TW202542158A - Fused-ring compounds, pharmaceutical compositions containing them, and their uses - Google Patents

Abstract

This invention relates to compounds of formula (I), pharmaceutical compositions comprising the same, and their use in the prevention or treatment of diseases.

Description

Fused-ring compounds, pharmaceutical compositions containing them, and their uses

Field of Invention This invention relates to fused-ring compounds, pharmaceutical compositions comprising them, and their use in the prevention or treatment of diseases.

Background of the Invention Microsatellite instability (MSI) is a type of genomic damage caused by mismatch repair deficiency (dMMR). Despite progress in the treatment of microsatellite highly unstable (MSI-H) cancers, tumor evolution and drug resistance remain major causes of treatment failure and death in cancer patients. For example, approximately half of dMMR colorectal cancer patients treated with PD-1 and PD-L1 checkpoint inhibitors experience primary resistance. See, for example, Overman MJ et al., J Clin Oncol. 2018 Mar 10;36(8):773-779. For patients who are refractory to currently available treatments, there remains a clinical need for new treatment options.

Werner helicase (WRN) is considered a synthetic lethal target in MSI-H cancer (see, for example, Chan EM et al., Nature. 2019 Apr;568(7753):551-556). WRN is a member of the RecQ family of DNA helicases and plays an important role in maintaining genome stability, DNA repair, replication, transcription, and telomere maintenance. Studies on the WRN-dependent mechanism have found that dinucleotide TA repeats undergo large-scale amplification in MSI cells. These amplified TA repeats form secondary DNA structures that require WRN helicase for unwinding (see, for example, van Wietmarschen N et al., Nature. 2020 Oct;586(7828):292-298). In the absence of WRN (or with WRN helicase inhibition), the amplified TA repeat sequences in MSI cells are cleaved by nucleases, ultimately leading to chromosome fragmentation. Therefore, inhibiting WRN helicase is an effective strategy for treating cancers characterized by microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR), including colorectal cancer, gastric cancer, or endometrial cancer.

Summary of the Invention This application provides compounds for use as WRN inhibitors, which can be used for the prevention or treatment of cancers characterized by microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR). Furthermore, the compounds of the invention possess excellent physicochemical properties (e.g., solubility, physical and/or chemical stability), favorable pharmacokinetic properties (e.g., improved bioavailability, good metabolic stability, suitable half-life and duration of action), good safety (lower toxicity (e.g., reduced cardiotoxicity) and/or fewer side effects), and a lower likelihood of inducing drug resistance.

One aspect of the invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug thereof, wherein said compound has the structure of formula (I): (I) Wherein: --- indicates a single key or a double key, provided that the two double keys are not directly connected; ^W1 , ^W2 , ^W3 and ^W4 are each independently C or N, provided that C is connected to a double key; preferably, at least one of ^W1 and ^W2 is N, and/or at least one of ^W3 and ^W4 is N; Selected from , , and ^R1 , ^R3 , ^R21 , and ^R22 are each independently selected from H, deuterium, halogen, -OH, _-NH2 , -CN, _-NO2 , _-SF5 , = _CH2 , _C1-6 alkyl, deuterated _C1-6 alkyl, halogenated _C1-6 alkyl, _C2-6 alkenyl, _C2-6 ynyl, C3-6 cycloalkyl, 3-10 membered heterocyclic, _C6-10 aryl, _5-14 membered heteroaryl, _C6-12 aralkyl, -C(=O) ^Ra , -OC(=O)Ra, -C(=O) ^ORa , ^-ORa , ^-SRa , -S(=O) ^{Ra ,} -S(=O) _2Ra , -S( ₌ O) ^{2NR a} R ^b , -S(=O)(=NR ^a )R ^b , -NR ^a R ^b , -C(=O)NR ^a R ^b , -NR ^a -C(=O)R ^b , -NR ^a -C(=O)OR ^b , -NR ^a -S(=O) ₂ -R ^b , -NR ^a -C(=O)-NR ^a R ^b , -P(=O)R ^a R ^b , -C _1-6 alkylene-R ^a , -C _1-6 alkylene-OR ^a , -C _1-6 alkylene-NR ^a R ^b , -OC _1-6 alkylene-NR ^a R ^b , (-C _3-6 cycloalkylene)-CN and (-C _3-6 cycloalkylene)-C _1-6 alkyl; When m is greater than 1, two Rs located on the same ring atom or adjacent ring atoms ^3, together with any of its connected groups, constitutes a _C3-6 hydrocarbon ring, a 3-10 member heterocyclic ring, a _C6-10 aromatic ring, or a 5-14 member heterocyclic ring; ^R4 is... ^L2 is selected from -O-, -C(=O)-, -NRC(=O)-, -S-, -S(=O)-, -S(=O) _2- , _C1-6 alkylene, and -O-( _C1-6 alkylene)-; ^R41 is selected from _C3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; R, ^Ra , and ^Rb are each independently selected from H, _C1-6 alkyl, _C3-10 cycloalkyl, 3-10 heterocyclic, _C6-10 aryl, 5-14 heteroaryl, and _C6-12 aralkyl when they appear independently; ring B, ring X, and ring Z are each independently selected from C _3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; ring Y is absent or selected from _C3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; when ring Y is absent, ^R22 is also absent; the above-mentioned alkylene, alkyl, alkenyl, alkynylene, cycloalkylene, cycloalkylene, hydrocarbon, hydrocarbon, heterocyclic, heterocyclic, aryl, aromatic, heteroaryl, heteroaryl, and aralkyl groups are each optionally substituted with one or more substituents independently selected from the following: deuterium, halogen, -OH, =O, _-NH2 -CN, _-NO₂ , = _CH₂ , C₁ _-6 alkyl, deuterated C₁ _-6 alkyl, C₂ _-6 alkenyl, C₂ _-6 ynyl, C₃ _-6 cycloalkyl, 3-10 membered heterocyclic, C₆ _-10 aryl, 5-14 membered heteroaryl, C₆ _-12 aralkyl, -C(=O) ^Rc , -OC(=O) ^Rc , -C(=O) ^ORc , ^-ORc , ^-SRc , -S(=O) ^Rc , -S(=O) _₂Rc ^, -S(=O) _₂NRcRd , ^-NRcRd , ^-C (=O) ^{NRcRd , -NRc} - ^C (=O) ^Rd , -NRc- ^C (=O) ^ORd , -NRc- ^S (=O) _₂ ^- R ^d , -NRc- ^C (=O) ^-NRcRd , _-C1-6 alkylene- ^ORc , _-C1-6 alkylene- ^NRcRd and _-OC1-6 alkylene ^{- NRcRd} , wherein the alkylene, ^alkyl , alkenyl, = _CH2 , alkynyl, cycloalkane, heterocyclic, aryl, heteroaryl and aralkyl are ^each further optionally substituted by one or more substituents independently selected from the following: halogen, -OH, =O, -C(=O)O-tert-butyl, _-NH2 , -CN, _-NO2 , _C1-6 alkyl, _C1-6 halogenated alkyl, C3-6 cycloalkane, _3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, C _6-12 aralkyl, -C _1-6 alkylene-C _3-6 cycloalkyl, -OC _1-6 alkyl, and -C _1-6 alkylene-OC _1-6 alkyl; ^Rc and ^Rd are each independently selected from H, C _1-6 alkyl, C _3-10 cycloalkyl, 3-10 membered heterocyclic, C _6-10 aryl, 5-14 membered heteroaryl, and C _6-12 aralkyl, wherein the alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, and aralkyl groups are further optionally substituted by one or more substituents independently selected from the following: halogen, -OH, =O, -C(=O)O-tert-butyl, _-NH2 , -CN, _-NO2 , C _1-6 alkyl, C _1-6 halogenated alkyl, C _3-6 cyclic hydrocarbons, 3-10 member heterocyclic hydrocarbons, _C6-10 aryl hydrocarbons, 5-14 member heteroaryl hydrocarbons, _C6-12 aralkyl hydrocarbons and _-C1-6 alkylene- _OC1-6 alkyl hydrocarbons; and p, q and m are each independently an integer selected from 1, 2 or 3.

Another aspect of the invention provides a pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug, and one or more pharmaceutically acceptable carriers.

Another aspect of the invention provides the use of the compound of the invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug or pharmaceutical composition thereof in the preparation of a medicament used as a WRN inhibitor.

Another aspect of the invention provides compounds of the invention or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvents, metabolites, isotopically labeled compounds or prodrugs or pharmaceutical compositions of the invention, which are used as WRN inhibitors.

Another aspect of the invention provides a method for the prevention or treatment of cancer (preferably cancers characterized by microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR)), said method comprising administering to an individual in need an effective amount of the compound of the invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug or pharmaceutical composition of the invention. Detailed definition of the invention

Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technology herein refer to technology as commonly understood in the art, including variations or equivalent substitutions of technology that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are provided to better explain the invention.

The terms “including,” “contains,” “have,” “includes,” or “involves,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon, preferably a saturated divalent hydrocarbon having 1, 2, 3, 4, 5, or 6 carbon atoms, such as methylene, ethylene, propylene, or butylene.

As used herein, the term “alkyl” is defined as a straight-chain or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, for example, 1 to 6 carbon atoms. For example, as used herein, the term “ _C1-6 alkyl” refers to a linear or branched group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, dibutyl, tributyl, n-pentyl, or n-hexyl) of 1 to 6 carbon atoms, optionally substituted with one or more (e.g., 1 to ₃ ) suitable substituents such as halogens (in which case the group is referred to as “halogenated alkyl”) ₍ e.g., _CF3 , _C2F5 , _CHF2 , _CH2F , _CH2CF3 , _CH2Cl , or _{-CH2CH2CF3 , etc.} ). The term " _C1-4 alkyl" refers to a linear or branched aliphatic hydrocarbon chain with 1 to 4 carbon atoms (i.e., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, dibutyl, or tert-butyl).

As used herein, the term "alkenyl" refers to a linear or branched monovalent hydrocarbon containing one or more double bonds and having 2-6 carbon atoms (" _C2-6 alkenyl"). The alkenyl group is, for example, -CH= _CH2 , _-CH2CH = _CH2 , -C( _CH3 )= _CH2 , -CH2-CH=CH- _{CH3, 2 -} pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl. When the compounds of the present invention contain an alkenyl group, the compounds may exist in pure E (iso-sided), pure Z (iso-sided), or any mixture thereof. The term "alkenyl" refers to the corresponding divalent group, including, for example, " _C2-6 alkenyl", " _C2-4 alkenyl", etc. Specific examples include, but are not limited to: -CH=CH-, _-CH2CH =CH-, -C( _CH3 )=CH-, butenyl, pentenyl, hexenyl, etc.

As used herein, the term "alkynyl" refers to a monovalent hydrocarbon containing one or more triple bonds, preferably having 2, 3, 4, 5, or 6 carbon atoms, such as ethynyl, 2-propynyl, 2-butynyl, 1,3-butadiynyl, etc. The alkynyl group is optionally substituted by one or more (e.g., 1 to 3) identical or different substituents. The term "ethynyl" refers to the corresponding divalent group, including, for example, " _C2-8 ethynyl,"" _C2-6 ethynyl,"" _C2-4 ethynyl," etc. Examples include, but are not limited to, _[ …]. , , , The alkyne group is optionally substituted by one or more (such as 1 to 3) identical or different substituents.

As used in this article, the term "fused ring" or "dense ring" refers to a ring system formed by two or more ring structures sharing two adjacent atoms.

As used in this article, the term "spiral ring" refers to a ring system consisting of two or more ring structures that share a single ring atom.

As used in this article, the term “bridge ring” refers to a ring system formed by two or more ring structures sharing two non-directly connected atoms.

As used herein, the terms “cycloene hydrocarbon,” “cycloalkyl,” and “hydrocarbon” refer to a saturated (i.e., “cycloene alkyl” and “cycloalkyl”) or partially unsaturated (i.e., having one or more double and/or triple bonds within the ring) monocycle having, for example, 3 to 10 (suitably 3 to 8, more preferably 3 to 6) cyclic carbon atoms. It may contain multiple cyclic hydrocarbons (including spirocyclic, fused-ring, or bridged-ring systems), including but not limited to (-)cyclopropyl, (-)cyclobutyl, (-)cyclopentyl, (-)cyclohexyl, (-)cycloheptyl, (-)cyclooctyl, (-)cyclononyl, (-)cyclohexenyl, etc.

As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic (e.g., bicyclic) hydrocarbon (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spirocyclic, fused, or bridged systems (e.g., bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, bicyclic [3.2.1]octyl, or bicyclic [5.2.0]nonyl, decahydronaphthyl, etc.) optionally substituted with one or more (e.g., one to three) suitable substituents. The cycloalkyl group has 3 to 15 carbon atoms. For example, the term "C _"3-6 cycloalkyl" refers to a saturated monocyclic or polycyclic (e.g., bicyclic) hydrocarbon (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) with 3 to 6 cyclic carbon atoms, which is optionally substituted with one or more (e.g., 1 to 3) suitable substituents, such as methyl-substituted cyclopropyl.

As used herein, the term "heterocyclic group" (or "heterocycle") refers to a saturated or partially unsaturated monocyclic or bicyclic group having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms and one or more (e.g., one, two, three, or four) heteroatoms selected from O, S, N, and P, and the "heterocyclic group" (or "heterocycle") may contain -C (=O)- as a ring member. The heterocyclic group may be linked to the remainder of the molecule via the carbon atoms and/or heteroatoms (if present). In particular, 3-10 member heterocyclic groups are groups having 3-10 carbon atoms and heteroatoms in a ring, such as, but not limited to, ethylene oxide, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolinyl, pyrrolidyl, pyrrolidoneyl, imidazodinyl, pyrazolidyl, pyrrololinyl, tetrahydropyranyl, piperidinyl, succinyl, dithianyl, thiosuccinyl, piperyl, or trithianyl.

As used herein, the term “heterocyclic base” (or “heterocyclic”) encompasses a fused-ring structure, wherein the connection point between the fused-ring structure and other groups can be on any ring within the fused-ring structure. Therefore, the heterocyclic groups of the present invention also include, but are not limited to, heterocyclic and heterocyclic groups, heterocyclic and cycloalkyl groups, monocyclic and monocyclic groups, monocyclic and monocyclic groups, aryl and heterocyclic groups, heteroaryl and heterocyclic groups, such as 3-7 member (mono) heterocyclic and 3-7 member (mono) heterocyclic groups, 3-7 member (mono) heterocyclic and (mono) cycloalkyl groups, 3-7 member (mono) heterocyclic and _C4-6 (mono) cycloalkyl groups, ... _6-10 aryl 3-7 member heterocyclic, 5-6 member heteroaryl 3-7 member heterocyclic, examples of which include, but are not limited to, pyrrolidinyl cyclopropyl, cyclopentyl azidocyclopropyl, pyrrolidinyl cyclobutyl, pyrrolidinyl pyrrolidinyl, pyrrolidinyl piperidinyl, pyrrolidinyl piperidinyl, piperidinyl pyrolinyl, , or .

As used in this article, the term “heterocyclic basis” (or “heterocyclic”) encompasses both bridged heterocyclic basis (bridged heterocyclic) and spirocyclic basis (spirocyclic).

As used herein, the term "bridged heterocycle" refers to a ring structure consisting of two rings sharing two non-directly connected ring atoms, containing one or more heteroatoms (e.g., 1, 2, 3, or 4) (e.g., oxygen, nitrogen, and/or sulfur atoms), including but not limited to 7-10 ribbed bridged heterocycles, 8-10 ribbed bridged heterocycles, 7-10 ribbed nitrogen-containing bridged heterocycles, 7-10 ribbed oxygen-containing bridged heterocycles, 7-10 ribbed sulfur-containing bridged heterocycles, etc., for example... , , , , , , , , , , , , , , , , , , , , , The "nitrogen-containing bridged heterocycle", "oxygen-containing bridged heterocycle", and "sulfur-containing bridged heterocycle" may optionally also contain one or more other heteroatoms selected from oxygen, nitrogen, and sulfur.

As used herein, the term "spiroheterocycle" refers to a ring structure consisting of two or more rings sharing a single ring atom, containing one or more heteroatoms (e.g., oxygen, nitrogen, sulfur atoms), including but not limited to 5-10 membered spiroheterocycles, 6-10 membered spiroheterocycles, 6-10 membered nitrogen-containing spiroheterocycles, 6-10 membered oxygen-containing spiroheterocycles, 6-10 membered sulfur-containing spiroheterocycles, etc., for example... , , , , , , , , , , , , , , , , , , and The "nitrogen-containing spirocyclic", "oxygen-containing spirocyclic", and "sulfur-containing spirocyclic" may optionally also contain one or more other heteroatoms selected from oxygen, nitrogen, and sulfur. The term "6-10-member nitrogen-containing spirocyclic group" refers to a spirocyclic group containing a total of 6-10 ring atoms, of which at least one ring atom is a nitrogen atom.

As used herein, the terms “(aryl)aryl” and “aromatic ring” refer to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated π-electron system. For example, as used herein, the terms “C _6-10 (aryl)aryl” and “C _6-10 aromatic ring” mean an aromatic group containing 6 to 10 carbon atoms, such as (aryl)phenyl (phenyl ring) or (aryl)naphthyl (naphthyl ring). The (aryl)aryl and aromatic ring are optionally substituted with one or more (e.g., 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, _-NO₂ , C _1-6 alkyl, etc.).

The term "aralkyl" refers to an aryl-substituted alkyl group, wherein the aryl group and the alkyl group are as defined herein. Typically, the aryl group may have 6-14 carbon atoms, and the alkyl group may have 1-6 carbon atoms. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, phenylpropyl, and phenylbutyl.

As used herein, the terms “(sub)heteroaryl” and “heteroary ring” refer to monocyclic, bicyclic, or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, particularly 1, 2, 3, 4, 5, 6, 9, or 10 carbon atoms, and containing at least one heteroatom that may be the same or different (the heteroatom being, for example, oxygen, nitrogen, or sulfur), and additionally, in each case, may be benzene-fused. Specifically, "(hybrid)aryl" or "hybrid ring" is selected from (hybrid)thienyl(ring), (hybrid)furanyl(ring), (hybrid)pyrroleyl(ring), (hybrid)azolyl(ring), (hybrid)thiazolyl(ring), (hybrid)imidazolyl(ring), (hybrid)pyrazolyl(ring), (hybrid)isoazolyl(ring), (hybrid)isothiazolyl(ring). (Ring), (Pyridyl), (Triazolyl), (Thiadiazolyl), etc., and their benzo[derivatives]; or (Pyridyl), (Pyridyl), (Pyrimidinyl), (Pyridyl), (Pyridyl), (Tripyridyl), etc., and their benzo[derivatives].

As used in this article, the term "halogenated" or "halogenated" group is defined as including F, Cl, Br, or I.

As used herein, the term "alkylthio" refers to an alkyl group as defined above, which is attached to a portion of the parent molecule by a sulfur atom. Representative examples of _C1-6 alkylthio groups include, but are not limited to, methylthio, ethylthio, tert-butylthio, and hexylthio.

