Crystal form of fused-ring nitrogen-containing compound, preparation method therefor and medical use thereof

Abstract

The present disclosure relates to a crystal form of a fused-ring nitrogen-containing compound, a preparation method therefor and the medical use thereof. Specifically, the present disclosure relates to a crystal form of a compound represented by general formula (I), a preparation method therefor, a pharmaceutical composition containing a therapeutically effective amount of the crystal form, and the pharmaceutical use thereof for treating diseases or disorders related to a specific target.

Description

Crystal form of a fused-ring nitrogen-containing compound, its preparation method and pharmaceutical uses Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to the crystal form of a fused-ring nitrogen-containing compound, its preparation method, and its applications. Background Technology

[0002] WRN is a synthetic lethal target in dMMR (mismatch repair deficient) / MSI-H (microsatellite instability-high) cancers. During DNA replication, normal cells mainly rely on mismatch repair proteins (such as MSH2 or MLH1) to repair mismatches. However, MSI-H tumor cells, due to a lack of mismatch repair genes or systemic defects, have a large number of (TA)n repeat sequences. When WRN enzyme activity is inhibited, a cross-shaped structure forms at the (TA)n repeat sequences, inducing DNA double-strand breaks and leading to apoptosis.

[0003] However, research on WRN inhibitors remains unsatisfied. This disclosure aims to provide WRN inhibitors and compounds for treating WRN-mediated diseases.

[0004] PCT / CN2024 / 102582 discloses the structures of a series of fused-ring nitrogen-containing compounds. In subsequent research and development, in order to facilitate the handling, filtration and drying of the products, and to seek suitable crystals that are easy to store and have long-term product stability, this disclosure conducts a comprehensive study on the crystal forms of the above-mentioned compounds. Summary of the Invention

[0005] All contents relating to patent PCT / CN2024 / 102582 are incorporated herein by reference.

[0006] This application provides a crystal form of the compound represented by general formula (I), and the structure of the compound is shown below:

[0007] in:

[0008] X1 and X2 are independently selected from CH or N;

[0009] R1 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0010] R2 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0011] R3 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0012] R4 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0013] R5 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0014] R6 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0015] R7 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0016] R8 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0017] R9 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl;

[0018] R 10 Selected from heterocyclic groups, wherein the heterocyclic group is a 5- or 6-membered fully saturated or partially unsaturated group, comprising a cyclic carbon atom and one or two cyclic heteroatoms selected from N, O and S, respectively.

[0019] In some embodiments of this disclosure, X1 and X2 are N;

[0020] R1 is selected from C1-C3 alkyl groups;

[0021] R2 is selected from F, Cl, Br, and I;

[0022] R3 is selected from C 1-3 Halogenated alkyl groups, preferably CF3;

[0023] R4 is selected from hydrogen;

[0024] R5 is selected from OH;

[0025] R6 is selected from C1-C3 alkyl groups;

[0026] R7 is selected from hydrogen;

[0027] R8 is selected from H or C1-C3 alkyl groups;

[0028] R9 is selected from C1-C3 alkyl groups;

[0029] R10 is selected from

[0030] In certain embodiments of this disclosure, the general formula (I) is selected from compound 1 or 2:

[0031] In certain embodiments of this disclosure, the crystal form is a solvent-containing or solvent-free crystal form, wherein the solvent is selected from water, methanol, acetone, ethyl acetate, acetonitrile, ethanol, 88% acetone, 2-methyltetrahydrofuran, dichloromethane, 1,4-dioxane, benzene, toluene, isopropanol, n-butanol, isobutanol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, n-propanol, tert-butanol, 2-butanone, 3-pentanone, n-heptane, ethyl formate, isopropyl acetate, cyclohexane, methyl tert-butyl ether, or isopropyl ether; preferably, the number of solvents is 0.2-3, more preferably 0.2, 0.5, 1, 1.5, 2, 2.5, or 3, and most preferably 0.5, 1, 2, or 3;

[0032] The crystal form is more preferably an anhydrous form.

[0033] In a preferred embodiment of this disclosure, a crystal form of compound 1 is provided, and the structure of compound 1 is as follows:

[0034] In a preferred embodiment of this disclosure, the crystal form of compound 1 is crystal form G.

[0035] In a further preferred embodiment of this disclosure, the powder X-ray diffraction pattern of the crystal form G has a characteristic peak at 2θ = 10.4 ± 0.2°, or at 2θ = 17.4 ± 0.2°, or at 2θ = 20.9 ± 0.2°, or at 2θ = 6.9 ± 0.2°, or at 2θ = 8.9 ± 0.2°, or at 2θ = 9.5 ± 0.2°, or at 2θ = 10.4 ± 0.2°. The characteristic peak is present at θ = 7.7 ± 0.2°, or at 2θ = 11.1 ± 0.2°, or at 2θ = 11.9 ± 0.2°, or at 2θ = 13.9 ± 0.2°, or at 2θ = 15.8 ± 0.2°, or at 2θ = 17.0 ± 0.2°; preferably, the characteristic peak is present at any of 2, 4, 6, 8, 10 or 12 of these locations.

[0036] The powder X-ray diffraction pattern of crystal form G has one or more characteristic peaks at 10.4±0.2°, 17.4±0.2°, 20.9±0.2° or 6.9±0.2°; preferably including 2-4 of these peaks, more preferably including 3-4, and most preferably including 4. Optionally, it may further include one or more characteristic peaks at 2θ of 8.9±0.2°, 9.5±0.2°, 7.7±0.2°, 11.1±0.2° or 11.9±0.2°, preferably including 2, 3, 4 or 5 of these peaks.

[0037] For example, the X-ray powder diffraction pattern of crystal form G shows diffraction peaks at the following positions with a 2θ value:

[0038] At 10.4±0.2°, 17.4±0.2° and 20.9±0.2°,

[0039] Alternatively, at 10.4±0.2°, 17.4±0.2°, 20.9±0.2°, and 6.9±0.2°,

[0040] Alternatively, at 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°.

[0041] In a further preferred embodiment of this disclosure, the X-ray powder diffraction pattern of crystal form G includes one or more diffraction peaks located at 2θ of 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°, 7.7±0.2° and 11.1±0.2°, 11.9±0.2° and 13.9±0.2°, 15.8±0.2°, and 17.0±0.2°; preferably, it includes diffraction peaks at any of 4, 6, 8, or 10 locations.

[0042] For example, the X-ray powder diffraction pattern of crystal form G shows diffraction peaks at the following positions with a 2θ value:

[0043] Alternatively, at 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°, 7.7±0.2° and 11.1±0.2°, 11.9±0.2° and 13.9±0.2°,

[0044] Alternatively, at 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°, 7.7±0.2° and 11.1±0.2°, 11.9±0.2° and 13.9±0.2°, 15.8±0.2°, and 17.0±0.2°,

[0045] The X-ray powder diffraction pattern of compound 1, crystal form G, expressed as 2θ angle and interplanar spacing d using Cu-Kα radiation is shown in Figure 1.

