Thiazololactamspiroheterocyclic compounds and their applications

Abstract

Thiazololactamspiroheterocyclic 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

[0001] This application claims the following priority:

[0002] CN202110722003.6, application date: June 28, 2021;

[0003] CN202111673614.2, application date: December 31, 2021;

[0004] CN202210693548.3, application date: June 17, 2022. Technical Field

[0005] This invention relates to a class of thiazolactamspiroheterocyclic compounds and their use in the preparation of medicaments for treating related diseases. Specifically, it relates to compounds of formula (I) and their pharmaceutically acceptable salts. Background Technology

[0006] The Ras / Raf / MEK / ERK pathway is a classic mitogen-activated protein kinase (MAPK) signaling cascade pathway that participates in the signal transduction of various growth factors, cytokines, mitogens, and hormone receptors after activation. It is one of the most important signal transduction pathways controlling cell growth, differentiation, and survival.

[0007] Studies have shown that abnormal activation of the Ras / Raf / MEK / ERK pathway caused by mutations or amplifications is a decisive factor in the development of various cancers. In human tumors, the mutation rate of RAS is approximately 22%, BRAF approximately 7%, and MEK approximately 1%. Therefore, key node proteins in this pathway have become important targets for cancer therapy (CancerDiscov. 2019, 9, 329-341). Currently, several BRAF inhibitors and MEK1 / 2 inhibitors, as well as their combination regimens, have been approved by the US FDA for the treatment of melanoma, BRAFV600E-mutant non-small cell lung cancer, and other cancers. However, the use of these upstream node BRAF and MEK inhibitors often leads to rapid drug resistance due to mutations or pathway reactivation, significantly limiting their clinical application.

[0008] Extracellular regulated protein kinases (ERKs), particularly ERK1 and ERK2 kinases, are key participants and downstream nodes in the Ras / Raf / MEK / ERK pathway, and their overactivation is found in many human cancers. As the terminal signaling kinase of this pathway, ERK has not yet exhibited drug resistance mutations. Therefore, drugs targeting ERK kinases hold promise for overcoming drug resistance issues arising from upstream inhibitor therapy, representing a more promising therapeutic strategy. However, to date, research on ERK inhibitors remains in the clinical stage, and no ERK inhibitor has been approved for marketing.

[0009] In conclusion, there is an urgent need to develop safe and effective ERK inhibitor drugs to meet the needs of cancer treatment. Summary of the Invention

[0010] This invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0011]

[0012] in,

[0013] R1 and R2 are independently selected from H and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. a replace;

[0014] R4, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. c replace;

[0015] n is 0 or 1;

[0016] m is 1 or 2;

[0017] Ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl groups are optionally surrounded by 1, 2 or 3 R groups. d replace;

[0018] R a and R c They were each independently selected from D, F, Cl, Br, and I;

[0019] R d Selected from F, Cl, Br, I, C 1-3 Alkyl and C 1-3 Alkoxy, the C 1-3 Alkyl and C 1-3The alkoxy group can be optionally substituted with 1, 2 or 3 R groups;

[0020] R is selected from F, Cl, Br and I.

[0021] This invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0022]

[0023] in,

[0024] R1 and R2 are independently selected from H and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. a replace;

[0025] R4, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. c replace;

[0026] n is 0 or 1;

[0027] m is 1 or 2;

[0028] Ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl groups are optionally surrounded by 1, 2 or 3 R groups. d replace;

[0029] R a and R c They were each independently selected from D, F, Cl, Br, and I;

[0030] R d Selected from F, Cl, Br, I, C 1-3 Alkyl and C 1-3 Alkoxy, the C 1-3 Alkyl and C 1-3 The alkoxy group can be optionally substituted with 1, 2 or 3 R groups;

[0031] R is selected from F, Cl, and Br.

[0032] In some embodiments of the present invention, R1 and R2 are independently selected from H, CH3, and CH2CH3, respectively, wherein CH3 and CH2CH3 are optionally converted by 1, 2, or 3 R... a Replacement, other variables as defined in this invention.

[0033] In some embodiments of the present invention, R1 and R2 are independently selected from H, CH3, CHF2, CD3 and CH2CH3, respectively, and other variables are as defined in the present invention.

[0034] In some embodiments of the present invention, R4, R5, R6, and R7 are each independently selected from H, F, Cl, Br, I, and CH3, wherein CH3 is optionally converted by 1, 2, or 3 Rs. c Replacement, other variables as defined in this invention.

[0035] In some embodiments of the present invention, R4, R5, R6 and R7 are independently selected from H, F, Cl, Br, I and CH3, respectively, and other variables are as defined in the present invention.

[0036] In some embodiments of the present invention, the above-mentioned R d The ingredients are selected from F, Cl, Br, I, CH3 and OCH3, wherein CH3 and OCH3 are optionally replaced by 1, 2 or 3 R, and other variables are as defined in this invention.

[0037] In some embodiments of the present invention, the above-mentioned R d Selected from CH3 and OCH3, other variables are as defined in this invention.

[0038] In some embodiments of the present invention, the ring A is selected from... The Choose 1, 2, or 3 Rs d Replacement, other variables as defined in this invention.

[0039] In some embodiments of the present invention, the ring A is selected from... Other variables are as defined in this invention.

[0040] In some embodiments of the present invention, the above-mentioned structural unit Selected from Other variables are as defined in this invention.

[0041] The present invention also includes some solutions derived from arbitrary combinations of the above variables.

[0042] In some embodiments of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from:

[0043]

[0044] in,

[0045] R2, R6 and R7 are as defined in this invention.

[0046] The present invention also provides compounds of the following formula or pharmaceutically acceptable salts thereof.

[0047]

[0048] The present invention also provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating solid tumors.

[0049] The present invention provides 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°, 18.8429±0.2000°, 20.6217±0.2000°;

[0050]

[0051] In some embodiments of the present invention, the above-mentioned crystal form A, in its X-ray powder diffraction pattern, represented by a 2θ angle, contains at least four or five diffraction peaks selected from the following: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°.

[0052] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal 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°, 25.4797±0.2000°.

[0053] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A, represented by a 2θ angle, contains 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°.

[0054] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal 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°, 25.4797±0.2000°.

[0055] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A, represented by a 2θ angle, contains 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°, 25.4797±0.2000°.

[0056] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal 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°.

[0057] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal 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 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°.

[0058] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal 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°, 26.9544°.

[0059] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystal form A is essentially as follows:Figure 1 As shown.

[0060] In some embodiments of the present invention, the XRPD spectrum analysis data of the above-mentioned crystal form A are shown in Table 1:

[0061] Table 1. XRPD pattern analysis data of WX001 crystal form A

[0062]

[0063]

[0064] 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℃.

[0065] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is as follows: Figure 2 As shown.

[0066] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A shows a weight loss of 0.83% at 150.0 ± 3.0 °C.

[0067] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is as follows: Figure 3 As shown.

[0068] The present invention also provides the application of the above-mentioned crystal form A in the preparation of a medicament for treating solid tumors.

[0069] Technical effect

[0070] The compounds of this invention exhibit superior inhibitory activity against ERK1 and ERK2 enzymes; the compounds of this invention exhibit superior inhibitory activity against HT29 cell proliferation; the compounds of this invention have good solubility under different pH conditions; the compounds of this invention have excellent pharmacokinetic properties and antitumor effects.

[0071] Definitions and Explanations

[0072] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0073] The term “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0074] The term "pharmaceutically acceptable salt" refers to the salt of the compounds of this invention, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this invention, with a specific substituent. When the compounds of this invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of this invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of this invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0075] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0076] Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereo isomers, enantiomers, optical isomers, diastereomers and tautomers.

[0077] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0078] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0079] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0080] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0081] Unless otherwise stated, "(+)" indicates right-handed rotation, "(-)" indicates left-handed rotation, and "(±)" indicates racemic rotation.

[0082] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key Or straight dashed key

[0083] Unless otherwise stated, the terms "tautomer" or "tautomer form" refer to isomers with different functional groups in dynamic equilibrium at room temperature, capable of rapidly interconverting into each other. Tautomers can be chemically equilibrated if possible (e.g., in solution). For example, proton tautomers (also called prototropic tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions involving the rearrangement of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the tautomers pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0084] Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the 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%.

[0085] Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

[0086] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, 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 desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0087] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium. 3 H), Iodine-125 125 I) or C-14 14 C). For example, deuterium can be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0088] The terms “optional” or “optionally” refer to events or conditions that may occur but are not required to occur as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0089] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which can include deuterium and hydrogen variants, provided that 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 it may or may not be substituted, unless otherwise specified, and the type and number of substituents can be arbitrary on a chemically feasible basis.

[0090] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0091] When the number of a linking group is 0, such as -(CRR)0-, it indicates that the linking group is a single bond.

[0092] 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.

