CN116574088A - 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, and preparation method and application thereof - Google Patents

6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, and preparation method and application thereof Download PDF

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CN116574088A
CN116574088A CN202310534519.7A CN202310534519A CN116574088A CN 116574088 A CN116574088 A CN 116574088A CN 202310534519 A CN202310534519 A CN 202310534519A CN 116574088 A CN116574088 A CN 116574088A
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trifluoromethyl
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张发光
秦沛
马军安
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Tianjin University
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Abstract

The invention provides a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound, which is characterized by being shown as a formula (I); wherein R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclyl; r' is selected from C1-C10 alkyl; the substituent groups in the substituted phenyl and the substituted heterocyclic groups 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-triazosin-3-amide compound provided by the invention contains trifluoromethylThe azosin skeleton has better tyrosine kinase inhibition activity, and can be further applied to the synthesis and development of drug molecules for blocking tyrosine kinase.

Description

6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compound, and a preparation method and application thereof.
Background
Chronic myelogenous leukemia is a malignant myeloproliferative disease of the blood system, the most common chronic leukemia, abbreviated CML, mainly caused by uncontrolled proliferation of hematopoietic stem cells. At present, the leukemia is a very important disease in the field, has very serious harm to human bodies and even causes the occurrence of life failure phenomena (pharmaceutical report 2021,56, 414-431;ACS Med.Chem.Lett.2019,10,153-160;AnnuRev Biochem.2006,75, 93-109). Researchers have discovered by studying their pathogenesis that it is the result of the reciprocal translocation of chromosome 9 and chromosome 22 and that it leads to the generation of the BCR-ABL fusion gene. The tyrosine kinase coded by the gene is not controlled by other molecules and is always in an active state, so that uncontrolled cell division is caused, and cancer is caused.
In a project directed to Protein Kinase C (PKC), researchers have found that a 2-anilinopyrimidine derivative exhibits the potential to inhibit both serine/threonine and tyrosine kinases. Based on this compound, researchers have made a series of synthetic attempts to continuously optimize the properties of this molecule: the addition of a pyridine group at position 3 of pyrimidine increases its intracellular activity; the benzamide group added on the benzene ring can enhance the inhibition capability on tyrosine kinase; modification of the 6 th 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 the molecule, making oral administration possible. Through a series of designs and modifications, the molecule shows extremely high specific inhibition capability, and as long as the cell expresses BCR-Abl protein, the growth of the cell is inhibited by the molecule, which is the later Imatinib (Imatinib, trade name Glivec, chinese name Glivec). After the batch, the researchers completed the work of phase 3 clinical trials. It shows a remarkable therapeutic effect on all indexes compared with standard therapy (New England Journalof medicine.2017,376, 982; J.Med. Chem.2022,65, 1047-1131). Only 30% of patients with chronic myelogenous leukemia survive 5 years after diagnosis before imatinib is born. Imatinib increased this number from 30% to 89% and after 5 years, complete hematological remissions were achieved in the older 98% of patients.
The presence of imatinib successfully turns chronic myelogenous leukemia into a chronic disease like diabetes or hypertension. On the basis, subsequent researchers have carried out corresponding structural improvements on the molecular basis of imatinib, and developed second-generation drugs of Nilotinib (Nilotinib, novartis in 2007), radatinib (Radotinib, IL-yan Pharm in 2012) and third-generation drugs of panatinib (Ponatinib, ariad in 2012) which contain a trifluoromethyl aniline pyrimidine skeleton, and are mainly used for adult patients with Chronic Myelogenous Leukemia (CML) which are ineffective or resistant to treatment with imatinib. Therefore, the development of new imatinib analogs and methods for their preparation have been devised to remain of great value for the development of new anticancer drugs.
Triazazines are an important class of six-membered aromatic heterocyclic compounds having 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, the technology has attracted more and more attention and application in the fields of medicines, pesticides, chemical biology and the like. Notably, diels-Alder cycloaddition reactions with inverse electron requirements are widely used as a unique and efficient method for nitrogen-containing heterocycle synthesis, natural product preparation, and bioorthogonal chemistry. Therefore, by combining the trifluoromethyl triazene unit with the molecular skeleton of imatinib, a new organic small molecule tyrosine kinase inhibitor can be designed and developed and further applied to the research and development of anticancer drugs.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, a preparation method and application thereof, wherein the preparation method has good yield, and the obtained 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound has good tyrosine kinase inhibition activity, and can be further applied to synthesis and development of drug molecules for blocking tyrosine kinase.
