Dimethyl-substituted thiazololactam compounds and uses thereof

Abstract

Dimethyl-substituted thiazololactam compounds and their use in the preparation of medicaments for treating related diseases are provided. Specifically, compounds represented by formula (I) and pharma- ceutically acceptable salts thereof are provided: [C1] TIFF2024526243000102.tif34156

Description

Technical Field

[0001] This application claims priority to the specification of Chinese Patent Application Publication No. 202110723288.5, filed on June 28, 2021, the specification of Chinese Patent Application Publication No. 202111669920.9, filed on December 31, 2021, and the specification of Chinese Patent Application Publication No. 202210693547.9, filed on June 17, 2022.

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

Background Art

[0003] The Ras / Raf / MEK / ERK pathway is a classical mitogen-activated protein kinase (MAPK) signaling 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 mutations or amplifications 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 mutant non-small cell lung cancer and other cancers. However, the use of BRAF inhibitors and MEK inhibitors for these upstream nodes can quickly cause problems of drug resistance due to mutations or pathway reactivation, which may significantly 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 the 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 C 1~3 alkyl is optionally substituted with 1, 2, or 3 R a and is

[0010] Each R4 is 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 and is

[0011] n is selected from 1 and 2,

[0012] Ring A is selected from pyrazolyl and tetrahydropyranyl, where the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2, or 3 R d and is

[0013] R a and R c are each independently selected from D, F, Cl, Br, and I,

[0014] R d is selected from F, Cl, Br, I, C 1~3 alkyl, and C 1~3 alkoxy, where the C 1~3 alkyl and C 1~3 alkoxy are optionally substituted with 1, 2, or 3 R,

[0015] R is selected from F, Cl, Br, and I.

[0016] The present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. [Chemical formula]

[0017] In the formula,

[0018] R1 and R2 are each independently selected from H and C 1~3 alkyl, where the C 1~3 alkyl is optionally substituted with 1, 2, or 3 R a and

[0019] each R4 is 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 and

[0020] n is selected from 1 and 2,

[0021] ring A is selected from pyrazolyl and tetrahydropyranyl, where the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2, or 3 R d and

[0022] R a and R c are each independently selected from D, F, Cl, Br, and I,

[0023] R d is selected from F, Cl, Br, I, C 1~3 alkyl, and C 1~3 alkoxy, where the C 1~3 alkyl and C 1~3 alkoxy are optionally substituted with 1, 2, or 3 R,

[0024] R is selected from F, Cl, and Br.

[0025] 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 and the other variables are as defined in the present disclosure.

[0026] 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.

[0027] In some embodiments of the present disclosure, the above-mentioned R4 is independently selected from H, F, Cl, Br, I, and CH3, where the above CH3 is optionally substituted with 1, 2, or 3 R c and the other variables are as defined in the present disclosure.

[0028] In some embodiments of the present disclosure, the above-mentioned R4 is independently selected from H, F, Cl, Br, I, and CH3, and the other variables are as defined in the present disclosure.

[0029] 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.

[0030] 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.

[0031] In some embodiments of the present disclosure, the above-mentioned ring A is

Chemical formula

Chemical formula

[0032] In some embodiments of the present disclosure, the above-mentioned ring A is

Chemical Formula

[0033] In some embodiments of the present disclosure, the above-mentioned structural moiety

Chemical Formula

Chemical Formula

[0034] In some embodiments of the present disclosure, the above-mentioned structural moiety

Chemical Formula

Chemical Formula

[0035] The present disclosure also includes some embodiments obtained by combining any of the above-mentioned variables.

[0036] In some embodiments of the present disclosure, the above-mentioned compound or a pharmaceutically acceptable salt thereof is disclosed, wherein the compound is selected from the following.

Chemical Formula

[0037] (wherein R2 and R4 are as defined in the present disclosure)

[0038] The present disclosure also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof.

Chemical formula

[0039] 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.

Advantages of the Invention

[0040] 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, the compounds of the present disclosure have excellent pharmacokinetic properties and tumor inhibitory effects, the compounds of the present disclosure have a weak inhibitory effect on hERG potassium channel current, a low risk of cardiotoxicity, high safety, and the compounds of the present disclosure have moderate to high plasma protein binding.

[0041] Definitions and Terms Unless otherwise defined, the following terms and phrases used in this specification 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 and 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.

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

[0043] The term "pharmaceutically acceptable salt" means a salt of a compound disclosed herein prepared by reacting a compound having the specific substituents 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 in a pure solution or a suitable inert solvent with a sufficient amount of a base. When a compound disclosed herein contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound in a pure solution or a suitable inert solvent with a sufficient amount of an acid. Certain compounds disclosed herein contain both basic and acidic functional groups and can be converted to any base or acid addition salt.

[0044] 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.

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

[0046] 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, as well as 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.

[0047] Unless otherwise specified, the terms "enantiomer" or "optical isomer" mean stereoisomers that are mirror images of each other.

[0048] Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" are caused by the fact that double or single bonds between ring-forming carbon atoms cannot rotate freely.

[0049] Unless otherwise specified, "diastereomer" means stereoisomers that contain two or more chiral centers in the molecule and are not mirror images of each other between molecules.

[0050] Unless otherwise specified, "(+)" means the dextrorotatory isomer, "(-)" means the levorotatory isomer, and "(±)" means the racemate.

