Dimethyl substituted thiazolide compounds and uses thereof

Abstract

Provided are dimethyl-substituted thiazolide compounds and use thereof in the preparation of drugs for treating related diseases, in particular, compounds represented by formula (I) and pharmaceutically acceptable salts thereof.

Description

[0001] This application claims the following priority:

[0002] CN202110723288.5, application date: June 28, 2021;

[0003] CN202111669920.9, application date: December 31, 2021;

[0004] CN202210693547.9, application date: June 17, 2022. Technical Field

[0005] This invention relates to a class of dimethyl-substituted thiazolactam 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] Each R4 is independently selected from H, F, Cl, Br, I, and C. 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 selected from 1 and 2;

[0016] 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;

[0017] R a and R c They were each independently selected from D, F, Cl, Br, and I;

[0018] 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;

[0019] R is selected from F, Cl, Br and I.

[0020] This invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0021]

[0022] in,

[0023] 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;

[0024] Each R4 is independently selected from H, F, Cl, Br, I, and C. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. c replace;

[0025] n is selected from 1 and 2;

[0026] 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;

[0027] R a and R c They were each independently selected from D, F, Cl, Br, and I;

[0028] 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;

[0029] R is selected from F, Cl, and Br.

[0030] 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.

[0031] 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.

[0032] In some embodiments of the present invention, the R4 is independently selected from H, F, Cl, Br, I, and CH3, wherein the CH3 is optionally converted by 1, 2, or 3 Rs. c Replacement, other variables as defined in this invention.

[0033] In some embodiments of the present invention, the R4 is independently selected from H, F, Cl, Br, I and CH3, and other variables are as defined in the present invention.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In some embodiments of the present invention, the ring A is selected from... Other variables are as defined in this invention.

[0038] In some embodiments of the present invention, the above-mentioned structural unit Selected from Other variables are as defined in this invention.

[0039] In some embodiments of the present invention, the above-mentioned structural unit Selected from Other variables are as defined in this invention.

[0040] The present invention also includes some solutions derived from arbitrary combinations of the above variables.

[0041] In some embodiments of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from:

[0042]

[0043] Wherein, R2 and R4 are as defined in this invention.

[0044] The present invention also provides compounds of the following formula or pharmaceutically acceptable salts thereof.

[0045]

[0046] The present invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating solid tumors.

[0047] Technical effect

[0048] 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; the compounds of this invention have weak inhibitory effect on hERG potassium channel current, lower risk of cardiotoxicity, and higher safety; the compounds of this invention have moderate to high plasma protein binding.

[0049] Definitions and Explanations

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereo isomers, enantiomers, optical isomers, diastereomers and tautomers.

[0055] 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.

[0056] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0057] 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.

[0058] 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.

[0059] Unless otherwise stated, "(+)" indicates right-handed rotation, "(-)" indicates left-handed rotation, and "(±)" indicates racemic rotation.

[0060] 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

[0061] 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.

[0062] 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%.

[0063] 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%.

[0064] 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).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] When the number of a linking group is 0, such as -(CRR)0-, it indicates that the linking group is a single bond.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] The solvent used in this invention is commercially available.

[0081] 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; Cbz represents benzyloxycarbonyl, an amine protecting group; BOC represents tert-butyloxycarbonyl, an amine protecting group; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; Boc2O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; iPrOH represents 2-propanol; mp represents melting point.

[0082] Compounds are named according to conventional naming principles in the field or using Software naming conventions are used; commercially available compounds use supplier catalog names.

[0083] Instruction manual illustrations

[0084] Figure 1 Tumor growth curves of human melanoma A375 model animals after administration of solvent and WX001, respectively;

[0085] Figure 2 The rate of weight change in human melanoma A375 model animals during drug administration. Detailed Implementation

[0086] 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.

[0087] Reference Example 1

[0088]

[0089] Step 1: Synthesis of compound A-1-2.

[0090] Add A-1-1 (500 g, 2.12 mol, 1 eq), water (1875 mL), and tetrahydrofuran (1875 mL) to a reaction flask. After purging with nitrogen, add lithium hydroxide monohydrate (97.76 g, 2.33 mol, 1.1 eq). React the mixture at 25°C for 3 hours. After the reaction is complete, concentrate to remove the organic solvent, then add ice water (2 L), followed by slowly adding 4N hydrochloric acid solution (600 mL) to adjust the pH to 2-3. Stir for 20 minutes, filter, wash the filter cake with water (1 L) and acetonitrile (500 mL), and collect the filter cake. Add acetonitrile (1 L) to the filter cake and stir for 0.5 hours. Filter, wash the filter cake with acetonitrile (500 mL), collect the filter cake, and dry it to obtain A-1-2. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 13.32 (br s, 1H), 8.46 (s, 1H).

[0091] Step 2: Synthesis of compound A-1-3.

