RU2805569C1 - Thiazolactam compounds as erk inhibitors and their applications - Google Patents

Abstract

FIELD: thiazolactam compounds.

SUBSTANCE: invention is related to thiazolactam compounds of formula (III), where the values of R₁-R₅, n, m are defined in the claims.

EFFECT: used in preparation of drugs for treatment of diseases associated with ERK.

16 cl, 2 dwg, 6 tbl, 13 ex

Description

Field of technology to which the invention relates

The present invention relates to a class of thiazolactam compounds and their use in the preparation of drugs for the treatment of diseases associated with ERK. In particular, the present invention relates to the compounds represented by formula (III) or pharmaceutically acceptable salts thereof.

State of the art

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

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 RAS mutation rate is about 22%, the BRAF mutation rate is about 7%, and the MEK mutation rate is about 1%. Therefore, proteins at key nodes in this pathway have become important targets for cancer treatment (Cancer Discov. 2019, 9, 329-341). Currently, a number of BRAF inhibitors and MEK1/2 inhibitors, as well as their combination regimens, are approved by the US FDA for the treatment of melanoma, BRAFV600E mutation-positive non-small cell lung cancer, and other cancers. However, the use of BRAF and MEK inhibitors on these upstream nodes can quickly cause problems of drug resistance due to mutation or reactivation of the pathway, severely limiting their clinical use.

Extracellular signal-regulated protein kinases (ERKs) (especially ERK1 and ERK2 kinases) are major players and downstream key nodes of the Ras/Raf/MEK/ERK pathway and are over-activated 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 kinases should overcome the problem of drug resistance caused by upstream target inhibitors and will become a more promising therapeutic strategy. But for now, research on ERK inhibitors is still in the clinical phase, and no ERK inhibitor has yet been approved for marketing as a drug.

In summary, there is an urgent need to develop safe and effective ERK inhibitor drugs to meet the need for cancer treatment.

The essence of the invention

The present invention provides the compounds represented by formula (III), or pharmaceutically acceptable salts thereof:

where: R ₁ means H, C _1-3 -alkyl or C _3-5 -cycloalkyl, and C _1-3 -alkyl or C _3-5 -cycloalkyl is optionally substituted with 1, 2 or 3 R _a ;

R ₂ and R ₃ are each independently C _1-3 alkyl, wherein C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

R ₄ is H, F, Cl, Br, I or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _c ;

R ₅ is F, Cl, Br, I or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _e ;

m is 0, 1 or 2;

n is 0, 1 or 2;

ring A is pyrazolyl or tetrahydropyranyl, wherein the pyrazolyl or tetrahydropyranyl is optionally substituted with 1, 2 or 3 R _d ;

R _a , R _b , R _c and R _e are each independently D, F, Cl, Br, I, OH or OCH ₃ ;

R _d means F, Cl, Br, I, CH ₃ or OCH ₃ .

In some embodiments of the present invention, the above R ₁ is H, CH ₃ or cyclopropyl, wherein CH ₃ or cyclopropyl is optionally substituted with 1, 2 or 3 R _a and other variables are already defined herein.

In some embodiments of the present invention, the above R ₁ is H, CH ₃ , CHF ₂ , CD ₃ , CH ₂ CH ₂ OCH ₃ or cyclopropyl, and other variables are already defined herein.

In some embodiments of the present invention, the above R ₂ and R ₃ are each independently CH ₃ or CH ₂ CH ₃ , wherein CH ₃ or CH ₂ CH ₃ is optionally substituted with 1, 2 or 3 R _b and other variables are already defined herein.

In some embodiments of the present invention, the above R ₂ and R ₃ are each independently CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₄ is H, F, Cl, Br, I or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _c and other variables are already defined herein.

In some embodiments of the present invention, the above R ₄ is H, F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is F, Cl, Br, I or CH ₃ , where CH ₃ is optionally substituted with 1, 2 or 3 R _e and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is H, F, Cl, Br, I or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _e and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is H, F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above ring A is , or , and , or optionally 1, 2 or 3 R _d is substituted, and other variables are already defined herein.

In some embodiments of the present invention, the above ring A is , , or , and other variables are already defined in this description.

In some embodiments of the present invention, the above structural grouping means , or , and other variables are already defined in this description.

In some embodiments of the present invention, the above structural grouping means , or , and other variables are already defined in this description.

The present invention provides the compounds represented by formula (III), or pharmaceutically acceptable salts thereof:

where: R ₁ is H or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _a ;

R ₂ and R ₃ are each independently C _1-3 alkyl, wherein C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

R ₄ is H, F, Cl, Br, I or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _c ;

R ₅ is F, Cl, Br, I or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _e ;

m is 0, 1 or 2;

n is 0, 1 or 2;

ring A is pyrazolyl or tetrahydropyranyl, wherein the pyrazolyl or tetrahydropyranyl is optionally substituted with 1, 2 or 3 R _d ;

R _a , R _b , R _c and R _e are each independently F, Cl, Br, I or OH;

R _d means F, Cl, Br, I or CH ₃ .

In some embodiments of the present invention, the above R ₁ is H or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _a and other variables are already defined herein.

In some embodiments of the present invention, the above R ₁ is CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₂ and R ₃ are each independently CH ₃ or CH ₂ CH ₃ , wherein CH ₃ or CH ₂ CH ₃ is optionally substituted with 1, 2 or 3 R _b and other variables are already defined herein.

In some embodiments of the present invention, the above R ₂ and R ₃ are each independently CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₄ is H, F, Cl, Br, I or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _c and other variables are already defined herein.

In some embodiments of the present invention, the above R ₄ is H, F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is F, Cl, Br, I or CH ₃ , where CH ₃ is optionally substituted with 1, 2 or 3 R _e and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is H, F, Cl, Br, I or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _e and other variables are already defined herein.

In some embodiments of the present invention, the above R ₅ is H, F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above ring A is , or , and , or optionally 1, 2 or 3 R _d is substituted, and other variables are already defined herein.

In some embodiments of the present invention, the above ring A is or , and other variables are already defined in this description.

In some embodiments of the present invention, the above structural grouping means , or , and other variables are already defined in this description.

In some embodiments of the present invention, the above structural grouping means , or , and other variables are already defined in this description.

The present invention provides the compounds represented by formula (I), or pharmaceutically acceptable salts thereof:

where: R ₁ is H or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _a ;

R ₂ and R ₃ are each independently C _1-3 alkyl, wherein C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

R ₄ is H, F, Cl, Br, I or C _1-3 -alkyl, wherein C _1-3 -alkyl is optionally substituted with 1, 2 or 3 R _c ;

m is 0, 1 or 2;

ring A is pyrazolyl or tetrahydropyranyl, wherein the pyrazolyl or tetrahydropyranyl is optionally substituted with 1, 2 or 3 R _d ;

R _a , R _b and R _c are each independently F, Cl, Br, I or OH;

R _d means F, Cl, Br, I or CH ₃ .

In some embodiments of the present invention, R ₁ is H or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _a and other variables are already defined herein.

In some embodiments of the present invention, the above R ₁ is CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₂ and R ₃ are each independently CH ₃ or CH ₂ CH ₃ , wherein CH ₃ or CH ₂ CH ₃ is optionally substituted with 1, 2 or 3 R _b and other variables are already defined herein.

In some embodiments of the present invention, the above R ₂ and R ₃ are each independently CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above R ₄ is H, F, Cl, Br, I or CH ₃ , wherein CH ₃ is optionally substituted with 1, 2 or 3 R _c and other variables are already defined herein.

In some embodiments of the present invention, the above R ₄ is H, F, Cl, Br, I or CH ₃ and other variables are already defined herein.

In some embodiments of the present invention, the above ring A is , or , and , or optionally 1, 2 or 3 R _d is substituted, and other variables are already defined herein.

In some embodiments of the present invention, the above ring A is or , and other variables are already defined in this description.

In some embodiments of the present invention, the above structural grouping means , or , and other variables are already defined in this description.

In some embodiments of the present invention, the above structural grouping means , or , and other variables are already defined in this description.

The present invention also includes certain embodiments that are obtained by combining any of the above variables.

Some embodiments of the present invention provide the above compounds, or pharmaceutically acceptable salts thereof, which are selected from the following compounds:

, ,

, ,

where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and m are already defined in the present description.

The present invention also provides compounds represented by the following formulas, or pharmaceutically acceptable salts thereof:

, ,

, ,

, ,

, ,

, ,

, ,

.

Some embodiments of the present invention provide for the use of the above compounds or pharmaceutically acceptable salts thereof in the manufacture of medicaments for the treatment of diseases associated with ERK.

In some embodiments of the present invention, the above use is characterized in that the agents related to ERK inhibitors are drugs for the treatment of solid tumors.

Technical effect

The compounds of the present invention exhibit excellent ERK2 kinase inhibitory activity. In addition, the compounds of the present invention exhibit excellent HT29 cell proliferation inhibition activity. The compounds of the present invention exhibit excellent oral exposure and bioavailability. The compounds of the present invention can significantly inhibit the growth of tumors. When administered, there is no significant reduction in the body weight of animals; the tolerability is good.