[0051] Unless otherwise specified, the wedge-shaped solid line bond

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[0052] Unless otherwise specified, the terms "tautomer" or "tautomeric form" mean that different functional groups in the isomers are in dynamic equilibrium and can readily convert to 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.

[0053] Unless otherwise specified, the terms "one isomer is concentrated", "concentrated isomer", "one enantiomer is concentrated" or "concentrated 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.

[0054] Unless otherwise specified, the term "isomer excess" or "enantiomeric excess" means the difference in the relative proportions of two isomers or two enantiomers. For example, if 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 enantiomeric excess (ee value) is 80%.

[0055] 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 wants to obtain one enantiomer of a specific 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 diastereoisomers are generally isolated by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization methods (such as carbamates generated from amines).

[0056] 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 this disclosure, regardless of radioactivity.

[0057] The term "optionally" or "optionally selected" means that a subsequent event or state may occur but is not essential, and this term includes cases where the event or state occurs and cases where the event or state does not occur.

[0058] 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 with 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.

[0059] When an arbitrary variable (e.g., R) appears multiple times in the composition 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. Furthermore, a combination of substituents and / or their variants is only allowed if the combination results in a stable compound.

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

[0061] 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.

[0062] 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.

[0063] 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, when L in A-L-Z represents a single bond, the structure of A-L-Z is actually A-Z.

[0064] When the bond of a substituent can bridge two or more atoms on a ring, such a substituent can be bonded to any atom on the ring. For example, for the structural moiety

Chem.

[0065] When the listed linking group does not indicate its linking direction, the linking direction is arbitrary. For example,

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[0066] Unless otherwise specified, if 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 a connectable site, if the connectable site with the H atom is connected to the chemical bond, the number of H atoms at this site will decrease accordingly as the number of connected chemical bonds increases, and the group will become 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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[0067] 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.

[0068] 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. C 1~3 Examples of C 1~2 alkyl groups include C 2~3 alkyl groups, C 1~3 alkyl groups, etc. It can be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methenyl). Examples of C 1~3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

[0069] 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 Examples of C 1~2 alkoxy groups include C 2~3 alkoxy groups, C 1~3 alkoxy groups, C3 alkoxy groups, C2 alkoxy groups, etc. Examples of C 1~3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropyl), etc.

[0070] 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 in this specification.

[0071] The solvents used in this disclosure are commercially available.

[0072] The following abbreviations are used in this 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, Cbz represents benzyloxycarbonyl which is an amine protecting group, BOC represents tert-butoxycarbonyl which is an amine protecting group, 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.

[0073] Compounds are named according to the general naming principles in the art or by ChemDraw® software, and commercially available compounds are named by their vendor directory names.

Brief Description of the Drawings

[0074]

Figure 1

[0075]

Figure 2

Modes for Carrying Out the Invention

[0076] Hereinafter, the present disclosure will be described in detail with reference to examples. However, it is not intended that these examples impose any adverse limitations 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 of the disclosure set forth herein. Reference Example 1

Chemical Formula

Chemical Formula

[0077] Step 1: Synthesis of Compound A-1-2

[0078] To the reaction flask were added A-1-1 (500 g, 2.12 mol, 1 eq), water (1875 mL) and tetrahydrofuran (1875 mL). The atmosphere was replaced with nitrogen gas, and then lithium hydroxide monohydrate (97.76 g, 2.33 mol, 1.1 eq) was added, and the mixture was reacted at 25 °C for 3 hours. After completion of the reaction, the mixture was concentrated to remove the organic solvent, and then ice water (2 L) was added. Then, a 4N hydrochloric acid solution (600 mL) was slowly added to adjust the pH to 2 - 3. The mixture was stirred for 20 minutes and then filtered. The filter cake was washed with water (1 L) and acetonitrile (500 mL). The filter cake was recovered. The filter cake was added to acetonitrile (1 L), and the mixture was stirred for 0.5 hour. The mixture was filtered, and the filter cake was washed with acetonitrile (500 mL). The filter cake was recovered and dried by baking to obtain A-1-2.

Number

[0079] Step 2: Synthesis of Compound A-1-3

[0080] To the reaction flask were added A-1-2 (175 g, 823.55 mmol, purity 97.9%, 1 eq) and 2-methyltetrahydrofuran (1.75 L). The atmosphere was replaced with nitrogen gas, and the mixture was cooled to -30 °C. Lithium diisopropylamide (2 M, 905.90 mL, 2.2 eq) was slowly added dropwise, and the mixture was stirred at -30 °C for an additional 1 hour. Then, a solution of acetone (95.66 g, 1.65 mol, 121.09 mL, 2 eq) and 2-methyltetrahydrofuran (175 mL) was slowly added dropwise, and the mixture was reacted at -30 °C for 1 hour. After the reaction was completed, the reaction solution was quenched with a saturated aqueous ammonium chloride solution (1750 mL) and adjusted to pH 3 - 4 with 4N hydrochloric acid (about 2 L). Each layer was separated, and the aqueous phase was extracted with ethyl acetate (3000 mL × 2). The organic phase was washed with saturated brine (1500 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was collected and concentrated to dryness 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. n-Hexane (3.5 L) was added, and the mixture was stirred for an additional 4 hours. The mixture was filtered, and the filter cake was collected to obtain A-1-3.