[0092] Add A-1-2 (175 g, 823.55 mmol, 97.9% purity, 1 eq) and 2-methyltetrahydrofuran (1.75 L) to the reaction flask. After purging with nitrogen, cool to -30 °C and slowly add diisopropylaminolithium (2 M, 905.90 mL, 2.2 eq). Stir at -30 °C for 1 hour. Then slowly add acetone (95.66 g, 1.65 mol, 121.09 mL, 2 eq) and a solution of 2-methyltetrahydrofuran (175 mL). React the mixture at -30 °C for 1 hour. After the reaction is complete, quench the reaction solution with saturated ammonium chloride aqueous solution (1750 mL) and adjust the pH to 3-4 with 4N hydrochloric acid (approximately 2 L). Separate the solution, and extract the aqueous phase with ethyl acetate (3000 mL x 2). The organic phase was washed with saturated brine (1500 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product. Methyl tert-butyl ether (3.5 L) was added to the crude product, and the mixture was stirred for 30 minutes. Then, n-hexane (3.5 L) was added, and the mixture was stirred for another 4 hours. The mixture was filtered, and the filter cake was collected to obtain A-1-3. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 13.28 (br s, 1H), 6.67-5.90 (br s, 1H), 1.62 (s, 6H).

[0093] Step 3: Synthesis of compound A-1-4.

[0094] Add A-1-3 (200 g, 668.89 mmol, 89% purity, 1 eq) and acetonitrile (2 L) to a reaction flask. After purging with nitrogen, add boron trifluoride diethyl ether solution (265.82 g, 1.87 mol, 231.15 mL, 2.8 eq). React the mixture at 60 °C for 8 hours. After the reaction is complete, add ethanol (200 mL) to the reaction solution and concentrate under reduced pressure to obtain a crude product. Then, slowly pour the crude product into water (2000 mL), stir for 30 minutes, filter, and collect the filter cake. Add anhydrous ethanol (600 mL) to the filter cake, stir for 30 minutes, filter, wash the filter cake with ethanol (200 mL), collect the filter cake, and dry it to obtain A-1-4. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 2.49 (s, 3H), 1.77 (s, 6H).

[0095] Step 4: Synthesis of compound A-1.

[0096] Add A-1-4 (150 g, 481.93 mmol, 92.9% purity, 1 eq) and ethanol (750 mL) to a reaction flask. After purging with nitrogen, slowly add hydrobromic acid (1.07 kg, 5.30 mol, 719.67 mL, 40% purity, 11 eq). React the mixture at 50 °C for 24 hours. After the reaction is complete, add dichloromethane (1.5 L) and ice water (500 mL) to the reaction solution, and adjust the pH to 7-8 with approximately 1500 mL of 4N sodium hydroxide aqueous solution. Separate the solutions. Extract the aqueous phase with dichloromethane (1000 mL x 2) to obtain the organic phase. Wash the organic phase with saturated brine (1000 mL) and dry it with anhydrous sodium sulfate. Filter the solution and concentrate the filtrate under reduced pressure to obtain the crude product. Add ethyl acetate (225 mL) and n-hexane (225 mL) to the crude product and stir for 2 hours. Filter and collect the filter cake to obtain A-1. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.88 (s, 1H), 1.51 (s, 6H).

[0097] See Example 2

[0098]

[0099] Step 1: Synthesis of compound B-1-2.

[0100] Sodium hydroxide (590.8 g, 14.8 mol, 1.05 eq), 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).

[0101] Step 2: Synthesis of compound B-1-3.

[0102] 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).

[0103] Step 3: Synthesis of compound B-1.

[0104] 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 (12000 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).

[0105] See Example 3

[0106]

[0107] Step 1: Synthesis of compound D-1-2.

[0108] Sodium acetate (4.54 g, 55.39 mmol, 5 eq), potassium persulfate (13.62 g, 22.16 mmol, 2 eq), and water (46 mL) were added to a dry reaction flask. The mixture was cooled to 0 °C, and a solution of D-1-1 (4.6 g, 11.08 mmol, 1 eq), methanol (46 mL), and tetrahydrofuran (46 mL) was added. The reaction was carried out at 25 °C for 12 hours. After the reaction was complete, the reaction solution was quenched with a saturated sodium sulfite aqueous solution (50 mL), extracted with dichloromethane (50 mL x 3), and the organic phases were combined. The organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a water pump to obtain D-1-2. 1 H NMR (400MHz, CDCl3) δ (ppm) = 8.66-8.67 (d, J = 4.60Hz, 1H), 7.64-7.65 (d, J = 4.82Hz, 1H), 3.37(s,3H),1.36-1.57(m,6H),1.33-1.35(m,6H),1.21-1.23(m,6H),0.88-0.95(m,9H).

[0109] Step 2: Synthesis of compound D-1.

[0110] Add D-1-2 (4.68 g, 10.46 mmol, 1 eq), D-1-3 (1.22 g, 12.56 mmol, 1.2 eq), and tetrahydrofuran (70 mL) to a dry reaction flask. Replace the atmosphere with nitrogen, and add hexamethyldisilamide lithium (1 M, 21.98 mL, 2.1 eq) dropwise at -30 °C. React at -30 °C for 2 hours. After the reaction is complete, pour the reaction mixture into a saturated ammonium chloride aqueous solution (100 mL), extract with ethyl acetate (100 mL x 3), and combine the organic phases. Wash the organic phase with saturated brine (100 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. Purify the crude product by column chromatography to obtain D-1. 1 HNMR (400MHz, CDCl3) δ (ppm) = 8.17 (d, J = 4.65Hz, 1H), 7.45 (d, J = 1.96Hz, 1H), 6.91 (d, J = 4.65Hz, 1H), 6.79 (br s,1H),6.31(d,J=1.96Hz,1H),3.78(s,3H),1.43-1.64(m,6H),1.24-1.38(m,6H),1.07-1.14(m,6H),0.89(t,J=7.34Hz,9H).