As used herein, the term "nitrogen-containing heterocycle" refers to a saturated or partially unsaturated monocyclic or bicyclic group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms and at least one nitrogen atom in the ring, and optionally also comprising one or more (e.g., one, two, three, or four) ring members selected from N, O, S, S=O, and S(=O) _₂ ; the nitrogen-containing heterocycle is connected to the remainder of the molecule through any one of the ring members. The nitrogen-containing heterocycle is preferably a saturated nitrogen-containing monocyclic ring. In particular, 3 to 14-membered nitrogen-containing heterocycles are groups having 3 to 14 carbon atoms and heteroatoms (at least one of which is a nitrogen atom) in the ring, including but not limited to three-membered nitrogen-containing heterocycles (such as aziridinyl), four-membered nitrogen-containing heterocycles (such as aziridinyl butyl), five-membered nitrogen-containing heterocycles (such as pyrroleyl, pyrrolidinyl (pyrrolidinyl ring), pyrrolinyl, pyrrolidinoneyl, imidazolyl, imidazodinyl, imidazolinyl, pyrazolyl, pyrazolyl), six-membered nitrogen-containing heterocycles (such as piperidinyl (piperidinyl ring), succinyl, thiosuccinyl, piperidinyl), and seven-membered nitrogen-containing heterocycles.

The term "substitution" refers to the selective replacement of one or more (e.g., one, two, three, or four) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and that the substitution forms a stable compound. Combinations of substituents and/or variations are permitted only if such combinations form a stable compound.

If a substituent is described as “optionally substituted,” then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more of the substituents in the list, then one or more hydrogen atoms on the carbon (to the extent that any hydrogen atoms are present) may be substituted individually and/or together by independently selected optional substituents. If the nitrogen of the substituent is described as being optionally substituted by one or more of the substituents in the list, then one or more hydrogen atoms on the nitrogen (to the extent that any hydrogen atoms are present) may each be substituted by independently selected optional substituents.

If a substituent is described as being "selected independently" from a group, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from the other substituent.

As used in this article, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, or ten.

Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable position of the substituent.

When a substituent bond is shown to be a bond that passes through two atoms in the ring, such a substituent can be bonded to any cyclic atom in the substituted ring.

This invention also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include (but are not limited to) isotopes of hydrogen (e.g., deuterium (D, ^2H ), tritium (T, ^3H )); isotopes of carbon (e.g., ^11C , ^13C , and ^14C ); isotopes of chlorine (e.g., ^36Cl ); isotopes of fluorine (e.g., ^18F ); isotopes of iodine (e.g., ^123I and ^125I ); isotopes of nitrogen (e.g., ^13N and ^15N ); isotopes of oxygen (e.g., ^15O , ^17O , and ^18O ); isotopes of phosphorus (e.g., ^32P ); and isotopes of sulfur (e.g., ^35S ). Certain isotope-labeled compounds of the present invention (e.g., those doped with radioactive isotopes) may be used in drug and/or recipient tissue distribution studies (e.g., analysis). Radioactive isotopes tritium (i.e., ^3H ) and carbon-14 (i.e., ^14C ) are particularly suitable for this purpose due to their ease of doping and detection. Substitution with positron-emitting isotopes (e.g., ^11C , ^18F , ^15O , and ^13N ) can be used to examine receptor occupancy in positron emission tomography (PET) studies. The isotopically labeled compounds of the invention can be prepared by using suitable isotopically labeled reagents instead of previously used unlabeled reagents, by methods similar to those described in the accompanying routes and/or embodiments and preparations. Pharmaceutically acceptable solvents of the invention include those in which the crystalline solvent can be isotopically substituted, for example, _D₂O , acetone- _d₆ , or DMSO- _d₆ .

The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, mixtures of diastereomers, and single diastereomers can occur. Individual molecules may also exist as geometric isomers (cis/trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and so on. It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

Solid lines can be used in this article. ), real wedge ( ) or virtual wedge ( Describe the carbon-carbon bonds of the compounds of the invention. Solid lines are used to depict bonds to asymmetrical carbon atoms, indicating all possible stereoisomers, including those at that carbon atom (e.g., specific enantiomers, racemic mixtures, etc.). Real or imaginary wedges are used to depict bonds to asymmetrical carbon atoms, indicating the presence of the shown stereoisomers. When present in racemic mixtures, real and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist in the form of stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

Rotation-restricted isomers are compounds that can be separated into rotation-restricted isomers.

It should also be understood that certain compounds of the present invention may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvents, metabolites, or prodrugs that, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention, their metabolites, or residues. Therefore, when referring to "compounds of the present invention" herein, it is also intended to encompass the aforementioned derivative forms of the compounds.

Pharmaceutically acceptable salts of the compounds of this invention include their acid addition salts and base addition salts.

For a summary of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts for the compounds of the present invention are known to those skilled in the art.

As used herein, the term "ester" means an ester derived from the various general formula compounds of this application, including physiologically hydrolyzable esters (compounds of the invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the invention may themselves be esters.

The compounds of the present invention may exist in the form of solvent compounds (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly such as water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, may be present in a stoichiometric or non-stoichiometric ratio.

The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body upon administration of the compounds of this invention. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amination, deamination, esterification, delipidation, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds prepared by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

Within its scope, this invention further includes prodrugs of the compounds of this invention, which are certain derivatives of the compounds of this invention that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted into the compounds of this invention having the desired activity through, for example, hydrolysis and cleavage. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the desired therapeutically active compounds. Further information on the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (E. B. Roche, ed., American Pharmaceutical Association). The prodrug of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compound of the present invention with certain portions known to those skilled in the art as “pro-moiety” (e.g., “Design of Prodrugs”, H. Bundgaard (Elsevier, 1985)).

This invention also covers compounds of the invention containing protecting groups. In any process of preparing compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and/or desirable, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in *Protective Groups in Organic Chemistry*, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

As used herein, the term "about" means within ±10%, preferably ±5%, and more preferably ±2% of the stated value. Compound

In some embodiments, this disclosure provides compounds or pharmaceutically acceptable salts, esters, stereoisomers, transconjugated isomers, tautomers, polymorphs, solvents, metabolites, isotopically labeled compounds or prodrugs thereof, wherein said compounds have the structure of formula (I): (I) Wherein: --- indicates a single key or a double key, provided that the two double keys are not directly connected; ^W1 , ^W2 , ^W3 and ^W4 are each independently C or N, provided that C is connected to a double key; preferably, at least one of ^W1 and ^W2 is N, and/or at least one of ^W3 and ^W4 is N; Selected from , , and ^R1 , ^R3 , ^R21 , and ^R22 are each independently selected from H, deuterium, halogen, -OH, _-NH2 , -CN, _-NO2 , _-SF5 , = _CH2 , _C1-6 alkyl, deuterated _C1-6 alkyl, halogenated _C1-6 alkyl, _C2-6 alkenyl, _C2-6 ynyl, C3-6 cycloalkyl, 3-10 membered heterocyclic, _C6-10 aryl, _5-14 membered heteroaryl, _C6-12 aralkyl, -C(=O) ^Ra , -OC(=O)Ra, -C(=O) ^ORa , ^-ORa , ^-SRa , -S(=O) ^{Ra ,} -S(=O) _2Ra , -S( ₌ O) ^{2NR a} R ^b , -S(=O)(=NR ^a )R ^b , -NR ^a R ^b , -C(=O)NR ^a R ^b , -NR ^a -C(=O)R ^b , -NR ^a -C(=O)OR ^b , -NR ^a -S(=O) ₂ -R ^b , -NR ^a -C(=O)-NR ^a R ^b , -P(=O)R ^a R ^b , -C _1-6 alkylene-R ^a , -C _1-6 alkylene-OR ^a , -C _1-6 alkylene-NR ^a R ^b , -OC _1-6 alkylene-NR ^a R ^b , (-C _3-6 cycloalkylene)-CN and (-C _3-6 cycloalkylene)-C _1-6 alkyl; When m is greater than 1, two Rs located on the same ring atom or adjacent ring atoms ^3, together with any of its connected groups, constitutes a _C3-6 hydrocarbon ring, a 3-10 member heterocyclic ring, a _C6-10 aromatic ring, or a 5-14 member heterocyclic ring; ^R4 is... ^L2 is selected from -O-, -C(=O)-, -NRC(=O)-, -S-, -S(=O)-, -S(=O) _2- , _C1-6 alkylene, and -O-( _C1-6 alkylene)-; ^R41 is selected from _C3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; R, ^Ra , and ^Rb are each independently selected from H, _C1-6 alkyl, _C3-10 cycloalkyl, 3-10 heterocyclic, _C6-10 aryl, 5-14 heteroaryl, and _C6-12 aralkyl when they appear independently; ring B, ring X, and ring Z are each independently selected from C _3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; ring Y is absent or selected from _C3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; when ring Y is absent, ^R22 is also absent; the above-mentioned alkylene, alkyl, alkenyl, alkynylene, cycloalkylene, cycloalkylene, hydrocarbon, hydrocarbon, heterocyclic, heterocyclic, aryl, aromatic, heteroaryl, heteroaryl, and aralkyl groups are each optionally substituted with one or more substituents independently selected from the following: deuterium, halogen, -OH, =O, _-NH2 -CN, _-NO₂ , = _CH₂ , C₁ _-6 alkyl, deuterated C₁ _-6 alkyl, C₂ _-6 alkenyl, C₂ _-6 ynyl, C₃ _-6 cycloalkyl, 3-10 membered heterocyclic, C₆ _-10 aryl, 5-14 membered heteroaryl, C₆ _-12 aralkyl, -C(=O) ^Rc , -OC(=O) ^Rc , -C(=O) ^ORc , ^-ORc , ^-SRc , -S(=O) ^Rc , -S(=O) _₂Rc ^, -S(=O) _₂NRcRd , ^-NRcRd , ^-C (=O) ^{NRcRd , -NRc} - ^C (=O) ^Rd , -NRc- ^C (=O) ^ORd , -NRc- ^S (=O) _₂ ^- R ^d , -NRc- ^C (=O) ^-NRcRd , _-C1-6 alkylene- ^ORc , _-C1-6 alkylene- ^NRcRd and _-OC1-6 alkylene ^{- NRcRd} , wherein the alkylene, ^alkyl , alkenyl, = _CH2 , alkynyl, cycloalkane, heterocyclic, aryl, heteroaryl and aralkyl are ^each further optionally substituted by one or more substituents independently selected from the following: halogen, -OH, =O, -C(=O)O-tert-butyl, _-NH2 , -CN, _-NO2 , _C1-6 alkyl, _C1-6 halogenated alkyl, C3-6 cycloalkane, _3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, C _6-12 aralkyl, -C _1-6 alkylene-C _3-6 cycloalkyl, -OC _1-6 alkyl, and -C _1-6 alkylene-OC _1-6 alkyl; ^Rc and ^Rd are each independently selected from H, C _1-6 alkyl, C _3-10 cycloalkyl, 3-10 membered heterocyclic, C _6-10 aryl, 5-14 membered heteroaryl, and C _6-12 aralkyl, wherein the alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, and aralkyl groups are further optionally substituted by one or more substituents independently selected from the following: halogen, -OH, =O, -C(=O)O-tert-butyl, _-NH2 , -CN, _-NO2 , C _1-6 alkyl, C _1-6 halogenated alkyl, C _3-6 cyclic hydrocarbons, 3-10 member heterocyclic hydrocarbons, _C6-10 aryl hydrocarbons, 5-14 member heteroaryl hydrocarbons, _C6-12 aralkyl hydrocarbons and _-C1-6 alkylene- _OC1-6 alkyl hydrocarbons; and p, q and m are each independently an integer selected from 1, 2 or 3.

In preferred embodiments, this disclosure provides compounds or pharmaceutically acceptable salts, esters, stereoisomers, transtransfer isomers, tautomers, polymorphs, solvents, metabolites, isotopically labeled compounds or prodrugs thereof, wherein said compounds have the structure of the following formula: (II) (III) (IV) (V) (VI) (VII) (VIII) (IX) (X) (XI).

In some implementation schemes, ring B is a C _3-6 hydrocarbon ring or a 3-10 hybrid ring.

In the preferred embodiment, ring B is a cyclopentene ring, a cyclohexene ring, a pyrrolidine ring, an oxazolidinyl ring, a piperidine ring, a cycloporin ring, or a nitrogen-containing cycloheptane ring.

In some embodiments, ^R1 is independently selected from H, halogen, _C1-6 alkyl, _C3-6 cycloalkyl, 3-10 membered heterocyclic, C6-10 aryl, 5-14 membered heteroaryl, -S(=O) _2Ra , ^{-ORa ,} and ^{-NRaRb , where Ra and Rb are independently} selected from H, _C1-6 alkyl, _C3-10 cycloalkyl, _3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, and _C6-12 aralkyl each time they appear; preferably, ^R1 is independently selected from 3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, and ^-NRaRb each time they appear ^. The alkyl, cycloalkane, heterocyclic, aryl, and heteroaryl groups are each optionally substituted by one or more substituents independently selected from the following: halogen, -S(=O) _2Rc , _C1-6 alkyl, _C2-6 alkenyl, = ^CH2 , _C3-6 cycloalkane, 3-10 membered heterocyclic, _C6-10 aryl _, and 5-14 membered heteroaryl; the alkyl, alkenyl, = _CH2 , cycloalkane, heterocyclic, aryl, and heteroaryl groups are further optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl, _C3-6 cycloalkane, 3-10 membered heterocyclic, and _-C1-6 alkylene-C _3-6 cyclic carboxylic acid groups.

In some embodiments, ^R1 is independently selected from H, halogen, _C1-6 alkyl, _C3-6 cycloalkyl, 3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, ^{-ORa ,} and ^-NRaRb each time it appears; preferably, ^R1 is independently selected from 3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl ^, and ^-NRaRb each time it appears; wherein the alkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are each optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl, _C2-6 alkenyl, = _CH2 , C _3-6 cycloalkane, 3-10 member heterocyclic, _C6-10 aryl, and 5-14 member heteroaryl; the alkyl, alkenyl, = _CH2 , cycloalkane, heterocyclic, aryl, and heteroaryl groups are each further optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl, _C3-6 cycloalkane, 3-10 member heterocyclic, and _-C1-6 alkylene- _C3-6 cycloalkane.

In some implementations, ^R1 is independently _C6-10 aryl, -NR ^{a Rb} each time it appears.

In some implementations, ^R1 is independently -NR ^a R ^b each time it occurs.

In the preferred embodiment, ^R1 is H, methyl, halogen, methoxy, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

In the preferred embodiment, ^R1 is H, methyl, halogen, methoxy, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or The preferred option is , , , , , or .

In the preferred embodiment, ^R1 is H, methyl, halogen, methoxy, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or The preferred option is , , , , , or .

In some implementation schemes, ^R1 is , , or .

In some implementation schemes, ring X is a benzene ring, a 5-6 member heterocyclic ring, or a 5-6 member heteroaromatic ring, and ring Y is absent.

In some implementation schemes, ring X is a benzene ring and ring Y is a _C3-6 hydrocarbon ring, a benzene ring, a 5-6 member heterocyclic ring, or a 5-6 member heteroaromatic ring.

In some implementation schemes, for , , , , , , , , , , , , , , The preferred option is .

In some implementation schemes, for , , , , , , , , The preferred option is .

In some embodiments, R ²¹ and R ²² are each independently selected from H, halogen, -SF ₅ , _C1-6 alkyl, _C3-6 cycloalkyl, 3-10 member heterocyclic, -O-( _C1-6 alkyl), -S(=O) _2- ( _C1-6 alkyl), -S(=O) _2- ( _C3-6 cycloalkyl), -S(=O)(=NR ^a )R ^b , -P(=O)( _C1-6 alkyl) ₂ , ( _-C3-6 cycloidene)-CN and ( _-C3-6 cycloidene)-C _1-6 alkyl, wherein the alkyl, cyclohexylene, cyclohexyl and heterocyclic groups are each optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl and halogenated _C1-6 alkyl.

In a preferred embodiment, R ²¹ and R ²² are each independently selected from H, halogen, _-SF5 , _C1-6 alkyl, _C3-6 cycloalkyl, 3-10 member heterocyclic and -O-( _C1-6 alkyl), wherein each alkyl, cycloalkyl and heterocyclic is optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl and halogenated _C1-6 alkyl.

In some implementation schemes, Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

In some implementation schemes, Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

In some embodiments, ^R3 is H, _C1-6 alkyl, ^-ORa or ^-SRa ; preferably, ^R3 is H or _C1-6 alkyl.

In preferred embodiments, ^R3 is H, methyl, ethyl, -O- _CH3 or -S- _CH3 ; most preferably, ^R3 is H or methyl.

In a preferred embodiment, when m is greater than 1, two ^R3s located on the same ring atom or adjacent ring atoms, together with the groups they are attached to, optionally constitute a _C3-6 hydrocarbon ring (preferably a cyclopropyl ring), wherein the hydrocarbon ring is optionally substituted by one or more substituents independently selected from the following: halogens, _C1-6 alkyl groups, and halogenated _C1-6 alkyl groups.

In some implementation schemes, ring Z is a 3-10 member heterocyclic ring or a benzene ring; preferably a 5-10 member heterocyclic ring; more preferably a 5-6 member heterocyclic ring; and...

The heterocyclic ring and the benzene ring are each optionally substituted with one or more substituents independently selected from the following: halogen, _C1-6 alkyl and halogenated _C1-6 alkyl, each time they appear.

In some implementation schemes, circumference Z is... , , , , , , , , , , , , , , , , , , , or .

In some embodiments, ^L2 is -C(=O)-, _C1-6 alkylene, or -NRC(=O)-, where R is H or _C1-6 alkyl.

In some embodiments, ^L2 is -C(=O)- or -NRC(=O)-, where R is H or _C1-6 alkyl.

In the preferred implementation, ^L2 is -C(=O)-, _-CH2- , or _-CD2- .

In the preferred implementation, ^L2 is -C(=O)-.

In some embodiments, R ⁴¹ is selected from 3-10 member heterocyclic rings, C _6-10 aromatic rings, and 5-14 member heterocyclic rings, each of which is optionally substituted by one or more substituents independently selected from the following: halogen, -OH, C _1-6 alkyl, -OC _1-6 alkyl, and -SC _1-6 alkyl. Preferably, the heterocyclic ring, aromatic ring, and heterocyclic ring are substituted by at least -OH or -OC _1-6 alkyl.

In some implementation schemes, -L ² -R ⁴¹ is... , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

In some implementation schemes, -L ² -R ⁴¹ is... , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

In some implementation schemes, -L ² -R ⁴¹ is... , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

In some implementations, R ⁴¹ is a 5-6 member heterocyclic ring, preferably a 6-membered heterocyclic ring, and more preferably a pyridine or pyrimidine ring, which is substituted with at least one -OH group.

In some implementation schemes, -L ² -R ⁴¹ is... , or .

This disclosure covers technical solutions/compounds obtained by arbitrarily combining various embodiments.