[0093] When a substituent is vacant, it means that the substituent does not exist. For example, if X is vacant in AX, it means that the structure is actually A.

[0094] When one of the variables is selected as a single bond, it means that the two groups it connects to are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0095] When a substituent can be cross-bonded to two or more atoms on a ring, this substituent can bond with any atom on that ring, for example, structural units. This indicates that the substituent R can be substituted at any position on the cyclohexyl or cyclohexadiene. When the listed substituents do not specify which atom they are attached to the substituted group, such substituents can be bonded to any of their atoms. For example, a pyridyl group as a substituent can be attached to the substituted group through any carbon atom on the pyridine ring.

[0096] When the listed linking groups do not specify their linking direction, the linking direction is arbitrary, for example, The linker group L is -MW-. In this case, -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form a ring. Alternatively, rings A and B can be connected in the opposite direction to the left-to-right reading order to form a ring. The combination of linking groups, substituents, and / or their variants is permitted only if such a combination produces a stable compound.

[0097] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be connected to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds connected, resulting in a group with a corresponding valence. The chemical bonds connecting the site to other groups can be straight solid line bonds. Straight dashed key or wavy line For example, a straight solid line bond in -OCH3 indicates that the oxygen atom in that group is connected to other groups; The straight dashed bond in the diagram indicates that the group is connected to other groups through both ends of the nitrogen atom in the group; The wavy lines in the text indicate that the phenyl group is connected to other groups through the carbon atoms at positions 1 and 2 of the phenyl group. This indicates that any connectable site on the piperidinyl group can be linked to other groups via a single chemical bond, including at least... Even if H atoms are drawn on -N- in these four connection methods, Still includes In this type of linkage, when a chemical bond is attached, the number of hydrogen atoms at that site is reduced by one, resulting in a monovalent piperidinyl group.

[0098] Unless otherwise specified, the number of atoms in a ring is usually defined as the elemental number of the ring. For example, a “5-7 elemental ring” refers to a “ring” with 5-7 atoms arranged around it.

[0099] Unless otherwise specified, the term "C" 1-3 "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl groups, etc.; they can be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). C 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

[0100] Unless otherwise specified, the term "C" 1-3 "Alkoxy" refers to alkyl groups containing 1 to 3 carbon atoms that are attached to the rest of the molecule by an oxygen atom. The C 1-3 Alkoxy groups include C 1-2 C 2-3 C3 and C2 alkoxy groups, etc. 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), etc.

[0101] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0102] Throughout this specification, the terms "an embodiment," "an embodiment," "in another embodiment," or "in some embodiments" refer to including, in at least one embodiment, a specific reference element, structure, or feature related to that embodiment. Therefore, the phrases "in an embodiment," "in another embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0103] In this invention, Exo Up in the DSC spectrum indicates upward exothermic reaction.

[0104] The structures of the compounds of this invention can be confirmed by conventional methods well known to those skilled in the art. If this invention relates to the absolute configuration of a compound, that absolute configuration can be confirmed by conventional techniques in the art. For example, single-crystal X-ray diffraction (SXRD) is used, where the cultured single crystal is used to collect diffraction intensity data using a Bruker D8 venture diffractometer with CuKα radiation as the light source. The scanning method is as follows: After scanning and collecting relevant data, the crystal structure can be further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0105] The present invention will be described in detail below through embodiments, which are not intended to limit the present invention in any way.

[0106] All solvents used in this invention are commercially available and can be used without further purification.

[0107] The solvent used in this invention is commercially available.

[0108] The following abbreviations are used in this invention: 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-butyldicarbonate; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; iPrOH represents 2-propanol; mp represents melting point.

[0109] Compounds are named according to conventional naming principles in the field or using Software naming conventions are used; commercially available compounds are named according to the supplier's catalog. This invention relates to a powder X-ray diffractometer (XRPD) method.

[0110] Instrument Model: PANalytical X'Pert 3 X-ray diffractometer

[0111] Test method: Approximately 10 mg of sample was used for XRPD detection.

[0112] Table 2 XRPD Test Parameters

[0113]

[0114]

[0115] This invention relates to a differential scanning calorimeter (DSC) method.

[0116] Instrument Model: TA 2500 Differential Scanning Calorimeter

[0117] Table 3 DSC Instrument Parameters and Test Methods

[0118] parameter Setting value method linear heating Sample tray Aluminum disc, capped / uncapped Temperature range 25℃ - Set the final temperature Scan rate (°C / min) 10 Protective gas Nitrogen

[0119] The present invention relates to a thermogravimetric analysis (TGA) method.

[0120] Instrument Model: TA 5500 Thermogravimetric Analyzer

[0121] Table 4. TGA Instrument Parameters and Test Methods

[0122] parameter Setting value method linear heating Sample tray Aluminum tray, open Temperature range Room temperature - Set endpoint temperature Scan rate (°C / min) 10 Protective gas Nitrogen

[0123] This invention presents a method for dynamic vapor adsorption analysis (DVS).

[0124] Dynamic moisture adsorption (DVS) curves were acquired using a DVSIntrinsic plus from SMS (Surface Measurement Systems). Relative humidity at 25°C was corrected for the deliquescence points of LiCl, Mg(NO3)2, and KCl.

[0125] Table 5 DVS Test Parameters

[0126]

[0127]

[0128] Table 6 Classification of Hygroscopicity Evaluation

[0129] Hygroscopic classification ΔW% deliquescence Absorbs sufficient water to form a liquid Highly hygroscopic ΔW% ≥ 15% Hygroscopic 15% > ΔW% ≥ 2% Slightly hygroscopic 2% > ΔW% ≥ 0.2% None or almost none of the hygroscopic properties ΔW%<0.2%

[0130] Note: ΔW% represents the moisture gain of the test sample at 25±1℃ and 80±2%RH.

[0131] Instruction manual illustrations

[0132] Figure 1 XRPD spectrum of Cu-Kα radiation in WX001 crystal form A;

[0133] Figure 2 DSC spectrum of WX001 crystal form A;

[0134] Figure 3 TGA spectrum of WX001 crystal form A;

[0135] Figure 4 DVS spectrum of WX001 crystal form A;

[0136] Figure 5 Tumor growth curves of human melanoma A375 model animals after administration of solvent and WX001, respectively;

[0137] Figure 6 The rate of weight change in human melanoma A375 model animals during drug administration. Detailed Implementation

[0138] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0139] Reference Example 1

[0140]

[0141] Step 1: Synthesis of compound A-1-2.

[0142] 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) to a reaction flask. Under nitrogen protection, add sodium borohydride (48.07 g, 1.27 mol, 2 eq), and react the mixture at 20 °C for 15 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure, dilute the concentrate with 15% citric acid aqueous solution (4000 mL), and extract with ethyl acetate (4000 mL x 3). Combine the organic phases, wash with saturated brine (2000 mL), and dry to anhydrous sodium sulfate. Filter, concentrate the filtrate under reduced pressure to obtain the crude product. Purify the crude product by column chromatography to obtain A-1-2. 1 ¹H NMR (400MHz, DMSO-d⁶) δ (ppm) = 7.47 (s, 1H), 5.58 (br s, 1H), 4.52 (s, 2H). Step 2: Synthesis of compound A-1-4.

[0143] Add A-1-2 (102 g, 525.64 mmol, 1 eq) and 2-methyltetrahydrofuran (1000 mL) to the reaction flask. After purging with nitrogen, cool to -70 °C and slowly add diisopropylaminolithium (2 M, 525.64 mL, 2.0 eq). Stir at -70 °C for 30 minutes, then slowly add A-1-3 (138.18 g, 788.46 mmol, 1.5 eq) in 2-methyltetrahydrofuran (400 mL), and continue the reaction at -70 °C for 1 hour. After the reaction is complete, quench the reaction solution with saturated ammonium chloride aqueous solution (2000 mL), extract with ethyl acetate (2000 mL x 4), and combine the liquids to obtain the organic phase. Wash the organic phase with saturated brine (1000 mL) and dry with anhydrous sodium sulfate. Filter, concentrate the filtrate under reduced pressure to dryness to obtain the crude product. The crude product was first purified by column chromatography, and then purified by methyl tert-butyl ether slurry to obtain A-1-4. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 6.41 (s, 1H), 5.43 (t, J = 5.6Hz, 1H), 5.00-4.84 (m, 4H), 4.38 (d, J = 5.6Hz, 2H), 1.12 (s, 9H).

[0144] Step 3: Synthesis of compound A-1-5.

