A 6-trifluoromethyl-1,2,4-triazine-3-amide compound, preparation method and application thereof

Abstract

The present invention provides a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, characterized in that, as shown in formula (I), wherein R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group; R' is selected from C1-C10 alkyl; the substituents in the substituted phenyl and substituted heterocyclic group are selected from C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano. One or more. Compared with the prior art, the 6-trifluoromethyl-1,2,4-triazine-3-amide compound provided by the present invention contains a trifluoromethyl triazine skeleton, has good tyrosine kinase inhibitory activity, and can be further applied to the synthesis and development of drug molecules that block tyrosine kinase.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, a preparation method and application thereof. Background Art

[0002] Chronic myeloid leukemia is a malignant myeloproliferative disease of the blood system. It is the most common chronic leukemia, referred to as CML, which is mainly caused by the uncontrolled proliferation of hematopoietic stem cells. It is currently a very important disease in the field of leukemia. It is very harmful to the human body and may even lead to the occurrence of life failure (Acta Pharmaceutica Sinica 2021, 56, 414-431; ACSMed.Chem.Lett.2019, 10, 153-160; AnnuRev Biochem.2006, 75, 93-109). Researchers have found through studying its pathogenic mechanism that it is the result of the reciprocal translocation of chromosomes 9 and 22, and leads to the production of the BCR-ABL fusion gene. The tyrosine kinase encoded by this gene is not controlled by other molecules and has been in an active state, leading to uncontrolled cell division and causing cancer.

[0003] In a project targeting protein kinase C (PKC), researchers found that a 2-phenylaminopyrimidine derivative showed drug potential and could inhibit both serine / threonine kinases and tyrosine kinases. Based on this compound, researchers made a series of synthetic attempts to continuously optimize the properties of this molecule: the pyridine group added to the 3rd position of pyrimidine can increase its activity in cells; the benzamide group added to the benzene ring can enhance the inhibitory ability to tyrosine kinase; the modification of the 6th position of the anilino benzene ring further enhances the inhibition of tyrosine kinase; the addition of the side chain of N-methylpiperazine greatly improves the solubility of this molecule, making oral administration possible. After a series of designs and modifications, this molecule demonstrated extremely high specific inhibitory ability. As long as the cell expresses the BCR-Abl protein, its growth will be inhibited by this molecule. This is the later imatinib (Imatinib, trade name Glivec, Chinese name Gleevec). After approval, researchers completed the work of Phase 3 clinical trials. Compared with standard therapy, it has demonstrated significant efficacy in all indicators (New England Journal of Medicine. 2017, 376, 982; J. Med. Chem. 2022, 65, 1047-1131). Before the birth of imatinib, only 30% of chronic myeloid leukemia patients could survive for 5 years after diagnosis. Imatinib increased this number from 30% to 89%, and after 5 years, 98% of patients still achieved complete hematological remission.

[0004] The emergence of imatinib has successfully turned chronic myeloid leukemia into a chronic disease like diabetes or hypertension. On this basis, subsequent researchers have made corresponding structural improvements on the molecular basis of imatinib, and developed the second-generation drugs Nilotinib (Nilotinib, Novartis in 2007), Radotinib (Radotinib, IL-YANG Pharm in 2012) and the third-generation drug Ponatinib (Ponatinib, Ariad in 2012) containing a trifluoromethylaniline pyrimidine skeleton, which are mainly used for adult patients with chronic myeloid leukemia (CML) who have been ineffective or resistant to imatinib treatment. Therefore, the design and development of new imatinib analogs and their preparation methods are still of great value for the development of new anticancer drugs.

[0005] Triazine heterocycles are an important class of six-membered aromatic heterocyclic compounds with three nitrogen atoms, forming three regioisomers: 1,2,4-triazine (a-triazine), 1,2,3-triazine (v-triazine) and 1,3,5-triazine (s-triazine). In recent years, they have attracted more and more attention and applications in the fields of medicine, pesticides, chemical biology, etc. It is worth noting that the reverse electron demand Diels-Alder cycloaddition reaction is widely used as a unique and effective method in the synthesis of nitrogen-containing heterocycles, preparation of natural products and bioorthogonal chemistry. Therefore, the combination of trifluoromethyl triazine units with the molecular skeleton of imatinib can be used to design and develop new organic small molecule tyrosine kinase inhibitors, and further applied to the research and development of anticancer drugs. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, a preparation method and application thereof. The preparation method has a good yield, and the obtained 6-trifluoromethyl-1,2,4-triazine-3-amide compound has good tyrosine kinase inhibitory activity, and can be further applied to the synthesis and development of drug molecules that block tyrosine kinase.

[0007] The present invention provides a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, as shown in formula (I):

[0008]

[0009] Wherein, R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group;

[0010] The substituents in the substituted phenyl and substituted heterocyclic groups are selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano;

[0011] R' is selected from C1-C10 alkyl groups.

[0012] Preferably, the heterocyclic group is selected from thienyl, furyl or pyridyl.

[0013] Preferably, R is selected from phenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-methylphenyl, 4-methoxyphenyl, 3,5-ditrifluoromethylphenyl, 3,4,5-trifluorophenyl, thiophene-2-yl, 5-methyl-thiophene-2-yl, 5-methyl-furan-2-yl or 6-trifluoromethyl-pyridin-3-yl.

[0014] Preferably, one or more of the formulas (1) to (14) are used:

[0015]

[0016] The present invention also provides a method for preparing a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, comprising the following steps:

[0017] Amination reaction is performed on the acyl halide compound represented by formula (II) and the compound represented by formula (III) to obtain the 6-trifluoromethyl-1,2,4-triazine-3-amide compound represented by formula (I);

[0018]

[0019] Wherein, X is a halogen;

[0020] R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group;

[0021] The substituents in the substituted phenyl and substituted heterocyclic groups are selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano;

[0022] R' is selected from C1-C10 alkyl groups.

