Thiazole-lactam-spiro heterocyclic compound and its application

Abstract

Thiazole-lactam-spiro heterocycle compounds and their application in the preparation of medicaments for treating related diseases.Specifically disclosed are compounds represented by formula (I) and pharma- ceutically acceptable salts thereof: [C1] TIFF2024526242000101.tif36156

Description

Technical Field

[0001] This application claims priority to the specification of Chinese Patent Application Publication No. 202110722003.6, filed on June 28, 2021, the specification of Chinese Patent Application Publication No. 202111673614.2, filed on December 31, 2021, and the specification of Chinese Patent Application Publication No. 202210693548.3, filed on June 17, 2022.

[0002] <Technical Field> The present disclosure relates to a class of thiazololactamospiro heterocyclic compounds and their use in the manufacture of pharmaceuticals for treating related diseases. Specifically, the present disclosure relates to compounds represented by formula (I) and pharmaceutically acceptable salts thereof.

Background Art

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

[0004] Studies have shown that abnormal activation of the Ras / Raf / MEK / ERK pathway caused by mutation or amplification is a determinant of various cancers. In human tumors, the incidence of RAS mutations is approximately 22%, the incidence of BRAF mutations is approximately 7%, and the incidence of MEK mutations is approximately 1%. Therefore, important nodal proteins on this pathway have become important targets for cancer treatment (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-mutated non-small cell lung cancer, and other cancers. However, the use of BRAF inhibitors and MEK inhibitors for these upstream nodes immediately causes problems of drug resistance due to mutations or pathway reactivation, which may severely limit their clinical applications.

[0005] Extracellular regulated protein kinases (ERKs) (especially ERK1 and ERK2 kinases) are major players and important downstream nodes in the Ras / Raf / MEK / ERK pathway, and their overactivation can be found in many human cancers. ERK, as the terminal signaling kinase of this pathway, has not yet been found to have mutations that cause drug resistance. Therefore, drugs targeting ERK kinase are expected to overcome the problem of drug resistance caused by treatment with upstream target inhibitors and become a more promising treatment strategy. However, so far, research on ERK inhibitors is still in the clinical stage, and no ERK inhibitor has been approved for sale as a drug.

[0006] In summary, it is urgent to develop safe and effective ERK inhibitors to meet the needs of tumor treatment.

Summary of the Invention

Means for Solving the Problems

[0007] The present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

Chemical formula

[0008] In the formula,

[0009] R1 and R2 are each independently selected from H and C 1~3 alkyl, where the above C 1~3 alkyl is optionally substituted with 1, 2 or 3 R a and is substituted,

[0010] R4, R5, R6 and R7 are each independently selected from H, F, Cl, Br, I and C 1~3 alkyl, where the above C 1~3 alkyl is optionally substituted with 1, 2 or 3 R c and is substituted,

[0011] n is 0 or 1,

[0012] m is 1 or 2,

[0013] Ring A is selected from pyrazolyl and tetrahydropyranyl, where the above pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2 or 3 R d and is substituted,

[0014] R a and R c are each independently selected from D, F, Cl, Br and I,

[0015] R d is selected from F, Cl, Br, I, C 1~3 alkyl and C 1~3 alkoxy, where the above C 1~3 alkyl and C 1~3 alkoxy are optionally substituted with 1, 2 or 3 R,

[0016] R is selected from F, Cl, Br and I.

[0017] The present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

Chemical formula

[0018] In the formula,

[0019] R1 and R2 are each independently selected from H and C 1~3 alkyl, wherein the C 1~3 alkyl is optionally substituted with 1, 2, or 3 R a ;

[0020] R4, R5, R6, and R7 are each independently selected from H, F, Cl, Br, I, and C 1~3 alkyl, wherein the C 1~3 alkyl is optionally substituted with 1, 2, or 3 R c ;

[0021] n is 0 or 1;

[0022] m is 1 or 2;

[0023] Ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2, or 3 R d ;

[0024] R a and R c are each independently selected from D, F, Cl, Br, and I;

[0025] R d is selected from F, Cl, Br, I, C 1~3 alkyl, and C 1~3 alkoxy, wherein the C 1~3 alkyl and C 1~3 alkoxy are optionally substituted with 1, 2, or 3 R;

[0026] R is selected from F, Cl, and Br.

[0027] In some embodiments of the present disclosure, the above-mentioned R1 and R2 are each independently selected from H, CH3, and CH2CH3, where the above CH3 and CH2CH3 are optionally substituted with 1, 2, or 3 R a as defined in the present disclosure, and the other variables are as defined in the present disclosure.

[0028] In some embodiments of the present disclosure, the above-mentioned R1 and R2 are each independently selected from H, CH3, CHF2, CD3, and CH2CH3, and the other variables are as defined in the present disclosure.

[0029] In some embodiments of the present disclosure, the above-mentioned R4, R5, R6, and R7 are each independently selected from H, F, Cl, Br, I, and CH3, where the above CH3 is optionally substituted with 1, 2, or 3 R c as defined in the present disclosure, and the other variables are as defined in the present disclosure.

[0030] In some embodiments of the present disclosure, the above-mentioned R4, R5, R6, and R7 are each independently selected from H, F, Cl, Br, I, and CH3, and the other variables are as defined in the present disclosure.

[0031] In some embodiments of the present disclosure, the above-mentioned R d is selected from F, Cl, Br, I, CH3, and OCH3, where the above CH3 and OCH3 are optionally substituted with 1, 2, or 3 R, and the other variables are as defined in the present disclosure.

[0032] In some embodiments of the present disclosure, the above-mentioned R d is selected from CH3 and OCH3, and the other variables are as defined in the present disclosure.

[0033] In some embodiments of the present disclosure, the above-mentioned ring A is

Chemical formula

Chemical formula

[0034] In some embodiments of the present disclosure, the above-mentioned ring A is

Chemical formula

[0035] In some embodiments of the present disclosure, the above-mentioned structural moiety

Chemical formula

Chemical formula

[0036] The present disclosure also includes some embodiments obtained by combining any of the above-mentioned variables.

[0037] In some embodiments of the present disclosure, the above-mentioned compound or its pharmaceutically acceptable salt is disclosed, and the compound is

Chemical formula

[0038] wherein,

[0039] R2, R6 and R7 are as defined in the present disclosure.

[0040] The present disclosure also provides a compound represented by the following formula or its pharmaceutically acceptable salt.

Chemical formula

[0041] The present disclosure also provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating solid tumors.

[0042] The present disclosure provides crystalline form A of WX001, which is characterized by a powder X-ray diffraction pattern having diffraction peaks characteristic of 2θ angles of 10.2080 ± 0.2000°, 18.8429 ± 0.2000°, and 20.6217 ± 0.2000°. [Chemical formula]

[0043] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-mentioned crystalline form A includes at least 4 or 5 diffraction peaks selected from 10.2080 ± 0.2000°, 18.8429 ± 0.2000°, 20.6217 ± 0.2000°, 25.0767 ± 0.2000°, and 25.4797 ± 0.2000° when represented by 2θ angle.

[0044] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-mentioned crystalline form A has diffraction peaks characteristic of 2θ angles of 10.2080 ± 0.2000°, 18.8429 ± 0.2000°, 20.6217 ± 0.2000°, 25.0767 ± 0.2000°, and 25.4797 ± 0.2000°.

[0045] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-mentioned crystalline form A includes at least 6, 7, or 8 diffraction peaks selected from 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° when represented by 2θ angle.

[0046] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-described crystalline form A has diffraction peaks characteristic of 2θ angles of 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°.

[0047] In some embodiments of the present disclosure, when represented by 2θ angles, the powder X-ray diffraction pattern of the above-described crystalline form A includes at least 9, 10, 11, or 12 diffraction peaks selected from 10.2080 ± 0.2000°, 14.4684 ± 0.2000°, 15.2687 ± 0.2000°, 17.6747 ± 0.2000°, 18.8429 ± 0.2000°, 20.6217 ± 0.2000°, 21.0531 ± 0.2000°, 21.5713 ± 0.2000°, 22.0420 ± 0.2000°, 22.4540 ± 0.2000°, 25.0767 ± 0.2000°, and 25.4797 ± 0.2000°.

[0048] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-described crystalline form A has diffraction peaks characteristic of 2θ angles of 10.2080 ± 0.2000°, 14.4684 ± 0.2000°, 15.2687 ± 0.2000°, 17.6747 ± 0.2000°, 18.8429 ± 0.2000°, 20.6217 ± 0.2000°, 21.0531 ± 0.2000°, 21.5713 ± 0.2000°, 22.0420 ± 0.2000°, 22.4540 ± 0.2000°, 25.0767 ± 0.2000°, and 25.4797 ± 0.2000°.

[0049] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-described crystalline form A has characteristic diffraction peaks at 2θ angles of 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°.

[0050] In some embodiments of the present disclosure, the powder X-ray diffraction pattern of the above-described crystalline form A has diffraction peaks characteristic of 2θ angles of 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°.

[0051] In some embodiments of the present disclosure, the XRPD pattern of the above-described crystalline form A is substantially as shown in FIG. 1.

