplate. 40 nL of compounds of different concentrations were added to each well using Echo, and placed in a 37°C, 5% CO ₂ constant temperature incubator for 5 days. 40 μL of CTG solution (Promega, Cat No. G7573) was added to each well and placed in a 37°C, 5% CO ₂ constant temperature incubator incubated in the dark for 30 minutes. The luminescence value was read using an Envision multifunctional microplate reader (Perkin Elmer, catalog number Envision 2104). The light signal was proportional to the amount of ATP in the system, and the ATP content directly characterized the number of viable cells in the system.

_IC50 value calculation:

Y = lower platform signal + (upper platform signal - lower platform signal) / (1 + 10^((LogIC ₅₀ -X) × Hill slope))

X: log value of compound concentration

Y: Inhibition rate (%)

Table 2.2: 2D antiproliferative effects of compounds on MSS colon cancer HT-29 cell line

N.D. = Not Tested

The above results show that the molecule of the present invention has no inhibitory effect on MSS tumor cells, reflecting the high selectivity brought by the selective inhibition of WRN by the molecule of the present invention.

Embodiment 6:

The liver microsome stability test of the compound is as follows:

The compounds of the present invention were subjected to a liver microsome stability test. The compounds to be tested were co-incubated with liver microsomes of different species with or without the addition of NADPH. The final concentration of the compounds to be tested in the test system was 1 μM, the final concentration of NADPH was 1 mM, and the final concentration of liver microsomes was 0.5 mg/mL. The compound concentrations in the incubation supernatant at different time points within 60 minutes were detected and pharmacokinetic parameters (e.g., clearance Cl _int ) were calculated.

The results show that the molecules of the present invention have good metabolic stability (especially in the human body, they have good metabolic stability, and the metabolism of compounds P13, P14 and P25 is more stable).

Table 3: Results of in vitro stability test of compounds on human liver microsomes

Embodiment 7:

Mouse pharmacokinetic evaluation experiment

CD1 female mice were used as test animals and the drug was administered orally/intravenously (oral dosage was 10 mg/kg, intravenous dosage was 2 mg/kg).

Experimental plan: 3 mice in each oral group (solvent: 10% Hβ-CD-pH7.4), 3 mice in each intravenous group. Oral: Collect plasma samples before (0h) and after (0.25, 0.5, 1, 2, 4, 8, 24h) administration; Intravenous: Collect plasma samples before (0h) and after (0.083, 0.25, 0.5, 1, 2, 4, 8, 24h) administration; Use LC/MS/MS method to determine the blood concentration of plasma after oral and intravenous administration of mice, and the collected data are calculated by AB Sciex QTRAP 6500 software. The experimental results are as follows:

Table 4: PK results of compounds in mice

The above experimental results show that the compound of the present invention has a good oral absorption effect. Compared with the control molecule A1, it has better oral absorption and a higher in vivo exposure, and is expected to bring higher therapeutic effects.

Claims (26)

1. A compound of formula (A), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:
   

   in,

   Ring B or Ring C is a 5-6 membered heteroaryl or a 5-6 membered heterocyclic group, and at least one ring exists;

   Y is selected from CH or N;

   R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 R _x ;

   _R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

   R ₃ is independently selected from H, D, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atoms where they are located to form a 3-6 membered spiro ring or bridged ring;

   R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

   Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

   R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

   _Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

   R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

   m is selected from 0, 1, 2 or 3;

   n is selected from 0, 1 or 2;