[0046] In a further preferred embodiment of this disclosure, the crystal form described above is a solvent-free or solvent-containing crystal form, wherein the solvent is selected from water, methanol, acetone, ethyl acetate, acetonitrile, ethanol, 88% acetone, 2-methyltetrahydrofuran, dichloromethane, 1,4-dioxane, benzene, toluene, isopropanol, n-butanol, isobutanol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, n-propanol, tert-butanol, 2-butanone, 3-pentanone, n-heptane, ethyl formate, isopropyl acetate, cyclohexane, methyl tert-butyl ether, or isopropyl ether, preferably the crystal is a hydrate.

[0047] In a further preferred embodiment of this disclosure, the number of solvents is 0.2-3, preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3.

[0048] This disclosure also provides a method for preparing crystal form G of the compound 1, specifically comprising the following steps:

[0049] Specifically, the steps include the following:

[0050] 1) Weigh an appropriate amount of free radical and dissolve it in a mixture of a good solvent and a poor solvent;

[0051] 2) Obtained by slowly evaporating a clear solution in a ventilated area;

[0052] in:

[0053] The preferred solvent is selected from tetrahydrofuran and dichloromethane;

[0054] The undesirable solvent is selected from n-heptane.

[0055] Another object of this disclosure is to provide a pharmaceutical composition comprising a therapeutically effective amount of the crystal form of the compound described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0056] Another object of this disclosure is to provide a method for preparing a pharmaceutical composition containing the aforementioned compound, comprising the step of mixing a crystal form of the aforementioned compound with one or more pharmaceutically acceptable carriers, diluents or excipients.

[0057] This disclosure further relates to the use of a crystal form of any of the foregoing compounds or the pharmaceutical composition thereof in the preparation of a medicament for treating cancer.

[0058] This disclosure further relates to the use of a crystal form of any of the foregoing compounds or the pharmaceutical composition thereof in the preparation of a medicament for treating a condition or disease that can be treated by WRN inhibition.

[0059] Furthermore, the cancer or condition or disease is selected from the group consisting of microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR), preferably, microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) selected from colorectal cancer, stomach cancer, prostate cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, and ovarian cancer. Attached Figure Description

[0060] Figure 1 shows the XRPD diagram of crystal form G of compound 1. Detailed Implementation

[0061] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0062] In this disclosure, alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc.

[0063] The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group. The present disclosure preferably includes methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuteralkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl. The hydroxy-substituted alkyl group may be 2-hydroxyisopropyl or 1-hydroxyethyl.

[0064] In this disclosure, a heterocyclic group refers to a saturated or partially unsaturated monocyclic or polycyclic heterocyclic group containing 3 to 20 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 10 ring atoms; even more preferably, it contains 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolyl, pyrrolidone, piperidin-2-keto, 3,4-dihydropyridine-2(1H)-keto, 4,5-dihydropyridazin-3(2H)-keto, azahexyl, oxacyclobutyl, oxacyclobutyl, oxacyclohexyl, imidazolyl, tetrahydrofuranyl, tetrahydrothiophene, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, etc.; preferably pyrrolidone, piperidin-2-keto, 3,4-dihydropyridine-2(1H)-keto, 4,5-dihydropyridazine-3(2H)-keto, aziridine, oxazine, dihydropyrrolidone, tetrahydrofuranyl, pyrazolyl, morpholinyl, etc. Piperazinyl and pyranyl; more preferably dihydropyrrolyl, pyrrolidone, piperidin-2-keto, 3,4-dihydropyridin-2(1H)-keto, 4,5-dihydropyridazin-3(2H)-keto, aziridine, oxetane, oxetane, morpholinyl, piperidinyl, piperazinyl Pyranyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups; wherein the spirocyclic, fused-ring, and bridged-ring heterocyclic groups involved are optionally connected to other groups by single bonds, or further cyclically linked to other cycloalkyl, heterocyclic, aryl, and heteroaryl groups by any two or more atoms on the ring.

[0065] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0066] In this disclosure, aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, more preferably 6- to 8-membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:

[0067] In this disclosure, alkoxy groups refer to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl is defined as described above, preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, and most preferably alkyl containing 1 to 3 carbon atoms. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group;

[0068] Non-limiting examples of alkoxy groups include propion-2-oxy, etc.

[0069] In this disclosure, alkyl halogroup refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above. Non-limiting examples of alkyl halogroups include: trifluoromethyl, trifluoroethyl;

[0070] Non-limiting examples of haloalkyl groups also include: difluoromethyl, 1,1,2,2-tetrafluoroethyl, perfluoroethyl, etc.

[0071] In this disclosure, a haloalkoxy group refers to an alkoxy group substituted with one or more halogens, wherein the alkoxy group is as defined above;

[0072] The halogenated alkoxy group can be fully halogenated or partially halogenated, and the number of halogens can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.; the halogen is preferably F, Cl, Br, I; for example, it can be trifluoromethoxy, difluoromethoxy, 1,1,2,2-tetrafluoroethoxy, perfluoroethoxy, etc.

[0073] In this disclosure, hydroxyalkyl means an alkyl group substituted with a hydroxyl group, wherein the alkyl group is as defined above.

[0074] In this disclosure, a haloalkyl means an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above.

[0075] In this disclosure, a haloalkoxy group refers to an alkoxy group substituted with one or more halogens, wherein the alkoxy group is as defined above.

[0076] "Hydroxy" refers to the -OH group.

[0077] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0078] "Amino" refers to -NH2.

[0079] “Cyano” refers to -CN.

[0080] "Nitro" refers to -NO2.

[0081] "THF" refers to tetrahydrofuran.

[0082] “EtOAc” refers to ethyl acetate.

[0083] "DMSO" refers to dimethyl sulfoxide.

[0084] "LDA" refers to lithium diisopropylamine.

[0085] "DMAP" refers to 4-dimethylaminopyridine.

[0086] “EtMgBr” refers to ethyl magnesium bromide.

[0087] “HOSu” refers to N-hydroxysuccinimide.

[0088] “EDCl” refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0089] "IPA" refers to isopropyl alcohol.

[0090] “MeOH” refers to methanol.

[0091] “EtOH” refers to ethanol.

[0092] "DMF" refers to N,N-dimethylformamide.

[0093] "DIPEA" refers to N,N-diisopropylethylamine.

[0094] “HEPES” refers to 4-hydroxyethylpiperazine ethanesulfonic acid.

[0095] The different terms such as "X is selected from A, B, or C", "X is selected from A, B, and C", "X is A, B, or C", and "X is A, B, and C" all express the same meaning, that is, X can be any one or more of A, B, and C.

[0096] "Optional" or "optionally" means that the event or situation described below may, but does not have to, occur, and the description includes the circumstances under which the event or situation may or may not occur.

[0097] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen atoms may be unstable when bonded to a carbon atom with an unsaturated bond (such as an alkene).