[0052] In some embodiments of the present disclosure, the analysis data of the XRPD pattern of the above-described crystalline form A is as shown in Table 1.

Table 1

[0053] In some embodiments of the present disclosure, the above-described crystalline form A has a differential scanning calorimetry curve having an endothermic peak starting point at 241.0 ± 3.0 °C.

[0054] In some embodiments of the present disclosure, the DSC curve of the above-described crystalline form A is as shown in FIG. 2.

[0055] In some embodiments of the present disclosure, the above-described crystalline form A has a thermogravimetric analysis curve having a maximum weight loss of 0.83% at 150.0 ± 3.0 °C.

[0056] In some embodiments of the present disclosure, the TGA curve of the above-described crystalline form A is as shown in FIG. 3.

[0057] The present disclosure also provides the use of the above-described crystalline form A in the manufacture of a medicament for treating solid tumors.

Advantages of the Invention

[0058] The compounds of the present disclosure exhibit excellent inhibitory activity against ERK1 and ERK2 enzymes, the compounds of the present disclosure exhibit excellent inhibitory activity against HT29 cell proliferation, the compounds of the present disclosure have good solubility under various pH conditions, and the compounds of the present disclosure have excellent pharmacokinetic properties and tumor inhibitory effects.

[0059] Definitions and Terms Unless otherwise defined, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered ambiguous or unclear in the absence of a specific definition, but should be understood in its conventional meaning. When a trade name appears in this specification, it is intended to refer to the corresponding product or its active ingredient.

[0060] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are commensurate with a reasonable benefit / risk ratio, are without excessive toxicity, irritation, allergic reaction or other problems or complications, and are suitable for use in contact with human and animal tissues within the scope of reliable medical judgment.

[0061] The term "pharmaceutically acceptable salt" as used herein means a salt of a compound disclosed herein prepared by reacting a compound having a specific substituent disclosed herein with a relatively non-toxic acid or base. When a compound disclosed herein contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. When a compound disclosed herein contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of an acid in a pure solution or a suitable inert solvent. Some specific compounds disclosed herein contain both basic and acidic functional groups and can be converted to any base or acid addition salt.

[0062] The pharmaceutically acceptable salts disclosed herein can be prepared from the parent compounds containing acidic or basic moieties by conventional chemical methods. In general, such salts can be prepared by reacting the compound in the free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture thereof.

[0063] Unless otherwise defined, the term "isomer" is intended to include geometric isomers, cis- or trans-isomers, stereoisomers, enantiomers, optical isomers, diastereomers, and tautomers.

[0064] The compounds disclosed herein may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis- and trans-isomers, (-)-enantiomers and (+)-enantiomers, (R)-enantiomers and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures, such as mixtures enriched in enantiomers or diastereoisomers, all of which are included within the scope of this disclosure. Substituents such as alkyl may further have asymmetric carbon atoms. All of these isomers and their mixtures are included within the scope of this disclosure.

[0065] Unless otherwise defined, the term "enantiomer" or "optical isomer" means stereoisomers that are mirror images of each other.

[0066] Unless otherwise defined, the term "cis-trans isomer" or "geometric isomer" results from the inability of a double or single bond between ring-forming carbon atoms to rotate freely.

[0067] Unless otherwise defined, the term "diastereomer" means stereoisomers that contain two or more chiral centers in a molecule and are not mirror images of each other.

[0068] Unless otherwise specified, “(+)” means the dextrorotatory isomer, “(-)” means the levorotatory isomer, and “(±)” means the racemate.

[0069] Unless otherwise specified, the wedge-shaped solid line bond

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[0070] Unless otherwise specified, the terms "tautomer" or "tautomeric form" mean that each functional group in the isomers is in dynamic equilibrium and can be readily converted into each other at room temperature. When tautomers are possible (in solution), the chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions by proton transfer such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the two tautomers pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0071] Unless otherwise specified, the terms "one isomer is enriched", "enriched isomer", "one enantiomer is enriched" or "enriched enantiomer" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0072] Unless otherwise specified, the terms "isomer excess" or "enantiomer excess" mean the difference in the relative proportions of two isomers or two enantiomers. For example, when one isomer or enantiomer is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomer or enantiomer excess (ee value) is 80%.

[0073] The optically active (R)- and (S)-isomers, or D and L isomers, can be prepared using chiral synthesis or chiral reagents or other prior art. If one wishes to obtain a particular enantiomer of a compound disclosed herein, the pure desired enantiomer can be obtained by separating the resulting mixture of diastereomers following asymmetric synthesis or the derivatization of a chiral auxiliary and cleaving the auxiliary group. Alternatively, if the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), the compound reacts with a suitable optically active acid or base to form salts of diastereomeric isomers, which are then subjected to diastereomeric resolution by conventional methods in the art to obtain the pure enantiomer. Further, enantiomers and diastereomers are generally isolated by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization methods (such as carbamates generated from amines).

[0074] The compounds disclosed herein may contain unnatural proportions of atomic isotopes in one or more of the atoms that make up the compound. For example, the compound can be labeled with a radioactive isotope such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). In another example, hydrogen can be replaced with deuterium to form a deuterated drug. The bond between deuterium and carbon is stronger than the bond between normal hydrogen and carbon. Compared to the non-deuterated drug, the deuterated drug has the advantages of reduced toxic side effects of the drug, increased drug stability, improved efficacy, and extended biological half-life. All variations in the isotope composition of the compounds disclosed herein are included within the scope of the present disclosure, regardless of radioactivity.

[0075] The term "optional" or "optionally" means that the subsequent event or state may occur but is not essential, and this term includes instances where the event or state occurs and instances where the event or state does not occur.

[0076] The term "substituted" means that one or more hydrogen atoms on a particular atom are substituted by a substituent including deuterium and hydrogen variants, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted. The position on the aromatic ring cannot be substituted by oxo. The term "optionally substituted" means that an atom may or may not be substituted by a substituent, and unless otherwise specified, the type and number of substituents can be arbitrary as long as they are chemically achievable.

[0077] When an arbitrary variable (e.g., R) appears multiple times in the constitution or structure of a compound, the definition of the variable at each occurrence is independent. Thus, for example, when a group is substituted with 0 to 2 Rs, the group may optionally be substituted with up to 2 Rs at most, and the definition of R at each occurrence is independent. Further, combinations of substituents and / or their variants are only permitted if the combination results in a stable compound.

[0078] When the number of linking groups is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0079] When the number of substituents is 0, it means that the substituent does not exist. For example, -A-(R)0 means that its structure is actually -A.

[0080] When a substituent is empty, it means that the substituent does not exist. For example, in A-X, when X is empty, the structure of A-X is actually A.

[0081] When one of the variables is a single bond, it means that the two groups linked by the single bond are directly connected. For example, in A-L-Z, when L represents a single bond, the structure of A-L-Z is actually A-Z.

[0082] When the bond of a substituent can bridge two or more atoms on the ring, such a substituent can be bonded to any atom on the ring. For example, the structural moiety

Chemical formula

[0083] When the listed linking group does not indicate its linking direction, the linking direction is arbitrary. For example,

Chemical formula

Chemical formula

Chemical formula

[0084] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups via chemical bonds. The connection position of the chemical bond is variable. When there is an H atom at the connectable site, when the connectable site with the H atom is connected to the chemical bond, the number of H atoms at this site decreases accordingly as the number of connected chemical bonds increases, and the group becomes a group with the corresponding valence. The chemical bond between that site and another group is represented by a straight solid line bond

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[0085] Unless otherwise specified, the number of atoms in the ring is generally defined as the number of ring members. For example, "5- to 7-membered ring" refers to a "ring" of 5 to 7 atoms arranged in the circumferential direction.

[0086] Unless otherwise specified, the term "C 1~3 alkyl" is used to indicate a straight-chain or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. C 1~3 As the C 1~2 alkyl group, C 2~3 alkyl group, C 1~3 alkyl group, etc. may be mentioned. It may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methenyl). Examples of the C 1~3 alkyl group include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

[0087] Unless otherwise specified, the term "C 1~3 alkoxy" refers to an alkyl group containing 1 to 3 carbon atoms and bonded to the rest of the molecule by an oxygen atom. C 1~3 As the C 1~2 alkoxy group, C 2~3 alkoxy group, C3 alkoxy group, C2 alkoxy group, etc. may be mentioned. Examples of the C 1~3 alkoxy group include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropyl), etc.

[0088] The compounds disclosed in this specification can be prepared by various synthetic methods well known to those skilled in the art, such as the embodiments listed below, the embodiments formed by the embodiments listed below in combination with other chemical synthesis methods, and equivalent alternatives well known to those skilled in the art. Alternative embodiments include, but are not limited to, the examples disclosed herein.

[0089] Throughout this specification, references to "an embodiment", "embodiment", "in another embodiment", or "in some embodiments" mean that the specifically recited element, structure, or feature described in connection with that embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment", "in an embodiment", "in another embodiment", or "in some embodiments" that appear in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the specific elements, structures, or features can be combined in any suitable manner in one or more embodiments.