[0111] See Example 4

[0112]

[0113] Step 1: Synthesis of compound E-1-3.

[0114] Potassium tert-butoxide (4.83 g, 43.04 mmol, 2.5 eq) and tetrahydrofuran (16 mL) were added to the reaction flask. After purging with nitrogen, a tetrahydrofuran solution of E-1-1 (2 g, 17.22 mmol, 2.30 mL, 1 eq) and E-1-2 (2.55 g, 34.44 mmol, 2.77 mL, 2 eq) was slowly added dropwise. The mixture was reacted at 25 °C for 3 hours. After the reaction was complete, the solution was concentrated to obtain E-1-3.

[0115] Step 2: Synthesis of compound E-1-5.

[0116] E-1-3 (2.24 g, 17.21 mmol, 1 eq) and isopropanol (140 mL) were added to the reaction flask. After purging with nitrogen, E-1-4 (2.62 g, 34.42 mmol, 2 eq) was added, and the mixture was reacted at 90 °C for 12 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain the crude product. 20 mL of water was added to the crude product, the pH was adjusted to 4 with acetic acid, and the mixture was filtered. The filter cake was collected. The filter cake was purified by column chromatography to obtain E-1-5. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 12.25 (br s, 2H), 7.22 (s, 1H), 2.21 (q, J = 7.5Hz, 2H), 1.01 (t, J = 7.5Hz, 3H).

[0117] Step 3: Synthesis of compound E-1-6.

[0118] Sodium hydroxide (1.34 g, 33.61 mmol, 1.05 eq) and water (50 mL) were added to the reaction flask, followed by compound E-1-5 (5 g, 32.01 mmol, 1 eq). After purging with nitrogen, the mixture was cooled to 10 °C, and iodomethane (5.68 g, 40.01 mmol, 2.49 mL, 1.25 eq) was slowly added. The mixture was reacted at 10 °C for 0.5 hours, then slowly heated to 25 °C and reacted for another 2.5 hours. After the reaction was complete, the reaction solution was cooled to 0–5 °C, and the pH was adjusted to 7–8 with 6 N hydrochloric acid. The mixture was filtered, the filter cake was collected, and dried to obtain E-1-6. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 12.64 (br s, 1H), 7.70 (br s, 1H), 2.45 (s, 3H), 2.29 (q, J = 7.4Hz, 2H), 1.06 (t, J = 7.4Hz, 3H).

[0119] Step 4: Synthesis of compound E-1-7.

[0120] Add E-1-6 (7.4 g, 43.47 mmol, 1 eq) and acetonitrile (75 mL) to a reaction flask. After purging with nitrogen, slowly add phosphorus oxychloride (8.33 g, 54.34 mmol, 5.05 mL, 1.25 eq). React the mixture at 62 °C for 2.5 hours. After the reaction is complete, pour the reaction solution into water (100 mL) and adjust the pH to 6–7 with solid sodium carbonate. Extract the aqueous phase with ethyl acetate (50 mL x 2). Combine the organic phases, wash with saturated brine (50 mL), and dry with anhydrous sodium sulfate. Filter and concentrate the filtrate under reduced pressure to obtain E-1-7. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.58 (s, 1H), 2.63 (q, J = 7.5Hz, 2H), 2.51 (s, 3H), 1.17 (t, J = 7.5Hz, 3H).

[0121] Step 5: Synthesis of compound E-1.

[0122] Add E-1-7 (3.5 g, 18.55 mmol, 1 eq) and acetonitrile (40 mL) to the reaction flask. After purging with nitrogen, add sodium iodide (18.52 g, 123.54 mmol, 6.66 eq), trimethylchlorosilane (6.71 g, 61.77 mmol, 7.84 mL, 3.33 eq), and water (802.26 mg, 44.52 mmol, 802.26 μL, 2.4 eq) sequentially. React the mixture at 25 °C for 12 hours. After the reaction is complete, add dichloromethane (50 mL) and water (50 mL) sequentially to the reaction solution, and then add sodium bicarbonate solid to adjust the pH to 6-7. After separation, extract the aqueous phase with dichloromethane (50 mL) and combine the organic phases. Wash the organic phase with saturated sodium sulfite aqueous solution (50 mL) and saturated brine (50 mL), respectively, and dry with 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 E-1. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.32 (s, 1H), 2.58-2.52 (m, 2H), 2.48 (s, 3H), 1.13 (t, J = 7.5Hz, 3H).

[0123] Example 1

[0124]

[0125] Synthesis route:

[0126]

[0127] Step 1: Synthesis of WX001-1

[0128] Add A-1 (70 g, 283.27 mmol, 1 eq), dichloromethane (1400 mL), 4-dimethylaminopyridine (38.07 g, 311.60 mmol, 1.1 eq), and di-tert-butyl dicarbonate (123.65 g, 566.54 mmol, 130.15 mL, 2 eq) to a dry reaction flask. Replace with nitrogen and react at 20 °C for 12 hours. After the reaction is complete, add water (300 mL) to the reaction solution to separate the organic and aqueous phases. Extract the aqueous phase three times with dichloromethane (400 mL). Combine the organic phases, wash with saturated brine (300 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. Pulverize the crude product with n-hexane (140 mL), filter, collect the filter cake, and dry to obtain WX001-1. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 1.75 (s, 6H), 1.52 (s, 9H).