Definitions and terms

Unless otherwise specified, the following terms and expressions in the present invention shall have the following meanings. Specific terms or expressions are not to be considered vague or unclear in the absence of a specific definition, but are to be understood in the generally accepted sense. In this case, trade names serve to designate the corresponding products or their active ingredients.

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

The term “pharmaceutically acceptable salt” means those salts of the compounds set forth herein that are prepared by reacting compounds containing certain substituents set forth herein with relatively non-toxic acids or bases. When the compounds herein contain relatively acidic functional groups, salts with bases can be prepared by treating the compounds with sufficient base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable salts with bases include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds herein contain relatively alkaline functional groups, acid salts can be prepared by treating the compounds with sufficient acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable salts with acids include salts of inorganic acids, the inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonates, phosphoric acid, monophosphates, diphosphates, sulfuric acid, bisulfates, hydroiodic acid, phosphinic acid, etc.; and salts of organic acids, the organic acids including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and the like; as well as salts of amino acids (such as arginine, etc.) and salts of organic acids such as glucuronic acid, etc. Some of the compounds given here contain both alkaline and acidic functional groups and can be converted into any salts with bases or acids.

The pharmaceutically acceptable salts provided herein can be prepared from starting compounds containing acidic or alkaline moieties by standard chemical methods. Typically, such salts can be prepared by reacting the free acid or base compound with a stoichiometric amount of the corresponding base or acid in water or an organic solvent or a mixture thereof.

Unless otherwise specified, the term “isomer” includes geometric isomers, cis or trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

The compounds described herein may be present as specific geometric or stereoisomers. All such compounds are contemplated by the present invention, including cis and trans isomers, (-) and (+) enantiomers, (R) and (S) enantiomers, diastereoisomers, (D) isomers, (L) isomers , racemic mixtures and other mixtures, for example, mixtures enriched in enantiomers or diastereoisomers, which are all within the scope of the present invention. Substituents such as alkyls may contain additional asymmetric carbon atoms. All of these isomers and mixtures thereof are within the scope of the present invention.

Unless otherwise indicated, the terms “enantiomer” or “optical isomer” mean those stereoisomers that are in mirror image relationship with each other.

Unless otherwise noted, the terms “cis-trans isomer” or “geometric isomer” arise from the lack of free rotation of the double bond or single bond between the ring carbon atoms.

Unless otherwise specified, the term “diastereomer” means a stereoisomer in which the molecule contains two or more chiral centers that are not in a mirror image relationship between the molecules.

Unless otherwise indicated, “(+)” means the dextro isomer, “(-)” means the left isomer, and “(±)” means the racemate.

Unless otherwise specified, wedge-shaped solid bond ( ) and wedge-shaped dotted connection ( ) denote the absolute configuration of the stereocenter; direct continuous connection ( ) and direct dotted connection ( ) indicate the relative configuration of the stereocenter; wavy line ( ) means a wedge-shaped continuous connection ( ) or wedge-shaped dotted connection ( ); or a wavy line ( ) means direct continuous connection ( ) or direct dotted connection ( ).

Unless otherwise specified, the term “single isomer enriched,” “isomer enriched,” “single enantiomer enriched,” or “enantiomer enriched” means that the content of one isomer or enantiomer is less than 100%, and the isomer or enantiomer content is 60% and more or 70% or more or 80% or more or 90% or more or 95% 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.

Unless otherwise specified, the term “isomeric excess” or “enantiomeric excess” means the difference between the relative percentages 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%, then the isomeric excess or enantiomeric excess (ee value) is 80%.

Optically active (R)- and (S)-isomers or D- and L-isomers can be prepared using chiral synthesis or chiral reagents or other standard methods. If one of the enantiomers of a particular compound described herein is desired, the desired pure enantiomer can be obtained by asymmetric synthesis or derivatization of a chiral auxiliary, followed by resolution of the resulting diastereomeric mixture and elimination of the auxiliary group. On the other hand, 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 is reacted with an appropriate optically active acid or base to form a salt of the diastereomeric isomer, which is then subjected to separation of the diastereomers by an art-accepted method. to obtain the pure enantiomer. In addition, enantiomers and diastereoisomers are usually isolated by chromatographic methods using a chiral stationary phase, optionally in combination with a chemical derivatization method (eg, preparation of a carbamate from an amine).

The compounds given here may contain unnatural proportions of atomic isotopes at one or more of the atoms that make up the compound. For example, a compound may be labeled with a radioisotope such as tritium ( ^3H ), iodine-125 ( ^125I ), or C-14 ( ^14C ). Alternatively, the hydrogen may be replaced by heavy hydrogen to produce a deuterated drug. The bond between deuterium and carbon is stronger than that between simple hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, increased efficacy, and prolonged biological half-life of drugs. All changes in the isotopic composition of the compounds shown here, regardless of radioactivity, are within the scope of the present invention.

The term “optional” or “optional” means that a subsequent event or condition may occur, but does not have to, and the term includes cases in which the event or condition occurs and cases in which the event or condition does not occur.

The term “substituted” means that one or more hydrogen atoms on a particular atom are replaced by a substituent, including deuterium and hydrogen variants, as long as the valency of that atom remains normal and the substituted compound is stable. If the substituent is oxo (i.e. =O), then this means that two hydrogen atoms are substituted. Positions on the aromatic ring cannot be replaced by an oxo group. The term “optionally substituted” means that the atom may or may not be replaced by a substituent unless otherwise indicated, and the type and number of substituents may be arbitrary as long as chemically feasible.

When any variable (of type R) appears more than once in the structure or structure of a compound, the definition of the variable in each case is independent. Thus, for example, if a group is substituted with 0-2 R, then it may optionally be substituted with a maximum of two R, and the definition of R in each case will be independent. In addition, combinations of substituents and/or variants thereof are permissible only if these combinations provide stable compounds.

When the number of connecting groups is 0 type -(CRR) ₀ -, it means that the connecting group is a simple bond.

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

When a substitute is vacant, it means that the substitute does not exist. For example, when X is vacant in A-X, then the structure A-X is actually A.

When one of the variables is a simple link, it means that two groups connected by a simple link are connected directly. For example, when the L in A-L-Z stands for a simple relationship, then the A-L-Z structure actually represents A-Z.

When the bond of a substituent can be cross-linked to two or more atoms on the ring, the substituent can be bonded to any atom on the ring. For example, in the structural grouping or a substituent R is provided which can be inserted at any position of the cyclohexyl or cyclohexadiene. When a numbered substituent does not indicate through which atom it is connected to the group being replaced, such a substituent can be connected through any of its atoms. For example, a pyridyl group as a substituent can be linked to a substituted group through any of the carbon atoms on the pyridine ring.

When a numbered connecting group does not have a bond direction specified, its bond direction is arbitrary. For example, when the connecting group L in is represented by -MW-, then -MW- can connect to ring A and ring B in the same direction as when read from left to right, making , or may connect to ring A and ring B in the opposite direction than when reading from left to right, making . Combinations of connecting groups, substituents and/or variants thereof are permitted only if such combinations can yield stable compounds.

Unless otherwise specified, when a group contains one or more connectable regions, any one or more regions of that group may be connected to other groups through chemical bonds. If the position of the connection at a chemical bond is variable and the site of connection contains H atoms, then when connecting sites containing H atoms are connected to a chemical bond, the number of H atoms at this site will correspondingly decrease as the number of chemical bonds connected increases and the group will become group c corresponding valency. The chemical bond between this site and other groups can be represented as a direct continuous bond ( ), direct dotted connection ( ) or wavy line ( ). For example, a straight continuous bond in -OCH ₃ means that this group connects to other groups through an oxygen atom in the group; direct dotted connection in means that this group connects to other groups through the two ends of the nitrogen atom in the group; wavy line in means that this group is connected to other groups through the 1st and 2nd carbon atoms of the phenyl group; means that any linkable portion of a piperidinyl group can link to other groups through a single chemical bond, including at least four joining methods: , , And ; even if -N- represents an H atom, still includes connection method ; Simply, when combined with one chemical bond, the amount of H in this place will decrease by one and this group will turn into the corresponding monovalent piperidinyl group.

Unless otherwise specified, the number of atoms in a ring is usually defined in terms of the number of ring members, for example, “5-7 membered ring” means a “ring” of 5-7 atoms arranged in a circle.

Unless otherwise specified, the term “C _1-3 -alkyl” is used to designate a linear or branched saturated hydrocarbon group consisting of 1-3 carbon atoms. C _1-3 alkyl groups include C _1-2 and C _2-3 alkyl groups and the like. They may be monovalent (eg methyl), divalent (eg methylene) or polyvalent (eg methenyl). Examples of C _1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, “C _3-5 -cycloalkyl” means a saturated cyclic hydrocarbon group consisting of 3-5 carbon atoms, which is a monocyclic ring system; C _3-5 -cycloalkyl groups include C _3-4 - and C _4-5 -cycloalkyl groups and the like; they can be monovalent, divalent or polyvalent. Examples of C _3-5 cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and the like.