[0023] Preferably, the acyl halide compound represented by formula (II) is prepared according to the following steps:

[0024] S1) hydrolyzing the compound represented by formula (IV) to obtain a carboxylic acid compound represented by formula (V);

[0025] S2) subjecting the carboxylic acid compound represented by formula (V) to a halogenation reaction with a halogenating agent to obtain an acyl halide compound represented by formula (II);

[0026]

[0027] Wherein, R" is selected from C1 to C5 alkyl.

[0028] Preferably, the hydrolysis in step S1) is carried out under alkaline conditions; the base used in the alkaline conditions is selected from one or more of potassium hydroxide, lithium hydroxide and sodium hydroxide; the hydrolysis is carried out in a mixed solvent; the mixed solvent includes an organic solvent and water; the organic solvent is selected from tetrahydrofuran and / or methanol; the volume ratio of the organic solvent to water is 1:(1-3); the temperature of the hydrolysis is 20°C-30°C; the time of the hydrolysis is 1-3h;

[0029] The halogenation reagent in step S2) is selected from oxalyl halide and / or dihalosulfoxide; the halogenation reaction is carried out in the presence of a catalyst; the catalyst is N,N-dimethylformamide; the halogenation reaction temperature is 20°C to 30°C; and the halogenation reaction time is 1 to 2 hours.

[0030] Preferably, the amination reaction is carried out in a solvent; the solvent includes N,N-dimethylformamide and dichloromethane; the volume ratio of N,N-dimethylformamide to dichloromethane is 1:(2-3); the temperature of the amination reaction is 20°C-30°C; the time of the amination reaction is 3-4h.

[0031] The present invention also provides a use of the above 6-trifluoromethyl-1,2,4-triazine-3-amide compound as a tyrosine kinase inhibitor.

[0032] Preferably, the 6-trifluoromethyl-1,2,4-triazine-3-amide compound inhibits tyrosine kinase by promoting the expression level of Caspase-9 protein.

[0033] The present invention provides a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, characterized in that, as shown in formula (I), R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group; R' is selected from C1-C10 alkyl; the substituents in the substituted phenyl and substituted heterocyclic group are selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano. Compared with the prior art, the 6-trifluoromethyl-1,2,4-triazine-3-amide compound provided by the present invention contains a trifluoromethyl triazine skeleton, has good tyrosine kinase inhibitory activity, and can be further applied to the synthesis and development of drug molecules that block tyrosine kinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound represented by formula (I) obtained in Example 1 of the present invention;

[0035] Figure 2is the nuclear magnetic resonance fluorine spectrum of the compound represented by formula (I) obtained in Example 1 of the present invention;

[0036] Figure 3 It is a bar graph showing the effect of the compounds obtained in the examples of the present invention on the expression level of protein Caspase 9. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] The present invention provides a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, as shown in formula (I):

[0039]

[0040] Wherein, R is a substituted or unsubstituted phenyl group, a substituted or unsubstituted heterocyclic group; the heterocyclic group is preferably a five-membered heterocyclic group or a six-membered heterocyclic group; the heteroatoms in the heterocyclic group preferably include but are not limited to one or more of S, O and N; in the present invention, most preferably, the heterocyclic group is a thienyl group, a furyl group or a pyridyl group.

[0041] The substituents in the substituted phenyl and substituted heterocyclic groups are one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano, preferably one or more of C1-C5 alkyl, C1-C5 alkoxy, C1-C5 haloalkyl, halogen and cyano, more preferably one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen and cyano, and more preferably one or more of C1-C2 alkyl, C1-C2 alkoxy, C1-C2 haloalkyl, halogen and cyano; the haloalkyl is The haloalkyl group well known to those skilled in the art can be used without any special limitation. In the present invention, it preferably includes but is not limited to one or more of fluoroalkyl, chloroalkyl, bromoalkyl and iodoalkyl; the haloalkyl group includes but is not limited to one or more of monohaloalkyl, dihaloalkyl and trihaloalkyl; the halogen is preferably one or more of F, Cl, Br and I; the present invention has no special limitation on the number of substituents in the substituted phenyl and substituted heterocyclic group, preferably 1 to 3; the present invention has no special limitation on the position of the substituents in the substituted phenyl and substituted heterocyclic group, which can be para, ortho or meta.

[0042] According to the present invention, most preferably, R is phenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-methylphenyl, 4-methoxyphenyl, 3,5-ditrifluoromethylphenyl, 3,4,5-trifluorophenyl, thiophen-2-yl, 5-methyl-thiophen-2-yl, 5-methyl-furan-2-yl or 6-trifluoromethyl-pyridin-3-yl.

[0043] R' is a C1-C10 alkyl group, preferably a C1-C5 alkyl group, more preferably a C1-C3 alkyl group, and still more preferably a methyl group or an ethyl group.

[0044] According to the present invention, most preferably, the 6-trifluoromethyl-1,2,4-triazine-3-amide compound is one or more of formula (1) to formula (14):

[0045]

[0046] The present invention also provides a method for preparing the above-mentioned 6-trifluoromethyl-1,2,4-triazine-3-amide compound, characterized in that it comprises the following steps:

[0047] Amination reaction is performed on the acyl halide compound represented by formula (II) and the compound represented by formula (III) to obtain the 6-trifluoromethyl-1,2,4-triazine-3-amide compound represented by formula (I);

[0048]

[0049]

[0050] Wherein, X is a halogen, preferably including but not limited to F, Cl or Br;

[0051] R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group;

[0052] The substituents in the substituted phenyl and substituted heterocyclic groups are selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano;

[0053] R' is selected from C1-C10 alkyl groups.