[0098] "Stereoheterogeneity" includes three categories: geometric heterogeneity (cis-trans heterogeneity), optical heterogeneity, and conformational heterogeneity.

[0099] As used herein, the name of a compound is intended to cover all possible isomer forms, including stereoisomers of the compound (e.g., enantiomers, diastereomers, racemic mixtures or mixtures thereof and any mixture thereof).

[0100] All hydrogen atoms described in this disclosure can be replaced by their isotope deuterium, and any hydrogen atom in the compounds of the embodiments involved in this disclosure can also be replaced by a deuterium atom.

[0101] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0102] Those skilled in the art will understand that the composition comprises at least one pharmaceutically acceptable excipient selected from fillers, disintegrants, lubricants, humectants, binders, preservatives, sweeteners, flavoring agents, emulsifiers, suspending agents, flow aids or thickeners, pharmaceutical solvents, stabilizers, or antioxidants. This means that the composition may contain one or more functional ingredients, such as fillers and disintegrants, or it may refer to one or more substances containing a single functional ingredient, such as two different types of fillers.

[0103] X-ray powder diffraction (XRPD) refers to the experimentally observed diffraction pattern or parameters derived from it, characterized by peak positions (x-axis) and peak intensities (y-axis). Those skilled in the art will understand that experimental errors depend on instrument conditions, sample preparation, and sample purity. In particular, it is known to those skilled in the art that X-ray diffraction patterns typically change with instrument conditions, and appropriate error tolerances for XRPD are: 2θ ± 0.5°; 2θ ± 0.4°; 2θ ± 0.3°; 2θ ± 0.2°. It is particularly important to note that the relative intensities of X-ray diffraction patterns can also vary with experimental conditions, so the order of peak intensities cannot be considered the sole or decisive factor. Furthermore, the influence of experimental factors such as sample height can cause an overall shift in peak angles, which is generally permissible. Therefore, those skilled in the art will understand that any crystal form with characteristic peaks identical or similar to those of the patterns disclosed herein falls within the scope of this disclosure.

[0104] "TGA" refers to thermogravimetric analysis (TGA) experiments.

[0105] "DSC" refers to the Differential Scanning Calorimetry (DSC) experiment.

[0106] "HPLC" refers to High Performance Liquid Chromatography (HPLC) experiments.

[0107] "PK" refers to pharmacokinetic (PK) experiments.

[0108] "KF" refers to the Karl Fischer moisture determination (KF) experiment.

[0109] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0110] Preparation of compounds

[0111] The following examples are used to explain this disclosure, but should not be considered as limiting the scope of this disclosure. If specific conditions for the experimental methods are not specifically described in the examples of this disclosure, they are generally based on the conventional or recommended conditions of the raw material and product manufacturers. Reagents not specifically named are commercially available conventional reagents.

[0112] Preparation Example 1

[0113] (R,E)-N-[2-chloro-4-(trifluoromethyl)phenyl]-2-[2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-[[1-(5-hydroxy-6-methylpyrimidin-4-carbonyl)-5-methylpyrrolidine-3-ylidene]methyl]-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl]acetamide, compound 1

[0114] Step 1: (R)-2-methyl-4-((4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methylene)pyrrolidine-1-carboxylic acid tert-butyl ester, 1-2

[0115] n-BuLi (2.5 M, 19.4 mL) was added to a solution of 2,2,6,6-tetramethylpiperidine (6.85 g, 48.5 mmol) in tetrahydrofuran (THF, 100 mL), and stirred at -30 °C for 0.5 h. After cooling to -78 °C, a solution of 4,4,5,5-tetramethyl-2-[(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methyl]-1,3,2-dioxaborane (10 g, 37.3 mmol) in THF (5 mL) was added and stirred for 0.5 h. Then, a solution of 1-1 (8.3 g, 44.8 mmol) in THF (50 mL) was added dropwise to the mixture, and the mixture was gradually heated to room temperature and stirred for 3 h. The mixture was then added to an aqueous solution of NH4Cl and stirred for 1 h. The mixture was filtered and concentrated under reduced pressure to remove THF. The residue was diluted with ethyl acetate (EA) and water, extracted once with EA, the organic layer was washed with brine, dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography to give 1–2 (8.6 g, yield 74.53%, Z / E mixture, Z / E ratio approximately 75 / 25). Mass calc C 16 H 28 BNO4 for 309.21 found:310.26(M+H) + ESI.

[0116] Step 2: (R,E)-2-methyl-4-((4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methylene)pyrrolidine-1-carboxylic acid tert-butyl ester, 1-2A

[0117] 1-2A was obtained by supercritical fluid chromatography (SFC) with a yield of 2 grams and a yield of 23.26%.

[0118] Preparative separation method: Instrument: WATERS150 preparative SFC (SFC-26); Column: ChiralPak AD, 250×30mm inner diameter, 10 μm; Mobile phase: A is carbon dioxide, B is ethanol, gradient: B 8%; Flow rate: 120 mL / min; Back pressure: 100 bar; Column temperature: 38℃; Wavelength: 220 nm.

[0119] Step 3: (R,E)-4-((4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)methylene)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester, 1-3

[0120] Int-2 (500 mg, 0.89 mmol) was suspended in 1,4-dioxane (15 mL), and H2O (1 mL), K3PO4 (567.81 mg, 2.67 mmol), and 1-2A (288.22 mg, 0.89 mmol) were added. The mixture was then degassed under argon for 10 min, followed by the addition of Pd(dppf)Cl2·DCM (36.14 mg, 44.58 μmol), and stirred overnight at 85 °C. The mixture was diluted with ethyl acetate (EA) and water, the organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography to give 1-3 (260 mg, 43.06% yield).

[0121] Mass calc C 32 H 36 ClF3N6O5 for 676.24,679.24 found:577.29,579.3(M-Boc+H) + ESI.

[0122] Step 4: (R,E)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-((5-methylpyrrolidine-3-ylidene)methyl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide, 1-4

[0123] 1-3 (80 mg, 0.05 mmol) was suspended in dichloromethane (DCM, 2 mL), and trifluoroacetic acid (TFA, 0.5 mL) was added. The mixture was stirred overnight at room temperature. The mixture was washed with an aqueous sodium bicarbonate solution, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product (RM) was concentrated under reduced pressure and used without purification.

[0124] Mass calc C 27 H 28 ClF3N6O3 for 576.19,578.18 found:577.3,579.4(M+H) + ESI.

[0125] Step 5: (R,E)-N-[2-chloro-4-(trifluoromethyl)phenyl]-2-[2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-[[1-(5-hydroxy-6-methylpyrimidin-4-carbonyl)-5-methylpyrrolidine-3-ylidene]methyl]-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl]acetamide, 1

[0126] 5-Hydroxy-6-methylpyrimidine-4-carboxylic acid (9.35 mg, 0.06 mmol), HOBT (16.39 mg, 0.12 mmol), and EDCI (23.26 mg, 0.12 mmol) were added to 1 mL of dichloromethane (DCM), followed by the addition of pyridine (14.39 mg, 0.18 mmol) at 0 °C. After stirring at 0 °C for 0.5 hours, 1-4 (35 mg, 0.06 mmol) was added and the mixture was stirred at room temperature for 2 hours. The crude product (RM) was purified by reversed-phase column chromatography (C18 column) to give product 1 (4.88 mg, yield 11.28%).