In preferred embodiments, this disclosure provides compounds or pharmaceutically acceptable salts, esters, stereoisomers, transisomers, tautomers, polymorphs, solvents, metabolites, isotopically labeled compounds or prodrugs thereof, wherein said compounds are selected from: Compound number Structure Compound number Structure C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35 C36 C37 C38 C39 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 C51 C52 C53 C54 C55 C56 C57 C58 C59 C60 C61 C62 C63 C64 C65 C66 C67 C68 C69 C70 C71 C72 C73 C74 C75 C76 C77 C78 C79 C80 C81 C82 C83 C84 C85 C86 C87 C88 C89 C90 C91 C92 C93 C94 C95 C96 C97 C98 C99 C100 C101 C102 C103 C104 C105 C106 C107 C108 C109 C110 C111 C112 C113 C114 C115 C116 C117 C118 C119 C120 C121 C122 C123 C124 C125 C126 C127 C128 C129 C130 C131 C132 C133 C134 C135 C136 C137 C138 C139 C140 C141 C142 C143 C144 C145 C146 C147 C148 C149 C150 . Drug combinations and treatments

In some embodiments, the present invention provides a pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug, and one or more pharmaceutically acceptable carriers. The pharmaceutical composition is preferably a solid formulation, semi-solid formulation, liquid formulation, or gaseous formulation. In some embodiments, the pharmaceutical composition may also comprise one or more other therapeutic agents.

In some embodiments, the present invention provides the use of the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug or pharmaceutical composition thereof in the preparation of a medicament used as a WRN inhibitor.

In some embodiments, the present invention provides the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug or pharmaceutical composition thereof, which is used as a WRN inhibitor.

In some embodiments, the present invention provides a method for the prevention or treatment of cancer (preferably cancer characterized by microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR)), said method comprising administering to an individual in need an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug or pharmaceutical composition of the present invention.

In some embodiments, the cancers include colorectal cancer, stomach cancer, endometrial cancer, uterine cancer, adrenocortical carcinoma, cervical cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, and ovarian cancer.

In this invention, "pharmaceuticalally acceptable carrier" means a diluent, excipient, adjuvant, or mediator administered with a treatment, which, to a reasonable medical judgment, is suitable for contact with human and/or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit/risk ratio.

Unless otherwise stated, as used herein, the term “treatment” means to reverse, alleviate, or inhibit the progression of a disease or condition to which the term applies, or one or more symptoms of such a disease or condition, or to prevent such a disease or condition, or one or more symptoms of such a disease or condition.

As used herein, “person” includes humans or non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. In this invention, “non-human animals” includes all vertebrates, such as non-mammalians (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

In another embodiment, the pharmaceutical composition of the invention may also comprise one or more additional therapeutic or preventative agents. General synthetic route: Route 1 Route 2 In Route 1 and Route 2, PG is the protection group, m is 1 or 2, and the other groups are defined as in this paper.

Detailed Description of Preferred Embodiments The invention is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

The abbreviations used in this invention have the following meanings: abbreviation Meaning ACN Acetonitrile AgBF ₄ Silver tetrafluoroborate Boc Tertiary butoxycarbonyl CMPI 2-Chloro-1-methylpyridinium iodide DCE 1,2-Dichloroethane DCM dichloromethane DIAD diisopropyl azodicarboxylate DIEA/DIPEA N,N-Diisopropylethylamine Diox/dioxane 1,4-Dioxane DMF N,N-Dimethylformamide DMSO dimethyl monoxide DPPA diphenyl phosphate EDCI 1-Ethyl-(3-dimethylaminopropyl)carbodiimide Et Ethyl Et ₃ N/NEt ₃ Triethylamine EtOAc/EA Ethyl acetate EtOH ethanol FA Formic acid h/hrs hour HCl hydrochloric acid Hex hexane HOAt N-Hydro-7-azabenzotriazole HOBT 1-Hydrobenzotriazole HPLC High performance liquid chromatography _{K₂CO₃ ​} Potassium carbonate LCMS Liquid chromatography-mass spectrometry LDA Diisopropylaminolithium MeOH methanol min minute NaOH Sodium hydroxide NBS N-bromosuccinimide NCS N-chlorosuccinimide n-BuLi n-Butyl lithium ON overnight PE petroleum ether p-TsOH p-Toluenesulfonic acid Pd(dppf)Cl ₂ 1,1-Bis(diphenylphosphine)ferrocene palladium dichloride t-BuOH Tertiary butanol TEA Triethylamine TEMPO 2,2,6,6-Tetramethylpiperidine oxide TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin-layer chromatography rt/rt room temperature Implementation Example 1 : (C87-P1-A , C87-P1-B , C87-P2-A , C87-P2-B) 1) Synthesis of intermediate C87-2 :

Compound C87-1 (18.46 g, 128.2 mmol) was added to tetrahydrofuran (600 mL), cooled to 0°C, and sodium hydroxide (5.12 g, 128.2 mmol) was slowly added. The mixture was purged with nitrogen three times and stirred at 0°C for half an hour. Then, n-butyllithium solution (80 mL, 128.2 mmol) was slowly added dropwise, and the mixture was stirred at 0°C for half an hour under nitrogen protection. Next, ethyl propionate (25 g, 128.2 mmol) was added to the reaction solution, and the mixture was stirred at 0°C for half an hour before being slowly heated to room temperature and reacted overnight. TLC showed that the reactants were basically completely reacted. The reaction solution was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, and the organic phase was washed with saturated salt water and dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (PE/EA=90/10) to obtain a yellow oily substance C87-2 (11.96 g, yield 36.2%). ^¹H NMR (400 MHz, _CDCl₃ ): δ 4.21–4.10 (m, 2H), 4.05–3.98 (m, 2H), 3.77 (s, 2H), 3.71–3.65 (m, 1H), 2.83–2.76 (m, 1H), 1.94–1.85 (m, 1H), 1.60–1.51 (m, 1H), 1.35 (s, 3H), 1.28 (m, 3H), 1.23 (t, J = 9.6 Hz, 3H), 1.11 (d, J = 9.6 Hz, 3H). 2) Synthesis of intermediate C₈₇₄ :

Compounds C87-2 (11.96 g, 46.3 mmol), C87-3 (7.4 g, 46.3 mmol), and anhydrous p-toluenesulfonic acid (0.80 g, 4.63 mmol) were added to n-butanol (15 mL). The reaction solution was stirred at 140 °C for 16 hours under nitrogen protection. LC-MS showed that the starting material reacted completely. After cooling, the reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (DCM/MeOH = 85/15) to give a white solid C87-4 (10.1 g, yield 69.6%). LCMS (ESI) m/z: 315.0 [M+H] ⁺ . 3) Synthesis of intermediate C87-5 :

Compound C87-4 (10.1 g, 32.1 mmol) and triphenylphosphine (12.6 g, 48.2 mmol) were added to tetrahydrofuran (300 mL), cooled to 0 °C, and then DIAD (9.57 g, 48.2 mmol) was slowly added dropwise. The mixture was then slowly heated to room temperature under a nitrogen atmosphere and stirred overnight. LC-MS showed that the reactants reacted completely. The reaction solution was cooled and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 85/15) to give a white solid C87-5 (7.46 g, yield 78.3%). ^¹H NMR (400 MHz, DMSO- _d₆ ): δ 8.14–8.12 (m, 2H), 7.55–7.52 (m, 3H), 5.95–5.95 (m, 1H), 5.03–5.98 (m, 1H), 4.76–4.74 (m, 1H), 4.49–4.10 (m, 1H), 3.72–3.67 (m, 1H), 3.54–3.36 (m, 1H), 2.62–2.45 (m, 1H), 2.11–1.90 (m, 1H), 1.39–1.31 (m, 3H). 4) Synthesis of intermediate C₈₇-6 :

TEMPO (0.83 g, 5.3 mmol) was added to an aqueous solution _{of NaH₂PO₄} (200 mL, 133.1 mmol, 0.67 M), followed by a suspension of compound C87-5 (7.88 g, 26.6 mmol) in acetonitrile (50 mL). Finally, an aqueous solution of _NaClO₂ (4.79 g, 53.2 mmol) and NaClO solution (0.8 mL) (40 mL) was added. The mixture was purged three times with nitrogen, then heated to 50 °C and stirred overnight under nitrogen atmosphere. LC-MS showed complete reaction of the starting material. The reaction solution was cooled to room temperature and purified by reversed-phase column chromatography ( _H₂O /ACN = 85/15) to obtain a white solid C87-6 (5.0 g, yield 61.1%). ^¹H NMR (400 MHz, DMSO- _d⁶ ): δ 8.15–8.12 (m, 2H), 7.58–7.53 (m, 3H), 7.31–7.05 (m, 2H), 5.97 (s, 1H), 4.84–4.80 (m, 1H), 2.83–2.73 (m, 1H), 2.02–1.97 (m, 1H), 1.34 (d, J = 9.6 Hz, 3H). 5) Synthesis of intermediate C₈₇₈ :

Compound C87-6 (500 mg, 1.61 mmol) and two drops of DMF were added to DCE (15 mL), followed by the slow addition of _POCl3 (500 mg, 3.22 mmol). The mixture was purged with nitrogen three times, heated to 80 °C, and stirred under nitrogen atmosphere for 1 hour. LC-MS showed that the starting material had reacted completely. The reaction solution was cooled to room temperature, and then pyridine (2 mmol) of compound C87-7 (1.58 g, 8.06 mmol) was slowly added. The solution was heated to 80°C and stirred for 1 hour under a nitrogen atmosphere. LC-MS showed that the reaction of the starting material was complete. The reaction solution was cooled to room temperature, washed with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated salt water, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE/EA=50/50) to give a yellow solid C87-8 (340 mg, yield 43.3%). ^¹H NMR (400 MHz, DMSO- _d⁶ ): δ 10.60 (s, ¹H), 8.23–8.10 (m, 2H), 8.04–8.02 (m, 2H), 7.77–7.75 (m, ¹H), 7.61–7.54 (m, 4H), 5.70 (d, J = 10.8 Hz, ¹H), 3.56–3.45 (m, ¹H), 2.78–2.71 (m, 1H), 2.48–2.41 (m, 1H), 1.41 (d, J = 9.2 Hz, 3H). 6) Synthesis of intermediate C87-9 :

Compound C87-8 (340 mg, 0.70 mmol) was added to chloroform (15 mL), and NBS (248 mg, 1.39 mmol) was slowly added at room temperature. The mixture was purged with nitrogen three times. The reaction solution was heated to 50 °C and stirred overnight under nitrogen atmosphere. LC-MS showed complete reaction of the starting material. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was purified by column chromatography (PE/EA = 65/35) to give a yellow solid C87-9 (360 mg, yield 74.7%). LCMS (ESI) m/z: 566.0 [M+H] ⁺ . 7) Synthesis of intermediate C87-10 :

Compound C87-9 (218 mg, 0.38 mmol), piperazine (664 mg, 7.7 mmol), and _AgBF4 (150 mg, 0.77 mmol) were added to DMSO (5 mL). The mixture was purged with nitrogen three times. The reaction solution was heated to 120 °C and stirred under nitrogen atmosphere for 2 hours. LC-MS showed that the starting material was completely reacted. The reaction solution was cooled to room temperature, washed with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine and dried with anhydrous sodium sulfate. The solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 85/15, 0.5% _NH3 · _H2O ) to give a yellow solid C87-10 (123 mg, yield 55.9%). LCMS (ESI) m/z: 572.2 [M+H] ⁺ . 8) Synthesis of C87-P1 and C87-P2 :

Compound C87-11 (24 mg, 0.15 mmol) was added to DMF (2 mL). HOBT (17 mg, 0.12 mmol) and EDCI (32 mg, 0.16 mmol) were added at room temperature, and the mixture was stirred at room temperature for 2 hours under nitrogen protection. Then, compound C87-10 (63 mg, 0.11 mmol) and DIEA (43 mg, 0.32 mmol) were added. The mixture was replaced with nitrogen three times, and stirred at room temperature for 2 hours under nitrogen protection. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (RP-PREP-3 SunFire C18 5um 19*150mm 18min-55-65B, A: _H₂O (0.2% FA), B: ACN, UV: 214). C87-P1 (8.0 mg, yield 10.2%, retention time 10.79 min) and C87-P2 (10.2 mg, yield 13.0%, retention time 11.59 min) were obtained by filtration at a flow rate of 15 ml/min (nm).

C87- P1 ¹ H NMR (400 MHz, DMSO- d ₆ ): δ 10.56 (s, 1 H), 8.55 (s, 1 H), 8.08-8.06 (m, 2 H), 7.98-7.96 (m, 2 H), 7.74-7.72 (m, 1 H), 7.51-7.50 (m, 3 H), 5.71-5.67 (m, 1 H), 3.78-3.73 (m, 1 H), 3.38-3.10 (m, 9 H), 2.65-2.62 (m, 1 H), 2.44-2.41 (m, 4 H), 1.52 (d, J =7.2 Hz, 3 H). LCMS (ESI) m/z: 707.9 [M+H] ⁺ .

C87- P2 ¹ H NMR (400 MHz, DMSO- d ₆ ): δ 10.56 (s, 1 H), 8.55 (s, 1 H), 8.10-8.07 (m, 2 H), 7.98-7.94 (m, 2 H), 7.75-7.73 (m, 1 H), 7.52-7.50 (m, 3 H), 5.62-5.59 (m, 1 H), 3.77-3.73 (m, 1 H), 3.34-3.10 (m, 9 H), 3.07-2.99 (m, 1 H), 2.44 (s, 3 H), 2.15 (d, J =13.6 Hz, 1 H), 1.44 (d, J =7.2 Hz, 3 H). LCMS (ESI) m/z: 707.9 [M+H] ⁺ . 9) Synthesis of C87-P1-A , C87-P1-B , C87-P2-A , and C87-P2-B :

Compound C87- P1 (8.0 mg) was chirally resolved (IBN, ACN:IPA:TFA=90:10:0.3, 25 ml/min, 254 nm) to give C87-P1 -A (2.8 mg, yield 35.0%, retention time 6.98 min) and C87-P1 -B (2.6 mg, yield 32.5%, retention time 12.46 min).

C87-P1 -A ¹ H NMR (400 MHz, CD ₃ OD): δ 8.75 (s, 1 H), 8.15 (s, 2 H), 8.08 (d, J =7.6 Hz, 1 H), 7.82 (s, 1 H), 7.60 (d, J =7.2 Hz, 1 H), 7.46 (s, 3 H), 5.70 (s, 1 H), 3.87 (s, 1 H), 3.87-3.31 (m, 4 H), 2.78 (s, 1 H), 2.78-2.61 (m, 3 H), 2.52 (s, 1 H), 1.99-1.65 (m, 4 H), 1.28 (s, 3 H). LCMS (ESI) m/z: 707.4 [M+H] ⁺ .

C87-P1 -B ¹ H NMR (400 MHz, CD ₃ OD): δ 8.74 (s, 1 H), 8.14 (d, J =7.2 Hz, 2 H), 8.08 (d, J =8.4 Hz, 1 H), 7.82 (s, 1 H), 7.60 (d, J =8.0 Hz, 1 H), 7.46-7.44 (m, 3 H), 5.71-5.69 (m, 1 H), 3.86 (s, 1 H), 3.85-3.31 (m, 4 H), 2.77 (s, 1 H), 2.76-2.60 (m, 3 H), 2.52 (s, 1 H), 1.99-1.65 (m, 4 H), 1.29 (d, J =8.4 Hz, 3 H). LCMS (ESI) m/z: 707.4 [M+H] ⁺ .

Compound C87-P2 (10.2 mg) was chirally resolved (IBN, ACN:IPA:TFA=70:30:0.3, 25 ml/min, 254 nm) to give C87-P2-A (3.4 mg, yield 33.3%, retention time 6.02 min) and C87-P2-B (3.2 mg, yield 31.3%, retention time 12.52 min).

C87-P2-A ¹ H NMR (400 MHz, CD ₃ OD): δ 8.57 (s, 1 H), 8.17-8.16 (m, 2 H), 8.08 (d, J =8.4 Hz, 1 H), 7.82 (s, 1 H), 7.62 (d, J =8.4 Hz, 1 H), 7.47-7.46 (m, 3 H), 5.63 (d, J =8.8 Hz, 1 H), 3.87-3.84 (m, 1 H), 3.51-3.30 (m, 8 H), 3.13-3.08 (m, 1 H), 2.52 (s, 3 H), 2.32 (d, J =13.6 Hz, 1 H), 1.55 (d, J =7.2 Hz, 3 H). LCMS (ESI) m/z: 707.5 [M+H] ⁺ .

C87-P2-B ¹ H NMR (400 MHz, CD ₃ OD): δ 8.40 (s, 1 H), 8.08-8.06 (m, 2 H), 7.98 (d, J =8.4 Hz, 1 H), 7.73-7.72 (m, 1 H), 7.54-7.52 (m, 1 H), 7.37-7.35 (m, 3 H), 5.55-5.51 (m, 1 H), 3.78-3.74 (m, 1 H), 3.38-3.20 (m, 8 H), 3.06-2.98 (m, 1 H), 2.41 (s, 3 H), 2.24-2.20 (m, 1 H), 1.46 (d, J =7.6 Hz, 3 H). LCMS (ESI) m/z: 707.5 [M+H] ⁺ . Implementation Example 2 : (C88-P1 , C88-P2 , C88-P3) 1) Synthesis of intermediate C88-2 :

Compound C87-9 (35 mg, 0.062 mmol), compound C88-1 (104.07 mg, 0.93 mmol), and silver tetrafluoroborate (24.18 mg, 0.124 mmol) were added to dimethyl sulfoxide (4 mL). The reaction solution was heated to 120 °C and reacted for 2 hours. LC-MS showed that the reaction of the starting materials was complete. The reaction was quenched dropwise with aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated salt water, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 10/1) to give a grayish-white solid compound C88-2 (15 mg, yield 40%). LCMS (ESI) m/z = 598.1 [M+H] ⁺ . 2) Synthesis of C88-P1 , C88-P2 , and C88-P3 :

Compound C87-11 (8.78 mg, 0.06 mmol) was added to DMF (2 mL). HOBT (5.94 mg, 0.044 mmol) and EDCI (11.52 mg, 0.06 mmol) were added at room temperature, and the mixture was stirred at room temperature for 2 hours under nitrogen protection. Then, compound C88-2 (25 mg, 0.04 mmol) and DIEA (15.48 mg, 0.12 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for another 2 hours. LC-MS showed complete reaction of the starting materials. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters-PREP-8 SunFire C18 5µm 19*150mm). The reaction was carried out at 18 min - 60 - 75 B, A: _H₂O (0.2% FA), B: ACN, UV: 214 nm, flow rate 15 ml/min to give C88-P1 (1.3 mg, yield 4.2%, retention time 9.57 min), C88-P2 (3.0 mg, yield 9.8%, retention time 10.65 min), and C88-P3 (4.2 mg, yield 13.7%, retention time 11.27 min). C88-P1 and C88-P2 were both monomeric compounds, while C88-P3 was a mixture of two diastereomers.

C88-P1 ( confirmed configuration by crystal structure, C88-P1 is compound C10): LCMS (ESI) m/z = 734.2 [M+H] ⁺ . ^1H NMR (400 MHz, _CD3 OD): δ 8.51 (s, 1H), 8.17-8.14 (m, 2H), 8.10 (d, J =8.4 Hz, 1H), 7.83 (s, 1H), 7.62 (d, J =9.2 Hz, 1H), 7.47-7.45 (m, 3H), 5.69-5.66 (m, 1H), 4.58-4.56 (m, 1H), 3.86-3.51 (m, 6H), 2.79-2.76 (m, 1H), 2.50 (s, 3H), 2.18-2.17 (m, 1 H), 1.62-1.59 (m, 7 H).