[0090] In the present disclosure, ExoUp in the DSC curve indicates an upward exotherm.

[0091] The structures of the compounds disclosed in this specification can be confirmed by conventional methods well known to those skilled in the art. When the present disclosure relates to the absolute configuration of a compound, its absolute configuration can be confirmed by prior art in the technical field, such as single crystal X-ray diffraction (SXRD). In single crystal X-ray diffraction (SXRD), the diffraction intensity data of the cultured single crystal is collected using a Bruker D8 Venture diffractometer equipped with a CuKα radiation light source in the scanning mode of φ / ω scan. After collecting the data, the crystal structure is further analyzed by the direct method (Shelxs97) to confirm the absolute configuration.

[0092] Hereinafter, the present disclosure will be described in detail by way of examples. These examples do not imply any limitation to the present disclosure.

[0093] All solvents used in the present disclosure are commercially available and can be used without further purification.

[0094] The solvents used in the present disclosure are commercially available.

[0095] The following abbreviations are used in the present disclosure. aq represents aqueous, eq represents equivalent or equivalents, DCM represents dichloromethane, PE represents petroleum ether, DMSO represents dimethyl sulfoxide, EtOAc represents ethyl acetate, EtOH represents ethanol, MeOH represents methanol, BOC represents the amine protecting group tert-butoxycarbonyl, r.t. represents room temperature, O / N represents overnight, THF represents tetrahydrofuran, Boc2O represents di-tert-butyl dicarbonate, TFA represents trifluoroacetic acid, DIPEA represents diisopropylethylamine, iPrOH represents 2-propanol, and mp represents melting point.

[0096] Compounds are named according to the general naming principles in the art or by ChemDraw® software. Commercially available compounds are named by their vendor directory names.

[0097] Powder X-ray diffractometer (XRPD) method used in the present disclosure Instrument model: X’Pert of PANalytical 3 X-ray diffractometer

[0098] Test method: Approximately 10 mg of the sample was used for XRPD detection. [Table 2]

[0099] Differential scanning calorimeter (DSC) method used in the present disclosure Instrument model: TA2500 differential scanning calorimeter [Table 3]

[0100] Thermogravimetric analyzer (TGA) method used in the present disclosure Machine model: TA5500 Thermogravimetric Analyzer

Table 4

[0101] The dynamic vapor sorption (DVS) method used in this disclosure The dynamic vapor sorption (DVS) curve was collected using a DVS Intrinsic plus from Surface Measurement Systems (SMS). The relative humidity at 25 °C was corrected at the deliquescence points of LiCl, Mg(NO3)2, and KCl.

Table 5

Table 6

[0102] Note: ΔW% represents the weight increase of the test sample due to moisture absorption at 25 ± 1 °C and 80 ± 2% RH.

Brief Description of the Drawings

[0103]

Figure 1

[0104]

Figure 2

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Figure 3

[0106]

Figure 4

[0107]

Figure 5

[0108]

Figure 6

Mode for Carrying Out the Invention

[0109] Hereinafter, the present disclosure will be described in detail by way of examples. However, it is not intended that these examples impose any unfavorable limitation on the present disclosure. The present disclosure is described in detail herein, and embodiments are also disclosed herein. It will be apparent to those skilled in the art that various changes and modifications can be made to the embodiments disclosed herein without departing from the spirit and scope disclosed herein. Reference Example 1

Chemical formula

Chemical formula

[0110] Step 1: Synthesis of Compound A-1-2

[0111] To a reaction flask, 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) were added. Under nitrogen, sodium borohydride (48.07 g, 1.27 mol, 2 eq) was added, and the mixture was reacted at 20 °C for 15 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The concentrate was diluted with 15% aqueous citric acid solution (4000 mL) and extracted with ethyl acetate (4000 mL × 3). The organic phases were combined, washed with saturated brine (2000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain A-1-2.

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[0112] Step 2: Synthesis of Compound A-1-4

[0113] To the reaction flask were added A-1-2 (102 g, 525.64 mmol, 1 eq) and 2-methyltetrahydrofuran (1000 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to -70 °C, and lithium diisopropylamide (2 M, 525.64 mL, 2.0 eq) was slowly added dropwise. The mixture was stirred at -70 °C for 30 minutes. Then, a solution of A-1-3 (138.18 g, 788.46 mmol, 1.5 eq) dissolved in 2-methyltetrahydrofuran (400 mL) was slowly added dropwise, and the mixture was reacted at -70 °C for an additional 1 hour. After the reaction was completed, the reaction solution was quenched with a saturated aqueous ammonium chloride solution (2000 mL) and extracted with ethyl acetate (2000 mL × 4). Each layer was separated. The organic phases were combined, washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain a crude product. The crude product was first purified by column chromatography and then purified by slurrying with methyl tert-butyl ether to obtain A-1-4.

Number

[0114] Step 3: Synthesis of Compound A-1-5

[0115] To the reaction flask, A-1-4 (50 g, 135.39 mmol, 1 eq), dipiperidyl azodicarboxylate (40.99 g, 162.47 mmol, 1.2 eq) and tetrahydrofuran (500 mL) were added. The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0 °C, and a solution prepared by dissolving tributylphosphine (32.87 g, 162.47 mmol, 40.09 mL, 1.2 eq) in tetrahydrofuran (100 mL) was slowly added dropwise. The mixture was reacted at 0 °C for 1 hour. After completion of the reaction, water (500 mL) and saturated brine (500 mL) were successively added to the reaction solution. The mixture was extracted with ethyl acetate (500 mL × 2). Each layer was separated. The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain a crude product. The crude product was slurried in 500 mL of methyl tert-butyl ether and filtered. The filtrate was collected and concentrated to obtain a crude product. The crude product was slurried in 50 mL of n-hexane and filtered. The filter cake was collected and dried to obtain A-1-5.

Number

[0116] Step 4: Synthesis of Compound A-1-6

[0117] To the reaction flask, A-1-5 (29 g, 82.55 mmol, 1 eq), tetrahydrofuran (250 mL) and water (50 mL) were added. The atmosphere was replaced with nitrogen gas. Iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) was added, and the mixture was reacted at 50 °C for 18 hours. Then, iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) was further added, and the mixture was reacted at 50 °C for an additional 6 hours. After completion of the reaction, a solution of crude A-1-6 was obtained and used as it was in the next step.

[0118] Step 5: Synthesis of Compound A-1-7

[0119] To the solution containing crude A-1-6, sodium carbonate (17.50 g, 165.11 mmol, 2 eq) was added. The atmosphere was replaced with nitrogen gas. 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 completion of the reaction, the reaction solution was poured into water (200 mL), and then extracted with ethyl acetate (300 mL × 3). Each layer was separated. The organic phases were combined, washed with saturated brine (300 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain A-1-7.

Number

[0120] Step 6: Synthesis of Compound A-1-8

[0121] To the reaction flask, A-1-7 (26.5 g, 76.32 mmol, 1 eq), glacial acetic acid (1.37 g, 22.90 mmol, 1.31 mL, 0.3 eq), and acetonitrile (260 mL) were added. The atmosphere was replaced with nitrogen gas. The mixture was heated to 50 °C, and a solution prepared by dissolving sodium chlorite (32.48 g, 305.28 mmol, purity 85%, 4 eq) in water (70 mL) was added dropwise. After completion of the addition, the mixture was reacted at 50 °C for an additional 12 hours. Then, sodium chlorite (8.97 g, 99.21 mmol, 1.3 eq) and glacial acetic acid (458.31 mg, 7.63 mmol, 436.49 μL, 0.1 eq) were further added, and the mixture was reacted at 50 °C for an additional 6 hours. After completion of the reaction, the reaction solution was quenched with a saturated aqueous solution of sodium bisulfite (150 mL), and water (90 mL) was added. The mixture was allowed to stand, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (90 mL). The combined organic phases were washed with saturated brine (90 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain a crude product. The crude product was slurried with ethyl acetate:n-hexane (1:5, 120 mL) with stirring and filtered. The filter cake was recovered and dried to obtain A-1-8.

Number

[0122] Step 7: Synthesis of Compound A-1

[0123] To a dried 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. The mixture was reacted at 0 °C for 0.5 h. After completion of the reaction, the reaction solution was slowly poured into an aqueous saturated sodium bicarbonate solution (1000 mL) to adjust the pH to 7 - 8. The mixture was extracted with dichloromethane (1000 mL × 3). Each layer was separated. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain A-1.

Number

Chemistry

Chemistry

[0124] Step 1: Synthesis of Compound B-1-2

[0125] To a reaction flask, 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. Then, methyl iodide (2495.80 g, 17.59 mol, 1.25 eq) was added and the mixture was reacted at 25 °C for 2 h. After completion of the reaction, 6N aqueous hydrochloric acid solution was slowly added to the reaction flask to adjust the pH to 6 - 7. The mixture was stirred for 0.5 h and filtered. The filter cake was recovered. Acetonitrile (500 mL) was added to the filter cake. The mixture was stirred for 0.5 h and filtered. The filter cake was recovered and dried by baking to obtain B-1-2.

Number

[0126] Step 2: Synthesis of Compound B-1-3

[0127] Acetonitrile (15 L) and B-1-2 (1500.00 g, 9.60 mol, 1 eq) were added to the reaction flask at 25°C. Then, phosphorus oxychloride (1840.00 g, 12.0 mol, 1.25 eq) was added. The mixture was slowly heated to 62°C and reacted 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 mixture was extracted with ethyl acetate (10.5 L), and each layer was separated to obtain the organic phase. The organic phase was washed with saturated brine (7.5 L), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain B-1-3.

Number

[0128] Step 3: Synthesis of Compound B-1

[0129] To the reaction flask, 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) were added. The atmosphere was replaced with nitrogen gas. Sodium iodide (571.59 g, 3.81 mol, 6.66 eq) and trimethylchlorosilane (186.61 g, 1.72 mol, 218.00 mL, 3 eq) were sequentially added, and the mixture was reacted at 20 °C for 14 hours. After completion of the reaction, dichloromethane (800 mL) and water (1200 mL) were sequentially added to the reaction solution. Then, solid sodium bicarbonate was added to adjust the pH to 6 - 7. Each layer was separated, and the aqueous phase was extracted once with dichloromethane (500 mL). The organic phases were combined, washed sequentially with saturated aqueous sodium sulfite solution (500 mL) and saturated brine (500 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. n-Heptane (0.5 L) was added to the crude product, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was recovered to obtain B-1.

Number

Chemistry

[0130] Synthesis route:

Chemistry

[0131] Step 1: Synthesis of WX001-2

[0132] To the reaction flask, A-1 (500 mg, 1.92 mmol, 1 eq), WX001-1 (427.54 mg, 2.30 mmol, 1.2 eq) and N,N-dimethylformamide (3 mL) were added. The atmosphere was replaced with nitrogen gas. Cesium carbonate (935.92 mg, 2.87 mmol, 1.5 eq) was added, and the mixture was reacted at 25 °C for 16 hours. After completion of the reaction, the reaction solution was poured into water (20 mL), and the mixture was extracted with ethyl acetate (30 mL × 3). Each layer was separated. The organic phases were combined, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by thin-layer chromatography on a silica gel plate to obtain WX001-2.

Number

[0133] Step 2: Synthesis of WX001-3

[0134] In a pre-dried reaction flask, the atmosphere was replaced with nitrogen, and wet palladium on carbon (0.1 g, 819.15 μmol, purity 10%, 1 eq) and ethanol (20 mL) were added. Then, WX001-2 (300 mg, 819.15 μmol, 1 eq) was added. The atmosphere was replaced with nitrogen three times, and the mixture was stirred and reacted at 50 °C and 50 Psi for 24 hours. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by thin-layer chromatography on a silica gel plate to obtain WX001-3.

Number

[0135] Step 3: Synthesis of WX001-4

[0136] To a dried reaction flask, WX001-3 (60 mg, 208.81 μmol, 1 eq), tetrahydrofuran (1 mL), and zinc chloride solution (0.7 M, 298.31 μL, 1 eq) were added. The mixture was cooled to -78 °C, and lithium hexamethyldisilazide (1 M, 417.63 μL, 2 eq) was added. The mixture was reacted at 20 °C for 1 hour to obtain Reaction Solution 1.

[0137] A mixture of B-1 (55.57 mg, 208.81 μmol, 1 eq) and tetrakis(triphenylphosphine)palladium (7.24 mg, 6.26 μmol, 0.03 eq) in N,N-dimethylacetamide (1 mL) was heated to 50 °C under nitrogen, and then Reaction Solution 1 was added dropwise. After the addition was complete, the mixture was reacted at 50 °C for another 1 hour. After the reaction was complete, the reaction solution was poured into water (5 mL), and then extracted with dichloromethane (30 mL × 3). Each layer was separated. The organic phases were combined, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by thin-layer chromatography on a silica gel plate to obtain WX001-4.

Number

[0138] Step 4: Synthesis of WX001-5

[0139] To the reaction flask, WX001-4 (100 mg, 235.00 μmol, 1 eq), acetonitrile (1 mL) and water (0.5 mL) were added. The atmosphere was replaced with nitrogen gas three times, and potassium persulfate (288.95 mg, 470.01 μmol, 2 eq) was added. The mixture was reacted at 20 °C for 14 hours. After completion of the reaction, the reaction solution was poured into a saturated aqueous sodium thiosulfate solution (5 mL), and the mixture was extracted with dichloromethane (30 mL × 3). Each layer was separated. The organic phases were combined, washed successively with a saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by thin layer chromatography on a silica gel plate to obtain WX001-5.

Number

[0140] Step 5: Synthesis of WX001

[0141] To the dried reaction flask, WX001-5 (40 mg, 87.43 μmol, 1 eq), C-1 (16.98 mg, 174.85 μmol, 2 eq) and tetrahydrofuran (0.5 mL) were added. The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0 °C, and lithium hexamethyldisilazide (1 M, 166.11 μL, 1.9 eq) was added dropwise. The mixture was reacted at 0 °C for 1 hour. After completion of the reaction, the reaction solution was poured into water (5 mL), and the mixture was extracted with dichloromethane (30 mL × 3). Each layer was separated. The organic phases were combined, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (column: Waters Xbridge BEH C18 100*25mm*5μm, mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile], B (acetonitrile)%: 20%-50%, 10 minutes) to obtain WX001.

Number

Chem.

[0142] Step 1: Synthesis of Compound I-1-3

[0143] Tetrahydrofuran (12 L) and I-1-1 (1200 g, 5.69 mol) were added to a reaction kettle. Tetramethylethylenediamine (661.59 g, 5.69 mol) was slowly added to the reaction kettle. The atmosphere was replaced with nitrogen gas. The mixture was cooled to -70 °C (internal temperature). Lithium diisopropylamide (2 M, 6.83 L) was slowly added dropwise, and the mixture was stirred at -70 °C for 0.5 h. A solution of I-1-2 (1.76 kg, 9.39 mol) dissolved in tetrahydrofuran (4.8 L) was slowly added dropwise (1.5 h), and the mixture was reacted at -70 °C for 1 h. After completion of the reaction, water (6 L) was added dropwise to the reaction solution to quench the reaction mixture. The reaction kettle was washed with water (2.4 L). The mixtures were combined and stirred. The mixture was warmed to 10 °C. Each layer was separated, and the aqueous phase was extracted with ethyl acetate (6 L). The remaining aqueous phase was adjusted to pH 3 - 4 with an aqueous solution of potassium bisulfate (15.6 L) and then extracted three times with ethyl acetate (6 L). The organic phases were combined and 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. Dichloromethane (2.4 L) and isopropyl ether (7.2 L) were added to the crude product, and the mixture was stirred at room temperature for 30 min. The mixture was filtered, and the filter cake was recovered to obtain I-1-3.

No.

[0144] Step 2: Synthesis of Compound I-1-4

[0145] Dichloromethane (1330 mL), I-1-3 (1330 g, 1.99 mol, crude product), and 4-dimethylaminopyridine (41.37 g, 338.61 mmol) were sequentially added to the reaction kettle. N,N'-carbonyldiimidazole (419.86 g, 2.59 mol) was added portionwise, and the mixture was reacted at 50 °C for 36 hours. After completion of the reaction, 2N aqueous hydrochloric acid solution (5.32 L) was added to the reaction solution to adjust the pH to 3 - 4. The mixture was stirred for 0.5 hour, and then water (5.32 L) was added. The mixture was concentrated to remove the organic solvent. The residue was filtered to obtain a filter cake. The filter cake was stirred with an aqueous sodium bicarbonate solution (5.32 L) for 0.5 hour, then filtered and washed with water (2.66 L). The filter cake was recovered. Anhydrous ethanol (10.64 L) was added to the crude product. The mixture was stirred for 2 hours and filtered. The filter cake was washed with anhydrous ethanol (1.3 L). The filter cake was recovered and dried to obtain I-1-4.

Number

[0146] Step 3: Synthesis of Compound I-1-6

[0147] To the reaction flask were added I-1-4 (283 g, 1.03 mol), cesium carbonate (505.36 g, 1.55 mol), and N,N'-dimethylformamide (2800 mL). The atmosphere was replaced with nitrogen gas. I-1-5 (221.24 g, 1.19 mol) was added, and the mixture was reacted at 20 °C for 12 hours. After completion of the reaction, the reaction solution was slowly poured into ice water (14 L). The mixture was stirred for 1 hour and filtered. The filter cake was recovered. Methyl tert-butyl ether (5 L) was added to the crude product, the mixture was stirred for 2 hours, and then filtered. The filter cake was recovered and dried to obtain I-1-6.

Number

[0148] Step 4: Synthesis of Compound I-1

[0149] Three reactions were carried out in parallel. To the reaction flask, I-1-6 (183 g, 468.60 mmol) and tetrahydrofuran (2745 mL) were added. The atmosphere was replaced with nitrogen gas. Diisopropylethylamine (181.69 g, 1.41 mol) and diethyl phosphite (194.14 g, 1.41 mol) were slowly added dropwise at 20 °C, and the mixture was reacted at 40 °C for 16 hours. After the reaction was completed, the reaction solution was diluted with water (915 mL), and the mixture was extracted with dichloromethane (1830 mL × 3). The organic phase was washed with saturated brine (915 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated at 45 °C and dried to obtain a crude product. The crude product was added to a mixed solvent of methyl tert-butyl ether and n-hexane (total 1098 mL, volume ratio 1:5), and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was recovered and dried. The filter cake was added to water (5400 mL), the mixture was stirred for 1 hour, and filtered. The filter cake was recovered and dried to obtain I-1.

Number

Chemistry

[0150] Step 5: Synthesis of Compound I-3

[0151] Into two reaction flasks, solutions prepared by dissolving I-1 (120 g, 403.05 mmol) and zinc chloride (0.7 M, 575.79 mL) in tetrahydrofuran (1200 mL) were added respectively. The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0 °C, and lithium hexamethyldisilazide (1 M, 806.11 mL) was slowly added dropwise. After the mixture was warmed to 20 °C, it was stirred for 1 hour to obtain Reaction Solution 1. In another reaction flask, B-1 (107.25 g, 403.05 mmol), tetrakis(triphenylphosphine)palladium (13.97 g, 12.09 mmol) and N,N'-dimethylformamide (600 mL) were added, and the reaction solution was heated to 50 °C to obtain Reaction Solution 2. Reaction Solution 1 was slowly added dropwise to Reaction Solution 2, and the mixture was reacted at 50 °C for 1 hour. After the reaction was completed, the reaction mixture was quenched with 0.1 M disodium ethylenediaminetetraacetate (10800 mL) and stirred for 30 minutes. n-Heptane (4800 mL) was added, and the mixture was stirred for 0.5 hour. The mixture was filtered, and the filter cake was recovered and dried to obtain a crude product. The crude product was slurried with ethanol (7200 mL) at 20 °C for 2 hours and filtered. The filter cake was recovered and dried to obtain I-3.

Number

[0152] Step 6: Synthesis of Compound I-4

[0153] Into a reaction flask, I-3 (120 g, 282.00 mmol), acetonitrile (110 mL) and water (550 mL) were added. The atmosphere was replaced with nitrogen gas. 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 completed, 200 mL of ice-water mixture was added to the reaction solution. Then, saturated aqueous sodium bicarbonate solution and saturated aqueous sodium thiosulfate solution (600 mL each) were added, and then 600 mL of water was added. The mixture was filtered, and the filter cake was recovered and dried to obtain a crude product. 600 mL of absolute ethanol was added to the crude product, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was recovered to obtain I-4.

Number

[0154] Process 7: Synthesis of Crystal Form A of WX001

[0155] 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. The atmosphere was replaced with nitrogen gas. Lithium hexamethyldisilazide (1 M, 246.54 mL, 2.4 eq) was added dropwise at -5 °C (controlling the internal temperature at -5 - 3 °C), and the mixture was reacted at 0 °C for 0.5 h. After the reaction was completed, the mixture was quenched with deionized water (470 mL). The mixture was concentrated to remove the organic solvent and filtered. The filter cake was recovered. The filter cake was stirred with deionized water (1000 mL) at room temperature for 30 min and filtered. The filter cake was recovered. The filter cake was stirred with acetonitrile (1000 mL) at room temperature for 30 min and filtered. The filter cake was recovered to obtain Crystal Form A of WX001.

Number

[0156] Example 3: Polymorph Screening of WX001 1. Vapor-Solid Penetration

[0157] Approximately 20 mg of Crystal Form A of WX001 was weighed into a 3 mL vial for each run, and approximately 4 mL of solvent was added to a 20 mL vial. The 3 mL (open) vial was placed into the 20 mL vial, and the 20 mL vial was sealed. The sample adsorbed the solvent and was partially dissolved or left standing at room temperature for 7 days, then the solid was recovered and tested by XRPD. The test results are shown in Table 7.

Table 7

[0158] 2. Gas-liquid diffusion

[0159] Approximately 20 mg of crystalline form A of WX001 per run was weighed into a 3 mL vial and dissolved in 1.2 - 1.8 mL of a solvent. Approximately 4 mL of a poor solvent was added to a 20 mL vial. The 3 mL (open) vial containing the clear solution was placed into the 20 mL vial, and then the 20 mL vial was sealed and allowed to stand at room temperature. The obtained solid was collected and tested by XRPD. The test results are shown in Table 8.

Table 8

[0160] * indicates that a solid was obtained by volatilization at room temperature.

[0161] 3. Slow volatilization

[0162] 15 - 20 mg of crystalline form A of WX001 per run was weighed into a 3 mL vial and dissolved in 1.0 - 3.0 mL of a solvent. The vial was sealed with a sealing film and four pinholes were made in the sealing film. This solution was allowed to stand at room temperature and slowly volatilized. The obtained solid was collected and tested by XRPD. The test results are shown in Table 9.

Table 9

[0163] 4. Slow cooling

[0164] 15 - 35 mg of crystalline form A of WX001 per run was weighed into a 3 mL vial and dissolved in 1.0 - 3.0 mL of a solvent. The solution was stirred at 50 °C for about 3.5 hours for equilibration and then filtered. The supernatant was collected. The obtained supernatant was placed in a biochemical incubator and cooled from 50 °C to 5 °C at 0.1 °C / min and then maintained at a constant temperature of 5 °C. The precipitated solid was collected and tested by XRPD. The test results are shown in Table 10.

Table 10

[0165] * indicates that the sample was transparent at 5 °C and -20 °C, and a solid was obtained by volatilization at room temperature.

[0166] 5. Stirring Using Temperature Cycle

[0167] Approximately 25 mg of crystalline form A of WX001 was weighed into an HPLC vial each time, and 0.5 mL of solvent was added thereto. The resulting suspension was subjected to a temperature cycle program while being magnetically stirred (1000 rpm) (the sample was heated to 50 °C, then cooled to 5 °C at a rate of 0.1 °C / min, then this cycle was repeated, and finally, the sample was maintained at 5 °C). The solid was collected by centrifugation and tested by XRPD. The test results are shown in Table 11.

Table 11

[0168] 6. Stirring of Suspension at Room Temperature

[0169] Approximately 25 mg of crystalline form A of WX001 was weighed into an HPLC vial each time, and 0.5 mL of solvent was added thereto. The resulting turbid solution was magnetically stirred (1000 rpm) at room temperature for 3 days. The solid was collected by centrifugation and tested by XRPD. The test results are shown in Table 12.

Table 12

[0170] 7. Stirring of Suspension at 50 °C

[0171] Approximately 25 mg of crystalline form A of WX001 was weighed into an HPLC vial each time, and 0.5 mL of solvent was added thereto. The resulting suspension was magnetically stirred (1000 rpm) at 50 °C for 3 days. The solid was collected by centrifugation and tested by XRPD. The test results are shown in Table 13.

Table 13

[0172] 8. Addition of poor solvent

[0173] Weighed approximately 15 mg of crystalline form A of WX001 into 20 mL vials, added 0.7 - 1.0 mL of solvent to completely dissolve the solid. While stirring (1000 rpm), the poor solvent was added dropwise to the clear solution until the solid precipitated or the total volume of the poor solvent reached 10 mL. Samples without solid precipitation were stirred at 5 °C until the solid precipitated. Then, the clear samples were stirred at -20 °C until the solid precipitated. Then, the still clear samples were volatilized at room temperature. The precipitated solid was separated and tested by XRPD. The results are shown in Table 14.

Table 14

[0174] * indicates that the sample was clear at room temperature and a solid was obtained by stirring at 5 °C.

[0175] Example 4: Examination of the hygroscopicity of crystalline form A of WX001 Materials for the assay:

[0176] SMS DVS Advantage dynamic vapor sorption device

[0177] Method for the assay:

[0178] Weighed 10 - 30 mg of crystalline form A of WX001 and placed it in a DVS sample pan for testing.

[0179] Results of the assay:

[0180] The DVS pattern of crystalline form A of WX001 is shown in Figure 4, with △W = 0.1134%.

[0181] Conclusions of the assay:

[0182] The weight increase of crystalline form A of WX001 due to moisture absorption at 25°C and 80% RH was 0.1134%, indicating that crystalline form A of WX001 has little hygroscopicity.

[0183] Example 5: Stability test of crystalline form A of WX001 Twelve samples of crystalline form A of WX001 were weighed in parallel, and each sample was approximately 5 mg. The samples were placed at the bottom of HPLC vials to form a thin layer. The samples were placed in a chamber under constant temperature and humidity conditions of 60°C / 75% RH and in a 92.5% RH desiccator. The vials were sealed with a sealing film. Several small holes were made in the sealing film so that the samples could be in complete contact with the ambient air. The caps of the sample vials placed at 60°C were tightened under light conditions and under light-shielding conditions (the samples under light-shielding conditions were wrapped with tinfoil). The test results are shown in Table 15 below.

Table 15

[0184] #: ICH conditions

[0185] Conclusion: Crystalline form A of WX001 has good stability.