Number

[0081] Step 3: Synthesis of Compound A-1-4

[0082] To the reaction flask, A-1-3 (200 g, 668.89 mmol, purity 89%, 1 eq) and acetonitrile (2 L) were added. The atmosphere was replaced with nitrogen gas, and boron trifluoride in diethyl ether (265.82 g, 1.87 mol, 231.15 mL, 2.8 eq) was added. The mixture was reacted at 60 °C for 8 hours. After the reaction was completed, ethanol (200 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure to obtain a crude product. Subsequently, the crude product was slowly poured into water (2000 mL). The mixture was stirred for 30 minutes and filtered. The filter cake was recovered. Absolute ethanol (600 mL) was added to the filter cake, the mixture was stirred for 30 minutes and filtered. The filter cake was washed with ethanol (200 mL). The filter cake was recovered and dried to obtain A-1-4.

Number

[0083] Step 4: Synthesis of Compound A-1

[0084] To the reaction flask, A-1-4 (150 g, 481.93 mmol, purity 92.9%, 1 eq) and ethanol (750 mL) were added. The atmosphere was replaced with nitrogen gas, and hydrogen bromide (1.07 kg, 5.30 mol, 719.67 mL, purity 40%, 11 eq) was slowly added dropwise. The mixture was reacted at 50 °C for 24 hours. After the reaction was completed, dichloromethane (1.5 L) and ice water (500 mL) were added to the reaction solution, and the mixture was adjusted to pH 7 - 8 with 4N aqueous sodium hydroxide solution (about 1500 mL). Each layer was separated, the aqueous phase was extracted with dichloromethane (1000 mL × 2) to obtain an organic phase, which was washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. Ethyl acetate (225 mL) and n-hexane (225 mL) were added to the crude product, and the mixture was stirred for 2 hours. The mixture was filtered, and the filter cake was recovered to obtain A-1.

Number

Chemistry

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[0085] Process 1: Synthesis of Compound B-1-2

[0086] 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 the reaction flask. Subsequently, methyl iodide (2495.80 g, 17.59 mol, 1.25 eq) was added, and the mixture was reacted at 25 °C for 2 hours. After the reaction was completed, 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 hour and filtered. The filter cake was collected. Acetonitrile (500 mL) was added to the filter cake. The mixture was stirred for 0.5 hour and filtered. The filter cake was collected and dried by calcination to obtain B-1-2.

Math.

[0087] Process 2: Synthesis of Compound B-1-3

[0088] Acetonitrile (15 L) and B-1-2 (1500.00 g, 9.60 mol, 1 eq) were added to the reaction flask at 25 °C. Subsequently, 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.

Math.

[0089] Step 3: Synthesis of Compound B-1

[0090] 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 (12000 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 bisulfite 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.

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Chemistry

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[0091] Step 1: Synthesis of Compound D-1-2

[0092] 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 dried 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 mixture was reacted at 25 °C for 12 hours. After the reaction was completed, the reaction solution was quenched with a saturated aqueous sodium sulfite solution (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure using a water pump to obtain D-1-2.

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[0093] Step 2: Synthesis of Compound D-1

[0094] 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) were added to a dried reaction flask. The atmosphere was replaced with nitrogen gas. Lithium hexamethyldisilazide (1 M, 21.98 mL, 2.1 eq) was added dropwise at -30 °C. The mixture was reacted at -30 °C for 2 hours. After the reaction was completed, the reaction solution was poured into a saturated aqueous ammonium chloride solution (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL), 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 D-1.

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[0095] Step 1: Synthesis of Compound E-1-3

[0096] Potassium tert-butoxide (4.83 g, 43.04 mmol, 2.5 eq) and tetrahydrofuran (16 mL) were added to a reaction flask. The atmosphere was replaced with nitrogen gas. A 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) dissolved in tetrahydrofuran (16 mL) was slowly added dropwise. The mixture was reacted at 25 °C for 3 hours. After the reaction was completed, the mixture was concentrated to obtain E-1-3.

[0097] Step 2: Synthesis of Compound E-1-5

[0098] E-1-3 (2.24 g, 17.21 mmol, 1 eq) and isopropanol (140 mL) were added to a reaction flask. The atmosphere was replaced with nitrogen gas. 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 completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. 20 mL of water was added to the crude product, the mixture was adjusted to pH 4 with acetic acid, and then filtered. The filter cake was collected. The filter cake was purified by column chromatography to obtain E-1-5.

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[0099] Step 3: Synthesis of Compound E-1-6

[0100] Sodium hydroxide (1.34 g, 33.61 mmol, 1.05 eq) and water (50 mL) were added to the reaction flask, and then compound E-1-5 (5 g, 32.01 mmol, 1 eq) was added. The atmosphere was replaced with nitrogen gas. The mixture was cooled to 10 °C, and methyl iodide (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 h, slowly heated to 25 °C, and further reacted for 2.5 h. After completion of the reaction, the reaction solution was cooled to 0 - 5 °C, adjusted to pH 7 - 8 with 6N hydrochloric acid, and filtered. The filter cake was recovered and dried to obtain E-1-6.

