CN113912591A - Biaryl compounds - Google Patents

Abstract

The invention discloses biaryl compounds, and particularly discloses a compound shown as a formula (I) and pharmaceutically acceptable salts thereof.

Description

Biaryl compounds

The following priority is claimed in the present application:

CN202010652149.3, filing date 2020, month 07, 08;

PCT/CN2020/133933, filing date 2020, 12/04.

Technical Field

The present invention discloses biaryl compounds, their pharmaceutically acceptable salts, which are inhibitors of Pan-RAF kinase and their use in the treatment of diseases associated with cancer.

Background

The mitogen-activated protein kinase (MAPK) pathway is an important signal transduction pathway in cells, and the pathway transfers signals from the outside of cells into cell nuclei through specific cascade phosphorylation of RAS/RAF/MEK/ERK, finally leads to activation of specific genes, and causes cell proliferation, apoptosis or differentiation. This pathway overactivation is closely related to the development of multiple tumors. RAF is a serine/threonine protein kinase that is important in the RAS/RAF/MEK/ERK signaling pathway, is located downstream of RAS, and can be activated by RAS. The RAF family includes three subtypes ARAF, BRAF and CRAF (RAF-1), and has high homology and similar structural domains. Wild-type RAF is capable of producing three subtypes of homo-or heterodimers. ARAF and CRAF mutations occur less frequently and BRAF mutation rates are higher. BRAF mutations are present in about 5% to 10% of malignant tumors, including 66% of melanoma patients. RAF kinase as a key signal protein downstream of RAS has important research significance in treating RAS mutant gene mutant tumors. Through inhibiting RAF kinase, MAPK signal transduction is regulated, so that the tumor cell proliferation promoter plays a role in RAS mutant tumor cell proliferation. Therefore, RAF kinase has become an important target for clinical treatment of tumors.

The first generation BRAF kinase inhibitors Vemurafenib and Dabrafinib have been FDA approved for the development of B-Raf^V600ETreatment of mutant cancers. Although Vemurafenib and Dabrafinib are in B-Raf^V600EThe treatment of mutant melanoma shows promising efficacy but still has certain limitations. Most patients using both drugs initially have a tumor that shrinks but recurs within a year (acquired resistance); the primary mechanism for developing this resistance is the reactivation of the MAPK signaling pathway. Research finds that on the basis of V600E mutation of BRAF, NRAS mutation can cause MAPK pathway activation in the presence of an inhibitor to generate drug resistance. Mutant N-Ras promotes B-Raf^V600EAnd C-Raf form homo-or heterodimers. The inhibitor binds to one monomer of the dimer, reducing the affinity of the drug for the other monomer, and promoting phosphorylation of the monomer without the effect of the inhibitor, resulting in activation of MEK. At present, the main method for clinically overcoming drug resistance caused by reactivation of MAPK pathway is to block 2 key sites of MAPK pathway by combined use of Raf and MEK inhibitors so as to delay drug resistance generation. In addition, development of a new generation of pan-Raf inhibitors, which can inhibit the activity of dimers and block abnormal activation of the pathway (paradoxcal activation), thereby reducing drug resistance, to overcome drug resistance and to expand the scope of clinical application is under development. The pan-Raf dimer inhibitors in clinical research mainly comprise HM95573, TAK-580, BGB-283, LXH254, LY3009120 and the like, and the development of the novel RAF inhibitors is expected to overcome the drug resistance of the first-generation inhibitors and further expand the clinical application.

Disclosure of Invention

The present invention provides a compound of formula (I), N- (3- (2- ((1R,5S) -3-oxabicyclo [3.1.0] hex-1-yl) -6- (2-hydroxyethoxy) pyridin-4-yl) -4-methylphenyl) -2- (trifluoromethyl) isonicotinamide or a pharmaceutically acceptable salt thereof,

the present invention also provides compounds of the formula:

the invention also provides

In the preparation of compounds of formula (I)

The use of (1).

The invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a RAF kinase inhibitor.

The invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.

In some embodiments of the invention, the cancer is colon cancer or lung cancer.

Technical effects

The compound has good drug properties, good RAF enzyme inhibition activity and various cell antiproliferative activities, and simultaneously has good in-vivo drug effect and good safety. Is expected to solve the current BRAF^V600EResistance to mutant cancer treatments, and provide effective treatments for RAS mutant-type cancers.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amines or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid, either in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid and the like. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.

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

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

Unless otherwise indicated, the term "cis-trans isomer" or "geometric isomer" results from the inability of a double bond or a single bond to rotate freely within a ring-forming carbon atom.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecules have two or more chiral centers and a non-mirror image relationship between the molecules.

Unless otherwise indicated, "(+)" means dextrorotation, "(-) -means levorotation," (±) "means racemization.

Using solid wedge keys, unless otherwise indicated

And wedge dotted bond

Showing the absolute configuration of a solid centre, by means of straight solid keys

And straight dotted line bond

Showing the relative configuration of the centres of solids, by wavy lines

Shows a solid wedge lineKey with a key body

Or wedge dotted bond

Or by wavy lines

Indicating straight solid-line keys

And straight dotted line bond

Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be rapidly interconverted. If tautomers are possible (e.g., in solution), then the chemical equilibrium of the tautomers can be reached. For example, proton tautomers (prototropic tautomers), also known as proton transfer tautomers (prototropic tautomers), include interconversions by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valencetatomer) include interconversion by recombination of some of the bonding electrons. A specific example of where keto-enol tautomerism is the interconversion between two tautomers of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "isomer enriched", "enantiomer enriched" or "enantiomeric enrichment" refer to a content of one isomer or enantiomer of less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the term "isomeric excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80%.

Optically active (R) -and (S) -isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one of the enantiomers of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines).

The term "protecting group" includes, but is not limited to, "amino protecting group," hydroxyl protecting group, "or" thiol protecting group. The term "amino protecting group" refers to a protecting group suitable for use in preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: a formyl group; acyl, for example alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr), 1-bis- (4' -methoxyphenyl) methyl; silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for use in preventing side reactions of a hydroxy group. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups, such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.

The compounds of the present invention may be structurally confirmed by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of the compound, the absolute configuration may be confirmed by means of conventional techniques in the art. For example, single crystal X-ray diffraction method (SXRD), diffraction intensity data of the cultured single crystal is collected by a Bruker D8 venture diffractometer, a light source is CuK alpha radiation, and a scanning mode is as follows:

after scanning and collecting relevant data, the crystal structure is further analyzed by a direct method (Shelxs97), so that the absolute configuration can be confirmed. The absolute configuration of the compound can also be confirmed by the chiral structure of the starting material and the reaction mechanism of the asymmetric synthesis.

The solvent used in the present invention can be commercially available.

The invention employs the following abbreviations: HATU stands for 2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate; HOBt represents 1-hydroxybenzotriazole; t is₃P represents tri-n-propyl cyclic phosphoric anhydride; pd₂dba₃Represents tris-diphenylpropanone-dipalladium; ruphos represents 2-dicyclohexyl-phosphorus-2 ',6' -diisopropoxy-1, 1' -biphenyl; brettphos for (2-dicyclohexylphosphine-3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl; TBAF for tetrabutylammonium fluoride, [ Ir (COD) OMe]₂Represents cyclooctadiene methoxy iridium dimer; tmphen represents 3,4,7, 8-tetramethyl-1, 10 phenanthroline; dtbpy represents 4,4 '-di-tert-butyl-2, 2' -bipyridine. Pd (dppf) Cl₂Represents diphenylphosphinoferrocene palladium dichloride; pd (dppf) Cl₂DCM for diphenylphosphinoferrocene palladium dichloride complex with dichloromethane; DIAD represents diisopropyl azodicarboxylate; DIPEA or DIEA for diisopropylethylamine; PPh₃Represents triphenylphosphine; TFA represents trifluoroacetic acid; THP represents 2-tetrahydropyran; OTHP represents 2-oxotetrahydropyran; TBSCl represents tert-butyldimethylsilyl chloride; TBS represents tert-butyldimethylsilyl group; OTBS stands for tert-butyl dimethylsiloxy; DMF represents N, N-dimethylformamide; DMSO represents dimethyl sulfoxide; EA represents ethyl acetate; PE represents petroleum ether; EtOH stands for ethanol; MeOH represents methanol; DME represents ethylene glycol dimethyl ether; DCM represents dichloromethane; THF represents tetrahydrofuran; MeCN represents acetonitrile; NMP stands for N-methylpyrrolidone; PE/EA represents the volume ratio of petroleum ether to ethyl acetate; DCM/MeOH represents the volume ratio of dichloromethane to methanol; HCOOH-MeCN-H₂O represents a separation system of formic acid-acetonitrile-water; IPA stands for isopropanol; me represents methyl, OMs represents methanesulfonyloxy.

Drawings

FIG. 1 is a schematic representation of a crystal of a compound of formula (I).

FIG. 2 is the tumor growth curve of mouse with tumor of human colon cancer Colo-205 cell subcutaneous xenograft tumor model.

FIG. 3 is the body weight change curve of mouse with human colon cancer Colo-205 cell subcutaneous xenograft tumor model.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the invention without departing from the spirit and scope of the invention.