C88-P2: LCMS (ESI) m/z = 734.2 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD): δ 8.49 (s, 1H), 8.17-8.15 (m, 2H), 8.06 (d, J =8.8 Hz, 1H), 7.82 (s, 1H), 7.62 (d, J =8.8 Hz, 1H), 7.47-7.43 (m, 3H), 5.59-5.56 (m, 1H), 4.74-4.60 (m, 1H), 3.96-3.59 (m, 6H), 3.13-3.05 (m, 1H), 2.49 (s, 3H), 2.34-2.30 (m, 1 H), 2.21-2.17 (m, 1 H), 1.56-1.53 (m, 6 H).

C88-P3: LCMS (ESI) m/z=734.1 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD): δ 8.53 (s, 1H), 8.18-8.14 (m, 2H), 8.10-8.07 (m, 1H), 7.82 (s, 1H), 7.64-7.59 (m, 1H), 7.47-7.44 (m, 3H), 4.76-4.60 (m, 1H), 5.70-5.60 (m, 1H), 3.87-3.52 (m, 6H), 3.23-3.10 (m, 0.5H), 2.78-2.67 (m, 0.5H), 2.47 (s, 3H), 2.28-2.20 (m, 1H), 1.71-1.59 (m, 7H). Example 3 : (C89-P1 , C89-P2 , C89-P3) 1) Synthesis of intermediate C89-2 :

C89-1 (9.0 g, 44.53 mmol) and anhydrous tetrahydrofuran (225 mL) were added sequentially to a 1000 mL three-necked flask. The mixture was stirred at -70 to -75 ^° C under nitrogen protection. A solution of n-butyllithium in n-hexane (2.5 mol/L, 48 mL, 120 mmol) was slowly added. The mixture was stirred at -70 to ^{-75 °} C for 20 minutes. The temperature was then slowly raised to -5 to 0 ^° C and stirred for 70 minutes. The temperature was lowered to -70 to -75 ^{° C} . Hexachloroethane (31.6 g, 133.6 mmol) was added at -70 to -75 ^° C and stirred for 40 minutes. The mixture was then slowly restored to room temperature and stirred for another 40 minutes. The reaction was quenched with 10% w/w citric acid aqueous solution, extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to give C89-2 (3.5 g, 33%). 2) Synthesis of intermediate C89-3 :

Diphenyl azidophosphate (4.08 g, 14.84 mmol) was added to an anhydrous terbutanol (30 mL) solution of C89-2 (3 g, 12.68 mmol). After the addition was complete, N,N-diisopropylethylamine (1.92 g, 19.02 mmol) was added to the reaction solution. The reaction solution was heated to 80 °C and stirred overnight. The reaction solution was concentrated under reduced pressure, and the residue was extracted with saturated sodium bicarbonate solution and ethyl acetate (3 × 50 mL). The organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to give a white solid compound C89-3 (2.8 g, 71%). 3) Synthesis of intermediate C89-4 :

C89-3 (450 mg, 1.46 mmol) was dissolved in dichloromethane (3 mL) solution, and trifluoroacetic acid (1 mL) was added. The reaction solution was stirred at 25 °C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to give a brown oily substance C89-4 (300 mg, mixture, yield: 98.8%). LCMS (ESI) m/z = 208.1 [M+H] ⁺ . 4) Synthesis of intermediate C89-5 :

Compound C87-6 (240 mg, 0.77 mmol) was added to dichloromethane (10 mL), and CMPI (217.16 mg, 0.85 mmol), DIEA (299.61 mg, 2.32 mmol), and C89-4 (281.82 mg, 1.16 mmol) were added at 0 °C. The reaction solution was heated to 50 °C and reacted for 2 hours. LC-MS showed that the reactants were completely reacted. The reaction was quenched by slow addition of aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated salt water, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 100/1) to give a yellow solid compound C89-5 (180 mg, yield 47%). LCMS (ESI) m/z = 500.1 [M+H] ⁺ . 5) Synthesis of intermediate C89-6 :

C89-5 (180 mg, 0.36 mmol) was added to dichloromethane (15 mL), and NBS (128.16 mg, 0.72 mmol) was added at 0 °C. The mixture was then heated to 50 °C and stirred overnight. LC-MS showed that the reaction of the starting material was complete. The reaction was quenched dropwise with aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 100/1) to give a pale yellow solid compound C89-6 (160 mg, yield 77%). LCMS (ESI) m/z = 579.8 [M+H] ⁺ . 6) Synthesis of intermediate C89-7 :

Compound C89-6 (160 mg, 0.28 mmol), compound C88-1 (464.25 mg, 4.15 mmol), and silver tetrafluoroborate (109.2 mg, 0.56 mmol) were added to dimethyl sulfoxide (4 mL). The reaction solution was heated to 120 °C and reacted for 2 hours. LC-MS showed that the reaction of the starting materials was complete. The reaction was quenched dropwise with aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated salt water, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/ _{H₂O.NH₃ =} 10/1/0.01) to give a grayish-white solid compound C89-7 (100 mg, yield 59%). LCMS (ESI) m/z = 610.1 [M+H] ⁺ . 7) Synthesis of C89-P1 , C89-P2 , and C89-P3 :

Compound C87-11 (34.5 mg, 0.23 mmol) was added to DMF (5 mL). HOBT (23.76 mg, 0.18 mmol) and EDCI (46.08 mg, 0.24 mmol) were added at room temperature. The mixture was stirred at room temperature for 2 hours under nitrogen protection. Then, compound C89-7 (100 mg, 0.16 mmol) and DIEA (61.92 mg, 0.48 mmol) were added. The mixture was replaced with nitrogen three times and stirred at room temperature for 4 hours under nitrogen protection. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (RP-PREP-3 SunFire C18 5um 19*150mm 18min-55-70B, A: _H₂O (0.2% FA)). B: ACN, UV: 214 nm, flow rate 15 ml/min) yielded white solids C89-P1 (10.5 mg, yield 8.6%, retention time 10.52 min), C89-P2 (11.2 mg, yield 9.2%, retention time 11.31 min), and C89-P3 (16.6 mg, yield 13.6%, retention time 12.56 min). C89-P1 and C89-P2 were both monomeric compounds, while C89-P3 was a mixture of two diastereomers.

C89-P1 (configuration confirmed by crystal structure, C89-P1 is compound C2 ) : LCMS (ESI) m/z = 744.1 [MH] ^- . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.49 (brs, 1H), 8.48 (s, 1H), 8.10-8.08 (m, 2H), 7.53-7.52 (m, 3H), 7.43 (d, J =8.8 Hz, 1H), 7.35 (d, J =8.8 Hz, 1H), 5.59-5.58 (m, 1H), 4.45-4.40 (m, 1H), 3.71-3.69 (m, 2H), 3.08-2.71 (m, 5H), 2.41 (s, 3H), 2.09-2.01 (m, 1H), 1.68-1.63 (m, 1H), 1.60–1.51 (m, 6H).

C89-P2 : LCMS (ESI) m/z = 744.1 [MH] ^- . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.54 (brs, 1H), 8.54 (s, 1H), 8.11-8.09 (m, 2H), 7.54-7.53 (m, 3H), 7.44 (d, J =8.8 Hz, 1H), 7.35 (d, J =8.8 Hz, 1H), 5.48-5.46 (m, 1H), 4.43-4.39 (m, 1H), 3.72-3.71 (m, 2H), 3.08-3.05 (m, 5H), 2.43 (s, 3H), 2.02-1.96 (m, 1H), 1.68-1.61 (m, 1H), 1.60–1.51 (m, 6H).

C89-P3 : LCMS (ESI) m/z = 744.1 [MH] ^- . ^¹H NMR (400 MHz, DMSO_d⁶ ): δ 10.53 (brs, ¹H), 8.51 (s, ¹H), 8.11–8.09 (m, 2H), 7.54–7.53 (m, 3H), 7.42 (d, J = 8.8 Hz, _¹H ), 7.33 (d, J = 8.8 Hz, ¹H), 5.56–5.53 (m, ¹H), 4.46–4.43 (m, 1H), 3.81–3.69 (m, 2H), 3.09–3.01 (m, 5H), 2.42 (s, 3H), 2.03–2.01 (m, 1H), 1.64–1.53 (m, 6H). Example 4 : (C90-P1 , C90-P2 , C90-P3)

Referring to the synthetic method of compounds C87-P1 and C87-P2 in Example 1, C87-7 was replaced with C89-4 to synthesize C90 . C90 was chirally resolved (IBN, MeOH:EtOH:FA=50:50:0.3, 25 ml/min, 254 nm) to give C90- P1 (2.0 mg, retention time 9.55 min), C90- P2 (2.3 mg, retention time 23.31 min), and C90- P3 (2.1 mg, retention time 32.67 min). C90-P1 is a mixture of two diastereomers, while C90-P2 and C90-P3 are both monomeric compounds.

C90-P1 : LCMS (ESI) m/z = 720.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.48 (brs, 1H), 8.56 (s, 1H), 8.12-8.08 (m, 2H), 7.55-7.52 (m, 3H), 7.44 (d, J =8.8 Hz, 1H), 7.36 (d, J =8.8 Hz, 1H), 5.48-5.46 (m, 1H), 3.77-3.73 (m, 1H), 3.08-3.02 (m, 6H), 2.51-2.50 (m, 1H), 2.46-2.44 (m, 4H), 2.01-1.99 (m, 2H), 1.53-1.44 (m, 3H).

C90-P2 : LCMS (ESI) m/z = 720.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.50 (brs, 1H), 8.49 (s, 1H), 8.33 (brs, 1H), 8.11-8.09 (m, 2H), 7.53-7.52 (m, 3H), 7.43 (d, J =8.8 Hz, 1H), 7.36 (d, J =8.8 Hz, 1H), 5.56-5.33 (m, 1H), 3.78-3.72 (m, 1H), 3.15-2.94 (m, 6H), 2.75-2.66 (m, 2H), 2.47 (s, 3H), 2.03-1.96 (m, 2H), 1.52-1.46 (m, 3H).

C90-P3 : LCMS (ESI) m/z = 720.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.49 (brs, 1H), 8.47 (s, 1H), 8.35 (brs, 1H), 8.12-8.10 (m, 2H), 7.54-7.53 (m, 3H), 7.44 (d, J =8.8 Hz, 1H), 7.34 (d, J =8.8 Hz, 1H), 5.49-5.47 (m, 1H), 3.77-3.75 (m, 1H), 3.08-2.97 (m, 6H), 2.53-2.49 (m, 2H), 2.41 (s, 3H), 2.03-1.96 (m, 2H), 1.46-1.44 (m, 3H). Embodiment 5 : (C93-P1 , C93-P2 , C93-P3 , C93-P4)

Referring to the synthesis method of Example 4, C93-1 was used instead of C87-3 , and C88-1 was used instead of piperazine. The final step was purified by preparative high performance liquid chromatography (Waters-PREP-8 SunFire C18 5um 19*150mm 18min-45-55B, A: _H2O (0.2% FA), B: ACN, UV: 214nm, flow rate 15 ml/min) to obtain C93-P1 (1.4 mg, yield 2.9%, retention time 9.83 min), C93-P2 (1.9 mg, yield 3.9%, retention time 10.70 min), C93-P3 (1.6 mg, yield 3.3%, retention time 11.57 min) and C93-P4 (1.2 mg, yield 2.5%, retention time 12.03 min).

C93 -P1: LCMS (ESI) m/z = 747.0 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD): δ 8.69 (d, J =4.4 Hz, 1H), 8.53 (s, 1H), 8.30 (d, J =8.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.55-7.48 (m, 2H), 7.18 (d, J =8.8 Hz, 1H), 5.64-5.61 (m, 1H), 5.35-5.33 (m, 1H), 3.35-3.33 (m, 7H), 2.80-2.76 (m, 1H), 2.53 (s, 3H), 2.03-2.01 (m, 1H), 1.79-1.65 (m, 6H).

C93 -P2: LCMS (ESI) m/z = 747.0 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD): δ 8.69 (d, J =3.6 Hz, 1H), 8.48 (s, 1H), 8.30 (d, J =7.6 Hz, 1H), 8.00-7.96 (m, 1H), 7.55-7.52 (m, 1H), 7.46 (d, J =8.8 Hz, 1H), 7.19 (d, J =8.8 Hz, 1H), 5.58-5.55 (m, 1H), 5.35-5.33 (m, 1H), 3.35-3.33 (m, 7H), 2.48 (s, 3H), 2.37-2.36 (m, 1H), 2.03-2.01 (m, 1H), 1.52-1.50 (m, 6H).

C93 -P3: LCMS (ESI) m/z = 747.0 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD): δ 8.69 (d, J =4.4 Hz, 1H), 8.48 (s, 1H), 8.31 (d, J =8.0 Hz, 1H), 8.00-7.97 (m, 1H), 7.55-7.52 (m, 1H), 7.47 (d, J =8.8 Hz, 1H), 7.20 (d, J =8.8 Hz, 1H), 5.61-5.58 (m, 1H), 5.35-5.33 (m, 1H), 3.32-3.31 (m, 7H), 2.49 (s, 3H), 2.34-2.31 (m, 1H), 2.03-1.97 (m, 1H), 1.58-1.56 (m, 6H).

C93 -P4: LCMS (ESI) m/z = 747.0 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD): δ 8.60 (d, J =3.2 Hz, 1H), 8.43 (s, 1H), 8.21 (d, J =7.6 Hz, 1H), 7.91-7.87 (m, 1H), 7.45-7.42 (m, 1H), 7.38 (d, J =8.8 Hz, 1H), 7.07 (d, J =8.4 Hz, 1H), 5.55-5.52 (m, 1H), 5.25-5.23 (m, 1H), 3.32-3.31 (m, 7H), 2.66-2.69 (m, 1H), 2.41 (s, 3H), 1.94-1.92 (m, 1H), 1.57-1.49 (m, 6H). Example 6 : (C43-P1 , C43-P2 , C43-P3)

Referring to the synthesis method of Example 3, C43-1 was used instead of C89-4 . The final step was purification by preparative high performance liquid chromatography (Waters-PREP-8 SunFire C18 5um 19*150mm 18min-42-78 B, A: H ₂ O (0.2% FA), B: ACN, UV: 214 nm, flow rate 15 ml/min) to obtain C43- P1 (11.1 mg, yield 5.7%, retention time 11.82 min), C43- P3 (14.1 mg, yield 7.3%, retention time 13.25 min) and crude C43- P2 (30 mg, retention time 12.82 min). Crude C43- P2 was purified by chiral resolution (IBN, Hex:EtOH:DEA=50:50:0.3, 25 ml/min, 230 nm) to obtain C43- P2 (12 mg, yield 6.2%, retention time 7.997 min).

C43- P1 (hydrochloride, single configuration compound) : ^¹H NMR (400 MHz, _CD₃OD ): δ 8.80 (s, ¹H), 8.12–8.10 (m, ²H), 7.68 (s, ¹H), 7.47–7.41 (m, ³H), 7.31–7.28 (m, ¹H), 7.18–7.16 (m, ¹H), 5.50–5.47 (m, ¹H), 4.62–4.59 (m, ¹H), 4.00–3.44 (m, ⁶H), 2.71–2.70 (m, ¹H), 2.64 (s, ³H), 2.52–2.45 (m, ¹H), 1.83–1.62 (m, 6 H), 1.42-1.39 (m, 1 H). LCMS (ESI) m/z=712.1 [M+H] ⁺ .

C43-P2 (free base, single configuration compound) : ^¹H NMR (400 MHz, DMSO- _d⁶ ): δ 10.86 (s, ¹H), 8.54 (s, ¹H), 8.05–8.03 (m, ²H), 7.74 (s, ¹H), 7.50–7.48 (m, ³H), 7.41–7.39 (m, ¹H), 7.32–7.29 (m, ¹H), 5.36 (d, J = 9.2 Hz, ¹H), 4.40–4.38 (m, ¹H), 3.71–3.70 (m, ²H), 3.24–2.95 (m, ⁶H), 2.43 (s, ³H), 2.10 (d, J = 9.2 Hz, ¹H). =13.6 Hz, 1 H), 1.75-1.34 (m, 6 H), 1.23-1.17 (m, 1 H). LCMS (ESI) m/z=712.1 [M+H] ⁺ .

C43-P3 (hydrochloride, a mixture of two diastereomers) : ^¹H NMR (400 MHz, _CD₃OD ): δ 8.86 (s, ¹H), 8.13–8.10 (m, ²H), 7.68–7.66 (m, ¹H), 7.47–7.41 (m, ³H), 7.30–7.26 (m, ¹H), 7.18–7.15 (m, ¹H), 5.49–5.42 (m, ¹H), 4.71–4.68 (m, ¹H), 3.79–3.54 (m, ⁶H), 3.13–3.03 (m, ¹H), 2.69 (s, ³H), 2.50–2.43 (m, ¹H). 2.01-1.55 (m, 6 H), 1.40-1.37 (m, 1 H). LCMS (ESI) m/z=712.1 [M+H] ⁺ . Implementation Example 7 : (C142-P1 , C142-P2 , C142-P3-A , C142-P3-B) 1) Synthesis of intermediate C142-2 :

Compound C142-1 (0.6 g, 2.38 mmol, prepared according to the synthetic method disclosed in patent application WO2008082484) and methylboronic acid (1.42 g, 23.8 mmol) were dissolved in a mixed solution of 1,4-dioxane (5 mL) and water (0.5 mL). Potassium carbonate (0.99 g, 7.14 mmol) was added under stirring. After three aeration cycles, 1,1-bis(diphenylphosphine)ferrocene palladium(II) dichloride (0.17 g, 0.24 mmol) was added, and the reaction was carried out at 80 °C for 16 hours with stirring. LCMS indicated the reaction was complete. The reaction solution was concentrated and purified by column chromatography (ethyl acetate/petroleum ether) to give a white solid C142-2 (0.4 g, yield: 89.8%). m/z [M+H] ⁺ = 188.1. 2) Synthesis of the product:

Referring to the synthesis method of Example 3, C142-2 was used instead of C89-4 . The final step was purified by preparative high performance liquid chromatography (RP-PREP-3 SunFire C18 5um 19*150mm 18min-50-75B, A: H ₂ O (0.2% FA), B: ACN, UV: 214nm, flow rate 15ml/min) to obtain C142-P1 (33.6 mg, yield 12.4%, retention time 10.13 min), C142-P2 (25.2 mg, yield 9.3%, retention time 10.74 min) and C142-P3 (54.1 mg, yield 19.9%, retention time 11.53 min). C142 -P1 and C142 -P2 are both monoconfigurations, while C142 -P3 is a mixture of two diastereomers. C142 -P3 was chirally resolved (IBN, Hex:EtOH:TFA=50:50:0.2, 25 ml/min, 254 nm) to give C142-P3-A (10.7 mg, retention time 11.1 min) and C142-P3-B (14.1 mg, retention time 37.7 min).