[0054] The present invention has no special restrictions on the sources of all raw materials, which can be commercially available or homemade; the R and R' are the same as described above and will not be repeated here.

[0055] In the present invention, the acyl halide compound represented by formula (II) is prepared according to the following steps: S1) hydrolyzing the compound represented by formula (IV) to obtain the carboxylic acid compound represented by formula (V); S2) halogenating the carboxylic acid compound represented by formula (V) with a halogenating agent to obtain the acyl halide compound represented by formula (II);

[0056]

[0057] Wherein, R" is a C1-C5 alkyl group, preferably a methyl group, an ethyl group or a propyl group, and more preferably a methyl group or an ethyl group.

[0058] The compound represented by formula (IV) is hydrolyzed to obtain the carboxylic acid compound represented by formula (V); the hydrolysis is preferably carried out under alkaline conditions; the base used in the alkaline conditions is preferably one or more of potassium hydroxide, lithium hydroxide and sodium hydroxide; the molar ratio of the compound represented by formula (IV) to the base is preferably 1: (1-1.5), more preferably 1: (1.2-1.5), and more preferably 1: 1.3; the hydrolysis is carried out in a mixed solvent; the mixed solvent includes an organic solvent and water; the organic solvent is preferably tetrahydrofuran and / or methanol; the volume ratio of the organic solvent to water is preferably 1: (1-3), more preferably 1: (1.5-2.5), and more preferably 1: 2; in the present invention, it is preferred to firstly The product is mixed with an organic solvent, and then alkali and water are added to perform hydrolysis; the hydrolysis temperature is preferably 20°C to 30°C; the hydrolysis time is 1 to 3 hours; after hydrolysis, the pH value of the reaction solution is preferably adjusted to 6 to 7, and the organic phase is extracted with ethyl acetate; in the present invention, the pH value of the reaction solution is preferably adjusted with an aqueous hydrochloric acid solution; the mass concentration of the aqueous hydrochloric acid solution is preferably 10% to 15%; after extracting the organic phase, it is preferably washed with water and saturated brine to obtain a carboxylic acid compound represented by formula (V); the number of washings with water and saturated brine is preferably 2 to 5 times, more preferably 3 to 4 times, each independently; after washing, it is preferably dried with a desiccant, filtered, and concentrated to obtain a carboxylic acid compound represented by formula (V); the desiccant is preferably anhydrous sodium sulfate.

[0059] The carboxylic acid compound represented by formula (V) is subjected to a halogenation reaction with a halogenating agent to obtain an acyl halide compound represented by formula (II); the halogenation reaction is preferably carried out in a protective atmosphere; the protective atmosphere is any protective atmosphere known to those skilled in the art and is not particularly limited, and is preferably argon in the present invention; the halogenating agent is preferably oxalyl halide and / or dihalosulfoxide; the specific type of the oxalyl halide and / or dihalosulfoxide can be selected according to the type of X in the compound represented by formula (II), and in the present invention, it can be oxalyl chloride and / or dichloride sulfoxide; in order to make the carboxylic acid represented by formula (V) The compound is converted into the corresponding acyl halide compound as much as possible, and the amount of the halogenating agent is preferably excessive; the halogenating reaction is preferably carried out in the presence of a catalyst; the catalyst is preferably N,N-dimethylformamide; the halogenating reaction is preferably carried out in an organic solvent; the organic solvent is preferably dichloromethane; in the present invention, it is preferred to first mix the carboxylic acid compound represented by formula (V) with an organic solvent, and then add the halogenating agent and the catalyst in sequence to carry out the halogenating reaction; the temperature of the halogenating reaction is preferably 20°C to 30°C; the time of the halogenating reaction is preferably 1 to 2 hours. In the present invention, the product after the halogenating reaction can be directly added to the compound represented by formula (III) for amination reaction without post-treatment.

[0060] The acyl halide compound represented by formula (II) is subjected to an amination reaction with the compound represented by formula (III) to obtain the 6-trifluoromethyl-1,2,4-triazine-3-amide compound represented by formula (I); the amination reaction is preferably carried out in a solvent; the solvent preferably includes N,N-dimethylformamide and dichloromethane; the volume ratio of N,N-dimethylformamide (DMF) to dichloromethane is preferably 1:(2-3), more preferably 1:2.5; in the present invention, the compound represented by formula (III) is preferably first mixed with DMF and then added to the reaction system; the temperature of the amination reaction is preferably 20°C to 30°C; The amination reaction time is preferably 3 to 4 hours; after the amination reaction, some solids are precipitated, preferably DMF and / or DMSO is added until the precipitated solids are just dissolved, and then extracted with ethyl acetate; after extraction, it is preferably washed with water and saturated brine; the number of water washing and saturated brine washing is preferably 2 to 5 times, more preferably 3 to 4 times, respectively; after washing, preferably a desiccant is added for drying, filtering, and concentration to obtain a crude product; the desiccant is preferably anhydrous sodium sulfate; in the present invention, the crude product is preferably washed with ethyl acetate to obtain a 6-trifluoromethyl-1,2,4-triazine-3-amide compound represented by formula (I).

[0061] The present invention uses 6-trifluoromethyl-1,2,4-triazine-3-carboxylate compounds as raw materials, and obtains fourteen 6-trifluoromethyl-1,2,4-triazine-3-amide compounds through three-step functional group transformation. The method has good yield, and the obtained 6-trifluoromethyl-1,2,4-triazine-3-amide compounds have good tyrosine kinase inhibitory activity and can be further applied to the synthesis and development of drug molecules that block tyrosine kinase.

