Thiazololactam-spiroheterocyclic compound and application thereof

Abstract

Thiazolone spirocyclic compounds, and their use in the preparation of medicaments for treating related diseases, specifically disclose the compound shown in formula (I) and its pharmaceutically acceptable salt.

Description

Technical Field

[0001] The present invention relates to a class of thiazolyllactamide and spiroheterocyclic compounds and their use in preparing drugs for treating related diseases, particularly compounds represented by formula (I) and pharmaceutically acceptable salts thereof. Prior Art

[0002] CN202110722003.6, application date: June 28, 2021; CN202111673614.2, application date: December 31, 2021; CN202210693548.3, application date: June 17, 2022.

[0003] The Ras / Raf / MEK / ERK pathway is a classic mitogen-activated protein kinase (MAPK) signaling cascade pathway that is involved in signal transduction after activation of various growth factors, cytokines, mitogens, and hormone receptors. It is one of the most important signal transduction pathways for controlling cell growth, differentiation, and survival.

[0004] Research has shown that abnormal activation of the Ras / Raf / MEK / ERK pathway, caused by mutation or amplification, is a key determinant of the development of various cancers. In human tumors, RAS mutations occur in approximately 22%, BRAF mutations in approximately 7%, and MEK mutations in approximately 1%. Therefore, key node proteins in this pathway have become important targets for cancer treatment (Cancer Discov. 2019, 9, 329-341). Currently, several BRAF and MEK1 / 2 inhibitors, as well as their combination regimens, have been approved by the US FDA for the treatment of cancers such as melanoma and BRAFV600E-mutant non-small cell lung cancer. However, the use of BRAF and MEK inhibitors targeting these upstream nodes can rapidly lead to drug resistance due to mutations or pathway reactivation, significantly limiting their clinical application.

[0005] Extracellular regulated protein kinases (ERKs), particularly ERK1 and ERK2, are key players and downstream nodes in the Ras / Raf / MEK / ERK pathway, and their overactivation is found in many human cancers. As the terminal signaling kinase in this pathway, ERK has yet to harbor drug-resistant mutations. Therefore, drugs targeting ERK kinases have the potential to overcome drug resistance that can arise after treatment with upstream inhibitors, making them a promising therapeutic strategy. However, research on ERK inhibitors remains in the clinical stage, and no ERK inhibitors have been approved for marketing.

[0006] In summary, there is an urgent need to develop safe and effective ERK inhibitors to meet the needs of tumor treatment. Summary of the Invention

[0007] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, R 1 and R 2 are independently selected from H and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R a; R 4, R 5, R 6 and R 7 are independently selected from H, F, Cl, Br, I and C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R c; n is 0 or 1; m is 1 or 2; Ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2 or 3 R d; R a and R c are independently selected from D, F, Cl, Br and I; R d is selected from F, Cl, Br, I, C 1-3 alkyl and C 1-3 alkoxy, wherein the C 1-3 alkyl and C 1-3 alkoxy are optionally substituted by 1, 2 or 3 R; R is selected from F, Cl, Br and I.

[0008] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, R 1 and R 2 are independently selected from H and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R a; R 4, R 5, R 6 and R 7 are independently selected from H, F, Cl, Br, I and C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R c; n is 0 or 1; m is 1 or 2; Ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2 or 3 R d; R a and R c are independently selected from D, F, Cl, Br and I; R d is selected from F, Cl, Br, I, C 1-3 alkyl and C 1-3 alkoxy, wherein the C 1-3 alkyl and C 1-3 alkoxy are optionally substituted by 1, 2 or 3 R; R is selected from F, Cl and Br.

[0009] In some embodiments of the present invention, R1 and R2 are independently selected from H, CH3 and CH2CH3, and CH3 and CH2CH3 are optionally substituted by 1, 2 or 3 Ras, and other variables are as defined in the present invention.

[0010] In some embodiments of the present invention, R1 and R2 are independently selected from H, CH3, CHF2, CD3 and CH2CH3, and other variables are as defined in the present invention.

[0011] In some embodiments of the present invention, R4, R5, R6 and R7 are independently selected from H, F, Cl, Br, I and CH3, and CH3 is optionally substituted by 1, 2 or 3 Rc, and other variables are as defined in the present invention.

[0012] In some embodiments of the present invention, R4, R5, R6 and R7 are independently selected from H, F, Cl, Br, I and CH3, and other variables are as defined in the present invention.

[0013] In some embodiments of the present invention, the above-mentioned R d is selected from F, Cl, Br, I, CH 3 and OCH 3, and the CH 3 and OCH 3 are optionally substituted by 1, 2 or 3 R, and other variables are as defined in the present invention.

[0014] In some embodiments of the present invention, the above-mentioned R d is selected from CH 3 and OCH 3, and the other variables are as defined in the present invention.

[0015] In some embodiments of the present invention, the ring A is selected from 、 and , said 、 and Optionally substituted by 1, 2 or 3 R d, and other variables are as defined in the present invention.

[0016] In some embodiments of the present invention, the ring A is selected from 、 and , other variables are as defined in the present invention.

[0017] In some embodiments of the present invention, the above structural unit Selected from , other variables are as defined in the present invention.

[0018] Some other solutions of the present invention are obtained by any combination of the above variables.

[0019] In some embodiments of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from: in, R 2, R 6 and R 7 are as defined herein.

[0020] The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof: .

[0021] The present invention also provides the use of the compound or a pharmaceutically acceptable salt thereof in preparing a drug for treating solid tumors.

[0022] The present invention provides a crystalline form A of WX001, characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°; .

[0023] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A, expressed as 2θ angle, contains at least 4 or 5 diffraction peaks selected from the following: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.

[0024] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A has characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.

[0025] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A, expressed in 2θ angles, comprises at least 6, 7 or 8 diffraction peaks selected from the following: 10.2080±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.

[0026] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A has characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.

[0027] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A, expressed in 2θ angles, comprises at least 9, 10, 11 or 12 diffraction peaks selected from the following: 10.2080±0.2000°, 14.4684±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 21.5713±0.2000°, 22.0420±0.2000°, 22.4540±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.

[0028] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A has characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 14.4684±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 21.5713±0.2000°, 22.0420±0.2000°, 22.4540±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°.

[0029] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A has characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 18.8429±0.2000°, and / or 20.6217±0.2000°, and / or 9.4003±0.2000°, and / or 10.4856±0.2000°, and / or 14.4684±0.2000°, and / or 15.0133±0.2000°, and / or 15.2687±0.2000°, and / or 15.6003±0.2000°, and / or 15.9518±0.2000°, and / or 16.6214±0.2000°, and / or 17.6747±0.2000°, and / or 17.9514±0.2000°, and / or 18.4703±0.2000°, and / or 19.1531±0.2000°, and / or 19.6571±0.2000°, and / or or 21.0531±0.2000°, and / or 21.2894±0.2000°, and / or 21.5713±0.2000°, and / or 22.0420±0.2000°, and / or 22.4540±0.2000°, and / or 23.1098±0.2000°, and / or 24.5027±0.2000°, and / or 25.0767±0.2000°, and / or 25.4797±0.2000°, and / or 25.8919±0.2000°, and / or 26.3255±0.2000°, and / or 26.9544±0.2000°, and / or 28.3997±0.2000°, and / or 29.0345±0.2000°, and / or 29.3507±0.2000°, and / or 33.5390±0.2000°, and / or 34.2457±0.2000°, and / or 37.9776±0.2000°.

[0030] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A has characteristic diffraction peaks at the following 2θ angles: 10.2080°, 10.4856°, 14.4684°, 15.0133°, 15.2687°, 15.9518°, 16.6214°, 17.6747°, 17.9514°, 18.4703°, 18.8429°, 19.1531°, 20.6217°, 21.0531°, 21.2894°, 21.5713°, 22.0420°, 22.4540°, 25.0767°, 25.4797°, 26.3255°, and 26.9544°.

[0031] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form A is basically as shown in Figure 1.

[0032] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A are shown in Table 1: Table 1 XRPD pattern analysis data of WX001 Form A serial number 2θ angle (°) Interplanar spacing (Å) Relative strength (%) serial number 2θ angle (°) Interplanar spacing (Å) Relative strength (%) 1 9.4003 9.41 4.31 18 21.2894 4.17 12.24 2 10.2080 8.67 100.00 19 21.5713 4.12 16.68 3 10.4856 8.44 18.12 20 22.0420 4.03 18.34 4 14.4684 6.12 12.63 twenty one 22.4540 3.96 15.00 5 15.0133 5.90 18.36 22 23.1098 3.85 2.96 6 15.2687 5.80 19.23 23 24.5027 3.63 4.08 7 15.6003 5.68 7.48 24 25.0767 3.55 37.76 8 15.9518 5.56 11.70 25 25.4797 3.50 23.82 9 16.6214 5.33 11.12 26 25.8919 3.44 9.21 10 17.6747 5.02 19.86 27 26.3255 3.39 12.81 11 17.9514 4.94 10.14 28 26.9544 3.31 7.34 12 18.4703 4.80 10.70 29 28.3997 3.14 5.21 13 18.8429 4.71 56.52 30 29.0345 3.08 7.36 14 19.1531 4.63 10.90 31 29.3507 3.04 4.04 15 19.6571 4.52 8.63 32 33.5390 2.67 3.43 16 20.6217 4.31 41.16 33 34.2457 2.62 7.39 17 21.0531 4.22 18.31 34 37.9776 2.37 2.33

[0033] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has an endothermic peak starting point at 241.0±3.0°C.