[0186] Assay example 1. In vitro kinase activity assay 1. Purpose of the assay:

[0187] The ability of compounds to inhibit ERK1 and ERK2 kinase activities was measured.

[0188] 2. Assay buffer:

[0189] 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM ethylene bis(oxyethylenenitrilo) tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg / mL bovine serum albumin (BSA), 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT), 1% DMSO.

[0190] 3. Treatment of the compound:

[0191] The assay compound was dissolved in 100% DMSO to prepare a stock solution at a specific concentration. The compound was serially diluted with the DMSO solution using an Integra Viaflo Assist smart pipette.

[0192] 4. Assay method:

[0193] 1) The substrate MBP was prepared with freshly prepared reaction buffer.

[0194] 2) ERK1 (or ERK2) kinase was added to the above MBP solution and gently mixed.

[0195] 3) The compound dissolved in 100% DMSO was added to the kinase reaction system using ultrasonic technology (Echo550, nanoliter range), and the mixture was incubated at room temperature for 20 minutes.

[0196] 4) 33 P-ATP (at a specific concentration of 10 μCi / μL) was added, and the reaction started at this point.

[0197] 5) The mixture was incubated at room temperature for 2 hours.

[0198] 6) The radioactivity was detected by the filter binding method.

[0199] 7) The ERK1 (or ERK2) kinase activity was calculated as the ratio of the remaining kinase activity in the assay sample to the kinase activity in the control group (treated with DMSO). The curve was fitted using Prism (GraphPad software), and the IC 50 value was calculated.

[0200] 5. The assay results are shown in Table 16 and Table 17.

Table 16

[0201] Conclusion: The compounds of the present disclosure exhibit excellent inhibitory activity against ERK1 kinase.

Table 17

[0202] Conclusion: The compounds of the present disclosure exhibit excellent inhibitory activity against ERK2 kinase.

[0203] Assay Example 2. In Vitro Cell Proliferation Inhibition Assay 1. Purpose of the assay:

[0204] To measure the ability of compounds to inhibit the proliferation of HT29 tumor cells.

[0205] 2. Treatment of the compound:

[0206] The assay compound was dissolved in 100% DMSO to prepare a 10 mM stock solution.

[0207] 3. Assay method and procedure:

[0208] 1) Turn on the UV light in the biological safety cabinet and count down for 30 minutes.

[0209] 2) Preheat RPMI1640 medium and trypsin in a 37 °C water bath.

[0210] 3) After the UV irradiation is completed, open the biological safety cabinet. Wipe the preheated medium, trypsin, and phosphate buffered saline (PBS) with alcohol and place them in the biological safety cabinet.

[0211] 4) Take out the HT29 cells from the incubator and remove the old medium in the biological safety cabinet. Add 10 mL of PBS. Gently shake the mixture and then remove the PBS.

[0212] 5) 1.5 ml of preheated 0.25% trypsin was added. The culture vessel was shaken horizontally so that the trypsin evenly covered the cells at the bottom and placed in the incubator for 2 minutes.

[0213] 6) Cell digestion was stopped with complete medium, and the cell suspension was pipetted until uniform and counted.

[0214] 7) According to the results of cell counting, the density of the cell suspension was adjusted to 1500 cells / well, and the cell suspension was seeded at 50 μl / well.

[0215] 8) The stock solution of the compound was serially diluted with DMSO solution, and the compound was added to the cell plate using Tecan.

[0216] 9) The cell plates with compound added and CellTiterGlo were equilibrated at room temperature, and then 25 microliters of CellTiterGlo was added to each well. The cell plates were shaken for 1 - 2 minutes and then left standing for 10 minutes. Then, the signal values were detected. The data was analyzed using XL - Fit, and the IC 50 of each compound was calculated.

[0217] 4. The assay results are shown in Table 18.

Table 18

[0218] Conclusion: The compounds of the present disclosure exhibit excellent inhibitory activity against HT29 cell proliferation.

[0219] Assay Example 3. In Vivo PK Test in Mice 1. Purpose of the assay:

[0220] Female BALB / c mice were used as assay animals to determine the blood concentration of the compound after single administration and evaluate the pharmacokinetic behavior.

[0221] 2. Procedure of the assay:

[0222] Four healthy adult female BALB / c mice were selected. Two mice were used as the intravenous injection group, and two mice were used as the oral group. The vehicle for the intravenous injection group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of the intravenous injection vehicle, vortexed, and sonicated to prepare a clear solution of 0.5 mg / mL. The clear solution was filtered through a microporous membrane and made ready for use immediately. The vehicle for the oral group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, vortexed, and sonicated to prepare a solution of 0.3 mg / mL. After intravenous administration at 1 mg / kg or oral administration at 3 mg / kg to the mice, whole blood was collected at regular intervals. Plasma was prepared. The drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

[0223] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0224] 3. The assay results are shown in Table 19.

Table 19

[0225] Note: C max is the maximum concentration, F% is the oral bioavailability, DNAUC is AUC PO / Dose, AUC PO is the oral exposure, Dose is the drug dose, Vd ss is the volume of distribution, Cl is the clearance rate, T 1 / 2 is the half-life, and NA means not available.

[0226] Conclusion: The compounds of the present disclosure show excellent oral exposure and bioavailability.

[0227] Assay Example 4. Solubility Test 1. Purpose of the assay:

[0228] The solubility of the compound was measured to evaluate the dissolution characteristics of the compound.

[0229] 2. Assay solution:

[0230] 1) Buffer A (pH 2.0): 50 mM phosphate buffer, pH 2.0; Buffer B (pH 6.5): 50 mM phosphate buffer, pH 6.5; Buffer C (pH 7.4): 50 mM phosphate buffer, pH 7.4

[0231] 2) Preparation of standard solution:

[0232] a) A 50% acetonitrile solution and 50% buffer were mixed to obtain a diluent.

[0233] b) A 10 mM (10 μL / compound) compound stock solution was added to the diluent (490 μL / compound) to obtain a 200 μM detection standard solution.

[0234] c) The 200 μM UV detection standard solution was diluted with 10-fold and 200-fold volumes of the diluent to obtain 20 μM and 1 μM UV standard solutions, respectively.

[0235] d) 1 μM, 20 μM, and 200 μM UV standard solutions were used as standard solutions for the solubility assay.

[0236] 3. Assay method:

[0237] 1) The compound was dissolved in DMSO to prepare a 10 mM stock solution. Amiodarone hydrochloride, carbamazepine, and chloramphenicol were used as controls for the solubility assay.

[0238] 2) The assay compound and the control stock solution (10 μL each) were placed in a 96-well plate, and 490 μL of three different dissolution media (buffer A, B, C) were added respectively. The pH of the corresponding solubility solutions was 2.0, 6.5, and 7.4 respectively. The theoretically maximum concentration of the assay compound was 200 μM in 2% DMSO.

[0239] 3) The plate was shaken at 600 rpm for 24 hours on a shaker at room temperature (25 ± 2 °C).

[0240] 4) 200 μL of this solution was pipetted from the 96-well plate, suction filtered with a vacuum filtration device, and transferred as an assay sample to a new 96-well plate.

[0241] 5) The compound concentration was determined using HPLC-UV. The HPLC conditions are as shown in Table 20.

Table 20

[0242] 6) Three UV standard solutions (1 μM, 20 μM, 200 μM) from low concentration to high concentration were injected into the HPLC, and then the assay samples of the compounds to be assayed were injected.

[0243] 7) The UV chromatography peaks were integrated and the solubility of the samples was calculated.

[0244] 4. The assay results are shown in Table 21.

Table 21

[0245] Conclusion: The compounds of the present disclosure have good solubility under various pH conditions.

[0246] Assay Example 5. In Vivo Efficacy Assay in a Mouse Model of Human Melanoma A375: 1. Purpose of the assay:

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

[0248] 2. Assay animals:

[0249] Species: Mouse

[0250] Strain: BALB / c nude mice

[0251] Age: 6 - 8 weeks old

[0252] Gender: Female

[0253] Body weight: 18 - 22 grams

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

[0255] 3. Breeding environment:

[0256] Animals were bred in an SPF - grade animal house IVC (independent ventilation cage system, constant temperature and humidity) cage (3 animals per cage) at a temperature of 20 - 26°C and a humidity of 40 - 70%.

[0257] Cage: The cage was made of polycarbonate and had a volume of 375 mm × 215 mm × 180 mm. The bedding material was corn cob, which was changed once a week.

[0258] Diet: Assay animals were allowed free access to diet (sterilized dry pellet diet by irradiation) throughout the assay period.

[0259] Drinking water: Assay animals were allowed free access to sterilized water.

[0260] Cage identification: The animal information card for each cage shall indicate the number of animals in the cage, gender, strain, receipt date, assay number of the dosing schedule, group, and start date of the assay.

[0261] Animal identification: The assay animals were identified by ear tags.

[0262] 4. Assay procedure:

[0263] 1) Assay cells and culture: Human melanoma A375 cells were cultured in vitro as a monolayer. The culture conditions were DMEM medium + 10% fetal bovine serum and a 5% CO2 incubator at 37 °C. Routine digestion with trypsin-EDTA was performed twice a week for passage. When the cell saturation was 80% - 90% and the amount reached the required amount, the cells were harvested, counted, and inoculated.