Example 1: preparation of Compounds of formula (I)

Scheme 1:

compound 2

To a solution of compound 1(10 g, 51.96 mmol) in dichloromethane (100mL) at-78 deg.C was added n-butyllithium (23mL, 2.5M), and the mixture was stirred for 30 minutes. To this mixture was added 1a (4.92 g, 57.16 mmol) at-78 ℃ and the mixture was stirred at 25 ℃ for 0.5 hour. To the reaction mixture was added a saturated ammonium chloride solution (40mL), and extraction was performed with dichloromethane (40mL × 2), and the combined extracts were dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (PE/EA ═ 10/1 to 5/1, V/V) to obtain compound 2.¹HNMR(400MHz,CDCl₃)δ7.72(t,J＝7.8Hz,1H),7.47(d,J＝7.2Hz,1H),7.28(d,J＝7.2Hz,1H),4.35(s,1H),4.24-4.14(m,2H),4.05-4.00(m,1H),3.96-3.92(m,1H),2.45(td,J＝8.8,13.0Hz,1H),2.32-2.22(m,1H)。

Compound 3

To a solution of compound 2(6 g, 30.06 mmol) in toluene (80mL) was added p-toluenesulfonic acid (11.5 g, 60.46 mmol), and the mixture was stirred for 16 hours while warming to 110 ℃. To the reaction mixture were added a saturated sodium bicarbonate solution (40mL) and ethyl acetate (100mL), and the mixture was separated, washed with a saturated sodium bicarbonate solution (50mL × 2) with a camera, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (PE/EA ═ 50/1 to 20/1, V/V) to obtain compound 3.¹HNMR(400MHz,CDCl₃)δ7.64(t,J＝7.8Hz,1H),7.23(dd,J＝5.2,7.8Hz,2H),6.68(quin,J＝2.0Hz,1H),5.08(dt,J＝2.0,5.0Hz,2H),4.91(dt,J＝2.0,5.0Hz,2H)。

Compound 4

To a mixture of potassium tert-butoxide (3.71 g, 33.04 mmol), compound 2a (7.3 g, 33.17 mmol) was added DMSO (50 mL). The reaction mixture was stirred at 25 ℃ under nitrogenStirring for 1 hour. To the reaction was added a solution of 3(1 g, 5.51 mmol) in DMSO (5mL), and the reaction mixture was heated to 70 ℃ and stirred for 6 hours. Water (180mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 × 3mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (PE/EA ═ 20/1, V/V) to give compound 4.¹HNMR(400MHz,CDCl₃)δ7.46(t,J＝7.8Hz,1H),7.05(d,J＝7.8Hz,1H),6.88(d,J＝7.6Hz,1H),4.11-4.04(m,2H),3.89-3.78(m,2H),2.08(ddd,J＝2.8,5.2,8.0Hz,1H),1.32(dd,J＝4.4,8.0Hz,1H),1.07(t,J＝4.6Hz,1H)。

Compound 5

Compound 3a (1.08 g, 4.25 mmol), [ Ir (COD) OMe]₂(70 mg, 105.60 micromoles) and tmphen (50 mg, 211.59 micromoles) were placed in methyl tertiary-butyl ether (20 mL). The reaction mixture was stirred under nitrogen with 4(770 mg, 3.94 mmol) added and heated to 80 ℃ for 3 hours. Filtering the reaction solution, and concentrating the filtrate to obtain a crude product of the compound 5. MS (ESI) M/z240.3[ M-84+2H]⁺。

Compound 6

Compound 5(1.2 g, 3.73 mmol), 4a (1.4 g, 3.90 mmol) and Pd (dppf) Cl₂DCM (300 mg, 367.36 micromoles) and sodium carbonate (800 mg, 7.55 mmol) in dioxane (50mL) and water (10 mL). The reaction mixture was heated to 100 ℃ and stirred for 3 hours under nitrogen. The reaction mixture was filtered, the filtrate was concentrated, ethyl acetate (20mL) and water (10mL) were added, the layers were separated, the aqueous layer was extracted with ethyl acetate (10mL × 3), the combined organic layers were dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (PE/EA ═ 10/1 to 5/1) to give compound 6.¹HNMR(400MHz,CDCl₃)δ8.92(d,J＝5.0Hz,1H),8.20(s,1H),8.11(s,1H),7.94(d,J＝5.0Hz,1H),7.60-7.51(m,2H),7.32(d,J＝7.8Hz,1H),7.10(d,J＝1.2Hz,1H),6.90(d,J＝1.2Hz,1H),4.19-4.14(m,2H),3.95-3.87(m,2H),2.27(s,3H),2.24-2.17(m,1H),1.48-1.41(m,1H),1.20-1.15(m,1H)。

Compound 7

To a solution of compound 6(900 mg, 1.90 mmol) and compound 5a (400 mg, 2.27 mmol) in toluene (30mL) was addedInto Pd₂dba₃(180 mg, 196.57 micromoles), Brettphos (200 mg, 372.60 micromoles) and cesium carbonate (1.26 g, 3.87 mmol). The reaction mixture was heated to 110 ℃ and stirred for 4 hours under nitrogen. The reaction solution was filtered, and the filtrate was concentrated and separated by silica gel column chromatography (PE/EA ═ 20/1 to 6/1) to obtain compound 7.¹HNMR(400MHz,DMSO-d₆)δ10.65(s,1H),8.95(d,J＝5.0Hz,1H),8.31(s,1H),8.14(d,J＝4.6Hz,1H),7.70(dd,J＝2.0,8.2Hz,1H),7.59(d,J＝2.2Hz,1H),7.29(d,J＝8.4Hz,1H),6.68(s,1H),6.49(s,1H),4.31(br d,J＝2.8Hz,1H),4.07-3.97(m,2H),3.90-3.86(m,2H),3.79-3.69(m,2H),2.17(s,3H),2.15-2.09(m,1H),1.33(dd,J＝3.8,7.8Hz,1H),1.13(t,J＝7.2Hz,1H),0.96-0.91(m,1H),0.80(s,9H),0.00(s,6H)。

Compound 8

To compound 7(300 mg, 488.81 micromoles) in tetrahydrofuran (10mL) was added hydrochloric acid (1mL, 4M), and the reaction was stirred at 25 ℃ for 1 hour. The reaction mixture was diluted with ethyl acetate (20mL), the pH was adjusted to 8 with a saturated solution of sodium hydrogencarbonate, and the mixture was extracted with ethyl acetate (10 × 3mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated, followed by separation on a thin-layer silica gel plate (PE/EA 1/1, V/V) to obtain compound 8.¹HNMR(400MHz,DMSO-d₆)δ10.70(s,1H),9.06-8.95(d,J＝5.0Hz,1H),8.37(s,1H),8.23-8.18(d,J＝4.2Hz,1H),7.77-7.71(dd,J＝2.2,8.4Hz,1H),7.68-7.64(d,J＝2.2Hz,1H),7.39-7.31(d,J＝8.4Hz,1H),6.73(s,1H),6.59-6.54(d,J＝1.0Hz,1H),4.86-4.81(t,J＝5.2Hz,1H),4.35-4.30(t,J＝5.2Hz,2H),4.13-4.03(m,2H),3.83-3.70(m,4H),2.24(s,3H),2.22-2.08(ddd,J＝2.4,5.0,7.8Hz,1H),1.40-1.32(dd,J＝3.6,7.8Hz,1H),1.02-0.96(t,J＝4.4Hz,1H)。MS(ESI)m/z:500.4[M+H]⁺。

A compound of formula (I)

Compound 8 was separated chirally by SFC (chiral column DAICEL CHIRALCEL OJ-H (250mm 30mm,5 μm), mobile phase A: ethanol (containing 0.05% diisopropylethylamine), mobile phase B: carbon dioxide) to give 8A (retention time 1.487min) and the compound of formula (I) (retention time 1.590 min). 8A is an enantiomer of a compound of formula (I).

Compound 8A:¹HNMR(400MHz,DMSO-d₆)δ10.70(s,1H),9.06-8.95(d,J＝5.0Hz,1H),8.37(s,1H),8.23-8.18(d,J＝4.2Hz,1H),7.77-7.71(dd,J＝2.2,8.4Hz,1H),7.68-7.64(d,J＝2.2Hz,1H),7.39-7.31(d,J＝8.4Hz,1H),6.73(s,1H),6.59-6.54(d,J＝1.0Hz,1H),4.86-4.81(t,J＝5.2Hz,1H),4.35-4.30(t,J＝5.2Hz,2H),4.13-4.03(m,2H),3.83-3.70(m,4H),2.24(s,3H),2.22-2.08(ddd,J＝2.4,5.0,7.8Hz,1H),1.40-1.32(dd,J＝3.6,7.8Hz,1H),1.02-0.96(t,J＝4.4Hz,1H)。MS(ESI)m/z:500.4[M+H]⁺，100％(ee％)。

a compound of formula (I):¹HNMR(400MHz,DMSO-d₆)δ10.70(s,1H),9.06-8.95(d,J＝5.0Hz,1H),8.37(s,1H),8.23-8.18(d,J＝4.2Hz,1H),7.77-7.71(dd,J＝2.2,8.4Hz,1H),7.68-7.64(d,J＝2.2Hz,1H),7.39-7.31(d,J＝8.4Hz,1H),6.73(s,1H),6.59-6.54(d,J＝1.0Hz,1H),4.86-4.81(t,J＝5.2Hz,1H),4.35-4.30(t,J＝5.2Hz,2H),4.13-4.03(m,2H),3.83-3.70(m,4H),2.24(s,3H),2.22-2.08(ddd,J＝2.4,5.0,7.8Hz,1H),1.40-1.32(dd,J＝3.6,7.8Hz,1H),1.02-0.96(t,J＝4.4Hz,1H)。MS(ESI)m/z:500.4[M+H]⁺，99.7％(ee％)。