[0034] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in Figure 2.

[0035] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form A shows a weight loss of 0.83% at 150.0±3.0°C.

[0036] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is shown in Figure 3.

[0037] The present invention also provides the use of the above-mentioned crystal form A in the preparation of drugs for treating solid tumors.

[0038] Technical Effects

[0039] The compound of the present invention exhibits relatively good inhibitory activity against ERK1 and ERK2 enzymes; the compound of the present invention exhibits relatively good inhibitory activity against HT29 cell proliferation; the compound of the present invention has good solubility under different pH conditions; the compound of the present invention has excellent pharmacokinetic properties and tumor inhibition effects.

[0040] Definition and Description

[0041] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0042] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0043] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in neat solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in neat solution or a suitable inert solvent. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0044] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0045] Unless otherwise indicated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

[0046] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are within the scope of the present invention.

[0047] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0048] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" refers to the inability of a compound to rotate freely about double bonds or single bonds of ring carbon atoms.

[0049] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other.

[0050] Unless otherwise specified, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

[0051] Unless otherwise specified, the key is a solid wedge ( ) and dotted wedge bonds ( ) represents the absolute configuration of a stereocenter, with a straight solid bond ( ) and straight dashed bond ( ) indicates the relative configuration of the stereocenter, and a wavy line ( ) represents a wedge-shaped solid bond ( ) or a dotted wedge key ( ), or use a wavy line ( ) represents a straight solid bond ( ) or a straight dashed key ( ).

[0052] Unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to functional group isomers that are in dynamic equilibrium at room temperature and readily interconvert into each other. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via reorganization of some of the bonding electrons. A specific example of keto-enol tautomerism is the interconversion between pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0053] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0054] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0055] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If a single enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by using chromatography using a chiral stationary phase, optionally combined with chemical derivatization (e.g., formation of carbamates from amines).

[0056] The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more atoms comprising the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3H), iodine-125 ( 125I), or carbon-14 ( 14C). Another example is that deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced therapeutic efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0057] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0058] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent. Substituents may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that the group may or may not be substituted. Unless otherwise specified, the type and number of substituents may be any chemically feasible.

[0059] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0060] When the number of a linking group is 0, such as -(CRR) 0-, it means that the linking group is a single bond.

[0061] When the number of a substituent is 0, it means that the substituent does not exist. For example, -A-(R) 0 means that the structure is actually -A.

[0062] When a substituent is vacant, it means that the substituent is not present. For example, when X in AX is vacant, it means that the structure is actually A.

[0063] When one of the variables is a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0064] When a substituent's bond can cross-link to two or more atoms in a ring, the substituent can be bonded to any atom in the ring, e.g. or Indicates that the substituent R can be substituted at any position on the cyclohexyl group or cyclohexadiene. When the listed substituent does not specify the atom through which it is bonded to the substituted group, the substituent can be bonded through any atom. For example, a pyridyl substituent can be bonded to the substituted group through any carbon atom on the pyridine ring.

[0065] When the linking group is listed without specifying its linking direction, its linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form , you can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0066] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease accordingly with the number of connected chemical bonds, and become a group with a corresponding valence. The chemical bond connecting the site to other groups can be represented by a straight solid bond ( ), straight dashed line key( ), or a wavy line ( ) represents. For example, the straight solid bond in -OCH 3 represents the connection to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy line indicates that the phenyl group is connected to other groups through the 1 and 2 carbon atoms; Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least 、 、 、 These four connection methods, even if the H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become a corresponding monovalent piperidine group.

[0067] Unless otherwise specified, the number of atoms in a ring is generally defined as the number of ring members; for example, a "5-7 membered ring" refers to a "ring" having 5-7 atoms arranged around it.

[0068] Unless otherwise specified, the term "C 1-3 alkyl" refers to a linear or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of C 1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0069] Unless otherwise specified, the term "C 1-3 alkoxy" refers to an alkyl group containing 1 to 3 carbon atoms that is attached to the rest of the molecule through an oxygen atom. The C 1-3 alkoxy group includes C 1-2, C 2-3, C 3, and C 2 alkoxy groups. Examples of C 1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

[0070] The compounds of the present invention can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0071] Reference throughout this specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that at least one embodiment includes the particular referenced elements, structures, or characteristics described in connection with that embodiment. Thus, appearances of the phrases "in one embodiment," "in an embodiment," "in another embodiment," or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0072] In the present invention, Exo Up in the DSC spectrum indicates upward heat release.

[0073] The structures of the compounds of the present invention can be confirmed using conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed using conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) involves collecting diffraction intensity data from a cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation and a φ / ω scanning mode. After collecting the relevant data, the crystal structure can be further analyzed using a direct method (Shelxs97) to confirm the absolute configuration.

[0074] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0075] All solvents used in the present invention were commercially available and used without further purification.

[0076] The solvent used in the present invention is commercially available.

[0077] The present invention uses the following abbreviations: aq represents water; eq represents equivalent; DCM represents dichloromethane; PE represents petroleum ether; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; BOC represents tert-butyloxycarbonyl, which is an amine protecting group; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; Boc2O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; iPrOH represents 2-propanol; and mp represents melting point.

[0078] Compounds were named according to conventional nomenclature in the field or using ChemDraw® software. Commercially available compounds were named according to the supplier's catalog name.

[0079] The powder X-ray diffractometer (XRPD) method of the present invention Instrument model: PANalytical (PANalytical) X'Pert 3 X-ray diffractometer Test Method: Approximately 10 mg of sample was used for XRPD analysis. Table 1 XRPD test parameters parameter Setting value model X'Pert 3 X-ray Cu,kα,Kα1 (Å):1.540598;Kα2 (Å):1.544426;Kα2 / Kα1:0.50 X-ray tube settings 45 kV, 40 mA Diverging slits 1 / 8° Scan Mode continuous Scanning range (°2Theta) 3~40 Scan time per step (s) 46.7 Scan step (°2Theta) 0.0263 Testing time About 5 minutes

[0080] Differential Scanning Calorimeter (DSC) method of the present invention Instrument model: TA 2500 differential scanning calorimeter Table 3 DSC instrument parameters and test methods parameter Setting value method Linear temperature rise Sample tray Aluminum tray, with or without cover Temperature range 25℃-Set the end point temperature Scan rate (℃ / min) 10 Shielding gas Nitrogen

[0081] Thermogravimetric analysis (TGA) method of the present invention Instrument model: TA 5500 Thermogravimetric Analyzer Table 4 TGA instrument parameters and test methods parameter Setting value method Linear temperature rise Sample tray Aluminum tray, open Temperature range Room temperature - set the end point temperature Scan rate (℃ / min) 10 Shielding gas Nitrogen

[0082] Dynamic Vapor Sorption (DVS) method of the present invention Dynamic moisture sorption (DVS) curves were collected on a DVS Intrinsic plus from SMS (Surface Measurement Systems). Relative humidity at 25°C was calibrated using the deliquescent points of LiCl, Mg(NO₃)₂, and KCl. Table 5 DVS test parameters parameter Setting value temperature 25℃ Sample size 10-20 mg Shielding gas and flow rate N2, 200 mL / min dm / dt 0.002 % / min Minimum dm / dt balance time 10 minutes Maximum balancing time 180 minutes RH test range 0%RH-95%RH RH gradient 10% (0%RH-90%RH,90%RH-0%RH) 5% (90%RH-95%RH,95%RH-90%RH) Table 6 Moisture absorption evaluation classification Hygroscopicity classification ΔW% deliquescence Absorb enough water to form a liquid Highly hygroscopic ΔW%≥15% Hygroscopic 15%>ΔW%≥2% Slightly hygroscopic 2%>ΔW%≥0.2% No or almost no hygroscopicity ΔW% <0.2% Note: ΔW% indicates the weight gain of the test sample at 25 ± 1°C and 80 ± 2% RH. Simple diagram description

[0083] FIG1 shows the XRPD spectrum of WX001 Form A using Cu-Kα radiation. Figure 2 shows the DSC spectrum of WX001 Form A. Figure 3 shows the TGA spectrum of WX001 Form A. Figure 4 shows the DVS spectrum of WX001 Form A. FIG5 shows the tumor growth curves of human melanoma A375 model animals after administration of solvent and WX001, respectively. FIG6 shows the body weight change rate of human melanoma A375 model animals during drug administration. Implementation Method

[0084] The present invention is described in detail below by way of examples, but this is not intended to limit the present invention in any way. While the present invention has been described in detail herein, including specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0085] Reference Example 1

[0086] Step 1: Synthesis of compound A-1-2. To a reaction flask, add A-1-1 (150 g, 635.36 mmol, 1 eq), calcium chloride (70.51 g, 635.36 mmol, 1 eq), tetrahydrofuran (500 mL), and ethanol (1000 mL). Under nitrogen, sodium borohydride (48.07 g, 1.27 mol, 2 eq) was added, and the mixture was reacted at 20°C for 15 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, diluted with 15% aqueous citric acid (4000 mL), and extracted with ethyl acetate (4000 mL x 3). The organic phases were combined, washed with saturated brine (2000 mL), and dried over anhydrous sodium sulfate. Filter, and the filtrate was concentrated under reduced pressure to dryness to obtain the crude product. The crude product was purified by column chromatography to obtain A-1-2. 1H NMR (400 MHz, DMSO- d 6) δ (ppm) = 7.47 (s, 1H), 5.58 (br s, 1H), 4.52 (s, 2H).