[0129] Step 2: Synthesis of WX001-2

[0130] Add WX001-1 (10 g, 28.80 mmol, 1 eq), tetrahydrofuran (133 mL), zinc chloride solution (0.7 M, 41.14 mL, 1 eq), and tetramethylethylenediamine (3.35 g, 28.80 mmol, 4.35 mL, 1 eq) to a dry reaction flask. Cool to -78 °C and add n-butyllithium (2.5 M, 17.28 mL, 1.5 eq), stirring for 10 minutes. Add n-butyllithium (2.5 M, 5.76 mL, 0.5 eq), stirring for another 10 minutes. Add n-butyllithium (2.5 M, 3.46 mL, 0.3 eq), and continue reacting at 20 °C for 1 hour to obtain reaction solution 1.

[0131] A mixture of B-1 (7.66 g, 28.80 mmol, 1 eq), tetrakis(triphenylphosphine)palladium (998.39 mg, 863.99 μmol, 0.03 eq), and N'N-dimethylformamide (67 mL) was heated to 50 °C to obtain reaction solution 2. Reaction solution 1 was added dropwise to reaction solution 2, and the mixture was reacted at 50 °C for 40 minutes. After the reaction was complete, 450 mL of 0.1 M disodium ethylenediaminetetraacetate aqueous solution was added to the reaction solution, and the mixture was stirred for 30 minutes. The mixture was filtered, and the filter cake was collected to obtain the crude product. The crude product was purified by column chromatography to obtain WX001-2. 1 HNMR (400MHz, DMSO-d6) δ (ppm) = 8.72 (s, 1H), 2.59 (s, 3H), 2.58 (s, 3H), 1.82 (s, 6H), 1.54 (s, 9H).

[0132] Step 3: Synthesis of WX001-3

[0133] Add WX001-2 (10 g, 24.60 mmol, 1 eq), DCM (100 mL), and trifluoroacetic acid (36.49 g, 320.03 mmol, 23.69 mL, 13.01 eq) to a dry reaction flask, and react at 20 °C for 1 hour. After the reaction is complete, concentrate the reaction solution and then remove the residual trifluoroacetic acid by chloroform (30 mL x 3) to obtain WX001-3. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.89 (s, 1H), 8.72 (s, 1H), 2.61 (s, 3H), 2.59 (s, 3H), 1.57 (s, 6H).

[0134] Step 4: Synthesis of WX001-5

[0135] Add WX001-3 (150 mg, 489.55 μmol, 1 eq), N'N-dimethylformamide (1.5 mL), cesium carbonate (239.26 mg, 734.32 μmol, 1.5 eq), and WX001-4 (109.30 mg, 587.46 μmol, 1.2 eq) to a dry reaction flask. Replace the atmosphere with nitrogen and react at 25 °C for 16 hours. After the reaction is complete, add water (10 mL) to the reaction solution and filter to obtain a filter cake. Dissolve the filter cake in dichloromethane (10 mL) and wash with saturated brine (15 mL). Separate the liquid to obtain the organic phase. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain WX001-5. 1 H NMR (400MHz, DMSO-d6) δ = 8.74 (s, 1H), 7.62 (t, J = 7.8Hz, 1H), 7.18-7.06 (m, 2H), 4.72 (s, 2H), 2.64 (s, 3H), 2.59 (s, 3H), 2.45 (s, 3H), 1.54 (s, 6H).

[0136] Step 5: Synthesis of WX001-6

[0137] Add WX001-5 (130 mg, 315.88 μmol, 1 eq), acetonitrile (3 mL), water (1.5 mL), and potassium persulfate (388.39 mg, 631.77 μmol, 2 eq) to a dry reaction flask. Replace with nitrogen and react at 20 °C for 16 hours. After the reaction is complete, add saturated sodium thiosulfate solution (10 mL) to the reaction solution, followed by extraction with dichloromethane (10 mL x 3). Combine the organic phases, wash successively with saturated sodium bicarbonate aqueous solution (20 mL x 2) and saturated brine (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain WX001-6. 1H NMR (400MHz, DMSO-d6) δ = 9.18 (s, 1H), 7.62 (t, J = 7.7Hz, 1H), 7.13 (d, J = 7.7Hz, 2H), 4.73 (s, 2H), 3.48 (s, 3H), 2.82 (s, 3H), 2.45 (s, 3H), 1.57 (s, 6H).

[0138] Step 6: Synthesis of WX001

[0139] Add WX001-6 (140 mg, 315.64 μmol, 1 eq), D-1-3 (61.31 mg, 631.28 μmol, 2 eq), dichloromethane (1 mL), and tetrahydrofuran (1 mL) to a dry reaction flask. Replace the nitrogen atmosphere and cool the reaction solution to 0 °C. Add hexamethyldisilamide lithium (1 M, 599.72 μL, 1.9 eq) dropwise. After the addition is complete, continue the reaction at 0 °C for 2 hours. After the reaction is complete, quench the reaction with water (10 mL), followed by extraction with dichloromethane (10 mL x 3). Combine the organic phases, wash with saturated brine (15 mL), dry to anhydrous sodium sulfate, and filter. Concentrate the filtrate under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography (column: Waters Xbridge BEH C18 100*30mm*10μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; B (acetonitrile)%: 25%-55%, 8 minutes) to obtain WX001. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 9.61 (s, 1H), 8.60 (s, 1H), 7.61 (t, J = 7.69Hz, 1H), 7.39 (d, J = 1.88Hz, 1H), 7.12 (dd, J=7.57,3.56Hz,2H),6.34(d,J=1.88Hz,1H),4.71(s,2H),3.72(s,3H),2.58(s,3H),2.45(s,3H),1.53(s,6H); LCMS m / z:461[M+H] + .