[0145] Add A-1-4 (50 g, 135.39 mmol, 1 eq), azodicarbonyl dipiperidine (40.99 g, 162.47 mmol, 1.2 eq), and tetrahydrofuran (500 mL) to the reaction flask. After purging with nitrogen, cool to 0 °C, and slowly add a tetrahydrofuran (100 mL) solution of tributylphosphine (32.87 g, 162.47 mmol, 40.09 mL, 1.2 eq). React the mixture at 0 °C for 1 hour. After the reaction is complete, add water (500 mL) and saturated brine (500 mL) to the reaction solution sequentially, then extract with ethyl acetate (500 mL x 2). Combine the liquid and liquid phases to obtain the organic phase. Wash the organic phase with saturated brine (300 mL) and dry with anhydrous sodium sulfate. Filter, concentrate the filtrate under reduced pressure to dryness to obtain the crude product. Add 500 mL of methyl tert-butyl ether to the crude product, stir, filter, collect the filtrate, concentrate the filtrate to obtain the crude product. Add 50 mL of n-hexane to the crude product, stir, filter, collect the filter cake and dry to obtain A-1-5. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 5.29 (d, J = 7.5Hz, 1H), 4.88-4.75 (m, 3H), 4.60 (d, J = 12.9Hz, 1H), 4.22 (d, J = 12.9Hz, 1H), 1.26 (s, 9H).

[0146] Step 4: Synthesis of compound A-1-6.

[0147] Add A-1-5 (29 g, 82.55 mmol, 1 eq), tetrahydrofuran (250 mL), and water (50 mL) to the reaction flask. After purging with nitrogen, add iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) and react the mixture at 50 °C for 18 hours. Then add more iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) and continue reacting at 50 °C for another 6 hours. After the reaction is complete, the crude solution of A-1-6 is used directly in the next step.

[0148] Step 5: Synthesis of compound A-1-7.

[0149] Sodium carbonate (17.50 g, 165.11 mmol, 2 eq) was added to a crude solution containing A-1-6. After purging with nitrogen, di-tert-butyl carbonate (27.03 g, 123.83 mmol, 28.45 mL, 1.5 eq) was added. The mixture was reacted at 20 °C for 12 hours. After the reaction was complete, the reaction solution was poured into water (200 mL) and extracted with ethyl acetate (300 mL x 3). The liquid and liquid phases 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 solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain A-1-7.1 H NMR (400MHz, DMSO-d6) δ (ppm) = 5.46 (d, J = 5.9Hz, 1H), 5.32 (d, J = 6.3Hz, 1H), 4. 64(d,J=6.1Hz,1H), 4.55(d,J=5.8Hz,1H), 4.49(d,J=9.4Hz,2H), 1.53(s,9H).

[0150] Step 6: Synthesis of compound A-1-8.

[0151] Add 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) to the reaction flask. After purging with nitrogen, heat to 50 °C. Add a 70 mL solution of sodium chlorite (32.48 g, 305.28 mmol, 85% purity, 4 eq) in water. After the addition is complete, continue the reaction at 50 °C for 12 hours. Then add 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), and continue the reaction at 50 °C for 6 hours. After the reaction was complete, the reaction solution was quenched with saturated sodium sulfite aqueous solution (150 mL), then water (90 mL) was added, and the mixture was allowed to stand to separate the organic phase. 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 collected and dried to obtain A-1-8. 1 H NMR (400MHz, CDCl3) δ (ppm) = 5.61 (d, J = 6.6 Hz, 2H), 4.76 (d, J = 6.6 Hz, 2H), 1.66 (s, 9H).

[0152] Step 7: Synthesis of compound A-1.

[0153] In a dry reaction flask, A-1-8 (10 g, 27.68 mmol, 1 eq) and dichloromethane (100 mL) were added. Trifluoroacetic acid (41.04 g, 359.90 mmol, 26.65 mL, 13 eq) was added at 0 °C, and the reaction was continued at 0 °C for 0.5 h. After the reaction was complete, the reaction solution was slowly poured into a saturated sodium bicarbonate aqueous solution (1000 mL), and the pH was adjusted to 7–8. Extraction was performed with dichloromethane (1000 mL x 3), and the liquid and liquid 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 obtain A-1. 1HNMR (400MHz, DMSO-d6) δ (ppm) = 9.59 (s, 1H), 4.99-4.78 (m, 4H).

[0154] See Example 2

[0155]

[0156] Step 1: Synthesis of compound B-1-2.

[0157] Sodium hydroxide (590.8 g, 14.8 mol, 1.05 eq), ice water (20 L), and B-1-1 (2000.00 g, 14.07 mol, 1 eq) were added to a reaction flask. Then, iodomethane (2495.80 g, 17.59 mol, 1.25 eq) was added, and the reaction was carried out at 25 °C for 2 hours. After the reaction was completed, 6 N glacial hydrochloric acid solution was slowly added to the reaction flask to adjust the pH to 6-7, and the mixture was stirred for 0.5 hours. The mixture was filtered, and the filter cake was collected. Acetonitrile (500 mL) was added to the filter cake, and the mixture was stirred for 0.5 hours. The mixture was then filtered, the filter cake was collected, and dried to obtain B-1-2. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 12.69 (br s, 1H), 7.74 (br s, 1H), 2.45 (s, 3H), 1.86 (s, 3H).

[0158] Step 2: Synthesis of compound B-1-3.

[0159] Acetonitrile (15 L), B-1-2 (1500.00 g, 9.60 mol, 1 eq) were added to a reaction flask at 25 °C, followed by phosphorus oxychloride (1840.00 g, 12.0 mol, 1.25 eq). The temperature was slowly raised to 62 °C, and the reaction was carried out at 62 °C for 12 hours. The reaction solution was poured into water (10.5 L), and solid sodium bicarbonate was added to adjust the pH to 6-7. The solution was extracted with ethyl acetate (10.5 L), and the organic phase was separated. The organic phase was washed with saturated brine (7.5 L) and dried over anhydrous sodium sulfate. The solution was filtered, and the filtrate was concentrated under reduced pressure to obtain B-1-3. 1 HNMR (400MHz, DMSO-d6) δ (ppm) = 8.54 (s, 1H), 2.50 (s, 3H), 2.22 (s, 3H).

[0160] Step 3: Synthesis of compound B-1.

[0161] 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) to the reaction flask. After purging with nitrogen, add sodium iodide (571.59 g, 3.81 mol, 6.66 eq) and trimethylchlorosilane (186.61 g, 1.72 mol, 218.00 mL, 3 eq) sequentially. React the mixture at 20 °C for 14 hours. After the reaction is complete, add dichloromethane (800 mL) and water (1200 mL) sequentially to the reaction solution. Then add sodium bicarbonate solid to adjust the pH to 6-7. Separate the solutions. Extract the aqueous phase once with dichloromethane (500 mL). Combine the organic phases and wash them sequentially with saturated sodium sulfite aqueous solution (500 mL) and saturated brine (500 mL). Dry the solution with anhydrous sodium sulfate. Filter the solution and concentrate the filtrate under reduced pressure to obtain a crude product. Add n-heptane (0.5 L) to the crude product, stir for 1 hour, filter, and collect the filter cake to obtain B-1. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.34 (s, 1H), 2.48 (s, 3H), 2.21 (s, 3H).

[0162] Example 1

[0163]

[0164] Synthesis route:

[0165]

[0166] Step 1: Synthesis of WX001-2

[0167] Add 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) to the reaction flask. After purging with nitrogen, add cesium carbonate (935.92 mg, 2.87 mmol, 1.5 eq) and react at 25 °C for 16 hours. After the reaction is complete, pour the reaction solution into water (20 mL), extract with ethyl acetate (30 mL x 3), and combine the liquids to obtain the organic phase. Wash the organic phase with saturated brine (30 mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. Purify the crude product by thin-layer chromatography using silica gel plates to obtain WX001-2. LCMS (m / z): 366, 368 [M+H] + .

[0168] Step 2: Synthesis of WX001-3

[0169] Argon gas was purged in a pre-dried reaction flask, and wet palladium on carbon (0.1 g, 819.15 μmol, 10% purity, 1 eq) and ethanol (20 mL) were added. Then WX001-2 (300 mg, 819.15 μmol, 1 eq) was added, and hydrogen gas was purged three times. The reaction was stirred at 50 °C and 50 Psi for 24 hours. After the reaction was complete, 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 using silica gel plates to obtain WX001-3. 1 H NMR (400MHz, CDCl3) δ (ppm) = 8.96 (s, 1H), 7.58 (t, J = 7.6Hz, 1H), 7.19 (d, J = 7.6Hz, 1H), 7.0 9(d,J=7.5Hz,1H), 5.26(d,J=7.6Hz,2H), 5.14(s,2H), 4.80(d,J=7.6Hz,2H), 2.55(s,3H).

[0170] Step 3: Synthesis of WX001-4

[0171] 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) to a dry reaction flask. Cool to -78 °C and add hexamethyldisilamide lithium (1 M, 417.63 μL, 2 eq). React at 20 °C for 1 hour to obtain reaction solution 1.