   p is selected from 0, 1 or 2.
2. The compound of claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein only one of ring B and ring C exists.
3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein Y is N.
4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein for Wherein M 1 and M ₂ are each independently selected from CH or N. When one of M ₁ or M ₂ is _{selected from N, M 1 and M 2 form a monomer; M 3 , M 4 and M 5 are each} independently selected from CH, O, S or N, and M ₃ , M ₄ and M ₅ contain at least one N atom; Y is selected from CH or N.
5. The compound of claim 4, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein for Wherein _M1 and _M2 are each independently selected from CH or N.
6. The compound of claim 4, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein for Wherein R ₅ is H or C _1-6 alkyl.
7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein R ₁ is selected from a 5-6 membered monocyclic heteroaryl containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur, a 5-6 membered monocyclic heterocyclic group containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur, a 7-12 membered bicyclic heteroaryl containing 1 or 2 or 3 or 4 heteroatoms selected from oxygen, nitrogen and sulfur, or a 7-12 membered bicyclic heterocyclic group containing 1 or 2 or 3 or 4 heteroatoms selected from oxygen, nitrogen and sulfur, each of which may be substituted by 1, 2 or 3 R _x , R _x is selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ ) _p -OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a , wherein R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl.
8. The compound of claim 7, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 R _x , wherein R _x is selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, CN, NH ₂ , -C(O)R _a , -C(O)OR _a , -(CH ₂ ) _p -OR _a , -P(O)-(R _a ) ₂ or -S(O) ₂ -R _a .
9. The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein R ₂ is selected from C _1-4 alkyl, C _1-4 haloalkyl, C 1-4 alkoxy, C _1-4 alkylthio, _cyclopropyl , cyclobutyl, cyclopentyl and cyclohexyl.
10. The compound of claim 9, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein R ₂ is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
11. The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein R ₃ is selected from H or C _1-6 alkyl.
12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein Ring A is absent, and for wherein R _4a , R _4b and R _4c are independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio or C _3-6 cycloalkyl.
13. The compound of claim 12, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein Ring A is absent, and for wherein R _4a is halogen or C _1-6 haloalkyl, and R _4b is halogen or C _1-6 alkyl.
14. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein for wherein R _4a is selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio or C _3-6 cycloalkyl, wherein ring A is a 5- or 6-membered heteroaryl or heterocyclic group, each of which contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur.
15. The compound of claim 14, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein for wherein R _4a is selected from halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio or C _3-6 cycloalkyl, and ring A is
16. The compound of claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, having the structure of formula (I):
    

    in,

    M ₁ and M ₂ are each independently selected from CH or N. When one of M ₁ or M ₂ is selected from N, M ₁ and M ₂ form a monomer;

    M ₃ , M ₄ and M ₅ are each independently selected from CH, O, S or N, and M ₃ , M ₄ and M ₅ contain at least one N atom;

    Y is selected from CH or N;

    R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

    R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

    R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

    Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

    _R5 is selected from H, halogen, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

    _Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

    R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

    m is selected from 0, 1, 2 or 3;

    n is selected from 0, 1 or 2.
17. The compound of claim 16, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, which is a compound of formula (II):
    

    in,

    R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

    R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

    R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

    Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

    _R5 is selected from H, halogen, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

    _Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

    R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

    m is selected from 0, 1, 2 or 3;

    n is selected from 0, 1 or 2.
18. The compound of claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, having a structure of formula (III) or formula (IV):
    

    in,

    M ₁ and M ₂ are each independently selected from CH or N;

    R ₁ is selected from 5-12 membered heteroaryl or 5-12 membered heterocyclyl; said R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from H, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C1-6 alkylthio or _C3-6 cycloalkyl;

    R ₃ is independently selected from H, halogen, CN, C _1-6 alkyl, C _1-6 haloalkyl or C _3-6 cycloalkyl; or two R ₃ are connected to the carbon atom where they are located to form a 3-6 membered spiro ring or bridged ring;

    R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

    Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

    R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

    _Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

    R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

    m is selected from 0, 1, 2 or 3;

    n is selected from 0, 1 or 2.
19. The compound of claim 18, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

    in,

    R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from methyl, ethyl, trifluoroethyl, methylthio or cyclopropyl;

    R ₃ is independently selected from H, F, CN, methyl, ethyl, trifluoromethyl, cyclopropyl; or two R ₃ are connected to the carbon atom where they are located to form a cyclopropyl or cyclobutyl group;