[0087] Step 2: Synthesis of compound A-1-4. Add A-1-2 (102 g, 525.64 mmol, 1 eq) and 2-methyltetrahydrofuran (1000 mL) to a reaction flask. After purging the nitrogen atmosphere, cool the mixture to -70°C and slowly add lithium diisopropylamide (2 M, 525.64 mL, 2.0 eq). Stir at -70°C for 30 minutes, then slowly add a solution of A-1-3 (138.18 g, 788.46 mmol, 1.5 eq) in 2-methyltetrahydrofuran (400 mL) dropwise. Continue the reaction at -70°C for 1 hour. After completion of the reaction, quench the reaction mixture with saturated aqueous ammonium chloride (2000 mL) and extract with ethyl acetate (2000 mL x 4). The separated phases are combined to obtain the organic phase. The organic phase is washed with saturated brine (1000 mL) and dried over anhydrous sodium sulfate. Filter the mixture, and concentrate the filtrate under reduced pressure to obtain the crude product. The crude product was first purified by column separation and then purified by beating with methyl tert-butyl ether to obtain A-1-4. 1H NMR (400 MHz, DMSO- d 6) δ (ppm) = 6.41 (s, 1H), 5.43 (t, J = 5.6 Hz, 1H), 5.00 - 4.84 (m, 4H), 4.38 (d, J = 5.6 Hz, 2H), 1.12 (s, 9H).

[0088] Step 3: Synthesis of compound A-1-5. To a reaction flask, add A-1-4 (50 g, 135.39 mmol, 1 eq), azodicarbonyldipiperidine (40.99 g, 162.47 mmol, 1.2 eq), and tetrahydrofuran (500 mL). After purging with nitrogen, cool the mixture to 0°C and slowly add a solution of tributylphosphine (32.87 g, 162.47 mmol, 40.09 mL, 1.2 eq) in tetrahydrofuran (100 mL) dropwise. The mixture is allowed to react at 0°C for 1 hour. After completion of the reaction, water (500 mL) and saturated brine (500 mL) are added sequentially to the reaction solution, followed by extraction with ethyl acetate (500 mL x 2). The separated phases are combined to obtain the organic phase. The organic phase is washed with saturated brine (300 mL) and dried over anhydrous sodium sulfate. Filter, and the filtrate is concentrated under reduced pressure to obtain the crude product. The crude product was slurried with 500 mL of methyl tert-butyl ether, filtered, and the filtrate was collected and concentrated to obtain the crude product. The crude product was further slurried with 50 mL of n-hexane, filtered, and the filter cake was collected and dried to obtain A-1-5. 1H NMR (400 MHz, DMSO- d 6) δ (ppm) = 5.29 (d, J = 7.5 Hz, 1H), 4.88 - 4.75 (m, 3H), 4.60 (d, J = 12.9 Hz, 1H), 4.22 (d, J = 12.9 Hz, 1H), 1.26 (s, 9H).

[0089] Step 4: Synthesis of compound A-1-6. To a reaction flask, add A-1-5 (29 g, 82.55 mmol, 1 eq), tetrahydrofuran (250 mL), and water (50 mL). After purging the nitrogen atmosphere, add iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq), and react at 50°C for 18 hours. Then, add additional iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq), and continue the reaction at 50°C for another 6 hours. Upon completion of the reaction, the crude solution of A-1-6 was used directly in the next step.

[0090] Step 5: Synthesis of compound A-1-7. To the crude product solution of A-1-6, sodium carbonate (17.50 g, 165.11 mmol, 2 eq) was added. After purging the nitrogen atmosphere, di-tert-butyl carbonate (27.03 g, 123.83 mmol, 28.45 mL, 1.5 eq) was added. The mixture was allowed to react at 20°C for 12 hours. After completion, the reaction solution was poured into water (200 mL) and extracted with ethyl acetate (300 mL x 3). The separated layers were combined to obtain the organic phase. The organic phase was washed with saturated brine (300 mL x 3) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by column chromatography to obtain A-1-7. 1H NMR (400 MHz, DMSO- d 6) δ (ppm) = 5.46 (d, J= 5.9 Hz, 1H), 5.32 (d, J= 6.3 Hz, 1H), 4.64 (d, J= 6.1 Hz, 1H), 4.55 (d, J= 5.8 Hz, 1H), 4.49 (d, J= 9.4 Hz, 2H), 1.53 (s, 9H).

[0091] Step 6: Synthesis of compound A-1-8. A-1-7 (26.5 g, 76.32 mmol, 1 eq), glacial acetic acid (1.37 g, 22.90 mmol, 1.31 mL, 0.3 eq), and acetonitrile (260 mL) were added to a reaction flask. After purging with nitrogen, the mixture was heated to 50°C and a solution of sodium chlorite (32.48 g, 305.28 mmol, 85% purity, 4 eq) in water (70 mL) was added dropwise. After the addition was complete, the reaction was continued at 50°C for 12 hours. Additional sodium chlorite (8.97 g, 99.21 mmol, 1.3 eq) and glacial acetic acid (458.31 mg, 7.63 mmol, 436.49 μL, 0.1 eq) were then added, and the reaction was continued at 50°C for 6 hours. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium sulfite (150 mL), followed by the addition of water (90 mL). The organic phase was allowed to stand and separated, and the aqueous phase was extracted with ethyl acetate (90 mL). The combined organic phases were washed with saturated brine (90 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was homogenized with ethyl acetate:n-hexane (1:5, 120 mL), filtered, and the filter cake was dried to yield A-1-8. 1H NMR (400 MHz, CDCl 3) δ (ppm) = 5.61 (d, J = 6.6 Hz, 2H), 4.76 (d, J = 6.6 Hz, 2H), 1.66 (s, 9H).

[0092] Step 7: Synthesis of compound A-1. A-1-8 (10 g, 27.68 mmol, 1 eq) and dichloromethane (100 mL) were added to a dry reaction flask. Trifluoroacetic acid (41.04 g, 359.90 mmol, 26.65 mL, 13 eq) was then added at 0°C. The mixture was then allowed to react at 0°C for 0.5 hours. After completion, the reaction solution was slowly poured into a saturated aqueous sodium bicarbonate solution (1000 mL) and the pH was adjusted to 7-8. The mixture was extracted with dichloromethane (1000 mL x 3), and the separated phases were combined to obtain the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield A-1. 1H NMR (400 MHz, DMSO-d 6) δ (ppm) = 9.59 (s, 1H), 4.99-4.78 (m, 4H).

[0093] Reference Example 2

[0094] Step 1: Synthesis of compound B-1-2. To a reaction flask, add sodium hydroxide (590.8 g, 14.8 mol, 1.05 eq) and ice water (20 L), along with B-1-1 (2000.00 g, 14.07 mol, 1 eq), followed by iodomethane (2495.80 g, 17.59 mol, 1.25 eq). The mixture was reacted at 25°C for 2 hours. After completion of the reaction, 6N glacial hydrochloric acid was slowly added to the reaction flask, the pH adjusted to 6-7, and the mixture was stirred for 0.5 hour. The mixture was filtered and the filter cake collected. Acetonitrile (500 mL) was added to the filter cake, stirred for 0.5 hour, filtered, and the filter cake collected and dried to yield B-1-2. 1H NMR (400 MHz, DMSO- d 6) δ (ppm) = 12.69 (br s, 1H), 7.74 (br s, 1H), 2.45 (s, 3H), 1.86 (s, 3H).

[0095] Step 2: Synthesis of compound B-1-3. To a reaction flask at 25°C, add acetonitrile (15 L), B-1-2 (1500.00 g, 9.60 mol, 1 eq), and then phosphorus oxychloride (1840.00 g, 12.0 mol, 1.25 eq). Slowly raise the temperature to 62°C and react at 62°C for 12 hours. Pour the reaction solution into water (10.5 L), and adjust the pH to 6-7 with solid sodium bicarbonate. Extract with ethyl acetate (10.5 L), and separate the layers to obtain the organic phase. The organic phase is washed with saturated brine (7.5 L) and dried over anhydrous sodium sulfate. Filter, and concentrate the filtrate under reduced pressure to obtain B-1-3. 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 8.54 (s, 1H), 2.50 (s, 3H), 2.22 (s, 3H).