[0172] Under nitrogen protection, a mixture of B-1 (55.57 mg, 208.81 μmol, 1 eq), tetrakis(triphenylphosphine)palladium (7.24 mg, 6.26 μmol, 0.03 eq) and N'N-dimethylacetamide (1 mL) was heated to 50 °C, followed by the dropwise addition of reaction solution 1. After the addition was complete, the mixture was allowed to react at 50 °C for 1 hour. After the reaction was complete, the reaction solution was poured into water (5 mL) and extracted with dichloromethane (30 mL x 3). The extracts 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 using silica gel plates to obtain WX001-4. LCMS (m / z): 426.0 [M+H] + .

[0173] Step 4: Synthesis of WX001-5

[0174] Add WX001-4 (100 mg, 235.00 μmol, 1 eq), acetonitrile (1 mL), and water (0.5 mL) to the reaction flask, purge with nitrogen three times, then add potassium persulfate (288.95 mg, 470.01 μmol, 2 eq), and react the mixture at 20 °C for 14 hours. After the reaction is complete, pour the reaction solution into a saturated sodium thiosulfate aqueous solution (5 mL), extract with dichloromethane (30 mL x 3), and combine the liquids to obtain the organic phase. Wash the organic phase with a saturated sodium bicarbonate aqueous solution (20 mL) and a saturated brine solution (30 mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. Purify the crude product by thin-layer chromatography using silica gel plates to obtain WX001-5. LCMS (m / z): 458.0 [M+H] + .

[0175] Step 5: Synthesis of WX001

[0176] 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) to a dry reaction flask. Purge with nitrogen, cool to 0 °C, and add hexamethyldisilamide lithium (1 M, 166.11 μL, 1.9 eq) dropwise. React at 0 °C for 1 hour. After the reaction is complete, pour the reaction solution into water (5 mL), extract with dichloromethane (30 mL x 3), and combine the liquids to obtain the organic phase. Wash the organic phase with saturated brine (30 mL x 3), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid preparative chromatography (column: Waters Xbridge BEH C18 100*25mm*5μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; B (acetonitrile)%: 20%-50%, 10 min) to obtain WX001. 1 H NMR(400MHz,DMSO-d6)δ=9.67(br s,1H),8.62(s,1H),7.64(t,J=7.7Hz,1H),7.43(d,J=1.8Hz,1H),7.14(dd,J=2.9,7.7Hz,2H),6.37(d,J=1.5Hz,1H),5. 12(d,J=7.2Hz,2H),5.02(s,2H),4.85(d,J=7.2Hz,2H),3.74(s,3H),2.57(s,3H),2.42(s,3H); LCMS(m / z):475.0[M+H] + .

[0177] Example 2: Preparation of WX001 crystal form A

[0178]

[0179] Step 1: Synthesis of compound I-1-3.

[0180] Tetrahydrofuran (12 L) and I-1-1 (1200 g, 5.69 mol) were added to the reaction vessel. Tetramethylethylenediamine (661.59 g, 5.69 mol) was slowly added to the reaction vessel, and the mixture was purged with nitrogen and cooled to -70 °C (internal temperature). Diisopropylaminolithium (2 M, 6.83 L) was slowly added dropwise, and the mixture was stirred at -70 °C for 0.5 hours. I-1-2 (1.76 kg, 9.39 mol) in tetrahydrofuran (4.8 L) solution was slowly added dropwise over 1.5 hours, and the mixture was reacted at -70 °C for 1 hour. After the reaction was complete, water (6 L) was added dropwise to quench the reaction, and the reaction flask was rinsed with water (2.4 L). The mixtures were combined and stirred. After the temperature rose to 10 °C, the mixtures were separated, and the aqueous phase was extracted with ethyl acetate (6 L). The remaining aqueous phase was adjusted to pH 3–4 with 15.6 L of potassium bisulfate solution, and then 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 obtain a crude product. The crude product was added to dichloromethane (2.4 L) and isopropyl ether (7.2 L) and stirred at room temperature for half an hour. The mixture was filtered, and the filter cake was collected to obtain I-1-3. 1 H NMR (400MHz, DMSO-d6) δ = 13.18 (br s, 1H), 6.20 (br s, 1H), 4.93-4.82 (m, 4H), 1.09 (s, 9H).

[0181] Step 2: Synthesis of compound I-1-4.

[0182] Dichloromethane (1330 mL), I-1-3 (1330 g, 1.99 mol, crude product), and 4-dimethylaminopyridine (41.37 g, 338.61 mmol) were added sequentially to a reaction vessel. N,N'-carbonyldiimidazole (419.86 g, 2.59 mol) was added in portions, and the mixture was reacted at 50 °C for 36 hours. After the reaction was complete, the pH of the reaction solution was adjusted to 3-4 by adding 5.32 L of 2N hydrochloric acid aqueous solution, and the mixture was stirred for 0.5 hours. Then, water (5.32 L) was added to concentrate the solution and remove the organic solvent. The residue was filtered to obtain a filter cake. The filter cake was stirred in sodium bicarbonate aqueous solution (5.32 L) for 0.5 hours, filtered, washed with water (2.66 L), and the filter cake was collected. Add 10.64 L of anhydrous ethanol to the crude product, stir for 2 hours, filter, wash the filter cake with 1.3 L of anhydrous ethanol, collect the filter cake, and dry it to obtain I-1-4. 1HNMR (400MHz, DMSO-d6) δ = 9.58 (br s, 1H), 4.94-4.84 (m, 4H).

[0183] Step 3: Synthesis of compound I-1-6.

[0184] I-1-4 (283 g, 1.03 mol), cesium carbonate (505.36 g, 1.55 mol), and N,N'-dimethylformamide (2800 mL) were added to a reaction flask. After purging with nitrogen, I-1-5 (221.24 g, 1.19 mol) was added, and the mixture was reacted at 20 °C for 12 hours. After the reaction was complete, the reaction solution was slowly poured into ice water (14 L), stirred for 1 hour, and the filter cake was collected. Methyl tert-butyl ether (5 L) was added to the crude product, stirred for 2 hours, filtered, the filter cake was collected, and dried to obtain I-1-6. 1 H NMR (400MHz, DMSO-d6) δ = 7.63 (t, J = 7.7Hz, 1H), 7.13 (dd, J = 2.8, 7.7Hz, 2H), 5.04 (d, J = 7.4Hz, 2H), 4.98 (s, 2H), 4.86 (d, J = 7.4Hz, 2H), 2.41 (s, 3H).

[0185] Step 4: Synthesis of compound I-1.

[0186] Three parallel reactions were performed. I-1-6 (183 g, 468.60 mmol) and tetrahydrofuran (2745 mL) were added to the 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 the reaction was complete, the reaction solution was diluted with water (915 mL), extracted with dichloromethane (1830 mL * 3), the organic phase was washed with saturated brine (915 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and dried at 45 °C to obtain the crude product. The crude product was added to a mixed solvent of methyl ether and n-hexane (total 1098 mL, volume ratio 1:5), stirred for 1 hour, filtered, and the filter cake was collected and dried. The filter cake was added to water (5400 mL), stirred for 1 hour, filtered, and the filter cake was collected and dried to obtain I-1. 1 HNMR (400MHz, DMSO-d6) δ = 9.32 (s, 1H), 7.64 (t, J = 7.6Hz, 1H), 7.13 (t, J = 8.4H z, 2H), 5.10 (d, J = 7.4Hz, 2H), 4.99 (s, 2H), 4.81 (d, J = 7.5Hz, 2H), 2.42 (s, 3H).

[0187]

[0188] Step 5: Synthesis of compound I-3.

[0189] Add 1200 mL of tetrahydrofuran solution of I-1 (120 g, 403.05 mmol) and zinc chloride (0.7 M, 575.79 mL) to two reaction flasks respectively. After replacing the nitrogen gas, cool to 0 °C and slowly add hexamethyldisilamide lithium (1 M, 806.11 mL). Heat naturally to 20 °C and stir for 1 hour. This is reaction solution 1. Add B-1 (107.25 g, 403.05 mmol), tetrakis(triphenylphosphine)palladium (13.97 g, 12.09 mmol), and N,N'-dimethylformamide (600 mL) to another reaction flask and heat to 50 °C to obtain reaction solution 2. Slowly add reaction solution 1 dropwise to reaction solution 2 and react the mixture at 50 °C for 1 hour. After the reaction is complete, quench the reaction with 0.1 M disodium ethylenediaminetetraacetate (10800 mL) and stir for 30 min. Add n-heptane (4800 mL) and stir for 0.5 hours. Filter, collect the filter cake, and dry to obtain the crude product. Pulverize the crude product with ethanol (7200 mL) at 20 °C for 2 hours, filter, collect the filter cake, and dry the filter cake to obtain I-3. 1 H NMR (400MHz, CDCl3) δ = 8.54 (s, 1H), 7.54 (t, J = 7.2Hz, 1H), 7.19 (d, J = 7.4Hz, 1H), 7.07 (d, J = 7.3Hz, 1 H), 5.29 (d, J = 7.5Hz, 2H), 5.11 (s, 2H), 4.84 (d, J = 7.6Hz, 2H), 2.73 (s, 3H), 2.66 (s, 3H), 2.52 (s, 3H).