The invention provides a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound, which is shown as a formula (I):
wherein R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclyl;
the substituent groups in the substituted phenyl and the 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 C1-C10 alkyl.
Preferably, the heterocyclic group is selected from thienyl, furyl or pyridyl.
Preferably, 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-furan-2-yl or 6-trifluoromethyl-pyridin-3-yl.
Preferably, one or more of the formulae (1) to (14):
the invention also provides a preparation method of the 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compound, which comprises the following steps:
subjecting an acyl halide compound shown in a formula (II) and a compound shown in a formula (III) to amination reaction to obtain a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound shown in a formula (I);
wherein X is halogen;
r is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic;
the substituent groups in the substituted phenyl and the 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 C1-C10 alkyl.
Preferably, the acid halide compound represented by the formula (II) is prepared according to the following steps:
s1) hydrolyzing a compound shown in a formula (IV) to obtain a carboxylic acid compound shown in a formula (V);
s2) carrying out halogenation reaction on a carboxylic acid compound shown in a formula (V) and a halogenating reagent to obtain an acyl halide compound shown in a formula (II);
wherein R' is selected from C1-C5 alkyl.
Preferably, the hydrolysis in step S1) is performed under alkaline conditions; the alkali used in the alkaline condition 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 comprises an organic solvent and water; the organic solvent is selected from tetrahydrofuran and/or methanol; the volume ratio of the organic solvent to the water is 1: (1-3); the temperature of the hydrolysis is 20-30 ℃; the hydrolysis time is 1-3 h;
the halogenating agent 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 temperature of the halogenation reaction is 20-30 ℃; the halogenation reaction time is 1-2 h.
Preferably, the amination reaction is carried out in a solvent; the solvent comprises N, N-dimethylformamide and dichloromethane; the volume ratio of the N, N-dimethylformamide to the dichloromethane is 1: (2-3); the temperature of the amination reaction is 20-30 ℃; the amination reaction time is 3-4 h.
The invention also provides application of the 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compound as a tyrosine kinase inhibitor.
Preferably, the 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound inhibits tyrosine kinase by promoting the expression level of Caspase-9 protein.
The invention provides a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound, which is characterized by being shown as a formula (I); wherein R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclyl; r' is selected from C1-C10 alkyl; the substituent groups in the substituted phenyl and the substituted heterocyclic groups 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-triazosin-3-amide compound provided by the invention contains a trifluoromethyl triazosin skeleton, has better tyrosine kinase inhibition activity, and can be further applied to the synthesis and development of drug molecules for blocking tyrosine kinase.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (I) obtained in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance fluorine spectrum of the compound represented by the formula (I) obtained in example 1 of the present invention;
FIG. 3 is a bar graph showing the effect of compounds obtained in the examples of the present invention on the expression level of protein Caspase 9.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound, which is shown as a formula (I):
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 heterocyclyl 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 thienyl, furyl or pyridyl.
The substituent of the substituted phenyl and the substituted heterocyclic group is 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, still more preferably one or more of C1-C2 alkyl, C1-C2 alkoxy, C1-C2 haloalkyl, halogen and cyano; the haloalkyl group is a haloalkyl group well known to those skilled in the art, and is not particularly limited, and preferably includes one or more of fluoroalkyl group, chloroalkyl group, bromoalkyl group and iodoalkyl group; the haloalkyl 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 number of substituents in the substituted phenyl group and the substituted heterocyclic group is not particularly limited, and is preferably 1 to 3; the present invention is not particularly limited in the position of the substituent in the substituted phenyl group and the substituted heterocyclic group, and may be para, ortho or meta.
According to the 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-furan-2-yl or 6-trifluoromethyl-pyridin-3-yl.
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 or ethyl group.
According to the present invention, most preferably, the 6-trifluoromethyl-1, 2, 4-triazin-3-amide compound is represented by one or more of the formulae (1) to (14):
the invention also provides a preparation method of the 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compound, which is characterized by comprising the following steps:
subjecting an acyl halide compound shown in a formula (II) and a compound shown in a formula (III) to amination reaction to obtain a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound shown in a formula (I);
wherein X is halogen, preferably including but not limited to F, cl or Br;
r is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic;
the substituent groups in the substituted phenyl and the 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 C1-C10 alkyl.
The invention has no special limitation on the sources of all raw materials, and can be sold in the market or self-made; the R and R' are the same as described above, and are not described in detail herein.
In the present invention, the acid halide compound represented by the formula (II) is prepared according to the following steps: s1) hydrolyzing a compound shown in a formula (IV) to obtain a carboxylic acid compound shown in a formula (V); s2) carrying out halogenation reaction on a carboxylic acid compound shown in a formula (V) and a halogenating reagent to obtain an acyl halide compound shown in a formula (II);
wherein R' is a C1-C5 alkyl group, preferably methyl, ethyl or propyl, more preferably methyl or ethyl.
Hydrolyzing a compound shown in a formula (IV) to obtain a carboxylic acid compound shown in a formula (V); the hydrolysis is preferably carried out under alkaline conditions; the alkali used in the alkaline condition is preferably one or more of potassium hydroxide, lithium hydroxide and sodium hydroxide; the molar ratio of the compound of formula (IV) to the base is preferably 1: (1 to 1.5), more preferably 1: (1.2 to 1.5), and more preferably 1:1.3; the hydrolysis is carried out in a mixed solvent; the mixed solvent comprises 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 to 3), more preferably 1: (1.5 to 2.5), and more preferably 1:2; in the present invention, it is preferable to mix the compound represented by the formula (IV) with an organic solvent, and then add a base and water to hydrolyze; the temperature of the hydrolysis is preferably 20-30 ℃; the hydrolysis time is 1-3 h; after hydrolysis, the pH value of the reaction solution is preferably adjusted to 6-7, and the organic phase is extracted by ethyl acetate; in the invention, the pH value of the reaction solution is preferably adjusted by adopting hydrochloric acid aqueous solution; the mass concentration of the hydrochloric acid aqueous solution is preferably 10% -15%; after the organic phase is extracted, washing with water and saturated brine are preferably further performed to obtain a carboxylic acid compound represented by formula (V); the number of times of washing with water and washing with saturated saline is preferably 2 to 5 times, more preferably 3 to 4 times, each independently; after washing, preferably drying by adding a drying agent, filtering, and concentrating to obtain a carboxylic acid compound represented by the formula (V); the dryer is preferably anhydrous sodium sulfate.
Carrying out halogenation reaction on a carboxylic acid compound shown in a formula (V) and a halogenating reagent to obtain an acyl halide compound shown in a formula (II); the halogenation reaction is preferably carried out in a protective atmosphere; the protective atmosphere is a protective atmosphere well known to those skilled in the art, and is not particularly limited, and argon is preferred in the present invention; the halogenating agent is preferably oxalyl halide and/or dihalosulfoxide; the specific type of the oxalyl halide and/or the dihalide sulfoxide can be selected according to the type of X in the compound shown in the formula (II), and in the invention, the oxalyl chloride and/or the dihalide sulfoxide can be specific; in order to convert the carboxylic acid compound represented by the formula (V) into the corresponding acid halide compound as much as possible, the halogenating agent is preferably used in an excess amount; the halogenation reaction is preferably carried out in the presence of a catalyst; the catalyst is preferably N, N-dimethylformamide; the halogenation reaction is preferably carried out in an organic solvent; the organic solvent is preferably dichloromethane; in the present invention, it is preferable to mix the carboxylic acid compound represented by the formula (V) with an organic solvent, and then sequentially add a halogenating agent and a catalyst to carry out a halogenation reaction; the temperature of the halogenation reaction is preferably 20-30 ℃; the halogenation reaction time is preferably 1 to 2 hours. In the present invention, the product after the halogenation reaction can be directly added to the compound represented by the formula (III) for the amination reaction without any post-treatment.
Subjecting an acyl halide compound shown in a formula (II) and a compound shown in a formula (III) to amination reaction to obtain a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound shown in a formula (I); the amination reaction is preferably carried out in a solvent; the solvent preferably comprises N, N-dimethylformamide and dichloromethane; the volume ratio of the N, N-Dimethylformamide (DMF) to the dichloromethane is preferably 1: (2-3), more preferably 1:2.5; in the present invention, the compound represented by the formula (III) is preferably added to the reaction system after being mixed with DMF; the temperature of the amination reaction is preferably 20-30 ℃; the time of the amination reaction is preferably 3-4 hours; part of the solid is separated out after the amination reaction, preferably DMF and/or DMSO is added until the separated solid is just dissolved, and then ethyl acetate is used for extraction; preferably, the extraction is further washed with water and saturated saline water; the number of times of washing with water and washing with saturated saline is preferably 2 to 5 times, more preferably 3 to 4 times, each independently; after washing, preferably adding a drying agent for drying, filtering and concentrating to obtain a crude product; the dryer is preferably anhydrous sodium sulfate; in the present invention, the crude product is preferably further washed with ethyl acetate to give a 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound represented by the formula (I).
The invention takes 6-trifluoromethyl-1, 2, 4-triazosin-3-carboxylate compound as raw material, fourteen 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compounds are obtained through three-step functional group conversion, the method has good yield, and the obtained 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compound has good tyrosine kinase inhibition activity, and can be further applied to synthesis and development of drug molecules for blocking tyrosine kinase.
The invention also provides application of the 6-trifluoromethyl-1, 2, 4-triazosin-3-amide compound as a tyrosine kinase inhibitor.
The 6-trifluoromethyl-1, 2, 4-triazene-3-amide compounds inhibit tyrosine kinase by promoting the expression level of Caspase-9 protein.
In order to further illustrate the present invention, the following describes in detail a 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, its preparation method and application provided in the present invention with reference to examples.
The reagents used in the examples below are all commercially available.
Example 1:5- (4-methoxyphenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
5- (4-methoxyphenyl) -6-trifluoromethyl-1, 2, 4-triazazine-3-methyl ester (939.7 mg,3 mmol) was dissolved in 5mL of tetrahydrofuran, and then lithium hydroxide (163.6 mg,3.9 mmol) and 10mL of water were added to the system. Stirring for 1-2 hours at room temperature, and monitoring the complete reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% aqueous hydrochloric acid to ph=6-7. The organic phase was then extracted with ethyl acetate, washed 3 times with water and 3 times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 5- (4-methoxyphenyl) -6-trifluoromethyl-1, 2, 4-triazosin-3-carboxylic acid (yield 85%).
Subsequently, 5- (4-methoxyphenyl) -6-trifluoromethyl-1, 2, 4-triazazine-3-carboxylic acid was dissolved in 5mL of anhydrous dichloromethane, the sealing plug was covered, the argon shield was replaced (argon balloon was inserted on the sealing plug), then oxalyl chloride (456.9 mg,3.6 mmol) was added with a 1mL disposable syringe, then 1 to 2 drops of anhydrous DMF was added dropwise, the reaction was carried out at room temperature for 1 to 2 hours, and the completion of the reaction of the starting materials was monitored by thin layer silica gel chromatography (TLC).
2- (5-amino-2-methylaniline) -4- (3-pyridine) pyrimidine (832 mg,3 mmol) was dissolved in 2mL anhydrous DMF, and then the solution was added dropwise to the above reaction system by syringe, reacted at room temperature for 3 to 4 hours, and a part of solid was precipitated. At this time, 4 to 5mL of DMF or DMSO was added to the system until a part of the solid was just dissolved, followed by transfer to a separating funnel and extraction with an appropriate amount of ethyl acetate, washing with water 3 to 5 times, washing with saturated saline water 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating to obtain a crude product, finally washing the crude product with ethyl acetate 8 to 10 times, pumping the solvent by vacuum pump to obtain a compound represented by formula (1) as a bright yellow solid (1.05 g, total yield 74%).
Analyzing the compound shown in the formula (1) obtained in the example 1 by nuclear magnetic resonance to obtain a nuclear magnetic resonance hydrogen spectrogram shown in the figure 1 and a nuclear magnetic resonance fluorine spectrogram shown in the figure 2; 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)。
Example 2:5- (4-methylphenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
5- (4-methylphenyl) -6-trifluoromethyl-1, 2, 4-triazazine-3-methyl ester (891.7 mg,3 mmol) was dissolved in 5mL of methanol, and then lithium hydroxide (163.6 mg,3.9 mmol) and 10mL of water were added to the system. Stirring for 1-2 hours at room temperature, and monitoring the complete reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% aqueous hydrochloric acid to ph=6-7. Then extracting the organic phase with ethyl acetate, respectively washing with water for 3 times and saturated saline water for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain 5- (4-methylphenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-formic acid.
Subsequently, 5- (4-methylphenyl) -6-trifluoromethyl-1, 2, 4-triazazine-3-carboxylic acid was dissolved in 5mL of anhydrous dichloromethane, the sealing plug was covered, the argon shield was replaced (argon balloon was inserted on the sealing plug), then oxalyl chloride (456.9 mg,3.6 mmol) was added with a 1mL disposable syringe, then 1 to 2 drops of anhydrous DMF was added dropwise, the reaction was carried out at room temperature for 1 to 2 hours, and the completion of the reaction of the starting materials was monitored by thin layer silica gel chromatography (TLC).
2- (5-amino-2-methylaniline) -4- (3-pyridine) pyrimidine (832 mg,3 mmol) was dissolved in 2mL anhydrous DMF, and then the solution was added dropwise to the above reaction system by syringe, reacted at room temperature for 3 to 4 hours, and a part of solid was precipitated. At this time, 4-5 ml of LDMF or DMSO was added to the system until a part of the solid was just dissolved, then transferred to a separating funnel and extracted with an appropriate amount of ethyl acetate, washed 3-5 times with water, washed 3 times with saturated saline, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, and finally the crude product was washed 8-10 times with ethyl acetate, and the solvent was pumped with a vacuum pump to give a compound (70%) represented by formula (2) as a bright yellow solid.
The compound represented by the formula (2) obtained in example 2 was analyzed by nuclear magnetic resonance to obtain 1 H 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＝8.0,4.8Hz,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)。
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-triazene-3-amide
5- (3, 5-bis (trifluoromethyl) phenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester (1.3 g,3 mmol) was dissolved in 5mL tetrahydrofuran, then potassium hydroxide (72.9 mg,3.9 mmol) and 10mL water were added to the system. Stirring for 1-2 hours at room temperature, and monitoring the complete reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% aqueous hydrochloric acid to ph=6-7. Then extracting the organic phase with ethyl acetate, washing with water for 3 times and saturated saline water for 3 times respectively, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain 5- (3, 5-bis (trifluoromethyl) phenyl) -6-trifluoromethyl-1, 2, 4-triazazine-3-formic acid.
5- (3, 5-bis (trifluoromethyl) phenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-carboxylic acid was then dissolved in 5mL of anhydrous dichloromethane, the sealing plug was covered, the argon shield was replaced (argon balloon was inserted on the sealing plug), then oxalyl chloride (456.9 mg,3.6 mmol) was added with a 1mL disposable syringe, followed by dropwise addition of 1-2 drops of anhydrous DMF, and the reaction was carried out at room temperature for 1-2 h, and completion of the starting material was monitored by thin layer silica gel chromatography (TLC).
2- (5-amino-2-methylaniline) -4- (3-pyridine) pyrimidine (832 mg,3 mmol) was dissolved in 2mL anhydrous DMF, and then the solution was added dropwise to the above reaction system by syringe, reacted at room temperature for 3 to 4 hours, and a part of solid was precipitated. At this time, 4-5 ml of LDMF or DMSO was added to the system until a part of the solid was just dissolved, then transferred to a separating funnel and extracted with an appropriate amount of ethyl acetate, washed 3-5 times with water, washed 3 times with saturated saline, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, and finally the crude product was washed 8-10 times with ethyl acetate, and the solvent was pumped in vacuo to give a compound (49%) represented by formula (3) as a bright yellow solid.
The compound represented by the 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)。
Example 4:5- (4-chlorophenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
5- (4-chlorophenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester (1.6 g,5 mmol) was dissolved in 5mL of tetrahydrofuran, and then sodium hydroxide (260 mg,6.5 mmol) and 10mL of water were added to the system. Stirring for 1-2 hours at room temperature, and monitoring the complete reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% aqueous hydrochloric acid to ph=6-7. Then extracting the organic phase with ethyl acetate, respectively washing with water for 3 times and saturated saline water for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain 5- (4-chlorophenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-formic acid.
Subsequently, 5- (4-chlorophenyl) -6-trifluoromethyl-1, 2, 4-triazazine-3-carboxylic acid was dissolved in 7.5mL of anhydrous dichloromethane, the sealing plug was covered, the argon shield was replaced (argon balloon was inserted on the sealing plug), then oxalyl chloride (456.9 mg,3.6 mmol) was added with a 1mL disposable syringe, then 1 to 2 drops of anhydrous DMF was added dropwise, and the reaction was carried out at room temperature for 1 to 2 hours, and the completion of the reaction of the starting materials was monitored by thin layer silica gel chromatography (TLC).
2- (5-amino-2-methylaniline) -4- (3-pyridine) pyrimidine (1.4 g,5 mmol) was dissolved in 3mL of anhydrous DMF, and then the solution was added dropwise to the above reaction system by syringe, reacted at room temperature for 3 to 4 hours, and a part of solid was precipitated. At this time, 5-6 ml of LDMF or DMSO was added to the system until a part of the solid was just dissolved, then transferred to a separating funnel and extracted with an appropriate amount of ethyl acetate, washed 3-5 times with water, washed 3 times with saturated saline, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, and finally the crude product was washed 8-10 times with ethyl acetate, and the solvent was pumped with a vacuum pump to give a compound (68%) represented by formula (4) as a bright yellow solid.
The compound represented by the 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)。
Example 5:5- (4-bromophenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
In analogy to example 1, 5- (4-bromophenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out according to the method described in example 1 to give the compound represented by formula (5) (48%) as a bright yellow solid.
The compound represented by the 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)。
Example 6:5- (4-fluorophenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
In analogy to example 1, 5- (4-fluorophenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was performed as described in example 1 to give the compound represented by formula (6) (64%) as a bright yellow solid.
The compound represented by the 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)。 19 F NMR(376MHz,DMSO-d6)δ(ppm):-60.63(s,3F)-108.32–-108.44(m,1F)。
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-triazene-3-amide
In analogy to example 1, 5- (3, 4, 5-trifluorophenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out as described in example 1 to give the compound represented by formula (7) (60%) as a bright yellow solid.
The compound represented by the 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)。
Example 8:5- (4-trifluoromethylphenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
In analogy to example 1, 5- (4-trifluoromethylphenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out as described in example 1 to give the compound of formula (8) (47%) as a bright yellow solid.
The compound represented by the 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)。
Example 9: 5-phenyl-N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
In analogy to example 1, 5-phenyl-6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out according to the method described in example 1 to give the compound represented by formula (9) (69%) as a bright yellow solid.
The compound represented by the 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)。
Example 10:5- (4-cyanophenyl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
In analogy to example 1, 5- (4-cyanophenyl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out as described in example 1 to give the compound of formula (10) (40%) as a bright yellow solid.
The compound represented by the 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)。
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-triazin-3-amide
In analogy to example 1, 5- ((6-trifluoromethylpyridin-3-yl) -1,2, 4-triazin-3-yl) methyl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out as described in example 1 to give the compound of formula (11) (43%) as a bright yellow solid.
The compound represented by the formula (11) obtained in example 11 was analyzed by nuclear magnetic resonance to obtain 1 HNMR(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),8.58–8.44(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)。
Example 12:5- (thiophen-2-yl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
In analogy to example 1, 5- (thiophen-2-yl) -6-trifluoromethyl-1, 2, 4-triazin-3-yl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out according to the method described in example 1 to give the compound (61%) of the formula (12) as a yellowish brown solid.