[0140] Example 2

[0141]

[0142] Synthesis route:

[0143]

[0144] Step 1: Synthesis of WX002

[0145] WX001-6 (180 mg, 405.82 μmol, 1 eq), WX002-1 (246.29 mg, 2.43 mmol, 6 eq), and DMSO (1 mL) were added to a dry reaction flask. The reaction mixture was reacted at 100 °C for 12 hours. After the reaction was complete, the reaction solution was directly purified by high performance liquid chromatography (HPLC) (column: Phenomenex C18 80*40 mm*3 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B (acetonitrile)%: 25%-55%, 8 min) to obtain WX002. 1 HNMR (DMSO-d6, 400MHz) δ = 8.40 (s, 1H), 7.61 (t, J = 7.7Hz, 1H), 7.2-7.4 (m, 1H), 7.12 (dd, J = 4.8, 7.7Hz, 2H), 4.6-4.8 (m, 2H), 3.8-4.0(m,3H),3.4-3.5(m,2H),2.52(s,3H),2.45(s,3H),1.89(dd,J=1.5,12.0Hz,2H),1.5-1.6(m,2H),1.52(s,6H); LCMS m / z:465[M+H] + .

[0146] Example 3

[0147]

[0148] Synthetic route

[0149]

[0150] Step 1: Synthesis of WX003-1

[0151] Add A-1 (500 mg, 2.02 mmol, 1 eq), N'N-dimethylformamide (5 mL), cesium carbonate (988.88 mg, 3.04 mmol, 1.5 eq), and WX001-4 (451.74 mg, 2.43 mmol, 1.2 eq) to a dry reaction flask. Replace the gas with nitrogen and react at 25 °C for 16 hours. After the reaction is complete, add water (20 mL) to the reaction solution and filter to obtain a filter cake. Dissolve the filter cake in dichloromethane (10 mL) and wash with saturated brine (15 mL x 3). Separate the liquid to obtain the organic phase. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain WX003-1. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 7.60 (t, J = 7.7Hz, 1H), 7.09 (dd, J = 7.7, 13.8Hz, 2H), 4.66 (s, 2H), 2.44 (s, 3H), 1.48 (s, 6H).

[0152] Step 2: Synthesis of WX003

[0153] Add WX003-1 (150 mg, 425.84 μmol, 1 eq), D-1 (217.46 mg, 468.42 μmol, 1.1 eq), and toluene (3 mL) to a dry reaction flask. Replace the gas with nitrogen, then add tetrakis(triphenylphosphine)palladium (98.42 mg, 85.17 μmol, 0.2 eq), and heat to 110 °C for 12 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure to obtain the crude product. The crude product is first purified by thin-layer chromatography on silica gel plates, and then purified by high-performance liquid chromatography (HPLC) (column: Waters Xbridge BEH C18 100*25 mm*5 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B (acetonitrile)%: 20%-50%, 10 min) to obtain WX003. 1 HNMR (400MHz, DMSO-d6) δ = 9.79 (s, 1H), 8.69 (d, J = 5.0Hz, 1H), 7.61 (t, J = 7.7Hz, 1H), 7.54 (d, J = 5.0Hz, 1H), 7.40 (d, LCMS m / z:447[M+H] + .

[0154] Example 4

[0155]

[0156] Synthetic route

[0157]

[0158] Step 1: Synthesis of WX004-2

[0159] Add WX001-1 (500.00 mg, 1.44 mmol, 1 eq), tetrahydrofuran (6.5 mL), zinc chloride solution (0.7 M, 2.06 mL, 1 eq), and tetramethylethylenediamine (167.33 mg, 1.44 mmol, 217.32 μL, 1 eq) to a dry reaction flask. Purge with nitrogen, cool to -78 °C, and add n-butyllithium (2.5 M, 863.99 μL, 1.5 eq) dropwise. Stir for 10 minutes, then add n-butyllithium (2.5 M, 288.00 μL, 0.5 eq). Stir for 10 minutes, then add n-butyllithium (2.5 M, 172.80 μL, 0.3 eq). After the addition is complete, react at 20 °C for 1 hour to obtain reaction solution 1.

[0160] Under nitrogen protection, a mixture of E-1 (403.38 mg, 1.44 mmol, 1 eq), tetrakis(triphenylphosphine)palladium (49.92 mg, 43.20 μmol, 0.03 eq), and N'N-dimethylformamide (3.5 mL) was heated to 50 °C, and then reaction solution 1 was added. The reaction was continued at 50 °C for 40 minutes. After the reaction was complete, the reaction solution was quenched with saturated ammonium chloride aqueous solution (20 mL) and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), 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 column chromatography to obtain WX004-2. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.79 (s, 1H), 3.14 (d, J = 7.34Hz, 2H), 2.60 (s, 3H), 1.82 (s, 6H), 1.54 (s, 9H), 1.22 (t, J = 7.40Hz, 3H).