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[0101] Step 4: Synthesis of compound E-1-7

[0102] E-1-6 (7.4 g, 43.47 mmol, 1 eq) and acetonitrile (75 mL) were added to the reaction flask. After replacing the atmosphere with nitrogen gas, phosphorus oxychloride (8.33 g, 54.34 mmol, 5.05 mL, 1.25 eq) was slowly added dropwise. The mixture was reacted at 62 °C for 2.5 h. After completion of the reaction, the reaction solution was poured into water (100 mL), and solid sodium carbonate was added to adjust the pH to 6 - 7. The aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain E-1-7.

Number

[0103] Step 5: Synthesis of compound E-1

[0104] To the reaction flask, E-1-7 (3.5 g, 18.55 mmol, 1 eq) and acetonitrile (40 mL) were added. The atmosphere was replaced with nitrogen gas. 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) were added sequentially. The mixture was reacted at 25 °C for 12 hours. After the reaction was completed, dichloromethane (50 mL) and water (50 mL) were added to the reaction solution sequentially, and the pH was adjusted to 6 - 7 by adding solid sodium bicarbonate. Each layer was separated. The aqueous phase was extracted with dichloromethane (50 mL), and the organic phases were combined. The combined organic phase was washed with saturated aqueous sodium sulfite solution (50 mL) and saturated brine (50 mL) respectively, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain E-1.

Number

Chemistry

[0105] Synthesis route:

Chemistry

[0106] Step 1: Synthesis of WX001-1

[0107] To a dried reaction flask were added 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). The atmosphere was replaced with nitrogen gas. The mixture was reacted at 20 °C for 12 hours. After completion of the reaction, water (300 mL) was added to the reaction solution, and the organic phase and the aqueous phase were separated. The aqueous phase was extracted three times with dichloromethane (400 mL). The combined organic phases were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was slurried with n-hexane (140 mL) and filtered. The filter cake was recovered and dried to obtain WX001-1.

Number

[0108] Step 2: Synthesis of WX001-2

[0109] To a dried reaction flask were added 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). The mixture was cooled to -78 °C. n-Butyllithium (2.5 M, 17.28 mL, 1.5 eq) was added, and the mixture was stirred for 10 minutes. Further, n-butyllithium (2.5 M, 5.76 mL, 0.5 eq) was added, and the mixture was stirred for another 10 minutes. Further, n-butyllithium (2.5 M, 3.46 mL, 0.3 eq) was added, and the mixture was reacted at 20 °C for another 1 hour to obtain Reaction Solution 1.

[0110] 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 completion of the reaction, an aqueous solution of 0.1 M disodium ethylenediaminetetraacetate (450 mL) was added to the reaction solution. The mixture was stirred for 30 minutes and filtered. The filter cake was recovered to obtain a crude product. The crude product was purified by column chromatography to obtain WX001-2.

Number

[0111] Step 3: Synthesis of WX001-3

[0112] To a dried reaction flask, 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) were added. The mixture was reacted at 20 °C for 1 hour. After completion of the reaction, the reaction solution was concentrated and then extracted with chloroform (30 mL × 3) to remove residual trifluoroacetic acid to obtain WX001-3.

Number

[0113] Step 4: Synthesis of WX001-5

[0114] To a dried reaction flask, 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) were added. The atmosphere was replaced with nitrogen gas. The mixture was reacted at 25 °C for 16 hours. After completion of the reaction, water (10 mL) was added to the reaction solution. The mixture was filtered to obtain a filter cake. The filter cake was dissolved in dichloromethane (10 mL) and washed with saturated brine (15 mL). Each layer was separated to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain WX001-5.

Number

[0115] Step 5: Synthesis of WX001-6

[0116] To a dried reaction flask, 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) were added. The atmosphere was replaced with nitrogen gas. The mixture was reacted at 20 °C for 16 hours. After completion of the reaction, saturated sodium thiosulfate solution (10 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL × 3). The organic phases were combined, washed successively with saturated aqueous sodium bicarbonate solution (20 mL × 2) and saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain WX001-6.

Number

[0117] Step 6: Synthesis of WX001

[0118] To a dried reaction flask, 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) were added. The atmosphere was replaced with nitrogen gas. The reaction solution was cooled to 0 °C, and lithium hexamethyldisilazide (1 M, 599.72 μL, 1.9 eq) was added dropwise. After the addition was complete, the mixture was reacted at 0 °C for an additional 2 hours. After the reaction was complete, the reaction solution was quenched by adding water (10 mL), and then extracted with dichloromethane (10 mL × 3). The organic phases were combined, washed with saturated brine (15 mL), 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 high performance liquid chromatography (column: Waters Xbridge BEH C18 100*30mm*10μm, mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile], B (acetonitrile)%: 25%-55%, 8 minutes) to obtain WX001.

Number

Chemistry

[0119] Synthesis route:

Chemistry

[0120] Step 1: Synthesis of WX002

[0121] To a dried reaction flask, 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. The mixture was reacted at 100 °C for 12 hours. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: Phenomenex C18 80*40mm*3μm, mobile phase: [water (ammonium bicarbonate)-acetonitrile], B (acetonitrile)%: 25%-55%, 8 minutes) to obtain WX002.

Number

Chemistry

[0122] Synthesis route

Chemistry

[0123] Step 1: Synthesis of WX003-1

[0124] To a dried reaction flask, 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) were added. The atmosphere was replaced with nitrogen gas. The mixture was reacted at 25 °C for 16 hours. After the reaction was completed, water (20 mL) was added to the reaction solution and the mixture was filtered. The filter cake was dissolved in dichloromethane (10 mL) and washed with saturated brine (15 mL×3). Each layer was separated to obtain the organic phase. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain WX003-1.

Number

[0125] Step 2: Synthesis of WX003

[0126] To a dried reaction flask, WX003-1 (150 mg, 425.84 μmol, 1 eq), D-1 (217.46 mg, 468.42 μmol, 1.1 eq) and toluene (3 mL) were added. The atmosphere was replaced with nitrogen gas. Then, tetrakis(triphenylphosphine)palladium (98.42 mg, 85.17 μmol, 0.2 eq) was added. The mixture was heated to 110 °C and reacted for 12 hours. After completion of the reaction, the reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was first purified by thin layer chromatography on a silica gel plate and then separated by high performance liquid chromatography (column: Waters Xbridge BEH C18 100*25mm*5μm, mobile phase: [water (ammonium bicarbonate)-acetonitrile], B (acetonitrile) %: 20%-50%, 10 minutes) to obtain WX003.

Number

Chemistry

[0127] Synthesis route

Chemistry

[0128] Step 1: Synthesis of WX004-2

[0129] To a dried reaction flask were added 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). The atmosphere was replaced with nitrogen gas. The mixture was cooled to -78 °C. n-Butyllithium (2.5 M, 863.99 μL, 1.5 eq) was added dropwise, and the mixture was stirred for 10 minutes. Further, n-butyllithium (2.5 M, 288.00 μL, 0.5 eq) was added, and the mixture was stirred for another 10 minutes. Further, n-butyllithium (2.5 M, 172.80 μL, 0.3 eq) was added. After the addition was completed, the mixture was reacted at 20 °C for 1 hour to obtain Reaction Solution 1.

[0130] 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 under nitrogen, and then the above-mentioned Reaction Solution 1 was added. The mixture was reacted at 50 °C for another 40 minutes. After the reaction was completed, the reaction solution was quenched with a saturated aqueous ammonium chloride solution (20 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 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 WX004-2.

Number

[0131] Step 2: Synthesis of WX004-3

[0132] To the reaction flask, WX004-2 (200 mg, 475.57 μmol, 1 eq) and dichloromethane (5 mL) were added. The mixture was cooled to 0 °C. Trifluoroacetic acid (108.45 mg, 951.14 μmol, 70.42 μL, 2 eq) was added to the reaction flask, and the mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was slowly poured into an aqueous solution of saturated sodium bicarbonate (20 mL) and adjusted to pH 7 - 8. The mixture was extracted with dichloromethane (10 mL × 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain WX004-3.

Number

[0133] Step 3: Synthesis of WX004-4

[0134] To the reaction flask, 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) were added. The atmosphere was replaced with nitrogen gas. WX001-4 (156.77 mg, 842.61 μmol, 1.13 mL, 1.2 eq) was added, and the mixture was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction solution was poured into an ice-water mixture (200 mL). The mixture was stirred for 0.5 hour and filtered. The filter cake was co-evaporated with toluene (5 mL × 3) to obtain WX004-4.

Number

[0135] Step 4: Synthesis of WX004-5

[0136] To a dried reaction flask, WX004-4 (100 mg, 234.98 μmol, 1 eq), water (1.5 mL), and acetonitrile (3 mL) were added. The atmosphere was replaced with nitrogen. The mixture was cooled to 0 °C, and potassium persulfate (288.92 mg, 469.96 μmol, 2 eq) was added portionwise. The mixture was reacted at 25 °C for 12 hours. After completion of the reaction, a saturated aqueous sodium sulfite solution (10 mL) was added to the reaction solution. The mixture was stirred for 0.5 hour and then extracted with ethyl acetate (10 mL × 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain WX004-5.

Number

[0137] Step 5: Synthesis of WX004

[0138] 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 a reaction flask. The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0 °C. Lithium hexamethyldisilazide (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 completion of the reaction, the reaction solution was quenched by adding water (10 mL) and then extracted with ethyl acetate (10 mL × 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was first purified by thin layer chromatography on a silica gel plate and then separated 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.

Number

[0139] Assay Example 1. In Vitro Kinase Activity Assay 1. Purpose of the assay:

[0140] The ability of compounds to inhibit ERK1 and ERK2 kinase activities was measured.

[0141] 2. Assay buffer:

[0142] 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.

[0143] 3. Treatment of the compound:

[0144] 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.

[0145] 4. Method of the assay:

[0146] a) The substrate MBP was prepared with freshly prepared reaction buffer.

[0147] b) ERK1 (or ERK2) kinase was added to the above MBP solution and gently mixed.

[0148] c) 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.

[0149] d) 33 P-ATP (specific activity 10 μCi / μL) was added, and the reaction started at this point.

[0150] e) The mixture was incubated at room temperature for 2 hours.

[0151] f) The radiation energy was detected by the filter binding method.

[0152] g) The ERK1 (or ERK2) kinase activity was calculated as the ratio of the remaining kinase activity in the assay sample to the kinase activity of the control group (treated with DMSO). Curves were fitted using Prism (GraphPad software), and the IC 50 value was calculated.

[0153] 5. The assay results are shown in Tables 1 and 2.

Table 1

[0154] Conclusion: The compounds of the present disclosure exhibit excellent inhibitory activity against ERK1 kinase.

Table 2

[0155] Conclusion: The compounds of the present disclosure exhibit excellent inhibitory activity against ERK2 kinase.

[0156] Assay Example 2. In Vitro Cell Proliferation Inhibition Assay 1. Purpose of the assay:

[0157] To measure the ability of compounds to inhibit the proliferation of HT29 tumor cells.

[0158] 2. Treatment of the compound:

[0159] The assay compound was dissolved in 100% DMSO to prepare a 10 mM stock solution.

[0160] 3. Method and procedure of the assay:

[0161] a) Turned on the UV light of the biological safety cabinet and counted down for 30 minutes.

[0162] b) Preheated RPMI1640 medium and trypsin in a 37°C water bath.

[0163] c) After the UV irradiation was completed, the biological safety cabinet was opened. The preheated medium, trypsin, and phosphate-buffered saline (PBS) were wiped with alcohol and placed in the biological safety cabinet.

[0164] d) Removed HT29 cells from the incubator and removed the old medium in the biological safety cabinet. Added 10 mL of PBS. The mixture was gently shaken and then the PBS was removed.

[0165] e) Added 1.5 ml of preheated 0.25% trypsin. 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.

[0166] f) Stopped cell digestion with complete medium, pipetted the cell suspension until it was uniform, and counted the cells.

[0167] g) According to the result of cell counting, adjusted the density of the cell suspension to 1500 cells / well and seeded the cell suspension at 50 μl / well.

[0168] h) Serial diluted the stock solution of the compound with DMSO solution and added the compound to the cell plate using Tecan.

[0169] i) Equilibrated the cell plate with the compound added and CellTiterGlo at room temperature, then added 25 microliters of CellTiterGlo to each well. The cell plate was shaken for 1 - 2 minutes and then left standing for 10 minutes. Then, the signal value was detected. The data was analyzed using XL-Fit and the IC 50 of each compound was calculated.

[0170] 4. The assay results are shown in Table 3. [Table 3]

[0171] Conclusion: The compounds of the present disclosure exhibit excellent inhibitory activity against HT29 cell proliferation.

[0172] Assay Example 3. In Vivo PK Test in Mice 1. Purpose of the assay:

[0173] 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.

[0174] 2. Procedure of the assay:

[0175] 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 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 used 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).

[0176] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0177] 3. The assay results are shown in Table 4. [Table 4]

[0178] 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 dosage, Vd ss is the volume of distribution, Cl is the clearance rate, T 1 / 2 is the half-life.

[0179] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

[0180] Assay Example 4. Solubility Test 1. Purpose of the assay:

[0181] The solubility of the compound was measured to evaluate the dissolution characteristics of the compound.

[0182] 2. Assay solution:

[0183] 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

[0184] 2) Preparation of the standard solution:

[0185] a) A diluent was obtained by mixing a 50% acetonitrile solution and a 50% buffer.

[0186] 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.

[0187] 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.

[0188] d) As standard solutions for the solubility assay, 1 μM, 20 μM, and 200 μM UV standard solutions were used.

[0189] 3. Assay method:

[0190] a) The compounds were dissolved in DMSO to prepare 10 mM stock solutions. Amiodarone hydrochloride, carbamazepine, and chloramphenicol were used as controls for the solubility assay.

[0191] b) Stock solutions of the assay compounds and controls (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 with 2% DMSO.

[0192] c) The plate was shaken at 600 rpm for 24 hours using a shaker at room temperature (25 ± 2 °C).

[0193] d) 200 μL of this solution was pipetted from the 96-well plate, suction filtered using a vacuum filtration device, and transferred as an assay sample to a new 96-well plate.

[0194] e) The compound concentration was determined using HPLC-UV. The HPLC conditions were as shown in Table 5.

Table 5

[0195] f) 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.

[0196] g) The UV chromatography peaks were integrated to calculate the solubility of the samples.

[0197] 4. The assay results are shown in Table 6.

Table 6

[0198] Conclusion: The compounds of the present disclosure have good solubility under various pH conditions.

[0199] Assay Example 5. In Vivo Efficacy Assay in a Mouse Model of Human Melanoma A375: 1. Purpose of the assay:

[0200] The antitumor effect of WX001 was evaluated using a subcutaneous xenograft tumor model of human melanoma A375 cells in nude mice.

[0201] 2. Assay animals:

[0202] Species: Mouse

[0203] Strain: BALB / c nude mice

[0204] Age: 6 - 8 weeks old

[0205] Gender: Female

[0206] Weight: 18 - 22 grams

[0207] Supplier: Vital River Laboratory Animal Technology Co., Ltd.

[0208] 3. Breeding environment:

[0209] The animals were bred in an SPF - grade animal house IVC (Independent Ventilated Cage, constant temperature and humidity) cage (3 animals per cage) at a temperature of 20 - 26°C and a humidity of 40 - 70%.

[0210] 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.

[0211] Food: The assay animals were allowed free access to food (sterilized dry pellet food by irradiation) throughout the assay period.

[0212] Drinking water: The assay animals were allowed free access to sterilized water.

[0213] Cage identification: The animal information card for each cage shall indicate the number of animals, sex, strain, receipt date, assay number of the dosing schedule, group, and start date of the assay in the cage.

[0214] Animal identification: The assay animals were identified by ear tags.

[0215] 4. Assay procedure:

[0216] 1) Assay cells and culture: Human melanoma A375 cells were cultured in a monolayer in vitro. 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 subculture. When the cell saturation reached 80% - 90% and the amount reached the required amount, the cells were harvested, counted, and inoculated.

[0217] 2) Tumor tissue inoculation and grouping: 0.1 mL (5×10 5 ) of A375 cells were subcutaneously inoculated under the right armpit of each mouse. When the average tumor volume reached 170 mm 3 , the animals were randomly divided into 4 groups and dosing was started. The assay grouping and dosing schedule are shown in Table 7.

Table 7

[0218] 3) Daily observation of assay animals: The development and any modification of this assay protocol were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC). The use and welfare of the assay animals were conducted in accordance with the regulations 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), weight change (weight measurement twice a week), the effects of drug treatment on signs of appearance 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.

[0219] 4) Formulation of assay compounds

[0220] a) Vehicle group: 5% DMSO + 95% (20% HP-β-CD)

[0221] 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 uniform suspension.

[0222] 5) Tumor measurement and assay indicators:

[0223] a) The tumor diameter was measured twice a week with calipers. The calculation formula for tumor volume was TV = 1 / 2 × a × b 2 (a is the long diameter of the tumor, b is the short diameter of the tumor).

[0224] 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%.

[0225] 5. Assay results:

[0226] 1) As shown in Table 8 and Figure 1, 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 5 mg / kg, 10 mg / kg, and 20 mg / kg, the TGI was 36%, 81%, and 104%, respectively.

[0227] 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 2, when administered until day 21, the body weights of all animals in the vehicle control group and the WX001 group did not decrease significantly, and there were no cases of morbidity or death.

Table 8

[0228] Conclusion: WX0 can inhibit tumor growth in a dose-dependent manner at three doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg. During administration, no significant decrease in the body weight of the animals was observed, and the tolerance was good.

[0229] Assay Example 6. In Vivo PK Test in SD Rats 1. Purpose of the assay:

[0230] Male SD rats were used as assay animals. After single administration, the plasma concentration of the compound was measured to evaluate the pharmacokinetic behavior.

[0231] 2. Procedure of the assay:

[0232] Six healthy adult male SD rats were selected, with three rats as the intravenous injection group and three rats 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 vehicle for intravenous injection, 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 solution of 1 mg / mL. 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).

[0233] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0234] 3. The assay results are shown in Table 9.

[0235]

Table 9

[0236] 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.

[0237] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

[0238] Assay Example 7. In Vivo PK Test in Cynomolgus Monkeys 1. Purpose of the assay:

[0239] Male cynomolgus monkeys were used as assay animals. After single administration, the plasma concentration of the compound was measured to evaluate the pharmacokinetic behavior.

[0240] 2. Assay procedure:

[0241] 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 the 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).

[0242] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0243] 3. The assay results are shown in Table 10.

[0244]

Table 10

[0245] 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 ssis the volume of distribution, Cl is the clearance rate, and T 1 / 2 is the half-life.

[0246] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

[0247] Assay Example 8. In Vivo PK Test in Beagle Dogs 1. Purpose of the assay:

[0248] Male Beagle dogs were used as assay animals. After single-dose administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

[0249] 2. Procedure of the assay:

[0250] Five healthy adult male Beagle dogs were selected. Two were in the intravenous injection group and three 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. 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).

[0251] Note: DMSO: Dimethyl sulfoxide, HP-β-CD: Hydroxypropyl-β-cyclodextrin

[0252] 3. The assay results are shown in Table 11.

Table 11

[0253] Annotation: C max is the maximum concentration, F% is the oral bioavailability, and DNAUC is AUC PO / Dose, and AUC PO is the oral exposure, Dose is the drug dosage, and Vd ss is the volume of distribution, Cl is the clearance rate, and T 1 / 2 is the half-life.

[0254] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

[0255] Assay Example 9. hERG Assay 1. Purpose of the assay:

[0256] Using the fully automated patch-clamp method, the effect of the compound on hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) current was assayed.

[0257] 2. Method of the assay:

[0258] 2.1 Cell preparation

[0259] 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, 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 so that the cell density became 2 - 5×10 6 / mL.

[0260] 2.2 Solution preparation

[0261] 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.

[0262] 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.

[0263] 2.3 Electrophysiological recording process

[0264] The processes of single-cell high-impedance sealing and whole-cell patterning were all automatically performed by a Qpatch device. 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, and then the cells were repolarized to -50 mV for 5 seconds and then returned to -80 millivolts. This voltage stimulus was applied every 15 seconds and recorded for 2 minutes. Then, the extracellular solution was given and recorded for 5 minutes. Then, the drug administration process was started. The compound concentration started from the lowest assay concentration, and each assay concentration was given for 2.5 minutes. After all concentrations were given continuously, 3M cisapride, a positive control compound, was given. At least 3 cells were assayed for each concentration (n ≧ 3).