The compound represented by the 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＝3.7Hz,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)。
Example 13:5- (5-methylthiophene-2-yl) -N- (4-methyl-3- (4- (pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) -6- (trifluoromethyl) -1,2, 4-triazin-3-amide
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5- (5-methylthiophene-2-yl) -6-trifluoromethyl-1, 2, 4-triazene-3-methyl ester (909.8 mg,3 mmol) was dissolved in 5mL of tetrahydrofuran, and then lithium hydroxide (163.6 mg,3.9 mmol) and 10mL of water were added to the system. Stirring for 1-2 hours at room temperature, and monitoring the complete reaction of the raw materials by thin layer silica gel chromatography (TLC). Acidify with 10% aqueous hydrochloric acid to ph=6-7. Then extracting the organic phase with ethyl acetate, respectively washing with water for 3 times and saturated saline water for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain 5- (5-methylthiophene-2-yl) -6-trifluoromethyl-1, 2, 4-triazoxide-3-formic acid.
5- (5-methylthiophene-2-yl) -6-trifluoromethyl-1, 2, 4-triazene-3-carboxylic acid was then dissolved in 5mL of anhydrous dichloromethane, the sealing plug was covered, the argon shield was replaced (argon balloon was inserted on the sealing plug), thionyl chloride (428.3 mg,3.6 mmol) was then added with a 1mL disposable syringe, followed by dropwise addition of 1-2 drops of anhydrous DMF, the reaction was carried out at room temperature for 1-2 h, and the completion of the starting material was monitored by thin layer silica gel chromatography (TLC).
2- (5-amino-2-methylaniline) -4- (3-pyridine) pyrimidine (832 mg,3 mmol) was dissolved in 2mL anhydrous DMF, and then the solution was added dropwise to the above reaction system by syringe, reacted at room temperature for 3 to 4 hours, and a part of solid was precipitated. At this time, 4 to 5mL of DMF or DMSO was added to the system until a part of the solid was just dissolved, followed by transfer to a separating funnel and extraction with an appropriate amount of ethyl acetate, water washing 3 to 5 times, saturated saline water washing 3 times, drying the organic phase with anhydrous sodium sulfate, filtration, concentration to obtain a crude product, finally washing the crude product with ethyl acetate 8 to 10 times, and pumping the solvent in vacuo to obtain a compound represented by formula (13) (63%) as a yellowish brown solid.
The compound represented by the formula (13) obtained in example 13 was analyzed by nuclear magnetic resonance to obtain 1 HNMR(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)。
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-triazin-3-amide
In analogy to example 13, 5- (5-methylfuran-2-yl) -6-trifluoromethyl-1, 2, 4-triazin-3-yl ester was dissolved in 5mL of tetrahydrofuran solvent and the subsequent reaction was carried out according to the method described in example 13 to give the compound of formula (14) (52%) as a tan solid.
The compound represented by the formula (14) obtained in example 14 was subjected to nuclear magnetic resonanceAnalysis is carried out 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)。
Results of Activity test
In vitro experiments are carried out based on THP-1 (human monocytic leukemia), and whether the compound has promotion effect on pro-apoptotic protein Caspase 9 is explored.
The experimental process comprises the following steps:
1) Cell culture
THP-1 cells were cultured in 1640 medium containing 10% Fetal Bovine Serum (FBS), when the cell density reached 80% -90%, centrifuged at 1000rpm for five minutes, the supernatant was aspirated, fresh medium was added, and the ratio was 1: passaging was performed at 3 density.
2) Cell plating
Adding the prepared cell solution into 12-well plate according to 1mL standard per well, placing in 37 deg.C incubator and 5% CO 2 Overnight.
3) Cell administration
The compound and imatinib were dissolved in DMSO separately and prepared to a concentration of 100mM as a mother solution for use. The compound, the positive control imatinib, was dissolved to a final concentration of 10 μm with 1640 medium as diluent.
Adding 1mL of the prepared solution into each pore plate, placing into a 37 ℃ incubator and 5% CO 2 After incubation for 24 hours, the cells were removed.
4) Protein extraction and Western Blot
Taking out the pore plate, washing with Phosphate Buffered Saline (PBS) once, adding 100 mu L of protein lysate RIPA, reacting at low temperature for 30 min, sucking and adding into a centrifuge tube, centrifuging at 13000rpm for 10min, sucking supernatant, quantifying protein, leveling protein concentration, preparing protein into corresponding loading volume, adding loading buffer, boiling, and placing into a refrigerator at 4 ℃ for standby.