[0062] The present invention also provides a use of the above 6-trifluoromethyl-1,2,4-triazine-3-amide compound as a tyrosine kinase inhibitor.

[0063] The 6-trifluoromethyl-1,2,4-triazine-3-amide compound inhibits tyrosine kinase by promoting the expression level of Caspase-9 protein.

[0064] In order to further illustrate the present invention, a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, a preparation method thereof and an application thereof provided by the present invention are described in detail below in conjunction with examples.

[0065] The reagents used in the following examples are all commercially available.

[0066] Example 1: 5-(4-methoxyphenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0067]

[0068] Dissolve 5-(4-methoxyphenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester (939.7 mg, 3 mmol) in 5 mL of tetrahydrofuran, then add lithium hydroxide (163.6 mg, 3.9 mmol) and 10 mL of water to the system. Stir at room temperature for 1 to 2 hours, and monitor the reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify to pH = 6 to 7 with 10% hydrochloric acid aqueous solution. Then extract the organic phase with ethyl acetate, wash with water 3 times and saturated brine 3 times, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain 5-(4-methoxyphenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid (yield 85%).

[0069] Subsequently, 5-(4-methoxyphenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid was dissolved in 5 mL of anhydrous dichloromethane, covered with a sealing stopper, replaced with argon protection (an argon balloon was inserted into the sealing stopper), and then oxalyl chloride (456.9 mg, 3.6 mmol) was added with a 1 mL disposable syringe, followed by 1 to 2 drops of anhydrous DMF. The reaction was carried out at room temperature for 1 to 2 hours, and the reaction of the raw materials was monitored by thin layer silica gel chromatography (TLC).

[0070] 2-(5-amino-2-methylaniline)-4-(3-pyridine)pyrimidine (832 mg, 3 mmol) was dissolved in 2 mL of anhydrous DMF, and then the solution was added dropwise to the above reaction system with a syringe, and reacted at room temperature for 3 to 4 hours, and some solids precipitated. At this time, 4 to 5 mL of DMF or DMSO was added to the system until some solids were just dissolved, and then transferred to a separatory funnel and extracted with an appropriate amount of ethyl acetate, washed with water 3 to 5 times, and washed with saturated brine 3 times. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Finally, the crude product was washed with ethyl acetate 8 to 10 times, and the solvent was removed by a vacuum pump to obtain the compound represented by formula (1) as a bright yellow solid (1.05 g, total yield 74%).

[0071] The compound represented by formula (1) obtained in Example 1 was analyzed by nuclear magnetic resonance, and its hydrogen nuclear magnetic resonance spectrum was obtained as follows: Figure 1 As shown, the NMR fluorine spectrum is as follows Figure 2 As shown; the result is 1 HNMR (400MHz, DMSO-d6), δ (ppm): 11.17 (s, 1H), 9.28 (d, J = 2.3Hz, 1H), 9.07 (s, 1H) ),8.68(dd,J=4.7,1.6Hz,1H),8.56–8.44(m,2H),8.22(d,J=2.1Hz,1H),7.87(d,J =8.5Hz,2H),7.63(dd,J=8.2,2.2Hz,1H),7.51(dd,J=8.0,4.8Hz,1H),7.45(d,J=5 .2Hz,1H),7.28(d,J=8.3Hz,1H),7.20(d,J=8.6Hz,2H),3.88(s,3H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.65 (s, 3F).

[0072] Example 2: 5-(4-methylphenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0073]

[0074] Dissolve 5-(4-methylphenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester (891.7 mg, 3 mmol) in 5 mL of methanol, then add lithium hydroxide (163.6 mg, 3.9 mmol) and 10 mL of water to the system. Stir at room temperature for 1 to 2 hours, and monitor the reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% hydrochloric acid aqueous solution to pH = 6 to 7. Then extract the organic phase with ethyl acetate, wash with water 3 times and saturated brine 3 times, respectively, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain 5-(4-methylphenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid.

[0075] Subsequently, 5-(4-methylphenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid was dissolved in 5 mL of anhydrous dichloromethane, covered with a sealing stopper, replaced with argon protection (an argon balloon was inserted into the sealing stopper), and then oxalyl chloride (456.9 mg, 3.6 mmol) was added with a 1 mL disposable syringe, followed by 1 to 2 drops of anhydrous DMF. The reaction was carried out at room temperature for 1 to 2 hours, and the reaction of the raw materials was monitored by thin layer silica gel chromatography (TLC).

[0076] 2-(5-amino-2-methylaniline)-4-(3-pyridine)pyrimidine (832 mg, 3 mmol) was dissolved in 2 mL of anhydrous DMF, and then the solution was added dropwise to the above reaction system with a syringe, and reacted at room temperature for 3 to 4 hours, and some solids precipitated. At this time, 4 to 5 mL of DMF or DMSO was added to the system until some solids were just dissolved, and then transferred to a separatory funnel and extracted with an appropriate amount of ethyl acetate, washed with water 3 to 5 times, and washed with saturated brine 3 times. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Finally, the crude product was washed with ethyl acetate 8 to 10 times, and the solvent was removed by a vacuum pump to obtain the compound represented by formula (2) (70%) as a bright yellow solid.

[0077] The compound represented by formula (2) obtained in Example 2 was analyzed by nuclear magnetic resonance to obtain 1H NMR (400MHz, DMSO-d6) δ (ppm): 11.14 (s, 1H), 9.29 (d, J = 2.3Hz, 1H), 9.07 (s, 1H), 8.68 (dd, J = 4.7, 1.6Hz, 1H), 8.57–8.44 (m, 2H), 8.25 (d, J = 2.4Hz, 1H) ),7.75(d,J=7.9Hz,2H),7.64(dd,J=8.3,2.2Hz,1H),7.51(dd,J=8.0,4.8H z,1H),7.48–7.41(m,3H),7.29(d,J=8.3Hz,1H),2.43(s,3H),2.28(s,3H), 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.52 (s, 3F).