[0127] Mass calc C 33 H 32 ClF3N8O5 for 712.21,714.21 found:711.32,713.30(MH) - ESI.

[0128] 1 H NMR(400MHz,DMSO-d6)δ:11.60(br,1H),10.40(br,1H),8.64-8.61(m,1H),8.10-8 .06(m,1H),7.96(s,1H),7.74-7.72(m,1H),6.87-6.84(m,1H),6.31-6.25(m,1H), 5.40-5.34(m,2H),4.80-4.59(m,2H),4.28-4.27(m,3H),3.84-3.79(m,2H),3.08- 2.99(m,1H),2.87-2.73(m,2H),2.55-2.54(m,3H),2.40(s,3H),1.23-1.08(m,6H).

[0129] Preparation Example 2

[0130] (E)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-((1-(5-hydroxy-6-methylpyrimidin-4-carbonyl)-5,5-dimethylpyrrolidine-3-ylidene)methyl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide, compound 2

[0131] Step 1: 2,2-Dimethyl-4-oxo-pyrrolidine-1-carboxylic acid tert-butyl ester, 2-2

[0132] 2-1 (5 g, 23.22 mmol) and DMP (14.78 g, 34.84 mmol) were mixed in dichloromethane (DCM, 10 mL) and stirred overnight at room temperature. The mixture was quenched with water, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give 2-2 (4.1 g, 19.22 mmol, yield 82.78%).

[0133] Detection was performed by thin-layer chromatography (TLC) 2-2.

[0134] Step 2: 2-(3-(allyloxy)but-1-en-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane, 2-3

[0135] 2,2,6,6-Tetramethylpiperidine (3.18 g, 22.51 mmol) was dissolved in tetrahydrofuran (THF, 15 mL). After cooling to -30 °C, n-butyllithium (n-BuLi, 1.1 mL) was added and stirred for 30 min. After cooling to -78 °C, a solution of bis(4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl)methane (6.03 g, 22.51 mmol) in tetrahydrofuran (THF, 15 mL) was added and stirred for 30 min. Then, a solution of 2-3 (4 g, 18.76 mmol) in tetrahydrofuran (THF, 15 mL) was added dropwise to the mixture, and the mixture was stirred overnight at room temperature. The mixture was quenched to 0 °C with aqueous NH4Cl solution and stirred for 30 min. After the reaction, the mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give 2-3 (5.5 g, yield 86.95%, Z / E mixture, Z / E ratio approximately 65 / 35). 2-3 was detected by thin-layer chromatography (TLC).

[0136] Step 3: (E)-2,2-dimethyl-4-((4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methylene)pyrrolidine-1-carboxylic acid tert-butyl ester, 2-3A

[0137] 2-3A was obtained by supercritical fluid chromatography (SFC) with a yield of 1.5 g and a yield of 32.73%.

[0138] Preparative separation method: Instrument: MGⅡ preparative SFC (SFC-14); Column: ChiralPak IC, 250×30 mm inner diameter, 5 μm; Mobile phase: A is carbon dioxide, B is ethanol; Gradient: B 10%; Flow rate: 120 mL / min; Back pressure: 100 bar; Column temperature: 38℃; Wavelength: 220 nm; Cycle time: approximately 4 min.

[0139] Step 4: (E)-4-((4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)ylide)-2,2-dimethylpyrrolidine-1-carboxylic acid tert-butyl ester, 2-4

[0140] 2-[6-bromo-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]-N-[2-chloro-4-(trifluoromethyl)phenyl]acetamide (1 g, 1.78 mmol), (4E)-2,2-dimethyl-4-[(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) [Methyl]pyrrolidine-1-carboxylic acid tert-butyl ester (721.73 mg, 2.14 mmol), K3PO4 (1.14 g, 5.35 mmol), and Pd(dppf)Cl2.DCM (72.28 mg, 89.17 μmol) were dissolved in 1,4-dioxane (20 mL) and H2O (2 mL), degassed three times with N2 gas, and then reacted under N2 atmosphere at 85 °C with stirring. The mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by reversed-phase column chromatography (C18 column) to give products 2-4.

[0141] Mass calc C 33 H 38 O5N6ClF3 for 690.25,found:591.32(M-Boc+H) + ESI.

[0142] Step 5: (E)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-((5,5-dimethylpyrrolidone-3-ylidene)methyl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide, 2-5

[0143] Trifluoroacetic acid (TFA, 28.95 mg, 253.93 μmol, 0.5 mL) was added to a solution of 2-4 (390 mg, 253.93 μmol) in dichloromethane (DCM, 2 mL), and the mixture was stirred at room temperature for 3 hours. The mixture was extracted twice with an aqueous sodium bicarbonate solution, washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure to obtain the residue. The crude product was used directly in the next reaction without purification.

[0144] Mass calc C 28 H 30 O3N6ClF3 for 590.20,found:591.31(M+H) + ESI.

[0145] Step 6: (E)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-((1-(5-hydroxy-6-methylpyrimidin-4-carbonyl)-5,5-dimethylpyrrolidine-3-ylidene)methyl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide, compound 2

[0146] Pyridine (Py, 40.15 mg, 507.59 μmol, 40.89 μL) was added to a solution of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (39.12 mg, 253.80 μmol), HOBT (45.72 mg, 338.40 μmol), and EDCI (64.87 mg, 338.40 μmol) in dichloromethane (DCM, 10.06 mL) at 0 °C. The resulting solution was stirred at 0 °C for 15 min. Then, 2-5 (100.00 mg, 169.20 μmol) was added, and the resulting solution was stirred at room temperature for 18 h. The mixture was concentrated under reduced pressure and purified by reversed-phase column chromatography (C18 column) to give product compound 2 (50 mg, yield 40.64%).

[0147] Mass calc C 34 H 34 O5N8ClF3 for 726.23,found:725.20(MH) - ESI.

[0148] 1H NMR(400MHz,DMSO-d6)δ:10.901(br,1H),10.390(br,1H),8.614(s,1H),8. 079-8.058(m,1H),7.979-7.976(m,1H),7.742-7.716(m,1H),6.851(s,1H) ,6.179(s,1H),5.350(s,2H),4.414-4.266(m,4H),3.831-3.804(m,2H),2. 776-2.673(m,4H),2.464-2.389(m,5H),1.512(s,6H),1.145-1.127(m,3H).