[0161] Step 2: Synthesis of WX004-3

[0162] Add WX004-2 (200 mg, 475.57 μmol, 1 eq) and dichloromethane (5 mL) to the reaction flask. After cooling to 0 °C, add trifluoroacetic acid (108.45 mg, 951.14 μmol, 70.42 μL, 2 eq) to the reaction flask and stir at 25 °C for 1 hour. After the reaction is complete, slowly pour the reaction solution into a saturated sodium bicarbonate aqueous solution (20 mL) and adjust the pH to 7–8. Extract with dichloromethane (10 mL x 2) and combine the organic phases. Wash the organic phase with saturated brine (20 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain WX004-3. 1H NMR (400MHz, DMSO-d6) δ (ppm) = 8.90 (s, 1H), 8.75 (s, 1H), 3.15 (q, J = 7.40Hz, 2H), 2.59 (s, 3H), 1.58 (s, 6H), 1.22 (t, J = 7.40Hz, 3H).

[0163] Step 3: Synthesis of WX004-4

[0164] Add WX004-3 (225 mg, 702.18 μmol, 1 eq), cesium carbonate (343.17 mg, 1.05 mmol, 1.5 eq), and N,N-dimethylformamide (5 mL) to the reaction flask. After purging with nitrogen, add WX001-4 (156.77 mg, 842.61 μmol, 1.13 mL, 1.2 eq), and react the mixture at 25 °C for 2 hours. After the reaction is complete, pour the reaction solution into an ice-water mixture (200 mL), stir for 0.5 hours, and filter to obtain a filter cake. Concentrate the filter cake with toluene (5 mL x 3) using an azeotropic method to obtain WX004-4. 1 H NMR (400MHz, DMSO-d6) δ (ppm) = 8.77 (s, 1H), 7.60-7.64 (t, J = 7.67Hz, 1H), 7.11-7.13 (d, J = 7.67Hz, 2H ), 4.72 (s, 2H), 3.18 (q, J = 7.38Hz, 2H), 2.60 (s, 3H), 2.45 (s, 3H), 1.54 (s, 6H), 1.24 (t, J = 7.45Hz, 3H).

[0165] Step 4: Synthesis of WX004-5

[0166] Add WX004-4 (100 mg, 234.98 μmol, 1 eq), water (1.5 mL), and acetonitrile (3 mL) to a dry reaction flask. After purging with nitrogen, cool to 0 °C, add potassium persulfate (288.92 mg, 469.96 μmol, 2 eq) in portions, and then react at 25 °C for 12 hours. After the reaction is complete, add saturated sodium sulfite aqueous solution (10 mL) to the reaction solution and stir for 0.5 hours, then extract with ethyl acetate (10 mL x 2). Combine the organic phases, wash with saturated brine (20 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain WX004-5. 1H NMR (400MHz, DMSO-d6) δ (ppm) = 8.77 (s, 1H), 7.62 (t, J = 7.7Hz, 1H), 7.12 (d, J = 7.7Hz, 2H), 4.7 2(s,2H),3.18(q,J=7.4Hz,2H),2.60(s,3H),2.45(s,3H),1.54(s,6H),1.24(t,J=7.5Hz,3H).

[0167] Step 5: Synthesis of WX004

[0168] WX004-5 (55 mg, 120.20 μmol, 1 eq), D-1-3 (23.35 mg, 240.40 μmol, 2 eq), dichloromethane (1 mL), and tetrahydrofuran (1 mL) were added to the reaction flask. Nitrogen gas was introduced, and the mixture was cooled to 0 °C. Hexamethyldisilamide lithium (1 M, 228.38 μL, 1.9 eq) was added dropwise to the reaction flask, and the mixture was stirred at 0 °C for 1 hour. After the reaction was complete, water (10 mL) was added to the reaction solution to quench the reaction, and then the mixture was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was first purified by thin-layer chromatography on silica gel plates, and then separated and purified by high-performance liquid chromatography (column: Waters Xbridge BEH C18 100*30mm*10μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B (acetonitrile)%: 25%-55%, 8 minutes) to obtain WX004. 1 H NMR (DMSO-d6, 400MHz) δ (ppm) = 9.64 (s, 1H), 8.63 (s, 1H), 7.61 (t, J = 7.7Hz, 1H), 7.39 (d, J = 1.8Hz, 1H), 7.12 (d, J = 7.8Hz, 2H), 6.34(d,J=1.8Hz,1H),4.71(s,2H),3.72(s,3H),3.12(q,J=7.3Hz,2H),2.45(s,3H),1.53(s,6H),1.22(t,J=7.4Hz,3H); LCMS m / z:475[M+H] + .

[0169] Experimental Example 1: In vitro enzyme activity test

[0170] 1. Experimental objective:

[0171] The ability of compounds to inhibit the activity of ERK1 and ERK2 kinases was measured.

[0172] 2. Experimental buffer solution:

[0173] 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.

[0174] 3. Compound treatment:

[0175] 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.

[0176] 4. Experimental Methods:

[0177] a) Prepare substrate MBP in freshly prepared reaction buffer;

[0178] b) Add the ERK1 (or ERK2) kinase to the above MBP solution and mix gently;

[0179] c) 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;

[0180] d) will 33 When P-ATP (at a specific concentration of 10 μCi / μL) is added to the reaction system, the reaction begins.