[0190] Step 6: Synthesis of compound I-4.

[0191] I-3 (120 g, 282.00 mmol), acetonitrile (110 mL), and water (550 mL) were added to a reaction flask. After purging with nitrogen, potassium persulfate (329.40 g, 535.81 mmol) was added, and the mixture was reacted at 30 °C for 12 hours. After the reaction was complete, 200 mL of an ice-water mixture was added to the reaction solution, followed by 600 mL each of saturated sodium bicarbonate aqueous solution and saturated sodium thiosulfate aqueous solution, and then 600 mL of water. The mixture was filtered, the filter cake was collected, and dried to obtain the crude product. 600 mL of anhydrous ethanol was added to the crude product, and the mixture was stirred for 1 hour. After filtration, the filter cake was collected to obtain I-4. 1H NMR (400MHz, CDCl3) δ = 8.90 (s, 1H), 7.55 (t, J = 7.7Hz, 1H), 7.20 (d, J = 7.7Hz, 1H), 7.06 (d, J = 7.7Hz, 1 H), 5.31 (d, J = 7.6Hz, 2H), 5.11 (s, 2H), 4.84 (d, J = 7.6Hz, 2H), 3.44 (s, 3H), 2.92 (s, 3H), 2.50 (s, 3H).

[0192] Step 7: Synthesis of WX001 crystal form A.

[0193] I-4 (47 g, 102.73 mmol), C-1 (25.94 g, 267.09 mmol), dichloromethane (470 mL), and tetrahydrofuran (470 mL) were added to the reaction flask. After purging with nitrogen, hexamethyldisilamide lithium (1 M, 246.54 mL, 2.4 eq) was added dropwise at -5 °C (internal temperature controlled between -5 and -3 °C). The mixture was reacted at 0 °C for 0.5 hours. After the reaction was complete, 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 the filter cake was collected by filtration to obtain WX001 crystal form A. 1 ¹H NMR (400MHz, CDCl₃) δ=8.44(s, 1H), 7.60-7.50(m, 2H), 7.18(d, J=7.4Hz, 1H), 7.06(d, J=7.6Hz, 1H), 6.88(s, 1H), 6.42(d, J=1.9Hz, 1H), 5.27(d, J=7.6Hz, 2H), 5.12(s, 2H), 4.85(d, J=7.6Hz, 2H), 3.85(s, 3H), 2.69(s, 3H), 2.52(s, 3H). The XRPD spectrum of WX001 crystal form A is shown below. Figure 1 As shown, the DSC spectrum is as follows Figure 2 As shown, the TGA spectrum is as follows Figure 3 As shown.

[0194] Example 3: Polymorph Screening of WX001

[0195] 1. Gas-solid permeation

[0196] Weigh approximately 20 mg of WX001 crystal form A into a 3 mL vial. Add approximately 4 mL of solvent to a 20 mL vial. Place the 3 mL vial open inside the 20 mL vial, then seal the 20 mL vial. Collect the solid after the sample has partially dissolved in the adsorbed solvent or after standing at room temperature for 7 days, and perform XRPD testing. The experimental results are shown in Table 7.

[0197] Table 7 Summary of Gas-Solid Permeation Test

[0198] solvent Test results MeOH Crystal form A acetone Crystal form A EtOAc Crystal form A MTBE Crystal form A ACN Crystal form A Toluene Crystal form A 1,4-Dioxane Crystal form A <![CDATA[H2O]]> Crystal form A

[0199] 2. Gas-liquid diffusion

[0200] Weigh approximately 20 mg of WX001 crystal form A into a 3 mL vial, dissolve it in 1.2–1.8 mL of solvent, and add approximately 4 mL of antisolvent to a separate 20 mL vial. Place the 3 mL vial containing the clear solution open over the 20 mL vial, seal the 20 mL vial, and allow it to stand at room temperature. Collect the obtained solid and perform XRPD testing. The experimental results are shown in Table 8.

[0201] Table 8 Summary of Gas-Liquid Diffusion Experiments

[0202]

[0203] * indicates that the solid was obtained by volatilization at room temperature.

[0204] 3. Slow evaporation

[0205] Weigh 15–20 mg of WX001 crystal form A into a 3 mL vial, add 1.0–3.0 mL of solvent to dissolve it, seal the vial with sealing film, and poke 4 pinholes in the film. Allow it to evaporate slowly at room temperature. Collect the resulting solid and perform XRPD testing. The experimental results are shown in Table 9.

[0206] Table 9 Summary of Slow Evaporation Test

[0207] Solvent (v / v) Test results MeOH / DCM (1:1) Crystal form A <![CDATA[THF / H2O(1:1)]]> Crystal form A <![CDATA[CHCl3]]> Crystal form A 1,4-Dioxane Crystal form A

[0208] 4. Slowly cool down

[0209] Weigh 15–35 mg of WX001 crystal form A into a 3 mL vial, add 1.0–3.0 mL of solvent, stir at 50 °C for approximately 3.5 hours to equilibrate, then filter and collect the supernatant. Place the supernatant in a biochemical incubator and cool from 50 °C to 5 °C at a rate of 0.1 °C / min, then maintain a constant temperature of 5 °C. Collect the precipitated solid and perform XRPD testing. The experimental results are shown in Table 10.

[0210] Table 10 Summary of the Slow Cooling Experiment

[0211] Solvent (v / v) Test results <![CDATA[CHCl3]]> Crystal form A* <![CDATA[THF / H2O(1:1)]]> Crystal form A* <![CDATA[ACN / H2O(1:1)]]> Crystal form A*

[0212] * indicates that the sample was clarified at 5℃ and -20℃, and the solid was obtained by volatilization at room temperature.

[0213] 5. Temperature circulation stirring

[0214] Weigh approximately 25 mg of WX001 crystal form A into an HPLC vial, add 0.5 mL of solvent, and magnetically stir the resulting suspension under temperature cycling (heating the sample to 50°C, then cooling it to 5°C at a rate of 0.1°C / min, repeating this cycle, and finally maintaining the sample at 5°C). Centrifuge to collect the solid and perform XRPD analysis. The experimental results are shown in Table 11.

[0215] Table 11 Summary of Temperature Cyclic Stirring Test

[0216] Solvent (v / v) Test results MeOH Crystal form A MIBK Crystal form A EtOAc Crystal form A <![CDATA[THF / H2O(1:1)]]> Crystal form A <![CDATA[ACN / H2O(1:1)]]> Crystal form A <![CDATA[DMAc / H2O(1:1)]]> Crystal form A

[0217] 6. Room temperature suspension stirring

[0218] Weigh approximately 25 mg of each WX001 crystal form A into an HPLC vial, add 0.5 mL of solvent, and place the resulting turbid solution in a room temperature environment with magnetic stirring (1000 rpm) for 3 days. Then, centrifuge to collect the solid and perform XRPD testing. The experimental results are shown in Table 12.

[0219] Table 12 Summary of Room Temperature Suspension and Stirring Test

[0220] Solvent (v / v) Test results EtOH Crystal form A EtOAc Crystal form A THF Crystal form A DCM Crystal form A n-Heptane Crystal form A <![CDATA[H2O]]> Crystal form A <![CDATA[EtOH / H2O(0.97:0.03,a w ~0.2)]]> Crystal form A <![CDATA[EtOH / H2O(0.93:0.07,a w ~0.4)]]> Crystal form A <![CDATA[EtOH / H2O(0.86:0.14,a w ~0.6)]]> Crystal form A <![CDATA[EtOH / H2O(0.71:0.29,a w ~0.8)]]> Crystal form A ACN Crystal form A

[0221] 7.50℃ Suspension stirring

[0222] Weigh approximately 25 mg of WX001 crystal form A into an HPLC vial, add 0.5 mL of solvent, and stir the resulting suspension magnetically at 50°C (1000 rpm) for 3 days. Then, centrifuge to collect the solid and perform XRPD testing. The experimental results are shown in Table 13.

[0223] Table 13 Summary of the 50℃ Suspension Stirring Test

[0224] Solvent (v / v) Test results IPA Crystal form A <![CDATA[Acetone / H2O (1:1)]]> Crystal form A IPAc Crystal form A MTBE Crystal form A 2-MeTHF Crystal form A 1,4-Dioxane Crystal form A <![CDATA[CHCl3 / n - heptane (1:1)]]> Crystal form A Toluene Crystal form A <![CDATA[DMSO / H2O(1:1)]]> Crystal form A ACN Crystal form A

[0225] 8. Antisolvent addition method

[0226] Weigh approximately 15 mg of WX001 crystal form A into 20 mL vials and dissolve the solid completely with 0.7–1.0 mL of solvent. While stirring (1000 rpm), add the antisolvent dropwise to the clear solution until solid precipitates, or until the total volume of antisolvent reaches 10 mL. For samples without solid precipitation, suspend and stir at 5 °C; for clear samples, transfer to -20 °C and suspend and stir; for samples still clear, allow to evaporate at room temperature. Separate the precipitated solid and perform XRPD testing. The results are shown in Table 14.