[0265] 2.4 Compound formulation

[0266] A stock solution of the compound at 20.00 mM was diluted with DMSO. 10 μL of the stock solution of the compound was added to 20 μL of the DMSO solution and serially diluted 3-fold to obtain six DMSO concentrations. 4 μL of the compound with each of the six DMSO concentrations was added to 396 μL of the extracellular solution and serially diluted 100-fold to obtain six intermediate concentrations. 80 μL of the compound with each of the six intermediate concentrations was added to 320 μL of the extracellular solution and serially diluted 5-fold to obtain the final concentration for the assay. The highest assay concentration was 40 μM, and there were a total of six 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 using a Bravo instrument.

[0267] 2.5 Data analysis

[0268] The assay data were analyzed using GraphPad Prism 5.0 software.

[0269] 2.6 Quality control

[0270] Environment: Humidity 20 - 50%, temperature 22 - 25 °C

[0271] Reagents: The assay reagents used were purchased from Sigma and had a purity of over 98%.

[0272] The assay data in the report must meet the following criteria.

[0273] Whole-cell seal impedance > 100 MΩ

[0274] Tail current amplitude > 300 pA

[0275] Pharmacological parameters:

[0276] The inhibitory effects of multiple concentrations of cisapride on the hERG channel were measured as a positive control.

[0277] 3. The assay results are shown in Table 12. [Table 12]

[0278] 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.

[0279] Assay Example 10. Plasma Protein Binding (PPB) Assay 1. Purpose of the assay:

[0280] The binding degree of the assay compound to human / mouse / rat / dog / monkey plasma albumin was tested.

[0281] 2. Procedure of the assay:

[0282] 1) Preparation of the matrix: On the day of the assay, the 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.

[0283] 2) Dilution procedure of the assay compound: The assay compound was dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with concentrations of 10 mM and 2 mM respectively. A working solution of 40 μM was prepared by diluting 2 μL of the stock solution (2 mM) with 98 μL of DMSO. A working solution of 400 μM 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 well mixed with the blank matrix (995 μL) at a ratio of 1:200 to prepare the loading matrix.

[0284] 3) Analysis step:

[0285] a) 30 μL of equal-volume loading matrix (n = 2) was transferred to a sample collection plate to prepare a 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 stop solution containing an internal standard were added to the T0 sample of the assay compound. It was then stored at 2 - 8 °C with other samples for further processing.

[0286] b) The remaining plasma samples were pre-incubated in a carbon dioxide incubator at 37 ± 1 °C for 30 minutes. All samples of protein-free sample (F sample) and loading matrix (230 μL) were transferred to polycarbonate tubes (n = 2) and ultracentrifuged at 37 °C and 155,000 × g (35,000 rpm) for 4 hours.

[0287] c) To prepare the T sample (assay sample), additional matrix-containing samples were transferred to another 96-well plate (sample incubation plate) and incubated at 37 °C for 4 hours.

[0288] 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 collection plate. Each sample was mixed with the corresponding blank buffer or matrix to a final volume of 60 μL and a matrix:buffer volume ratio of 1:1. 60 μL of 4% H3PO4 aqueous solution and 480 μL of stop solution (containing an 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.

[0289] 3. The assay results are shown in Table 13.

Table 13

[0290] Conclusion: The compounds of the present disclosure have moderate to high 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, Each R ４ is 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 groups, n is selected from 1 and 2, Ring A is selected from pyrazolyl and tetrahydropyranyl, where the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2 or 3 R ｄ groups 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, where the above C １～３ alkyl and C １～３ alkoxy is optionally substituted with one, two or three Rs, R is selected from F, Cl, Br, and I) or a pharmaceutically acceptable salt thereof.

2. The compound as defined below: i) R １ and R ２ are each independently selected from H, CH ３ and CH ２ CH ３ wherein said CH ３ and CH ２ CH ３ are optionally substituted with 1, 2 or 3 R ａ groups; ii) R1 and R2 are each independently selected from H, CH3, CHF2, CD3, and CH2CH3; iii) R4 is independently selected from H, F, Cl, Br, I, and CH3, wherein said CH3 is optionally substituted with 1, 2, or 3 Rc; iv) R4 is independently selected from H, F, Cl, Br, I, and CH3; v) Rd is selected from F, Cl, Br, I, CH3, and OCH3, wherein said CH3 and OCH3 are optionally substituted with 1, 2, or 3 R; vi) Rd is selected from CH3 and OCH3; vii) Ring A is 【Chemical 2】 selected from, wherein said 【Chemical 3】 is optionally substituted with 1, 2, or 3 Rd; viii) Ring A is 【Chemical Formula 4】 selected from; ix) The structural moiety 【Chemical Formula 5】 is 【Chemical Formula 6】 selected from; x) The structural moiety [Chemical Formula 7] is 【Chemical 8】 selected from; xi) Rd is selected from F, Cl, Br, I, and C1-3 alkyl, wherein said C1-3 alkyl is optionally substituted with 1, 2, or 3 R; xii) Rd is selected from F, Cl, Br, I, and CH3, wherein said CH3 is optionally substituted with 1, 2, or 3 R; xiii) Rd is selected from CH3; xiv) Ring A is 【Chemical Formula 9】 selected from, wherein said 【Chemical Formula 10】 is optionally substituted with 1, 2, or 3 Rd; The compound according to claim 1 or a pharmaceutically acceptable salt thereof having one or more of the above. 【Claim ​ 【Chemical 11】 ​ R ２ is as defined in claim 1, R ４ is as defined in claim 1) ​ ​ ​ 【Chemical 12】 ​ ​ ​

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