C142-P1 : LCMS (ESI) m/z = 726.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.24 (s, 1H), 8.53 (s, 1H), 8.10-8.02 (m, 2H), 7.56-7.48 (m, 3H), 7.23 (d, J =8.8 Hz, 1H), 7.10 (d, J =8.8 Hz, 1H), 5.49-5.45 (m, 1H), 4.40-4.38 (m, 1H), 3.76-3.73 (m, 2H), 3.35-3.17 (m, 4H), 2.67-2.52 (m, 1H), 2.51-2.47 (m, 1H), 2.45 (s, 3 H), 2.22 (s, 3 H), 1.67-1.66 (m, 1H), 1.65-1.57 (m, 3H), 1.49-1.42 (m, 2H), 1.23-1.22 (m, 2H).

C142-P2 : LCMS (ESI) m/z = 726.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.28 (s, 1H), 8.50 (s, 1H), 8.10-8.08 (m, 2H), 7.54-7.52 (m, 3H), 7.24 (d, J =8.4 Hz, 1H), 7.10 (d, J =8.4 Hz, 1H), 5.41-5.38 (m, 1H), 4.40-4.38 (m, 1H), 3.74-3.72 (m, 2H), 3.31-3.23 (m, 4H), 3.04-3.01 (m, 1H), 2.42 (s, 3 H), 2.23 (s, 3 H), 2.41-2.11 (m, 1H), 1.68-1.66 (m, 1H), 1.51-1.40 (m, 5H), 1.23-1.22 (m, 2H).

C142-P3-A : LCMS (ESI) m/z = 726.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.28 (s, 1H), 8.56 (s, 1H), 8.12-8.05 (m, 2H), 7.58-7.50 (m, 3H), 7.25-7.21 (m, 1H), 7.11 (d, J =8.4 Hz, 1H), 5.50-5.45 (m, 1H), 4.45-4.42 (m, 1H), 3.76-3.71 (m, 1H), 3.61-3.45 (m, 5H), 2.63-2.58 (m, 1H), 2.50-2.35 (m, 4H), 2.21 (s, 3H), 1.66-1.63 (m, 1H), 1.49 (d, J =6.8 Hz, 3H), 1.44-1.16 (m, 4H).

C142-P3-B (configuration confirmed by crystal structure analysis, C142-P3-B is compound C100 ): LCMS (ESI) m/z = 726.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.26 (s, 1H), 8.56 (s, 1H), 8.12-8.05 (m, 2H), 7.58-7.50 (m, 3H), 7.24 (d, J =8.4 Hz, 1H), 7.08 (d, J =8.4 Hz, 1H), 5.41 (d, J =8.4 Hz, 1H), 4.47-4.42 (m, 1H), 3.80-3.73 (m, 2H), 3.61-3.45 (m, 4H), 3.10-3.02 (m, 1H), 2.44 (s, 3H), 2.22 (s, 3H), 2.10 (d, J =13.6 Hz, 1H), 1.68-1.63 (m, 1H), 1.48 (d, J =6.8 Hz, 3H), 1.44-1.16 (m, 4H). Embodiment 8 : (C143-P1 , C143-P2 , C143-P3 , C143-P4)

Referring to the synthesis method of Example 5, C142-2 was used instead of C89-4 . The final step was purified by preparative high performance liquid chromatography (Waters-PREP-8 SunFire C18 5um 19*150mm 18min-45-50B, A: H ₂ O (0.2% FA), B: ACN, UV: 214nm, flow rate 15ml/min) to obtain C143-P1 (2.4 mg, yield 3%, retention time 9.88 min), C143-P2 (2.1 mg, yield 2.6%, retention time 10.25 min), C143-P3 (3.6 mg, yield 4.5%, retention time 12.07 min) and C143-P4 (3.6 mg, yield 4.5%, retention time 12.55 min).

C143-P1 : LCMS (ESI) m/z=727.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.28 (s, 1H), 8.73 (d, J =4.0 Hz, 1H), 8.48 (s, 1H), 8.14 (d, J =8.0 Hz, 1H), 8.01-7.97 (m, 1H), 7.55-7.52 (m, 1H), 7.23-7.20 (m, 1H), 7.16-7.13 (m, 1H), 5.51-5.47 (m, 1H), 4.48-4.46 (m, 1H), 3.76-3.64 (m, 2H), 2.99-2.96 (m, 4H), 2.67-2.58 (m, 1H), 2.33 (s, 3H), 2.21 (s, 3H), 2.03-1.97 (m, 1H), 1.55-1.35 (m, 7H).

C143-P2 : LCMS (ESI) m/z=727.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.33 (s, 1H), 8.72 (d, J =4.0 Hz, 1H), 8.47 (s, 1H), 8.14 (d, J =8.0 Hz, 1H), 8.01-7.96 (m, 1H), 7.54-7.51 (m, 1H), 7.24-7.20 (m, 1H), 7.16-7.12 (m, 1H), 5.50-5.44 (m, 1H), 4.41-4.40 (m, 1H), 3.73-3.64 (m, 2H), 3.17-3.05 (m, 4H), 2.44 (s, 3H), 2.40-2.39 (m, 1H), 2.27 (s, 3H), 2.03-1.89 (m, 1H), 1.59-1.45 (m, 7H).

C143-P3 : LCMS (ESI) m/z=727.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.32 (s, 1H), 8.73 (d, J =4.4 Hz, 1H), 8.43 (s, 1H), 8.15 (d, J =8.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.55-7.52 (m, 1H), 7.25-7.23 (m, 1H), 7.15-7.11 (m, 1H), 5.46-5.44 (m, 1H), 4.45-4.40 (m, 1H), 3.80-3.64 (m, 2H), 3.17-3.01 (m, 4H), 2.46-2.44 (m, 1H), 2.34 (s, 3H), 2.21 (s, 3H), 2.03-1.90 (m, 1H), 1.53-1.40 (m, 7H).

C143-P4 : LCMS (ESI) m/z=727.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO_ d ₆ ): δ 10.28 (s, 1H), 8.74 (d, J =4.0 Hz, 1H), 8.49 (s, 1H), 8.14 (d, J =8.0 Hz, 1H), 8.01-7.97 (m, 1H), 7.55-7.52 (m, 1H), 7.23-7.20 (m, 1H), 7.16-7.13 (m, 1H), 5.51-5.47 (m, 1H), 4.45-4.43 (m, 1H), 3.76-3.67 (m, 2H), 3.10-2.93 (m, 4H), 2.64-2.59 (m, 1H), 2.41 (s, 3H), 2.20 (s, 3H), 2.03-1.97 (m, 1H), 1.56-1.34 (m, 7H). Example 9 : (C144-P1 , C144-P2 , C144-P3-A , C144-P3-B) 1) Synthesis of intermediate C144-2:

Compound C144-1 (50 g, 341.9 mmol) was added to acetonitrile (500 mL), followed by 2 mol/L dimethylaminetetrahydrofuran solution (200 mL), and reacted at 50 ^° C for 4 hours. Then, hydrazine hydrate (51.35 g, 820.6 mmol) was added, and the mixture was refluxed overnight. LC-MS showed the reaction was complete. The reaction solution was returned to room temperature, concentrated under reduced pressure to a remaining 230 mL, and a solid precipitated. The precipitate was filtered to obtain a filter cake, which was dried to give C144-2 (39 g, yield: 89.7%). m/z [M+H] ⁺ = 128.0. 2) Synthesis of intermediate C144-3:

Compound C144-2 (32 g, 251.7 mmol) was added to acetic acid (210 mL), followed by methyl acetate (39.3 g, 302.0 mmol), and the reaction was carried out overnight at 90 ^° C. LC-MS showed the reaction was complete. The reaction solution was returned to room temperature, and the acetic acid was removed by reduced pressure concentration. Ethyl acetate (180 mL) and petroleum ether (110 mL) were then added, and the mixture was ground and stirred for 1 hour. The mixture was filtered to obtain a filter cake, which was dried to give C144-3 (50 g, yield: 95.9%). m/z [M+H]+ = 208.1. 3) Synthesis of intermediate C144-4:

Compound C144-3 (4.5 g, 21.72 mmol) was added to tetrahydrofuran (110 mL) and 1,4-dioxane (110 mL). Under nitrogen protection, LDA (2 M, 14 mL) was added and the reaction was carried out at room temperature for 30 minutes. LDA (2 M, 14 mL) was added again at 0 ^° C and the mixture was stirred at 0 ^° C for 30 minutes. The mixture was then stirred at room temperature for another 30 minutes. S-epoxychloropropane (5.22 g, 56.47 mmol) was added at room temperature and the mixture was stirred overnight at room temperature. LCMS showed the reaction was complete. The reaction solution was added to an ammonium chloride aqueous solution, and the organic phase was separated. The aqueous phase was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to obtain C144-4 (4 g, yield: 69.96%). m/z [M+H] ⁺ = 264.2. 4) Synthesis of intermediate C144-5:

Compound C144-4 (3.5 g, 13.29 mmol) was added to dichloromethane (40 mL), and NBS (2.60 g, 14.62 mmol) was added at 0 °C. The reaction was carried out at room temperature for 6 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with dichloromethane, and 10% sodium sulfite solution was added. The mixture was extracted by separation, and extracted twice with dichloromethane. The organic phases were combined and washed twice with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give C144-5 (4 g, yield: 87.94%). m/z [M+H]+ = 342.1. 5) Synthesis of intermediate C144-6:

Compound C144-5 (4 g, 11.69 mmol) was added to dichloromethane (80 mL), followed by an aqueous solution of sodium bicarbonate (9.82 g, 116.90 mmol) (100 mL), potassium bromide (0.14 g, 1.17 mmol), trioctylmethylammonium chloride (0.24 g, 0.58 mmol), TEMPO (0.091 g, 0.58 mmol), and a 10% sodium hypochlorite aqueous solution (43.51 g, 58.45 mmol) at 0 °C. The reaction was allowed to proceed at room temperature for 2.5 hours. LCMS indicated the reaction was complete. Dichloromethane was added to the reaction solution, and after extraction and separation, an aqueous phase was obtained. Dichloromethane was added again to the aqueous phase, and after extraction and separation, the organic phases were combined. A 10% sodium bicarbonate aqueous solution was added to the combined organic phase, and after extraction and separation, an aqueous phase was obtained. The two aqueous phases were combined, and dichloromethane was added to the aqueous phase. The pH was adjusted to 3-4 with dilute hydrochloric acid, and the solution was extracted three times with dichloromethane. The dichloromethane phase was combined, washed once with saturated brine, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to dryness to obtain C144-6 (2.5 g, yield: 60.05%). m/z [M+H]+ = 356.1. 6) Synthesis of intermediate C144-7:

Compound C144-6 (2.15 g, 6.04 mmol) was added to dichloromethane (40 mL), followed by 2-chloro-1-methylpyridinium iodide (1.70 g, 6.64 mmol), DIEA (3.51 g, 27.18 mmol), and C142-2 (1.76 g, 7.85 mmol). The mixture was stirred at room temperature for 4 hours. LC-MS showed the reaction was complete. Dichloromethane and water were added to the reaction mixture, and the mixture was extracted separately. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed twice with dilute hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and subjected to column chromatography to give C144-7 (2.6 g, yield: 82.0%). m/z [M+H]+ = 525.1. 7) Synthesis of intermediate C144-8:

Compound C144-7 (700 mg, 1.33 mmol) was added to DMSO (3 mL), followed by C88-1 (0.75 g, 6.65 mmol) and DIPA (0.67 g, 6.65 mmol). Silver tetrafluoroborate (0.39 g, 2.00 mmol) was added under nitrogen protection, and the mixture was microwaved at 105 °C for 2 hours under nitrogen protection. LCMS showed the reaction was complete. The reaction solution was brought to room temperature, diluted with dichloromethane, filtered, and the filtrate was concentrated under reduced pressure and purified by reverse-phase column chromatography to give C144-8 (270 mg, yield: 36.41%). m/z [M+H] ⁺ = 557.2 8) Synthesis of product C144:

Compound C144-8 (0.10 g, 0.66 mmol) and HOAT (0.10 g, 0.76 mmol) were added to acetonitrile (3 mL), followed by EDCI (0.19 g, 0.98 mmol), and stirred at room temperature for 1 hour. Then, C87-11 (270 mg, 0.49 mmol) and DIEA (0.22 g, 1.71 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. LCMS indicated the reaction was complete. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain C144 (110 mg, yield: 32.74%). Further purification by preparative high-performance liquid chromatography (SHIMADZU LC 20, Agilent 10 Prep-C18, 250*21.2 mm, 10 μm, A: _H₂O (0.1% FA), B: ACN, UV: 214 nm, flow rate 20 ml/min) yielded C144-P1 (19.8 mg, yield 4.3%, retention time 13.1 min), C144-P2 (7.3 mg, yield 1.6%, retention time 14.0 min), and C144 - P3 (retention time 16.2 min). C144 -P1 and C144 -P2 are both monoconfigurations, while C144 -P3 is a mixture of two diastereomers. C144 -P3 was chirally resolved (Waters SFC-150 mgm, Daicel OJ, 30*250 mm, 10 μm, _CO2 /MEOH=75/25, 50 ml/min, 254 nm) to give C144-P3-A (8 mg, retention time 12.01 min) and C144-P3-B (19.7 mg, retention time 16.19 min).

C144 -P1: LCMS m/z [M+H] ⁺ = 693.3. ¹ H NMR (400 MHz, chloroform- d ) δ 11.46 (s, 1H), 8.69 (s, 1H), 8.59 (s, 1H), 7.24 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 5.20 (d, J = 8.2 Hz, 1H), 4.66 (s, 1H), 4.12 (d, J = 66.5 Hz, 2H), 3.70 (dd, J = 15.7, 6.7 Hz, 2H), 3.08 (s, 6H), 3.06 – 2.98 (m, 1H), 2.54 (s, 3H), 2.43 – 2.27 (m, 1H), 2.24 – 2.16 (m, 1H), 2.14 (s, 3H), 2.07 – 1.95 (m, 1H), 1.68 – 1.57 (m, 1H), 1.55 – 1.38 (m, 4H), 1.37 – 1.28 (m, 1H), 1.20 – 1.09 (m, J = 9.7 Hz, 1H).

C144 -P2: LCMS m/z [M+H] ⁺ = 693.3. ¹ H NMR (400 MHz, chloroform- d ) δ 11.46 (s, 1H), 8.69 (s, 1H), 8.59 (s, 1H), 7.24 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 5.20 (d, J = 8.2 Hz, 1H), 4.66 (s, 1H), 4.12 (d, J = 66.5 Hz, 2H), 3.70 (dd, J = 15.7, 6.7 Hz, 2H), 3.08 (s, 6H), 3.06 – 2.98 (m, 1H), 2.54 (s, 3H), 2.43 – 2.27 (m, 1H), 2.24 – 2.16 (m, 1H), 2.14 (s, 3H), 2.07 – 1.95 (m, 1H), 1.68 – 1.57 (m, 1H), 1.55 – 1.38 (m, 4H), 1.37 – 1.28 (m, 1H), 1.20 – 1.09 (m, J = 9.7 Hz, 1H).

C144-P3-A: LCMS m/z [M+H] ⁺ = 693.3. ¹ H (400 MHz, chloroform -d ) δ 11.49 (s, 1H), 9.20 (s, 1H), 8.59 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 5.23 (d, J = 9.1 Hz, 1H), 4.71 (s, 1H), 4.09 (s, 2H), 3.74 – 3.50 (m, 2H), 3.10 (s, 6H), 2.81 (d, J = 13.1 Hz, 1H), 2.68 (dd, J = 20.1, 8.8 Hz, 1H), 2.54 (s, 3H), 2.35 (q, J = 9.6 Hz, 1H), 2.16 (s, 3H), 2.06 – 1.97 (m, 1H), 1.68 – 1.57 (m, 1H), 1.55 – 1.38 (m, 4H), 1.37 – 1.28 (m, 1H), 1.20 – 1.09 (m, J = 9.7 Hz, 1H).

C144-P3-B: LCMS m/z [M+H] ⁺ = 693.3. ¹ H (400 MHz, chloroform -d ) δ 11.49 (s, 1H), 9.20 (s, 1H), 8.59 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 5.23 (d, J = 9.1 Hz, 1H), 4.71 (s, 1H), 4.09 (s, 2H), 3.74 – 3.50 (m, 2H), 3.10 (s, 6H), 2.81 (d, J = 13.1 Hz, 1H), 2.68 (dd, J = 20.1, 8.8 Hz, 1H), 2.54 (s, 3H), 2.35 (q, J = 9.6 Hz, 1H), 2.16 (s, 3H), 2.06 – 1.97 (m, 1H), 1.68 – 1.57 (m, 1H), 1.55 – 1.38 (m, 4H), 1.37 – 1.28 (m, 1H), 1.20 – 1.09 (m, J = 9.7 Hz, 1H). Embodiment 10 : (C146-P1 , C146-P2 , C146-P3-A , C146-P3-B)

Referring to the synthetic method of Example 9, tetrahydropyrrole was used instead of dimethylamine hydrochloride in the first step, and the final step was purified by preparative high performance liquid chromatography (SHIMADZU LC 20, Agilent 10 Prep-C18, 250*21.2 mm, 10 μm, A: H ₂ O (0.1% FA), B: ACN, UV: 214 nm, flow rate 20 ml/min) to obtain C146-P1 (retention time 10.5 min), C146-P2 (retention time 11.3 min), and C146-P3 (retention time 12.6 min). C146 -P1 and C146 -P2 are both monomeric compounds, while C146 -P3 is a mixture of two diastereomers. C146 -P3 was chirally resolved (Waters SFC-150 mgm, Daicel OD, 30*250 mm, 10 μm, _CO2 /MEOH=65/35, 50 ml/min, 214 nm) to obtain C146-P3-A (5.81 min) and C146-P3-B (11.14 min).

C146-P1 : LCMS m/z [M+H] ⁺ = 719.3, ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.17 (s, 1H), 8.54 (s, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 5.30 (dq, J = 8.3, 5.0 Hz, 1H), 4.36 (d, J = 13.0 Hz, 1H), 3.65 (dq, J = 22.0, 7.2 Hz, 3H), 3.54 – 3.43 (m, 4H), 3.25 – 3.10 (m, 3H), 2.96 (dt, J = 13.5, 9.9 Hz, 1H), 2.42 (s, 3H), 2.35 (ddd, J = 12.6, 8.1, 5.9 Hz, 1H), 2.19 (s, 3H), 1.99 (dt, J = 13.0, 7.0 Hz, 1H), 1.90 (t, J = 5.9 Hz, 4H), 1.61 (d, J = 8.5 Hz, 1H), 1.51 (d, J = 7.1 Hz, 1H), 1.39 (dd, J = 20.7, 10.2 Hz, 3H), 1.15 (t, J = 8.9 Hz, 1H).

