CN110669038B - Pyrimidine FGFR4V550LInhibitor, preparation method and application thereof - Google Patents

Abstract

The invention disclosesA pyrimidine FGFR4 is disclosed^V550LInhibitor, preparation and application thereof, pyrimidine FGFR4^V550LThe structure of the inhibitor is shown in a formula (I), Ar is substituted or unsubstituted aryl, and the substituent on the aryl is alkyl, alkoxy, halogen or 4-methylpiperazin-1-yl. Test results show that the 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound can effectively inhibit FGFR4^V550LCan be used as FGFR4 with high potential inhibitory activity^V550A potent compound.

Description

Pyrimidine FGFR4V550LInhibitor, preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a 2-N-aryl-4-N-aryl-5-trifluoromethylpyrimidine compound and a preparation method and application thereof.

Background

Fibroblast Growth Factor Receptors (FGFRs) are a class of transmembrane receptor tyrosine kinases, and currently known FGFRs mainly include 4 types, namely FGFR1, FGFR2, FGFR3 and FGFR4, and FGFR family members play an important role in signal pathways controlling cell proliferation and differentiation. Among 4 FGFR members, FGFR4 and FGFR 1-3 have larger difference in structure and function. Structurally, FGFR4 has the least homology with other three types, the difference of protein domains is large, and the D3 domain structure of the extracellular part of FGFR4 is also different from FGFR 1-3. Functionally, the high expression of the FGFR1 gene is commonly found in squamous non-small cell lung cancer, breast cancer, ovarian cancer, bladder cancer and the like, the high expression of FGFR2 and 3 is related to gastric cancer, breast cancer and bladder cancer, and the high expression of FGFR4 is closely related to liver cancer. In addition, the knockout of FGFR4 does not lead to early embryonic lethality, and is not involved in regulating skeletal development as FGFR 1-3 does. Therefore, a small molecule inhibitor that selectively targets FGFR4 would be relatively more advantageous, without significant side effects, to treat tumors.

A partial FGFR4 inhibitor has been reported or entered into clinical trials in succession. Currently, important FGFR4 inhibitor compounds are known: pyrazoles such as AZD454714 and CH 518328415; pyrimidine derivatives such as NVP-BGJ 39816; indazole compound LY287445517 and compound Ponatiniib with imidazo [1,2-b ] pyridazine as main pharmacophore. These inhibitors show varying degrees of inhibitory activity and selectivity against FGFR4 and its family members. British Blueprint pharmaceutical company publishes a first FGFR4 selective inhibitor BLU9931, and the compound takes quinazoline as a parent nucleus and shows good FGFR4 selective inhibition effect in vivo and in vitro tests. However, BLU9931 eventually fails to enter clinical research stage due to drug-induced deficiency such as unreasonable physicochemical parameters and poor water solubility (HAGEL M, MIDUTURU C, SHEETS M, equivalent. first selective small molecule inhibitor of FGFR4 for the treatment of the refractory fibrous pathogens with FGFR activated 4signaling pathway [ J ]. CancerDiscov,2015,5,424-. BLU554, which has a modified chemical structure, has better solubility than BLU9931, and has been used as a liver cancer therapeutic drug in the clinical phase I research stage (Lin X, Yosaatmadja Y, KalyukinaM, et al.

Although the FGFR4 inhibitor develops rapidly, besides the inhibition capability and selectivity of most of the inhibitors are not high enough, the existing better inhibitors are often subjected to the problems of drug-induced deficiency, metabolism or off-target effect and the like, and only few inhibitors can be finally clinically researched. In addition, secondary resistance is another important reason limiting the clinical utility of FGFR4 inhibitors (GALLH, NELSON KN, MEYER AN, et al]Cytokine growth factor Rev,2015,26(4): 425-449.). The first-generation FGFR4 inhibitor or a plurality of multi-target inhibitors (such as sorafenib and the like) usually have obvious secondary drug resistance (such as V550L residue dislocation mutation) after clinical medication for 9-12 months, thereby greatly limiting the survival of tumor patientsProlongation of time (GAO L, WANG X, TANG Y, et al FGF19/FGFR4signaling constraints to the resistance of a hepatocellular carbon sorbent [ J]J ExpClin Cancer Res,2017,36(1): 8.). However, as yet, it has not been shown to be useful for reversing FGFR4^V550LLiterature reports of drug-resistant small molecule inhibitors.

Disclosure of Invention

The invention provides a pyrimidine FGFR4^V550LInhibitor, preparation and application thereof, and pyrimidine FGFR4^V550LThe inhibitor can be used as a potential anticancer drug.