[0181] e) Incubate at room temperature for 2 hours;

[0182] f) Detect the amount of radioactivity using a filtration-binding method;

[0183] g) 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 was performed using Prism (GraphPad software), and IC50 was calculated. 50 value.

[0184] 5. The experimental results are shown in Tables 1 and 2:

[0185] Table 1 Results of ERK1 enzyme activity test

[0186]

[0187] Conclusion: The compounds of this invention exhibit superior inhibitory activity against ERK1 enzyme.

[0188] Table 2 Results of ERK2 enzyme activity test

[0189]

[0190] Conclusion: The compounds of this invention exhibit superior inhibitory activity against ERK2 enzyme.

[0191] Experiment Example 2: In vitro cell proliferation inhibition experiment

[0192] 1. Experimental objective:

[0193] The ability of the compound to inhibit the proliferation of HT29 tumor cells was measured.

[0194] 2. Compound treatment:

[0195] The test compound was dissolved in 100% DMSO to prepare a 10 mM stock solution.

[0196] 3. Experimental steps and methods:

[0197] a) Turn on the UV light in the biosafety cabinet and start the 30-minute countdown;

[0198] b) Preheat RPMI 1640 medium and trypsin in a 37°C water bath;

[0199] c) After UV irradiation is complete, open the biosafety cabinet, wipe the preheated culture medium, trypsin, phosphate buffered saline solution (PBS) with alcohol and place them in the biosafety cabinet;

[0200] d) 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;

[0201] e) 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;

[0202] f) Terminate cell digestion with complete culture medium and pipette until a homogeneous cell suspension is formed for counting;

[0203] g) 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;

[0204] h) The compound stock solution was continuously diluted in DMSO solution, and the compound was added to the cell plate using Tecan;

[0205] i) 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 .

[0206] 4. The experimental results are shown in Table 3:

[0207] Table 3 Results of in vitro cell viability test

[0208]

[0209]

[0210] Conclusion: The compounds of this invention exhibit superior inhibitory activity against the proliferation of HT29 cells.

[0211] Experimental Example 3: PK Study in Mice

[0212] 1. Experimental objective:

[0213] 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.

[0214] 2. Experimental Procedure:

[0215] 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.

[0216] 3. The experimental results are shown in Table 4:

[0217] Table 4. PK test results of compounds

[0218]

[0219] 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 is the volume of distribution. 1 / 2 It is the half-life.

[0220] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0221] Experiment Example 4: Solubility Study

[0222] 1. Experimental objective:

[0223] To determine the solubility of a compound and to evaluate its solubility.

[0224] 2. Test solution:

[0225] 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;

[0226] 2) Preparation of standard solutions:

[0227] a) Mix a 50% acetonitrile solution and a 50% buffer solution to obtain a diluted solution;

[0228] b) Mix 10 mM (10 μL / compound) compound stock solution with diluent (490 μL / compound) to prepare a 200 μM detection standard solution;

[0229] 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;

[0230] d) 1 μM, 20 μM and 200 μM UV standard solutions were used as standard solutions for solubility tests.

[0231] 3. Experimental methods:

[0232] a) 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.

[0233] b) 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;

[0234] c) Shake in a shaker at 600 revolutions per minute for 24 hours at room temperature (25±2℃);

[0235] d) Pipette 200 μL of the solution into a 96-well plate vacuum filtration apparatus and then transfer it to a new 96-well plate as a test sample;

[0236] e) The concentration of the compound was determined by HPLC-UV, and the HPLC conditions are shown in Table 5:

[0237] Table 5 HPLC conditions

[0238]

[0239] f) Inject three UV standard solutions into the HPLC from low to high concentrations (1 μM, 20 μM, 200 μM), and then inject the test sample of the compound to be tested.

[0240] g) Integrate the ultraviolet chromatographic peaks and calculate the solubility of the sample.

[0241] 4. The experimental results are shown in Table 6:

[0242] Table 6. Results of compound solubility tests

[0243]

[0244] Conclusion: The compounds of this invention exhibit good solubility under different pH conditions.

[0245] Experimental Example 5: In vivo efficacy study of a human melanoma A375 mouse model

[0246] 1. Experimental objective:

[0247] The antitumor effect of WX001 was evaluated using a nude mouse model of subcutaneous xenograft of human melanoma A375 cells.

[0248] 2. Laboratory animals:

[0249] Species: Mouse

[0250] Strain: BALB / c nude mice

[0251] Age: 6-8 weeks

[0252] Sex: Female

[0253] Weight: 18-22 grams

[0254] Supplier: Vital River Laboratory Animal Technology Co., Ltd.

[0255] 3. Rearing environment:

[0256] 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%.

[0257] Cage: Made of polycarbonate, with dimensions of 375mm x 215mm x 180mm, and corn cob bedding that is changed weekly;

[0258] Food: Laboratory animals can eat freely throughout the experimental period (sterilized by irradiation, dry granular food);

[0259] Drinking water: Laboratory animals may drink sterilized water freely;

[0260] 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;

[0261] Animal identification: Laboratory animals are identified by ear tags.

[0262] 4. Experiment Content:

[0263] 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.

[0264] 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 7.

[0265] Table 7 Grouping and Dosing Regimens of Experimental Animals

[0266]

[0267]

[0268] 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.