    R ₄ is independently selected from H, F, Cl, Br, CN, SF ₅ , methyl, ethyl, trifluoromethyl, difluoromethyl, methylthio or cyclopropyl;

    Ring A is present or absent, and ring A is selected from a 5-6 membered heteroaryl group or a 5-7 membered heterocyclic group;

    R ₅ is selected from H, F, Cl, methyl, ethyl, trifluoromethyl, methoxy, methylthio or cyclopropyl;

    _Rx is selected from H, _C1-6 alkyl, C1-6 haloalkyl, _C1-6 alkoxy, CN, NH2, -C(O) _Ra , -C(O) _ORa , -( _CH2 ) _p - _ORa , _-P (O)-( _Ra ) ₂ or _-S (O) ₂ - _Ra ;

    R _a is selected from H, C _1-6 alkyl or C _3-6 cycloalkyl;

    m is selected from 0, 1, 2 or 3:

    n is selected from 0, 1 or 2.
20. The compound of claim 18, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, having the structure of formula (III-1):
    

    in,

    _M1 is selected from CH or N;

    R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from H, methyl, ethyl, trifluoromethyl, trifluoroethyl, methoxy, methylthio or cyclopropyl;

    R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

    Ring A is selected from 5-6 membered heteroaryl or Ring A is absent;

    R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

    _Rx is selected from H, methyl, trifluoromethyl, methoxy, CN, _NH2 , -C(O)Me, -C(O)OMe, -( _CH2 )-OH or -S(O) _2- Me;

    m is selected from 0, 1, 2 or 3.
21. The compound of claim 18, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, having the structure of formula (IV-1):
    

    in,

    R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from H, methyl, ethyl, trifluoromethyl, trifluoroethyl, methoxy, methylthio or cyclopropyl;

    R ₄ is independently selected from H, halogen, CN, SF ₅ , C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _3-6 cycloalkyl or 4-10 membered heterocyclyl;

    Ring A is selected from 5-6 membered heteroaryl or Ring A is absent;

    R ₅ is selected from H, halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 alkylthio or C _3-6 cycloalkyl;

    _Rx is selected from H, methyl, trifluoromethyl, methoxy, CN, _NH2 , -C(O)Me, -C(O)OMe, -( _CH2 )-OH or -S(O) _2- Me;

    m is selected from 0, 1, 2 or 3.
22. The compound of claim 21, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

    in,

    R ₁ is selected from The R ₁ may be substituted by 1, 2 or 3 R _x ;

    _R2 is selected from H, methyl, ethyl, trifluoromethyl, trifluoroethyl, methoxy, methylthio or cyclopropyl;

    R ₄ is independently selected from H, Cl, Br, CN, SF ₅ , methyl, ethyl, trifluoromethyl, difluoromethyl or cyclopropyl;

    Ring A is selected from or ring A is absent;

    R ₅ is selected from H, Cl, F, methyl, trifluoromethyl, methoxy, methylthio or cyclopropyl;

    _Rx is selected from H, methyl, trifluoromethyl, methoxy, CN, _NH2 , -C(O)Me, -C(O)OMe, -( _CH2 )-OH or -S(O) _2- Me;

    m is selected from 0, 1, 2 or 3.
23. Compound, or its tautomer, stereoisomer, wherein the compound is selected from:
    
    
    
24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 23, or a pharmaceutically acceptable salt, tautomer, stereoisomer or isotopic variant thereof, and a pharmaceutically acceptable excipient; preferably, it further comprises other therapeutic agents.
25. Use of a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt, enantiomer, diastereomer or isotopic variant thereof or a pharmaceutical composition according to claim 24 in the preparation of a medicament for treating and/or preventing a WRN-mediated disease.
26. The use of a compound or composition according to claim 25, wherein the WRN-mediated disease is cancer, and the cancer is selected from: colorectal cancer (e.g., colon cancer, rectal cancer, large intestine adenocarcinoma), gastric cancer (e.g., gastric adenocarcinoma) or lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma).