CN115433231A - Biaryl compounds - Google Patents

Biaryl compounds Download PDF

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CN115433231A
CN115433231A CN202210626503.4A CN202210626503A CN115433231A CN 115433231 A CN115433231 A CN 115433231A CN 202210626503 A CN202210626503 A CN 202210626503A CN 115433231 A CN115433231 A CN 115433231A
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刘希乐
丁照中
陈曙辉
胡利红
周成亮
孙德恒
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Qilu Pharmaceutical Co Ltd
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Medshine Discovery Inc
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    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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Abstract

A biaryl compound, specifically discloses a compound shown in a formula (II) or a pharmaceutically acceptable salt thereof, and applications of the compound and the pharmaceutically acceptable salt thereof in treating related diseases.

Description

Biaryl compounds
The following priority is claimed in the present application:
application No. CN202110624336.5, application date 2021, 06 month 04.
Technical Field
The invention discloses a biaryl compound, and particularly discloses a compound shown as a formula (II) or a pharmaceutically acceptable salt thereof, and application of the compound and the pharmaceutically acceptable salt thereof in treatment of related diseases.
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 the RAS, and can be activated by the 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, a key signal protein downstream of RAS, has important research significance in the treatment of RAS mutant gene mutant tumors. Through inhibiting RAF kinase, MAPK signal transduction is regulated, so that the RAF inhibitor has an effect on proliferation of RAS mutant tumor cells. Therefore, RAF kinase has become an important target for clinical treatment of tumors.
The first generation BRAF kinase inhibitors Vemurafenib, dabrafinib and Encorafenib have been approved by the FDA for the development of B-Raf V600E Treatment of mutant cancers. Although Vemurafenib, dabrafinib and Encorafenib are in B-Raf V600E The 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. The group which is found to generate V600E mutation in BRAFIn addition, NRAS mutations can lead to activation of the MAPK pathway in the presence of inhibitors, resulting in drug resistance. Mutant N-Ras promotes B-Raf V600E And 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 of inhibitors and further expand the clinical application.
Disclosure of Invention
The present invention provides compounds of formula (II)
Figure BDA0003677844870000021
The invention also provides the application of the compound shown in the formula (II) in the preparation of the RAF kinase inhibitor,
Figure BDA0003677844870000022
the invention also provides the application of the compound shown in the formula (II) in the preparation of the medicine for treating lung cancer,
Figure BDA0003677844870000023
technical effects
The compound of the invention has good drug property, good RAF enzyme inhibition activity and muchThe seed cell has antiproliferative activity and good in-vivo drug effect. Hopefully solving the current BRAF V600E Resistance to mutant cancer treatments, and provide effective treatments for RAS mutant-type cancers.
Definitions and explanations
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 (SXRD), diffraction intensity data of the cultured single crystal is collected by Bruker D8 vision diffractometer, the light source is CuK α radiation, and the scanning mode:
Figure BDA0003677844870000024
after scanning and collecting relevant data, the crystal structure is further analyzed by a direct method (Shelxs 97), so that the absolute configuration can be confirmed.
The solvent used in the present invention can be commercially available.
The invention employs the following abbreviations:
Pd(dppf)Cl 2 DCM represents [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride-dichloromethane complex, [ Ir (COD) OMe] 2 Represents cyclooctadiene methoxyiridium dimer, tmphen represents 3,4,7, 8-tetramethyl-1, 10 phenanthroline, pd 2 dba 3 Represents tris-diphenylpropanedione-dipalladium, brettphos represents (2-dicyclohexylphosphine-3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl, PE represents petroleum ether, EA represents ethyl acetate, DMSO represents dimethyl sulfoxide.
Detailed Description
The present invention is described in detail below by way of examples, but is not meant to be limited 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 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 present invention without departing from the spirit and scope of the invention.
Example 1: preparation of Compounds of formula (I)
Figure BDA0003677844870000031
Compound 2
To a solution of compound 1 (10 g, 51.96 mmol) in dichloromethane (100 mL) was added n-butyllithium (23ml, 2.5 m) at-78 ℃ and 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 (40 mL), and extracted with dichloromethane (40 mL × 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 isolate 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 2 (6 g, 30.06 mmol) in toluene (80 mL) 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 (40 mL) and ethyl acetate (100 mL), and the mixture was separated, washed with a 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 obtain 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), 2a (7.3 g, 33.17 mmol) was added DMSO (50 mL). The reaction mixture was stirred at 25 ℃ for 1 hour under nitrogen. Adding into the reaction solutionA solution of 3 (1 g, 5.51 mmol) in DMSO (5 mL) was added and the reaction mixture was heated to 70 ℃ and stirred for 6 hours. Water (180 mL) was added to the reaction mixture, 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 obtain 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
Mixing 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-tert-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/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 micromoles) 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 ℃ and stirred for 3 hours under nitrogen. The reaction mixture was filtered, the filtrate was concentrated, ethyl acetate (20 mL) and water (10 mL) were added, liquid separation was performed, the aqueous phase was extracted with ethyl acetate (10 mL × 3), the combined organic phase was dried over anhydrous sodium sulfate and concentrated, and separation was performed by silica gel column chromatography (PE/EA =10/1 to 5/1) to obtain 6. 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 6 (900 mg, 1.90 mmol) and 5a (400 mg, 2.27 mmol) in toluene (30 mL) was added Pd 2 dba 3 (180 mg, 196.57. Mu. Mol), 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. 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
Hydrochloric acid (1mL, 4M) was added to 7 (300 mg, 488.81. Mu.mol) of tetrahydrofuran (10 mL), and the reaction mixture was stirred at 25 ℃ for 1 hour. The reaction solution was diluted with ethyl acetate (20 mL), adjusted to pH =8 with a saturated solution of sodium hydrogencarbonate, extracted with ethyl acetate (10 × 3 mL), the organic phase was dried over anhydrous sodium sulfate and concentrated, and separated on a thin layer silica gel plate (PE/EA =1/1, V/V) to obtain 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)
Compound 8 was subjected to SFC chiral separation (chiral column DAICEL CHIRALCEL OJ-H (250mm 30mm,5 μm), mobile phase A: ethanol (0.05% DIEA; mobile phase B: carbon dioxide) to give 8A (retention time 1.487 min) and the compound of formula (I) (retention time 1.590 min). 8A is an enantiomer of the 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％)。
EXAMPLE 2 preparation of Compound of formula (II)
Figure BDA0003677844870000061
Compound II-1
To a compound of formula (I) (1.0 g, 2.0 mmol), II-1A (1.4 g, 4.04 mmol) in dichloromethane (40 mL) was added tetrazole (284.0 mg, 4.05 mmol) and the reaction mixture was heated to 40 ℃ under nitrogen blanket and stirred for 16 h. The reaction solution is directly used for the next reaction to obtain a crude product of the compound II-1.
Compound II-2
To compound II-1 (1.5 g, 2.02 mmol) in dichloromethane (40 mL) was added II-2A (0.8 mL, 4.05 mmol, 5M n-hexane solution) and the reaction mixture was stirred under nitrogen at 25 ℃ for 45 min. The reaction solution was separated by silica gel column chromatography (PE/EA =5/1 to 2/1, containing 20% dichloromethane) to obtain compound II-2. 1 H NMR(400MHz,DMSO-d 6 )δ10.69(s,1H),8.99(br d,J＝5.0Hz,1H),8.36(s,1H),8.19(br d,J＝4.6Hz,1H),7.82-7.71(m,1H),7.64(s,1H),7.33(s,11H),6.76(s,1H),6.56(s,1H),5.13-4.88(m,4H),4.47(br s,2H),4.33(br s,2H),4.14-4.02(m,2H),3.82-3.67(m,2H),2.25-2.16(m,3H),2.16-2.11(m,1H),1.36(br dd,J＝3.6,7.8Hz,1H),1.00-0.93(m,1H)。
Compound II-3
To compound II-2 (1.4 g, 1.84 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (10 mL, 135.06 mmol) and water (1 mL, 55.49 mmol), and the reaction mixture was stirred at 25 ℃ for 8 hours. The reaction solution was concentrated, dissolved in ethyl acetate (30 mL), washed with water (20 mL × 2), a saturated sodium carbonate solution (30 mL) was added to the organic phase, the organic phase was discarded after separation, the aqueous phase was washed with ethyl acetate (15 mL × 2), hydrochloric acid (2M) was added to the aqueous phase to adjust pH =3-4, extraction was performed with ethyl acetate (20 mL × 3), and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude compound II-3. 1 HNMR(400MHz,DMSO-d 6 )δ10.70(s,1H),8.99(d,J＝5.0Hz,1H),8.36(s,1H),8.19(d,J＝5.0Hz,1H),7.75(dd,J＝2.0,8.4Hz,1H),7.65(d,J＝2.0Hz,1H),7.34(d,J＝8.4Hz,1H),6.76(s,1H),6.60(s,1H),4.50-4.40(m,2H),4.20-4.13(m,2H),4.11-4.03(m,2H),3.84-3.73(m,2H),2.23(s,3H),2.19(ddd,J＝2.6,5.0,7.8Hz,1H),1.39(dd,J＝3.8,7.8Hz,1H),0.98(t,J＝4.4Hz,1H)。
A compound of formula (II)
To compound II-3 (1.0 g, 1.73 mmol) in tetrahydrofuran (10 mL) was added an aqueous solution (10 mL) of sodium hydroxide (140 mg, 3.50 mmol) and the reaction mixture was stirred at 25 ℃ for 1 hour. The reaction was concentrated, dissolved in acetonitrile (10 mL) and water (40 mL), and lyophilized under vacuum to give the compound of formula (II). 1 HNMR(400MHz,DMSO-d 6 )δ11.08(s,1H),8.99-8.92(d,J＝5.0Hz,1H),8.38(s,1H),8.33-8.25(d,J＝5.0Hz,1H),7.88-7.80(dd,J＝1.6,8.4Hz,1H),7.72-7.65(d,J＝1.8Hz,1H),7.33-7.26(d,J＝8.4Hz,1H),6.72(s,1H),6.59(s,1H),4.37-4.31(br t,J＝4.8Hz,2H),4.11-4.02(m,2H),3.98-3.92(br d,J＝4.8Hz,2H),3.80-3.75(m,2H),2.21(s,3H),2.18-2.10(ddd,J＝2.4,4.8,7.6Hz,1H),1.39-1.32(dd,J＝4.0,7.6Hz,1H),0.98-0.93(t,J＝4.4Hz,1H)。MS(ESI)m/z:580.1[M+3H-2Na] + 。
Example 3: in vitro enzyme Activity assay
Purpose of the experiment:
the inhibitory effect of the compounds on the cRAF enzyme activity was examined.
Experimental materials:
TABLE 1
Experimental Material Brand goods number
cRAF protein Creative BioMart-RAF1-416H
MEK1 protein Invitrogen-PR3984A
ADP-Glo kinase detection kit Promega-V9102
Tris-HCl,pH 7.4 Sigma-T2663-1L
MgCl 2 Sigma-63020-1L
NaCl Sigma-S5150
DTT Invitrogen-P2325
Triton X-100 Sigma-X100
H 2 O Gibco-15230-162
384 middle plate Greiner-781280
384 experiment board PerkinElmer-6007299
The experimental steps are as follows:
(1) Preparation of compound:
test and reference compounds were diluted to 100 μ M with DMSO and 3-fold gradient dilutions of compounds were performed with Echo to give 11 concentration-gradient target plates.