[0096] Step 3: Synthesis of compound B-1. To a reaction flask, add B-1-3 (100 g, 572.57 mmol, 1 eq), water (24.76 g, 1.37 mol, 24.76 mL, 2.4 eq), and acetonitrile (1000 mL). After purging the nitrogen atmosphere, sodium iodide (571.59 g, 3.81 mol, 6.66 eq) and trimethylsilyl chloride (186.61 g, 1.72 mol, 218.00 mL, 3 eq) were added sequentially. The mixture was reacted at 20°C for 14 hours. After completion of the reaction, dichloromethane (800 mL) and water (1200 mL) were added sequentially. Solid sodium bicarbonate was then added to adjust the pH to 6-7. The mixture was separated, and the aqueous phase was extracted once with dichloromethane (500 mL). The organic phases were combined and washed sequentially with saturated aqueous sodium sulfite (500 mL) and saturated brine (500 mL), then dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. n-heptane (0.5 L) was added to the crude product, stirred for 1 hour, filtered, and the filter cake collected to obtain B-1. 1H NMR (400 MHz, DMSO- d 6) δ (ppm) = 8.34 (s, 1H), 2.48 (s, 3H), 2.21 (s, 3H).

[0097] Example 1 Synthesis route:

[0098] Step 1: Synthesis of WX001-2 A-1 (500 mg, 1.92 mmol, 1 eq), WX001-1 (427.54 mg, 2.30 mmol, 1.2 eq), and N'N-dimethylformamide (3 mL) were added to the reaction flask. After purging the atmosphere with nitrogen, cesium carbonate (935.92 mg, 2.87 mmol, 1.5 eq) was added and the mixture was allowed to react at 25°C for 16 hours. After completion of the reaction, the reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL x 3). The separated phases were combined to obtain the organic phase. The organic phase was washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography on silica gel to obtain WX001-2. LCMS (m / z): 366, 368 [M+H] +.

[0099] Step 2: Synthesis of WX001-3 In a pre-dried reaction flask, the argon atmosphere was replaced with wet palladium on carbon (0.1 g, 819.15 μmol, 10% purity, 1 eq) and ethanol (20 mL). WX001-2 (300 mg, 819.15 μmol, 1 eq) was then added. The hydrogen atmosphere was replaced three times, and the reaction was stirred at 50°C and 50 psi for 24 hours. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography on silica gel to obtain WX001-3. 1H NMR (400 MHz, CDCl 3) δ (ppm) = 8.96 (s, 1H), 7.58 (t, J= 7.6 Hz, 1H), 7.19 (d, J= 7.6 Hz, 1H), 7.09 (d, J= 7.5 Hz, 1H), 5.26 (d, J= 7.6 Hz, 2H), 5.14 (s, 2H), 4.80 (d, J= 7.6 Hz, 2H), 2.55 (s, 3H).

[0100] Step 3: Synthesis of WX001-4 To a dry reaction flask, add WX001-3 (60 mg, 208.81 μmol, 1 eq), tetrahydrofuran (1 mL), and zinc chloride solution (0.7 M, 298.31 μL, 1 eq). Cool the mixture to -78°C and add lithium hexamethyldisilazane (1 M, 417.63 μL, 2 eq). React at 20°C for 1 hour to obtain reaction solution 1. Under nitrogen, a mixture of B-1 (55.57 mg, 208.81 μmol, 1 eq) and tetrakistriphenylphosphine palladium (7.24 mg, 6.26 μmol, 0.03 eq) in N'N-dimethylacetamide (1 mL) was heated to 50°C and then added dropwise to reaction solution 1. After the addition was complete, the mixture was allowed to react at 50°C for 1 hour. After completion of the reaction, the reaction solution was poured into water (5 mL) and extracted with dichloromethane (30 mL x 3). The separated phases were combined to obtain the organic phase. The organic phase was washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography on silica gel to obtain WX001-4. LCMS (m / z): 426.0 [M+H] +.

[0101] Step 4: Synthesis of WX001-5 To a reaction flask, WX001-4 (100 mg, 235.00 μmol, 1 eq), acetonitrile (1 mL), and water (0.5 mL) were added. The atmosphere was purged with nitrogen three times, followed by the addition of potassium monopersulfate (288.95 mg, 470.01 μmol, 2 eq). The mixture was allowed to react at 20°C for 14 hours. After completion, the reaction mixture was poured into saturated sodium thiosulfate (5 mL) and extracted with dichloromethane (30 mL x 3). The separated phases were combined to obtain the organic phase. The organic phase was washed with saturated sodium bicarbonate (20 mL) and saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography on silica gel to obtain WX001-5. LCMS (m / z): 458.0 [M+H] +.

[0102] Step 5: Synthesis of WX001 To a dry reaction flask, add WX001-5 (40 mg, 87.43 μmol, 1 eq), C-1 (16.98 mg, 174.85 μmol, 2 eq), and tetrahydrofuran (0.5 mL). After replacing the nitrogen atmosphere, cool the reaction mixture to 0°C, and then add lithium hexamethyldisilazane (1 M, 166.11 μL, 1.9 eq) dropwise. The mixture reacts at 0°C for 1 hour. After completion, the reaction mixture is poured into water (5 mL) and extracted with dichloromethane (30 mL x 3). The organic phases are separated and combined. The organic phases are washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*25mm*5μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; B (acetonitrile)%: 20%-50%, 10 minutes) to obtain WX001. 1H NMR (400 MHz, DMSO- d 6) δ = 9.67 (br s, 1H), 8.62 (s, 1H), 7.64 (t, J= 7.7 Hz, 1H), 7.43 (d, J= 1.8 Hz, 1H), 7.14 (dd, J= 2.9, 7.7 Hz, 2H), 6.37 (d, J= 1.5 Hz, 1H), 5.12 (d, J= 7.2 Hz, 2H), 5.02 (s, 2H), 4.85 (d, J= 7.2 Hz, 2H), 3.74 (s, 3H), 2.57 (s, 3H), 2.42 (s, 3H);LCMS (m / z): 475.0 [M+H] +.

[0103] Example 2: Preparation of WX001 Form A

[0104] Step 1: Synthesis of compound I-1-3. Add tetrahydrofuran (12 L) and I-1-1 (1200 g, 5.69 mol) to a reactor. Slowly add tetramethylethylenediamine (661.59 g, 5.69 mol) to the reactor, purge the nitrogen atmosphere, and cool to -70°C (internal temperature). Slowly add lithium diisopropylamide (2 M, 6.83 L) dropwise, and stir at -70°C for 0.5 hour. Slowly add a solution of I-1-2 (1.76 kg, 9.39 mol) in tetrahydrofuran (4.8 L) dropwise over 1.5 hours, and allow the mixture to react at -70°C for 1 hour. After completion of the reaction, quench the reaction by adding water (6 L). Rinse the reaction flask with water (2.4 L), combine the mixed solutions, and stir. After the temperature rises to 10°C, separate the layers, and extract the aqueous phase with ethyl acetate (6 L). The remaining aqueous phase was adjusted to pH 3-4 with aqueous potassium bisulfate (15.6 L) and extracted three times with ethyl acetate (6 L). The combined organic phases were washed with saturated brine (6 L). The organic phase was dried over 1200 g of anhydrous sodium sulfate (m / m = 1:1) and concentrated under reduced pressure at 45°C to yield the crude product. Dichloromethane (2.4 L) and isopropyl ether (7.2 L) were added to the crude product and stirred at room temperature for half an hour. The filter cake was collected by filtration to yield I-1-3. 1H NMR (400 MHz, DMSO-d 6) δ = 13.18 (br s, 1H), 6.20 (br s, 1H), 4.93 - 4.82 (m, 4H), 1.09 (s, 9H).

[0105] Step 2: Synthesis of compound I-1-4. To the reactor, dichloromethane (1330 mL), I-1-3 (1330 g, 1.99 mol, crude), and 4-dimethylaminopyridine (41.37 g, 338.61 mmol) were added sequentially. N,N'-carbonyldiimidazole (419.86 g, 2.59 mol) was added portionwise, and the mixture was reacted at 50°C for 36 hours. After completion of the reaction, the reaction mixture was adjusted to a pH of 3-4 by adding 2 N aqueous hydrochloric acid (5.32 L). The mixture was stirred for 0.5 hour, and then water (5.32 L) was added to concentrate the mixture to remove the organic solvent. The residue was filtered to obtain a filter cake. The filter cake was stirred with aqueous sodium bicarbonate (5.32 L) for 0.5 hour, filtered, and washed with water (2.66 L). Anhydrous ethanol (10.64 L) was added to the crude product, stirred for 2 hours, filtered, and the filter cake was washed with anhydrous ethanol (1.3 L). The filter cake was collected and dried to give I-1-4. 1H NMR (400 MHz, DMSO- d 6) δ = 9.58 (br s, 1H), 4.94 - 4.84 (m, 4H).

[0106] Step 3: Synthesis of compound I-1-6. To a reaction flask, add I-1-4 (283 g, 1.03 mol), cesium carbonate (505.36 g, 1.55 mol), and N, N'-dimethylformamide (2800 mL). After purging the nitrogen atmosphere, add I-1-5 (221.24 g, 1.19 mol). The mixture is allowed to react at 20°C for 12 hours. After completion of the reaction, slowly pour the reaction solution into ice water (14 L), stir for 1 hour, and filter to collect the filter cake. Add methyl tert-butyl ether (5 L) to the crude product, stir for 2 hours, filter, collect the filter cake, and dry to obtain I-1-6. 1H NMR (400 MHz, DMSO- d 6) δ = 7.63 (t, J = 7.7 Hz, 1H), 7.13 (dd, J = 2.8, 7.7 Hz, 2H), 5.04 (d, J = 7.4 Hz, 2H), 4.98 (s, 2H), 4.86 (d, J = 7.4 Hz, 2H), 2.41 (s, 3H).