[0078] Example 3: 5-(3,5-bis(trifluoromethyl)phenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0079]

[0080] Dissolve 5-(3,5-bis(trifluoromethyl)phenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester (1.3g, 3mmol) in 5mL tetrahydrofuran, then add potassium hydroxide (72.9mg, 3.9mmol) and 10mL water to the system. Stir at room temperature for 1-2 hours, and monitor the reaction of the raw material by thin layer silica gel chromatography (TLC). Acidify to pH=6-7 with 10% hydrochloric acid aqueous solution. Then extract the organic phase with ethyl acetate, wash with water 3 times and saturated brine 3 times, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain 5-(3,5-bis(trifluoromethyl)phenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid.

[0081] Subsequently, 5-(3,5-bis(trifluoromethyl)phenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid was dissolved in 5 mL of anhydrous dichloromethane, covered with a sealing stopper, replaced with argon protection (an argon balloon was inserted into the sealing stopper), and then oxalyl chloride (456.9 mg, 3.6 mmol) was added with a 1 mL disposable syringe, followed by 1 to 2 drops of anhydrous DMF. The reaction was carried out at room temperature for 1 to 2 hours, and the reaction of the raw materials was monitored by thin layer silica gel chromatography (TLC).

[0082] 2-(5-amino-2-methylaniline)-4-(3-pyridine)pyrimidine (832 mg, 3 mmol) was dissolved in 2 mL of anhydrous DMF, and then the solution was added dropwise to the above reaction system with a syringe, and reacted at room temperature for 3 to 4 hours, and some solids precipitated. At this time, 4 to 5 mL of DMF or DMSO was added to the system until some solids were just dissolved, and then transferred to a separatory funnel and extracted with an appropriate amount of ethyl acetate, washed with water 3 to 5 times, and washed with saturated brine 3 times. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Finally, the crude product was washed with ethyl acetate 8 to 10 times, and the solvent was removed by a vacuum pump to obtain the compound represented by formula (3) (49%) as a bright yellow solid.

[0083] The compound represented by formula (3) obtained in Example 3 was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.32 (s, 1H), 9.28 (d, J = 2.3Hz, 1H), 9.07 (s, 1H), 8.67 (dd, J = 4.8, 1.6Hz, 1H), 8.52 (dd, J = 8.5, 3.5Hz, 1H), 8 .49(s,4H),8.24(d,J=2.1Hz,1H),7.63(dd,J=8.3,2.2Hz,1H),7.60–7.48(m,1H),7.45(t,J=5.1Hz,1H),7.29(d,J=8.3Hz,1H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.80 (s, 3F), -61.48 (s, 6F).

[0084] Example 4: 5-(4-chlorophenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0085]

[0086] Dissolve 5-(4-chlorophenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester (1.6 g, 5 mmol) in 5 mL of tetrahydrofuran, then add sodium hydroxide (260 mg, 6.5 mmol) and 10 mL of water to the system. Stir at room temperature for 1 to 2 hours, and monitor the reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% hydrochloric acid aqueous solution to pH = 6 to 7. Then extract the organic phase with ethyl acetate, wash with water 3 times and saturated brine 3 times, respectively, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain 5-(4-chlorophenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid.

[0087] Subsequently, 5-(4-chlorophenyl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid was dissolved in 7.5 mL of anhydrous dichloromethane, covered with a sealing stopper, replaced with argon protection (an argon balloon was inserted into the sealing stopper), and then oxalyl chloride (456.9 mg, 3.6 mmol) was added with a 1 mL disposable syringe, followed by 1 to 2 drops of anhydrous DMF. The reaction was carried out at room temperature for 1 to 2 hours, and the reaction of the raw materials was monitored by thin layer silica gel chromatography (TLC).

[0088] 2-(5-amino-2-methylaniline)-4-(3-pyridine)pyrimidine (1.4 g, 5 mmol) was dissolved in 3 mL of anhydrous DMF, and then the solution was added dropwise to the above reaction system with a syringe, and reacted at room temperature for 3 to 4 hours, and some solids precipitated. At this time, 5 to 6 mL of DMF or DMSO was added to the system until some solids were just dissolved, and then transferred to a separatory funnel and extracted with an appropriate amount of ethyl acetate, washed with water 3 to 5 times, washed with saturated brine 3 times, the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, and finally washed with ethyl acetate 8 to 10 times, and the solvent was removed by a vacuum pump to obtain the compound represented by formula (4) (68%) as a bright yellow solid.

[0089] The compound represented by formula (4) obtained in Example 4 was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.17 (s, 1H), 9.28 (d, J = 2.2Hz, 1H), 9.08 (s ,1H),8.72–8.65(m,1H),8.57–8.44(m,2H),8.22(d,J=2.2Hz,1H),7.84(d,J =8.3Hz,2H),7.74(d,J=8.4Hz,2H),7.63(dd,J=8.3,2.2Hz,1H),7.51(dd,J =8.0,4.8Hz,1H),7.45(d,J=5.2Hz,1H),7.29(d,J=8.3Hz,1H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.60 (s, 3F).

[0090] Example 5: 5-(4-bromophenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0091]

[0092] In a similar manner to Example 1, 5-(4-bromophenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (5) (48%) as a bright yellow solid.