The technical scheme of the invention is as follows:

pyrimidine FGFR4^V550LThe inhibitor is a compound 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound, and the structure of the compound is shown as the formula (I):

in the formula (I), Ar is substituted or unsubstituted aryl;

r is CF₃、-COOC₂H₅H or NO₂(ii) a R is preferably CF₃；

The substituent on the aryl is alkyl, alkoxy, halogen or 4-methylpiperazin-1-yl.

Preferably, the pyrimidine FGFR4^V550LThe inhibitor is a compound represented by the following formula:

the in vitro kinase inhibition activity test result shows that the compound has FGFR4 effect^V550LThe kinase has strong inhibitory activity and IC₅₀A value of 45.8nM higher than other similar compounds

The invention also provides a preparation method of the 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound, which comprises the following steps:

(1) under the action of N, N-dimethylformamide and alkali, 2, 4-dichloro-5-trifluoromethylpyrimidine reacts with 6-aminoindazole to obtain an intermediate;

(2) and (2) under the action of methanol and acid, reacting the intermediate obtained in the step (1) with arylamine to obtain the 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound.

Preferably, in step (1), the base is N, N-diisopropylethylamine.

Preferably, in step (2), the acid is trifluoroacetic acid.

Preferably, in the step (2), the arylamine is 4- (4-methylpiperazine) aniline.

The invention also provides a preparation method of the 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound, and the medicine is used for treating or preventing tumors.

Preferably, the medicament is used for selectively inhibiting FGFR 4.

Preferably, the medicament is used for inhibiting liver cancer cells.

The synthesized 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound is a new compound, and meanwhile, the result of in vitro antitumor kinase inhibition evaluation shows that the compound has FGFR4 effect^V550LThe kinase has the strongest inhibitory activity, and the inhibitory activity is improved by about 30 percent compared with that of a lead compound D3.

Detailed Description

Example 1

Synthesis of Compounds

The melting point was measured using an X-4 micro melting point apparatus (temperature not corrected); the mass spectrum was measured using an Agilent 1100 series single quadrupole LC Mass spectrometer.¹H-NMR、¹³C-NMR was determined using a BrukeraVANCE III 500 nuclear magnetic resonance apparatus (DMSO-d)₆As solvent, TMS as internal standard); GF for thin layer chromatography₂₅₄Silica gel powder (200-300 meshes) for silica gel plate and column chromatography is purchased from Aladdin reagent company (Shanghai Crystal pure and technology Co., Ltd.) and chemical reagent company of national drug group, respectively; other used reagents and solvents are all commercial analytical pure products, and are used after anhydrous drying treatment according to requirements.

The synthetic route is as follows:

1.1 general Synthesis of intermediates 3a to 3j (example 3a)

333.94mg (2.0mmol) of 2, 4-dichloro-5-fluoropyrimidine and 516.96mg (4.0mmol) of N, N-Diisopropylethylamine (DIPEA) were dissolved in 5mL of dry N, N-Dimethylformamide (DMF). 6-Aminoindazole (266.3mg, 2mmol) dissolved in 2mL DMF was added slowly with stirring in an ice bath. After 1 hour, the reaction was slowly warmed to room temperature and monitored by TLC. After the reaction is finished, a large amount of cold ultrapure water is added into the reaction solution, and precipitates are separated out. Suction filtration, ethanol washing and filter cake drying to obtain the light yellow solid (3a)55.8mg, yield 70.2%. The yield of 3 b-3 j is 10-95.1%.

1.24 Synthesis of (4-methylpiperazine) nitrobenzene (6)

1.0g (7.1mmol) of p-fluoronitrobenzene (4) and 1.42g (14.17mmol) of N-methylpiperazine (5) were dissolved together in 10mL of dry dimethyl sulfoxide (DMSO), and 1.96g (14.17mmol) of potassium carbonate was added to the mixed solution. Stirring and reacting for 5h at normal temperature, adding ice water into the reaction solution, separating out a precipitate, performing suction filtration, and drying a filter cake to obtain 1.18g of a yellow product (6), wherein the yield is 75.25%.

1.34 Synthesis of (4-methylpiperazine) aniline (7)

1.0g of 4- (4-methylpiperazine) nitrobenzene (6) is dissolved in 10mL of methanol, a catalytic amount of 10% Pd/C (mass fraction) is slowly added with stirring, hydrogen is introduced, and the reaction is carried out for 5h at normal temperature. The reaction solution was filtered with suction and the filter cake was washed with methanol. The filtrate was subjected to rotary drying under reduced pressure to remove methanol, and the residue was subjected to silica gel column chromatography to give 0.72g of 4- (4-methylpiperazine) aniline (7) as an off-white solid in 83.7% yield.

General Synthesis of target Compounds 8a to 8j (example 8 a)

263.66mg (1.0mmol) of intermediate 3a and 191.27mg (1.0mmol) of 4- (4-methylpiperazine) aniline (7) were dissolved in 5mL of methanol, 0.20mL of trifluoroacetic acid (TFA) was slowly added dropwise with stirring, and the reaction mixture was heated under reflux at 80 ℃ for 2 hours. After the reaction, the reaction mixture was cooled to room temperature, the reaction mixture was adjusted to neutral pH with saturated sodium bicarbonate solution, extracted with ethyl acetate (10mL × 3), the organic phases were combined, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate under reduced pressure, and separating with silica gel column chromatography to obtain brown yellow target product 8a112.4 mg with yield 95.1%. The yields, physicochemical properties and spectral data of the 9 target compounds synthesized are detailed in table 1.

TABLE 1

Example 2

Example 2 on FGFR4^V550LInhibition activity test for protein kinase

Testing target compounds 8a-8 j on FGFR4 by using Caliper EZ Reader drug screening platform^V550LInhibitory activity of protein kinases. The experimental steps are as follows: 1.25 Xkinase reaction buffer (62.5mmol/L HEPES, pH 7.5; 0.001875% Brij-35; 12.5mmol/L MgCl 2; 2.5mM DTT) and kinase reaction stop solution (100mmol/LHEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent #3) were prepared; to 5. mu.l of 5 Xconcentration compound solution (dissolved in DMSO, diluted 10-fold with water) was added 10. mu.l of 2.5 XFGFR 4^V550LKinase solution (kinase added to 1.25 Xkinase reaction buffer), incubated at room temperature for 10min, then 10. mu.l of 2.5 Xsubstrate peptide solution (FAM-labeled peptide and ATP added to 1.25 Xkinase reaction buffer) was added, and after a specified time at 28 ℃ 25. mu.l of kinase reaction stop solution was added. Data were collected by testing on a Caliper with a (max-conversion)/(max-min) 100 inhibition of kinase activity. "max" is DMSO control without compound addition and "min" is low control. Determination of IC₅₀The samples were set at 2 replicate wells for each of 5 dilutions, 3 replicates, and the results are shown in table 2.

TABLE 2

Wherein, the structure of the compound D3 is shown as the following formula:

the experimental results show that most of the target compounds are opposite to FGFR4^V550LKinase inhibition activity is poor, and particularly, compounds with large volume substituent groups on the optimized pyrimidine or pyridine parent nucleus, such as ester group (8a, 8i), methylthio (8h) and thiophene (8j) substituted compounds, such as FGFR4^V550LProtein kinases have little inhibitory activity. In addition, 4, 6-disubstituted aminopyrimidine series compounds 8f-8h have no inhibitory activity basically, while in 2, 4-disubstituted aminopyrimidine series compounds 8a-8e, a compound (8e) with a substituent at the 6-position and a substituent with larger volume at the 5-position are not beneficial to improving the inhibitory activity, and the compound with trifluoromethyl (8d) can greatly improve FGFR4^V550LIs effective against FGFR4^V550LInhibited IC₅₀The value was 45.8nM, and the inhibitory activity of the most active compound 8D was increased by approximately 5-fold over that of the lead compound D3.

Claims (4)

1. Pyrimidine FGFR4^V550LAn inhibitor characterized by being a compound represented by the following formula:

2. the pyrimidine FGFR4 of claim 1^V550LThe preparation method of the inhibitor is characterized by comprising the following steps:

(1) under the action of N, N-dimethylformamide and alkali, 2, 4-dichloro-5-trifluoromethylpyrimidine reacts with 6-aminoindazole to obtain an intermediate;

(2) under the action of methanol and acid, reacting the intermediate obtained in the step (1) with arylamine to obtain the 2-N-aryl-4-N-aryl-5-trifluoromethyl pyrimidine compound;

in the step (1), the alkali is N, N-diisopropylethylamine;

in the step (2), the acid is trifluoroacetic acid;

in the step (2), the arylamine is 4- (4-methylpiperazine) aniline.

3. The pyrimidine FGFR4 of claim 1^V550LUse of an inhibitor in the manufacture of a medicament for the treatment or prevention of a tumour.

4. Pyrimidine FGFR4 according to claim 3^V550LThe application of the inhibitor in the preparation of the medicament is characterized in that the medicament is used for inhibiting liver cancer cells.