scheme 2:

step 1: compound I-2

N, N-dimethylformamide (12.5 l) was charged into a 50 l reaction vessel at room temperature, and Compound I-1(2.75 kg, 20.91 mol) and Compound I-1-1(3.55 kg, 31.36 mol) were added and stirred until dissolved and clarified. Then, potassium carbonate (7.22 kg, 52.27 mol) was added thereto, and the mixture was heated to 75 to 80 ℃ and stirred for 16 hours. After the reaction is finished, cooling the reaction liquid to 25 ℃; adding water (27 liters) into a reaction kettle, and transferring the reaction solution of about 27 liters into a white barrel of 30 liters for temporary storage; hydrochloric acid solution (6 mol/l, 8.5 l) was slowly added to the remaining 27 l of reaction solution in the kettle, 0.3 l each time, and the pH was adjusted to 3-4. A yellow solid precipitated with a large amount of gas generation and a small exotherm. The solid-liquid mixture was filtered off with suction under reduced pressure and the filter cake was washed three times with 2 liters of water each time. The remaining 27 liters of the reaction solution were treated in the same manner. After the filter cakes are combined, the mixture is dried in vacuum at 45 DEGDrying for 24 hours to obtain the compound I-2.¹HNMR(400MHz,CDCl₃)δ14.32(br s,1H),7.42(dd,J＝7.4,9.0Hz,1H),7.12(d,J＝9.0Hz,1H),6.53(dd,J＝1.4,7.4Hz,1H),4.26-4.18(m,2H),1.28(t,J＝7.2Hz,3H)。

Step 2: compound I-3

Dimethyl sulfoxide (12L) was charged to a 50L reactor at room temperature, compound I-2(3.6 kg, 16.03 mol) was added, and the internal temperature was raised to 70 ℃. After the reaction solution was clarified, a solution of sodium chloride (1.87 kg, 32.05 mol) in water (6 l) was added, the internal temperature was raised to 80 to 85 ℃ and stirred for 16 hours. After completion of the reaction, the temperature was lowered to 25 ℃, water (20.5 liters) was added to the kettle, stirring was carried out for 5 minutes, and the reaction solution was extracted with ethyl acetate 2 times by 10 liters each. The combined organic phases were washed twice with 10 l each time of saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure at 45 degrees to give the product, compound I-3.¹HNMR(400MHz,CDCl₃)δ7.74(t,J＝7.8Hz,1H),7.43(d,J＝7.6Hz,1H),7.34(d,J＝8.0Hz,1H),3.94(s,2H)。

And step 3: compound I-4

Tetrahydrofuran (7.5 l) was charged to a 50 l reactor at room temperature, compound I-3(1.5 kg, 9.83 mol) and compound I-3-1(0.926 kg, 10 mol) were added and the internal temperature was lowered to-25 ℃. Hexamethylsilamidolithium (10 liters, 10 moles, tetrahydrofuran solution) was pumped into the kettle under nitrogen atmosphere, and the internal temperature was controlled not to exceed-15 ℃ for about 3 hours. The internal temperature rises between minus 8 ℃ and minus 5 ℃, and the reaction is carried out for 3 hours under the condition of heat preservation. The temperature was lowered to-25 ℃ and sodium hexamethyl-silazane (10 l, 10 mol, tetrahydrofuran solution) was added to the reactor by means of a peristaltic pump (addition was completed over 2 hours) with the internal temperature being controlled not to exceed-15 ℃. Raising the temperature to 20-25 ℃, and stirring for reacting for 16 hours. After completion of the reaction, water (0.883L, 49.1 mol) was slowly added to the reaction solution, and the mixture was stirred for 0.5 hour while controlling the internal temperature to be lower than 30 ℃. The reaction solution was distilled under reduced pressure at 40 ℃ in a kettle and concentrated to give a crude product solution (about 10 liters) of Compound I-4, which was used directly in the next reaction.

And 4, step 4: compound I-5

To a 50 liter reactor of crude compound I-4(10 liter solution) was added hydrogen hydroxide at room temperatureAdding an aqueous solution of potassium (9.6 liters, 19.2 moles) and ethanol (10 liters), raising the internal temperature to 75-80 ℃, and stirring for 5 hours. After the reaction is finished, cooling to 25-30 ℃, slowly adding a hydrochloric acid aqueous solution (8 liters and 48 moles) into the reaction solution, controlling the temperature not to exceed 40 ℃, heating the internal temperature to 60 ℃, and stirring for 1 hour. After completion of the reaction, concentration gave a crude product solution (about 2 l of the residue) which was extracted twice with 7.5 l each time of ethyl acetate. The extract was charged into a reaction vessel, silica gel (100 and 200 mesh, 750 g) was added, n-heptane (15L) was added, and stirring was carried out for 16 hours. Filtration was carried out, the filter cake was washed three times with 1 l each time of n-heptane/ethyl acetate (1:1), the organic phases were combined and concentrated under reduced pressure to give the crude product as a black solid. Heating the crude product with ethanol (3L) to an internal temperature of 70 deg.C, stirring to dissolve, cooling to 20 deg.C, precipitating a large amount of solid, adding water (9L) into the suspension, and stirring for 0.5 hr. Filtering, washing a filter cake twice with water, wherein each time is 1 liter; the filter cake was added to the mixed solution (n-heptane/ethanol, 4:1, 4.5 liters), stirred for 0.5 hour, filtered, washed 2 times with n-heptane/ethanol (4:1), 0.3 liters each, and dried under vacuum at 45 degrees for 16 hours to give compound I-5.¹HNMR(400MHz,CDCl₃)δ8.09(dd,J＝0.8,7.8Hz,1H),7.64(t,J＝7.8Hz,1H),7.18(dd,J＝0.8,7.8Hz,1H),4.43(dd,J＝4.6,9.2Hz,1H),4.31(d,J＝9.2Hz,1H),3.04-2.89(m,1H),2.14(dd,J＝4.4,7.8Hz,1H),1.56-1.44(m,1H)。

And 5: compound I-6

Tetrahydrofuran (16 l) was added to a 50 l kettle at room temperature, compound I-5(3.2 kg, 15.27 mol) was added, and stirred until dissolved and clear. Raising the internal temperature to 30 ℃, slowly adding a tetrahydrofuran solution of lithium borohydride (4.58 liters and 9.16 moles) into the kettle by using a peristaltic pump under the nitrogen atmosphere (after 1.5 hours of addition), controlling the temperature to be not more than 35 ℃, controlling the temperature of a jacket to be 10 ℃, controlling the internal temperature to be 25-35 ℃ after the feeding is finished, preserving the temperature for reaction for 2 hours, and stirring overnight for 16 hours. To the reaction solution, a saturated ammonium chloride (2.3 liters) solution was slowly added, and the mixture was stirred for 0.5 hour while controlling the internal temperature to be lower than 20 ℃. Adding n-heptane (8L) into the reaction solution, stirring for 0.5 hr, vacuum filtering the solid-liquid mixture, washing the filter cake with n-heptane/tetrahydrofuran (1:2) for 2 times (1.5L each time), filteringThe solution was concentrated under reduced pressure to about 8 liters, and 16 liters of ethyl acetate was added. The organic phase is washed 3 times with 3 liters each time and the aqueous phase is extracted 3 times with 2 liters each time with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate (2 kg) and concentrated under reduced pressure. The crude product was slurried with n-heptane/ethyl acetate (10:1, 6.4 liters), stirred for 0.5 hour, filtered, the filter cake was washed 1 time with n-heptane/ethyl acetate (10:1, 3.2 liters) and the filter cake was dried under vacuum for 16 hours to give compound I-6.¹HNMR(400MHz,DMSO-d₆)δ7.76(t,J＝7.8Hz,1H),7.59(d,J＝7.8Hz,1H),7.25(d,J＝7.8Hz,1H),4.84-4.62(m,2H),4.09(dd,J＝6.2,12.4Hz,1H),3.88-3.71(m,2H),3.50(ddd,J＝4.8,8.8,11.8Hz,1H),1.82-1.62(m,1H),1.29(dd,J＝4.0,8.8Hz,1H),0.92(dd,J＝4.0,6.2Hz,1H)。

Step 6: compound I-7

Tetrahydrofuran (22.5 l) was added to a 50 l kettle at room temperature, compound I-6(1.5 kg, 7.02 mol) was added, and stirred until dissolved and clear. Azodicarbonyldipiperidine (2.04 kg, 8.07 mol) was added, stirred until clear, and a solution of n-butylphosphine in tetrahydrofuran (1.63 kg, 8.07 mol) was added slowly to the kettle (over 1.5 hours) using a peristaltic pump under nitrogen, with the temperature being controlled to no more than 40 ℃. After the feeding is finished, controlling the internal temperature to be 15-25 ℃, reacting for 2 hours, and stirring overnight for 16 hours at room temperature. The reaction solution is filtered, the filter cake is washed with methyl tertiary butyl ether for 2 times, 3 liters each time, and the filtrate is concentrated to obtain a crude product. The crude product was dissolved with methyl tert-butyl ether (15 l), washed 3 times with hydrochloric acid (4 mol/l), 3 l each time, the aqueous phase was extracted 3 times with methyl tert-butyl ether, 3 l each time, the combined organic phases were washed 3 times with saturated brine, 5 l each time, dried over anhydrous sodium sulfate (2 kg), and concentrated under reduced pressure to give the crude product. The crude product was transferred to a kettle and n-heptane (7 liters) was added, warmed to an internal temperature of 70 ℃ and stirred for 0.5 hour to dissolve clear. The temperature is reduced to 15 ℃ of the internal temperature, and the mixture is stirred for 16 hours. Filtration, washing of the filter cake with n-heptane (1.4 l) 1 time, and vacuum drying of the filter cake for 16 h gave compound I-7.¹HNMR(400MHz,CDCl₃)δ7.46(t,J＝7.8Hz,1H),7.04(dd,J＝0.8,7.8Hz,1H),6.87(dd,J＝0.8,7.7Hz,1H),4.10-4.04(m,2H),3.86-3.78(m,2H),2.07(ddd,J＝2.8,5.2,8.0Hz,1H),1.32(dd,J＝4.4,8.2Hz,1H),1.07(t,J＝4.8Hz,1H)。