The term “protecting group” includes, without limitation, “amino-protecting groups”, “hydroxy-protecting groups” or “thio-protecting groups”. The term “amino protecting groups” means protecting groups useful for blocking side reactions at the amino nitrogen. Representative amino protecting groups include, but are not limited to: formyl; acyls such as alkanoyls (eg acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyls such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyls such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyls such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyls such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc. The term “hydroxy protecting groups” means protecting groups suitable for blocking side reactions at hydroxyl. Representative hydroxy protecting groups include, but are not limited to: alkyls such as methyl, ethyl and tert-butyl; acyls such as alkanoyls (eg acetyl); arylmethyls such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyls such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc.

The compounds provided herein can be prepared by various synthetic methods well known to those skilled in the art, including the following numbered embodiments, embodiments prepared by the following enumerated embodiments in combination with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Alternative embodiments include, without limitation, the embodiments given herein.

The structure of the compounds presented herein can be verified by standard methods well known to those skilled in the art. If the present invention provides an absolute configuration of a compound, the absolute configuration can be verified by standard methods such as single crystal X-ray diffraction (SXRD). In single crystal X-ray diffraction (SXRD), diffraction intensity data on a grown single crystal is obtained on a commercial Bruker D8 diffractometer with a CuKα radiation source in the ϕ/ω scanning mode; Once the relevant data are obtained, further analysis of the crystal structure by the direct method (Shelxs97) is carried out to verify the absolute configuration.

The solvents used in the present invention are commercially available.

In the present invention, the following abbreviations are used: aq means aqueous; eq means equivalent or equivalent; DCM means dichloromethane; PE means petroleum ether; DMSO means dimethyl sulfoxide; EtOAc means ethyl acetate; EtOH means ethanol; MeOH means methanol; Cbz means benzyloxycarbonyl, which is an amino protecting group; BOC means tert-butoxycarbonyl, which is an amino protecting group; O/N means overnight; THF means tetrahydrofuran; Boc2O means di-tert-butyl dicarbonate; TFA means trifluoroacetic acid; DIPEA means diisopropylethylamine; iPrOH means 2-propanol; k.t. means room temperature; m.p. means melting point.

Compounds are named according to general naming conventions in the art or ^ChemDraw® program, and commercially available compounds are named by their trade names.

Brief description of the figures

Fig. 1. Growth curve of human H358 non-small cell lung cancer tumors in an animal model after administration of vehicle and WX006, respectively.

Fig. 2. Extent of weight change (%) in the human H358 non-small cell lung cancer animal model upon administration.

Disclosure of the invention

In the following, the present invention is described in detail using examples. However, this does not mean that these examples have any unfavorable limitations on the present invention. The present invention is described in detail here, and embodiments are also set forth here. Those skilled in the art will appreciate that various changes and modifications may be made to the embodiments set forth herein without departing from the spirit and scope of the invention set forth herein.

Test case 1. Fragment A-1

Step 1. Synthesis of compound A-1-2

A solution of sodium acetate (4.64 g, 56.60 mmol, 5 eq.), potassium monopersulfate (13.92 g, 22.64 mmol, 2 eq.) and water (47 ml) was added to a dry one-neck flask. The mixture was cooled to 0°C. A solution of A-1-1 (4.7 g, 11.32 mmol, 1 eq), solvent tetrahydrofuran (47 ml) and methanol (47 ml) were added dropwise and the mixture was stirred at 0°C for 1 hour. The mixture was then stirred in an oil bath at 29°C for 15 hours. Upon completion of the reaction, the reaction solution was poured into water (200 ml) and the aqueous phase was extracted with ethyl acetate (3×50 ml). The organic phases were combined, and the combined organic phase was washed successively with saturated NaCl solution (200 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was collected and evaporated under reduced pressure to leave a residue. The residue was purified by flash column chromatography to give A-1-2. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm 8.67 (d, J = 4.9 Hz, 1H), 7.64 (d, J = 4.9 Hz, 1H), 3.37 (s , 3H), 1.63-1.53 (m, 6H), 1.39-1.30 (m, 6H), 1.26-1.12 (m, 6H), 0.90 (t, J = 7.3 Hz, 9H).

Step 2. Synthesis of compound A-1

A-1-2 (3.9 g, 8.72 mmol, 1 eq.), A-1-3 (1.02 g, 10.46 mmol, 1.2 eq.) and tetrahydrofuran ( 117 ml). The atmosphere was replaced with nitrogen gas and then lithium hexamethyldisilazide (1 M, 18.31 mL, 2.1 eq.) was added dropwise at -35°C. A solution of this mixture was incubated at -35°C for 10 minutes. Upon completion of the reaction, the reaction solution was quenched with saturated aqueous ammonium chloride (100 ml) and extracted with ethyl acetate (2×100 ml) and dichloromethane (100 ml). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness on a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography to give A-1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm 8.17 (d, J=4.85 Hz, 1H), 7.46 (d, J=1.76 Hz, 1H), 6.91 (d, J=4.63 Hz, 1H), 6.60 (s, 1H), 6.32 (d, J=1.98 Hz, 1H), 3.79 (s, 3H), 1.52-1.61 (m, 6H), 1.28-1.40 (m, 6H), 1.03-1.20 (m, 6H ), 0.89 (t, J=7.28 Hz, 9H).

Example 1

Synthesis circuit

Stage 1. Synthesis of WX001-3

A solution of lithium diisopropylamide (2M, 2.88 ml, 2.4 eq.) in tetrahydrofuran (5 ml) was added to the reaction flask at -78°C under a nitrogen atmosphere, and then a solution of WX001-1 (500 mg, 2.4 eq.) was added slowly. 40 mmol, 1 eq.) and tetramethylethylenediamine (418.94 mg, 3.61 mmol, 544.08 μl, 1.5 eq.) in tetrahydrofuran (1 ml). The mixture was incubated at -78°C for 0.5 hour, and then WX001-2 (279.18 mg, 4.81 mmol, 353.40 μl, 2 eq.) was added. The mixture was incubated at -78°C for an additional 2 hours. Upon completion of the reaction, the reaction solution was slowly poured into 30 ml of saturated aqueous ammonium chloride at 0°C, adjusted to pH 3-4 with hydrochloric acid (2 mol/l) and extracted with ethyl acetate (3 x 20 ml). The organic phases were combined, washed with saturated NaCl solution (3×20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain WX001-3.

Stage 2. Synthesis of WX001-4

WX001-3 (250 mg, 939.45 µmol, 1 eq) and acetonitrile (8 ml) were added to the reaction flask. The atmosphere was replaced with nitrogen gas, and then concentrated sulfuric acid (110.57 mg, 1.13 mmol, 60.09 μL, 1.2 eq.) was added at 0°C. A solution of this mixture was incubated at 25°C for 16 hours. Upon completion of the reaction, the reaction solution was diluted with 10 ml of water and extracted with ethyl acetate (3×30 ml). The organic phases were combined, washed with saturated NaCl solution (3×30 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain WX001-4.

Stage 3. Synthesis of WX001-5

WX001-4 (200 mg, 651.12 µmol, 1 eq.) and acetic anhydride (2 ml) were added to the dry reaction flask. The atmosphere was replaced with nitrogen gas and the mixture was then incubated at 90°C for 16 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure using an oil pump at 45°C to obtain WX001-5.

Stage 4. Synthesis of WX001-6

WX001-5 (60 mg, 207.51 µmol, 1 eq), hydrochloric acid (2 M, 2 ml, 19.28 eq) and ethanol (2 ml) were added to a dry reaction flask. The mixture was incubated at 50°C for 16 hours and then incubated at 70°C for another 4 hours. Upon completion of the reaction, the reaction solution was evaporated under reduced pressure using a water pump at 45°C and extracted with ethyl acetate (3×30 ml). The organic phases were combined, washed with saturated NaCl solution (3×30 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX001-6. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ ppm 8.85 (br s, 1H), 1.48 (s, 6H).

Stage 5. Synthesis of WX001-7

WX001-6 (60 mg, 242.80 µmol, 1 eq.) and N,N-dimethylformamide (1 ml) were added to the dry reaction flask. The atmosphere was replaced with nitrogen gas and then sodium hydride (14.57 mg, 364.21 µmol, 60% purity, 1.5 eq.) was added at 0°C. The mixture was incubated at 0°C for 0.5 hour, and then 3-chlorobenzyl bromide (49.89 mg, 242.80 μmol, 31.78 μl, 1 eq.) was added. The reaction solution was slowly heated to 25°C and incubated for another 2 hours. After completion of the reaction, 20 ml of water was added to the reaction solution and the mixture was extracted with ethyl acetate (3×10 ml). The organic phases were combined, washed with saturated NaCl solution (3×10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX001-7. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm 7.26 (s, 1H), 7.17 (s, 3H), 4.62 (s, 2H), 1.37 (s, 6H).

