CN113912591B

CN113912591B - Biaryl compounds

Info

Publication number: CN113912591B
Application number: CN202110772717.8A
Authority: CN
Inventors: 丁照中; 刘希乐; 陈曙辉; 胡利红; 周成亮
Original assignee: Qilu Pharmaceutical Co Ltd
Current assignee: Qilu Pharmaceutical Co Ltd
Priority date: 2020-07-08
Filing date: 2021-07-08
Publication date: 2023-10-20
Anticipated expiration: 2041-07-08
Also published as: CN113912591A

Abstract

The application discloses biaryl compounds, and particularly discloses compounds shown in a formula (I) and pharmaceutically acceptable salts thereof.

Description

Biaryl compounds

The present application claims the following priorities:

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

PCT/CN2020/133933, 12 th year, 04 th year of the filing date 2020.

Technical Field

Biaryl compounds, pharmaceutically acceptable salts thereof, as Pan-RAF kinase inhibitors, and their use in the treatment of cancer-related diseases are disclosed.

Background

The Mitogen Activated Protein Kinase (MAPK) pathway is an important signal transduction pathway in cells that transmits signals from outside cells into the nucleus through specific cascade phosphorylation of RAS/RAF/MEK/ERK, ultimately leading to activation of specific genes, causing proliferation, apoptosis or differentiation of cells. This pathway over-activation is closely related to the occurrence of a variety of tumors. RAF is a very important serine/threonine protein kinase in RAS/RAF/MEK/ERK signaling pathway, and is located downstream of RAS and can be activated by RAS. The RAF family includes three subtypes of ARAF, BRAF, CRAF (RAF-1), with a high degree of homology and similar domains. Wild-type RAF is capable of producing homo-or heterodimers of three subtypes. ARAF and CRAF mutations occur less frequently, with a higher BRAF mutation rate. About 5% to 10% of malignancies, including 66% of melanoma patients, present with BRAF mutations. RAF kinase is used as a key signal protein at the downstream of RAS, and has important research significance in the treatment of RAS mutant gene mutant tumor. MAPK signaling is regulated by inhibiting RAF kinase, thereby playing a role in proliferation of RAS mutant tumor cells. Thus, RAF kinase has become an important target for clinical treatment of tumors.

The first generation of BRAF kinase inhibitors Vemurafenib and Dabrafenib have been approved by the FDA for the generation of B-Raf ^V600E Treatment of mutant cancers. Although Vemurafenib and Dabrafenib are in B-Raf ^V600E The treatment of mutant melanoma has shown promising therapeutic efficacy, but certain limitations remain. Most patients with both drugs initially shrink their tumors, but relapse within one year (acquired resistance); the primary mechanism for this resistance is that the MAPK signaling pathway is reactivated. Studies have found that NRAS mutations can lead to MAPK pathway activation in the presence of inhibitors, resulting in resistance, based on the V600E mutation of BRAF. Mutant N-Ras promotes B-Raf ^V600E And C-Raf forms homo-or heterodimers. Binding of the inhibitor to one monomer of the dimer may reduce the affinity of the drug for the other monomer, promoting phosphorylation of the monomer without the inhibitor, resulting in activation of MEK. Currently, the primary approach to clinically overcoming the resistance caused by reactivation of the MAPK pathway is to block 2 key sites of the MAPK pathway by the combined use of Raf and MEK inhibitors to delay resistance development. In addition, the development of a new generation of pan-Raf inhibitors to overcome resistance and to expand the range of clinical applications is also under development, and pan-Raf inhibitors can inhibit dimer activity and block abnormal activation of pathways (paradoxcal activation), thereby reducing resistance. The pan-Raf dimer inhibitors in clinical studies are mainly HM95573, TAK-580, BGB-283, LX H254, LY3009120, etc., the development of these novel RAF inhibitors is expected to overcome the drug resistance of the first-generation inhibitors, further expanding 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 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 application of the compound shown in the formula (I) or pharmaceutically acceptable salt thereof in preparing a medicament for treating cancer.

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

Technical effects

The compound has good drug property, good RAF enzyme inhibition activity and various cell antiproliferative activity, good in-vivo drug effect and good safety. Hopefully solving the current BRAF ^V600E The problem of resistance to mutant cancer treatment provides an effective treatment for RAS mutant type cancers.

Definition and description

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

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 salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting such compounds with a sufficient amount of acid in pure 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 organic acid salts including acids such as 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, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; also included are salts of amino acids (e.g., arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts.

Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of 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 both.

The compounds of the 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, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present invention.

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

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

Unless otherwise indicated, the term "diastereoisomer" refers to stereoisomers of a molecule having two or more chiral centers and having a non-mirror relationship between the molecules.

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

Unless otherwise indicated, with solid wedge bondsAnd wedge-shaped dotted bond->Representing the absolute configuration of a solid centre, using straight solid keys +.>And straight dotted bond->Representing the relative configuration of the stereo centers, using wavy lines +.>Representing a wedge solid key +.>Or wedge-shaped dotted bond->Or by wave lines->Representing a straight solid line key->And straight dotted bond->

Unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to the fact that at room temperature, different functional group isomers are in dynamic equilibrium and are capable of rapid interconversion. If tautomers are possible (e.g., in solution), chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as proton tautomers) (prototropic tautomer) include interconversions by proton transfer, such as keto-enol isomerisation and imine-enamine isomerisation. Valence isomer (valance tautomer) includes the interconversion by recombination of some of the bond-forming electrons. A specific example of where keto-enol tautomerization 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", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100% and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

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

Optically active (R) -and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary wherein the resulting diastereomeric mixture is separated and the auxiliary group 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), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereomeric resolution is carried out by conventional methods well known in the art, and then the pure enantiomer is recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amine).

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "mercapto protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: a formyl group; acyl groups such as alkanoyl (e.g., acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl groups such as t-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 hydroxyl protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and t-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 set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.

The compounds of the present invention may be structured by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of a compound, the absolute configuration may be confirmed by conventional means in the art. For example, single crystal X-ray diffraction (SXRD), the grown single crystal is collected from diffraction intensity data using a Bruker D8 vent diffractometer, and the light source is cukα radiation, scanning:after scanning and collecting the relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by a direct method (Shellxs 97). The absolute configuration of the compounds can also be confirmed by the chiral structure of the starting materials and the reaction mechanism of the asymmetric synthesis.

The solvent used in the present invention is commercially available.

The invention adopts the following abbreviations: HATU represents 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate; HOBt represents 1-hydroxybenzotriazole; t (T) ₃ P represents tri-n-propyl cyclic phosphoric anhydride; pd (Pd) ₂ dba ₃ Represents tris-biphenylacetone-dipalladium; ruphos stands for 2-dicyclohexylphosphorus-2 ',6' -diisopropyloxy-1, 1' -biphenyl; brettphos stands for (2-dicyclohexylphosphine-3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl; TBAF stands for tetrabutylammonium fluoride; ir (COD) OMe] ₂ Represents cyclooctadiene AOxyiridium dimers; tmphen represents 3,4,7, 8-tetramethyl-1, 10 phenanthroline; dtbpy represents 4,4 '-di-tert-butyl-2, 2' -bipyridine. Pd (dppf) Cl ₂ Represents diphenylphosphine ferrocene palladium dichloride; pd (dppf) Cl ₂ DCM represents a diphenylphosphine ferrocene palladium dichloride and dichloromethane complex; DIAD stands for diisopropyl azodicarboxylate; DIPEA or DIEA represents diisopropylethylamine; PPh (PPh) ₃ Represents triphenylphosphine; TFA represents trifluoroacetic acid; THP represents 2-tetrahydropyran; OTHP represents 2-oxo-tetrahydropyran; TBSCl represents tert-butyldimethylchlorosilane; TBS represents t-butyldimethylsilyl; OTBS represents tert-butyldimethylsilyloxy; DMF represents N, N-dimethylformamide; DMSO represents dimethylsulfoxide; 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 represents 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-H ₂ O represents that the separation system is formic acid-acetonitrile-water; IPA represents isopropanol; me represents methyl and OMs represents methanesulfonyloxy.

Drawings

FIG. 1 shows a unit cell diagram of a single crystal of a compound of formula (I).

FIG. 2 is a graph showing tumor growth curves of tumor-bearing mice of a human colon carcinoma Colo-205 cell subcutaneous xenograft tumor model.

FIG. 3 is a graph showing the weight change of tumor-bearing mice in a model of human colon carcinoma Colo-205 cell subcutaneous xenograft.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limiting in any way. 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 set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

Example 1: preparation of Compounds of formula (I)

Synthetic route 1:

Compound 2

N-butyllithium (23 mL, 2.5M) was added to a solution of Compound 1 (10 g, 51.96 mmol) in dichloromethane (100 mL) at-78deg.C, and stirred for 30 min. To this mixture was added 1a (4.92 g, 57.16 mmol) at-78 ℃, and the mixture was stirred for 0.5 hour at 25 ℃. To the reaction mixture was added saturated ammonium chloride solution (40 mL), extracted with dichloromethane (40 ml×2), and the combined extracts were dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography (PE/ea=10/1 to 5/1, V/V) to obtain compound 2. ¹ HNMR(400MHz,CDCl ₃ )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 (80 mL) was added p-toluene sulfonic acid (11.5 g, 60.46 mmol), and the mixture was stirred at 110 ℃ for 16 hours. To the reaction mixture was added saturated sodium bicarbonate solution (40 mL) and ethyl acetate (100 mL), the mixture was separated, washed with saturated sodium bicarbonate solution (50 ml×2) with a camera, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (PE/ea=50/1 to 20/1, V/V) to give compound 3. ¹ HNMR(400MHz,CDCl ₃ )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) and compound 2a (7.3 g, 33.17 mmol) was added DMSO (50 mL). The reaction mixture was stirred at 25 ℃ under nitrogen for 1 hour. 3 (1) was added to the reaction solution Gram, 5.51 mmol) of DMSO (5 mL), the reaction mixture was heated to 70 ℃ and stirred for 6 hours. To the reaction mixture was added water (180 mL), extracted with ethyl acetate (30×3 mL), 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. ¹ HNMR(400MHz,CDCl ₃ )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. Mu. Mol) and tmphen (50 mg, 211.59. Mu. Mol) were placed in methyl t-butyl ether (20 mL). The reaction mixture was added 4 (770 mg, 3.94 mmol) under nitrogen and heated to 80 ℃ and stirred for 3 hours. The reaction solution is filtered, and the filtrate is concentrated to obtain the crude product of the compound 5. MS (ESI): m/z 240.3[ M-84+2H] ⁺ 。

Compound 6

Compound 5 (1.2 g, 3.73 mmol), 4a (1.4 g, 3.90 mmol) and Pd (dppf) Cl ₂ DCM (300 mg, 367.36. Mu. Mol) and sodium carbonate (800 mg, 7.55 mmol) were placed in dioxane (50 mL) and water (10 mL). The reaction mixture was heated to 100 ℃ under nitrogen and stirred for 3 hours. The reaction mixture was filtered, ethyl acetate (20 mL) and water (10 mL) were added after concentrating the filtrate, the liquid was separated, the aqueous phase was extracted with ethyl acetate (10 ml×3), and the combined organic phases were dried over anhydrous sodium sulfate and concentrated, and compound 6 was isolated by silica gel column chromatography (PE/ea=10/1 to 5/1). ¹ HNMR(400MHz,CDCl ₃ )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 (30 mL) was added Pd ₂ dba ₃ (180Milligram, 196.57 micromole), brettphos (200 milligram, 372.60 micromole) and cesium carbonate (1.26 gram, 3.87 millimole). The reaction mixture was heated to 110 ℃ under nitrogen and stirred for 4 hours. 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 give compound 7. ¹ HNMR(400MHz,DMSO-d ₆ )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 tetrahydrofuran (10 mL) of compound 7 (300 mg, 488.81. Mu. Mol) was added hydrochloric acid (1 mL, 4M), and the reaction mixture was stirred at 25℃for 1 hour. The reaction solution was diluted with ethyl acetate (20 mL), ph=8 was adjusted with a saturated solution of sodium hydrogencarbonate, extracted with ethyl acetate (10×3 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated, and a thin-layer silica gel plate (PE/ea=1/1, V/V) was separated to give compound 8. ¹ HNMR(400MHz,DMSO-d ₆ )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)

Chiral separation of compound 8 by SFC (chiral column DAICEL CHIRALCEL OJ-H (250 mm. Times.30 mm,5 μm), mobile phase A: ethanol (0.05% diisopropylethylamine; mobile phase B: carbon dioxide) gave 8A (retention time 1.487 min) and compound of formula (I) (retention time 1.590 min). 8A is an enantiomer of a compound of formula (I).

Compound 8A: ¹ HNMR(400MHz,DMSO-d ₆ )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): ¹ HNMR(400MHz,DMSO-d ₆ )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％)。

synthetic route 2:

step 1: compound I-2

N, N-dimethylformamide (12.5L) was added to a 50L 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. 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 completed, the reaction solution is cooled to 25 ℃; adding water (27 liters) into the reaction kettle, and transferring about 27 liters of reaction liquid into a 30 liter white barrel for temporary storage; hydrochloric acid solution (6 mol/l, 8.5 l) was slowly added to the remaining 27 l of the reaction solution in the autoclave, 0.3 l each time, and the pH was adjusted to 3 to 4. A yellow solid precipitated with a large amount of gas generation and a small amount of exotherm. The solid-liquid mixture was filtered off 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 was treated in the same manner. After the filter cakes were combined, they were dried at 45℃under vacuum for 24 hours to give Compound I-2. ¹ HNMR(400MHz,CDCl ₃ )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 added to a 50L reaction vessel at room temperature, and Compound I-2 (3.6 kg, 16.03 mol) was added and the 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 liters) was added, and 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℃and water (20.5 liters) was added to the vessel and stirred for 5 minutes, and the reaction solution was extracted with ethyl acetate 2 times, 10 liters each. The organic phases were combined and washed twice with 10 liters of saturated brine each. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure at 45℃to give the product compound I-3. ¹ HNMR(400MHz,CDCl ₃ )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)。

Step 3: compound I-4

Tetrahydrofuran (7.5 liters) was charged into a 50 liter reaction vessel at room temperature, and compound I-3 (1.5 kg, 9.83 moles) and compound I-3-1 (0.926 kg, 10 moles) were added and the internal temperature was lowered to-25 ℃. Lithium hexamethyl-silicon amide (10L, 10 mol, tetrahydrofuran solution) was pumped into the kettle under nitrogen atmosphere, and the internal temperature was controlled to be not more than-15℃and the addition was completed for about 3 hours. The internal temperature rise is kept between-8 ℃ and-5 ℃ and the reaction is carried out for 3 hours. The temperature was reduced to-25℃and sodium hexamethylsilamidate (10 liters, 10 moles, tetrahydrofuran solution) was added to the reaction vessel (2 hours of addition) using a peristaltic pump, and the internal temperature was controlled to not exceed-15 ℃. The internal temperature is raised to 20-25 ℃, and the reaction is stirred for 16 hours. After completion of the reaction, water (0.883 liter, 49.1 mol) was slowly added to the reaction mixture, and the mixture was stirred at an internal temperature of less than 30℃for 0.5 hour. The reaction mixture was distilled under reduced pressure at 40℃in a pot, and concentrated to give a crude product solution (about 10 liters) of Compound I-4, which was used directly in the next reaction.

Step 4: compound I-5

An aqueous solution of potassium hydroxide (9.6 liters, 19.2 moles) and ethanol (10 liters) were charged at room temperature to a 50 liter reaction vessel of crude compound I-4 (10 liters solution)) The internal temperature rises to 75-80 ℃ and is stirred for 5 hours. Cooling to 25-30 deg.c after the reaction, adding hydrochloric acid aqua (8L, 48 mol) slowly to the reaction liquid, controlling the temperature not to exceed 40 deg.c, heating to 60 deg.c and stirring for 1 hr. After completion of the reaction, the crude product solution (about 2 liters of the remaining solution) was concentrated, and extracted twice with 7.5 liters each time of ethyl acetate. The extract was added to the reaction vessel, silica gel (100-200 mesh, 750 g) was added, n-heptane (15 l) was added, and stirred for 16 hours. The mixture was filtered, and the filter cake was washed three times with n-heptane/ethyl acetate (1:1), 1 liter each, and the organic phases were combined and concentrated under reduced pressure to give a crude black solid. The crude product was heated to an internal temperature of 70℃with ethanol (3 liters), stirred to a clear solution, cooled to 20℃and after a large amount of solids had been precipitated, water (9 liters) was added to the suspension and stirred for 0.5 hour. Filtering, washing the filter cake twice with water, 1 liter each time; the filter cake was added to the mixed solution (n-heptane/ethanol, 4:1,4.5 liter), stirred for 0.5 hour, filtered, and the filter cake was washed 2 times with n-heptane/ethanol (4:1), 0.3 liter each, and the filter cake was dried at 45 degrees under vacuum for 16 hours to give compound I-5. ¹ HNMR(400MHz,CDCl ₃ )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)。

The method comprises the following steps: compound I-6

Tetrahydrofuran (16 l) was added to a 50 l reaction vessel at room temperature, compound I-5 (3.2 kg, 15.27 mol) was added and stirred until dissolved and clear. The internal temperature is raised to 30 ℃, a peristaltic pump is used for slowly adding a tetrahydrofuran solution of lithium borohydride (4.58 liters, 9.16 moles) into a kettle (after 1.5 hours), the temperature is controlled to be not more than 35 ℃, the jacket temperature is controlled to be 10 ℃, the internal temperature is controlled to be 25-35 ℃ after the charging is finished, the reaction is carried out for 2 hours under the heat preservation condition, and the stirring is carried out for 16 hours overnight. To the reaction solution was slowly added a saturated ammonium chloride (2.3 liters) solution, the internal temperature was controlled to be lower than 20 ℃, and the mixture was stirred for 0.5 hour. N-heptane (8L) was added to the reaction mixture, stirred for 0.5 hour, the solid-liquid mixture was suction-filtered under reduced pressure, the cake was washed with n-heptane/tetrahydrofuran (1:2) 2 times, 1.5L each time, the filtrate was concentrated under reduced pressure to about 8L, and ethyl acetate 16 was addedLifting. The organic phase is washed 3 times with 3 liters each time and the aqueous phase is extracted 3 times with ethyl acetate 2 liters each time. 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, 6.4L), stirred for 0.5 h, filtered, the filter cake washed 1 time with n-heptane/ethyl acetate (10:1, 3.2L) and the filter cake dried in vacuo for 16 h to give compound I-6. ¹ HNMR(400MHz,DMSO-d ₆ )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 liters) was added to a 50 liter reaction vessel at room temperature, compound I-6 (1.5 kg, 7.02 moles) was added and stirred until dissolved and clarified. Azodicarbonyl dipiperidine (2.04 kg, 8.07 moles) was added, stirred until dissolved and clarified, and a tetrahydrofuran solution of n-butylphosphine (1.63 kg, 8.07 moles) was slowly added to the kettle (1.5 hours over) with a peristaltic pump under nitrogen atmosphere, with the temperature controlled to not more than 40 ℃. After the material is fed, the internal temperature is controlled between 15 and 25 ℃, the reaction is carried out for 2 hours, and the mixture is stirred at room temperature for 16 hours overnight. The reaction solution was filtered, the filter cake was washed with methyl tert-butyl ether 2 times, 3 liters each time, and the filtrate was 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, n-heptane (7 liters) was added, the temperature was raised to 70℃and stirred for 0.5 hour until a clear solution was obtained. Cooling to 15 deg.C, stirring for 16 hr. Filtering, washing the filter cake with n-heptane (1.4L) for 1 time, and vacuum drying the filter cake for 16 hours to obtain the compound I-7. ¹ HNMR(400MHz,CDCl ₃ )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)。

Step 7: compound I-8

Dioxa-hexacyclic ring (12 liter) was charged into a 50 liter reaction vessel at room temperature, and Compound I-7-1 (2.11 kg, 17.89 mol) and potassium t-butoxide (2.51 kg, 22.36 mol) were added. The reaction solution was heated to 95℃and a dioxane (5.5L) solution of Compound I-7 (1.75 kg, 8.94 mol) was added to the reaction solution using a constant pressure dropping funnel, and the internal temperature was controlled at 90-100 ℃. After the addition for 30 minutes, the reaction was incubated for 30 minutes. After the reaction was completed, the temperature was lowered to 50 ℃, the mixture was filtered through celite, the cake was washed with n-heptane 2 times, 2 liters each time, and the filtrates were combined and dried by spin. The resulting yellow oil was dissolved in n-heptane (20 liters) and washed 3 times with 15 liters each, the dot panels showing no compound I-7-1 remaining. The organic phase was dried over anhydrous sodium sulfate (2.5 kg) and concentrated to give the crude compound I-8. ¹ HNMR(400MHz,CDCl ₃ )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)。

Step 8: compound 4a

Dichloromethane (13 l) was added to a 50 l reaction vessel at room temperature, and trifluoromethyliisonicotinic 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 slowly added using a constant pressure dropping funnel and the tail gas was taken up in lye. Stirring is carried out for 16 hours at 20℃under nitrogen. After the completion of the reaction, the solvent was distilled off from the reaction mixture, the obtained trifluoromethyl isonicotinyl chloride was dissolved in N, N-dimethylformamide (12 liters), and the solution was slowly added to a solution of 3-bromo-4-methylaniline (3.0 kg, 13.97 mol) and diisopropylethylamine (4.11 liters, 20.44 mol) in N, N-dimethylformamide (6 liters) with a constant pressure dropping funnel, and the internal temperature was controlled to 15 to 25 ℃. After the addition was complete, the reaction was continued for 0.5 hours. After the reaction is completed, the reaction solution is added into water (54 liters), solids are separated out, the temperature is controlled at 20-30 ℃, stirring is continued for 10 minutes after the addition is completed, the suspension is filtered by a suction filtration funnel under reduced pressure, a filter cake is washed once by water (2 liters), and the filter cake is collected. Transferring the filter cake into a reaction kettle filled with n-heptane (15L), and stirring at an internal temperature of 15-25 DEG C And 16 hours. The reaction solution was filtered, and the cake was washed with n-heptane (1 liter) 2 times, and the cake was dried in vacuo for 16 hours to give compound 4a. ¹ HNMR(400MHz,CDCl ₃ )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)。

Step 9: compound I-9

Tert-butyl methyl ether (18L) was added to a 50L reaction vessel at room temperature, and Compound I-8 (1.8 kg, 6.49 mol) and pinacol borate (1.73 kg, 6.81 mol) were added, and the mixture was stirred to clear and the system was evacuated to displace nitrogen three times. Tmphen (30.67 g, 0.13 mol) and [ Ir (COD) (OMe) ]2 (40.02 g, 65 mmol) were added to the reaction vessel in sequence, and the temperature was raised to 55-60℃under nitrogen flow protection, and the mixture was stirred for 2 hours under heat preservation. After the reaction was completed, the reaction solution was naturally cooled and stirred for 16 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product of the compound I-9.

Step 10: compound I-10

Dioxahexacyclic ring (16L) was added to a 50L reaction vessel at room temperature, compound 4a (2.2 kg, 6.12 mol) was added, stirred until clear, and Pd (dppf) Cl was added ₂ ·CH ₂ Cl ₂ (263.22 g, 0.32 mol) was added to the reaction vessel and replaced 3 times with nitrogen. Sodium carbonate (1.37 kg, 12.89 mol) was dissolved in water (5.2 l) to clarify and then added to the reaction vessel, and the temperature was raised to 70-75 ℃. N (N) ₂ Solution compound I-9 (2.6 kg, 6.45 mol) was added to the reaction vessel (100 ml/min) under protection using peristaltic pump, the internal temperature was controlled at 70-75 ℃, and stirring was performed for about 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature, the reaction solution was subjected to suction filtration under reduced pressure with celite, and the cake was washed with ethyl acetate 2 times each of 2.5 liters. After the filtrate was concentrated under reduced pressure, ethyl acetate (25 liters) and saturated brine (20 liters) were added thereto, and the filtrate was separated by suction filtration under reduced pressure with celite, and then the organic phase was dried with anhydrous sodium sulfate (2.5 kg). The organic phase was transferred to a reaction kettle, to which modified silica gel (3.6 kg) was added, heated to 60 degrees 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 material was added tert-butyl methyl ether (4.5L), and after 50℃solution was removed, the mixture was transferred to 50L To the autoclave, n-heptane (13.5 liters) was added and the temperature was raised to 60 ℃. And (5) turning off the heating, naturally cooling the beating liquid to 20 ℃, and stirring for 16 hours. Vacuum filtration, washing the filter cake with (tert-butyl methyl ether/n-heptane=1/3, 2.5 liter), and vacuum drying the filter cake for 16 hours to obtain a crude product. Ethanol/n-heptane (1/10, 12.5 l) was added to the 50 l reactor, and the crude product was heated to 65℃and stirred for 1 hour at constant temperature. And (5) turning off 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. ¹ HNMR(400MHz,CDCl ₃ )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

Formic acid (12L) was added to a 50L reaction vessel at room temperature, compound I-10 (2.4 kg, 4.32 mol) was added, the internal temperature was raised to 60 to 70℃and stirred for 4 to 5 hours. After the completion of the reaction, the 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 liters) and water (15 liters) were added to the crude product, sodium hydrogencarbonate solid was added to adjust the pH of the solution to 4 to 5, the solution was separated, and the organic phase was washed once with saturated brine (10 liters), dried over anhydrous sodium sulfate and dried by spinning. Transferring to an oven, and vacuum drying for 16 hours to obtain the compound I-11. ¹ HNMR(400MHz,CDCl ₃ )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 added to a 50 liter reaction vessel at room temperature. Compound I-11 (2.1 kg, 3.98 mol) was added and the solution was stirred until it was clear. Cooled to 5-10 ℃, a solution of sodium hydroxide (238.85 g, 5.97 mol) in pure water (2.1 liter) was slowly added and stirred for 0.5 hour. After completion of the reaction, ethyl acetate (20 liters) and pure water (10 liters) were added to the reaction solution, the solution was separated, and the organic phase was washed once with water (10 liters) and dried over anhydrous sodium sulfate. The organic phase was distilled off by spin-drying, and the obtained solid was transferred to an oven and dried in vacuo for 16 hours to give the compound of formula (I) (crude). ¹ HNMR(400MHz,CDCl ₃ )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 single crystals of the Compound of formula (I)

The experimental method comprises the following steps:

30 mg of the compound of formula (I) was dissolved in 2mL of acetone, and the dissolved solution was placed in 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 mouth of the pipe is sealed by a sealing film, and a small number of evaporation pores are opened. Standing at room temperature for 10 days, collecting crystals after most of the solvent volatilizes, and performing single crystal X-ray diffraction (SC-XRD) detection.

Experimental results:

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

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TABLE 2 atomic coordinates (. Times.10) of crystals of the Compound of formula (I) ⁴ ) And equivalent isotropic shift parameter/>

TABLE 3 bond lengths of crystals of the Compound of formula (I)And bond angle [ deg ]]/>

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TABLE 4 torsion Angle of crystals of the Compound of formula (I) [ deg ]

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Conclusion: the unit cell diagram of the single crystal is shown in figure 1, and the result shows that the structure of the compound shown in the formula (I) is N- (3- (2- ((1R, 5S) -3-oxabicyclo [3.1.0] hex-1-yl) -6- (2-hydroxyethoxy) pyridine-4-yl) -4-methylphenyl) -2- (trifluoromethyl) isonicotinamide.

Biological testing:

experimental example 1: in vitro enzyme Activity assay

The purpose of the experiment is as follows:

the inhibitory effect of the compounds on RAF kinase activity was examined.

The experimental steps are as follows:

(1) Compound preparation:

the test compound and the reference compound were diluted to 1. Mu.M with DMSO, and the compounds were subjected to 3-fold gradient dilution to obtain target plates of 10 concentration gradients. ATP concentration was 10. Mu.M.

(2) The experimental procedure is as follows:

1) Buffer solution preparation: 20mM HEPES (pH 7.5), 10mM MgCl ₂ ,1mM EGTA,0.01％Brij35,0.02mg/ml,BSA,0.1mM Na ₃ VO ₄ ,2mM DTT,1％DMSO；

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

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

4) Adding a DMSO solution of the compound to the solution using Echo 550;

5) Adding in ³³ P-ATP (specific activity 0.01. Mu. Ci/. Mu.l) initiates the reaction and incubate at room temperature for 2 hours;

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 remaining radio-phosphorylated substrate on the filter paper is detected.

Experimental results: table 5 provides the inhibitory activity of the compounds of the invention against RAF kinase.

In vitro enzymatic Activity of the compounds of Table 5

Conclusion: the compound has remarkable inhibitory activity on RAF enzyme.

Experimental example 2: calu-6 (Kras) ^Q61K ) Antiproliferative activity assay

Experimental materials:

1) Experimental reagent consumable

Name of the name	Brand goods number
		EMEM culture medium	Weisente-320-005-CL
Fetal bovine serum	Biosera-FB-1058/500
		0.25% trypsin	Source culture-S310 KJ
Double antibody (penicillin, streptomycin)	Procell-PB180120
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Experimental instrument

Name of the name	Brand goods number
		Cell counting plate	Refinement of the work
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) The original culture medium in the cell culture flask was removed, cells were digested with pancreatin, and then counted, and the cell suspension was diluted with the culture medium to a cell density of 3.75X10 as required for plating ⁴ Each cell per milliliter;

(3) 100. Mu.L of medium was added to each well around the cell plate, 80. Mu.L of cell suspension was added to the other wells, and the mixture was put into a well containing 5% CO ₂ Is cultured overnight in a 37 degree incubator.

Adding the medicine:

gradient dilution and dosing of the compounds with Echo, and then placing the cell plates back into the incubator for three days;

reading a plate and analyzing data:

add CTG and read plate: to each well of the cell plate, 20 μl celltiter glo was added and the plate read on a Victor Nivo with shaking for 10min in the dark.

Experimental results: 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(s)	Compounds of formula (I)
		IC50 (nM) for anti-proliferative activity of lung cancer Calu-6 cells	600
Colon cancer HCT-116 cell antiproliferative activity IC50 (nM)	1100
		Lung cancer Calu-6 cell ERK phosphorylation inhibitory activity IC50 (nM)	490
Colon cancer HCT-116 cell ERK phosphorylation inhibition activity IC50 (nM)	180

Experimental example 3: HCT-116 (Kras) ^G13D ) Antiproliferative activity assay

Experimental materials:

1) Experimental reagent consumable

Name of the name	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 antibody (penicillin, streptomycin)	Procell-PB180120
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Experimental instrument

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 in the cell culture flask, digesting the cells with pancreatin, counting, diluting the cell suspension with the culture medium to the cell density of 2.5X10 needed for plating ⁴ Each cell per milliliter;

Adding the medicine:

performing gradient dilution and dosing on the compound, and then placing the cell plates back into an incubator for culturing for three days;

reading a plate and analyzing data:

Experimental results: table 6 provides the antiproliferative activity of the compounds of the invention on HCT-116 cells.

Experimental example 4: HCT116 (Kras) ^G13D ) ERK phosphorylation inhibition assay

Experimental materials:

1. reagent consumable

Reagent(s)	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-well plate	COSTAR-3599
DMSO	Sigma-D2650-100mL
		0.05％Trypsin-EDTA	Gibco-25300-062

2. Main instrument

Instrument for measuring and controlling the intensity of light	Manufacturing factories	Model number
			Biological safety cabinet	AIRTECH	BSC-1304IIA2
Carbon dioxide incubator	Thermo	311
			Cell counter	BECKMAN	Vi-cellXR
Enzyme label instrument	PerkinElmer	Envision
			Centrifugal machine	Eppendorf	Centrifuge 5810R

3. Cell information

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

Experimental procedure and method:

1) Cells were recovered and cultured to log phase, digested with pancreatin, seeded in 96-well plates, and placed in incubator overnight.

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

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

4) mu.L of cell lysate was transferred to HTRF plates per well, followed by 4. Mu.L of the formulated antibody mix.

5) After overnight incubation, plates were read with Envision, fitted curves were obtained from ratio (ratio of Ex665/Ex615 fluorescence intensity) and from

Four-parameter fitting formula y=bottom+ (Top-Bottom)/(1+10 + (log EC 50-X) ×hillslope) for Graphpad was calculated to give EC ₅₀ . Experimental results: table 6 provides the inhibitory activity of the compounds of the present invention against HCT-116ERK phosphorylation.

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

Experimental materials:

1) Reagent consumable

2) Main instrument

3) Cell information

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

Experimental procedure and method:

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

Four-parameter fitting formula y=bottom+ (Top-Bottom)/(1+10 + (log EC 50-X) ×hillslope) for Graphpad was calculated to give EC ₅₀ 。

Experimental results: table 6 provides the inhibitory activity of the compounds of the present invention against Calu-6 ERK phosphorylation.

Conclusion: the compounds of formula (I) have good antiproliferative activity and good ERK phosphorylation inhibition activity on both Calu-6 cells and HCT-116 cells.

Experimental example 6: a375 (BRAF) ^V599E ) Antiproliferative activity assay

Experimental materials:

1) Experimental reagent consumable

Name of the name	Brand goods number
		EMEM culture medium	Weisente-320-005-CL
Fetal bovine serum	Biosera-FB-1058/500
		0.25% trypsin	Basal Media-S310KJ
Double antibody (penicillin, streptomycin)	Basal Media-S110JV
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Experimental instrument

Name of the name	Brand goods number
		EnVision	PerkinElmer

The experimental steps are as follows:

cell inoculation:

(2) Removing the original culture medium in the cell culture flask, digesting the cells with pancreatin, counting, diluting the cell suspension with the culture medium to 2000 cells per well with the cell density required for plating;

(3) mu.L of cell suspension was added to each well and the mixture was put into a well containing 5% CO ₂ Is cultured overnight in a37 degree incubator.

Adding the medicine:

mu.L of compound working solution was added to the cell plates per well, and incubation was continued for 5 days at 37℃in a 5% CO2 incubator.

Reading a plate and analyzing data:

add CTG and read plate: to each well of the cell plate 50 μl celltiter glo was added and shaken for 10min and tested using EnVision.

Experimental results: table 7 provides antiproliferative activity of the compounds of the invention on A375 cells.

In vitro cell Activity of the compounds of Table 7

Compounds of formula (I)	A375 cell IC50 (nM)	Colo-205 cell IC50 (nM)
			A compound of formula (I)	269.5	619.5

Experimental example 7: colo-205 (BRAF) ^V599E ) Antiproliferative activity is achievedVerification

Experimental materials:

1) Experimental reagent consumable

Name of the name	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 antibody (penicillin, streptomycin)	Basal Media-S110JV
		CellTiter Glo	Promega-G7573
Cell plate	Corning-3610

2) Experimental instrument

Name of the name	Brand goods number
		EnVision	PerkinElmer

The experimental steps are as follows:

cell inoculation:

(2) Removing the original culture medium in the cell culture flask, digesting the cells with pancreatin, counting, diluting the cell suspension to the cell density of 3000 cells per hole required for plating with the culture medium;

Adding the medicine:

mu.L of compound working solution was added to the cell plates per well, and incubation was continued for 3 days at 37℃in a 5% CO2 incubator.

Reading a plate and analyzing data:

Experimental results: table 7 provides the antiproliferative activity of the compounds of the invention on Colo-205 cells.

Conclusion: the compounds of the invention have significant antiproliferative activity on A375 cells and Colo-205 cells.

Experimental example 8: pharmacokinetic study of single intravenous and oral administration in mice

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

Sample collection and preparation:

after intravenous injection or oral administration, blood samples of animals were collected and the actual blood sampling time was recorded. Immediately after blood collection, the samples were transferred to a labeled K2-EDTA-containing centrifuge tube, followed by centrifugation to obtain plasma. The plasma was transferred to a pre-chilled centrifuge tube, quick 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 the compounds were processed in a non-compartmental model using pharmacokinetic software. Peak concentration (C) _max ) Peak time of sum (T) _max ) And the quantifiable end time, obtained directly from the plasma concentration-time curve. The following pharmacokinetic parameters were calculated using the log linear trapezoidal method: half-life (T) _1/2 ) Apparent distribution volume (V _dss ) And clearance (Cl), area under the time-plasma concentration curve (AUC) from 0 point to end time point _0-last ) Initial concentration (C) ₀ )。

Experimental results:

table 8 pharmacokinetic parameters of single intravenous and oral administration of the compounds of the invention in mice

Conclusion: the compound of formula (I) has better oral absorption in mice, lower clearance rate, longer half-life period and better bioavailability.

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

Experimental materials:

1.1 laboratory animals and feeding Environment

1.1.1 laboratory animals

Species: a mouse

Strain: BALB/c nude mice

Arrival week: 6-8 weeks of age

Gender: female

1.1.2 raising Environment

Animals were kept in SPF-class animal houses with IVC (independent air supply system), constant temperature and humidity cages (3-5 animals per cage)

Temperature: 20-26 DEG C

Humidity: 40-70%

1.2 Compound information

1.3 tumor tissue or cell information

And (3) cells: in vitro culturing of human lung cancer Calu-6 cells, adding 0.2Units/mL bovine insulin,10% fetal bovine serum, and 5% CO at 37deg.C into EMEM culture medium ₂ Incubator culture. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the saturation of the cells is 80% -90% and the number reaches the requirement, the cells are collected, counted and inoculated.

1.4 other reagent information

Name of the name	Manufacturing factories	Goods number	Preservation conditions
				Fetal bovine serum	Hyclone	SV30087.03	-20℃
Trypsin	gibco	25200-072	-20℃
				EMEM culture medium	ATCC	ATCC30-2003	2-8℃

1.5 instrument information

Name of the name	Manufacturing factories	Model number
			Carbon dioxide incubator	Sai Mo Feishi L (Thermo Fisher)	Heracell240i
Low-temperature high-speed centrifugal machine	Ai Bende (Eppendorf)	5810R
			Analytical balance	Sartolius (Sartorius)	SECURA225D-1CN
Common balance	Flat instruments Inc. of Changzhou day	EL-2KJ
			Digital vernier callipers	Sanfeng (three kinds of wind and power plant)	0～150mm

The experimental method comprises the following steps:

2.1 tumor cell seeding

Cell inoculation: 0.2mL of Calu-6 cells (1:1 to matrigel ratio) were inoculated subcutaneously on the right back of each mouse, with an average tumor volume of 173mm ³ The administration of the packets was started at that time.

2.2 grouping of laboratory animals and dosing regimen

Note that: 1, the number of mice in each group; 2, dosing volume parameters: based on the weight of the mice, 10. Mu.L/g. If the weight is reduced by more than 15%, stopping taking the medicine until the weight is recovered to be within 10%, and then taking the medicine; 3:0.5% MC (methylcellulose).

2.3 preparation of test substances

Note 1: the drug should be gently mixed well before administration to animals, with the solvent being polyethylene glycol-15-hydroxystearate and the PEG being polyethylene glycol.

2.4 tumor measurement and Experimental index

Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b ² A and b represent the major and minor diameters of the tumor, respectively.

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) =t _RTV /C _RTV ×100％(T _RTV : RTV mean of treatment group; c (C) _RTV : negative controlGroup RTV average). Relative tumor volume (relative tumor volume, RTV) is calculated from the result of the tumor measurement, calculated as rtv=v _t /V ₀ Wherein V is ₀ Is the tumor volume obtained by measurement of group administration (D0), V _t For tumor volume at a certain measurement, T _RTV And C _RTV The same day data was taken.

TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group)/(mean tumor volume at the end of treatment of solvent control group-mean tumor volume at the beginning of treatment of solvent control group) ]x100%.

2.5 statistical analysis

Statistical analysis was performed using SPSS software based on RTV data at the end of the test. The comparison between groups was analyzed by one-way ANOVA, with variance (significant differences in F values), and examined by using the gas-Howell method. p <0.05 was considered a significant difference.

3. Experimental results

3.1 inhibition of human Lung cancer nude mice subcutaneously transplanted tumor growth by test substances

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

3.2 weight change conditions

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

Conclusion: the compound has remarkable inhibition effect on the growth of tumor-bearing mice of a human lung cancer Calu-6 cell subcutaneous xenograft tumor model.

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

Experimental materials

1.1 laboratory animals and feeding Environment

1.1.1 laboratory animals

Species: a mouse

Strain: BALB/c nude mice

Arrival week: 6-8 weeks of age

Gender: female

1.1.2 raising Environment

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

Temperature: 20-26 DEG C

Humidity: 40-70%

1.2 Compound information

1.3 tumor tissue or cell information

And (3) cells: human colon cancer Colo-205 cells (ATCC-CCL-222) are cultivated in an in vitro adherence way under the condition that 10 percent of fetal bovine serum, 1 percent of penicillin/streptomycin/amphotericin B and 5 percent of CO at 37 ℃ are added into RPMI 1640 medium ₂ Incubator culture. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the saturation of the cells is 80% -90% and the number reaches the requirement, the cells are collected, counted and inoculated.

1.4 other reagent information

Name of the name	Manufacturing factories	Goods number	Preservation conditions
				Fetal bovine serum	Cellmax	SA211.02	-10℃
RPMI 1640 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 seeding and grouping

Cell inoculation: 0.1mL (5X 10) ⁶ Personal) Colo-205 cells were inoculated subcutaneously into the right back of each mouse with an average tumor volume of up to about 150mm ³ The administration of the packets was started at that time.

Grouping and dosing regimen for laboratory animals

Note that: n is the number of mice in each group; dosing volume parameters: based on the weight of the mice, 10. Mu.L/g. If the weight is reduced by more than 15%, stopping administration until the weight is recovered to be within 10% and then administration is performed.

2.3 preparation of test substances

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

2.4 tumor measurement and Experimental index

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) =t _RTV /C _RTV ×100％(T _RTV : RTV mean of treatment group; c (C) _RTV : negative control RTV mean). Relative tumor volume (relative tumor volume, RTV) is calculated from the result of the tumor measurement, calculated as rtv=v _t /V ₀ Wherein V is ₀ Is the tumor volume obtained by measurement of group administration (D0), V _t For tumor volume at a certain measurement, T _RTV And C _RTV The same day data was taken.

2.5 statistical analysis

3. Experimental results

3.1 inhibition of human colon cancer nude mice subcutaneously transplanted tumor growth by test substances

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

3.2 weight change conditions

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

Conclusion: the compound has remarkable inhibition effect on growth of colon cancer Colo-205 cell subcutaneous xenograft tumor model tumor-bearing mice.

Experimental example 11: repeated administration of SD rats by oral lavage for 4 weeks recovered 4 weeks toxicity test was accompanied by toxico-kinetic test

Experimental materials:

1.1 laboratory animals and feeding Environment

1.1.1 laboratory animals

Species: rat (rat)

Strain: SD rat

Age of animals: about 7 to 9 weeks of age

Gender: female and male

1.1.2 raising Environment

Temperature: 20-26 DEG C

Humidity: 40-70%

The cage is a suspension PC transparent rat rearing box on a stainless steel rearing frame, and the male and female parts are separately reared, and each box is not more than 5. The temperature of the animal raising room is controlled at 20-26 ℃ and the relative humidity is controlled at 40-70%. The light/dark is controlled for 12 hours to circulate (such as to avoid the requirement temporarily when the night operation is met).

1.2 Compound information

Name of the name	Molecular weight	Purity (%)	Content (%)	Preservation conditions
					Dispersions of compounds of formula (I)	499.49	97.6	93.1	Sealing at 2-8 deg.c

1.3 other reagent information

1.4 instrument information

Name of the name	Manufacturing factories	Model number
			Blood analyzer	Siemens medical diagnostic products (Shanghai) Inc	ADVIA 2120i
Full-automatic integrated biochemical analyzer	Siemens medical diagnostic products (Shanghai) Inc	Dimension EXL 200
			Electrolyte analyzer	Mideca medical device (su state) limited	EasyLyte PLUS
Full-automatic hemagglutination 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 method for preparing solvent preparation

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

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

3) Sealing and preserving for 3 months at the temperature of 2-8 ℃.

2.1.2 preparation method of blank adjuvant preparation

1) Measuring a solvent preparation with a target volume for standby;

2) Accurately weighing (not more than theoretical amount of + -1%) required blank auxiliary materials into a clean and dry mortar, adding a small amount of solvent preparation, grinding until no obvious particles are observed in eyes, transferring to a proper container, rinsing the mortar with the rest of solvent preparation, and transferring to the container together with rinsing liquid;

3) Stirring with magnetic stirrer for at least 1 hr until visual uniformity.

* Theoretical amount = theoretical concentration of high concentration test article preparation x volume/test article content x auxiliary material content in test article = theoretical concentration of high concentration test article preparation x volume/46.5% x50%.

2.1.3 preparation method of sample preparation

1) Measuring and preparing a volume solvent preparation of a target for standby;

2) Accurately weighing a required amount (not more than theoretical amount of +/-1.0%) of a test sample into a clean and dry mortar, adding a small amount of solvent preparation, grinding until no obvious particles are observed in eyes, transferring the mixture into a proper container, rinsing the mortar with the rest solvent preparation, and transferring the mixture into the container together with a rinsing liquid;

3) Dispersing by a dispersing machine until visual uniformity is achieved;

4) Stirring with magnetic stirrer for at least 1 hr until visual uniformity.

Note that: * Theoretical amount = theoretical concentration x volume/content = theoretical concentration x volume/46.5%.

2.2 grouping and administration of animals

2.2.1 grouping of animals

Rats were stratified randomly grouped into 8 groups based on recent animal body weight using the Provantis system according to random and body weight balance principles.

2.2.2 animal administration

Route of administration: oral lavage;

Administration period and frequency: dosing is carried out 1 time a day, continuous dosing is carried out for 28 days, and stopping dosing is carried out for 28 days;

pre-and in-dose requirements: the sample preparation and the adjuvant preparation are stirred for at least 30min before administration, and stirring is continued until the administration is completed.

Grouping and dosing regimen for laboratory animals

Note that: 1. animals with 11-15 last two animals numbered enter recovery phase.

2. Animal number 4, thousand indicated sex, "1" male, "2" female; the last two bits represent the intra-group sequence numbers.

3. If animals need to be kept separately for trauma or other reasons, the records are stored in the original record, only as the original record.

4. Dosages are calculated for the compound dispersion API of formula (I).

2.3 Experimental detection

2.3.1 clinical pathology sampling and detection

Blood: all animals were fasted overnight before blood collection, and were not limited to drinking water. Blood was collected via the abdominal aorta using a disposable vacuum negative pressure tube. EDTA-K for hematology ₂ An anticoagulant tube, the blood collection amount is about 1.0mL; the serum biochemistry/electrolyte adopts a separation gel/coagulant tube, and the blood collection amount is about 3.0mL; the blood coagulation is conventionally carried out by adopting a sodium citrate anticoagulation tube, and the blood collection amount is about 1.0mL.

Urine: placing the rat (single rat) in a clean metabolism cage, sucking not less than 0.5mL of urine by a syringe after a certain amount of urine is stored in a collecting pipe, transferring the urine into a clean urine collecting pipe, and recording information such as collecting time and volume.

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

2.3.2 toxicological kinetic sampling and evaluation

Blood samples of animals at each time point of day 1 and day 28 of administration were collected at about 0.2 mL/time point and a 1.5mL centrifuge tube containing 15. Mu.L of EDTA-K at a concentration of 30mg/mL ₂ Anticoagulant (EDTA-K) ₂ The preparation method comprises the following steps: to prepare 10mL 30mg/mL EDTA-K ₂ For example, 300mg EDTA-K was accurately weighed ₂ Put into a clean container, added with 10mL of ultrapure water, shaken uniformly, and stored in a closed state at 2-8 ℃ for standby).

The concentration of the compound of formula (I) in plasma was determined by LC-MS/MS method, and after analysis of the concentration of the compound of formula (I) in plasma by analytical 1.6.3 and Watson LIMS 7.4 software, concentration and time curves were drawn and at least the following parameters were calculated using WinNonlin 8.1: concentration-timeArea under the inter-curve (AUC) _0-t Or AUC _0-∞ ) Maximum peak concentration (C) _max ) Peak time (T) _max ). Reporting principal parameters (including blood concentration at various times, C _max 、T _max And AUC), average and standard deviation values, each parameter being calculated for each individual class.

2.4 data processing and analysis

The data collected directly by Provantis 10.2.3, and the data entered back into Provantis 10.2.3, were processed and statistically analyzed using SAS statistical software built in Provantis 10.2.3. The raw data of the LC-MS/MS measured toxin were derived using the analysis 1.6.3 software and further statistically analyzed using Microsoft Excel. The application software Agilent OpenLAB CDS 2.4.4 collects and processes data.

Experimental results:

no obvious abnormality was observed in clinical pathology and toxico-kinetics detection of female and male SD rats in each experimental group, and no abnormality was observed in serum biochemical total bilirubin.

Conclusion of experiment:

after the compound shown in the formula (I) is orally and gastrographically administrated to SD rats, the safety medicine is good, the male MTD is more than or equal to 300 mg/kg, and the female MTD is more than or equal to 100 mg/kg.

Experimental example 12: beagle dogs were repeatedly given by oral gavage for 4 weeks and 4 weeks of toxicity test was followed by pharmacokinetic test

Experimental materials:

1.1 laboratory animals and feeding Environment

1.1.1 laboratory animals

Species: dogs

Strain: beagle dog

Age of animals: 7-8 months of age

Gender: female and male

1.1.2 raising Environment

Temperature: 19-26 DEG C

Humidity: 40-70%

Animals were kept in stainless steel cages in single cages. The temperature of the raising room is controlled at 19-26 ℃ and the relative humidity is controlled at 40-70%. The light/dark is controlled for 12h circulation (such as to avoid the requirement temporarily when the night operation is met).

1.2 Compound information

1.3 other reagent information

Name of the name	Manufacturing factories	Preservation conditions
			Kollidon VA 64	BASF SE	Sealing at 10-30 deg.c
Methylcellulose and process for producing the same	Alfa elsa (china) chemical Co., ltd	Room temperature

1.4 instrument information

Experimental methods and procedures

2.1 preparation and preservation

2.1.1 method for preparing solvent preparation

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

3) Sealing and preserving for 3 months at the temperature of 2-8 ℃.

2.1.2 preparation method of blank adjuvant preparation

1) Measuring a solvent preparation with a target volume for standby;

3) Stirring with magnetic stirrer for at least 1 hr until visual uniformity.

2.1.3 preparation method of sample preparation

3) Dispersing by a dispersing machine until visual uniformity is achieved;

4) Stirring with magnetic stirrer for at least 1 hr until visual uniformity.

2.2 grouping and administration of animals

2.2.1 grouping of animals

The dogs were stratified randomly grouped into 4 groups based on recent animal weight using the Provantis system according to the random and weight balance principles.

2.2.2 animal administration

Route of administration: oral lavage;

administration period and frequency: dosing was 1 time daily, 4 weeks continuous, 4 weeks recovery from D29 withdrawal;

pre-and in-dose requirements: the animals were dosed about 1h after feeding. The pre-dosing formulation was stirred at room temperature for at least 1h and stirring was continued during dosing until dosing was completed.

Grouping and dosing

Note that: 1. animals with animal numbers end 4, 5 enter recovery phase.

3. Dosages are calculated for the compound dispersion API of formula (I).

2.3 Experimental detection

2.3.1 clinical pathology sampling and detection

Blood: all animals were collected before feeding (exception of moribund animals) and were not limited to drinking water. Blood was collected via the forelimb or hindlimb vein using a disposable vacuum negative pressure tube. EDTA-K for hematology ₂ An anticoagulant tube, the blood collection amount is about 1.0mL; the serum biochemistry/electrolyte adopts a separation gel/coagulant tube, and the blood sampling amount is about 3.0mL; the blood coagulation is conventionally carried out by adopting a sodium citrate anticoagulation tube, and the blood collection amount is about 1.0mL.

Urine: after fresh urine was collected with a clean stainless steel urine collection tray, about 1.0mL of urine was withdrawn with a disposable sterile syringe and transferred to a clean urine collection tube (identified by a corresponding label template inside the mechanism) and the information on its collection time and volume was recorded.

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

2.3.2 toxicological kinetic sampling and evaluation

Blood samples of animals were collected at each time point of day 1 and day 28 of administration, about 1mL, containing EDTA-K ₂ An anticoagulant blood collection tube.

After analysis of the concentration of the compound of formula (I) in plasma using analysis 1.6.3 and Watson LIMS 7.4 software, concentration and time curves were plotted and at least the following parameters were calculated using WinNonlin 8.1: area under concentration-time curve (AUC _0-t Or AUC _0-∞ ) Maximum peak concentration (C) _max ) Peak time (T) _max ). Reporting principal parameters (including blood concentration at various times, C _max 、T _max And AUC), mean and standard deviation values. The parameters are calculated for each different class.

2.4 data processing and analysis

Experimental results

No obvious abnormality was observed in clinical pathology and toxico-kinetic detection of female and male Beagle dogs in each experimental group, and no abnormality was observed in serum biochemical total bilirubin. Conclusion of experiment:

after the compound shown in the formula (I) is orally and gastrographically administrated to Beagle dogs, the safety medicine is good, and the MTD is more than or equal to 100 milligrams per kilogram.

Claims

1. 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,

2. a pharmaceutical composition comprising the compound N- (3- (2- ((1 r,5 s) -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) as defined in claim 1 or a salt thereof or a pharmaceutical composition as defined in claim 2 for the preparation of a RAF kinase inhibitor.