And 7: compound I-8

Dioxane (12L) was charged to a 50L reactor at room temperature, and Compound I-7-1(2.11 kg, 17.89 mol) and potassium tert-butoxide (2.51 kg, 22.36 mol) were added. The reaction solution was heated to 95 ℃ and a solution of compound I-7(1.75 kg, 8.94 mol) in dioxane (5.5 l) was added to the reaction solution via a constant pressure dropping funnel with an internal temperature of 90 to 100 ℃. After 30 minutes, the reaction was incubated for 30 minutes. After the reaction was complete, the temperature was reduced to 50 ℃, the pad was filtered through celite, the filter cake was washed 2 times with 2 liters each time with n-heptane, and the filtrates were combined and spin dried. The resulting yellow oil was dissolved in n-heptane (20 l) and washed 3 times with 15 l each time, and the dot plate showed no compound I-7-1 remaining. The organic phase was dried over anhydrous sodium sulfate (2.5 kg) and concentrated to give crude compound I-8.¹HNMR(400MHz,CDCl₃)δ7.45(t,J＝7.8Hz,1H),6.66-6.48(m,2H),4.45-4.29(m,2H),4.21-4.07(m,2H),3.95-3.81(m,2H),3.75-3.64(m,2H),2.13-2.02(m,1H),1.42-1.34(m,1H),1.25-1.17(m,9H),1.10-1.02(m,1H)。

And 8: compound 4a

Dichloromethane (13 l) was added to a 50 l kettle at room temperature, and trifluoromethyl isonicotinic acid (2.6 kg, 13.6 mol) and N, N-dimethylformamide (105 ml, 1.36 mol) were added and stirred until clear. Oxalyl chloride (1.67 l, 19.05 mol) was added slowly from an isopiestic dropping funnel and the off-gas was absorbed with lye. Stirring was carried out at 20 ℃ under a nitrogen atmosphere for 16 hours. After the reaction is completed, the solvent in the reaction solution is removed by rotary evaporation, the obtained trifluoromethyl isonicotinoyl chloride is dissolved by N, N-dimethylformamide (12 liters), the solution is slowly added into a solution of 3-bromo-4-methylaniline (3.0 kg, 13.97 moles) and diisopropylethylamine (4.11 liters, 20.44 moles) in N, N-dimethylformamide (6 liters) by using a constant-pressure dropping funnel, and the internal temperature is controlled to be 15-25 ℃. After the addition was complete, the reaction was continued for 0.5 hour. And after the reaction is finished, adding the reaction solution into water (54 liters), separating out solids, controlling the temperature to be 20-30 ℃, continuing stirring for 10 minutes after the addition is finished, filtering the suspension by using a suction filtration funnel under reduced pressure, washing the filter cake once by using water (2 liters), and collecting the filter cake. The filter cake was transferred to a column filled with n-heptane (15 l)) The reaction kettle (2) is stirred for 16 hours at an internal temperature of 15-25 ℃. The reaction was filtered, the filter cake was washed 2 times with n-heptane (1 l), and the filter cake was dried under vacuum for 16 hours to give compound 4 a.¹HNMR(400MHz,CDCl₃)δ8.93(d,J＝4.8Hz,1H),8.11(s,1H),8.01(br s,1H),7.96-7.86(m,2H),7.49(br d,J＝8.2Hz,1H),7.26(d,J＝8.2Hz,1H),2.41(s,3H)。

And step 9: compound I-9

At room temperature, tert-butyl methyl ether (18L) was charged into a 50L reactor, compound I-8(1.8 kg, 6.49 mol) and pinacol borate (1.73 kg, 6.81 mol) were added, the mixture was stirred to clarify, and the system was evacuated for three nitrogen replacements. Tmphen (30.67 g, 0.13 mol) and [ Ir (COD) (OMe) 2(40.02 g, 65 mmol) were added in turn to the reaction vessel, and the temperature was raised to 55-60 ℃ under the protection of nitrogen stream, and the mixture was stirred for 2 hours under heat. After the reaction was completed, the reaction solution was naturally cooled and stirred for 16 hours. And concentrating the reaction solution under reduced pressure to obtain a crude product, namely a compound I-9.

Step 10: compound I-10

At room temperature, dioxane (16L) was charged to a 50L reactor, compound 4a (2.2 kg, 6.12 mol) was added, stirring was continued until clear, and Pd (dppf) Cl was added₂·CH₂Cl₂(263.22 g, 0.32 mol) was charged into the autoclave and purged with nitrogen 3 times. Sodium carbonate (1.37 kg, 12.89 mol) was dissolved in water (5.2 l) to clarify, added to the reaction kettle and heated to 70-75 ℃. N is a radical of₂The solution of compound I-9(2.6 kg, 6.45 mol) was added to the reaction vessel (100 ml/min) with a peristaltic pump under protection and stirred for about 2 hours with internal temperature controlled at 70-75 ℃. After the reaction was completed, the reaction solution was cooled to room temperature, the reaction solution was filtered with celite under reduced pressure, and the filter cake was washed with ethyl acetate 2 times (2.5 liters each time). After the filtrate was concentrated under reduced pressure, ethyl acetate (25 l) and saturated brine (20 l) were added thereto, and suction filtration was performed under reduced pressure using celite, and after separating the filtrate, the organic phase was dried over anhydrous sodium sulfate (2.5 kg). The organic phase was transferred to a reaction kettle, to which was added modified silica gel (3.6 kg), heated to 60 ℃ and stirred for 16 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. To the yellow viscous mass was added tert-butyl methyl ether (4).5 l), is dissolved at 50 ℃, is transferred to a 50 l reaction kettle while hot, and is added with n-heptane (13.5 l) and heated to 60 ℃. And (4) closing the heating, naturally cooling the pulping liquid to 20 ℃, and stirring for 16 hours. The mixture was filtered under reduced pressure, the filter cake was washed with (tert-butyl methyl ether/n-heptane-1/3, 2.5 l) and the filter cake was dried under vacuum for 16 hours to give the crude product. Ethanol/n-heptane (1/10, 12.5 liters) was added to a 50 liter kettle and the crude product was added thereto, warmed to 65 degrees and stirred for 1 hour with constant temperature. And (4) closing the heating, pulping, naturally cooling to 20 ℃, and stirring for 16 hours. Vacuum filtering, washing the filter cake with ethanol/n-heptane (1:10, 2L), and vacuum drying the filter cake for 16 hr to obtain compound I-10.¹HNMR(400MHz,CDCl₃)δ8.90(d,J＝4.8Hz,1H),8.24(br s,1H),8.15(s,1H),7.95(br d,J＝3.8Hz,1H),7.63(br d,J＝8.2Hz,1H),7.44(s,1H),7.32-7.27(m,1H),6.53(s,1H),6.47(s,1H),4.41(t,J＝5.2Hz,2H),4.20-4.07(m,2H),3.94-3.84(m,2H),3.74(t,J＝5.2Hz,2H),2.25(s,3H),2.12(ddd,J＝2.6,5.0,7.8Hz,1H),1.41(dd,J＝4.2,8.0Hz,1H),1.24(s,9H),1.08(t,J＝4.4Hz,1H)。

Step 11: compound I-11

At room temperature, adding formic acid (12L) into a 50L reaction kettle, adding the compound I-10(2.4 kg, 4.32 mol), raising the internal temperature to 60-70 ℃, and stirring for 4-5 hours. After completion of the reaction, heating was stopped, the reaction solution was cooled to room temperature, and the reaction solution was concentrated under reduced pressure (45 degrees). Ethyl acetate (20 l) and water (15 l) were added to the crude product, sodium bicarbonate solid was added to adjust the pH of the solution to 4-5, the solution was separated, the organic phase was washed once with saturated brine (10 l), dried over anhydrous sodium sulfate and spin dried. Transferring to an oven for vacuum drying for 16 hours to obtain the compound I-11.¹HNMR(400MHz,CDCl₃)δ8.87(d,J＝4.8Hz,1H),8.41(s,1H),8.17-8.06(m,2H),7.93(br d,J＝4.4Hz,1H),7.62-7.52(m,1H),7.46(s,1H),7.32-7.21(m,1H),6.56(s,1H),6.50(s,1H),4.64-4.45(m,4H),4.22-4.03(m,2H),3.94-3.83(m,2H),2.25(s,3H),2.12(ddd,J＝2.6,5.0,7.8Hz,1H),1.40(dd,J＝4.2,7.8Hz,1H),1.09(t,J＝4.4Hz,1H)。

Step 12: a compound of formula (I)