[0107] Step 4: Synthesis of compound I-1. Three parallel reactions were performed. I-1-6 (183 g, 468.60 mmol) and tetrahydrofuran (2745 mL) were added to a reaction flask. After purging with nitrogen, diisopropylethylamine (181.69 g, 1.41 mol) and diethyl phosphite (194.14 g, 1.41 mol) were slowly added dropwise at 20°C. The mixture was reacted at 40°C for 16 hours. After completion of the reaction, the reaction mixture was diluted with water (915 mL) and extracted with dichloromethane (1830 mL x 3). The organic phase was washed with saturated brine (915 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness at 45°C to obtain the crude product. A mixture of tert-methyl ether and n-hexane (1098 mL total, 1:5 volume ratio) was added to the crude product, stirred for 1 hour, filtered, and the filter cake was collected and dried. Water (5400 mL) was added to the filter cake, stirred for 1 hour, filtered, and the filter cake was collected and dried to obtain I-1. 1H NMR (400 MHz, DMSO- d 6) δ = 9.32 (s, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.13 (t, J = 8.4 Hz, 2H), 5.10 (d, J = 7.4 Hz, 2H), 4.99 (s, 2H), 4.81 (d, J = 7.5 Hz, 2H), 2.42 (s, 3H).

[0108] Step 5: Synthesis of compound I-3. To two reaction flasks, add I-1 (120 g, 403.05 mmol) and a tetrahydrofuran solution (1200 mL) of zinc chloride (0.7 M, 575.79 mL), respectively. After nitrogen is purged, the temperature is lowered to 0 °C. Lithium hexamethyldisilazane (1 M, 806.11 mL) is slowly added dropwise. The temperature is naturally raised to 20 °C and stirred for 1 hour. This is reaction solution 1. To another reaction flask, B-1 (107.25 g, 403.05 mmol), tetrakistriphenylphosphine palladium (13.97 g, 12.09 mmol) and N,N'-dimethylformamide (600 mL) were added. The reaction solution was heated to 50 °C to obtain reaction solution 2. Reaction solution 1 was slowly added dropwise to reaction solution 2, and the mixture was reacted at 50 °C for 1 hour. After the reaction was completed, the reaction was quenched with 0.1M disodium ethylenediaminetetraacetic acid (10800 mL) and stirred for 30 min. n-heptane (4800 mL) was added and stirred for 0.5 hour. The mixture was filtered, the filter cake was collected, and the crude product was dried. The crude product was slurried with ethanol (7200 mL) at 20 °C for 2 hours, filtered, the filter cake was collected, and the filter cake was air-dried to obtain I-3. 1H NMR (400 MHz, CDCl 3) δ = 8.54 (s, 1H), 7.54 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.07 (d, J = 7.3 Hz, 1H), 5.29 (d, J = 7.5 Hz, 2H), 5.11 (s, 2H), 4.84 (d, J = 7.6 Hz, 2H), 2.73 (s, 3H), 2.66 (s, 3H), 2.52 (s, 3H).

[0109] Step 6: Synthesis of compound I-4. To a reaction flask, add I-3 (120 g, 282.00 mmol), acetonitrile (110 mL), and water (550 mL). After purging the nitrogen atmosphere, add potassium monopersulfate (329.40 g, 535.81 mmol), and incubate the mixture at 30°C for 12 hours. After completion, add 200 mL of ice-water mixture to the reaction solution, followed by 600 mL each of saturated sodium bicarbonate and sodium thiosulfate solutions, and then 600 mL of water. Filter, collect the filter cake, and dry to obtain the crude product. Add 600 mL of anhydrous ethanol to the crude product, stir for 1 hour, filter, and collect the filter cake to obtain I-4. 1H NMR (400 MHz, CDCl 3) δ = 8.90 (s, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.06 (d, J = 7.7 Hz, 1H), 5.31 (d, J = 7.6 Hz, 2H), 5.11 (s, 2H), 4.84 (d, J = 7.6 Hz, 2H), 3.44 (s, 3H), 2.92 (s, 3H), 2.50 (s, 3H).

[0110] Step 7: Synthesis of WX001 Form A. To a reaction flask, add I-4 (47 g, 102.73 mmol), C-1 (25.94 g, 267.09 mmol), dichloromethane (470 mL), and tetrahydrofuran (470 mL). After purging the nitrogen atmosphere, lithium hexamethyldisilazane (1 M, 246.54 mL, 2.4 eq) was added dropwise at -5°C (maintaining the internal temperature between -5 and 3°C). The mixture was allowed to react at 0°C for 0.5 h. After completion of the reaction, deionized water (470 mL) was added to quench the reaction. The organic solvent was removed by concentration. The filter cake was collected by filtration. The filter cake was stirred with deionized water (1000 mL) at room temperature for 30 minutes. The filter cake was collected by filtration. The filter cake was stirred with acetonitrile (1000 mL) at room temperature for 30 minutes and then filtered to obtain WX001 Form A. 1H NMR (400 MHz, CDCl 3) δ = 8.44 (s, 1H), 7.60 - 7.50 (m, 2H), 7.18 (d, J = 7.4 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 6.88 (s, 1H), 6.42 (d, J = 1.9 Hz, 1H), 5.27 (d, J = 7.6 Hz, 2H), 5.12 (s, 2H), 4.85 (d, J = 7.6 Hz, 2H), 3.85 (s, 3H), 2.69 (s, 3H), 2.52 (s, 3H). The XRPD spectrum of WX001 Form A is shown in Figure 1, the DSC spectrum is shown in Figure 2, and the TGA spectrum is shown in Figure 3.

[0111] Example 3: Polymorph screening of WX001

[0112] 1. Gas-solid permeability Approximately 20 mg of WX001 Form A was weighed into a 3 mL vial. Approximately 4 mL of solvent was added to a separate 20 mL vial. The 3 mL vial was then placed open inside the 20 mL vial, which was then sealed. After the sample partially dissolved in the solvent or allowed to stand at room temperature for 7 days, the solid was collected and analyzed by XRPD. The results are shown in Table 7. Table 7 Summary of gas-solid permeability test solvent Test results MeOH Form A acetone Form A EtOAc Form A MTBE Form A ACN Form A Toluene Form A 1,4-Dioxane Form A H2O Form A

[0113] 2. Gas-liquid diffusion Weigh approximately 20 mg of each portion of WX001 Form A into a 3 mL vial and dissolve it in 1.2-1.8 mL of solvent. Add approximately 4 mL of antisolvent to another 20 mL vial. Place the open 3 mL vial containing the clear solution into the 20 mL vial. Seal the 20 mL vial and let it sit at room temperature. Collect the resulting solid and perform XRPD analysis. The results are shown in Table 8. Table 8 Summary of gas-liquid permeation test solvent antisolvent Test results CHCl 3 acetone Form A DMSO EtOAc Form A* Toluene Form A* *Indicates solids obtained by evaporation at room temperature.

[0114] 3. Slow evaporation Weigh 15-20 mg of WX001 Form A into a 3 mL vial and dissolve it in 1.0-3.0 mL of solvent. Seal the vial with parafilm and poke four pinholes in the vial. Allow to evaporate slowly at room temperature. Collect the resulting solid and perform XRPD analysis. The results are shown in Table 9. Table 9 Summary of slow volatilization test Solvent (v / v) Test results MeOH / DCM (1:1) Form A THF / H 2O (1:1) Form A CHCl 3 Form A 1,4-Dioxane Form A

[0115] 4. Slow cooling Weigh 15-35 mg portions of WX001 Form A into 3 mL vials, add 1.0-3.0 mL of solvent, and equilibrate at 50°C with stirring for approximately 3.5 hours. Filter the supernatant. Place the resulting supernatant in a biochemical incubator, cool it from 50°C to 5°C at a rate of 0.1°C / min, and maintain it at 5°C. Collect the precipitated solid and perform XRPD analysis. The results are shown in Table 10. Table 10 Summary of slow cooling test Solvent (v / v) Test results CHCl 3 Form A* THF / H 2O (1:1) Form A* ACN / H2O (1:1) Form A* *Indicates that the sample was clarified at 5 ºC and -20 ºC, and the solid was evaporated at room temperature.