[0149] Biological Experiment

[0150] WRN helicase assay

[0151] To evaluate the inhibitory properties of the disclosed compounds on WRN helicase activity, a fluorescence assay using branched DNA was established. In the helicase assay, internally produced His-WRN was used. 527-1072Fluorescent forked DNA prepared by annealing with OLIGOA-BHQ2 (TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC-BHQ2) and OLIGOA-TAMRA (TAMRA-GAACGAACACATCGGGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT was used as substrate. The test compounds were prepared into 2.5-fold serial dilutions at 7 gradient points using DMSO, and then 1.5 μl of each concentration was added to 48.5 μl of assay buffer (25 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, and 2.5 μg / ml BSA) for further dilution. 5 μl of 45 nM WRN protein and 5 μl of each diluted compound solution were transferred to a 384-well assay plate and pre-incubated at room temperature for 30 min. Triples of each compound concentration were added to the plate. Controls were included in the assay to validate the assay. Instead of the compound solutions, assay buffer containing 3% DMSO was added to the positive control (no inhibition), and no protein was added to the negative control (maximum inhibition). Forked DNA and ATP were diluted to 300 nM and 6 mM, respectively, with assay buffer. To initiate the reaction, 5 μl of dsDNA and ATP solution were added to the assay plate. After incubation at room temperature for 60 minutes, the plate was transferred to a Victor Nivo multimode plate reader (Perkin Elmer, Waltham, MA) and fluorescence output was monitored. Dose-response curves were plotted using a GraphPad Prism 9 (GraphPad Software, San Diego, CA) to determine the IC50s.

[0152] WRN ATPase assay

[0153] To evaluate the inhibitory properties of the disclosed compounds against DNA-dependent WRN ATPase activity, an ATPase assay was established using the ADP-Glo ​​assay kit (Promega, Madison, WI). In the ATPase assay, the same WRN protein and DNA substrate as described in the helicase assay were used. Serial dilutions of the compounds in DMSO were the same as in the helicase assay. In further dilutions, 2 μl of each concentration was transferred to 48 μl of assay buffer. 5 μl of 100 nM WRN protein and 2.5 μl of each diluted compound solution were pre-incubated in a 384-well plate at room temperature for 30 min. All test wells were set in triplicate. Then, 2.5 μl of assay buffer containing 400 nM DNA substrate and 4 mM ATP was added to each well and incubated for 60 min. The assay included a protein-free control (maximum inhibition) and a compound-free control (no inhibition). To stop the reaction and consume excess ATP, 10 μl of reagent from the assay kit was added. TM The sample was incubated for 40 minutes. Then, 20 μl of ATP assay reagent was added. After incubation for 30 minutes, luminescence was measured using a Victor Nivo multimode plate reader (Perkin Elmer, Waltham, MA). The dose-response curve, spanning IC50s, was generated by a GraphPad Prism 9 (GraphPad Software, San Diego, CA).

[0154] Cell viability assay

[0155] The high microsatellite instability (MSI) cell line SW48 and the microsatellite stable MSS cell line SW480 were purchased from ATCC and cultured in DMEM GlutaMax medium (Gibco, 10564-011) consisting of penicillin-streptomycin (Gibco-15140122) and 10% fetal bovine serum (Gibco-10082-147) at 37°C in a humidified 5% CO2 incubator.

[0156] Cells were seeded at 3000 cells / well in white, clear-bottomed 384-well plates (PerkinElmer). The next day, twelve serially diluted (3-fold) aliquots of 10 mM DMSO stock solution were added to each replicate well. Blank wells contained cell-free culture medium, and DMSO control wells contained only cells and DMSO. After 72 hours of treatment, [the cells were then used...] Cell viability was analyzed using a 2.0 assay (Promega, G9243), and luminescence signals were detected on a VICTOR Nivo™ multimodal plate reader (PerkinElmer) after incubation at room temperature for 10 minutes. For data analysis, all data points were normalized by subtracting blank values, and the ratio of DMSO control values ​​was calculated. IC50 values ​​were calculated using a GraphPad Prism 9. The IC50 values ​​are shown in the table below.

[0157] Table 1

[0158] In vivo pharmacodynamic studies and pharmacokinetic / pharmacodynamic (PKPD) analysis

[0159] method

[0160] In vivo efficacy studies were conducted using 8- to 10-week-old female BALB / c nude mice (Shanghai Bikai Keyi Biotechnology Co., Ltd.). SW48 human colorectal cancer cells (CBP60018, COBIOER) were cultured in DMEM medium (11995-065, Gibco) supplemented with 10% FBS (A5669701, Gibco) at 37°C in a 5% CO2 incubator. A mixture of 3 million SW48 cells / Matrigel (356234, Corning) was subcutaneously injected into the right upper quadrant of the mice. Following inoculation, mice carrying tumors were monitored periodically, and when tumors reached an appropriate size, mice were randomly assigned to treatment groups (n = 6 or 7 per group).

[0161] The test compound was dissolved in a 10% 2-hydroxypropyl-β-cyclodextrin (HPBCD) solution. Mice were administered the test compound via oral gavage (PO) daily (QD) at a dose of 10 mpk, 20 mpk, or 40 mpk.

[0162] Mice were weighed twice weekly and tumor volume was measured. Tumor volume (mm) 3 According to the formula 0.5*D*d 2 Calculate, where D is the tumor length and d is the tumor width.

[0163] Tumor growth inhibition rate (TGI%) was calculated using the formula [1 - (△tumor volume in the treatment group / △tumor volume in the control group)] * 100; tumor regression rate was calculated using the formula - (△tumor volume in the treatment group / tumor volume in the treatment group on day 0) * 100, where △tumor volume represents the tumor volume on the measurement day minus the tumor volume on day 0. Drug efficacy was calculated using the formula (△tumor volume in the treatment group / △tumor volume in the control group) * 100. GraphPad Prism 10 was used to analyze tumor growth and weight change data.

[0164] Blood samples were collected in 1.5 ml EDTA-K2 tubes at various sampling time points following the last dose of the drug. Sampling time points were 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours. Blood samples were centrifuged at 8000 rpm for 5 minutes at room temperature. Plasma samples were transferred to pre-labeled tubes and stored at -80°C. For blood pharmacokinetic analysis, 5 μl of plasma sample was taken and 100 μl of internal standard (IS, glipizide, 50 ng / ml) in methanol was added. The mixture was vortexed for 1 minute and then centrifuged at 5228 g for 10 minutes. The supernatant was analyzed by LC-MS / MS-24 (Triple Quad 6500). Pharmacokinetic data were analyzed using a GraphPad Prism 10.

[0165] Tumors were collected, isolated, and processed using a dry ice rapid freezing method 4 hours or 24 hours after the last dose of the drug.

[0166] For tumor PK analysis, tumor samples were homogenized with 3 volumes (v / w) of PBS. 20 μl of sample was taken and 400 μl of internal standard (IS, glipizide, 50 ng / ml) in methanol was added. The mixture was vortexed for 1 min and then centrifuged at 5228 g for 10 min. The supernatant was analyzed by LC-MS / MS-24 (Triple Quad 6500). PK data were analyzed using a GraphPad Prism 10.