Stage 6. Synthesis of WX001

WX001-7 (35 mg, 94.17 µmol, 1 eq), A-1 (56.28 mg, 103.58 µmol, 1.1 eq) and toluene (2 ml) were added to the reaction flask and the atmosphere was replaced nitrogen gas. The mixture was heated to 125°C and then tetrakis(triphenylphosphine)palladium (21.76 mg, 18.83 μmol, 0.2 eq.) was slowly added. The mixture was incubated at 125°C for 48 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX001.

Example 2

Synthesis circuit

Stage 1. Synthesis of WX002-2

WX001-6 (50 mg, 202.34 µmol, 1 eq.) and dimethylformamide (1 ml) were added to a dry reaction flask. The atmosphere was replaced with nitrogen gas and then sodium hydride (12.14 mg, 303.51 µmol, 60% purity, 1.5 eq.) was added at 0°C. The mixture was incubated at 0°C for 0.5 hour, then WX002-1 (38.25 mg, 202.34 μmol, 24.84 μl, 1 eq.) was added. The reaction solution was slowly heated to 20°C and incubated for another 2 hours. After completion of the reaction, 20 ml of water was added to the reaction solution and the mixture was extracted with ethyl acetate (3×10 ml). The organic phases were combined, washed with saturated NaCl solution (3×10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX002-2. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 7.32-7.40 (m, 1H), 7.15-7.23 (m, 2H), 7.03-7.11 (m, 1H), 4.67 (s, 2H ), 1.48 (d, J = 3.8 Hz, 6H).

Stage 2. Synthesis of WX002

WX002-2 (60 mg, 168.91 µmol, 1 eq), A-1 (100.94 mg, 185.80 µmol, 1.1 eq) and toluene (1 ml) were added to the reaction flask and the atmosphere was replaced nitrogen gas. The mixture was heated to 125°C and then tetrakis(triphenylphosphine)palladium (39.04 mg, 33.78 μmol, 0.2 eq.) was slowly added. The mixture was incubated at 125°C for 48 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX002.

Example 3

Synthesis circuit

Stage 1. Synthesis of WX003-2

WX001-6 (80 mg, 325.04 µmol, 1 eq.) and dimethylformamide (4 ml) were added to the dry reaction flask. The atmosphere was replaced with nitrogen gas and then sodium hydride (19.50 mg, 487.56 µmol, 60% purity, 1.5 eq.) was added at 0°C. The mixture was incubated at 0°C for 0.5 hour, then WX003-1 (67.29 mg, 325.04 μmol, 41.54 μl, 1 eq.) was added. The reaction solution was slowly heated to 20°C and incubated for another 2 hours. After completion of the reaction, 20 ml of water was added to the reaction solution and the mixture was extracted with ethyl acetate (3×20 ml). The organic phases were combined, washed with saturated NaCl solution (3×20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by column chromatography to obtain WX003-2. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 7.33-7.47 (m, 2H), 7.23 (m, 1H), 4.65 (s, 2H), 1.41-1.52 (m, 6H).

Stage 2. Synthesis of WX003

WX003-2 (70 mg, 187.56 µmol, 1 eq), A-1 (112.09 mg, 206.32 µmol, 1.1 eq) and toluene (2 ml) were added to the reaction flask and the atmosphere was replaced nitrogen gas. The mixture was heated to 125°C and then tetrakis(triphenylphosphine)palladium (43.35 mg, 37.51 μmol, 0.2 eq.) was slowly added. The mixture was incubated at 125°C for 48 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX003.

Example 4

Synthesis circuit

Stage 1. Synthesis of WX004-1

WX002-2 (70 mg, 197.06 µmol, 1 eq.), A-1-2 (113.45 mg, 216.76 µmol, 1.1 eq.) and toluene (2 ml) were added to the reaction flask. replaced the atmosphere with nitrogen gas. The mixture was heated to 125°C and then tetrakis(triphenylphosphine)palladium (45.54 mg, 39.41 μmol, 0.2 eq.) was slowly added. The mixture was incubated at 125°C for 48 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX004-1. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 9.26 (d, J = 5.1 Hz, 1H), 8.43 (d, J = 5.3 Hz, 1H), 7.33-7.42 (m, 1H) , 7.16-7.28 (m, 2H), 7.08 (td, J = 8.6, 2.0 Hz, 1H), 4.73 (s, 2H), 3.50 (s, 3H), 1.55 (s, 6H).

Stage 2. Synthesis of WX004

WX004-1 (50 mg, 115.61 µmol, 1 eq.) and WX004-2 (11.69 mg, 115.61 µmol, 1 eq.) were added to a dry reaction flask, and then the mixture was dissolved in dimethyl sulfoxide (1 ml ). The mixture was incubated with stirring at 100°C for 14 hours. Upon completion of the reaction, the reaction solution was evaporated, and the residue was purified by preparative thin layer chromatography to obtain WX004.

Example 5

Synthesis circuit

Stage 1. Synthesis of WX005-2

WX001-7 (530 mg, 1.43 mmol, 1 eq), zinc chloride (0.7 M, 1.83 mL, 0.9 eq), and tetrahydrofuran (3.5 mL) were added to the reaction flask. The atmosphere was replaced with nitrogen gas and then n-butyllithium (2.5 M, 855.58 μL, 1.5 eq.) was added at -25°C. A solution of this mixture was incubated at 20°C for 1 hour. A solution of WX005-1 (379.45 mg, 1.43 mmol, 1 eq.), tris(dibenzylideneacetone)dipalladium (65.29 mg, 71.30 µmol, 0.05 eq.) was added dropwise at -25°C. and tri(2-furyl)phosphine (33.11 mg, 142.60 µmol, 0.1 eq.) in tetrahydrofuran (0.5 ml) and incubated the mixture at 20°C for 10 hours. More tetrakis(triphenylphosphine)palladium (82.5 mg, 71.5 µmol, 0.05 eq) was added and the mixture was incubated at 65°C for 20 hours. Upon completion of the reaction, the reaction solution was quenched with methanol and evaporated to obtain the crude product. The crude product was purified by preparative thin layer chromatography to give WX005-2. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 8.75 (s, 1H), 7.46 (s, 1H), 7.26-7.41 (m, 3H), 4.72 (s, 2H), 2.65 ( s, 3H), 2.59 (s, 3H), 1.53 (s, 6H).

Stage 2. Synthesis of WX005-3

WX005-2 (140 mg, 324.85 µmol, 1 eq.) and dichloromethane (6 ml) were added to the reaction flask. The atmosphere was replaced with nitrogen gas and then m-chloroperoxybenzoic acid (210.22 mg, 974.54 µmol, 80% purity, 3 eq.) was added at 0°C. A solution of this mixture was slowly heated to 20°C and incubated for 10 hours. Upon completion of the reaction, the reaction solution was quenched with saturated sodium sulfite (15 ml) and saturated aqueous sodium bicarbonate (15 ml) to an alkaline pH until the color of the KI indicator paper no longer changed, and the mixture was extracted with dichloromethane (3 x 3 ml) . The organic phase was evaporated to dryness under reduced pressure using a water pump at 45°C to obtain a brown solid. The brown substance was then triturated with ethyl acetate (3 ml) to obtain WX005-3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 9.18 (s, 1H), 7.47 (s, 1H), 7.30-7.39 (m, 3H), 4.73 (s, 2H), 3.48 ( s, 3H), 2.83 (s, 3H), 1.55 (s, 6H).

Stage 3. Synthesis of WX005

A-1-3 (15.73 mg, 162.00 µmol, 1.5 eq) and N,N-dimethylformamide (1 ml) were added to the reaction flask. The atmosphere was replaced with nitrogen gas and then sodium hydride (6.48 mg, 162.00 µmol, 60% purity, 1.5 eq.) was added at 0°C. After 0.5 hours, a solution of WX005-3 (50 mg, 108.00 μmol, 1 eq.) in N,N-dimethylformamide (1 ml) was added and the mixture was incubated at 20°C for 10 hours. Upon completion of the reaction, the reaction solution was quenched with saturated aqueous ammonium chloride (5 ml) and extracted with dichloromethane (3×1 ml). The organic phase was evaporated to dryness under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by thin layer chromatography to give WX005.

Example 6

Synthesis circuit

Stage 1. Synthesis of WX006-1

WX002-2 (0.456 g, 1.28 mmol, 1 eq), zinc chloride (0.7 M, 1.65 mL, 0.9 eq), and tetrahydrofuran (10.5 mL) were added to a dry reaction flask. The atmosphere was replaced with nitrogen gas, and then the reaction system was cooled to -25°C and n-butyllithium (2.5 M, 770.22 µl, 1.5 eq.) was added. A solution of this mixture was incubated with stirring at 20°C for 1 hour. A solution of WX005-1 (341.59 mg, 1.28 mmol, 1 eq.) and tetrakis(triphenylphosphine)palladium (74.17 mg, 64.18 μmol, 0.05 eq.) in tetrahydrofuran (1.5 mL) was added ) at -25°C. The mixture was incubated with stirring at 60°C for 12 hours. Upon completion of the reaction, the reaction solution was quenched with 3 mL of methanol and evaporated to dryness on a rotary evaporator to obtain the crude product. The crude product was purified by thin layer chromatography on a silica gel plate to obtain WX006-1.