Ethylene glycol dimethyl ether (10.5 liters) was charged to a 50 liter reactor at room temperature. Adding the compound I-11(2.1 kg, 3.98 mol) and stirred until the solution is clear. The temperature is reduced to 5-10 ℃, a pure water (2.1 liters) solution of sodium hydroxide (238.85 g, 5.97 mol) is slowly added, and the mixture is stirred for 0.5 hour. After completion of the reaction, ethyl acetate (20 l) and pure water (10 l) were added to the reaction solution, and the mixture was separated, and the organic phase was washed once with water (10 l) and dried over anhydrous sodium sulfate. The organic phase was rotary evaporated and the resulting solid transferred to an oven and dried in vacuo for 16 h to give the compound of formula (I) (crude).¹HNMR(400MHz,CDCl₃)δ8.93(d,J＝4.8Hz,1H),8.18(s,1H),8.14(s,1H),7.96(br d,J＝4.8Hz,1H),7.60(br d,J＝8.2Hz,1H),7.52(s,1H),7.32(d,J＝8.2Hz,1H),6.63(s,1H),6.57(s,1H),4.54-4.46(m,2H),4.20-4.11(m,2H),4.02-3.96(m,2H),3.96-3.86(m,2H),2.29(s,3H),2.16(ddd,J＝2.6,5.0,7.8Hz,1H),1.41(dd,J＝4.2,7.9Hz,1H),1.14(t,J＝4.4Hz,1H)。

Example 2: preparation of a Single Crystal of the Compound of formula (I)

The experimental method comprises the following steps:

30mg of the compound of the formula (I) was dissolved in 2mL of acetone, and the dissolved solution was put into a centrifuge tube (capacity: 10 mL). 2mL of petroleum ether is slowly added into the solution, the two solvents are obviously layered up and down, the pipe orifice is sealed by a sealing film, and a small evaporation hole is formed. Standing at room temperature for 10 days until most of the solvent is volatilized and crystals are separated out, collecting the crystals, and detecting by single crystal X-ray diffraction (SC-XRD).

The results are shown in tables 1-4:

TABLE 1 Crystal Structure data and measurement parameters for the Compounds of formula (I)

TABLE 2 atomic coordinates (. times.10) of the crystals of the compound of formula (I)⁴) And equivalent isotropic shift parameter

TABLE 3 bond length of crystals of the compound of formula (I)

And bond angle [ deg ]]

TABLE 4 twist angle [ deg ] of crystals of the compound of formula (I)

And (4) conclusion: the crystal unit diagram is shown in figure 1 and the results indicate that the compound of formula (I) has the structure N- (3- (2- ((1R,5S) -3-oxabicyclo [3.1.0] hex-1-yl) -6- (2-hydroxyethoxy) pyridin-4-yl) -4-methylphenyl) -2- (trifluoromethyl) isonicotinamide.

Biological test:

experimental example 1: in vitro enzyme Activity assay

Purpose of the experiment:

the inhibitory effect of the compounds on RAF kinase activity is tested.

The experimental steps are as follows:

(1) preparation of compound:

the test compound and the reference compound were diluted to 1 μ M with DMSO and the compounds were diluted in 3-fold gradient to obtain 10 concentration-gradient target plates. ATP concentration was 10. mu.M.

(2) The experimental process comprises the following steps:

1) preparing a buffer solution: 20mM HEPES (pH 7.5),10mM MgCl₂,1mM EGTA,0.01％Brij35,0.02mg/ml,BSA,0.1mM Na₃VO₄,2mM DTT,1％DMSO；

2) Preparing a specified substrate solution by using a newly prepared buffer solution, and adding a corresponding cofactor;

3) adding corresponding RAF kinase enzyme into the substrate solution, and uniformly mixing;

4) adding a DMSO solution of the compound to the above solution with Echo 550;

5) adding into³³P-ATP (specific activity 0.01. mu. Ci/. mu.l) elicited reactions, incubated for 2 hours at room temperature;

6) the reaction was labeled with P81 ion exchange paper (Whatman # 3698-915);

7) the filter was washed thoroughly with 0.75% phosphoric acid;

8) the residual radiophosphorylated substrate on the filter paper is detected.

The experimental results are as follows: table 5 provides the inhibitory activity of the compounds of the present invention on RAF kinase.

In vitro enzymatic Activity of the Compounds of Table 5

And (4) conclusion: the compound of the invention has obvious inhibitory activity on RAF enzyme.

Experimental example 2: calu-6 (Kras)^Q61K) Experiment for antiproliferative Activity

Experimental materials:

1) experimental reagent consumable

2) Laboratory apparatus

Name (R)	Brand goods number
		Cell counting plate	Refinement of the original
Victor Nivo	PerkinElmer

The experimental steps are as follows:

cell inoculation:

(1) cell culture medium: 89% EMEM, 10% fetal bovine serum and 1% penicillin-streptomycin;

(2) removing the original culture medium from the culture flask, digesting the cells with pancreatin, counting, diluting the cell suspension with the culture medium to the cell density of 3.75 × 10⁴Per ml of individual cells;

(3) add 100. mu.L of medium to each well around the cell plate, add 80. mu.L of cell suspension to the other wells, and put 5% CO₂And cultured overnight in a 37-degree incubator.

Adding medicine:

gradient dilution and dosing of the compounds with Echo were performed, and then the cell plates were returned to the incubator for three days;

reading the plate, analyzing data:

add CTG and read plate: add 20. mu.L of CellTiterGlo to each well of the cell plate, shake for 10min away from light, read the plate on Victor Nivo.

The experimental results are as follows: table 6 provides the antiproliferative activity of the compounds of the invention on Calu-6 cells.

In vitro Activity of the Compounds of Table 6

Type (B)	A compound of formula (I)
		Lung cancer Calu-6 cell antiproliferative activity IC50(nM)	600
Anti-proliferative Activity of Colon cancer HCT-116 cells IC50(nM)	1100
		Lung cancer Calu-6 cell ERK phosphorylation inhibitory Activity IC50(nM)	490
ERK phosphorylation inhibitory Activity of Colon cancer HCT-116 cells IC50(nM)	180

Experimental example 3: HCT-116 (Kras)^G13D) Experiment for antiproliferative Activity

Experimental materials:

1) experimental reagent consumable

Name (R)	Brand goods number
		Mc' Coy 5A Medium	BI-01-075-1ACS
Fetal bovine serum	Biosera-FB-1058/500
		0.25% Trypsin	Source culture-S310 KJ
Double resistance (penicillin, streptomycin)	Procell-PB180120
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Laboratory apparatus

Name (R)	Brand goods number
		Cell counting plate	Refinement of the original
Victor Nivo	PerkinElmer

The experimental steps are as follows:

cell inoculation:

(1) cell culture medium: 89% Mc' Coy 5A, 10% fetal bovine serum and 1% penicillin-streptomycin;

(2) removing the original culture medium from the culture flask, digesting the cells with pancreatin, counting, diluting the cell suspension with the culture medium to the cell density 2.5X 10 required for plating⁴Per ml of individual cells;

(3) add 100. mu.L of medium to each well around the cell plate, add 80. mu.L of cell suspension to the other wells, and put 5% CO₂And cultured overnight in a 37-degree incubator.

Adding medicine:

carrying out gradient dilution and dosing on the compound, and then putting the cell plate back to an incubator for culturing for three days;

reading the plate, analyzing data:

add CTG and read plate: add 20. mu.L of CellTiterGlo to each well of the cell plate, shake for 10min away from light, read the plate on Victor Nivo.

The experimental results are as follows: table 6 provides the antiproliferative activity of the compounds of the invention on HCT-116 cells.

Examples of the experiments4：HCT116(Kras^G13D) ERK phosphorylation inhibition assay

Experimental materials:

1. reagent consumable

2. Main instrument

Instrument for measuring the position of a moving object	Manufacturer of the product	Model number
			Biological safety cabinet	AIRTECH	BSC-1304IIA2
Carbon dioxide incubator	Thermo	311
			Cell counter	BECKMAN	Vi-cellXR
Enzyme-linked immunosorbent assay (ELISA) instrument	PerkinElmer	Envision
			Centrifugal machine	Eppendorf	Centrifuge 5810R

3. Cellular information

Cell name	Source	Goods number
			HCT116	ATCC	ATCC-HTB-132

Experimental procedures and methods:

1) the cells were revived and cultured to logarithmic phase, trypsinized, seeded in 96-well plates, and placed in an incubator for overnight incubation.

2) DMSO dissolved series of gradient compounds were added to 96-well plates and placed back into the incubator for 1 hour incubation.

3) The cell plates were removed, the supernatant removed, and cell lysates (containing 1% blocking peptide) added, incubated and lysed for 30min at room temperature.

4) Transfer 16 μ L of cell lysate to HTRF plate per well followed by 4 μ L of prepared antibody mix.

5) After overnight incubation, plates were read with Envision, fitted curves were obtained according to ratio (ratio of Ex665/Ex615 fluorescence intensity) and EC was calculated according to Graphpad's four-parameter fitting formula Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC 50-X): HillSlope))₅₀。

The experimental results are as follows: table 6 provides the inhibitory activity of the compounds of the invention on HCT-116ERK phosphorylation.