[0116] 5. Temperature circulation stirring Approximately 25 mg portions of WX001 Form A were weighed into HPLC vials, and 0.5 mL of solvent was added to each vial. The resulting suspension was subjected to a temperature cycle (heating the sample to 50°C, then cooling it to 5°C at a rate of 0.1°C / min, and then repeating this cycle, with the sample finally maintained at 5°C) with magnetic stirring (1000 rpm). The solid was collected by centrifugation and analyzed by XRPD. The results are shown in Table 11. Table 11 Summary of temperature cycle stirring test Solvent (v / v) Test results MeOH Form A MIBK Form A EtOAc Form A THF / H 2O (1:1) Form A ACN / H2O (1:1) Form A DMAc / H2O (1:1) Form A

[0117] 6. Suspension and stirring at room temperature Approximately 25 mg portions of WX001 Form A were weighed into HPLC vials. 0.5 mL of solvent was added to each vial. The resulting turbid solution was magnetically stirred (1000 rpm) at room temperature for 3 days. The solid was then collected by centrifugation and analyzed by XRPD. The test results are shown in Table 12. Table 12 Summary of room temperature suspension stirring test Solvent (v / v) Test results EtOH Form A EtOAc Form A THF Form A DCM Form A n-heptane Form A H2O Form A EtOH / H 2O (0.97:0.03, aw~0.2) Form A EtOH / H 2O (0.93:0.07, aw~0.4) Form A EtOH / H 2O (0.86:0.14, aw~0.6) Form A EtOH / H 2O (0.71:0.29, aw~0.8) Form A ACN Form A

[0118] 7. Suspend and stir at 50°C Approximately 25 mg portions of WX001 Form A were weighed into HPLC vials. 0.5 mL of solvent was added to each vial. The resulting suspension was magnetically stirred (1000 rpm) at 50°C for 3 days. The solid was then collected by centrifugation and analyzed by XRPD. The results are shown in Table 13. Table 13 Summary of 50°C Suspension Stirring Test Solvent (v / v) Test results IPA Form A Acetone / H2O (1:1) Form A IPAc Form A MTBE Form A 2-MeTHF Form A 1,4-Dioxane Form A CHCl 3 / n-heptane (1:1) Form A Toluene Form A DMSO / H 2O (1:1) Form A ACN Form A

[0119] 8. Antisolvent Addition Method Weigh approximately 15 mg of WX001 Form A into a 20 mL vial and completely dissolve the solid with 0.7–1.0 mL of solvent. Add the antisolvent dropwise to the clear solution while stirring (1000 rpm) until solid precipitates. Alternatively, when the total antisolvent volume reaches 10 mL, suspend and stir the sample without solid precipitation at 5°C. Transfer the clear sample to -20°C and suspend and stir. The remaining clear sample is then allowed to evaporate at room temperature. Isolate the precipitated solid and perform XRPD analysis. The results are shown in Table 14. Table 14 Summary of Antisolvent Addition Test Solvent (v / v) antisolvent Test results CHCl 3 MeOH Form A* acetone Form A EtOAc Form A MTBE Form A 2-MeTHF Form A ACN Form A n-heptane Form A Toluene Form A MeOH / DCM (1:1) MIBK Form A IPAc Form A MTBE Form A THF / H 2O (1:1) H2O Form A NMP H2O Form A DMAc H2O Form A * Indicates that the sample was clear at room temperature and the solid was obtained by stirring at 5 ºC.

[0120] Example 4: Study on the Hygroscopicity of WX001 Form A Experimental Materials: SMS DVS Advantage Dynamic Vapor Sorption Analyzer Experimental methods: Take 10-30 mg of WX001 crystal form A and place it in the DVS sample tray for testing. Experimental results: The DVS spectrum of WX001 Form A is shown in Figure 4, with ΔW = 0.1134%. Experimental conclusion: The moisture absorption weight gain of WX001 form A at 25°C and 80% RH is 0.1134%, indicating almost no hygroscopicity.

[0121] Example 5: Stability test of WX001 crystal form A Weigh 12 parallel portions of WX001 Form A sample, approximately 5 mg each, and place them on the bottom of an HPLC vial, spreading them into a thin layer. Seal the vials with sealing film and poke small holes in the vials, ensuring sufficient contact with ambient air, while placing the samples in a 60°C / 75% humidity chamber and a 92.5% RH desiccator. The vials were then tightly capped for the samples stored at 60°C under both light and shade conditions (the shaded sample was wrapped in tinfoil). The test results are shown in Table 15 below: Table 15 Solid stability test results of WX001 Form A Test conditions Conditions for taking points Crystal form 0 days -- Form A 60 ºC 5 days Form A 10 days Form A 92.5%RH 5 days Form A 10 days Form A Visible light# Illumination reaches 1.2E+06 Lux·hrs Form A Light-blocking control group Take points at the same time as the visible light group Form A Visible light + ultraviolet# Illumination reaches 200 W·hrs / m2 Form A Light-blocking control group Take points at the same time as the visible light + ultraviolet group Form A 60°C / 75%RH January Form A February Form A March Form A #:ICH conditions Conclusion: WX001 Form A has good stability.

[0122] Experimental Example 1: In vitro enzyme activity test 1. Purpose: The ability of compounds to inhibit ERK1 and ERK2 kinase activity was measured. 2. Experimental Buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg / mL bovine serum albumin (BSA), 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT), 1% DMSO. 3. Compound treatment: Test compounds were dissolved in 100% DMSO to prepare stock solutions of specific concentrations. Compounds were serially diluted in DMSO using an Integra Viaflo Assist pipette. 4. Experimental Methods: 1) Prepare the substrate MBP in freshly prepared reaction buffer; 2) Add ERK1 (or ERK2) kinase to the above MBP solution and mix gently; 3) Compounds dissolved in 100% DMSO were added to the kinase reaction using ultrasound (Echo550; nanoliter range) and incubated at room temperature for 20 minutes. 4) 33P-ATP (specific concentration 10 μCi / μL) is added to the reaction system, and the reaction begins; 5) Incubate at room temperature for 2 hours; 6) Detect the amount of radioactivity by filtration-binding method; 7) ERK1 (or ERK2) kinase activity was calculated as the ratio of the remaining kinase activity in the test sample to the kinase activity in the control (dimethylsulfoxide-treated) control. Curve fitting and IC50 values ​​were calculated using Prism (GraphPad software). 5. The experimental results are shown in Tables 16 and 17: Table 16 ERK1 enzyme activity test results Compound ERK1 IC 50 (nM) WX001 1.4 Conclusion: The compounds of the present invention exhibited excellent inhibitory activity against ERK1 enzyme. Table 17 ERK2 enzyme activity test results Compound ERK2 IC 50 (nM) WX001 0.54 Conclusion: The compounds of the present invention exhibit excellent inhibitory activity against ERK2 enzyme.

[0123] Experimental Example 2: In vitro cell proliferation inhibition experiment 1. Purpose: The ability of compounds to inhibit the proliferation of HT29 tumor cells was measured. 2. Compound treatment: The test compound was dissolved in 100% DMSO to prepare a 10 mM stock solution. 3. Experimental steps and methods: 1) Turn on the UV light of the biosafety cabinet and count down for 30 minutes; 2) Preheat RPMI1640 medium and trypsin in a 37°C water bath; 3) After UV irradiation, open the biosafety cabinet and wipe the preheated culture medium, trypsin, phosphate buffered saline (PBS), etc. with alcohol and place them in the biosafety cabinet; 4) Remove the HT29 cells from the incubator, remove the old culture medium in a biosafety cabinet, add 10 ml of PBS, shake gently, and remove the PBS; 5) Add 1.5 ml of preheated 0.25% trypsin, shake the culture flask horizontally to evenly cover the cells at the bottom, and place in the incubator for 2 minutes; 6) Terminate cell digestion with complete culture medium and pipette to a uniform cell suspension for counting; 7) Based on the cell count results, adjust the cell suspension density to 1500 cells per well and plate 50 μl per well; 8) Serially dilute the compound stock solution in DMSO solution and add the compound to the cell plate using Tecan; 9) Allow the cell plates and CellTiterGlo to equilibrate at room temperature. Add 25 μL of CellTiterGlo to each well, shake for 1-2 minutes, let stand for 10 minutes, and then measure the signal value. Analyze the data using XL-Fit and calculate the IC50 of each compound. 4. The experimental results are shown in Table 18: Table 18 In vitro cell activity test results Compound HT29 IC 50 (nM) WX001 66 Conclusion: The compounds of the present invention exhibited excellent inhibitory activity against HT29 cell proliferation.

[0124] Experimental Example 3: PK study in mice 1. Purpose: Female BALB / c mice were used as test animals. The blood concentrations of the compounds were measured after a single dose and the pharmacokinetic behavior was evaluated. 2. Experimental Procedure: Four healthy adult female BALB / c mice were selected: two were administered intravenously and two were administered orally. The intravenous vehicle for the intravenous group consisted of 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with an appropriate amount of the intravenous vehicle, vortexed, and sonicated to obtain a 0.5 mg / mL clear solution, which was then filtered through a microporous filter and used for later use. The oral vehicle for the oral group consisted of 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with the vehicle, vortexed, and sonicated to obtain a 0.3 mg / mL solution. Following intravenous administration of 1 mg / kg or oral administration of 3 mg / kg to mice, whole blood was collected at defined intervals and plasma was prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin. 3. The experimental results are shown in Table 19: Table 19 Compound PK test results Compound C max (nM) F% Oral DNAUC (nM.h / mpk) Vd ss(L / kg) Cl (mL / min / kg) T 1 / 2(h) WX001 4790 154 2935 1.23 18.9 1.1 Note: Cmax is the maximum concentration; F% is the oral bioavailability; DNAUC = AUCPO / Dose, AUCPO is the oral exposure, Dose is the drug dose; Vdss is the volume of distribution; Cl is the clearance; T1 / 2 is the half-life; NA means not tested. Conclusion: The compounds of the present invention exhibit excellent oral exposure and bioavailability.