[0269] 4) Preparation of the test substance

[0270] a) Vehicle group: 5% DMSO+95% (20% HP-β-CD).

[0271] 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.

[0272] 5) Tumor measurement and experimental indicators:

[0273] 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;

[0274] 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%.

[0275] 5. Experimental Results:

[0276] 1) As shown in Table 8 and Figure 1 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 36%, 81% and 104% at three doses of 5 mg / kg, 10 mg / kg and 20 mg / kg, respectively.

[0277] 2) The body weight of experimental animals serves as a reference indicator for indirectly determining drug toxicity. For example... Figure 2 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, and there were no cases of disease or death.

[0278] Table 8. Results of in vivo pharmacological experiments in mouse A375 model.

[0279] drug TGI WX001 (5mg / kg, PO, BID) 36％ WX001 (10mg / kg, PO, BID) 81％ WX001 (20mg / kg, PO, BID) 104％

[0280] Experimental conclusions: WX001 was able to inhibit tumor growth in a dose-dependent manner at three dosages of 5 mg / kg, 10 mg / kg and 20 mg / kg; no significant decrease in body weight was observed in the animals during the administration process, indicating good tolerability.

[0281] Experimental Example 6: In vivo PK study in SD rats

[0282] 1. Experimental objective:

[0283] 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.

[0284] 2. Experimental Procedure:

[0285] 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.

[0286] 3. The experimental results are shown in Table 9:

[0287] Table 9. PK test results of compounds

[0288]

[0289] 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 is the volume of distribution. 1 / 2 It is the half-life.

[0290] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0291] Experiment Example 7: PK Study in Crab-Eating Monkeys

[0292] 1. Experimental objective:

[0293] 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.

[0294] 2. Experimental Procedure:

[0295] 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).

[0296] Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0297] 3. The experimental results are shown in Table 10:

[0298] Table 10 PK test results of compounds

[0299]

[0300] 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 is the volume of distribution. 1 / 2 It is the half-life.

[0301] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0302] Experimental Example 8: PK Study in Beagle Dogs

[0303] 1. Experimental objective:

[0304] 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.

[0305] 2. Experimental Procedure:

[0306] 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, USA).

[0307] Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0308] 3. The experimental results are shown in Table 11:

[0309] Table 11 PK test results of compounds

[0310]

[0311] 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 is the volume of distribution. 1 / 2 It is the half-life.

[0312] Conclusion: The compounds of this invention exhibit excellent oral exposure and bioavailability.

[0313] Experiment Example 9: hERG Test

[0314] 1. Experimental objective:

[0315] 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.

[0316] 2. Experimental Methods:

[0317] 2.1 Cell Preparation

[0318] 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.

[0319] 2.2 Solution Preparation

[0320] 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.

[0321] Intracellular fluid formulation (mM): 140 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA and 10 HEPES, pH adjusted to 7.2 with KOH.

[0322] 2.3 Electrophysiological Recording Process

[0323] 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).

[0324] 2.4 Compound Preparation

[0325] 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.

[0326] 2.5 Data Analysis

[0327] Experimental data were analyzed using GraphPad Prism 5.0 software.

[0328] 2.6 Quality Control

[0329] Environment: Humidity 20-50%, temperature 22-25℃

[0330] Reagents: All experimental reagents used were purchased from Sigma-Aldrich and had a purity >98%.

[0331] The experimental data in the report must meet the following standards:

[0332] Whole-cell sealing impedance >100MΩ

[0333] Tail current amplitude >300pA

[0334] Pharmacological parameters:

[0335] The inhibitory effect of multiple concentrations of Cisapride on hERG channels was used as a positive control.

[0336] 3. The experimental results are shown in Table 12:

[0337] Table 12 hERG test results of compounds

[0338] compound IC50 (μM) WX001 >40

[0339] 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.

[0340] Experimental Example 10: Plasma Protein Binding Test (PPB)

[0341] 1. Experimental objective:

[0342] The study investigated the binding of the test compound to human / mouse / rat / dog / monkey plasma albumin.

[0343] 2. Experimental Procedure:

[0344] 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.

[0345] 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.

[0346] 3) Analysis steps:

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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 (matrix:buffer volume ratio 1:1). Add 60 μL of 4% H3PO4 aqueous solution and 480 μL of stop solution (containing internal standard) to all samples. Centrifuge the mixture at 4000 rpm for 20 min, and take 100 μL of the supernatant from each sample for LC-MS / MS analysis.

[0351] 3. The experimental results are shown in Table 13:

[0352] Table 13 Results of plasma protein binding tests for compounds

[0353]

[0354]

[0355] Conclusion: The compounds of this invention have medium to high 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; Structural unit Selected from ; 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 Independently selected from D, F, Cl, Br, and I; R d Selected from C 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally substituted with 1, 2, or 3 Rs; 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, R d Selected from CH3, wherein the CH3 is optionally replaced by 1, 2 or 3 R.

5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein, R d Selected from CH3.

6. The compound according to 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.

7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from and The and Choose 1, 2, or 3 Rs d replace.

8. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein, Ring A is selected from , and .

9. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from... , in, R2 is defined as in any one of claims 1 to 3; R4 is as defined in claim 1.

10. The compound shown in the following formula or a pharmaceutically acceptable salt thereof, 。 11. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10 in the preparation of a medicament for treating solid tumors.

12. A medicament comprising the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof.

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