CN115925632A - Imidazole phenyl aminobenzamide compound with antitumor activity - Google Patents
Imidazole phenyl aminobenzamide compound with antitumor activity Download PDFInfo
- Publication number
- CN115925632A CN115925632A CN202211372805.XA CN202211372805A CN115925632A CN 115925632 A CN115925632 A CN 115925632A CN 202211372805 A CN202211372805 A CN 202211372805A CN 115925632 A CN115925632 A CN 115925632A
- Authority
- CN
- China
- Prior art keywords
- hydrogen
- compound
- formula
- methyl
- alkyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides an imidazole phenyl aminobenzamide compound with anti-tumor activity, the structure of the imidazole phenyl aminobenzamide compound is shown as a general formula I, and the definition of each substituent group is described in the specification. The imidazole phenyl aminobenzamide compound has obvious effect in vitroThe Bcr-Abl1 kinase has obvious inhibition effect on K562, HL60 and MCF-7 tumor cells, can cause apoptosis and inhibit cell migration, has partial compound activity superior to that of imatinib, and is a potential molecule with antitumor activity.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to an imidazole phenyl aminobenzamide compound with anti-tumor activity.
Background
Protein Kinases (PKs) are enzymes that catalyze the phosphorylation of specific serine, threonine or tyrosine residues in cellular proteins. These post-translational modifications of substrate proteins serve as molecular switches that regulate cell proliferation, activation, and/or differentiation. Frequent or excessive PK activity has been observed in a number of disease states, including benign and malignant proliferative disorders. In many cases, diseases, such as proliferative diseases, have been treated by in vitro and in vivo use of PK inhibitors. Protein tyrosine kinases are a class of enzymes that catalyze the transfer of phosphate groups from ATP to tyrosine residues located on protein substrates. Protein tyrosine kinases play an important role in normal cell growth. Many growth factor receptor proteins function as tyrosine kinases and it is through this process that they affect signal transduction. The interaction of growth factors with these receptors is a prerequisite for normal cell growth regulation. However, in some cases, uncontrolled cell proliferation may lead to tumor growth due to mutation or expression, since phosphorylation is a necessary signal for cell division to occur, and overexpressed or mutated kinases are associated with tumor disease, in which process inhibitors, protein tyrosine kinase inhibitors, would be of therapeutic value for tumor disease and other diseases characterized by uncontrolled or abnormal cell growth. At present, a plurality of new anti-tumor drug targets become a research hotspot, wherein among various molecular targets, protein tyrosine kinase is an anti-tumor drug target which is researched more and has obvious effect at present.
The Philadelphia chromosome, which is a marker of Chronic Myelogenous Leukemia (CML), carries a fusion gene containing the N-terminal exon of the BCR gene and the major C-terminal portion of the ABL gene (exons 2-11). The gene product is a 210kD protein (p 210 BCR-ABL 1). BCR-ABL1 is constitutively activated in a BCR-ABL1 fusion protein. This deregulated tyrosine kinase interacts with a variety of cellular signaling pathways leading to cellular transformation and deregulated cell proliferation (Lugo et al, science 247, 1079[1990 ]).
Although drugs that inhibit the tyrosine kinase activity of BCR-ABL1 via an ATP-competitive mechanism, such as imatinib, nilotinib, dasatinib, are effective in treating CML, some patients relapse due to the emergence of drug-resistant clones, and therefore, the development of novel inhibitors of the BCR-ABL1 enzyme is of great interest.
Disclosure of Invention
The invention provides a compound shown as a formula I or a pharmaceutically acceptable salt thereof:
wherein R is 6 Is composed of
X is selected from CH 2 R 7 、NR 8 Or O, n is an integer of 0 to 4, and R is 6 Optionally substituted by one or more R 9 Substitution; the R is 9 Selected from the group consisting of hydrogen, C1-C6 alkyl, halogen, cyano, -NR 10 R 11 、NO 2 - (C1-C6 alkyl) -O-, are present in the animal or in the animal>
Acetyl or 1-3 halo-substituted C1-C6 alkyl;
R 1 、R 2 、R 3 、R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, halogen, cyano, -NR 10 R 11 、NO 2 (C1-C6 alkyl) -O-),
Acetyl, 1-3 halo-substituted C1-C6 alkyl;
R 8 and R 9 Each independently selected from hydrogen, C1-C6 alkyl,
Or an acetyl group;
R 10 and R 11 Each independently selected from H or C1-C6 alkyl.
In some embodiments, n is selected from an integer from 0 to 1.
In some embodiments, the compound of formula I has the structure shown in formula II,
wherein the hydrogen atom on the piperazine ring A may be substituted by one or more R 9 Substituted, R 1 、R 2 、R 3 、R 4 、R 5 、R 8 And R 9 As defined for compounds of formula I.
In some typical embodiments, R 1 Selected from hydrogen, C1-C6 alkyl or cyano, preferably, R 1 Selected from hydrogen, methyl or cyano, more preferably, R 1 Selected from methyl.
In some typical embodiments, R 2 Selected from hydrogen or C1-C6 alkyl, preferably, R 2 Selected from hydrogen or methyl, more preferably, R 2 Selected from hydrogen.