[0093] The compound represented by formula (5) obtained in Example 5 was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.15 (s, 1H), 9.28 (s, 1H), 9.05 (s, 1H), 8.68 (d, J = 4.9Hz, 1H), 8.51 (dd, J = 23.2, 6.5Hz, 2H), 8.23 ​​( s,1H),7.87(d,J=8.3Hz,2H),7.77(d,J=8.1Hz,2H),7.64(d,J=8.5Hz,1H),7.59–7.39(m,2H),7.29(d,J=8.2Hz,1H),2.27(s,3H). 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.59 (s, 3F).

[0094] Example 6: 5-(4-fluorophenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0095]

[0096] In a similar manner to Example 1, 5-(4-fluorophenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (6) (64%) as a bright yellow solid.

[0097] The compound represented by formula (6) obtained in Example 6 was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.17 (s, 1H), 9.29 (s, 1H), 9.07 (s, 1H), 8.68 (d, J = 4.8Hz, 1H), 8.59–8.38 (m, 2H), 8.25 ( d,J=2.2Hz,1H),7.91(dd,J=8.4,5.3Hz,2H),7.70–7.60(m,1H),7.60–7.40(m,4H),7.29(d,J=8.3Hz,1H),2.28(s,3H). 19F NMR(376MHz, DMSO-d6)δ(ppm):-60.63(s,3F)-108.32–-108.44(m,1F).

[0098] Example 7: 5-(3,4,5-trifluorophenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0099]

[0100] In a similar manner to Example 1, 5-(3,4,5-trifluorophenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (7) (60%) as a bright yellow solid.

[0101] The compound represented by formula (7) obtained in Example 7 was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.15 (s, 1H), 9.24 (d, J = 2.3Hz, 1H), 9.04 (s, 1H), 8.63 (dd, J = 4.8, 1.7Hz, 1H), 8.57–8.36 (m, 2H), 8.17(d,J=2.3Hz,1H),7.74(t,J=7.2Hz,2H),7.58(dd,J=8.2,2.3Hz,1H),7.53–7.34(m,2H),7.25(d,J=8.2Hz,1H),2.23(s,3H). 19 F NMR (376MHz, DMSO) δ (ppm): -60.80 (s, 3F), -133.54--133.62 (d, J = 29.6Hz, 2F), -156.32--156.45 (m, 1F).

[0102] Example 8: 5-(4-trifluoromethylphenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0103]

[0104] In a similar manner to Example 1, 5-(4-trifluoromethylphenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (8) (47%) as a bright yellow solid.

[0105] The compound represented by formula (8) obtained in Example 8 was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.20 (s, 1H), 9.29 (d, J = 2.3Hz, 1H), 9.06 (s, 1H), 8.68 (d, J = 4.8Hz, 1H), 8.60–8.39 (m, 2H), 8 .24(d,J=2.1Hz,1H),8.09–7.92(m,4H),7.64(dd,J=8.2,2.2Hz,1H),7.59–7.41(m,2H),7.29(d,J=8.3Hz,1H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.64 (s, 3F), -61.49 (s, 3F).

[0106] Example 9: 5-phenyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0107]

[0108] In a similar manner to Example 1, 5-phenyl-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (9) (69%) as a bright yellow solid.

[0109] The compound represented by formula (9) obtained in Example 9 was analyzed by nuclear magnetic resonance to obtain 1 HNMR(400MHz,DMSO-d6)δ(ppm):11.15(s,1H),9.29(d,J=2.2Hz,1H),9.06(s,1H),8.68(dd,J=4.8,1.6Hz,1H),8.58–8.41( m,2H),8.24(d,J=2.1Hz,1H),7.96–7.77(m,2H),7.76–7.59(m,4H),7.56–7.40(m,2H),7.29(d,J=8.3Hz,1H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.50 (s, 3F).

[0110] Example 10: 5-(4-cyanophenyl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0111]

[0112] In a similar manner to Example 1, 5-(4-cyanophenyl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (10) (40%) as a bright yellow solid.

[0113] The compound represented by formula (10) obtained in Example 10 was analyzed by nuclear magnetic resonance to obtain 1 HNMR(400MHz, DMSO-d6)δ(ppm):11.22(s,1H),9.28(d,J=2.3Hz,1H),9.06(s,1H),8.68(dd,J=4.8,1.6Hz,1H),8.56–8.41(m,2H),8.25(d,J= 2.3Hz,1H),8.14(d,J=8.1Hz,2H),7.98(d,J=8.1Hz,2H),7.64(dd,J=8.2,2.2Hz,1H),7.55–7.38(m,2H),7.29(d,J=8.4Hz,1H),2.28(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.63 (s, 3F).

[0114] Example 11: 5-((6-trifluoromethyl)pyridin-3-yl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0115]

[0116] In a similar manner to Example 1, 5-((6-trifluoromethylpyridin-3-yl)-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound represented by formula (11) (43%) as a bright yellow solid.

[0117] The compound represented by formula (11) obtained in Example 11 was analyzed by nuclear magnetic resonance to obtain 1HNMR (400MHz, DMSO-d6) δ (ppm): 11.24 (s, 1H), 9.28 (d, J = 2.3Hz, 1H), 9.16 (d, J = 2.0Hz, 1H), 9.07 (s, 1H), 8.68 (dd, J = 4.7, 1.7Hz, 1H), 8.58–8.4 4(m,3H),8.37–8.16(m,2H),7.66(dd,J=8.3,2.2Hz,1H),7.51(dd,J=8.0,4.9Hz,1H),7.45(d,J=5.2Hz,1H),7.30(d,J=8.3Hz,1H),2.28(s,3H). 19 F NMR (376MHz, DMSO-d6) δ (ppm): -60.70 (s, 3F), -66.81 (s, 3F).

[0118] Example 12: 5-(Thiophen-2-yl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0119]

[0120] In a similar manner to Example 1, 5-(Thiophen-2-yl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 1 to obtain the compound of formula (12) (61%) as a khaki solid.

[0121] The compound represented by formula (12) obtained in Example 12 was analyzed by nuclear magnetic resonance to obtain 1 HNMR (400MHz, DMSO-d6) δ (ppm): 11.10 (s, 1H), 9.28 (s, 1H), 9.05 (s, 1H), 8.69 (d, J = 4.4Hz, 1H), 8.52 (dd, J = 18.6, 6.6Hz, 2H), 8.23 ​​(d, J = 3.7H z,2H),8.00(d,J=3.9Hz,1H),7.61(dd,J=8.2,2.2Hz,1H),7.53(dd,J=8.0,4.8Hz,1H),7.49–7.36(m,2H),7.29(d,J=8.3Hz,1H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -63.91 (s, 3F).

[0122] Example 13: 5-(5-methylthiophen-2-yl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0123]

[0124] Dissolve 5-(5-methylthiophene-2-yl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester (909.8 mg, 3 mmol) in 5 mL of tetrahydrofuran, then add lithium hydroxide (163.6 mg, 3.9 mmol) and 10 mL of water to the system. Stir at room temperature for 1 to 2 hours, and monitor the reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify to pH = 6 to 7 with 10% hydrochloric acid aqueous solution. Then extract the organic phase with ethyl acetate, wash with water 3 times and saturated brine 3 times, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain 5-(5-methylthiophene-2-yl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid.

[0125] Subsequently, 5-(5-methylthiophene-2-yl)-6-trifluoromethyl-1,2,4-triazine-3-carboxylic acid was dissolved in 5 mL of anhydrous dichloromethane, covered with a sealing stopper, replaced with argon protection (an argon balloon was inserted into the sealing stopper), and then dichlorothionyl (428.3 mg, 3.6 mmol) was added with a 1 mL disposable syringe, followed by 1 to 2 drops of anhydrous DMF. The reaction was carried out at room temperature for 1 to 2 hours, and the reaction of the raw materials was monitored by thin layer silica gel chromatography (TLC).

[0126] 2-(5-amino-2-methylaniline)-4-(3-pyridine)pyrimidine (832 mg, 3 mmol) was dissolved in 2 mL of anhydrous DMF, and then the solution was added dropwise to the above reaction system with a syringe, and reacted at room temperature for 3 to 4 hours, and some solids precipitated. At this time, 4 to 5 mL of DMF or DMSO was added to the system until some solids were just dissolved, and then transferred to a separatory funnel and extracted with an appropriate amount of ethyl acetate, washed with water 3 to 5 times, and washed with saturated brine 3 times. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Finally, the crude product was washed with ethyl acetate 8 to 10 times, and the solvent was removed by a vacuum pump to obtain the compound represented by formula (13) (63%) as a khaki solid.

[0127] The compound represented by formula (13) obtained in Example 13 was analyzed by nuclear magnetic resonance to obtain 1HNMR (400MHz, DMSO-d6) δ (ppm): 11.02 (s, 1H), 9.28 (d, J = 2.2Hz, 1H), 9.04 ( s,1H),8.69(dd,J=4.8,1.7Hz,1H),8.60–8.40(m,2H),8.20(d,J=2.2Hz,1H) ,7.86(d,J=3.9Hz,1H),7.57(dd,J=8.1,3.5Hz,2H),7.46(d,J=5.2Hz,1H),7 .29(d,J=8.3Hz,1H),7.15(dd,J=4.0,1.2Hz,1H),2.60(s,3H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -64.37 (s, 3F).

[0128] Example 14: 5-(5-methylfuran-2-yl)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-6-(trifluoromethyl)-1,2,4-triazine-3-amide

[0129]

[0130] In a manner similar to Example 13, 5-(5-methylfuran-2-yl)-6-trifluoromethyl-1,2,4-triazine-3-methyl ester was dissolved in 5 mL of tetrahydrofuran solvent, and then the subsequent reaction was carried out according to the method described in Example 13 to obtain the compound of formula (14) (52%) as a gray-brown solid.

[0131] The compound represented by formula (14) obtained in Example 14 was analyzed by nuclear magnetic resonance to obtain 1 HNMR (400MHz, DMSO-d6) δ (ppm): 10.95 (s, 1H), 9.28 (d, J = 2.2Hz, 1H), 9.05 (s, 1H) ,8.69(dd,J=4.8,1.7Hz,1H),8.57–8.44(m,2H),8.19(d,J=2.1Hz,1H),8.02(d,J= 3.6Hz, 1H), 7.61 (dd, J=8.2, 2.2Hz, 1H), 7.53 (dd, J=8.0, 4.8Hz, 1H), 7.45 (d, J=5 .2Hz,1H),7.29(d,J=8.3Hz,1H),6.65(d,J=3.6Hz,1H),2.49(s,3H),2.27(s,3H); 19 F NMR (376MHz, DMSO-d6) δ (ppm): -64.38 (s, 3F).

[0132] Activity test results

[0133] In vitro experiments were conducted based on THP-1 (human monocytic leukemia) to explore whether this type of compound has a promoting effect on the pro-apoptotic protein Caspase 9.

[0134] Experimental process:

[0135] 1) Cell culture

[0136] THP-1 cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS). When the cell density reached 80% to 90%, the cells were centrifuged at 1000 rpm for five minutes, the supernatant was removed, new medium was added, and the cells were passaged at a density of 1:3.

[0137] 2) Cell plating

[0138] Add 1 mL of the prepared cell solution to each well of a 12-well plate and place in a 37°C incubator with 5% CO2 overnight.

[0139] 3) Cellular drug delivery

[0140] The compound and imatinib were dissolved in DMSO to a concentration of 100 mM as a stock solution for later use. The compound and positive control imatinib were dissolved in 1640 medium as a diluent to a final concentration of 10 μM.