[0167] For tumor PD analysis, tumor tissue was prepared in 3-volume RIPA buffer containing phosphatase / protease inhibitors and homogenized on ice. The homogenized samples were repeatedly mixed and incubated on ice for 30 minutes. Centrifugation at 14,000g for 20 minutes at 4°C was performed to precipitate cell debris. The supernatant was transferred to fresh tubes for protein quantification using the Pierce BCA Protein Quantification Kit (#23225) and followed by Western blot analysis.

[0168] 30 μg of tumor protein lysis buffer was loaded onto a pre-prepared gel (4-12% Bis-Tris Midi Gels, Thermofisher, WG1403BOX), transferred to a polyvinylidene fluoride (PVDF) membrane or a pre-wetted nitrocellulose (NC) membrane, blocked with 5% skim milk diluted in Tris-buffered saline / Tween (TBST) for 1 hour, and then incubated overnight at 4°C with diluted primary antibody. Antibodies used for detecting protein targets, including WRN antibody (8H3, #4665), phosphorylated histone H2A.X Ser139 antibody (20E3#9718), phosphorylated Chk2 (Thr68) antibody (C13C1#2197), and p21 Waf1 / Cip1 antibody (12D1#2947), were all purchased from Cell Signaling Technology. After washing in 0.1% TBST for 5 minutes, repeated three times, the membrane was incubated with secondary antibody diluted in TBST containing 5% skim milk powder in a spinner at room temperature for 1 hour. Following membrane washing, target proteins were detected using a Tanon 5200 chemiluminescence imaging system and the ECL method. Loading controls were scanned using an Odyssey infrared imager. Data on target protein expression levels in tumors were analyzed using a GraphPad Prism 10.

[0169] result

[0170] The compounds disclosed herein exhibit good tumor-suppressive activity, safety, and PK properties.

[0171] Compound 2, administered orally daily at 10 mg / kg, showed a significant tumor regression rate of approximately 47% on day 28. Under the experimental conditions described above, mice treated with a 10 mpk dose showed a body weight change on day 28 within ±15%, more preferably within ±10%. The AUClast (hr*ng / ml) of the disclosed compounds in plasma or tumor (10 mpk, PO, last dose in the efficacy study) ranged from 1000 to 40000. Tables 3-5 show detailed results for representative compounds. No significant weight loss was observed in any treatment group.

[0172] Table 2: Tumor Growth Inhibition Notes: i. * indicates tumor regression rate, meaning the tumor volume is less than the volume on day 0. ii. The formula for calculating the tumor growth inhibition rate (TGI%) is: [1 - (△tumor volume in the treatment group / △tumor volume in the control group)] * 100; iii. The formula for calculating the tumor regression rate is: -(△tumor volume in the treatment group / tumor volume in the treatment group on day 0) * 100, where △tumor volume represents the tumor volume on the measurement day minus the tumor volume on day 0.

[0173] Table 3: Weight Changes

[0174] 2. As shown in Table 4, the compounds disclosed herein have high exposure levels in plasma.

[0175] Table 4: PK Analysis of Efficacy Studies

[0176] Single-dose pharmacokinetics / pharmacodynamics (PKPD) of the SW48 xenograft model

[0177] Research Methods

[0178] The PKPD of the selected compounds was studied using the SW48 xenograft model.

[0179] SW48 human colorectal cancer cells (CBP60018, COBIOER) were cultured in DMEM medium (11995-065, Gibco) supplemented with 10% FBS (A5669701, Gibco) at 37°C in a 5% CO2 incubator.

[0180] BALB / c nude mice (female, 8-10 weeks old, Shanghai Bikai Keyi Biotechnology Co., Ltd.) were injected with a mixture of 3 million SW48 cells / Matrigel (356234, Corning). After inoculation, mice carrying tumors were monitored regularly, and when the tumors reached an appropriate size, the mice were randomly assigned to the treatment group (n=3 per group).

[0181] The test compound was dissolved in a 10% 2-hydroxypropyl-β-cyclodextrin (HPBCD) solution. Mice were administered the test compound via oral gavage (PO) daily (QD) at doses of 5 mpk, 10 mpk, 20 mpk, or 40 mpk.

[0182] Plasma PK sampling points were 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48 hours and 72 hours after drug administration. Tumor PK / PD sampling points were 0.5, 2, 4, 8, 24, 48 hours and 72 hours after drug administration.

[0183] At the PK sampling site, blood samples were collected into 1.5 ml EDTA-K2 tubes. Blood samples were centrifuged at 8000 rpm for 5 minutes at room temperature. Plasma samples were transferred to pre-labeled tubes and stored at -80°C. Tumor samples were collected, separated, and rapidly frozen with dry ice. For blood PK analysis, 5 μl of plasma sample was taken and 100 μl of internal standard (IS, glipizide, 50 ng / ml) in methanol was added. The mixture was vortexed for 1 minute and then centrifuged at 5228 g for 10 minutes. The supernatant was analyzed by LC-MS / MS-24 (Triple Quad 6500). PK data were analyzed using a GraphPad Prism 10.

[0184] At the tumor sampling point, the tumor was collected, separated, and processed using a dry ice rapid freezing method.

[0185] For tumor pharmacokinetic (PK) analysis, tumor samples were homogenized with 3 volumes (v / w) of PBS. 20 μl of sample was taken and 400 μl of internal standard (IS, glipizide, 50 ng / ml) in methanol was added. The mixture was vortexed for 1 min and then centrifuged at 5228 g for 10 min. The supernatant was analyzed by LC-MS / MS-24 (Triple Quad 6500). When the plasma concentration was below the limit of detection (1 ng / ml), a PK plot was constructed using half the limit of detection. Data were analyzed using a GraphPad Prism 10.

[0186] For tumor PD analysis, tumor tissue was prepared in 3-volume RIPA buffer containing phosphatase / protease inhibitors and homogenized on ice. The homogenized samples were repeatedly mixed and incubated on ice for 30 minutes. Centrifugation at 14,000g for 20 minutes at 4°C was performed to precipitate cell debris. The supernatant was transferred to fresh tubes for protein quantification using the Pierce BCA Protein Quantification Kit (#23225) and followed by Western blot analysis.

[0187] 30 μg of tumor protein lysis buffer was loaded onto a pre-prepared gel (4-12% Bis-Tris Midi Gels, Thermofisher, WG1403BOX), transferred to a polyvinylidene fluoride (PVDF) membrane or a pre-wetted nitrocellulose (NC) membrane, blocked with 5% skim milk diluted in Tris-buffered saline / Tween (TBST) for 1 hour, and then incubated overnight at 4°C with diluted primary antibody. Antibodies used for detecting protein targets, including WRN antibody (8H3, #4665), phosphorylated histone H2A.X Ser139 antibody (20E3#9718), phosphorylated Chk2 (Thr68) antibody (C13C1#2197), and p21 Waf1 / Cip1 antibody (12D1#2947), were all purchased from Cell Signaling Technology. After washing in 0.1% TBST for 5 minutes, repeated three times, the membrane was incubated with secondary antibody diluted in TBST containing 5% skim milk powder in a spinner at room temperature for 1 hour. Following membrane washing, target proteins were detected using a Tanon 5200 chemiluminescence imaging system and the ECL method. Loading controls were scanned using an Odyssey infrared imager. Data on target protein expression levels in tumors were analyzed using a GraphPad Prism 10.