[0227] Table 14 Summary of Antisolvent Addition Experiments

[0228]

[0229] * indicates that the sample was clear at room temperature and the solid was obtained by stirring at 5°C.

[0230] Example 4: Study on the hygroscopicity of WX001 crystal form A

[0231] Experimental materials:

[0232] SMSDVS Advantage Dynamic Vapor Adsorption Unit

[0233] Experimental methods:

[0234] Take 10-30 mg of WX001 crystal form A1 and place it in the DVS sample tray for testing.

[0235] Experimental results:

[0236] The DVS spectrum of WX001 crystal form A is as follows: Figure 4 As shown, △W = 0.1134%.

[0237] Experimental conclusion:

[0238] WX001 crystal form A has a moisture absorption weight gain of 0.1134% at 25℃ and 80%RH, and is almost non-hygroscopic.

[0239] Example 5: Stability test of WX001 crystal form A

[0240] Weigh out 12 parallel portions of WX001 crystal form A sample, approximately 5 mg each, and place them at the bottom of an HPLC vial, spreading them into a thin layer. Seal the vial openings of samples placed in a 60℃ / 75% humidity incubator and a 92.5% RH desiccator with sealing film, making small holes to ensure sufficient contact between the sample and ambient air. Tightly cap the vials of samples placed at 60℃ under both light and light-shielding conditions (the samples under light-shielding conditions were wrapped in aluminum foil). The experimental results are shown in Table 15 below:

[0241] Table 15 Results of solid stability tests for WX001 crystal form A

[0242]

[0243] #: ICH conditions

[0244] Conclusion: WX001 crystal form A has good stability.

[0245] Experimental Example 1: In vitro enzyme activity test

[0246] 1. Experimental objective:

[0247] The ability of compounds to inhibit the activity of ERK1 and ERK2 kinases was measured.

[0248] 2. Experimental buffer solution:

[0249] 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.

[0250] 3. Compound treatment:

[0251] The test compound was dissolved in 100% DMSO to prepare a stock solution of a specific concentration. The compound was then continuously diluted in the DMSO solution using the Integra ViafloAssist smart pipette.

[0252] 4. Experimental Methods:

[0253] 1) Prepare the substrate MBP in the freshly prepared reaction buffer;

[0254] 2) Add the ERK1 (or ERK2) kinase to the above MBP solution and mix gently;

[0255] 3) Using ultrasound technology (Echo 550; nanoliter range), the compound dissolved in 100% DMSO was added to the kinase reaction system and incubated at room temperature for 20 minutes;

[0256] 4) 33 When P-ATP (at a specific concentration of 10 μCi / μL) is added to the reaction system, the reaction begins.

[0257] 5) Incubate at room temperature for 2 hours;

[0258] 6) Detect the amount of radioactivity using a filtration-binding method;

[0259] 7) ERK1 (or ERK2) kinase activity is calculated as the ratio of the remaining kinase activity in the test sample to the kinase activity in the control group (treated with dimethyl sulfoxide). Curve fitting is performed using Prism (GraphPad software), and IC50 is calculated. 50 value.

[0260] 5. The experimental results are shown in Tables 16 and 17:

[0261] Table 16 Results of ERK1 enzyme activity test

[0262]

[0263] Conclusion: The compounds of this invention exhibit superior inhibitory activity against ERK1 enzyme.

[0264] Table 17 Results of ERK2 enzyme activity test

[0265]

[0266] Conclusion: The compounds of this invention exhibit superior inhibitory activity against ERK2 enzyme.

[0267] Experiment Example 2: In vitro cell proliferation inhibition experiment

[0268] 1. Experimental objective:

[0269] The ability of the compound to inhibit the proliferation of HT29 tumor cells was measured.

[0270] 2. Compound treatment:

[0271] The test compound was dissolved in 100% DMSO to prepare a 10 mM stock solution.

[0272] 3. Experimental steps and methods:

[0273] 1) Turn on the UV light in the biosafety cabinet and start the countdown in 30 minutes;

[0274] 2) Preheat RPMI 1640 medium and trypsin in a 37℃ water bath;

[0275] 3) After UV irradiation is complete, open the biosafety cabinet, wipe the preheated culture medium, trypsin, phosphate buffered saline (PBS) solution, etc. with alcohol and place them in the biosafety cabinet;

[0276] 4) Remove HT29 cells from the incubator, remove the old culture medium in a biosafety cabinet, add 10 ml of PBS, gently shake, and remove the PBS;

[0277] 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 it in the incubator for 2 minutes;

[0278] 6) Terminate cell digestion with complete culture medium and pipette until a homogeneous cell suspension is formed for cell counting;

[0279] 7) Based on the cell count results, adjust the cell suspension density to 1500 cells per well and seed the plate with 50 μL per well;

[0280] 8) Continuously dilute the compound stock solution in DMSO solution, and add the compound to the cell plate using Tecan;

[0281] 9) After adding the compound to the cell plate and CellTiterGlo, bring the mixture to room temperature for equilibration. Then 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.50 .

[0282] 4. The experimental results are shown in Table 18:

[0283] Table 18 Results of in vitro cell viability assay

[0284]

[0285] Conclusion: The compounds of this invention exhibit superior inhibitory activity against the proliferation of HT29 cells.

[0286] Experimental Example 3: PK Study in Mice

[0287] 1. Experimental objective:

[0288] Female BALB / c mice were used as test animals. After a single dose, the blood concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

[0289] 2. Experimental Procedure:

[0290] Four healthy adult female BALB / c mice were selected, with two receiving intravenous injection and two receiving oral administration. In the intravenous injection group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with an appropriate amount of the intravenous solvent, vortexed, and sonicated to prepare a clear solution of 0.5 mg / mL, which was then filtered through a microporous membrane for later use. In the oral administration group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with the solvent, vortexed, and sonicated to prepare a solution of 0.3 mg / mL. After intravenous administration of 1 mg / kg or oral administration of 3 mg / kg to the mice, whole blood was collected at a certain time to prepare plasma. Drug concentration was analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using PhoenixWinNonlin software (Pharsight Pharmaceuticals, USA). Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0291] 3. The experimental results are shown in Table 19:

[0292] Table 19 PK test results of compounds

[0293]

[0294] Note: C max Maximum concentration; F% is oral bioavailability; DNAUC = AUC PO / Dose, AUC PO This refers to oral exposure; Dose is the drug dose; Vd ss T is the distribution volume; Cl is the clearance rate; T 1 / 2 NA indicates half-life; NA indicates untested.

[0295] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0296] Experiment Example 4: Solubility Study

[0297] 1. Experimental objective:

[0298] To determine the solubility of a compound and to evaluate its solubility.

[0299] 2. Test solution:

[0300] 1) Buffer A (pH 2.0): 50mM phosphate buffer, pH 2.0; Buffer B (pH 6.5): 50mM phosphate buffer, pH 6.5; Buffer C (pH 7.4): 50mM phosphate buffer, pH 7.4;

[0301] 2) Preparation of standard solutions:

[0302] a) Mix a 50% acetonitrile solution and a 50% buffer solution to obtain a diluted solution;

[0303] b) Mix 10 mM (10 μL / compound) compound stock solution with diluent (490 μL / compound) to prepare a 200 μM detection standard solution;

[0304] c) Dilute the 200 μM UV detection standard solution with 10-fold and 200-fold diluents to obtain 20 μM and 1 μM UV standard solutions;

[0305] d) 1 μM, 20 μM and 200 μM UV standard solutions were used as standard solutions for solubility tests.

[0306] 3. Experimental methods:

[0307] 1) Dissolve the compound in DMSO to prepare a 10 mM stock solution. Amiodarone hydrochloride, carbamazepine, and chloramphenicol were used as controls in the solubility test.

[0308] 2) Stock solutions (10 μL each) of the test compound and control were placed in 96-well plates, and 490 μL of each of three different dissolving media (buffer A, B, C) were added, corresponding to pH values ​​of 2.0, 6.5, and 7.4, respectively. The theoretical maximum concentration of the experimental compound is 200 μM, containing 2% DMSO;

[0309] 3) Shake in a shaker at 600 revolutions per minute for 24 hours at room temperature (25±2℃);

[0310] 4) Pipette 200 μL of the solution into a 96-well plate vacuum filtration device and then transfer it to a new 96-well plate as a test sample.

[0311] 5) The concentration of the compound was tested by HPLC-UV, and the HLPC conditions are shown in Table 20.