[0125] Experimental Example 4: Solubility Study 1. Purpose: Determine the solubility of the compound and evaluate its solubility. 2. Test solution: 1) Buffer A (pH 2.0): 50 mM phosphate buffer, pH 2.0; Buffer B (pH 6.5): 50 mM phosphate buffer, pH 6.5; Buffer C (pH 7.4): 50 mM phosphate buffer, pH 7.4; 2) Preparation of standard solution: a) Mix 50% acetonitrile solution and 50% buffer solution to obtain a diluent; b) 10 mM (10 μL / compound) compound stock solution was added to the diluent (490 μL / compound) to prepare a 200 μM assay standard solution; c) Dilute the 200 μM UV detection standard solution with 10-fold and 200-fold diluent to obtain 20 μM and 1 μM UV standard solutions; d) 1 μM, 20 μM, and 200 μM UV standard solutions were used as standard solutions for solubility experiments. 3. Experimental Methods: 1) Dissolve the compound in DMSO to prepare a 10 mM stock solution. Amiodarone hydrochloride, carbamazepine, and chloramphenicol serve as controls for solubility experiments. 2) 10 μL of each stock solution of the test compound and control was placed in a 96-well plate. 490 μL of each of three different solubility media (Buffers A, B, and C) was added, corresponding to pH values ​​of 2.0, 6.5, and 7.4, respectively. The theoretical maximum concentration of the test compound was 200 μM in 2% DMSO. 3) Shake in a shaker at 600 rpm at room temperature (25 ± 2°C) for 24 hours; 4) Pipette 200 μL of the solution into a 96-well plate vacuum filtration device and transfer it to a new 96-well plate as the test sample; 5) Test the compound concentration using HPLC-UV. The HPLC conditions are shown in Table 20. Table 20 HPLC conditions Test methods HPLC-UV detection instrument Agilent 1200 Mobile phase A: Water + 0.37% trifluoroacetic acid B: Acetonitrile + 0.19% trifluoroacetic acid Chromatographic columns Waters Xbridge RP-C18 (2.1×50 mm, 5 µm) Proportion Time (min) 0.00 2.00 2.50 3.01 4.00 B% 5 90 90 5 5 Flow rate (mL / min) 1.0 1.0 1.0 1.0 1.0 6) Inject three UV standards into the HPLC from low to high concentration (1 µM, 20 µM, 200 µM), and then inject the test sample of the compound to be tested; 7) Integrate the UV chromatographic peak and calculate the solubility of the sample. 4. The experimental results are shown in Table 21: Table 21 Compound solubility test results Compound Solubility at different pH pH=2.0 pH=6.5 pH=7.4 WX001 199.5 μM 2.94 μM 2.64 μM Conclusion: The compounds of the present invention have good solubility under different pH conditions.

[0126] Experimental Example 5: In vivo efficacy study in a human melanoma A375 mouse model 1. Purpose: The anti-tumor effect of WX001 was evaluated using a nude mouse model with subcutaneous xenografts of human melanoma A375 cells. 2. Experimental Animals: Species: Mouse Strain: BALB / c nude mice Age: 6-8 weeks Gender: Female Weight: 18-22 grams Supplier: Weitong Lihua Laboratory Animal Technology Co., Ltd. 3. Rearing environment: The animals were housed in an SPF animal room in IVC (independent ventilation system, constant temperature and humidity) cages (3 per cage), with a temperature of 20-26°C and a humidity of 40-70%. Cages: Made of polycarbonate, 375 mm x 215 mm x 180 mm, littered with corn cobs, changed weekly. Food: Experimental animals were allowed to eat freely during the entire experimental period (irradiation sterilized, dry pelleted food); Drinking water: Experimental animals can drink sterile water freely; Cage identification: The animal information card for each cage should indicate the number of animals in the cage, sex, strain, receipt date, dosing regimen experiment number, group and experiment start date; Animal identification: Experimental animals were identified with ear tags. 4. Experimental Content: 1) Experimental Cell Culture: Human melanoma A375 cells were cultured as monolayers in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO₂ incubator. Twice weekly, cells were routinely digested and passaged using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated. 2) Tumor Tissue Inoculation and Grouping: 0.1 mL (5 × 10 5 cells) of A375 cells were subcutaneously inoculated into the right axilla of each mouse. When the average tumor volume reached 170 mm 3 , the animals were randomly divided into four groups and drug administration began. The experimental groups and dosing schedule are shown in Table 22: Table 22 Experimental animal groups and dosing regimen Group Number of animals drug Dosage (mg / kg) Dosing cycle Route and frequency of administration 1 6 Solvent control (Vehicle) -- 21 days Oral administration (PO), twice daily (BID) 2 6 WX001 12.5 21 days Oral administration (PO), twice daily (BID) 3 6 WX001 25 21 days Oral administration (PO), twice daily (BID) 4 6 WX001 50 21 days Oral administration (PO), twice daily (BID) 3) Daily Observation of Laboratory Animals: This experimental protocol and any modifications were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The use and welfare of experimental animals were carried out in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual assessment only), weight changes (measured twice weekly), and any other abnormalities. Within-group mortality and adverse reactions were recorded based on the number of animals in each group. 4) Preparation of test substance a) Vehicle group: 5% DMSO+95% (20% HP-β-CD). b) Test compound group: Weigh a fixed amount of the test compound into a dispensing bottle, add the corresponding volume of DMSO and vortex to obtain a clear solution, then add the corresponding volume of 20% HP-β-CD and vortex to obtain a homogeneous suspension. 5) Tumor Measurement and Experimental Indicators: a) Measure tumor diameter twice weekly with a vernier caliper. Tumor volume is calculated using the formula: TV = 1 / 2 × a × b², where a and b represent the major and minor diameters of the tumor, respectively. b) The tumor inhibition efficacy of the compound was evaluated using the TGI (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = {[1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of that treatment group at the start of dosing)] / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)} × 100%. 5. Experimental Results: 1) As shown in Table 23 and Figure 5, WX001 was able to dose-dependently inhibit tumor growth in a nude mouse model bearing subcutaneous human melanoma A375 cell xenografts after oral administration until day 21, with TGIs of 45%, 58%, and 102% at doses of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg, respectively. 2) Animal body weight serves as an indirect indicator for measuring drug toxicity. As shown in Figure 6, by day 21, no significant decrease in body weight was observed in either the solvent control group or the WX001 group, indicating good tolerability. Table 23 In vivo efficacy test results of mouse A375 model drug TGI WX001 (12.5 mg / kg, PO, BID) 45% WX001 (25 mg / kg, PO, BID) 58% WX001 (50 mg / kg, PO, BID) 102% Experimental conclusion: WX001 can inhibit tumor growth in a dose-dependent manner at three doses of 12.5 mg / kg, 25 mg / kg and 50 mg / kg; there was no significant weight loss in the animals during the administration process, and the animals had good tolerance.

[0127] Experimental Example 6: In vivo PK study in SD rats 1. Purpose: Male SD rats were used as test animals. The blood concentration of the compound was measured after a single dose and the pharmacokinetic behavior was evaluated. 2. Experimental Procedure: Six healthy adult male Sprague-Dawley rats were enrolled, with three assigned to the intravenous injection group and three to the oral administration group. The intravenous injection vehicle consisted of 5% DMSO combined with 95% (20% HP-β-CD). The test compound was mixed with the appropriate amount of the intravenous vehicle, vortexed, and sonicated to obtain a 0.2 mg / mL clear solution, which was then filtered through a microporous filter and used for later use. The oral administration vehicle consisted of 5% DMSO combined with 95% (20% HP-β-CD). The test compound was mixed with the vehicle, vortexed, and sonicated to obtain a 1 mg / mL solution. Following intravenous administration of 1 mg / kg or oral administration of 10 mg / kg to the Sprague-Dawley rats, whole blood was collected at defined intervals and plasma was prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin. 3. The experimental results are shown in Table 24: Table 24 Compound PK test results Compound C max (nM) F% Oral DNAUC (nM.h / mpk) Vd ss(L / kg) Cl (mL / min / kg) T 1 / 2(h) WX001 2500 49% 1445.79 1.08 13.4 3.77 Note: Cmax is maximum concentration; F% is oral bioavailability; DNAUC = AUCPO / Dose, AUCPO is oral exposure, Dose is drug dose; Vdss is distribution volume; Cl is clearance; T1 / 2 is half-life. Conclusion: The compounds of the present invention exhibit excellent oral exposure and bioavailability.

[0128] Experimental Example 7: In vivo PK study in cynomolgus monkeys 1. Purpose: Male cynomolgus monkeys were used as test animals. The blood concentration of the compound was measured after a single dose and the pharmacokinetic behavior was evaluated. 2. Experimental Procedure: Five healthy adult male cynomolgus monkeys were enrolled, two for intravenous administration and three for oral administration. In the intravenous administration group, the test compound was mixed with an appropriate amount of the intravenous solvent in a mixture of 5% DMSO and 95% (20% HP-β-CD) and stirred to dissolve, resulting in a 0.4 mg / mL clear solution. The solution was then filtered through a microporous filter and used for later use. In the oral administration group, the test compound was mixed with the solvent in a mixture of 5% DMSO and 95% (20% HP-β-CD) and stirred to dissolve, resulting in a 0.3 mg / mL solution. Following intravenous administration of 1 mg / kg or oral administration of 3 mg / kg to cynomolgus monkeys, whole blood was collected at defined intervals and plasma was prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin. 3. The experimental results are shown in Table 25: Table 25 Compound PK test results Compound C max (nM) F% Oral DNAUC (nM.h / mpk) Vd ss(L / kg) Cl (mL / min / kg) T 1 / 2(h) WX001 921 50% 1152.35 1.98 16.1 2.41 Note: Cmax is maximum concentration; F% is oral bioavailability; DNAUC = AUCPO / Dose, AUCPO is oral exposure, Dose is drug dose; Vdss is distribution volume; Cl is clearance; T1 / 2 is half-life. Conclusion: The compounds of the present invention exhibit excellent oral exposure and bioavailability.