[0188] result

[0189] The compounds disclosed herein exhibit superior PK properties.

[0190] The AUClast (hr*ng / ml) (10mpk, PO, last dose in a pharmacodynamic study) of the disclosed compounds in plasma or tumor ranges from 5,000 to 60,000, more preferably from 10,000 to 50,000.

[0191] In vivo pharmacodynamic studies and pharmacokinetic / pharmacodynamic (PKPD) analysis

[0192] method

[0193] HCT-116 human colorectal cancer cells (JNO-H0125, Guangzhou Genio Biotechnology Co., Ltd.) were cultured in McCoy's 5A medium (12230-031, Gibco) supplemented with 10% FBS (A5669701, Gibco) at 37°C in a 5% CO2 incubator.

[0194] In vivo efficacy studies of HCT-116 were conducted using 8-10 week old female BALB / c nude mice (Shanghai Bikai Keyi Biotechnology Co., Ltd.). 4 million human colorectal cancer HCT-116 cells were subcutaneously injected into the right upper quadrant of the mice. After inoculation, mice carrying tumors were monitored periodically, and when the tumors reached an appropriate size, the mice were randomly assigned to treatment groups (n=7 per group).

[0195] The test compound was dissolved in a 10% 2-hydroxypropyl-β-cyclodextrin (HPBCD) solution. Mice were administered the test compound via oral gavage (PO) daily (QD) at a dose of 10 mpk, 20 mpk, or 40 mpk.

[0196] Mice were weighed twice weekly and tumor volume was measured. Tumor volume (mm) 3 According to the formula 0.5*D*d 2 Calculate, where D is the tumor length and d is the tumor width.

[0197] Tumor growth inhibition rate (TGI%) was calculated using the formula [1 - (△tumor volume in the treatment group / △tumor volume in the control group)] * 100; tumor regression rate was calculated using the formula - (△tumor volume in the treatment group / tumor volume in the treatment group on day 0) * 100, where △tumor volume represents the tumor volume on the measurement day minus the tumor volume on day 0. Drug efficacy was calculated using the formula (△tumor volume in the treatment group / △tumor volume in the control group) * 100. GraphPad Prism 10 was used to analyze tumor growth and weight change data.

[0198] Blood samples were collected in 1.5 ml EDTA-K2 tubes at various sampling time points following the last dose of the drug. Sampling time points were 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours. Blood samples were centrifuged at 8000 rpm for 5 minutes at room temperature. Plasma samples were transferred to pre-labeled tubes and stored at -80°C. For blood pharmacokinetic analysis, 5 μl of plasma sample was taken and 100 μl of internal standard (IS, glipizide, 50 ng / ml) in methanol was added. The mixture was vortexed for 1 minute and then centrifuged at 5228 g for 10 minutes. The supernatant was analyzed by LC-MS / MS-24 (Triple Quad 6500). Pharmacokinetic data were analyzed using a GraphPad Prism 10.

[0199] Tumors were collected, isolated, and processed using a dry ice rapid freezing method 4 hours or 24 hours after the last dose of the drug.

[0200] For tumor PK analysis, tumor samples were homogenized with 3 volumes (v / w) of PBS. 20 μl of sample was taken and 400 μl of internal standard (IS, glipizide, 50 ng / ml) in methanol was added. The mixture was vortexed for 1 min and then centrifuged at 5228 g for 10 min. The supernatant was analyzed by LC-MS / MS-24 (Triple Quad 6500). PK data were analyzed using a GraphPad Prism 10.

[0201] For tumor PD analysis, tumor tissue was prepared in 3-volume RIPA buffer containing phosphatase / protease inhibitors and homogenized on ice. The homogenized samples were repeatedly mixed and incubated on ice for 30 minutes. Centrifugation at 14,000g for 20 minutes at 4°C was performed to precipitate cell debris. The supernatant was transferred to fresh tubes for protein quantification using the Pierce BCA Protein Quantification Kit (#23225) and followed by Western blot analysis.

[0202] 30 μg of tumor protein lysis buffer was loaded onto a pre-prepared gel (4-12% Bis-Tris Midi Gels, Thermofisher, WG1403BOX), transferred to a polyvinylidene fluoride (PVDF) membrane or a pre-wetted nitrocellulose (NC) membrane, blocked with 5% skim milk diluted in Tris-buffered saline / Tween (TBST) for 1 hour, and then incubated overnight at 4°C with diluted primary antibody. Antibodies used for detecting protein targets, including WRN antibody (8H3, #4665), phosphorylated histone H2A.X Ser139 antibody (20E3#9718), phosphorylated Chk2 (Thr68) antibody (C13C1#2197), and p21 Waf1 / Cip1 antibody (12D1#2947), were all purchased from Cell Signaling Technology. After washing in 0.1% TBST for 5 minutes, repeated three times, the membrane was incubated with secondary antibody diluted in TBST containing 5% skim milk powder in a spinner at room temperature for 1 hour. Following membrane washing, target proteins were detected using a Tanon 5200 chemiluminescence imaging system and the ECL method. Loading controls were scanned using an Odyssey infrared imager. Data on target protein expression levels in tumors were analyzed using a GraphPad Prism 10.

[0203] result

[0204] The compounds disclosed herein possess good tumor-suppressive activity, safety profile, and pharmacokinetic properties.

[0205] Regarding tumor suppression, under the experimental conditions described above, the tumor growth inhibition rate (TGI) of the 10 mpk dose group exceeded 50% on day 28. More preferably, the TGI exceeded 80% on day 28. Even more preferably, the tumor regression rate reached 10-80% on day 28.

[0206] Under the above experimental conditions, the weight change of mice treated with a 10 mpk dose on day 28 was within ±15%, more preferably within ±10%.

[0207] The AUClast (hr*ng / ml) of the disclosed compounds in plasma or tumor (10mpk, orally, last dose in a pharmacodynamic study) ranges from 1,000 to 50,000, more preferably from 5,000 to 40,000.

[0208] Table 5-7 shows detailed results for representative compounds.

[0209] Compound 1 and Compound 2 were administered orally at doses of 3, 10, and 20 mpk daily. On day 28, the 20 mpk dose of Compound 1 showed a 50.36% reduction, while the 3, 10, and 20 mpk doses of Compound 2 showed 44.56%, 41.32%, and 59.75% reductions, respectively. No significant weight loss was observed in any of the treatment groups.

[0210] Table 5: Tumor Growth Inhibition Notes: i. * indicates tumor regression rate, meaning the tumor volume is less than the volume on day 0. ii. The formula for calculating the tumor growth inhibition rate (TGI%) is: [1 - (△tumor volume in the treatment group / △tumor volume in the control group)] * 100; iii. The formula for calculating the tumor regression rate is: -(△tumor volume in the treatment group / tumor volume in the treatment group on day 0) * 100, where △tumor volume represents the tumor volume on the measurement day minus the tumor volume on day 0.