(2) The experimental process comprises the following steps:
1) Preparing a buffer solution: 50mM Tris-HCl (pH 7.4), 3.5mM MgCl 2 ，150mM NaCl，1mM DTT，0.02％Triton X-100，H 2 O；
2) Preparing a mixture of MEK1 and ATP with a buffer solution, and adding 5 μ L of the substrate mixture to the 384 intermediate plate;
3) The cRAF enzyme was diluted with buffer and 5 μ L was added to 384 intermediate plates;
4) Transfer 5 μ L of reaction mixture to 384 plates with Bravo, centrifuge for 15 seconds and incubate in a 23 ℃ incubator.
5) After 1 hour, 5. Mu.L ADP-Glo was added to the 384 assay plates, shaken, centrifuged for 15 seconds, and incubated in a 23 ℃ incubator.
6) After 40 minutes, 10. Mu.L of kinase assay reagent was added to the 384 assay plates, shaken, centrifuged for 15 seconds, and incubated in a 23 ℃ incubator.
7) After 1 hour, plates were read on Envision.
The experimental results are as follows: table 2 provides the inhibitory activity of the compounds of the present invention on cRAF enzyme.
Table 2: in vitro Activity of Compounds
Figure BDA0003677844870000081
Example 4: calu-6 (Kras) Q61K ) Experiment for antiproliferative Activity
Experimental materials:
TABLE 3 Experimental reagent consumables
Name (R) Brand goods number
EMEM culture medium Vickers-320-005-CL
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
TABLE 4 Experimental instruments
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 × 10 4 Per ml of individual cells;
(3) Adding 100. Mu.L of medium to each well around the cell plate, adding 80. Mu.L of cell suspension to the other wells, adding 5% CO 2 And 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 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 1 provides the antiproliferative activity of the compounds of the invention on Calu-6 cells.
Example 5: HCT-116 (Kras) G13D ) Experiment for antiproliferative Activity
Experimental materials:
TABLE 5 Experimental reagent consumables
Name(s) 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
TABLE 6 Experimental apparatus
Name(s) BrandGoods 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 plating 4 Per ml of individual cells;
(3) Adding 100. Mu.L of medium to each well around the cell plate, adding 80. Mu.L of cell suspension to the other wells, adding 5% CO 2 And the culture was carried out overnight in a 37 ℃ 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 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 2 provides the antiproliferative activity of compounds of formula (I) on HCT-116 cells.
Example 6: HCT116 (Kras) G13D ) ERK phosphorylation inhibition assay
Experimental materials:
TABLE 7 reagent consumables
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
TABLE 8 Main instruments
Instrument for measuring the position of a moving object Manufacturer of the product Type 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
TABLE 9 cell information
Cell name Source Goods number
Colon cancer HCT116 ATCC ATCC-HTB-132
Experimental procedures and methods:
1) The cells were revived and cultured to logarithmic growth phase, digested with trypsin, 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 30 min 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 and fitted to the ratio of ratio (Ex 665/Ex615 fluorescence intensity) according to
Calculating a four-parameter fitting formula Y = Bottom + (Top-Bottom)/(1 +10^ ((LogEC 50-X) × hillSlope)) of Graphpad to obtain EC 50 。
The experimental results are as follows: table 1 provides the inhibitory activity of the compounds of formula (I) on HCT-116ERK phosphorylation.
Example 7: calu-6 (Kras) Q61K ) ERK phosphorylation inhibition assay
Experimental materials:
TABLE 10 reagent consumables
Reagent Brand goods number
High-sensitivity detection kit for human ERK phosphorylated protein Cisbio-64AERPEH
RPMI1640 culture 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
Watch 11 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 mark instrument PerkinElmer Envision
Centrifugal machine Eppendorf Centrifuge 5810R
TABLE 12 cell 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 30 min at room temperature.