C146-P2 : LCMS m/z [M+H] ⁺ = 719.3, ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.17 (s, 1H), 8.54 (s, 1H), 7.23 (dd, J = 8.6, 1.9 Hz, 1H), 7.06 (dd, J = 11.0, 8.6 Hz, 1H), 5.31 – 5.22 (m, 1H), 4.38 (t, J = 16.1 Hz, 1H), 3.65 (dq, J = 22.0, 7.2 Hz, 3H), 3.54 – 3.43 (m, 4H), 3.25 – 3.10 (m, 3H), 2.96 (dt, J = 13.5, 9.9 Hz, 1H), 2.42 (s, 3H), 2.35 (ddd, J = 13.2, 8.8, 6.2 Hz, 1H), 2.19 (s, 3H), 2.01 (d, J = 13.7 Hz, 1H), 1.93 – 1.86 (m, 4H), 1.61 (d, J = 8.5 Hz, 1H), 1.51 (d, J = 7.1 Hz, 1H), 1.39 (dd, J = 20.7, 10.2 Hz, 3H), 1.15 (t, J = 8.9 Hz, 1H).

C146-P3-A: LCMS m/z [M+H] ⁺ = 719.3, ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.19 (s, 1H), 8.55 (s, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 5.31 (dt, J = 9.0, 5.1 Hz, 1H), 4.47 – 4.29 (m, 1H), 3.66 – 3.57 (m, 3H), 3.54 – 3.43 (m, 4H), 3.25 – 3.10 (m, 3H), 2.96 (dt, J = 13.5, 9.9 Hz, 1H), 2.42 (s, 3H), 2.35 (ddd, J = 13.2, 8.8, 6.2 Hz, 1H), 2.19 (s, 3H), 1.99 (dt, J = 13.0, 6.8 Hz, 1H), 1.93 – 1.86 (m, 4H), 1.61 (d, J = 8.5 Hz, 1H), 1.51 (d, J = 7.1 Hz, 1H), 1.39 (dd, J = 20.7, 10.2 Hz, 3H), 1.15 (t, J = 8.9 Hz, 1H).

C146-P3-B: LCMS m/z [M+H] ⁺ = 719.3, ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.18 (s, 1H), 8.55 (s, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 5.24 (d, J = 9.8 Hz, 1H), 4.42 (d, J = 9.1 Hz, 1H), 3.75 – 3.48 (m, 3H), 3.54 – 3.43 (m, 4H), 3.27 – 3.12 (m, 3H), 3.09 – 2.90 (m, 1H), 2.43 (s, 3H), 2.19 (s, 3H), 2.04 – 1.98 (m, 1H), 1.90 (t, J = 6.5 Hz, 4H), 1.62 (dd, J = 15.1, 7.2 Hz, 1H), 1.52 – 1.47 (m, 1H), 1.42 (d, J = 7.3 Hz, 3H), 1.34 (d, J = 8.4 Hz, 1H), 1.16 (t, J = 8.6 Hz, 1H). Example 11 : (C145 , C145-P1 , C145-P2) 1) Synthesis of intermediate C145-2 :

Compound C145-1 (0.1 g, 0.19 mmol, prepared according to the synthetic method disclosed in patent application WO2024079623) was added to dichloromethane (2 mL), followed by 2-chloro-1-methylpyridinium iodide (0.053 g, 0.21 mmol), DIEA (0.11 g, 0.85 mmol), and C142-2 (0.55 g, 0.25 mmol), and stirred at room temperature for 4 hours. LCMS showed the reaction was complete. Dichloromethane and water were added to the reaction solution, and the mixture was extracted separately. The dichloromethane extraction was performed twice, and the organic phases were combined. The organic phase was washed twice with dilute hydrochloric acid, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and subjected to column chromatography to obtain C145-2 (70 mg, yield: 54.0%). m/z [M+H] ⁺ = 696.1. 2) Synthesis of intermediate C145-3 :

Compound C145-2 (0.7 g, 0.1 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 1 hour. LCMS showed that the reaction was complete. The reaction mixture was filtered and concentrated to give crude yellow oil C145-3 (100 mg, trifluoroacetate, yield: 100%). m/z [M+H] ⁺ = 596.3. 3) Synthesis of product C145 :

Compound C87-11 (23 mg, 0.15 mmol) and HOAT (22 mg, 0.16 mmol) were added to DMF (1 mL), followed by EDCI (35 mg, 0.18 mmol), and the mixture was stirred at room temperature for 1 hour. Then, C145-3 (0.10 g crude product, 0.1 mmol) and DIEA (39 mg, 0.3 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. LCMS showed the reaction was complete. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to give C145 (40 mg, yield: 54.5%). LCMS m/z [M+H] ⁺ = 732.4. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.18 (d, J = 8.7 Hz, 1H), 8.55 (s, 1H), 7.27 – 7.19 (m, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.80 (dd, J = 4.6, 2.6 Hz, 1H), 5.35 (ddd, J = 21.9, 9.5, 4.3 Hz, 1H), 4.40 (t, J = 15.3 Hz, 1H), 4.26 (q, J = 2.9 Hz, 2H), 3.86 – 3.65 (m, 4H), 3.55 – 3.43 (m, 2H), 3.17 (s, 2H), 3.05 – 2.94 (m, 1H), 2.54 – 2.50 (m, 1H), 2.43 (s, 3H), 2.42 – 2.38 (m, 1H), 2.18 (s, 3H), 2.06 (dt, J = 12.9, 2.6 Hz, 1H), 1.62 (d, J = 9.9 Hz, 1H), 1.53 (d, J = 6.7 Hz, 1H), 1.45 (d, J = 7.0 Hz, 3H), 1.37–1.30 (m, 1H), 1.21–1.11 (m, 1H). 4) Synthesis of products C145-P1 and C145-P2 :

C145 was purified by preparative high-performance liquid chromatography (SHIMADZU LC 20, Agilent 10 Prep-C18, 250*21.2 mm, 10 μm, A: H ₂ O (0.1% FA), B: ACN, UV: 214 nm, flow rate 20 ml/min) to obtain C145-P1 (8 mg, retention time 10.675 min) and C145-P2 (18 mg, retention time 11.147 min).

C145-P1: LCMS m/z [M+H] ⁺ = 732.4. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.16 (s, 1H), 8.55 (s, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.85 – 6.72 (m, 1H), 5.38 (dd, J = 8.5, 5.7 Hz, 1H), 4.37 (d, J = 11.3 Hz, 1H), 4.31 – 4.22 (m, 2H), 3.81 (t, J = 5.7 Hz, 2H), 3.74 – 3.65 (m, 2H), 3.56 – 3.45 (m, 2H), 3.35 – 3.15 (m, 4H), 2.58 (q, J = 7.2 Hz, 1H), 2.43 (s, 3H), 2.38 (dd, J = 8.0, 5.3 Hz, 1H), 2.18 (s, 3H), 2.00 (dq, J = 13.0, 6.1 Hz, 1H), 1.71 – 1.60 (m, 1H), 1.54 (d, J = 7.0 Hz, 3H), 1.48 – 1.40 (m, 1H), 1.21 – 1.12 (m, 1H).

C145-P2: LCMS m/z [M+H] ⁺ = 732.4. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.22 (s, 1H), 8.48 (s, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 6.80 (s, 1H), 5.33 (d, J = 9.8 Hz, 1H), 4.40 (d, J = 13.1 Hz, 1H), 4.26 (d, J = 3.1 Hz, 2H), 3.85 – 3.65 (m, 4H), 3.51 (dd, J = 16.8, 7.4 Hz, 2H), 3.20 – 3.10 (m, 4H), 3.07 – 2.93 (m, 1H), 2.54 – 2.50 (m, 1H), 2.41 (s, 3H), 2.18 (s, 3H), 2.06 (d, J = 13.5 Hz, 1H), 1.63 (q, J = 6.6, 5.4 Hz, 1H), 1.45 (d, J = 7.2 Hz, 3H), 1.40 – 1.27 (m, 1H), 1.19 – 1.10 (m, 1H). Embodiment 12 : (C147)

Compound 100 mg (0.88 mmol) and HOAT (100 mg, 0.73 mmol) were added to DMF (1 mL), followed by EDCI (150 mg, 0.78 mmol), and the mixture was stirred at room temperature for 1 hour. Then, C142-5 (0.08 g, 0.14 mmol) and DIEA (78 mg, 0.6 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. LCMS showed the reaction was complete. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain C147 (25 mg, yield: 26.5%). LCMS m/z [M+H]+ = 698.4. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.30 (d, J = 9.2 Hz, 1H), 8.21 – 8.03 (m, 3H), 7.71 (d, J = 5.6 Hz, 1H), 7.57 (dd, J = 5.2, 2.0 Hz, 3H), 7.30 (dd, J = 23.9, 8.6 Hz, 1H), 7.14 (dd, J = 23.1, 8.6 Hz, 1H), 5.56 – 5.33 (m, 1H), 4.47 (t, J = 7.4 Hz, 1H), 3.89 (s, 3H), 3.80 – 3.69 (m, 1H), 3.65 – 3.49 (m, 2H), 3.43 (d, J = 5.3 Hz, 1H), 3.08 (dt, J = 13.3, 9.8 Hz, 1H), 2.25 (s, 3H), 2.15 (s, 3H), 2.11 (t, J = 2.6 Hz, 1H), 1.60 (d, J = 7.0 Hz, 1H), 1.50 (d, J = 7.2 Hz, 3H), 1.46 – 1.36 (m, 1H), 1.21–1.11 (m, 1H). Bioassay Section Experiment Example 1 Bioactivity Assay : Determination of ATPase Activity of WRN Helicase

The commercially available ADP-Glo test kit (Promega, #V9102) is used to detect the ADP content produced by the hydrolysis of ATP by WRN helicase, which reflects the ATPase activity of WRN helicase.

The 45 nt oligoDNA single-strand FLAP26 (TTTTTTTTTTTTTTTTTTTTCCAAGTAAAACGACGGCCAGTGC) was synthesized by Ascentage. See, for example, Brosh RM Jr et al., J Biol Chem, 2002 Jun;277(26):23236-45. Prepare reaction buffer (30 mM Tris pH 7.5, ₂ mM MgCl2, 0.02% BSA, 50 mM NaCl, 0.1% pluronic F127). Add 5 µL of 3× test compound (diluted to 0.5% DMSO in reaction buffer, final initial concentration of 10 µM, 1:3 serial dilution, for a total of 9 gradients) to a 384-well clear plate, 5 µL of 3× WRN recombinant protein and 3× ATP acceptor solution (diluted in reaction buffer, final concentrations of WRN and ATP of 10 nM and 300 µM, respectively), shake to mix well, and incubate at 37°C for 3 hours. Next, a 3 × FLAP26 solution was prepared using the reaction buffer. 5 µL of the solution (the final concentration of FLAP26 was 0.4 nM) was added to a 384-well transparent plate, shaken to mix, and the enzymatic reaction was started. The plate was then incubated at room temperature for 30 minutes.

Transfer 5 µL of the above mixture to a 384-well white plate, add 5 µL of ADP-Glo reagent, shake well, and incubate at room temperature in the dark for 40 minutes. Add 10 µL of kinase assay reagent to the above solution, shake well, and incubate at room temperature in the dark for 30 minutes. Record the chemiluminescence reading. Calculate the inhibition rate of the compound on enzyme activity. Fit the inhibition rate value and the logarithm of the compound concentration using nonlinear regression (dose response - variable slope) to obtain the _IC50 value of the test compound. Table 1: WRN ATPase activity data of the compounds in this application. compound The _IC50 (µM) inhibition of WRN ATPase activity is [value missing]. C89-P1 0.001 C89-P2 0.273 C89-P3 0.003 C88-P1 0.002 C88-P2 0.134 C88-P3 0.011 C87-P1-B 0.02 C87-P2-B 0.006 C90-P2 0.01 C90-P3 0.013 C93-P1 0.009 C93-P3 0.009 C43-P1 0.004 C43-P3 0.004 C142-P1 0.001 C142-P3-B 0.001 C143-P1 0.014 C143-P3 0.017 C144-P1 0.022 C144-P3-B 0.005 C145 0.002 C145-P1 0.007 C145-P2 0.005 C147 0.001

As shown in Table 1, the compounds in this application exhibit good inhibitory activity against WRN protein ATPase activity. Determination of tumor cell proliferation inhibitory activity. cell line Item Number Manufacturer HCT116 CBP60028 Nanjing Kebai SW48 FH0526 Fuheng DLD1 FH0017 Fuheng

The DLD1-WRN-KO cell line was constructed by stably knocking out the WRN gene in DLD1 cells using CRISPR/Cas9 technology to evaluate the potential off-target effects of the compound.

Human colonic adenocarcinoma cells SW48 were cultured in vitro in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and strobin at 37°C with 5% _CO2 . Human colonic adenocarcinoma cells HCT116 were also cultured in vitro in McCoy's 5A medium containing 10% fetal bovine serum, 1% penicillin, and strobin at 37°C with 5% _CO2 . DLD1-WRN-KO cells were cultured in vitro as a monolayer under the following conditions: 1640 medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin, at 37°C and 5% _CO2 . Cells were passaged twice weekly using trypsin digestion.

After treating microsatellite-instable SW48 and HCT116 cell lines, as well as the control cell line DLD1-WRN-KO, with the test compound for 4 days, ATP levels were measured using the CellCounting-Lite kit from Novartis to evaluate the inhibitory effect of the test compound on tumor cell line growth.

In this application, SW48, HCT116, and DLD1-WRN-KO cell lines were seeded at appropriate cell densities in 96-well cell culture plates. After 24 hours, the cells were treated with nine gradients of the test compound at a maximum concentration of 10 μM, diluted 1:3. A DMSO treatment group was also included. Cells were cultured at 37°C/5% _CO2 for 4 days. To test the inhibitory effect of the test compound on tumor cell proliferation, the cells were equilibrated at room temperature for 30 minutes, followed by the addition of 100 μL of CellCounting-Lite (CCL) cell proliferation assay reagent to each well. After shaking for 5 minutes, the cells were incubated in the dark for 10 minutes. The chemiluminescence values were read using a Thermo Varioskan LUX-3020 multifunctional microplate reader and converted into a proliferation index to calculate the inhibitory rate of the compound on tumor cell proliferation. The inhibition rate value and the logarithm of the compound concentration were combined using a nonlinear regression (dose response - variable slope) to obtain the _IC50 value of the compound. Table 2 shows the tumor cell proliferation inhibitory activity data of the compounds of this application. compound SW48 proliferation inhibition _IC50 (µM) HCT116 proliferation inhibition _IC50 (µM) DLD1-WRN-KO proliferation inhibition _IC50 (µM) C89-P1 0.0002 >10 C89-P2 0.24 >10 C89-P3 0.0002 >10 C88-P1 0.0004 >10 C88-P2 0.087 >10 C88-P3 0.002 >10 C87-P1-B 0.109 >10 C87-P2-B 0.001 >10 C90-P2 0.148 >10 C90-P3 0.029 >10 C93-P1 0.010 0.019 >10 C93-P3 0.0042 0.0054 >10 C43-P1 0.010 0.009 >10 C43-P3 0.011 0.004 >10 C142-P1 0.001 0.0025 >10 C142-P3-B 0.0002 0.0001 >10 C143-P1 0.012 0.0097 >10 C143-P3 0.0027 0.0072 >10 C144-P1 0.018 >10 C144-P3-B 0.006 >10 C146-P1 0.011 >10 C146-P3-B 0.008 >10 C145 0.014 >10 C145-P1 0.018 0.015 >10 C145-P2 0.012 0.007 >10 C147 0.2 0.11 >10

As shown in Table 2, the compounds in this application exhibit good inhibitory activity against the proliferation of microsatellite-instable SW48 and HCT116 cells, but no significant inhibitory activity against WRN-knockout DLD1 cells, demonstrating good selectivity. Experimental Example 2: Liver Microsomal Stability Test

To assess the phase-dependent metabolic stability of the test compound in liver microsomes of CD-1 mice, Sprague-Dawley rats, beagle dogs, cynomolgus monkeys, and humans. Experimental systems:

The animal and human liver microsomes used in this testing system were purchased from Xenotech, Corning, or other qualified suppliers and stored at a temperature below -60°C before use. Experimental overview:

The test sample and reference compound were incubated with animal and human liver microsomes at 37±1℃ for a specified time, with a maximum incubation time of 60 minutes. Samples were removed at designated time points, and the reaction was terminated with acetonitrile or other organic solvents containing an internal standard. After centrifugation, the supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Experimental methods: 1. Preparation of buffer solution

Dissolve 73.21 g of potassium dihydrogen phosphate trihydrate and 10.78 g of potassium dihydrogen phosphate in 4000 mL of ultrapure water. Adjust the pH of the solution to between 7.40 ± 0.10 using 10% phosphoric acid or 1 M potassium hydroxide, achieving a final concentration of 100 mM. 2. Preparation of the working solution

The test sample powder is prepared into a stock solution of a certain concentration using DMSO or other organic solvents, and then further diluted with a suitable organic solvent.

The control compounds testosterone, diclofenac, and propafenone were prepared into 10 mM stock solutions using DMSO, and then further diluted with suitable organic solvents. 3. Preparation of Liver Microsomal Solution

The microparticles of various species were diluted to a 2× working solution using 100 mM potassium phosphate buffer. The final concentration of microparticles in the reaction system was 0.5 mg/mL. 4. Preparation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system

Weigh appropriate amounts of nicotinamide adenine diphosphate dinucleotide (NADP) and isocitric acid (ISO) powder, dissolve them in magnesium chloride solution, and shake to mix thoroughly. Add an appropriate amount of isocitric acid dehydrogenase (IDH), and gently invert to mix thoroughly. The final concentrations in the reaction system are: 1 mM NADP, 1 mM magnesium chloride, 6 mM ISO, and 1 unit/mL IDH, respectively. 5. Preparation of Termination Solution

The termination solution is prepared using acetonitrile or other organic solvents containing an internal standard (tolbutamide or other suitable compound). The prepared termination solution is stored in a refrigerator at 2-8°C. 6. Incubation Process

Incubation will be performed in 96-well plates. Prepare eight incubation plates, named T0, T5, T15, T30, T45, T60, Blank60, and NCF60. The first six plates correspond to reaction time points of 0, 5, 15, 30, 45, and 60 minutes, respectively. Do not add the test or control compound to the Blank60 plate, and take a sample after 60 minutes of incubation. In the NCF60 plate, incubate for 60 minutes using potassium phosphate buffer instead of the NADPH regeneration system solution. All conditions are tested in triplicate.

Mix the microparticles with the test or control compound, and then pre-incubate the incubation plates (Blank60, T5, T15, T30, T45, and T60, except for T0 and NCF60) in a 37°C water bath for approximately 10 minutes. For incubation plate T0, add the stop solution first, followed by the NADPH regeneration system working solution. For incubation plate NCF60, add 98 μL of potassium phosphate buffer to each well to initiate the reaction. After pre-incubation of incubation plates Blank60, T5, T15, T30, T45, and T60, add 98 μL of NADPH regeneration system working solution to each well to initiate the reaction. The reaction temperature was 37±1℃, the final reaction volume was 200 μL, and the reaction system included 0.5 mg/mL microparticles, 1.0 μM acceptor, 1 mM NADP, 6 mM ISO and 1 unit/mL IDH.

At 5, 15, 30, 45 and 60 minutes, cold stop solution containing internal standard was added to the reaction plate to terminate the reaction.