[0211] Table 6: Weight Changes

[0212] Table 7: PK Analysis of Efficacy Studies

[0213] Crystal form study

[0214] 1.1 Experimental Apparatus

[0215] 1.1.1 Physicochemical detection instruments and parameters used

[0216] 1.2 Preparation of the crystal form of compound 1

[0217] Example 1: Preparation of free crystal form G

[0218] Add 5 mg of compound 1 to a vial, add 1.5 ml of dichloromethane / n-heptane (2:1), stir at room temperature to dissolve, filter, and place in a fume hood to slowly evaporate, to obtain the free crystalline form G of compound 1.

Claims

1. A crystal form of a compound represented by general formula (I), the structure of which is shown below: in, X1 and X2 are independently selected from CH or N; R1 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R2 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R3 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R4 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R5 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R6 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R7 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R8 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R9 is selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 haloalkyl; R 10 Selected from heterocyclic groups, wherein the heterocyclic group is a 5- or 6-membered fully saturated or partially unsaturated group, comprising a cyclic carbon atom and one or two cyclic heteroatoms selected from N, O and S, respectively.

2. The crystal form of the compound according to claim 1, characterized in that, X1 and X2 are N; R1 is selected from C1-C3 alkyl groups; R2 is selected from F, Cl, Br, and I; R3 is selected from C 1-3 Halogenated alkyl groups, preferably CF3; R4 is selected from hydrogen; R5 is selected from OH; R6 is selected from C1-C3 alkyl groups; R7 is selected from hydrogen; R8 is selected from H or C1-C3 alkyl groups; R9 is selected from C1-C3 alkyl groups; R10 is selected from 3. The crystal form of the compound according to claim 1, characterized in that, The compound is selected from either compound 1 or compound 2 below:

4. The crystal form of the compound according to any one of claims 1-3, characterized in that, The crystal form is a solvent-containing or solvent-free crystal form, wherein the solvent is selected from water, methanol, acetone, ethyl acetate, acetonitrile, ethanol, 88% acetone, 2-methyltetrahydrofuran, dichloromethane, 1,4-dioxane, benzene, toluene, isopropanol, n-butanol, isobutanol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, n-propanol, tert-butanol, 2-butanone, 3-pentanone, n-heptane, ethyl formate, isopropyl acetate, cyclohexane, methyl tert-butyl ether, or isopropyl ether; preferably, the number of solvents is 0.2-3, more preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, and most preferably 0.5, 1, 2 or 3; The crystal form is more preferably an anhydrous form.

5. The crystal form according to claim 3, characterized in that, The crystal form is crystal form G of compound 1, wherein: The powder X-ray diffraction pattern of crystal form G has a characteristic peak at 2θ = 10.4 ± 0.2°, or at 2θ = 17.4 ± 0.2°, or at 2θ = 20.9 ± 0.2°, or at 2θ = 6.9 ± 0.2°, or at 2θ = 8.9 ± 0.2°, or at 2θ = 9.5 ± 0.2°, or at 2θ = 7.7 ± 0.2°. The peak has a characteristic peak at 2θ, or at 2θ = 11.1 ± 0.2°, or at 2θ = 11.9 ± 0.2°, or at 2θ = 13.9 ± 0.2°, or at 2θ = 15.8 ± 0.2°, or at 2θ = 17.0 ± 0.2°; preferably, the peak has a characteristic peak at any of the following 2, 4, 6, 8, 10 or 12 locations.

6. The crystal form according to claim 5, characterized in that, The powder X-ray diffraction pattern of crystal form G has one or more characteristic peaks at 10.4±0.2°, 17.4±0.2°, 20.9±0.2° or 6.9±0.2°; preferably including 2-4 of these peaks, more preferably including 3-4, and most preferably including 4. Optionally, it may further include one or more characteristic peaks at 2θ of 8.9±0.2°, 9.5±0.2°, 7.7±0.2°, 11.1±0.2° or 11.9±0.2°, preferably including 2, 3, 4 or 5 of these peaks. For example, the X-ray powder diffraction pattern of crystal form G shows diffraction peaks at the following positions with a 2θ value: At 10.4±0.2°, 17.4±0.2° and 20.9±0.2°, Alternatively, at 10.4±0.2°, 17.4±0.2°, 20.9±0.2°, and 6.9±0.2°, Alternatively, at 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°.

7. The crystal form according to claim 5, characterized in that, The X-ray powder diffraction pattern of crystal form G includes one or more diffraction peaks located at 2θ of 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°, 7.7±0.2° and 11.1±0.2°, 11.9±0.2° and 13.9±0.2°, 15.8±0.2°, and 17.0±0.2°; preferably, it includes diffraction peaks at any of 4, 6, 8, or 10 selected locations. For example, the X-ray powder diffraction pattern of crystal form G shows diffraction peaks at the following positions with a 2θ value: Alternatively, at 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°, 7.7±0.2° and 11.1±0.2°, 11.9±0.2° and 13.9±0.2°, Alternatively, at 10.4±0.2°, 17.4±0.2° and 20.9±0.2° and 6.9±0.2°, 8.9±0.2° and 9.5±0.2°, 7.7±0.2° and 11.1±0.2°, 11.9±0.2° and 13.9±0.2°, 15.8±0.2°, and 17.0±0.2°.

8. The crystal form according to claim 5, characterized in that, The X-ray powder diffraction pattern of crystal form G is shown in Figure 1.

9. The crystal form according to any one of claims 5-8, characterized in that, The 2θ error between the positions of the top ten diffraction peaks with the highest relative peak intensities in the X-ray powder diffraction pattern of crystal form G and the corresponding diffraction peaks in Figures 1-6 is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and more preferably ±0.2°.

10. A method for preparing crystal form G of the compound according to any one of claims 5-9, specifically comprising the following steps: 1) Weigh an appropriate amount of free radical and dissolve it in a mixture of a good solvent and a poor solvent; 2) Obtained by slowly evaporating a clear solution in a ventilated area; in: The preferred solvent is selected from tetrahydrofuran and dichloromethane; The undesirable solvent is selected from n-heptane.

11. A pharmaceutical composition comprising a therapeutically effective amount of the crystal form of any one of claims 1-9, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

12. A method for preparing a pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, comprising the step of mixing a crystal form of the compound of any one of claims 1-9 with one or more pharmaceutically acceptable carriers, diluents or excipients.

13. Use of the crystal form of the compound according to any one of claims 1-9 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating cancer.

14. Use of the crystal form of the compound of any one of claims 1-9 or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating a condition or disease that can be treated by WRN inhibition.

15. The use according to claim 13 or 14, wherein the cancer or the condition or disease is selected from the group consisting of microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR), preferably, microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) selected from colorectal cancer, gastric cancer, prostate cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, and ovarian cancer.

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