Preparing corresponding lower glue according to the size of the protein, adding newly prepared electrophoresis liquid into the inner layer, adding recovered electrophoresis liquid into the outer layer, and converting into 120V after 20min, wherein 90min is the time. The transfer conditions were 200mA for 60 minutes. The membrane is taken out and placed in 5% concentration skimmed milk powder, and after shaking for 1 hour in a shaker, the corresponding antibody is added. Placed in a refrigerator at 4 ℃ overnight. The membrane was then removed and washed three times with TBST for 15 minutes each. Adding secondary antibody, shaking at room temperature for 1 hr, taking out, and washing the membrane with TBST three times for 15 min each time.
Experimental results:
a bar graph of protein Caspase 9 expression levels was obtained as shown in FIG. 3. As shown in FIG. 3, the promotion effect of 10. Mu.M compound and positive drug imatinib on protein Caspase 9 is shown, and compared with the model, the promotion effect of imatinib and compound shown in formula (8), compound shown in formula (9) and compound shown in formula (11) on protein Caspase 9 is obviously up-regulated. According to One-Way ANOVA analysis, the above compounds and imatinib all have statistical differences. The compound shown in the formula (8), the compound shown in the formula (9) and the compound shown in the formula (11) have good promotion effect on the pro-apoptosis protein Caspase 9 in the human monocytic leukemia, and the action effect of the compound 9 is obviously better than that of the positive control drug imatinib.

Claims (10)

1. A 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, characterized by being represented by formula (I):
wherein R is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclyl;
the substituent groups in the substituted phenyl and the 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 C1-C10 alkyl.
2. The 6-trifluoromethyl-1, 2, 4-triazin-3-amide compound according to claim 1, wherein the heterocyclic group is selected from thienyl, furyl or pyridyl.
3. The 6-trifluoromethyl-1, 2, 4-triazin-3-carboxamide compound according to claim 1, characterized in that 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-furan-2-yl or 6-trifluoromethyl-pyridin-3-yl.
4. The 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound according to claim 1, characterized by one or more of the formulae (1) to (14):
5. a process for the preparation of a 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, comprising the steps of:
subjecting an acyl halide compound shown in a formula (II) and a compound shown in a formula (III) to amination reaction to obtain a 6-trifluoromethyl-1, 2, 4-triazazine-3-amide compound shown in a formula (I);
wherein X is halogen;
r is selected from substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic;
the substituent groups in the substituted phenyl and the 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 C1-C10 alkyl.
6. The process according to claim 5, wherein the acid halide compound represented by the formula (II) is prepared by:
s1) hydrolyzing a compound shown in a formula (IV) to obtain a carboxylic acid compound shown in a formula (V);
s2) carrying out halogenation reaction on a carboxylic acid compound shown in a formula (V) and a halogenating reagent to obtain an acyl halide compound shown in a formula (II);
wherein R' is selected from C1-C5 alkyl.
7. The method according to claim 6, wherein the hydrolysis in step S1) is performed under alkaline conditions; the alkali used in the alkaline condition 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 comprises an organic solvent and water; the organic solvent is selected from tetrahydrofuran and/or methanol; the volume ratio of the organic solvent to the water is 1: (1-3); the temperature of the hydrolysis is 20-30 ℃; the hydrolysis time is 1-3 h;
the halogenating agent 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 temperature of the halogenation reaction is 20-30 ℃; the halogenation reaction time is 1-2 h.
8. The process according to claim 5, wherein the amination is carried out in a solvent; the solvent comprises N, N-dimethylformamide and dichloromethane; the volume ratio of the N, N-dimethylformamide to the dichloromethane is 1: (2-3); the temperature of the amination reaction is 20-30 ℃; the amination reaction time is 3-4 h.
9. Use of a 6-trifluoromethyl-1, 2, 4-triazin-3-amide compound as shown in any one of claims 1 to 4 or a 6-trifluoromethyl-1, 2, 4-triazin-3-amide compound prepared by the preparation method as claimed in any one of claims 5 to 8 as a tyrosine kinase inhibitor.
10. The use according to claim 9, wherein the 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound inhibits tyrosine kinase by promoting the expression level of Caspase-9 protein.
CN202310534519.7A 2023-05-12 2023-05-12 6-trifluoromethyl-1, 2, 4-triazene-3-amide compound, and preparation method and application thereof Pending CN116574088A (en)

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