Experimental example 5: calu-6 (Kras)^Q61K) ERK phosphorylation inhibition assay

Experimental materials:

1) reagent consumable

Reagent	Brand goods number
		High-sensitivity detection kit for human ERK phosphorylated protein	Cisbio-64AERPEH
RPMI1640 medium	Gibco-22400089
		Fetal bovine serum	Hyclone-SV30087.03
96HTRF microplate	Cisbio-66PL96025
		96 micro-porous plate	COSTAR-3599
DMSO	Sigma-D2650-100mL
		0.05％Trypsin-EDTA	Gibco-25300-062

2) Main instrument

Instrument for measuring the position of a moving object	Manufacturer of the product	Model number
			Biological safety cabinet	AIRTECH	BSC-1304IIA2
Carbon dioxide incubator	Thermo	311
			Cell counter	BECKMAN	Vi-cellXR
Enzyme-linked immunosorbent assay (ELISA) instrument	PerkinElmer	Envision
			Centrifugal machine	Eppendorf	Centrifuge 5810R

3) Cellular information

Cell name	Source	Goods number
			Calu6	ATCC	ATCC-HTB-56

Experimental procedures and methods:

1) the cells were revived and cultured to logarithmic phase, trypsinized, seeded in 96-well plates, and placed in an incubator for overnight incubation.

2) DMSO dissolved series of gradient compounds were added to 96-well plates and placed back into the incubator for 1 hour incubation.

3) The cell plates were removed and lysed by adding cell lysate (containing 1% blocking peptide) for 30min at room temperature.

4) Transfer 16 μ L of cell lysate to HTRF plate per well followed by 4 μ L of prepared antibody mix.

5) After overnight incubation, plates were read with Envision, fitted curves were obtained according to ratio (ratio of Ex665/Ex615 fluorescence intensity) and EC was calculated according to Graphpad's four-parameter fitting formula Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC 50-X): HillSlope))₅₀。

The experimental results are as follows: table 6 provides the inhibitory activity of the compounds of the present invention on Calu-6 ERK phosphorylation.

And (4) conclusion: the compound of the formula (I) has good antiproliferative activity and good ERK phosphorylation inhibition activity on Calu-6 cells and HCT-116 cells.

Experimental example 6: a375 (BRAF)^V599E) Experiment for antiproliferative Activity

Experimental materials:

1) experimental reagent consumable

Name (R)	Brand goods number
		EMEM medium	Vickers-320-005-CL
Fetal bovine serum	Biosera-FB-1058/500
		0.25% Trypsin	Basal Media-S310KJ
Double resistance (penicillin, streptomycin)	Basal Media-S110JV
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Laboratory apparatus

Name (R)	Brand goods number
		EnVision	PerkinElmer

The experimental steps are as follows:

cell inoculation:

(1) cell culture medium: 89% EMEM, 10% fetal bovine serum and 1% penicillin-streptomycin;

(2) removing the original culture medium in the cell culture bottle, digesting the cells with pancreatin, counting, and diluting the cell suspension with the culture medium to the cell density of 2000 cells per well required by the plate laying;

(3) add 80. mu.L of cell suspension to each well and put 5% CO₂And cultured overnight in a 37-degree incubator.

Adding medicine:

add 20. mu.L of compound working solution to each well in cell plates at 37 ℃ in a 5% CO2 incubator and incubate for an additional 5 days.

Reading the plate, analyzing data:

add CTG and read plate: to each well of the cell plate, 50. mu.L CellTiterGlo was added, shaken for 10min, and detected using EnVision. The experimental results are as follows: table 7 provides the antiproliferative activity of compounds of the invention on a375 cells.

In vitro cell Activity of the Compounds of Table 7

Compound (I)	A375 cells IC50(nM)	Colo-205 cells IC50(nM)
			A compound of formula (I)	269.5	619.5

Experimental example 7: colo-205 (BRAF)^V599E) Experiment for antiproliferative Activity

Experimental materials:

1) experimental reagent consumable

Name (R)	Brand goods number
		RPMI-1640 medium	Biological Industries-06-1055-57-1ACS
Fetal bovine serum	Biosera-FB-1058/500
		0.25% Trypsin	Basal Media-S310KJ
Double resistance (penicillin, streptomycin)	Basal Media-S110JV
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Laboratory apparatus

Name (R)	Brand goods number
		EnVision	PerkinElmer

The experimental steps are as follows:

cell inoculation:

(1) cell culture medium: 89% EMEM, 10% fetal bovine serum and 1% penicillin-streptomycin;

(2) removing the original culture medium in the cell culture bottle, digesting the cells with pancreatin, counting, and diluting the cell suspension with the culture medium to the cell density of 3000 cells per well required by the plate laying;

(3) add 80. mu.L of cell suspension to each well and put 5% CO₂And cultured overnight in a 37-degree incubator.

Adding medicine:

add 20. mu.L of compound working solution to each well in cell plates at 37 ℃ in a 5% CO2 incubator and incubate for an additional 3 days.

Reading the plate, analyzing data:

add CTG and read plate: to each well of the cell plate, 50. mu.L CellTiterGlo was added, shaken for 10min, and detected using EnVision.

The experimental results are as follows: table 7 provides the anti-proliferative activity of compounds of the invention on Colo-205 cells.

And (4) conclusion: the compound of the invention has obvious antiproliferative activity on A375 cells and Colo-205 cells.

Experimental example 8: pharmacokinetics studies of single intravenous and oral dosing in mice

This experiment was conducted to investigate the Pharmacokinetic (PK) profile of the compounds of formula (I) in mice following a single intravenous and single oral administration of the compounds.

Sample collection and preparation:

after intravenous or oral administration, animal blood samples were collected and the actual blood sampling time was recorded. Immediately after blood collection, the cells were transferred to labeled centrifuge tubes containing K2-EDTA, and then centrifuged to obtain plasma. The plasma was transferred to a pre-chilled centrifuge tube, snap frozen in dry ice and stored in an ultra low temperature freezer at-70 ± 10 ℃ until LC-MS/MS analysis was performed.

Pharmacokinetic data analysis:

plasma drug concentration data for compounds were processed in a non-compartmental model using pharmacokinetic software. Peak concentration (C)_max) And time to peak (T)_max) And quantifiable end times, obtained directly from blood concentration-time profiles. The following pharmacokinetic parameters were calculated using the log linear trapezoidal method: half life (T)_1/2) Apparent volume of distribution (V)_dss) And clearance (Cl), area under the time-plasma concentration curve from point 0 to the end point (AUC)_0-last) Initial concentration (C)₀)。

The results are shown in Table 8:

TABLE 8 pharmacokinetic parameters of single intravenous and oral administration of the Compounds of the invention in mice

And (4) conclusion: the compound of formula (I) is better absorbed in mice by oral administration, has lower clearance rate, longer half-life period and better bioavailability.

Experimental example 9: in vivo pharmacodynamic experiment of human source lung cancer Calu-6 cell subcutaneous xenograft tumor BALB/c nude mouse model

Experimental materials:

1.1 Experimental animals and raising environments

1.1.1 Experimental animals

The species are as follows: mouse

Strain: BALB/c nude mice

Arrival at week: 6-8 weeks old

Sex: female

1.1.2 Breeding Environment

Animals are raised in SPF animal room in IVC (independent air supply system, constant temperature and humidity) cages (3-5 per cage)

Temperature: 20-26 deg.C

Humidity: 40-70 percent

1.2 information on Compounds

1.3 tumor tissue or cell information

Cell: human lung cancer Calu-6 cell in vitro culture, adding 0.2Units/mL bovine insulin, 10% fetal calf serum, 5% CO at 37 deg.C into EMEM culture medium₂And (5) incubator culture. Passage was performed twice a week with conventional digestion treatment with pancreatin-EDTA. When the saturation degree of the cells is 80-90% and the quantity reaches the requirement, collecting the cells, counting and inoculating.

1.4 other reagent information

Name (R)	Manufacturer of the product	Goods number	Storage conditions
				Fetal bovine serum	Hyclone	SV30087.03	-20℃
Trypsin	gibco	25200-072	-20℃
				EMEM medium	ATCC	ATCC30-2003	2-8℃

1.5 Instrument information

Name (R)	Manufacturer of the product	Model number
			Carbon dioxide incubator	Saimo Feishale (Thermo Fisher)	Heracell240i
Low-temperature high-speed centrifuge	Aibende (Eppendorf)	5810R
			Analytical balance	Sadorius (Sartorius)	SECURA225D-1CN
Ordinary balance	Changzhou Tianping Instrument and Equipment Co Ltd	EL-2KJ
			Digital display vernier caliper	Sanfeng tea		0～150mm

The experimental method and the steps are as follows:

2.1 tumor cell inoculation

Cell inoculation: 0.2mL of Calu-6 cells (1:1 in matrigel) were subcutaneously inoculated to the right hind-dorsal aspect of each mouse, and the mean tumor volume reached 173mm³The grouped administration is started.

2.2 Experimental animal grouping and dosing regimens

Note: 1, the number of mice in each group; 2, administration volume parameter: according to the weight of the mouse, 10 mu L/g. Stopping administration if the body weight is reduced by more than 15% until the body weight is recovered to be within 10%; 3: 0.5% MC (methyl cellulose).

2.3 preparation of the test substances

Note 1: the medicine is required to be gently and fully mixed before being administered to animals, wherein the solutol is polyethylene glycol-15-hydroxystearate, and the PEG is polyethylene glycol.

2.4 tumor measurement and Experimental indices

Tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v is 0.5a × b²And a and b represent the major and minor diameters of the tumor, respectively.

The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%)T_RTV/C_RTV×100％(T_RTV: treatment group RTV mean; c_RTV: negative control RTV mean). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is that RTV is V_t/V₀In which V is₀The resulting tumor volume, V, was measured at the time of group administration (i.e., D0)_tTumor volume at a certain measurement, T_RTVAnd C_RTVThe same day data was taken.

TGI (%), reflecting the rate of tumor growth inhibition. TGI (%) × 100% (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group)).