After termination, shake all reaction plates well and centrifuge at 4°C, 3220 ×g for 20 minutes. Dilute the supernatant to a certain proportion and perform LC-MS/MS analysis. Sample Analysis

Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), excluding standard curves and quality control samples. Semi-quantitative determination was performed using the ratio of analyte peak area to internal standard peak area. Retention times of analytes and internal standards, chromatographic acquisition, and chromatographic integration were processed using Analyst software (Sciex, Framingham, Massachusetts, USA).

The CV of the internal standard peak area for each matrix in each analytical batch should be within 20%. (Data Analysis )

The in vitro elimination rate constant ke of the compound is obtained by converting the ratio of the compound's peak area to the internal standard peak area into a residual rate using the following formula: CL _{int (mic)} = 0.693 / T _1/2 / Microsomal protein content (microsomal concentration at incubation, mg/mL) CL _{int (liver)} = CL _{int (mic)} × Microsomal protein content in liver (mg/g) × Liver weight to body weight ratio

According to the well-stirred model, the intrinsic liver clearance and liver clearance rate can be converted using the following formula: CL _(liver) = (CL _int(liver) * _Qh ) / (CL _int(liver) + _Qh ). The parameters in the formula are shown in the table below. (Parameters in the data analysis formula) Species Liver weight to body weight ratio (g/kg body weight ) Hepatic blood flow (Q _h ) (mL/min/kg) Microsomal protein content (mg/g liver weight ) mice 88 90.0 45 rats 40 55.2 dog 32 30.9 monkey 30 43.6 people 20 20.7 Experimental Example 3: Hepatocyte Metabolic Stability Test

This experiment was used to test the metabolic stability of the compound in liver cells.

Prepare a 0.5 x ^10⁶ /mL hepatocyte suspension using preheated medium, and then add 198 μL of the preheated cell suspension to a 96-well plate. Add 2 μL of the test compound to each well of the 96-well plate to achieve a final concentration of 1 μM, and set up 2 duplicate wells. For samples at T=0 min, mix the compound and cells thoroughly for 1 min, and then immediately add 25 μL of the sample to 125 μL of termination solution (containing 200 ng/mL tolbutamide and 200 ng/mL labetalol in acetonitrile) in an ice bath and mix well. Simultaneously, place all plates in an incubator at 37°C with 5% _CO₂ and shake at 600 rpm. The samples were mixed at 15, 30, 60, and 90 minutes of incubation. 25 μL of the sample was added to 125 μL of termination solution (containing 200 ng/mL tolbutamide and 200 ng/mL labetalol in acetonitrile) in an ice bath, and the mixture was shaken at 500 rpm for 10 minutes. Subsequently, the samples were centrifuged at 3220 × g for 20 minutes at 4 °C. After centrifugation, 80 μL of supernatant from each well was transferred to another 96-well plate containing 240 μL of ultrapure water. The intrinsic clearance (CLint) and half-life (T1/2) were then analyzed and calculated by LC-MS/MS. Example 4: Rat Pharmacokinetic Assay

In this experiment, the pharmacokinetic behavior of the test compound in SD rats was investigated after administration by intravenous injection (IV) and oral gavage (PO).

On the day of drug administration, the actual body weight of the rats was measured and the volume of drug administered was calculated. Three rats were used in each group, and two groups were tested for each compound: one group received a single intravenous injection, and the other group received a single gavage administration. Whole blood samples were collected via jugular vein at specified times (0.25, 0.5, 1, 2, 4, 8, and 24 hours after drug administration). Immediately after collection, the blood samples were transferred to labeled commercially available tubes containing K2-EDTA (0.85–1.15 mg), centrifuged (3200 x g, 4°C, 10 minutes), and plasma was collected. The plasma was transferred to pre-cooled centrifuge tubes, rapidly frozen in dry ice, and then stored in an ultra-low temperature freezer at -60°C or lower until LC-MS/MS analysis was performed.

Plasma concentrations were determined using LC-MS/MS. WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process the plasma drug concentration data of the compounds using a non-compartmental model. Relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. Example 5 : Pharmacokinetic Testing in Mice

In this experiment, the pharmacokinetic behavior of the test compound in BALB/c mice was investigated after intravenous (IV) and oral (PO) administration.

On the day of drug administration, the actual body weight of the mice was measured and the volume of drug administered was calculated. Nine mice were used in each group, and two groups were tested for each compound: one group received a single intravenous injection, and the other group received a single gavage administration. Whole blood samples were collected via orbital sampling at specified times (0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after drug administration). Immediately after collection, the blood samples were transferred to labeled commercially available tubes containing K2-EDTA (0.85–1.15 mg), centrifuged (3200 x g, 4°C, 10 min), and plasma was collected. The plasma was transferred to pre-cooled centrifuge tubes, rapidly frozen in dry ice, and then stored in an ultra-low temperature freezer at -60°C or lower until LC-MS/MS analysis was performed.

Plasma concentrations were determined using LC-MS/MS. The plasma drug concentration data for the compounds were processed using a non-compartmental model with WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software. Relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. Compound number Drug delivery route Dosage (mg/kg) AUC _0-t (ng*h/mL) IV C _0h or P C _max (ng/mL) T _1/2 (h) C142-P3-B IV 2 9224 2360 5.6 PO 10 55042 6096 6.5 C146-P3-B IV 2 4530 2013 3.5 PO 10 28222 5299 4.1 Experimental Example 6 hERG Inhibition Test

HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg/mL G418 at 37°C and 5% _CO2 concentration. Cells were digested with TrypLE™ Express, centrifuged, and the cell density was adjusted to 2 × ^10⁶ cells/mL. The cells were then gently equilibrated on a shaker at room temperature for 15–20 min before patch-clamp detection. The prepared cell culture medium was replaced with extracellular fluid. Intracellular and extracellular fluids were aspirated from the liquid pool and added to the intracellular fluid pool and cell and test substance pool of the QPlate chip, respectively. Whole-cell patch-clamp recordings of whole-cell hERG potassium current voltage stimulation were performed, and the data were acquired and stored using Qpatch. The compound was started at 30 μM, diluted 3-fold, and six concentration sites were established, with each concentration administered twice over at least 5 minutes. The current detected in a compound-free extracellular solution was used as a control group, and each concentration was measured independently twice using at least two cells. All electrophysiological experiments were performed at room temperature.

Data analysis first standardized the current after each drug concentration was applied and the blank control current. Then calculate the inhibition rate corresponding to each drug concentration. Calculate the mean and standard error for each concentration, and calculate the half-inhibitory concentration (WIC) for each compound: The dose-dependent effect was nonlinearly fitted using the above equation, where Y represents the inhibition rate, C represents the test substance concentration, _IC50 is the half-inhibitory concentration, and HillSlope represents the Hill coefficient. Curve fitting and _IC50 calculation were performed using Graphpad software. Example 7: Cytochrome oxidase P450 inhibition test 1) Preparation of buffer solution:

100 mM K-Buffer: Mix 9.5 mL of stock solution A into 40.5 mL of stock solution B, adjust the total volume to 500 mL with ultrapure water, _and titrate the buffer with KOH or _H₃PO₄ to pH 7.4. Raw material A (1 M potassium dihydrogen phosphate): 136.5 g potassium dihydrogen phosphate in 1 L of water; Storage B (1 M potassium dihydrogen phosphate): 174.2 g potassium dihydrogen phosphate in 1 L of water. 2) Preparation of the test substance.

The test sample powder is prepared into a stock solution of a certain concentration using DMSO or other organic solvents, and then further diluted with a suitable organic solvent. 3) In vitro incubation

The in vitro incubation system for liver microsomes in CYP450 enzyme metabolic phenotype studies involves preparing liver microsomes, supplementing them with redox coenzymes, and then adding enzyme-specific selective inhibitors. Biochemical reactions are then conducted under simulated physiological temperature and environmental conditions. 4) Detection of the parent drug or metabolites

The concentrations of the parent drug or its metabolites in the incubation solution were determined by LC-MS/MS. Example 8: Mouse tumor pharmacodynamic model.

In this experimental example, the in vivo efficacy of the test compound in a mouse xenograft model was tested after administration by gavage (PO).

Human colonic adenocarcinoma cell line SW48 was cultured in vitro as a monolayer under the following conditions: DMEM medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin, at 37°C and 5% _CO2 . Cells were passaged twice weekly using trypsin digestion. When cell saturation reached 80%-90%, cells were harvested, counted, and seeded. 0.1 mL ( ^10⁷ cells) of SW48 cells were subcutaneously seeded into the right posterior back of each mouse. On day 14 post-seedling, when the average tumor volume reached approximately 200 ^mm³ , mice were randomly assigned to receive the drug via gavage once daily, and changes in body weight and tumor volume were recorded. The experiment was terminated after a certain number of days of drug administration. Changes in tumor volume and mouse body weight were statistically analyzed. Results showed that the compound in this application exhibits excellent tumor-inhibiting effects. Example 9: PXR Activation Test

In this experimental example, the activation effect of the test compound on PXR was investigated.

100 µL of DPX2 stable transgenic cells (4.5 × ^10⁵ cells/mL, prepared with Puracyp medium) were inoculated into each well of a 96-well cell culture plate, and the plate was then incubated at 37°C. After 24 hours, the 96-well plate was removed from the incubator, and the medium was replaced with 100 μL of the test compound (final concentrations of 10 µM and 30 µM), the positive control compound rifampin (final concentration of 10 µM), and the DMSO treatment group (final concentration of 0.1%), with 3 replicates. The plate was then returned to the incubator. After 24 hours, replace the medium with freshly prepared test compound and rifampicin solution, and return the test plate to the incubator. After 48 hours of compound treatment, remove the test plate from the incubator, discard the medium in the wells, wash twice with PBS, add 50 μL of medium containing 1x CellTiter-Fluor™ cell viability assay reagent, and incubate at 37°C for 30 minutes. Remove the test plate, equilibrate to room temperature, and detect the fluorescence signal under 400 nm excitation/505 nm emission conditions using a microplate reader. Subsequently, add 50 μL of medium containing ONE-Glo assay reagent to each well, shake to mix, incubate at room temperature for 5 minutes, and detect chemiluminescence using a microplate reader. Data were analyzed to evaluate the activation effect of the test compound on PXR. Results showed that the compound in this application did not exhibit significant PXR activation activity. Example 10 : Test of saturated solubility of the sample in FaSSIF solution. 1. Experimental procedure 1.1 Preparation of FaSSIF solution: Buffer (pH 6.5) preparation:

Weigh approximately 0.21 g of sodium hydroxide, approximately 2.24 g of sodium dihydrogen phosphate dihydrate, and 3.09 g of sodium chloride. Add 500 ml of water and dissolve. Adjust the pH to 6.5 with 1N sodium hydroxide or 1N hydrochloric acid.

Weigh 112 mg of FaSSIF solid into a 50 mL volumetric flask, dissolve it in the buffer solution described above, dilute to the mark, shake well, and let stand at room temperature for at least 2 hours. 1.2 Sample Preparation

Test sample: Take about 1 mg of the test sample, add 1 ml of FaSSIF solution, stir overnight at room temperature, centrifuge, and take the supernatant for analysis.

Reference solution: Accurately weigh approximately 1.5 mg of the test sample into a 50 mL volumetric flask, add DMF to dissolve and dilute to the mark, mix well to obtain the reference solution. 1.3 Preparation of mobile phase

Mobile phase A: Accurately measure 1000 mL of purified water, add 1 mL of formic acid, mix well, and degas by ultrasonication to obtain mobile phase A. Mobile phase B: Acetonitrile. 1.4 Chromatographic conditions Instruments HPLC Spectral Column Waters XBridge C18, 4.6*50 mm 3.5 um Sample tray (°C) off Column temperature (°C) 30 UV wavelength (nm) 254 Needle washing solution MeOH Flow rate (mL/min) 1.0 Running time(min) 6 Gradient elution table Time(min) Mobile phase A (%) mobile phase B (%) 0 90 10 0.5 90 10 6 10 90 7 10 90 7.1 90 10 10 90 10 Experimental Example 11 : Cocrystallization of small molecules with WRN protein

Purified recombinant WRN (500-945) protein (storage buffer: 50 mM HEPES, 500 mM NaCl, 0.5 mM TCEP, pH 7.5, 5% glycerol) was concentrated to 7 mg/ml and mixed with the corresponding compound dissolved in 100% DMSO to achieve a final concentration of 2 mM. After mixing, the mixture was incubated at 4°C for 2 hours and centrifuged at 12000 rpm for 10 minutes at 4°C on a benchtop eppendorf 5418 R cryogenic centrifuge. Crystallization was then performed on a Mosquito Xtal3 or Formulatrix NT8 pipette using commercially available kits such as Hampton Research's Crystal Screen/Crystal Screen 2, Index, PEG/Ion, PEGRx HT, etc. The initial crystals obtained were optimized around corresponding conditions. Finally, diffraction-ready crystals were obtained under the following conditions: 0.1 M sodium citrate pH 5.0; 8-12% w/v PEG 6,000, or 0.2 M potassium sodium tartrate; 0.1 M BIS-TRIS pH 6.5; 8-12% w/v PEG 10,000, or 0.1 M sodium citrate pH 5.0; 16-20% w/v PEG 20,000. After being cryoprotected with 20%-25% glycerol under crystallization conditions, the crystals were rapidly frozen in liquid nitrogen and sent to a synchrotron radiation source for data collection. Experimental Example 12 : CYP Induction Test 1) Preparation of Human Hepatocytes and Preparation of Spread Culture Medium

Prepare the following culture media in a biosafety cabinet and store them at 4°C before use:

Hepatocyte thawing medium (prepared by mixing the following components: Williams E medium, isotonic Percoll, DPBS, glutamic acid, HEPES, fetal bovine serum, human recombinant insulin, and dexamethasone).

Spread culture medium (prepared by mixing the following ingredients: Williams E medium, fetal bovine serum, dexamethasone, penicillin/streptomycin, human recombinant insulin, glutamic acid and HEPES).

Incubation medium (prepared by mixing the following components: Williams E medium, dexamethasone, ITS, penicillin/streptomycin, glutamic acid, and HEPES, serum-free). Hepatocyte thawing and processing.

Thaw a vial of frozen human liver cells in a 37°C water bath for 2 minutes. Wipe the vial with 70% alcohol in a biosafety cabinet. Transfer the liver cells to 50 mL of preheated liver cell thawing medium using a wide-mouth pipette tip. Add approximately 500 µL of liver cell thawing medium to thoroughly rinse the vial, cap it, and invert it several times.

Centrifuge at 150 g for 10 minutes at room temperature. Carefully aspirate the supernatant and dilute with plate medium to an inoculation density of 0.55 × 10⁶ cells/mL. Transfer 100 µL to each well of a collagen I-coated 96-well plate. Incubate the plate in an incubator at 37°C, 5% CO₂/95% air, and 95% relative humidity for 4–6 hours. Further treatment...

After incubation, observe cell morphology under a microscope. Gently shake the plate to loosen any debris and replace 125 µL of medium with 0.25 mg/mL Matrigel diluted in the incubation medium. Return the plate to the incubator and incubate for another 18 hours. The culture is then ready for induction studies. 2) Co- incubation with test compounds .

The test compound was prepared to a maximum working concentration of 1000× in DMSO and also to a maximum working concentration in incubation medium. The solubility of the compound in both solutions was determined visually. Stock solutions of the test compound, negative control, and positive control inducing agent at a final concentration of 1000× were then prepared in DMSO and diluted to their respective working concentrations using incubation medium preheated to 37°C. A negative control was prepared by adding 5 μL of DMSO to 5 mL of warm incubation medium. Sometimes, the test compound may be prepared using a higher concentration of DMSO, or directly to a final concentration in a medium containing 0.1% DMSO.

Test compound and positive control inducer (rifampin) concentration: 10 µM. Processing procedure.

Remove the liver cell plate from the incubator. Observe cell morphology under a microscope. Replace the medium in the corresponding wells with DMSO control, inducer, or test compound solution, with three replicates for each treatment. Incubate and change medium.

After 24 and 48 hours, the liver cell plates were removed from the incubator and the cell morphology was observed under a microscope. The medium was replaced with test compounds freshly diluted from DMSO stock solution. The plates were then returned to the incubator. The total incubation time was 72 hours. 3) mRNA preparation and RT-PCR mRNA preparation

mRNA was prepared and measured using the Cells-to-Ct kit purchased from Life Technologies. Residual CellTiter cell viability assay reagent was aspirated, and the cell monolayer was washed twice with 125 μL phosphate buffer, then the plate was placed on ice. DNase was added to the lysis buffer according to the instructions. 50 μL of lysis buffer was added to each well of the liver cell plate, and the lysis reaction was mixed by pipetting up and down 5 times. The lysis reaction was incubated at room temperature for 8 minutes, then 5 μL of termination buffer was added to each lysis reaction, and the mixture was mixed by pipetting up and down 5 times. The mixture was incubated at room temperature for another 2 minutes. The lysates can be stored at -20°C or -80°C for up to 5 months before RT reaction. Reverse transcription reaction.

Retrograde programming of the qPCR system: Retrograde (hold), 1 cycle, 37°C, 60 min; RT inactivation (hold), 1 cycle, 95°C, 5 min; Hold, 1 cycle, 4°C, indefinite time.

Prepare a mixture of 106 reactions in 15 mL tubes and then aliquot it into the corresponding wells of a 96-well PCR plate. Prepare one tube of mixture per plate.

Reverse transcriptase master mix: 2× RT buffer, 25 μL per reaction; 20× RT enzyme mixture, 2.5 μL per reaction; nuclease-free water, 7.5 μL per reaction; final volume of reverse transcriptase master mix: 35 μL.

Add 15 μL of sample lysate to each aliquot of the reverse transcriptase master mixture to bring the final reaction volume to 50 μL. The negative control (NC) is prepared by adding 15 μL of the mixture from the previous step (without cell incubation). Once assembled, gently mix the reaction mixture and then briefly centrifuge to collect the contents at the bottom of the reaction vessel.

Incubate samples using a qPCR system at 37°C for 60 minutes, then at 95°C for 5 minutes to inactivate the RT enzyme. Store the prepared RT samples at -20°C until the qPCR reaction is performed. Real-time PCR cycle .

Real-time PCR cycle planning for the qPCR system: Enzyme activation (hold), 1 cycle, 95°C, 5 min. PCR (cycle), 45 cycles, 95°C, 15 sec; 60°C, 1 min.

Prepare separate PCR cocktails for CYP 3A4; each cocktail contains a CYP-specific probe set and an ACTB probe set as an endogenous control gene.

PCR cocktail: Taqman gene expression master mix (2×), 10 μL per reaction; Taqman gene expression detection probe (20×, CYP3A4, FAM labeled), 1 μL per reaction; Taqman gene expression detection probe (20×, ACTB, VIC labeled), 1 μL per reaction; nuclease-free water, 4 μL per reaction; final volume of PCR cocktail, 16 μL.