Stage 2. Synthesis of WX006-2

WX006-1 (210 mg, 506.61 µmol, 1 eq) and dichloromethane (5 ml) were added to the dry reaction flask. The reaction system was cooled to 0°C and m-chloroperoxybenzoic acid (327.85 mg, 1.52 mmol, 80% purity, 3 eq.) was added. The mixture was slowly warmed to room temperature (20°C) and incubated with stirring for 12 hours. After completion of the reaction, the reaction solution was adjusted to pH about 8 with aqueous sodium bicarbonate (5 ml). Add a saturated solution of sodium sulfite until the indicator paper with starch and KI turns blue. A solution of this mixture was extracted with dichloromethane (3×10 ml). The organic phases were combined, washed with saturated NaCl solution (2×10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by thin layer chromatography on a silica gel plate to give WX006-2.

Stage 3. Synthesis of WX006

A-1-3 (26.10 mg, 268.75 µmol, 1.2 eq) and N,N-dimethylformamide (3 ml) were added to the dry reaction flask. The reaction system was cooled to 0°C. Sodium hydride (13.44 mg, 335.93 µmol, 60% purity, 1.5 eq.) was then added. The mixture was incubated with stirring at 0°C for 0.5 hours. WX006-2 (100 mg, 223.96 µmol, 1 eq) was added and the mixture was incubated with stirring at 0°C for 0.5 hour. Upon completion of the reaction, the reaction solution was quenched with water (5 ml) and extracted with ethyl acetate (3×5 ml). The organic phases were combined, washed with saturated NaCl solution (4×5 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by thin layer chromatography on a silica gel plate to give WX006.

Example 7

Synthesis circuit

Stage 1. Synthesis of WX007-1

WX001-7 (50 mg, 134.52 µmol, 1 eq.), A-1-2 (77.45 mg, 147.98 µmol, 1.1 eq.) and toluene (2 ml) were added to the reaction flask and replaced the atmosphere with nitrogen gas. The mixture was heated to 125°C and then tetrakis(triphenylphosphine)palladium (31.09 mg, 26.90 µmol, 0.2 eq.) was slowly added. The mixture was incubated at 125°C for 48 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure using a water pump at 45°C to obtain the crude product. The crude product was purified by thin layer chromatography on a silica gel plate to obtain WX007-1. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 9.26 (d, J = 5.1 Hz, 1H), 8.43 (d, J = 5.1 Hz, 1H), 7.47 (s, 1H), 7.28 -7.39 (m, 3H), 4.73 (s, 2H), 3.50 (s, 3H), 1.55 (s, 6H).

Stage 2. Synthesis of WX007

WX007-1 (30 mg, 66.82 µmol, 1 eq.) and WX004-2 (6.76 mg, 66.82 µmol, 1 eq.) were added to a dry reaction flask, and then the mixture was dissolved in dimethyl sulfoxide (1 ml ). The mixture was incubated with stirring at 100°C for 16 hours. Upon completion of the reaction, the reaction solution was evaporated, and the residue was purified by preparative thin layer chromatography to obtain WX007.

Example 8

Synthesis circuit

Stage 1. Synthesis of WX008-2

WX008-1 (9 g, 79.59 mmol, 1 eq), potassium carbonate (13.20 g, 95.51 mmol, 1.2 eq) and N,N-dimethylformamide (100 ml) were added to the reaction flask. and replaced the atmosphere with nitrogen gas. The mixture was heated to 120°C and stirred for 10 minutes. Sodium 2-chloro-2,2-difluoroacetate (24.27 g, 159.19 mmol, 2 eq.) was then added portionwise. A solution of this mixture was incubated at 120°C for 20 minutes. Upon completion of the reaction, the reaction solution was diluted with water (800 ml) and extracted with ethyl acetate (3×200 ml). The organic phase was washed with saturated NaCl solution (300 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump, and the residue was purified by thin layer chromatography on a silica gel plate to obtain WX008-2. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 7.96-8.28 (m, 2H), 7.50 (d, J = 2.0 Hz, 1H).

Stage 2. Synthesis of WX008-3

Palladium on carbon (50 mg, 367.91 µmol, 10% purity, 1 eq.) and methanol (1 ml) were added to the reaction flask. The atmosphere was replaced with nitrogen gas and then WX008-2 (60 mg, 367.91 µmol, 1 eq.) was added. The atmosphere was then replaced with hydrogen gas and a solution of this mixture was incubated at 15 psi in a hydrogen atmosphere (741.67 μg, 367.91 μmol, 1 eq.) at 25°C for 1 hour. After completion of the reaction, the reaction solution was filtered through celite, and the filtrate was evaporated to dryness under reduced pressure using a water pump to obtain WX008-3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 7.36-7.69 (m, 1H), 7.33 (s, 1H), 5.84 (br s, 2H), 5.31 (d, J = 1.3 Hz , 1H).

Stage 3. Synthesis of WX008

WX008-3 (17.88 mg, 134.37 µmol, 1.5 eq.), WX006-2 (40 mg, 89.58 µmol, 1 eq.), dichloromethane (0.5 ml) and tetrahydrofuran (0.5 ml). The atmosphere was replaced with nitrogen gas and the mixture was cooled to 0°C. Lithium hexamethyldisilazide (1 M, 188.12 μL, 2.1 eq.) was then slowly added dropwise and the solution of this mixture was incubated at 0°C for 0.5 hour. Upon completion of the reaction, the reaction solution was evaporated, and the residue was purified by preparative thin layer chromatography to obtain WX008.

Example 9

Synthesis circuit

Stage 1. Synthesis of WX009-3

WX009-1 (8.03 g, 75.67 mmol, 7.65 mL, 1 eq.), WX009-2 (10 g, 75.67 mmol, 1 eq.) and tetrahydrofuran (100 mL) were added to the reaction flask. . The atmosphere was replaced with nitrogen gas and the solution of this mixture was incubated at 25°C for 4 hours. Upon completion of the reaction, the reaction solution was evaporated to dryness on a rotary evaporator to obtain WX009-3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 10.86 (br s, 1H), 8.00 (s, 1H), 7.59 (br d, J = 6.6 Hz, 2H), 7.31-7.45 ( m, 3H), 1.47 (s, 9H).

Stage 2. Synthesis of WX009-4

WX009-3 (10 g, 45.40 mmol, 1 eq), potassium tert-butoxide (6.11 g, 54.48 mmol, 1.2 eq) and tetrahydrofuran (160 ml) were added to the reaction flask. The atmosphere was replaced with nitrogen gas and then deuterated iodomethane (7.90 g, 54.48 mmol, 3.39 mL, 1.2 eq.) was added slowly dropwise. A solution of this mixture was incubated at 25°C for 16 hours. Upon completion of the reaction, the reaction solution was diluted with water (50 ml) and extracted with ethyl acetate (3×100 ml). The organic phase was washed with saturated NaCl solution (100 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump to obtain WX009-4. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 7.81 (s, 1H), 7.64-7.72 (m, 2H), 7.32-7.46 (m, 3H), 1.50 (s, 9H).

Stage 3. Synthesis of WX009-5

Wet palladium on carbon (2 g, purity 10%) and methanol (100 ml) were added to the reaction flask. The atmosphere was replaced with hydrogen gas and then WX009-4 (10.5 g, 44.25 mmol, 1 eq.) was added. A solution of this mixture was incubated under hydrogen (89.19 mg, 44.25 mmol, 1 eq.) at 50 psi at 50°C for 48 hours. Upon completion of the reaction, the reaction solution was filtered through celite and washed with methanol (2×20 ml). The filtrate was evaporated to dryness under reduced pressure using a water pump to obtain WX009-5. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 4.48 (s, 2H), 1.40 (s, 9H).

Stage 4. Synthesis of WX009-6

WX009-5 (6.6 g, 44.23 mmol, 1 eq.) and a solution of hydrochloric acid in ethyl acetate (4 M, 33.18 ml, 3 eq.) were added to the reaction flask and the solution of this mixture was incubated at 25°C within 16 hours. The reaction solution was evaporated to dryness on a rotary evaporator to obtain WX009-6. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ ppm 4.95 (br s, 3H).

Stage 5. Synthesis of WX009-8

WX009-6 (1.5 g, 17.53 mmol, 1 eq., HCl), WX009-7 (2.51 g, 17.53 mmol, 2.63 ml, 1 eq.), acetic acid were added to the reaction flask acid (2.11 g, 35.07 mmol, 2.01 ml, 2 eq.), magnesium sulfate (5.02 g, 41.73 mmol, 2.38 eq.) and ethanol (75 ml). The atmosphere was replaced with nitrogen gas, and then the solution of this mixture was incubated at 90°C for 2 hours. Upon completion of the reaction, the reaction solution was diluted with saturated aqueous sodium bicarbonate (50 ml) and extracted with dichloromethane (3×50 ml). The organic phase was washed with saturated NaCl solution (50 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C to obtain a crude product, which was purified by column chromatography to obtain WX009-8. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 6.98 (d, J = 1.6 Hz, 1H), 5.24 (d, J = 1.8 Hz, 1H), 5.09 (br s, 2H).