2.5 statistical analysis

Statistical analysis was performed using SPSS software based on RTV data at the end of the experiment. Comparisons between groups were analyzed by one-way ANOVA, and variance (significant differences in F-values) was examined by the Games-Howell method. Significant differences were considered with p < 0.05.

3. Results of the experiment

3.1 inhibition of growth of subcutaneous transplanted tumor of human Lung cancer nude mouse

The experiment evaluates the efficacy of the test substance in a human lung cancer xenograft tumor model, with a solvent control group as a reference. The T/C of the compound (100mg/kg) of the formula (I) of the administration group is 11.4 percent, the TGI is 99.1 percent, and the compound has obvious tumor inhibition effect (P is less than 0.01).

3.2 weight Change

The weight and the state of the mice are not abnormal.

And (4) conclusion: the compound of the invention has obvious inhibition effect on the growth of a tumor-bearing mouse of a human lung cancer Calu-6 cell subcutaneous xenograft tumor model.

Experimental example 10: in vivo pharmacodynamic experiment of BALB/c nude mouse of human colon cancer Colo-205 cell subcutaneous xenograft tumor

Experimental Material

1.1 Experimental animals and raising environments

1.1.1 Experimental animals

The species are as follows: mouse

Strain: BALB/c nude mice

Arrival at week: 6-8 weeks old

Sex: female

1.1.2 Breeding Environment

Animals were kept in IVC (independent air supply system, constant temperature and humidity) cages (3 per cage) in SPF animal houses

Temperature: 20-26 deg.C

Humidity: 40-70 percent

1.2 information on Compounds

1.3 tumor tissue or cell information

Cell: human colon cancer Colo-205 cells (ATCC-CCL-222) are cultured in vitro adherent manner by adding 10% fetal bovine serum, 1% penicillin/streptomycin/amphotericin B into RPMI1640 culture medium and 5% CO at 37 DEG C₂And (5) incubator culture. Passage was performed twice a week with conventional digestion treatment with pancreatin-EDTA. When the saturation degree of the cells is 80-90% and the quantity reaches the requirement, collecting the cells, counting and inoculating.

1.4 other reagent information

Name (R)	Manufacturer of the product	Goods number	Storage conditions
				Fetal bovine serum	Cellmax	SA211.02	-10℃
RPMI1640 medium	gibco	22400-089	2℃-8℃
				Anti-Anti	Gibco	15240062	-20℃-5℃
Pancreatin	Gibco	25200072	-20℃-5℃
				PBS	Hyclone	SH30256.01	15℃-30℃

1.5 Instrument information

Experimental methods and procedures

2.1 tumor cell inoculation and grouping

Cell inoculation: 0.1mL (5X 10)⁶Seed) Colo-205 cells were inoculated subcutaneously into each litterThe average tumor volume of the right back of the mouse reaches about 150mm³The grouped administration is started.

Grouping and dosing regimen for laboratory animals

Note: n is the number of mice in each group; dosing volume parameters: according to the weight of the mouse, 10 mu L/g. If the weight loss exceeds 15%, stopping administration until the weight is recovered to within 10%.

2.3 preparation of the test substances

Note: the drug needs to be gently mixed well before administration to the animal.

2.4 tumor measurement and Experimental indices

Tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v is 0.5a × b²And a and b represent the major and minor diameters of the tumor, respectively.

The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) ═ T_RTV/C_RTV×100％(T_RTV: treatment group RTV mean; c_RTV: negative control RTV mean). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is that RTV is V_t/V₀In which V is₀The resulting tumor volume, V, was measured at the time of group administration (i.e., D0)_tTumor volume at a certain measurement, T_RTVAnd C_RTVThe same day data was taken.

TGI (%), reflecting the rate of tumor growth inhibition. TGI (%) × 100% (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group)).

2.5 statistical analysis

Statistical analysis was performed using SPSS software based on RTV data at the end of the experiment. Comparisons between groups were analyzed by one-way ANOVA, and variance (significant differences in F-values) was examined by the Games-Howell method. Significant differences were considered with p < 0.05.

3. Results of the experiment

3.1 inhibition of growth of subcutaneous transplanted tumors in nude mice with human Colon cancer

The experiment evaluates the efficacy of the test substance in a human lung cancer xenograft tumor model, with a solvent control group as a reference. The T/C of the compound (30mg/kg QD,100mg/kg QD) of the administration group formula (I) is 68.46% and 36.12% respectively, the TGI is 31.98% and 74.05% respectively, the compound has obvious tumor inhibition effect and better dose-effect relationship. Human colon carcinoma Colo-205 cell subcutaneous xenograft tumor model tumor growth curves of tumor-bearing mice after administration of the compound are shown in FIG. 2.

3.2 weight Change

The weight and the state of the mice are not abnormal. The effect of the test substance on the body weight of the mice is shown in FIG. 3.

And (4) conclusion: the compound has obvious inhibition effect on the growth of tumor-bearing mice of colon cancer Colo-205 cell subcutaneous xenograft tumor models.

Experimental example 11: SD rat oral gavage repeated administration for 4 weeks recovery 4 weeks toxicity test accompanied by pharmacokinetic test

Experimental materials:

1.1 Experimental animals and raising environments

1.1.1 Experimental animals

The species are as follows: rat

Strain: SD rat

Age of animal: about 7 to 9 weeks old

Sex: female and male

1.1.2 Breeding Environment

Temperature: 20-26 deg.C

Humidity: 40-70 percent

The cage is a suspension type PC transparent rat feeding box on a stainless steel feeding rack, male and female animals are separately fed, and each box is not more than 5 animals. The temperature of the animal raising room is controlled to be 20-26 ℃, and the relative humidity is controlled to be 40-70%. The light/dark is controlled for each 12h cycle (this requirement can be temporarily avoided if night operation is encountered).

1.2 information on Compounds

1.3 other reagent information

1.4 Instrument information

Experimental methods and procedures

2.1 preparation and preservation

2.1.1 solvent preparation preparing method

1) Measuring 1000mL of pure water into a clean and dry beaker;

2) accurately (not more than +/-1% of theoretical amount) weighing 0.5g of methylcellulose, adding into 1, and stirring on a magnetic stirrer until the methylcellulose is fully dissolved to obtain the required preparation;

3) sealed for 3 months at the temperature of 2-8 ℃.

2.1.2 preparation method of blank auxiliary material preparation

1) Measuring a solvent preparation with a target volume for later use;

2) accurately weighing (not more than +/-1% of theoretical amount) required blank auxiliary materials into a clean and dry mortar, adding a small amount of solvent preparation, grinding until no obvious particles appear on eyes, transferring the mixture into a proper container, rinsing the mortar with the rest standby solvent preparation, and transferring the rinsing solution into the container;

3) and (5) placing the mixture in a magnetic stirrer for stirring for at least 1h until the mixture is uniform visually, thus obtaining the eye-friendly eye-cream.

Theoretical concentration x volume/sample content x auxiliary material content in the sample is high concentration sample preparation theoretical concentration x volume/46.5% × 50%.

2.1.3 method for preparing test preparation

1) Measuring a volume solvent preparation for preparing a target for later use;

2) accurately weighing the required amount of the test sample (not more than +/-1.0% of theoretical amount), adding a small amount of solvent preparation into a clean and dry mortar, grinding until no obvious particles appear on the eye, transferring the mixture into a proper container, rinsing the mortar with the rest of the standby solvent preparation, and transferring the rinsed mortar and rinsing liquid into the container;

3) dispersing with a dispersing machine until the visual uniformity is achieved;

4) stirring in magnetic stirrer for at least 1 hr until it is uniform visually to obtain the desired preparation.

Note: theoretical concentration × volume/content ═ theoretical concentration × volume/46.5%.

2.2 animal grouping and administration

2.2.1 animal groups

Rats were divided into sex groups according to the principles of randomness and weight balance, and the rats were stratified into 8 groups using the Provantis system according to the recent weight of the animals.

2.2.2 animal administration

The administration route is as follows: performing oral gavage;

duration and frequency of administration: the medicine is taken 1 time a day for 28 days continuously, and the medicine is stopped and recovered for 28 days;

requirements before and during administration: the test preparation and the adjuvant preparation are stirred for at least 30min before administration, and are continuously stirred until the administration is finished.

Grouping and dosing regimen for laboratory animals

Note: 1. animals with the last two animal numbers of 11-15 enter the recovery period.

2. The animal number is 4, thousand represent sex, "1" is male, "2" is female; the last two bits represent the intra-group sequence number.

3. If the animals need to be raised separately due to trauma or other reasons, the records are recorded in original records, see a general evaluation table, and are only stored as the original records.

4. The dosages are based on the formula (I) compound dispersion API.

2.3 Experimental testing

2.3.1 clinical pathology sampling and testing

Blood: all animals were fasted overnight before blood sampling without restriction of drinking water. Blood was taken via the abdominal aorta using a disposable vacuum suction tube. The hematology adopts EDTA-K₂An anticoagulation tube, with a blood collection volume of about 1.0 mL; separating gel/coagulant tube is adopted for serum biochemistry/electrolyte, and the blood collection amount is about 3.0 mL; the blood coagulation is conventionally performed by using a sodium citrate anticoagulation tube, and the blood collection amount is about 1.0 mL.

Urine: the rat (single rat) is placed in a clean metabolism cage, a syringe is used for sucking not less than 0.5mL of urine after a certain amount of urine is stored in a collection tube, the urine is transferred to a clean urine collection tube, and information such as collection time, volume and the like of the rat is recorded.