[0129] Experimental Example 8: In vivo PK study in beagle dogs 1. Purpose: Male beagle dogs were used as test animals. The plasma concentrations of the compounds were measured after a single dose and the pharmacokinetic behavior was evaluated. 2. Experimental Procedure: Five healthy adult male beagle dogs were enrolled, two in the intravenous injection group and three in the oral administration group. In the intravenous injection group, the test compound was mixed with an appropriate amount of the intravenous injection vehicle, stirred and dissolved, and a 0.4 mg / mL clear solution was prepared. The solution was then filtered through a microporous filter and used for later use. In the oral administration group, the test compound was mixed with the vehicle in 5% DMSO and 95% 20% HP-β-CD, stirred and dissolved, and a 0.3 mg / mL solution was prepared. Following intravenous administration of 1 mg / kg or oral administration of 3 mg / kg to cynomolgus monkeys, whole blood was collected at defined intervals and plasma was prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin. 3. The experimental results are shown in Table 26: Table 26 Compound PK test results Compound C max (nM) F% Oral DNAUC (nM.h / mpk) Vd ss(L / kg) Cl (mL / min / kg) T 1 / 2(h) WX001 1769 60% 2307.32 1.29 10.1 3.48 Note: Cmax is maximum concentration; F% is oral bioavailability; DNAUC = AUCPO / Dose, AUCPO is oral exposure, Dose is drug dose; Vdss is distribution volume; Cl is clearance; T1 / 2 is half-life. Conclusion: The compounds of the present invention exhibit excellent oral exposure and bioavailability.

[0130] Experimental Example 9: hERG Test 1. Purpose of the study: The effects of compounds on hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) currents were tested using an automated patch clamp method. 2. Experimental Methods: 2.1 Cell Preparation CHO-hERG cells were cultured in a 175 cm² flask. When the cell density reached 60-80%, the culture medium was removed and the cells were washed once with 7 mL of PBS (Phosphate Buffered Saline). The cells were then digested with 3 mL of Detachin. After complete digestion, 7 mL of culture medium was added for neutralization. The cells were then centrifuged, the supernatant aspirated, and the cells were resuspended in 5 mL of culture medium to maintain a cell density of 2-5 × 10⁶ / mL. 2.2 Solution preparation Extracellular solution formula (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1.25 MgCl2, 10 HEPES and 10 Glucose, adjusted to pH 7.4 with NaOH. Intracellular solution formula (mM): 140 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA and 10 HEPES, adjusted to pH 7.2 with KOH. 2.3 Electrophysiological Recording Process The Qpatch instrument automatically performs the single-cell high-impedance sealing and whole-cell recording process. After acquiring whole-cell recording mode, the cell was clamped at -80 mV. A 50-millisecond pre-voltage of -50 mV was applied before a 5-second depolarizing stimulus of +40 mV was applied. The cell then repolarized to -50 mV for 5 seconds before returning to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular solution was added for 5 minutes. Drug administration then began, starting with the lowest tested concentration and administering each test concentration for 2.5 minutes. After all concentrations were administered, the positive control compound, 3 μM Cisapride, was administered. At least three cells were tested for each concentration (n ≥ 3). 2.4 Compound preparation A 20.00 mM stock solution of the compound was diluted with DMSO. 10 μL of the stock solution was added to 20 μL of DMSO solution, and serial dilutions were performed three-fold to six DMSO concentrations. 4 μL of each of the six DMSO concentrations was added to 396 μL of extracellular fluid and diluted 100-fold to six intermediate concentrations. 80 μL of each of the six intermediate concentrations was then added to 320 μL of extracellular fluid and diluted five-fold to the desired final concentration. The highest concentration tested was 40 μM, followed by six concentrations of 40, 13.3, 4.4, 1.48, 0.494, and 0.165 μM. The DMSO content in the final test concentration did not exceed 0.2%, as this concentration has no effect on hERG potassium channels. Compound preparation was performed using the Bravo instrument throughout the entire dilution process. 2.5 Data Analysis The experimental data were analyzed by GraphPad Prism 5.0 software. 2.6 Quality Control Environment: Humidity 20~50%, temperature 22~25℃ Reagents: All experimental reagents were purchased from Sigma, with a purity of >98%. The experimental data in the report must meet the following standards: Whole-cell seal impedance > 100 MΩ Tail current amplitude > 300 pA Pharmacological parameters: The inhibitory effects of various concentrations of Cisapride on hERG channels were set as positive controls. 3. The experimental results are shown in Table 27: Table 27 hERG test results of compounds Compound IC50 (μM) WX001 > 40 Conclusion: The compounds of the present invention have weak inhibitory effect on hERG potassium channel current, lower cardiotoxicity risk and higher safety.

[0131] Experimental Example 10: Plasma Protein Binding Test (PPB) 1. Purpose: Study the binding of the test compound to human / mouse / rat / dog / monkey plasma albumin. 2. Experimental Procedure: 1) Matrix Preparation: On the day of the experiment, thaw plasma in cold water and centrifuge at 3220 rpm for 5 minutes to remove any clots. Measure the pH of the resulting plasma and adjust it to 7.4 ± 0.1 using 1% phosphoric acid or 1N sodium hydroxide as needed. 2) Test Compound Dilution Procedure: Dissolve the test compound in dimethylsulfoxide (DMSO) to prepare stock solutions of 10 mM and 2 mM, respectively. Dilute 2 μL of the 2 mM stock solution with 98 μL of DMSO to prepare a 40 μM working solution. Dilute 10 μL of the stock solution with 240 μL of DMSO to prepare a 400 μM working solution of the control compound. Prepare the loading matrix by mixing the compound working solution (5 μL) with the blank matrix (995 μL) at a ratio of 1:200. 3) Analysis steps: a) Prepare time 0 (T0) samples for residue determination by transferring an equal amount of 30 μL of loading matrix (n=2) to a sample collection plate. Immediately match the samples with the corresponding blank buffer to a final volume of 60 μL, with a 1:1 plasma to buffer ratio in each well. Next, add 60 μL of 4% H₃PO₄ in H₂O and 480 μL of stop solution containing an internal standard to the T0 sample of the test compound. Store these samples with the other samples at 2-8°C until further processing. b) Preincubate the remaining plasma sample in a CO2 incubator at 37 ± 1°C for 30 min. Prepare the protein-free sample (F sample). Transfer both the protein-free sample and the matrix-loaded sample (230 μL) to a polycarbonate tube (n = 2) and ultracentrifuge at 37°C and 155,000 × g (35,000 rpm) for 4 h. c) To prepare the T sample (test sample), an additional aliquot of the matrix-containing sample was transferred to a separate 96-well plate (sample incubation plate) and incubated at 37°C for 4 h. d) After centrifugation, transfer 30 μL of the protein-free sample and 30 μL of the T sample from the second layer of the supernatant (below the upper layer) to a new sample collection plate. Mix each sample with the corresponding blank buffer or matrix to a final volume of 60 μL, with a 1:1 matrix:buffer ratio. Add 60 μL of a 4% aqueous H₃PO₄ solution and 480 μL of stop solution (containing an internal standard) to all samples. Centrifuge the mixture at 4000 rpm for 20 min. Remove 100 μL of the supernatant from each sample for LC-MS / MS analysis. 3. The experimental results are shown in Table 28: Table 28 Compound plasma protein binding test results Compound Plasma protein binding rate (unbound%) people mice rats dog monkey WX001 15.9% 11.7% 8.4% 14.3% 14.2% Conclusion: The compounds of the present invention have moderate plasma protein binding.

[0132] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Those skilled in the art may make modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended patent applications.

[0133] none

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are each independently selected from H and C1-3 alkyl groups; R4, R5, R6 and R7 are each independently selected from H and C1-3 alkyl groups; n is 0 or 1; m is 1 or 2; ring A is selected from pyrazolyl group, which is optionally substituted by 1, 2 or 3 Rd groups; Rd is selected from C1-3 alkyl groups.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are independently selected from H, CH3, and CH2CH3, respectively.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are independently selected from H and CH3, respectively.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R4, R5, R6 and R7 are independently selected from H and CH3, respectively.

5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein, R7 is CH3.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, Rd is CH3.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from and, wherein the and can be optionally replaced by 1, 2 or 3 Rd.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from and.

9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, The structural units are selected from [the relevant source].

10. A crystal form A of WX001, characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 10.4856±0.2000°, 15.0133±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 22.0420±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°; 11. The crystal form A of WX001 according to claim 10 has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ angles: 10.2080±0.2000°, 10.4856±0.2000°, 15.0133±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 22.0420±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°.

12. Use of a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or crystal form A of WX001 according to any one of claims 10 to 11, in the preparation of a medicament for treating solid tumors.

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