[0264] 2) Tumor tissue inoculation and grouping: 0.1 mL (5×10 5 ) of A375 cells were subcutaneously inoculated under the right axilla of each mouse. When the average tumor volume reached 170 mm 3 , the animals were randomly divided into 4 groups and administration was started. The assay grouping and administration schedule are shown in Table 22.

Table 22

[0265] 3) Daily observation of assay animals: The development and any modifications of this assay protocol were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC). The use and welfare of assay animals were conducted in accordance with the rules of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The animals were monitored daily for health and death. Routine examinations included observation of tumor growth, as well as the animals' daily behavior, such as activity of movement, food and water intake (visual inspection only), body weight changes (body weight measurement twice a week), and the effects of drug treatment on external signs or other abnormalities. The deaths and side effects of the animals in each group were recorded based on the number of animals in each group.

[0266] 4) Formulation of assay compounds

[0267] a) Vehicle group: 5% DMSO + 95% (20% HP-β-CD)

[0268] b) Assay compound group: A quantitative amount of the assay compound was weighed into a formulation bottle. The corresponding volume of DMSO was added, and then the mixture was vortexed to obtain a clear solution. The corresponding volume of 20% HP-β-CD was added, and then the mixture was vortexed to obtain a homogeneous suspension.

[0269] 5) Tumor measurement and assay index:

[0270] a) The tumor diameter was measured twice a week with calipers. The calculation formula for tumor volume was TV = 1 / 2 × a × b 2 (where a is the major axis of the tumor and b is the minor axis of the tumor).

[0271] b) The tumor inhibitory efficacy of the compound was evaluated by TGI (%). TGI (%) reflected the inhibition rate of tumor growth. TGI (%) was calculated as follows: TGI (%) = {[1 - (average tumor volume at the end of administration in the treatment group - average tumor volume at the start of administration in the treatment group)] / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)} × 100%.

[0272] 5. Assay results:

[0273] 1) As shown in Table 23 and Figure 5, in a subcutaneous xenograft tumor model of human melanoma A375 cells in nude mice, WX001 was able to inhibit tumor growth in a dose-dependent manner until day 21 after oral administration. At three doses of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg, the TGI values were 45%, 58%, and 102%, respectively.

[0274] 2) The body weights of the assay animals were used as a reference index for the indirect measurement of drug toxicity. As shown in Figure 6, when administered until day 21, the body weights of all animals in the solvent control group and the WX001 group did not decrease significantly, and the tolerance was good.

Table 23

[0275] Conclusion: WX001 can dose-dependently inhibit tumor growth at three doses of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg. During administration, no significant decrease in the body weight of animals was observed, and the tolerance was good.

[0276] Assay Example 6. In Vivo PK Test in SD Rats 1. Purpose of the assay:

[0277] Male SD rats were used as assay animals. After single-dose administration, the plasma concentration of the compound was measured to evaluate the pharmacokinetic behavior.

[0278] 2. Procedure of the assay:

[0279] Six healthy adult male SD rats were selected. Three rats were in the intravenous injection group and three rats were in the oral group. The vehicle for the intravenous injection group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of intravenous injection vehicle, vortexed, and sonicated to prepare a clear solution of 0.2 mg / mL. The clear solution was filtered through a microporous membrane and made ready for use immediately. The vehicle for the oral group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, vortexed, and sonicated to prepare a 1 mg / mL solution. After intravenous administration at 1 mg / kg or oral administration at 10 mg / kg to SD rats, whole blood was collected at regular intervals. Plasma was prepared. The drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

[0280] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0281] 3. The assay results are shown in Table 24.

Table 24

[0282] Annotation: C max is the maximum concentration, F% is the oral bioavailability, DNAUC is AUC PO / Dose, and AUC PO is the oral exposure, Dose is the drug dose, Vd ss is the volume of distribution, Cl is the clearance rate, and T 1 / 2 is the half-life.

[0283] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

[0284] Assay Example 7. In Vivo PK Test in Cynomolgus Monkeys 1. Purpose of the assay:

[0285] Male cynomolgus monkeys were used as assay animals. After single-dose administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

[0286] 2. Procedure of the assay:

[0287] Five healthy adult male cynomolgus monkeys were selected. Two animals were in the intravenous injection group and three animals were in the oral group. The vehicle for the intravenous injection group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of the intravenous injection vehicle and dissolved with stirring to prepare a clear solution of 0.4 mg / mL. The clear solution was filtered through a microporous membrane and made ready for use immediately. The vehicle for the oral group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle and dissolved with stirring to prepare a solution of 0.3 mg / mL. After intravenous administration at 1 mg / kg or oral administration at 3 mg / kg to cynomolgus monkeys, whole blood was collected at regular intervals. Plasma was prepared. The drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

[0288] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0289] 3. The assay results are shown in Table 25.

Table 25

[0290] Note: C max is the maximum concentration, F% is the oral bioavailability, DNAUC is AUC PO / Dose, AUC PO is the oral exposure, Dose is the drug dose, Vd ss is the volume of distribution, Cl is the clearance rate, T 1 / 2 is the half-life.

[0291] Conclusion: The compounds of the present disclosure show excellent oral exposure and bioavailability.

[0292] Assay Example 8. In Vivo PK Test in Beagle Dogs 1. Purpose of the assay:

[0293] Male beagle dogs were used as assay animals. After single administration, the plasma concentration of the compound was measured to evaluate the pharmacokinetic behavior.

[0294] 2. Assay procedure:

[0295] Five healthy adult male beagle dogs were selected, with 2 dogs in the intravenous injection group and 3 dogs in the oral group. The vehicle for the intravenous injection group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of the intravenous injection vehicle and dissolved with stirring to prepare a clear solution of 0.4 mg / mL. The clear solution was filtered through a microporous membrane and made ready for use immediately. The vehicle for the oral group was 5% DMSO + 95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle and dissolved with stirring to prepare a solution of 0.3 mg / mL. Beagle dog After intravenous administration at 1 mg / kg or oral administration at 3 mg / kg, whole blood was collected at regular intervals. Plasma was prepared. The drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

[0296] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0297] 3. The assay results are shown in Table 26.

Table 26

[0298] Note: C max is the maximum concentration, F% is the oral bioavailability, DNAUC is AUC PO / Dose, AUC PO is the oral exposure, Dose is the drug dose, Vd ss is the volume of distribution, Cl is the clearance rate, T 1 / 2 is the half-life.

[0299] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

[0300] Assay Example 9. hERG Assay 1. Purpose of the assay:

[0301] Using the whole-cell patch-clamp method, the effect of the compound on hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) current was assayed.

[0302] 2. Method of the assay:

[0303] 2.1 Cell preparation

[0304] CHO-hERG cells were cultured in a 175 cm 2 culture flask. When the cells grew to a density of 60 - 80%, the culture medium was removed. The cells were washed once with 7 mL of PBS (phosphate-buffered saline), and then 3 mL of Detachin was added for digestion. After digestion was completed, 7 mL of culture medium was added to neutralize, and then the mixture was centrifuged. The supernatant was aspirated, and then 5 mL of culture medium was added to resuspend the cells to a cell density of 2 - 5×10 6 / mL.

[0305] 2.2 Solution preparation

[0306] Extracellular solution formulation (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1.25 MgCl2, 10 HEPES, and 10 glucose; this formulation was adjusted to pH 7.4 with NaOH.

[0307] Intracellular solution formulation (mM): 140 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA, and 10 HEPES; this formulation was adjusted to pH 7.2 with KOH.

[0308] 2.3 Electrophysiological recording process

[0309] The single-cell high-impedance sealing and whole-cell patterning processes were all automatically performed by the Qpatch instrument. After obtaining the whole-cell recording mode, the cells were clamped at -80 mV. A pre-voltage of -50 mV for 50 milliseconds and a depolarizing stimulus of +40 mV for 5 seconds were sequentially applied to the cells, followed by repolarization to -50 mV for 5 seconds and then back to -80 millivolts. This voltage stimulus was applied every 15 seconds and recorded for 2 minutes. Then, the extracellular solution was applied and recorded for 5 minutes. Next, the drug administration process was initiated. The compound concentration started from the lowest assay concentration, and each assay concentration was applied for 2.5 minutes. After all concentrations were applied continuously, the positive control compound, 3M cisapride, was applied. At least 3 cells were assayed for each concentration (n≧3).

[0310] 2.4 Preparation of Compounds

[0311] The stock solution of the 20.00 mM compound was diluted with DMSO. 10 μL of the compound stock solution was added to 20 μL of the DMSO solution and serially diluted 3-fold to obtain 6 DMSO concentrations. 4 μL of the compound with 6 DMSO concentrations was added to 396 μL of the extracellular solution respectively and serially diluted 100-fold to obtain 6 intermediate concentrations. 80 μL of the compound with 6 intermediate concentrations was added to 320 μL of the extracellular solution respectively and serially diluted 5-fold to obtain the final concentrations for assay. The highest assay concentration was 40 μM, and there were a total of 6 concentrations: 40, 13.3, 4.4, 1.48, 0.494, and 0.165 μM respectively. The DMSO content at the final assay concentration did not exceed 0.2%. DMSO at this concentration did not affect the hERG potassium channel. All dilutions in the preparation of the compound were performed by the Bravo instrument.