[0312] Table 20 HPLC conditions

[0313]

[0314]

[0315] 6) Inject three UV standard solutions into the HPLC system at concentrations ranging from low to high (1 μM, 20 μM, 200 μM), and then inject the test sample containing the compound to be tested.

[0316] 7) Integrate the ultraviolet chromatographic peaks and calculate the solubility of the sample.

[0317] 4. The experimental results are shown in Table 21:

[0318] Table 21 Results of compound solubility test

[0319]

[0320] Conclusion: The compounds of this invention exhibit good solubility under different pH conditions.

[0321] Experimental Example 5: In vivo efficacy study of a human melanoma A375 mouse model

[0322] 1. Experimental objective:

[0323] The antitumor effect of WX001 was evaluated using a nude mouse model of subcutaneous xenograft of human melanoma A375 cells.

[0324] 2. Laboratory animals:

[0325] Species: Mouse

[0326] Strain: BALB / c nude mice

[0327] Age: 6-8 weeks

[0328] Sex: Female

[0329] Weight: 18-22 grams

[0330] Supplier: Vital River Laboratory Animal Technology Co., Ltd.

[0331] 3. Rearing environment:

[0332] Animals are housed in SPF-grade animal rooms in IVC (independent ventilation system, constant temperature and humidity) cages (3 animals per cage), with a temperature of 20-26℃ and a humidity of 40-70%.

[0333] Cage: Made of polycarbonate, with dimensions of 375mm x 215mm x 180mm, and corn cob bedding that is changed weekly;

[0334] Food: Laboratory animals can eat freely throughout the experimental period (sterilized by irradiation, dry granular food);

[0335] Drinking water: Laboratory animals may drink sterilized water freely;

[0336] Cage labeling: Each cage animal information card should indicate the number of animals in the cage, sex, strain, date of receipt, drug administration protocol experiment number, group, and experiment start date;

[0337] Animal identification: Laboratory animals are identified by ear tags.

[0338] 4. Experiment Content:

[0339] 1) Experimental Cells and Culture: Human melanoma A375 cells were cultured in vitro in a monolayer using DMEM medium supplemented with 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0340] 2) Tumor tissue inoculation and grouping: 0.1 mL (5 × 10⁻⁶) 5 A375 cells were subcutaneously injected into the right axilla of each mouse, and the average tumor volume reached 170 mmHg. 3 Animals were randomly divided into four groups and drug administration began. The experimental groupings and administration regimens are shown in Table 22.

[0341] Table 22 Grouping and Dosing Regimens of Experimental Animals

[0342]

[0343] 3) Daily Observation of Laboratory Animals: The formulation of this experimental protocol and any modifications thereof have been evaluated and approved by the International Association for the Management and Use of Laboratory Animals (IACUC). The use and welfare of laboratory animals are conducted in accordance with the regulations of the International Committee for the Evaluation and Accreditation of Laboratory Animals (AAALAC). Animal health and mortality are monitored daily. Routine examinations include observing the effects of tumor growth and drug treatment on daily behavior, such as activity levels, food and water intake (visual assessment only), weight changes (measured twice weekly), physical appearance, or other abnormalities. The number of deaths and side effects within each group are recorded based on the number of animals in each group.

[0344] 4) Preparation of the test substance

[0345] a) Vehicle group: 5% DMSO+95% (20% HP-β-CD).

[0346] b) Test compound group: Weigh a quantitative amount of the test compound into a reagent bottle, add the corresponding volume of DMSO and vortex to obtain a clear solution, add the corresponding volume of 20% HP-β-CD and vortex to obtain a homogeneous suspension.

[0347] 5) Tumor measurement and experimental indicators:

[0348] a) Measure the tumor diameter twice a week using calipers. The formula for calculating tumor volume is: TV = 1 / 2 × a × b 2 a and b represent the long and short diameters of the tumor, respectively;

[0349] b) The antitumor efficacy of the compound was evaluated using TGI (%). TGI (%) reflects the tumor growth inhibition rate. The calculation of TGI (%) is as follows: TGI (%) = {[1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment)] / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the beginning of treatment in the solvent control group)} × 100%.

[0350] 5. Experimental Results:

[0351] 1) As shown in Table 23 and Figure 5 As shown, in a nude mouse model of human melanoma A375 cell subcutaneous xenograft tumor, WX001 was able to inhibit tumor growth in a dose-dependent manner after oral administration up to day 21, with TGI of 45%, 58% and 102% at three doses of 12.5 mg / kg, 25 mg / kg and 50 mg / kg, respectively.

[0352] 2) The body weight of experimental animals serves as a reference indicator for indirectly determining drug toxicity. For example... Figure 6 As shown, by day 21 of administration, the body weight of all animals in the solvent control group and the WX001 group did not decrease significantly, indicating good tolerability.

[0353] Table 23 Results of in vivo pharmacological experiments in mouse A375 model

[0354] drug TGI WX001 (12.5 mg / kg, PO, BID) 45％ WX001 (25mg / kg, PO, BID) 58％ WX001 (50mg / kg, PO, BID) 102％

[0355] Experimental conclusions: WX001 was able to inhibit tumor growth in a dose-dependent manner at three dosages of 12.5 mg / kg, 25 mg / kg and 50 mg / kg; no significant decrease in body weight was observed in the animals during the administration process, indicating good tolerability.

[0356] Experimental Example 6: In vivo PK study in SD rats

[0357] 1. Experimental objective:

[0358] Male SD rats were used as test animals. After a single administration, the blood concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

[0359] 2. Experimental Procedure:

[0360] Six healthy adult male SD rats were selected, with three receiving intravenous injection and three receiving oral administration. In the intravenous injection group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with an appropriate amount of the intravenous solvent, vortexed, and sonicated to prepare a clear solution of 0.2 mg / mL, which was then filtered through a microporous membrane for later use. In the oral administration group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with the solvent, vortexed, and sonicated to prepare a solution of 1 mg / mL. After intravenous administration of 1 mg / kg or oral administration of 10 mg / kg to the SD rats, whole blood was collected at a certain time to prepare plasma. Drug concentration was analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using PhoenixWinNonlin software (Pharsight Pharmaceuticals, USA). Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0361] 3. The experimental results are shown in Table 24:

[0362] Table 24 PK test results of compounds

[0363]

[0364] Note: C max Maximum concentration; F% is oral bioavailability; DNAUC = AUC PO / Dose, AUC PO This refers to oral exposure; Dose is the drug dose; Vd ss T is the distribution volume; Cl is the clearance rate; T 1 / 2 It is the half-life.

[0365] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0366] Experimental Example 7: PK Study in Crab-Eating Monkeys

[0367] 1. Experimental objective:

[0368] Male cynomolgus monkeys were used as test animals. After a single dose, the blood concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

[0369] 2. Experimental Procedure:

[0370] Five healthy adult male cynomolgus macaques were selected, with two receiving intravenous injection and three receiving oral administration. In the intravenous injection group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with an appropriate amount of the intravenous solvent, stirred, and dissolved to prepare a clear solution of 0.4 mg / mL, which was then filtered through a microporous membrane for later use. In the oral administration group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with the solvent, stirred, and dissolved to prepare a solution of 0.3 mg / mL. After intravenous administration of 1 mg / kg or oral administration of 3 mg / kg to the cynomolgus macaques, whole blood was collected at a certain time to prepare plasma. Drug concentration was analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using PhoenixWinNonlin software (Pharsight Pharmaceuticals, USA).

[0371] Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0372] 3. The experimental results are shown in Table 25:

[0373] Table 25 PK test results of compounds

[0374]

[0375] Note: C max Maximum concentration; F% is oral bioavailability; DNAUC = AUC PO / Dose, AUC PO This refers to oral exposure; Dose is the drug dose; Vd ss T is the distribution volume; Cl is the clearance rate; T 1 / 2 It is the half-life.

[0376] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0377] Experimental Example 8: PK Study in Beagle Dogs

[0378] 1. Experimental objective:

[0379] Male beagle dogs were used as test animals. After a single dose, the blood concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

[0380] 2. Experimental Procedure:

[0381] Five healthy adult male beagle dogs were selected, with two receiving intravenous injection and three receiving oral administration. In the intravenous injection group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with an appropriate amount of the intravenous solvent, stirred, and dissolved to prepare a clear solution of 0.4 mg / mL, which was then filtered through a microporous membrane for later use. In the oral administration group, the solvent was 5% DMSO + 95% (20% HP-β-CD). The test compound was mixed with the solvent, stirred, and dissolved to prepare a solution of 0.3 mg / mL. After administration of 1 mg / kg intravenously or 3 mg / kg orally, whole blood was collected at a certain time to prepare plasma. Drug concentration was analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using PhoenixWinNonlin software (Pharsight Pharmaceuticals, USA).