[0141] Add 1 mL of the prepared solution to each well plate, place the plate in a 37°C incubator with 5% CO2 and incubate for 24 hours before taking it out.

[0142] 4) Protein extraction and Western Blot

[0143] The well plate was taken out, washed once with phosphate buffered saline (PBS), and then 100 μL of protein lysis buffer RIPA was added. After reacting at low temperature for 30 minutes, the sample was aspirated and added to a centrifuge tube. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was aspirated for protein quantification. After adjusting the protein concentration, the protein was configured into the corresponding loading volume, the loading buffer was added to cook the sample, and the sample was placed in a 4°C refrigerator for use.

[0144] According to the size of the protein, configure the corresponding lower layer gel, add the newly configured electrophoresis solution to the inner layer, and add the recycled electrophoresis solution to the outer layer. Run the gel at 90V for 20min and then switch to 120V for 90min. The transfer condition is 200mA for 60 minutes. Take out the membrane and place it in 5% skim milk powder. After shaking on a shaker for 1 hour, add the corresponding antibody. Place it in a 4℃ refrigerator overnight. Then take it out and wash the membrane three times with TBST, 15 minutes each time. Add the secondary antibody and shake it at room temperature for 1 hour, then take it out and wash the membrane three times with TBST, 15 minutes each time.

[0145] Experimental results:

[0146] The bar graph of protein Caspase 9 expression level is obtained as follows Figure 3 As shown. Figure 3 It can be seen that the 10 μM compound and the positive drug imatinib have a promoting effect on the protein Caspase 9. Compared with the model, imatinib and the compound represented by formula (8), the compound represented by formula (9), and the compound represented by formula (11) all have a significant upregulation of Caspase 9. According to the One-Way ANOVA analysis, the above compounds and imatinib are statistically different. It shows that the compound represented by formula (8), the compound represented by formula (9), and the compound represented by formula (11) have a good promoting effect on the apoptotic protein Caspase 9 in human monocytic leukemia, and the effect of compound 9 is significantly better than that of the positive control drug imatinib.

Claims

1. A 6-trifluoromethyl-1,2,4-triazine-3-amide compound, characterized in that: As shown in formula (I): Wherein, R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group; The substituents in the substituted phenyl and substituted heterocyclic groups are selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano; R' is selected from a C1-C10 alkyl group; The heterocyclic group is selected from thienyl, furanyl or pyridyl.

2. The 6-trifluoromethyl-1,2,4-triazine-3-amide compound according to claim 1, characterized in that The R is selected from phenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-methylphenyl, 4-methoxyphenyl, 3,5-ditrifluoromethylphenyl, 3,4,5-trifluorophenyl, thiophen-2-yl, 5-methyl-thiophen-2-yl, 5-methyl-furan-2-yl or 6-trifluoromethyl-pyridin-3-yl.

3. The 6-trifluoromethyl-1,2,4-triazine-3-amide compound according to claim 1, characterized in that As shown in one or more of formula (1) to formula (14):

4. A method for preparing a 6-trifluoromethyl-1,2,4-triazine-3-amide compound, characterized in that: The following steps are involved: Amination reaction is performed on the acyl halide compound represented by formula (II) and the compound represented by formula (III) to obtain the 6-trifluoromethyl-1,2,4-triazine-3-amide compound represented by formula (I); Wherein, X is a halogen; R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group; The substituents in the substituted phenyl and substituted heterocyclic groups are selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen and cyano; R' is selected from a C1-C10 alkyl group; The heterocyclic group is selected from thienyl, furanyl or pyridyl.

5. The preparation method according to claim 4, characterized in that: The acyl halide compound represented by the formula (II) is prepared according to the following steps: S1) hydrolyzing the compound represented by formula (IV) to obtain a carboxylic acid compound represented by formula (V); S2) subjecting the carboxylic acid compound represented by formula (V) to a halogenation reaction with a halogenating agent to obtain an acyl halide compound represented by formula (II); Wherein, R" is selected from C1 to C5 alkyl.

6. The preparation method according to claim 5, characterized in that: The hydrolysis in step S1) is carried out under alkaline conditions; the base used in the alkaline conditions is selected from one or more of potassium hydroxide, lithium hydroxide and sodium hydroxide; the hydrolysis is carried out in a mixed solvent; the mixed solvent includes an organic solvent and water; the organic solvent is selected from tetrahydrofuran and / or methanol; the volume ratio of the organic solvent to water is 1: (1-3); the temperature of the hydrolysis is 20°C-30°C; the time of the hydrolysis is 1-3h; The halogenation reagent in step S2) is selected from oxalyl halide and / or dihalosulfoxide; the halogenation reaction is carried out in the presence of a catalyst; the catalyst is N,N-dimethylformamide; the halogenation reaction temperature is 20°C to 30°C; and the halogenation reaction time is 1 to 2 hours.

7. The preparation method according to claim 4, characterized in that: The amination reaction is carried out in a solvent; the solvent includes N,N-dimethylformamide and dichloromethane; the volume ratio of N,N-dimethylformamide to dichloromethane is 1:(2-3); the temperature of the amination reaction is 20°C-30°C; and the time of the amination reaction is 3-4h.

8. Use of the 6-trifluoromethyl-1,2,4-triazine-3-amide compound as claimed in any one of claims 1 to 3 or the 6-trifluoromethyl-1,2,4-triazine-3-amide compound prepared by the preparation method of any one of claims 4 to 7 in the preparation of tyrosine kinase inhibitors.

9. The use according to claim 8, characterized in that: The 6-trifluoromethyl-1,2,4-triazine-3-amide compound inhibits tyrosine kinase by promoting the expression level of Caspase-9 protein.

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