Stage 6. Synthesis of WX009

WX009-8 (13.46 mg, 134.37 µmol, 1.5 eq.), WX006-2 (40 mg, 89.58 µmol, 1 eq.), dichloromethane (0.5 ml) and tetrahydrofuran (0.5 ml). The atmosphere was replaced with nitrogen gas and the mixture was cooled to 0°C. Lithium hexamethyldisilazide (1 M, 188.12 μL, 2.1 eq.) was then slowly added dropwise and the solution of this mixture was incubated at 0°C for 0.5 hour. Upon completion of the reaction, the reaction solution was diluted with saturated aqueous ammonium chloride (5 ml) and extracted with dichloromethane (3×5 ml). The organic phase was washed with saturated NaCl solution (5 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure using a water pump at 45°C, and the residue was purified by preparative thin layer chromatography to obtain WX009.

Example 10

Synthesis circuit

Stage 1. Synthesis of WX010

WX006-2 (30 mg, 67.19 µmol, 1 eq.) and WX010-1 (15.68 mg, 141.09 µmol, 2.1 eq.) were added to a tetrahydrofuran mixture (1.5 ml) into a dry reaction flask ) and dichloromethane (1.5 ml). The atmosphere was replaced with nitrogen gas and the mixture was cooled to 0°C. Lithium hexamethyldisilazide (1 M, 134.37 μL, 2 eq.) was added, the mixture was stirred at 0°C for 0.5 hour, heated to 25°C and stirred for an additional 1.5 hour. Upon completion of the reaction, the reaction solution was diluted with water (10 ml) and extracted with dichloromethane (3×10 ml). The layers were separated. The organic phase was then collected, which was successively washed with saturated NaCl solution (3×10 ml), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by preparative thin layer chromatography to give WX010.

Example 11

Synthesis circuit

Stage 1. Synthesis of WX011-2

WX009-7 (226.22 mg, 1.58 mmol, 237.13 µl, 1 eq.), magnesium sulfate (452.63 mg, 3.76 mmol, 2.38 eq.), acetic acid was added to the dry reaction flask. acid (189.75 mg, 3.16 mmol, 180.72 µl, 2 eq.) and ethanol (2.5 ml). The atmosphere was replaced with nitrogen gas and the mixture was incubated at 25°C for 1 hour. WX011-1 (200 mg, 1.58 mmol, 1 equiv, HCl) was then added and the mixture was incubated at 80°C for 2 hours and at 90°C for an additional 12 hours. After completion of the reaction, the reaction solution was poured into 3 ml of saturated aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane (2×3 ml). The organic phases were combined, washed with 3 ml saturated NaCl solution, dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the crude product. The crude product was purified by thin layer chromatography on a silica gel plate to give WX011-2. ¹ H-NMR (400 MHz, CH ₃ Cl-d): δ (ppm) 7.25 (d, J = 1.8 Hz, 1H), 5.50 (d, J = 2.0 Hz, 1H), 4.15-4.22 (m, 2H ), 3.83-4.11 (m, 2H), 3.68-3.73 (m, 2H), 3.34 (s, 3H).

Stage 2. Synthesis of WX011

WX006-2 (40 mg, 89.58 µmol, 1 eq.) and WX011-2 (26.56 mg, 188.12 µmol, 2.1 eq.) were added to a dry reaction flask into a mixture of tetrahydrofuran (2 ml) and dichloromethane (2 ml). The atmosphere was replaced with nitrogen gas and the mixture was cooled to 0°C. Lithium hexamethyldisilazide (1 M, 179.16 μL, 2 eq.) was added, the mixture was stirred at 0°C for 0.5 hour, heated to 25°C and stirred for an additional 1 hour. Upon completion of the reaction, the reaction solution was diluted with water (5 ml) and extracted with dichloromethane (3×5 ml). The layers were separated. The organic phase was then collected, which was successively washed with saturated NaCl solution (3×5 ml), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by preparative thin layer chromatography to give WX011.

Example 12

Synthesis circuit

Stage 1. Synthesis of WX012

WX006-2 (40 mg, 89.58 µmol, 1 eq), WX012-1 (16.55 mg, 134.37 µmol, 1.5 eq), dichloromethane (1 ml) and tetrahydrofuran ( 1 ml). The atmosphere was replaced with nitrogen gas and the mixture was cooled to 0°C. Lithium hexamethyldisilazide (1 M, 188.12 μL, 2.1 eq.) was slowly added dropwise and the solution of this mixture was incubated at 0°C for 0.5 hour. Upon completion of the reaction, the reaction solution was evaporated, and the residue was purified by preparative thin layer chromatography to obtain WX012.

Example 13

Synthesis circuit

Stage 1. Synthesis of WX013-2

WX013-1 (800 mg, 4.70 mmol, 1 eq.) and lithium hydroxide monohydrate (986.42 mg, 23.51 mmol, 5 eq.) were dissolved in a mixture of water (8 mL) and tetrahydrofuran (8 mL). The mixture was stirred at 25°C for 2 hours. Upon completion of the reaction, the reaction solution was directly evaporated to dryness on a rotary evaporator and then extracted with water (10 ml) and dichloromethane (3×10 ml). The organic phase was collected, washed with saturated NaCl solution, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness on a rotary evaporator to obtain WX013-2.

Stage 2. Synthesis of WX013-3

WX013-2 (500 mg, 3.20 mmol, 1 eq), diphenylphosphoryl azide (889.00 mg, 3.23 mmol, 700 μl, 1.01 eq) and triethylamine (1.45 g, 14.37) were dissolved mmol, 2 ml, 4.49 eq.) in tert-butanol (10 ml). The atmosphere was replaced with nitrogen gas three times, and then the mixture was stirred at 85°C for 16 hours. Upon completion of the reaction, the reaction solution was evaporated to dryness on a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography to obtain WX013-3. ¹ H-NMR (400 MHz, CD ₃ Cl): δ ppm 1.50 (s, 9H), 3.58 (s, 3H), 3.84 (s, 3H), 5.61 (br s, 1H).

Stage 3. Synthesis of WX013-4

WX013-3 (520 mg, 2.29 mmol, 1 eq.) and a solution of hydrochloric acid in ethyl acetate (4 M, 5 ml, 8.74 eq.) were added to the reaction flask and the mixture was stirred at 25°C for 2 hours . After completion of the reaction, the reaction solution was extracted with water (10 ml). The aqueous phase was collected and then evaporated to dryness on a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography to obtain WX013-4. ¹ H-NMR (400 MHz, CD ₃ Cl): δ ppm 3.53 (s, 3H), 3.82 (s, 3H), 5.01 (s, 1H).

Stage 4. Synthesis of WX013

WX006-2 (50 mg, 111.98 µmol, 1 eq), WX013-4 (29.90 mg, 235.15 µmol, 2.1 eq), dichloromethane (1 ml) and tetrahydrofuran were added to the dry reaction flask (1 ml). The atmosphere was replaced with nitrogen gas and the mixture was cooled to 0°C. Lithium hexamethyldisilazide (1 M, 223.96 μL, 2 eq.) was added dropwise. The mixture was incubated at 0°C for 0.5 hour and at 25°C for another 1 hour. Upon completion of the reaction, the reaction solution was quenched with 10 ml of water and extracted with 20 ml of dichloromethane. The layers were separated. The organic phase was collected and the aqueous phase was extracted with dichloromethane (3×20 ml). The organic phases were combined and washed successively with saturated NaCl solution (3×20 ml), dried over anhydrous sodium sulfate and evaporated under reduced pressure. Upon completion of evaporation, the residue was purified by preparative thin layer chromatography to obtain WX013.

Table 1 shows the ¹ H-NMR spectra and mass spectra from each example.