4) Transfer 16 μ L of cell lysate to the HTRF plate per well followed by 4 μ L of prepared antibody cocktail.
5) After overnight incubation, plates were read with Envision, fitted curves were obtained based on 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 2 provides the inhibitory activity of the compounds of formula (I) on Calu-6 ERK phosphorylation.
And (4) conclusion: the compound of the formula (I) has stronger inhibitory activity to cRAF enzyme, and simultaneously has better antiproliferative activity and ERK phosphorylation inhibitory activity to Calu-6 cells and HCT-116 cells.
Example 8: in vivo pharmacokinetic study experiment of mice
Purpose of the experiment
Detection of pharmacokinetic parameters of Compounds of the invention in mice
Experimental protocol
1) Experimental drugs: compounds of formula (I) and compounds of formula (II);
2) Experimental animals: 4 female CD-1 mice, divided into 2 groups of 2 mice each;
3) Preparing the medicine: appropriate amounts of drug were weighed and dissolved in aqueous solution of solutol (5% by volume), DMSO (5% by volume) and PEG-300 (25% by volume) for administration by injection. Appropriate amount of the drug is weighed and dispersed in 5% solutol aqueous solution (80% volume), then PEG-400 (20% volume) is added, and the mixture is mixed evenly for gastric lavage administration. The solutol is polyethylene glycol-15-hydroxystearate.
Experimental procedures
Animals in the first group were given a dose of 2mg/kg of compound at a concentration of 1mg/mL by intravenous injection. The animals collected plasma samples at 0.117, 0.333, 1, 2, 4,7 and 24 hours post-dose; a second group of animals was administered a compound at a dose of 100mg/kg and a concentration of 10mg/mL by gavage. The animals collected plasma samples at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dose; the drug concentration at each point was measured using LC-MS/MS method, giving the kinetic parameters of the tested drugs as shown in tables 13 and 14 below.
Watch 13
Figure BDA0003677844870000121
TABLE 14
Figure BDA0003677844870000122
Note: * the mass of the compound of the formula (II) is converted into the mass of the compound of the formula (I) when the compound of the formula (II) is administrated by gastric lavage, ** the measured values are parameters of the compounds of the formula (I).
The experimental conclusion is that: the compound of the invention has good in vivo pharmacokinetic properties in mice.
Example 9: in vivo pharmacokinetic study experiment of rats
Purpose of the experiment
Testing the pharmacokinetic Properties of the Compounds of the invention in rats
Experimental protocol
1) Experimental drugs: a compound of formula (II);
2) Experimental animals: 2 male SD rats;
3) Preparing the medicine: appropriate amount of the medicine is weighed and dissolved in water for intragastric administration.
Experimental procedures
Animals were administered a dose of 870mg/kg of compound at a concentration of 87mg/mL by gavage. The animals collected plasma samples at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dose; the concentrations of the compound of formula (II) and the compound of formula (I) in plasma at each time point were determined using LC-MS/MS method as in table 14 below.
Watch 15
Compound/time point 0.083h 0.25h 0.5h 1h 2h 4h 6h 8h 24h
Concentration (nM) of the Compound of formula (I) 336 4687 14439 15218 13303 11684 8838 9788 4060
Concentration (nM) of the Compound of formula (II) 2001 1112 727 311 55 179 98 82 25
And (4) experimental conclusion: the compound of the formula (II) can be rapidly metabolized into the compound of the formula (I) in a rat body, and has good pharmacokinetic properties.

Claims (3)

1. A compound of formula (II)
Figure FDA0003677844860000011
2. The use of a compound of formula (II) in the preparation of a RAF kinase inhibitor,
Figure FDA0003677844860000012
3. the application of the compound shown in the formula (II) in preparing the medicine for treating lung cancer,
Figure FDA0003677844860000013
CN202210626503.4A 2021-06-04 2022-06-02 Biaryl compounds Pending CN115433231A (en)

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