At room temperature, aliquot the PCR cocktails into the wells of a real-time PCR plate. Dilute the cDNA samples 3-fold with nuclease-free water. Add 4 μL of diluted cDNA sample to each PCR cocktail aliquot, bringing the final volume to 20 μL. Cover the plate and mix gently. Briefly centrifuge to collect the contents at the bottom of the well. Add 4 μL of RT mixture (free of cell lysates) to each well of the PCR cocktail as a negative control. The standard curve template was prepared by continuously diluting the cDNA sample mixture of the corresponding rifampicin-induced sample at a 3-fold concentration.

Place the reaction in a qPCR system and start the real-time PCR cycle program. 4) Data analysis: mRNA level determination

All calculations were performed using Microsoft Excel. The mRNA content in each vial is expressed as 2Ct (ACTB) - Ct (CYP). The induction fold mRNA level was determined using the following formula: Induction fold = mRNA (inducible) / mRNA (solvent control). The results showed that the compound in this application did not exhibit significant CYP-inducing activity. Experimental Example 13 : Caco-2 Permeability Test 1) Cell Culture:

Caco-2 cells were cultured in DMEM medium at 37°C and 5% _CO2 . The DMEM medium contained 10% fetal bovine serum, penicillin-streptomycin solution (100 U/mL and 0.1 mg/mL), 1% MEM non-essential amino acids, and 25 μM HEPES. Cells were seeded in Transwell-24-well cell plates at a density of 1.00 × ^10⁵ cells/mL. Cells were cultured in a CO2 incubator for 21 days before being used in transport experiments, with the medium changed every three days. 2) Solution preparation:

The administration solutions were roxithromycin solution (high efflux reference), metoprolol solution (high osmotic reference), atenolol solution (low osmotic reference), and the test compound solution, respectively. Administration and receiving solutions were prepared using HBSS.

Solution preparation for the Apical to Basal (A-B) drug transport experiment: The A-end 10 µM dosing solution contained 5 µM fluorescent yellow and 0.4% DMSO; the B-end receiving solution contained 0.4% DMSO.

Solution preparation for the Basal to Apical (B-A) drug transport experiment: A-end receiving solution: 5 µM fluorescent yellow and 0.4% DMSO; B-end 10 µM dosing solution containing 0.4% DMSO. 3) Incubation steps: ① Quality control test before permeability test:

Before the experiment, the transmembrane resistance of the cells was measured using a resistance meter, and the apparent transmembrane resistance of a single cell membrane was calculated. ② Permeability Test:

Before the experiment, the cell culture medium in the culture plate was aspirated and then washed three times with HBSS buffer preheated to 37°C (Apical and Basal ends).

Drug A-B transport experiment: Remove the buffer solution in the plate, add 800 μL of HBSS buffer solution preheated to 37°C to end B, and add 600 μL of roxithromycin solution, metoprolol solution, atenolol solution and the solution of the test compound preheated to 37°C to end A respectively. Take 100 μL of the solution from end A as the 0-minute A-end sample and store it at -20°C for testing.

Drug B-A transport experiment: Remove the buffer solution in the plate, add 500 μL of HBSS buffer solution preheated to 37°C to end A, and add 900 μL of roxithromycin solution, metoprolol solution, atenolol solution and test compound solution preheated to 37°C to end B respectively. Take 100 μL of the solution at end B as the 0-minute B-end sample and store it at -20°C for testing.

Place the culture plate in a cell culture incubator and incubate for 90 minutes.

After incubation, 100 μL of solution was taken from both the A and B ends of each sample as a 90-minute sample and stored at -20°C for testing.

Standard curves for erythromycin, metoprolol, atenolol, and the analyte were prepared. All samples were mixed with acetonitrile containing the internal standard and analyzed by LC-MS/MS. ③ Permeability test quality control test (checking cell membrane integrity):

At 0 minutes, 100 μL of solution was extracted from the A end of A-B and B-A respectively and transferred to a black 96-well plate; after incubation for 90 minutes, 100 μL of solution was extracted from the B end of A-B and B-A respectively and transferred to a black 96-well plate. Fluorescence intensity was measured using a fluorescent microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 527 nm. 4) Data Analysis

Drug permeability: Apparent permeability coefficient (P _{_app} ) = (Receiver solution volume / (Membrane surface area × Time)) × (Drug concentration at the receiver at 90 minutes × Dilution factor) / (Drug concentration at the delivery end at 0 minutes × Dilution factor)

The time is the total transit time in seconds.

Recovery rate: Recovery rate % = 100 × (Drug concentration at the receiving end at 90 minutes × volume of solution at the receiving end × dilution factor + Drug concentration at the delivery end at 90 minutes × volume of solution at the delivery end × dilution factor) / (Drug concentration at the delivery end at 0 minutes × volume of solution at the delivery end × dilution factor).

Effluent ratio (ER) = Apparent permeation coefficient from BA (P _app ) / Apparent permeation coefficient from AB (P _app )

In addition to those described herein, various modifications of the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended patent applications. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

(without)

Claims (15)

A compound or a pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug thereof, wherein said compound has the structure of formula (I): (I) Wherein: --- indicates a single key or a double key, provided that the two double keys are not directly connected; ^W1 , ^W2 , ^W3 and ^W4 are each independently C or N, provided that C is connected to a double key; preferably, at least one of ^W1 and ^W2 is N, and/or at least one of ^W3 and ^W4 is N; Selected from , , and ^R1 , ^R3 , ^R21 , and ^R22 are each independently selected from H, deuterium, halogen, -OH, _-NH2 , -CN, _-NO2 , _-SF5 , = _CH2 , _C1-6 alkyl, deuterated _C1-6 alkyl, halogenated _C1-6 alkyl, _C2-6 alkenyl, _C2-6 ynyl, C3-6 cycloalkyl, 3-10 membered heterocyclic, _C6-10 aryl, _5-14 membered heteroaryl, _C6-12 aralkyl, -C(=O) ^Ra , -OC(=O)Ra, -C(=O) ^ORa , ^-ORa , ^-SRa , -S(=O) ^{Ra ,} -S(=O) _2Ra , -S( ₌ O) ^{2NR a} R ^b , -S(=O)(=NR ^a )R ^b , -NR ^a R ^b , -C(=O)NR ^a R ^b , -NR ^a -C(=O)R ^b , -NR ^a -C(=O)OR ^b , -NR ^a -S(=O) ₂ -R ^b , -NR ^a -C(=O)-NR ^a R ^b , -P(=O)R ^a R ^b , -C _1-6 alkylene-R ^a , -C _1-6 alkylene-OR ^a , -C _1-6 alkylene-NR ^a R ^b , -OC _1-6 alkylene-NR ^a R ^b , (-C _3-6 cycloalkylene)-CN and (-C _3-6 cycloalkylene)-C _1-6 alkyl; When m is greater than 1, two Rs located on the same ring atom or adjacent ring atoms ^3, together with any of its connected groups, constitutes a _C3-6 hydrocarbon ring, a 3-10 member heterocyclic ring, a _C6-10 aromatic ring, or a 5-14 member heterocyclic ring; ^R4 is... ^L2 is selected from -O-, -C(=O)-, -NRC(=O)-, -S-, -S(=O)-, -S(=O) _2- , _C1-6 alkylene, and -O-( _C1-6 alkylene)-; ^R41 is selected from _C3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; R, ^Ra , and ^Rb are each independently selected from H, _C1-6 alkyl, _C3-10 cycloalkyl, 3-10 heterocyclic, _C6-10 aryl, 5-14 heteroaryl, and _C6-12 aralkyl when they appear independently; ring B, ring X, and ring Z are each independently selected from C _3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; ring Y is absent or selected from _C3-6 hydrocarbon rings, 3-10 heterocyclic rings, _C6-10 aromatic rings, and 5-14 heterocyclic rings; when ring Y is absent, ^R22 is also absent; the above-mentioned alkylene, alkyl, alkenyl, alkynylene, cycloalkylene, cycloalkylene, hydrocarbon, hydrocarbon, heterocyclic, heterocyclic, aryl, aromatic, heteroaryl, heteroaryl, and aralkyl groups are each optionally substituted with one or more substituents independently selected from the following: deuterium, halogen, -OH, =O, _-NH2 -CN, _-NO₂ , = _CH₂ , C₁ _-6 alkyl, deuterated C₁ _-6 alkyl, C₂ _-6 alkenyl, C₂ _-6 ynyl, C₃ _-6 cycloalkyl, 3-10 membered heterocyclic, C₆ _-10 aryl, 5-14 membered heteroaryl, C₆ _-12 aralkyl, -C(=O) ^Rc , -OC(=O) ^Rc , -C(=O) ^ORc , ^-ORc , ^-SRc , -S(=O) ^Rc , -S(=O) _₂Rc ^, -S(=O) _₂NRcRd , ^-NRcRd , ^-C (=O) ^{NRcRd , -NRc} - ^C (=O) ^Rd , -NRc- ^C (=O) ^ORd , -NRc- ^S (=O) _₂ ^- R ^d , -NRc- ^C (=O) ^-NRcRd , _-C1-6 alkylene- ^ORc , _-C1-6 alkylene- ^NRcRd and _-OC1-6 alkylene ^{- NRcRd} , wherein the alkylene, ^alkyl , alkenyl, = _CH2 , alkynyl, cycloalkane, heterocyclic, aryl, heteroaryl and aralkyl are ^each further optionally substituted by one or more substituents independently selected from the following: halogen, -OH, =O, -C(=O)O-tert-butyl, _-NH2 , -CN, _-NO2 , _C1-6 alkyl, _C1-6 halogenated alkyl, C3-6 cycloalkane, _3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, C _6-12 aralkyl, -C _1-6 alkylene-C _3-6 cycloalkyl, -OC _1-6 alkyl, and -C _1-6 alkylene-OC _1-6 alkyl; ^Rc and ^Rd are each independently selected from H, C _1-6 alkyl, C _3-10 cycloalkyl, 3-10 membered heterocyclic, C _6-10 aryl, 5-14 membered heteroaryl, and C _6-12 aralkyl, wherein the alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, and aralkyl groups are further optionally substituted by one or more substituents independently selected from the following: halogen, -OH, =O, -C(=O)O-tert-butyl, _-NH2 , -CN, _-NO2 , C _1-6 alkyl, C _1-6 halogenated alkyl, C _3-6 cyclic hydrocarbons, 3-10 member heterocyclic hydrocarbons, _C6-10 aryl hydrocarbons, 5-14 member heteroaryl hydrocarbons, _C6-12 aralkyl hydrocarbons and _-C1-6 alkylene- _OC1-6 alkyl hydrocarbons; and p, q and m are each independently an integer selected from 1, 2 or 3. The compound of claim 1 or a pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug thereof, wherein said compound has the structure of the following formula: (II) (III) (IV) (V) (VI) (VII) (VIII) (IX) (X) (XI). The compound of claim 1 or its pharmaceutically acceptable salt, ester, stereoisomer, transtransfer isomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug, wherein: ring B is a _C3-6 hydrocarbon ring or a 3-10 member heterocyclic ring; preferably, ring B is a cyclopentene ring, a cyclohexene ring, a pyrrolidine ring, an oxazolidinyl ring, a piperidine ring, a cyclophosphine ring or a nitrogen-containing heptane ring. The compound or its pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound, or prodrug, as described in any of claims 1-3, wherein: ^R1 is independently selected each time from H, halogen, _C1-6 alkyl, _C3-6 cyclic hydrocarbon, 3-10 membered heterocyclic, _C6-10 aryl, 5-14 membered heteroaryl, -S(=O) _2Ra , ^-ORa , and ^{-NRaRb ,} wherein ^Ra and ^Rb are independently selected each time from H, _C1-6 alkyl, _C3-10 cyclic hydrocarbon, 3-10 membered heterocyclic, C6-10 alkyl, C6 ^... _6-10 aryl, 5-14 member heteroaryl, and _C6-12 aralkyl; preferably, ^R1 is independently selected from 3-10 member heterocyclic, _C6-10 aryl, 5-14 member heteroaryl ^, and ^-NRaRb each time it appears; wherein the alkyl, cycloalkane, heterocyclic, aryl, and heteroaryl groups are each optionally substituted by one or more substituents independently selected from the following: halogen, -S(=O) _2Rc , ^C1-6 alkyl, _C2-6 alkenyl, = _CH2 , _C3-6 cycloalkane, _3-10 member heterocyclic, _C6-10 aryl, and 5-14 member heteroaryl; wherein the alkyl, alkenyl, = _CH2 The cycloalkyl, heterocyclic, aryl, and heteroaryl groups are each optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl, _C3-6 cycloalkyl, 3-10 membered heterocyclic, and _-C1-6 alkylene- _C3-6 cycloalkyl; preferably, ^R1 is independently _C6-10 aryl, ^-NRaRb , each time it ^appears ; preferably, ^R1 is H, methyl, halogen, methoxy, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or The preferred option is , , , , , or The optimal location is ^R1 . , , or . Compounds of any one of claims 1-4, or pharmaceutically acceptable salts, esters, stereoisomers, transisomers, tautomers, polymorphs, solvents, metabolites, isotopically labeled compounds, or prodrugs thereof, wherein ring X is a benzene ring, a 5-6 member heterocyclic ring, or a 5-6 member heteroaromatic ring and ring Y is absent; or ring X is a benzene ring and ring Y is a 5-6 member heterocyclic ring or a 5-6 member heteroaromatic ring; preferably, for , , , , , , , , , , , , , , The preferred option is . The compound or its pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, polymorph, solvent, metabolite, isotopically labeled compound or prodrug, as requested in any of items 1-5, wherein ^R21 and ^R22 are each independently selected from H, halogen, -SF5, _C1-6 alkyl, _C3-6 cyclic hydrocarbon, 3-10 member heterocyclic, -O-( _C1-6 alkyl), -S( ₌ O) _2- ( _C1-6 alkyl), -S(=O) _2- ( _C3-6 cyclic hydrocarbon), -S(=O)(= ^NRa ) ^Rb , -P(=O)( _C1-6 alkyl) ₂ , (-C _(-C3-6- cyclic hydrocarbon)-CN and ( _-C3-6- cyclic hydrocarbon) _-C1-6 alkyl, wherein the alkyl, cyclic hydrocarbon, cyclic hydrocarbon, and heterocyclic group are each optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl, and halogenated _C1-6 alkyl; preferably, ^R21 and ^R22 , each in each occurrence, are each independently selected from H, halogen, _-SF5 , C1-6 alkyl, C3-6 cyclic hydrocarbon, 3-10-membered heterocyclic group, and -O-(C1-6 alkyl), wherein the alkyl, cyclic hydrocarbon, and heterocyclic group are each optionally substituted by one or more substituents independently selected from the following: halogen, _C1-6 alkyl, _C3-6 cyclic hydrocarbon, 3-10-membered heterocyclic group, and -O-( _C1-6 alkyl), wherein the alkyl, cyclic hydrocarbon, and heterocyclic group are each optionally substituted by one or more substituents independently selected from the following: halogen, C1-6 alkyl, C1-6 alkyl, C1-6 alkyl, C3-6 cyclic hydrocarbon, 3-10-membered heterocyclic group, and -O-(C1-6 alkyl). _1-6 alkyl and halogenated C _1-6 alkyl. The compound or its pharmaceutically acceptable salt, ester, stereoisomer, transtransfer isomer, tautomer, polymorph, solvent, metabolite, isotope-labeled compound or prodrug, as described in any of claims 1-6, wherein... Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and . The compound or its pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug of any of claims 1-7, wherein ^R3 is H, _C1-6 alkyl, ^-ORa or ^-SRa ; preferably, R3 is H or _C1-6 alkyl; more preferably, ^R3 is H, methyl, ethyl, -O- _CH3 or -S- _CH3 ; most preferably, ^R3 is H or methyl; when m is greater than 1, two ^R3s located on the same ring atom or adjacent ring ^atoms , together with the groups they are attached to, optionally constitute C _3-6 hydrocarbon rings (preferably cyclopropyl rings), wherein the hydrocarbon rings are optionally substituted by one or more substituents independently selected from the following: halogens, _C1-6 alkyl groups and halogenated _C1-6 alkyl groups. The compound of any one of claims 1-8, or a pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound, or prodrug thereof, wherein ring Z is a 3-10 membered heterocyclic ring or a benzene ring; preferably a 5-10 membered heterocyclic ring; more preferably a 5-6 membered heterocyclic ring; and wherein the heterocyclic ring and the benzene ring are each optionally substituted with one or more substituents independently selected from the following: halogen, _C1-6 alkyl, and halogenated _C1-6 alkyl; preferably, ring Z is , , , , , , , , , , , , , , , , , , , or . The compound of any one of claims 1-9 or a pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug thereof, wherein ^L2 is -C(=O)-, _C1-6 alkylene or -NRC(=O)-, wherein R is H or _C1-6 alkyl; preferably, ^L2 is -C(=O)-, _-CH2- , _-CD2- . The compound of any one of claims 1-10, or its pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound, or prodrug, wherein R ⁴¹ is selected from 3-10 member heterocyclic rings, _C6-10 aromatic rings, and 5-14 member heterocyclic rings, wherein each heterocyclic, aromatic, and heterocyclic ring is optionally substituted by one or more substituents independently selected from the following: halogen, -OH, _C1-6 alkyl, _-OC1-6 alkyl, and _-SC1-6 alkyl; preferably, the heterocyclic, aromatic, and heterocyclic rings are substituted by at least -OH or _-OC1-6 alkyl; preferably, ^-L2 -R ⁴¹ is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or . The compound or pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug of any of claims 1-11, wherein R ⁴¹ is a 5-6 member heteroaryl ring, preferably a 6-membered heteroaryl ring, more preferably a pyridine ring or pyrimidine ring, wherein it is substituted by at least one -OH; preferably, -L ² -R ⁴¹ is , or . The compound or pharmaceutically acceptable salt, ester, stereoisomer, transisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug of any of claims 1-12, wherein the compound is selected from: Compound number Structure Compound number Structure C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35 C36 C37 C38 C39 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 C51 C52 C53 C54 C55 C56 C57 C58 C59 C60 C61 C62 C63 C64 C65 C66 C67 C68 C69 C70 C71 C72 C73 C74 C75 C76 C77 C78 C79 C80 C81 C82 C83 C84 C85 C86 C87 C88 C89 C90 C91 C92 C93 C94 C95 C96 C97 C98 C99 C100 C101 C102 C103 C104 C105 C106 C107 C108 C109 C110 C111 C112 C113 C114 C115 C116 C117 C118 C119 C120 C121 C122 C123 C124 C125 C126 C127 C128 C129 C130 C131 C132 C133 C134 C135 C136 C137 C138 C139 C140 C141 C142 C143 C144 C145 C146 C147 C148 C149 C150 . A pharmaceutical composition comprising a preventive or therapeutically effective amount of any one of claims 1-13 of the compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug, and a pharmaceutically acceptable carrier. Use of a compound of any one of claims 1-13 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvent, metabolite, isotopically labeled compound or prodrug, or a pharmaceutical composition of claim 14, in the preparation of a drug for use as a WRN inhibitor, preferably, the drug for the prevention or treatment of cancer (preferably, the cancer is characterized by microsatellite instability-high (MSI-H) or mismatch repair defect (dMMR)); preferably, the cancer is selected from colorectal cancer, gastric cancer, endometrial cancer, uterine cancer, adrenocortical carcinoma, cervical cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, and ovarian cancer.