Table 1 Example Compound NMR MS m/z 1 WX001 ^1H -NMR (400 MHz, DMSO- _d6 ) δ ppm 9.82 (br s, 1H), 8.70 (d, J = 4.9 Hz, 1H), 7.56 (d, J = 5 ,0 Hz, 1H), 7.46 (s, 1H), 7.41 (d, J = 1.6 Hz, 1H), 7.36 (s, 3H), 6.35 (s, 1H), 4.72 (s, 2H), 3.74 (s, 3H), 1.53 (s, 6H). 466
[M+H] ⁺ 2 WX002 ^1H -NMR (400 MHz, DMSO-d6) δ (ppm) 9.78 (br s, 1H), 8.68 (d, J = 4.9 Hz, 1H), 7.54 (d, J = 5.0 Hz, 1H), 7.31-7.42 (m, 2H), 7.16-7.24 (m, 2H), 7.07 (td, J = 8.6, 2.2 Hz , 1H), 6.33 (d, J = 1.6 Hz, 1H), 4.71 (s, 2H), 3.72 (s, 3H), 1.52 (s, 6H). 450
[M+H] ⁺ 3 WX003 ^1H -NMR (400 MHz, DMSO-d6) δ (ppm) 9.80 (br s, 1H), 8.69 (d, J = 5.0 Hz, 1H), 7.54 (d, J = 5.0 Hz, 1H), 7.32-7.50 (m, 3H), 7.25 (m, 1H), 6.33 (d, J = 1.4 Hz, 1H), 4.68 ( s, 2H), 3.72 (s, 3H), 1.52 (s, 6H). 468
[M+H] ⁺ 4 WX004 ¹ H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.52 (br d, J = 4.8 Hz, 1H), 7.61 (br s, 1H), 7.33-7.48 (m, 1H), 7.18-7.30 (m, 3H), 7.04-7.15 (m, 1H), 4.73 (s, 2H), 3.83-4.05 (m , 3H), 3.43 (m, 2H), 1.89 (br s, 2H), 1.49-1.64 (m, 8H). 454
[M+H] ⁺ 5 WX005 ^1H -NMR (400 MHz, DMSO-d6) δ (ppm) 9.61 (s, 1H), 8.60 (s, 1H), 7.46 (s, 1H), 7.39 (d, J = 1.9 Hz, 1H), 7.30-7.38 (m, 3H), 6.34 (d, J = 1.8 Hz, 1H), 4.71 (s, 2H), 3.72 (s, 3H), 2.59 (s, 3H), 1.51 (s, 6H). 480
[M+1] ⁺ 6 WX006 ¹ H-NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.60 (s, 1H), 7.40-7.33 (m, 2H), 7.25-7, 18 (m, 2H), 7.04-7.11 (m, 1H), 6.31-6.35 (m, 1H), 4.71 (s, 2H), 3.72 (s, 3H) , 2.59 (s, 3H), 1.52 (s, 6H). 464
[M+1] ⁺ 7 WX007 ¹ H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.51 (br d, J = 4.8 Hz, 1H), 7.51-7.64 (m, 1H), 7.46 ( s, 1H), 7.30-7.40 (m, 3H), 7.26 (d, J = 4.6 Hz, 1H), 4.71 (s, 2H), 3.85-4.04 (m, 3H), 3.42 (br t, J = 11.2 Hz, 2H), 1.89 (br d, J = 4.5 Hz, 2H), 1.48-1.64 (m, 8H). 470
[M+1] ⁺ 8 WX008 ¹ H-NMR (400 MHz, DMSO-d6) δ (ppm) 10.04 (br s, 1H), 8.65 (s, 1H), 7.59-7.94 (m, 2H), 7, 31-7.42 (m, 1H), 7.16-7.27 (m, 2H), 7.08 (br t, J = 8.6 Hz, 1H), 6.54 (s, 1H), 4.72 (s, 2H), 2.61 (s, 3H), 1.52 (s, 6H). 500
[M+1] ⁺ 9 WX009 ¹ H-NMR (400 MHz, DMSO-d6) δ (ppm) 9.62 (s, 1H), 8.60 (s, 1H), 7.32-7.41 (m, 2H), 7.17 -7.26 (m, 2H), 7.07 (td, J = 8.5, 2.4 Hz, 1H), 6.34 (d, J = 1.8 Hz, 1H), 4.71 ( s, 2H), 2.59 (s, 3H), 1.51 (s, 6H). 467
[M+1] ⁺ 10 WX010 ¹ H-NMR (400 MHz, DMSO-d6) δ (ppm) 9.52 (s, 1H), 8.59 (s, 1H), 7.31-7.42 (m, 1H), 7.17 -7.27 (m, 2H), 7.02-7.13 (m, 1H), 6.08 (s, 1H), 4.72 (s, 2H), 3.63 (s, 3H), 2.59 (s, 3H), 2.15 (s, 3H), 1.53 (s, 6H). 478
[M+1] ⁺ eleven WX011 ¹ H-NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.60 (s, 1H), 7.43 (d, J=2.0 Hz, 1H), 7.40 - 7.32 (m, 1H), 7.25-7.16 (m, 2H), 7.07 (dt, J=2.0, 8.6 Hz, 1H), 6.40 (d, J =1.8 Hz, 1H), 4.71 (s, 2H), 4.25 (t, J=5.6 Hz, 2H), 3.67 (t, J=5.7 Hz, 2H), 3.21 (s, 3H), 2.59 (s, 3H), 1.51 (s, 6H). 508
[M+1] ⁺ 12 WX012 ¹ H-NMR (400 MHz, DMSO-d6) δ ppm 0.88-0.95 (m, 2H), 0.96-1.03 (m, 2H), 1.51 (s, 6H), 2 .59 (s, 3H), 3.54 (tt, J=7.24, 3.71 Hz, 1H), 4.71 (s, 2H), 6.35 (d, J=1.63 Hz, 1H), 7.02-7.12 (m, 1H), 7.17-7.26 (m, 2H), 7.32-7.41 (m, 2H), 8.61 (s, 1H) , 9.54 (s, 1H). 490
[M+1] ⁺ 13 WX013 ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.66 (s, 1H), 8.61 (s, 1H), 7.31-7.43 (m, 1H), 7, 15-7.27 (m, 2H), 7.03-7.12 (m, 1H), 5.81 (s, 1H), 4.72 (s, 2H), 3.75 (s, 3H) , 3.57 (s, 3H), 2.59 (s, 3H), 1.52 (s, 6H). 494
[M+1] ⁺

Test example 1. In vitro kinase activity assay

1. Purpose of the study

Measuring the ability of compounds to inhibit ERK2 kinase activity.

2. Analysis buffer

20 mM Hepes (pH 7.5), 10 mM MgCl ₂ , 1 mM ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg/ml bovine serum albumin (BSA), 0.1 mM Na ₃ VO ₄ , 2 mM dithiothreitol (DTT), 1% DMSO.

3. Processing connections

The test compounds were dissolved in 100% DMSO to obtain stock solutions at a certain concentration. Serial dilutions of compounds were made in DMSO solution using the Integra Viaflo Assist smart pipette.

4. Research methodology

1) Prepare MBP substrate in freshly prepared reaction buffer.

2) ERK2 kinase was added to this MBP solution and mixed gently.

3) Compounds dissolved in 100% DMSO were added to the kinase reaction system using ultrasonic technology (Echo550; range: nanoliters), and the mixtures were incubated at room temperature for 20 minutes.

4) ³³ P-ATP (specific concentration: 10 µCi/‌µl) was added to the reaction system and the reaction was started immediately.

5) Incubate the mixtures at room temperature for 2 hours.

6) The level of radioactivity was determined by the filter binding method.

7) ERK2 kinase activity was calculated as the ratio of the kinase activity remaining in the test sample to the kinase activity in the control group (DMSO). Curves were generated using Prism (GraphPad software) and IC ₅₀ values were calculated.

5. The results of the study are presented in Table 2.

Table 2. Results of in vitro kinase activity assay Compound ERK2 IC ₅₀ (nM) WX001 0.24 WX002 0.35 WX003 0.37 WX004 0.25 WX005 0.05 WX006 0.30 WX007 0.39 WX008 0.52 WX009 0.64 WX0010 0.14 WX0011 0.98 WX0012 0.48 WX0013 0.19

Conclusion. The compounds of the present invention exhibit excellent ERK2 kinase inhibitory activity.

Test example 2: In vitro cell proliferation inhibition assay

1. Purpose of the study

Measuring the ability of compounds to inhibit proliferation of HT29 cancer cells.

2. Processing connections

The test compounds were dissolved in 100% DMSO to obtain 10 mM stock solutions.

3. Methodology and stages of research

1) Turn on the UV light in the biosafety cabin and start counting 30 minutes.

2) RPMI 1640 medium and trypsin were heated in a water bath at 37°C.

3) Upon completion of UV irradiation, the biological safety cabin was opened. Warmed medium, trypsin and phosphate buffered saline (PBS), etc. wiped with alcohol and placed in a biological safety cabin.

4) Remove HT29 cells from the incubator and remove old media in a biosafety cabin. 10 ml PBS was added. The mixture was shaken gently and then the PBS was removed.

5) Added 1.5 ml of warmed 0.25% trypsin. The culture vessel was shaken horizontally so that the trypsin evenly coated the cells at the bottom and placed in the incubator for 2 minutes.

6) Cell splitting was stopped by adding complete medium and the cell suspension was brought to homogeneity with a pipette and cells were counted.

7) Based on the results of cell counting, the density of the cell suspension was adjusted to 1500 cells per well and the cell suspension was seeded at 50 μl per well.

8) Serial dilutions of stock solutions of compounds in DMSO solution were made and compounds were added to the plate using Tecan.

9) Equilibrate the plate with cells and added compounds and CellTiterGlo at room temperature, and then 25 µl CellTiterGlo is added to each well. The plate with cells was shaken for 1-2 minutes and then left for 10 minutes. Then the signal values were determined. Data were analyzed using XL-Fit and the IC ₅₀ for each compound was calculated.

4. The results of the study are presented in Table 3.

Table 3. Results of in vitro cell activity assay Compound HT29 IC ₅₀ (nM) WX001 14 WX002 24 WX003 27 WX004 35 WX005 8 WX006 7 WX007 26 WX008 58 WX009 6 WX0010 27 WX0011 26 WX0012 37 WX0013 18

Conclusion. The compounds of the present invention exhibit excellent HT29 cell proliferation inhibitory activity.