After treatment, the parameters of blood and urine are analyzed by an instrument.

2.3.2 pharmacokinetic sampling and evaluation

Blood samples were collected from animals at each time point on day 1 and day 28 of dosing at approximately 0.2 mL/time point, and 15. mu.L of EDTA-K was added to a 1.5mL centrifuge tube at a concentration of 30mg/mL₂Anticoagulant (EDTA-K)₂The preparation method comprises the following steps: to prepare 10mL of EDTA-K30 mg/mL₂For example, 300mg of EDTA-K was accurately weighed₂Put into a clean container, added with 10mL of ultrapure water, shaken up, kept at 2-8 ℃ and sealed for later use).

Measurement by LC-MS/MS methodThe concentration of the compound of formula (I) in plasma is determined and analyzed using Analyst 1.6.3 and Watson LIMS 7.4 software, after which concentration and time curves are drawn and at least the following parameters are calculated using winnonlin 8.1: area under concentration-time Curve (AUC)_0-tOr AUC_0-∞) Maximum peak concentration (C)_max) Time to peak (T)_max). Report major parameters (including plasma concentrations at different times, C)_max、T_maxAnd AUC), each parameter was calculated for each sex.

2.4 data processing and analysis

Data collected directly by the Provantis 10.2.3 and data recorded back into the Provantis 10.2.3 are processed and analyzed statistically by SAS statistical software built in the Provantis 10.2.3. The viral raw data measured by LC-MS/MS were derived using Analyst 1.6.3 software and further statistically analyzed using Microsoft Excel. And acquiring and processing data by using Agilent OpenLAB CDS 2.4.

The experimental results are as follows:

no obvious abnormality is observed in clinical pathology and pharmacokinetic detection of female and male SD rats in each experimental group, and no abnormality is found in serum biochemical total bilirubin.

And (4) experimental conclusion:

after the compound of the formula (I) is orally administrated to SD rats by gavage, the compound is safe and good, the male MTD is more than or equal to 300mg per kilogram, and the female MTD is more than or equal to 100mg per kilogram. (MTD: maximum tolerated dose)

Experimental example 12: beagle dog oral gavage repeated administration for 4 weeks recovery 4 weeks toxicity test accompanied by pharmacokinetic test

Experimental materials:

1.1 Experimental animals and raising environments

1.1.1 Experimental animals

The species are as follows: dog

Strain: beagle dog

Age of animal: 7-8 months old

Sex: female and male

1.1.2 Breeding Environment

Temperature: 19-26 deg.C

Humidity: 40-70 percent

Animals were housed in a single cage using a stainless steel cage. The temperature of the breeding room is controlled at 19-26 ℃, and the relative humidity is controlled at 40-70%. The light/dark 12h cycle is controlled (this requirement can be temporarily waived if night operation is met).

1.2 information on Compounds

1.3 other reagent information

1.4 Instrument information

Name (R)	Manufacturer of the product	Model number
			Blood analyzer	Siemens medical diagnostic products (Shanghai) Co., Ltd	ADVIA 2120i
Full-automatic integrated biochemical analyzer	Siemens medical diagnostic products (Shanghai) Co., Ltd	Dimension EXL				200
			Electrolyte analyzer	Madi card medical devices (Suzhou) Inc	EasyLyte PLUS
Full-automatic blood coagulation analyzer	Heatson Meikang Medical Electronics (Shanghai) Co.,Ltd.	CA-1500
			Urine analyzer	URIT Medical Electronic Co.,Ltd.	URIT-500B

Experimental methods and procedures

2.1 preparation and preservation

2.1.1 solvent preparation preparing method

1) Measuring 1000mL of pure water into a clean and dry beaker;

2) accurately (not more than +/-1% of theoretical amount) weighing 0.5g of methylcellulose, adding into 1, and stirring on a magnetic stirrer until the methylcellulose is fully dissolved to obtain the required preparation;

3) sealed for 3 months at the temperature of 2-8 ℃.

2.1.2 preparation method of blank auxiliary material preparation

1) Measuring a solvent preparation with a target volume for later use;

2) accurately weighing (not more than +/-1% of theoretical amount) required blank auxiliary materials into a clean and dry mortar, adding a small amount of solvent preparation, grinding until no obvious particles appear on eyes, transferring the mixture into a proper container, rinsing the mortar with the rest standby solvent preparation, and transferring the rinsing solution into the container;

3) and (5) placing the mixture in a magnetic stirrer for stirring for at least 1h until the mixture is uniform visually, thus obtaining the eye-friendly eye-cream.

Theoretical concentration x volume/sample content x auxiliary material content in the sample is high concentration sample preparation theoretical concentration x volume/46.5% × 50%.

2.1.3 method for preparing test preparation

1) Measuring a volume solvent preparation for preparing a target for later use;

2) accurately weighing the required amount of the test sample (not more than +/-1.0% of theoretical amount), adding a small amount of solvent preparation into a clean and dry mortar, grinding until no obvious particles appear on the eye, transferring the mixture into a proper container, rinsing the mortar with the rest of the standby solvent preparation, and transferring the rinsed mortar and rinsing liquid into the container;

3) dispersing with a dispersing machine until the visual uniformity is achieved;

4) stirring in magnetic stirrer for at least 1 hr until it is uniform visually to obtain the desired preparation.

Note: theoretical concentration × volume/content ═ theoretical concentration × volume/46.5%.

2.2 animal grouping and administration

2.2.1 animal groups

According to the principle of randomness and weight balance, dogs are classified into 4 groups according to the recent weight of animals by using a Provantis system.

2.2.2 animal administration

The administration route is as follows: performing oral gavage;

duration and frequency of administration: 1 dose daily for 4 weeks, 4 weeks after discontinuation of D29;

requirements before and during administration: animals were dosed about 1h after feeding. The preparation is stirred for at least 1h in a room temperature environment before administration, and the stirring is continued during the administration until the administration is finished.

Grouping and dosing

Note: 1. animals with last animal number 4, 5 entered recovery.

2. The animal number is 4, thousand represent sex, "1" is male, "2" is female; the last two bits represent the intra-group sequence number.

3. The dosages are based on the formula (I) compound dispersion API.

2.3 Experimental testing

2.3.1 clinical pathology sampling and testing

Blood: all animals were bled before feeding (except for dying animals), without restriction on drinking water. Blood was collected via the forelimb or hindlimb veins with a disposable vacuum suction tube. The hematology adopts EDTA-K₂An anticoagulation tube, the blood collection volume is about 1.0 mL; separating gel/coagulant tube is adopted for serum biochemistry/electrolyte, and the blood collection amount is about 3.0 mL; the blood coagulation is conventionally performed by using a sodium citrate anticoagulation tube, and the blood collection amount is about 1.0 mL.

Urine: after collecting fresh urine by using a clean stainless steel urine collecting disc, about 1.0mL of urine is extracted by using a disposable sterile syringe, the urine is transferred into a clean urine collecting tube (identified according to a corresponding label template in the mechanism), and information such as collecting time, volume and the like of the urine is recorded.

After treatment, the parameters of blood and urine are analyzed by an instrument.

2.3.2 pharmacokinetic sampling and evaluation

Blood samples of approximately 1mL EDTA-K were collected from the animals at each time point on day 1 and day 28 of the administration₂Anticoagulant blood collection tube.

After determining the concentration of the compound of formula (I) in plasma by analysis using Analyst 1.6.3 and Watson LIMS 7.4 software, concentration and time curves are plotted and at least the following parameters are calculated using WinNonlin 8.1: area under concentration-time Curve (AUC)_0-tOr AUC_0-∞) Maximum peak concentration (C)_max) Time to peak (T)_max). Report major parameters (including plasma concentrations at different times, C)_max、T_maxAnd AUC), the mean and standard deviation values. The respective parameters were calculated for different sexes.

2.4 data processing and analysis

Data collected directly by the Provantis 10.2.3 and data recorded back into the Provantis 10.2.3 are processed and analyzed statistically by SAS statistical software built in the Provantis 10.2.3. The viral raw data measured by LC-MS/MS were derived using Analyst 1.6.3 software and further statistically analyzed using Microsoft Excel. And acquiring and processing data by using Agilent OpenLAB CDS 2.4.

Results of the experiment

No obvious abnormality is observed in clinical pathology and pharmacokinetic detection of female and male Beagle dogs in each experimental group, and no abnormality is found in serum biochemical total bilirubin.

And (4) experimental conclusion:

after the compound shown as the formula (I) is orally administrated to Beagle dogs by gastric lavage, the compound is safe and good, and the MTD is more than or equal to 100 milligrams per kilogram.

Claims (5)

1. A compound of formula (I) N- (3- (2- ((1R,5S) -3-oxabicyclo [3.1.0] hex-1-yl) -6- (2-hydroxyethoxy) pyridin-4-yl) -4-methylphenyl) -2- (trifluoromethyl) isonicotinamide or a pharmaceutically acceptable salt thereof,

2. a pharmaceutical composition comprising the compound N- (3- (2- ((1R,5S) -3-oxabicyclo [3.1.0] hex-1-yl) -6- (2-hydroxyethoxy) pyridin-4-yl) -4-methylphenyl) -2- (trifluoromethyl) isonicotinamide of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. Use of a compound of formula (I) and salts thereof according to claim 1 or a pharmaceutical composition according to claim 2 in the preparation of a RAF kinase inhibitor.

4. A compound of the formula:

5. use of a compound according to claim 4 in the preparation of a compound of formula (I)

The use of (1).

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