[0382] Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0383] 3. The experimental results are shown in Table 26:

[0384] Table 26 PK test results of compounds

[0385]

[0386] Note: C max Maximum concentration; F% is oral bioavailability; DNAUC = AUC PO / Dose, AUC PO This refers to oral exposure; Dose is the drug dose; Vd ss T is the distribution volume; Cl is the clearance rate; T 1 / 2 It is the half-life.

[0387] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0388] Experiment Example 9: hERG Test

[0389] 1. Experimental objective:

[0390] The effects of the compound on the current of the hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) were tested using a fully automated patch-clamp method.

[0391] 2. Experimental Methods:

[0392] 2.1 Cell Preparation

[0393] CHO-hERG cells were cultured at 175 cm⁻¹ 2In the culture flask, once the cell density reaches 60-80%, remove the culture medium, wash once with 7 mL of PBS (Phosphate Buffered Saline), and then add 3 mL of Detachin for digestion. After complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and resuspend in 5 mL of culture medium to ensure a cell density of 2-5 × 10⁶ cells / year. 6 / mL.

[0394] 2.2 Solution Preparation

[0395] Extracellular fluid formulation (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1.25 MgCl2, 10 HEPES and 10 Glucose, adjusted to pH 7.4 with NaOH.

[0396] Intracellular fluid formulation (mM): 140 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA and 10 HEPES, pH adjusted to 7.2 with KOH.

[0397] 2.3 Electrophysiological Recording Process

[0398] The entire process of single-cell high-impedance sealing and whole-cell pattern formation was automated by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +40 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds, and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular fluid was applied for 5 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2.5 minutes. After all concentrations were administered, the positive control compound 3MCisapride was administered. At least 3 cells were tested for each concentration (n≥3).

[0399] 2.4 Compound Preparation

[0400] The 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 the solution was serially diluted 3-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. Then, 80 μL of each of the six intermediate concentrations was added to 320 μL of extracellular fluid and diluted 5-fold to the final concentration to be tested. The highest test concentration was 40 μM, with subsequent 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 of DMSO has no effect on the hERG potassium channel. The entire dilution process was performed using a Bravo instrument.

[0401] 2.5 Data Analysis

[0402] Experimental data were analyzed using GraphPad Prism 5.0 software.

[0403] 2.6 Quality Control

[0404] Environment: Humidity 20-50%, temperature 22-25℃

[0405] Reagents: All experimental reagents used were purchased from Sigma-Aldrich and had a purity >98%.

[0406] The experimental data in the report must meet the following standards:

[0407] Whole-cell sealing impedance >100MΩ

[0408] Tail current amplitude >300pA

[0409] Pharmacological parameters:

[0410] The inhibitory effect of multiple concentrations of Cisapride on hERG channels was used as a positive control.

[0411] 3. The experimental results are shown in Table 27:

[0412] Table 27 hERG test results of compounds

[0413] compound IC50 (μM) WX001 >40

[0414] Conclusion: The compounds of this invention have a weak inhibitory effect on hERG potassium channel current, a lower risk of cardiotoxicity, and a higher safety profile.

[0415] Experimental Example 10: Plasma Protein Binding Test (PPB)

[0416] 1. Experimental objective:

[0417] The study investigated the binding of the test compound to human / mouse / rat / dog / monkey plasma albumin.

[0418] 2. Experimental Procedure:

[0419] 1) Matrix preparation: On the day of the experiment, the plasma was thawed in cold water and centrifuged at 3220 rpm for 5 min to remove all blood clots. The pH of the obtained plasma was measured and adjusted to 7.4 ± 0.1 as needed using 1% phosphoric acid or 1N sodium hydroxide.

[0420] 2) Dilution procedure for the test compounds: The test compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at concentrations of 10 mM and 2 mM. 2 μL of the stock solution (2 mM) was diluted with 98 μL of DMSO to prepare a 40 μM working solution. 10 μL of the stock solution was diluted with 240 μL of DMSO to prepare a 400 μM working solution of the control compound. The working solution of the compound (5 μL) was mixed thoroughly with the blank matrix (995 μL) at a ratio of 1:200 to prepare the loading matrix.

[0421] 3) Analysis steps:

[0422] a) Transfer equal volumes of 30 μL of loaded matrix (n = 2) to the sample collection plate to prepare the test time 0 (T0) sample for residue determination. Immediately match the sample with the corresponding blank buffer to a final volume of 60 μL, with a plasma-to-buffer volume ratio of 1:1 per well. Then, add 60 μL of 4% H₃PO₄ H₂O and 480 μL of stop solution containing the internal standard to the T0 samples for testing the compound. These samples are then stored together with other samples at 2–8 °C for further processing.

[0423] b) Pre-incubate the remaining plasma samples in a CO2 incubator at 37±1℃ for 30 min. Prepare protein-free samples (F samples). The samples with the matrix loaded (230 μL) were transferred to polycarbonate tubes (n=2) and ultracentrifuged at 37℃ and 155000×g (35000rpm) for 4 h.

[0424] c) To prepare the T sample (test sample), an additional matrix-containing sample was transferred to a separate 96-well plate (sample incubation plate) and incubated at 37°C for 4 hours.

[0425] d) After centrifugation, transfer 30 μL of protein-free sample and 30 μL of T sample from the second layer (below the top layer) of the supernatant 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 matrix:buffer volume ratio of 1:1. Add 60 μL of 4% H3PO4 aqueous solution and 480 μL of stop solution (containing internal standard) to all samples.

[0426] The mixture was centrifuged at 4000 rpm for 20 min, and 100 μL of the supernatant from each sample was taken for LC-MS / MS analysis.

[0427] 3. The experimental results are shown in Table 28:

[0428] Table 28 Results of plasma protein binding tests for compounds

[0429]

[0430] Conclusion: The compounds of this invention have moderate plasma protein binding.

Claims

1. The compound of formula (I) or a pharmaceutically acceptable salt thereof, in, R1 and R2 are independently selected from H and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. a replace; R4, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. c replace; n is 0 or 1; m is 1 or 2; Ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl groups are optionally surrounded by 1, 2 or 3 R groups. d replace; R a and R c They were each independently selected from D, F, Cl, Br, and I; R d Selected from F, Cl, Br, I, C 1-3 Alkyl and C 1-3 Alkoxy, the C 1-3 Alkyl and C 1-3 The alkoxy group can be optionally substituted with 1, 2 or 3 R groups; R is selected from F, Cl, Br and I.

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, wherein CH3 and CH2CH3 are optionally converted by 1, 2, or 3 Rs. a replace.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are independently selected from H, CH3, CHF2, CD3 and CH2CH3, 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, F, Cl, Br, I, and CH3, respectively, wherein CH3 is optionally divided by 1, 2, or 3 Rs. c replace.

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

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R d Selected from F, Cl, Br, I and C 1-3 Alkyl, the C 1-3 The alkyl group may be optionally substituted with 1, 2 or 3 Rs.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R d The ingredients are selected from F, Cl, Br, I, CH3 and OCH3, wherein CH3 and OCH3 are optionally replaced by 1, 2 or 3 R.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein, R d The ingredients are selected from F, Cl, Br, I and CH3, wherein the CH3 is optionally replaced by 1, 2 or 3 R.

9. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein, R d Selected from CH3 and OCH3.

10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein, R d Selected from CH3.

11. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from pyrazolyl groups, wherein the pyrazolyl group is optionally surrounded by 1, 2 or 3 R groups. d replace.

12. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from , and The , and Choose 1, 2, or 3 Rs d replace.

13. The compound of claim 12 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from The Choose 1, 2, or 3 Rs d replace.

14. The compound of claim 12 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from , and .

15. The compound of claim 14 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from and .

16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, Structural unit Selected from .

17. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, selected from... ， in, R2 is defined as in any one of claims 1 to 3; R6 and R7 are as defined in any one of claims 1, 4 or 5.

18. The compound shown in the following formula, or a pharmaceutically acceptable salt thereof, 。 19. 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°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°; 。 20. The crystal form A according to claim 19, wherein its X-ray powder diffraction pattern 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°.

21. The crystal form A according to claim 20, wherein its X-ray powder diffraction pattern 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°, 26.9544°.

22. The crystal form A according to claim 21, the XRPD pattern of which is basically as shown in Figure 1.

23. Crystal form A according to any one of claims 19 to 22, wherein the differential scanning calorimetry curve has an endothermic starting point at 241.0 ± 3.0 °C.

24. The DSC spectrum of crystal form A according to claim 23 is shown in Figure 2.

25. Crystal form A according to any one of claims 19 to 22, wherein the thermogravimetric analysis curve shows a weight loss of 0.83% at 150.0 ± 3.0 °C.

26. The TGA spectrum of crystal form A according to claim 25 is shown in Figure 3.

27. The use of the compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, or crystal form A according to any one of claims 19 to 26, in the preparation of ERK1 or ERK2 inhibitors.

28. A medicament comprising the compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, or crystal form A according to any one of claims 19 to 26.

Images