Test Example 3: In Vivo Study of DMPK

In vivo study of DMPK in mice.

1. Purpose of the study

Determination of blood concentrations of compounds and assessment of pharmacokinetic behavior after single administration using female BALB/c mice as study animals.

2. Research procedure

Eight healthy adult female BALB/c mice were recruited, with 4 mice in the intravenous group and 4 mice in the oral group. The vehicle in the IV group was 5% DMSO + 95% (20% HP-β-CD). The test compounds were mixed with an appropriate amount of vehicle for intravenous injection, vibrated and sonicated to obtain a clear solution of 0.5 mg/ml. The clear solution was filtered through a microporous membrane and prepared for use. The vehicle in the oral group was 5% DMSO + 95% (20% HP-β-CD). The compounds under study were mixed with the carrier, treated with a vibrating mixer and ultrasound, obtaining a solution of 0.3 mg/ml. Mice were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected over time. Plasma was isolated. Drug concentrations were analyzed by LC-MS/MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

Notes DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

3. The results of the study are presented in table 4.

Table 4. Results of PK analysis of compounds Compound C _max
(nM) F% Oral DNAUC
(nM h/mpk) Vd _ss
(l/kg) Cl
(ml/min/kg) _T½
(h) WX001 3355 86% 2153 1.1 14.3 0.9 WX005 1029 n/a 468 n/a n/a n/a WX006 1035 34% 530 1.7 23.0 1.0 WX009 1170 67% 820 1.7 28.0 1.7

Notes. C _max - maximum concentration; F% - oral bioavailability; DNAUC = AUC _PO /dose, where AUC _PO is AUC after oral administration, and dose is the dose of the drug; Vd _ss - volume of distribution; Cl - clearance rate; T _½ - half-life; and NA means the study was not conducted.

Conclusion. The compounds of the present invention exhibit excellent oral exposure and bioavailability.

Test Example 4: In Vivo Efficacy Analysis in H358 Mouse Model

1. Purpose of the study

Evaluation of the antitumor effect of WX006 in a model of subcutaneous xenografts of human non-small cell lung cancer H358 cells in nude mice.

2. Animals for research

Type: mouse; line: BALB/c nude mice; age: 6-7 weeks; gender: females; weight: 20 grams; supplier: Shanghai Sippe-Bk Lab Animal Co., Ltd.; animal certificate No. 20180006017149.

3. Conditions of detention

Animals were kept in IVC (independent air supply, constant temperature and humidity) cages (4 animals per cage) in an SPF class animal vivarium at a temperature of 20-26°C and a humidity of 40-70%. The cages were made of polycarbonate and had a volume of 300 mm × 180 mm × 150 mm. The bedding material was corn cob core and was changed once a week. The study animals had free access to food (irradiation-sterilized, dry food in granules) throughout the study. The animals had free access to drinking sterilized water. Cage identification: The animal information card for each cage must indicate the number, sex, strain, date of entry into the cage, study number, administration regimen, group and start date of the study. Study animals were identified by ear tags.

4. Research procedure

1) Cells and their cultivation. Human non-small cell lung cancer H358 cells were cultured in a monolayer in vitro. Culture conditions: 1640 medium plus 10% fetal calf serum and 37°C incubator with 5% CO ₂ . Usually, for subculture, cells were treated with trypsin with EDTA three times a week. When the cell confluency reached 80%-90% and their number met the requirement, the cells were collected, counted and plated.

2) Inoculation of tumor tissue and division into groups. 0.1 ml (5×10 ⁵ ) of H358 cells were inoculated subcutaneously into the right axilla of each mouse. When the average tumor volume reached 100 mm ³ , the animals were randomly divided into 2 groups and administration began. The scheme of distribution into groups and administration during the study is presented in Table 5.

Table 5. Scheme of grouping of animals and administration during the study Group Number of animals A drug Dosage (mg/kg) Administration cycle Method and frequency of administration 1 6 control for solvent (carrier) - 28 days orally (PO), once daily (QD) 2 6 WX006 thirty 28 days orally (PO), once daily (QD)

3) Daily observation of the studied animals. The development of this study method and its modifications was carried out with the approval of the Institutional Animal Care and Use Committee (IACUC). The use and welfare of study animals were monitored in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. Animals were examined daily for health and mortality. Routine examinations included monitoring tumor growth and the effect of drug administration on the animals' daily behavior such as behavioral activity, food and water intake (visual inspection only), weight changes (weight measured twice a week), external signs or other abnormalities. Animal deaths and side effects in each group were noted based on the number of animals in each group.

4) Formulations of the studied compounds. Vehicle group: 5% DMSO + 95% (20% HP-β-CD). Test compound group: weighed the test compound in a prescription bottle. An appropriate volume of DMSO was added and the mixture was then stirred until a clear solution was obtained. An appropriate volume of 20% HP-β-CD was added and the mixture was then stirred until a homogeneous suspension was obtained. Compounds were prepared every three days.

5) Measurement of tumors and parameters during the study. The diameter of the tumors was measured twice a week using a caliper. The volume of tumors was calculated using the formula: TV=1/2×a×b ² , where a and b are the long and short diameters of the tumor, respectively. The effectiveness of tumor inhibition by compounds was determined by TGI (%). TGI (%) reflects the degree of tumor growth inhibition. TGI (%) was calculated as follows: TGI (%) = {[1 - (average volume of tumors at the end of treatment in the experimental group - average volume of tumors at the beginning of administration in the same experimental group)]/(average volume of tumors at the end of treatment in solvent control group - the average volume of tumors at the beginning of treatment in the solvent control group)}×100%.

5. Research results

1) As can be seen from table 6 and fig. 1, in a model of subcutaneous xenografts of human non-small cell lung cancer H358 cells in nude mice, when administered orally before day 28, WX006 at a dose of 30 mg/kg had a significant inhibitory effect on tumor growth with a TGI value of 94%.

2) The body weight of the studied animals was used as an indicator for indirectly determining the toxicity of the drug. As can be seen from Fig. 2, when administered before day 28, all animals in the vehicle control group and the WX006 group had no significant reduction in body weight and no morbidity or mortality was observed.

Table 6. In vivo efficacy results in the H358 mouse model. A drug TGI WX006 (30 mg/kg, PO, QD) 94%

6. Conclusion. WX006 can significantly inhibit tumor growth at the administered dose. When administered, there is no significant reduction in the body weight of animals; the tolerability is good.

Claims (30)

1. The compound represented by formula (III), or a pharmaceutically acceptable salt thereof: where R ₁ means H, C _1-3 -alkyl or C _3-5 -cycloalkyl, and C _1-3 -alkyl or C _3-5 -cycloalkyl is optionally substituted with 1, 2 or 3 R _a ; R ₂ and R ₃ are each independently C _1-3 -alkyl; R ₄ is selected from F and Cl; R ₅ is selected from C _1-3 -alkyl; m is 0, 1 or 2; n is 0, 1 or 2; ring A is pyrazolyl or tetrahydropyranyl, wherein the pyrazolyl or tetrahydropyranyl is optionally substituted with 1, 2 or 3 R _d ; R _a is each independently D, F or OCH ₃ ; R _d is selected from CH ₃ or OCH ₃ . 2. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ₁ is H, CH ₃ or cyclopropyl, wherein CH ₃ or cyclopropyl is optionally substituted with 1, 2 or 3 R _a . 3. The compound or its pharmaceutically acceptable salt according to claim 2, wherein R ₁ means H, CH ₃ , CHF ₂ , CD ₃ , CH ₂ CH ₂ OCH ₃ or cyclopropyl. 4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R ₂ and R ₃ are each independently selected from CH ₃ or CH ₂ CH ₃ . 5. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R ₂ and R ₃ are each independently CH ₃ . 6. The compound or its pharmaceutically acceptable salt according to claim 1, wherein R ₄ means F. 7. The compound or its pharmaceutically acceptable salt according to claim 1, wherein R ₄ means Cl. 8. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ₅ is CH ₃ . 9. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein ring A represents , or , and , or optionally substituted with 1, 2 or 3 R _d . 10. The compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein ring A represents , , or . 11. The compound or its pharmaceutically acceptable salt according to claim 1, wherein the structural group means , or . 12. The compound or its pharmaceutically acceptable salt according to claim 11, wherein the structural group means , or . 13. The compound or its pharmaceutically acceptable salt according to any one of paragraphs. 1-8, wherein the connection is selected from: where R ₁ is already defined in any of paragraphs. 1-3; R ₂ and R ₃ are defined in any of paragraphs. 1, 4, 5; R ₄ is defined in any of paragraphs. 1, 6, 7; R ₅ is defined in paragraph 1 or 8; and m - in paragraph 1. 14. A compound represented by the following formula, or a pharmaceutically acceptable salt thereof: , ,
, ,
, , , , , , , , . 15. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-14 in the manufacture of a medicament for the treatment of diseases associated with ERK. 16. Use according to claim 15, characterized in that the drug for the treatment of diseases associated with ERK is a drug for the treatment of solid tumors.