[0312] 2.5 Data Analysis

[0313] The assay data were analyzed by GraphPad Prism 5.0 software.

[0314] 2.6 Quality Control

[0315] Environment: Humidity 20 - 50%, Temperature 22 - 25°C

[0316] Reagent: The assay reagent used was purchased from Sigma and had a purity of over 98%.

[0317] The assay data in the report must meet the following criteria.

[0318] Whole - cell seal impedance > 100 MΩ

[0319] Tail current amplitude > 300 pA

[0320] Pharmacological parameters:

[0321] The inhibitory effects of multiple concentrations of cisapride on the hERG channel were measured using a positive control.

[0322] 3. The assay results are shown in Table 27.

Table 27

[0323] Conclusion: The compounds of the present disclosure have a weak inhibitory effect on hERG potassium channel current, thereby reducing the risk of cardiotoxicity and improving safety.

[0324] Assay Example 10. Plasma Protein Binding (PPB) Assay 1. Purpose of the assay:

[0325] The binding degree of the assay compound to human / mouse / rat / dog / monkey plasma albumin was tested.

[0326] 2. Procedure of the assay:

[0327] 1) Preparation of matrix: On the day of the assay, plasma was thawed with cold water and centrifuged at 3220 rpm for 5 minutes to remove all blood clots. The pH of the obtained plasma was measured and adjusted to 7.4 ± 0.1 using 1% phosphoric acid or 1N sodium hydroxide as necessary.

[0328] 2) Dilution procedure of assay compound: The assay compound was dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at concentrations of 10 mM and 2 mM respectively. A 40 μM working solution was prepared by diluting 2 μL of the stock solution (2 mM) with 98 μL of DMSO. A 400 μM working solution of the control compound was prepared by diluting 10 μL of the stock solution with 240 μL of DMSO. The working solution of the compound (5 μL) was thoroughly mixed with the blank matrix (995 μL) at a ratio of 1:200 to prepare the loading matrix.

[0329] 3) Analysis step:

[0330] a) An equal volume of 30 μL of the loading matrix (n = 2) was transferred to a sample collection plate to prepare the time 0 (T0) sample for residue measurement. The sample was immediately combined with the corresponding blank buffer to a final volume of 60 μL, and the volume ratio of plasma to buffer in each well was set to 1:1. Then, 60 μL of 4% H3PO4 in H2O and 480 μL of the stop solution containing the internal standard were added to the T0 sample of the assay compound. Then, it was stored at 2 - 8 °C with other samples for further processing.

[0331] b) The remaining plasma samples were pre-incubated in a carbon dioxide incubator at 37 ± 1 °C for 30 minutes. Samples without protein (F samples) and samples of the loading matrix (230 μL) were all transferred to polycarbonate tubes (n = 2) and ultracentrifuged at 37 °C and 155,000 × g (35,000 rpm) for 4 hours.

[0332] c) To prepare the T sample (assay sample), an additional matrix-containing sample was transferred to another 96-well plate (sample incubation plate) and incubated at 37 °C for 4 hours.

[0333] d) At the end of centrifugation, 30 μL of protein-free sample and 30 μL of T sample were transferred from the second layer of the supernatant (below the top layer) to a new sample recovery plate. Each sample was mixed with the corresponding blank buffer or matrix to a final volume of 60 μL with a matrix:buffer volume ratio of 1:1. 60 μL of 4% aqueous H3PO4 solution and 480 μL of stop solution (containing internal standard) were added to all samples. The mixture was centrifuged at 4000 rpm for 20 minutes, and 100 μL of the supernatant of each sample was analyzed by LC-MS / MS.

[0334] 3. The assay results are shown in Table 28.

Table 28

[0335] Conclusion: The compounds of the present disclosure have moderate plasma protein binding.

Claims

1. A compound represented by formula (I) 【Chemical 1】 (wherein R １ and R ２ are each independently selected from H and C １～３ alkyl, where said C １～３ alkyl is optionally substituted with 1, 2 or 3 R ａ groups, R ４ 、 R ５ 、 R ６ and R ７ are each independently selected from H, F, Cl, Br, I, and C １～３ alkyl, where the C １～３ alkyl is optionally substituted with 1, 2, or 3 R ｃ s. n is 0 or 1, m is 1 or 2, Ring A is selected from pyrazolyl and tetrahydropyranyl, where the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2 or 3 R ｄ s, R ａ and R ｃ are each independently selected from D, F, Cl, Br, and I, R ｄ is selected from F, Cl, Br, I, C １～３ alkyl and C １～３ alkoxy, wherein the C １～３ alkyl and C １～３ alkoxy are optionally substituted with 1, 2 or 3 R's, R is selected from F, Cl, Br and I) or a pharmaceutically acceptable salt thereof.

2. R １ and R ２ are each independently H, CH ３ and CH ２ CH ３ selected from, where the above CH ３ and CH ２ CH ３ is optionally substituted with 1, 2 or 3 R ａ The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein R1 and R2 are each independently selected from H, CH3, CHF2, CD3 and CH2CH3.

4. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein R4, R5, R6 and R7 are each independently selected from H, F, Cl, Br, I and CH3, where the CH3 is optionally substituted with 1, 2 or 3 Rc.

5. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein R4, R5, R6 and R7 are each independently selected from H, F, Cl, Br, I and CH3.

6. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein Rd is selected from F, Cl, Br, I, CH3 and OCH3, where the CH3 and OCH3 are optionally substituted with 1, 2 or 3 R.

7. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein Rd is selected from F, Cl, Br, I and CH3, where the CH3 is optionally substituted with 1, 2 or 3 R.

8. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein Rd is selected from CH3 and OCH3.

9. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein Rd is selected from CH3.

10. The compound or a pharmaceutically acceptable salt thereof according to Claim 1, wherein ring A is selected from pyrazolyl, where the pyrazolyl is optionally substituted with 1, 2 or 3 Rd.

11. Ring A is [Chemical Formula 2] selected from 【Chemical Formula 3】 where the is optionally substituted with 1, 2 or 3 Rd, the compound or a pharmaceutically acceptable salt thereof according to Claim 1. 【Chemical 4】

12. Ring A is 【Chemical Formula 5】 selected from where the ​ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from

14. Ring A is 【Chemical 7】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from

15. The structural moiety 【Chemical Formula 8】 is 【Chemical Formula 9】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from

16. Rd is selected from F, Cl, Br, I and C1-3 alkyl, wherein the C1-3 alkyl is optionally substituted with 1, 2 or 3 Rs. The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

17. The above compound is 【Chemical 10】 (wherein R ２ is as defined in claim 1, R ６ and R ７ are as defined in claim 1) The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from

18. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is 【Chemical 11】 A compound or a pharmaceutically acceptable salt thereof.

19. The crystalline form A of WX001, characterized in that the powder X-ray diffraction pattern has diffraction peaks characteristic of 2θ angles of 10.2080 ± 0.2000°, 18.8429 ± 0.2000°, 20.6217 ± 0.2000°. 【Chemical Formula 12】

20. The crystalline form A of WX001 according to claim 19, wherein the above powder X-ray diffraction pattern has diffraction peaks characteristic of 2θ angles of 10.2080 ± 0.2000°, 18.8429 ± 0.2000°, 20.6217 ± 0.2000°, 25.0767 ± 0.2000°, 25.4797 ± 0.2000°.

21. The crystalline form A of WX001 according to claim 19, wherein the above powder X-ray diffraction pattern has diffraction peaks characteristic of 2θ angles of 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°.

22. The crystalline form A of WX001 according to claim 19, wherein the powder X-ray diffraction pattern has diffraction peaks characteristic of 2θ angles of 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°.

23. The crystalline form A of WX001 according to claim 19, wherein the powder X-ray diffraction pattern has diffraction peaks characteristic of 2θ angles of 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°.

24.

24. The crystalline form A of WX001 according to claim 19, having a differential scanning calorimetry curve with an endothermic peak starting point at 241.0 ± 3.0 °C.

25.

25. The crystalline form A of WX001 according to claim 19, having a thermogravimetric analysis curve with a maximum weight loss of 0.83% at 150.0 ± 3.0 °C.

26.

27. The crystalline form A of WX001 according to claim 24, having a thermogravimetric analysis curve with a maximum weight loss of 0.83% at 150.0 ± 3.0 °C.

28.

29. A pharmaceutical composition for treating solid tumors, comprising the compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, or the crystalline form A of WX001 according to any one of claims 19 to 26.

30. A pharmaceutical composition for treating cancer, comprising the compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, or the crystalline form A of WX001 according to any one of claims 19 to 26.

31. A pharmaceutical composition comprising the compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, or crystalline form A of WX001 according to any one of claims 19 to 26.

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