In some typical embodiments, R 3 Selected from hydrogen or C1-C6 alkyl, preferably, R 3 Selected from hydrogen or methyl, more preferablyOptionally, R 3 Selected from hydrogen.
In some typical embodiments, R 4 Selected from hydrogen or C1-C6 alkyl substituted by 1-3 halogens, preferably R 3 Selected from hydrogen or trifluoromethyl, more preferably, R 3 Selected from trifluoromethyl.
In some typical embodiments, R 5 Selected from hydrogen or (C1-C6 alkyl) -O-, preferably, R 5 Selected from hydrogen or methoxy, more preferably, R 5 Selected from hydrogen.
In some typical embodiments, R 8 Selected from hydrogen,
Acetyl, n-butyl, methyl, tert-butyl, ethyl or isopropyl, preferably R 8 Selected from n-butyl, methyl or tert-butyl.
In some typical embodiments, R 9 Selected from hydrogen.
In some embodiments, the compound of formula I has the structure shown in formula III,
wherein the hydrogen atoms of the morpholine ring B may be substituted by one or more R 9 Substituted, R 1 、R 2 、R 3 、R 4 、R 5 And R 9 As defined for compounds of formula I.
In some typical embodiments, R 1 、R 2 、R 3 、R 4 And R 5 Selected from hydrogen, C1-C6 alkyl or C1-C6 alkyl substituted by 1-3 halogens, preferably R 1 、R 2 、R 3 、R 4 And R 5 Selected from hydrogen, methyl or trifluoromethyl, more preferably, R 1 Is methyl, R 4 Is trifluoromethyl, R 2 、R 3 And R 5 Are all hydrogen.
In some typical embodiments, R 9 Is hydrogen or C1-C6 alkyl, preferably, R 9 Is hydrogen or methyl。
In some embodiments, the compound of formula I has the structure shown in formula IV,
wherein the hydrogen atoms on the pyrrolidine C may be substituted by one or more R 9 Substituted, R 1 、R 2 、R 3 、R 4 、R 5 And R 9 As defined for compounds of formula I.
In some typical embodiments, R 1 Is methyl, R 4 Is trifluoromethyl, R 2 、R 3 And R 5 Are all hydrogen.
In some typical embodiments, R 9 Is hydrogen.
In another aspect, the present invention provides the following compounds or pharmaceutically acceptable salts thereof:
preferably, the present invention provides the following compounds or pharmaceutically acceptable salts thereof:
in some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, II, III, or VI, or a pharmaceutically acceptable salt thereof.
In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, II, III, or VI, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The pharmaceutical compositions of the invention may be administered by any suitable route or method, for example, by oral or parenteral (e.g., intravenous) administration. A therapeutically effective amount of a compound of formula I, II, III or VI is from about 1mg to 1g/Kg body weight/day.
For oral administration, the pharmaceutical compositions of the present invention are typically provided in the form of tablets, capsules or solutions. Tablets may comprise a compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, diluents, disintegrants, binders, lubricants.
For parenteral administration, the pharmaceutical composition of the present invention may be administered by intravenous injection, intramuscular injection or subcutaneous injection. It is usually provided as a sterile aqueous solution or suspension or lyophilized powder, with appropriate adjustment of pH and isotonicity.
In another aspect, the present invention also provides the use of a compound of formula I, II, III or VI, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease responsive to inhibition of protein kinase activity.
In another aspect, the present invention also provides the use of a compound of formula I, II, III or VI, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament for the prevention and/or treatment of a neoplastic disease.
Preferably, the neoplastic disorder is selected from chronic myelogenous leukemia and acute lymphocytic leukemia.
In another aspect, the present invention provides a process for preparing compounds of formula I, including, but not limited to, the following synthetic schemes:
wherein R is 1 、R 2 、R 3 、R 4 、R 5 、R 9 And X is as defined for compounds of formula I, P and M are independently selected from Cl, br or I.
Carrying out coupling reaction on the compound of the formula 1 and the compound of the formula 2 in the presence of a catalyst to prepare a compound of a formula 3; carrying out amide forming reaction on the compound of the formula 3 and the compound of the formula 4 to obtain a compound of a formula 5; reacting the compound shown in the formula 5 with the compound shown in the formula 6 under alkaline conditions to obtain the compound shown in the formula 7.
Correlation definition
Unless specifically stated otherwise, the following terms used in the specification and claims have the following meanings:
the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Numerical ranges herein refer to each integer in the given range. For example, "C1-C6" means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
The term "substituted" or "substituted" means that any one or more hydrogen atoms on a specified group is replaced with a substituent, so long as the valence of the specified group is normal and the substituted compound is stable. For example, "substituted with a halogen" means that any one or more hydrogen atoms on a particular group is substituted with a halogen, provided that the valence of the particular group is normal and the substituted compound is stable.
The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight or branched chain saturated hydrocarbon groups having the number of carbon atoms shown. For example, the term "C1-C6 alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl, examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl or 3-hexyl and the like.
"halogen" (or halo) means chlorine, fluorine, bromine or iodine.
The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness of the free acids and bases of the particular compound without biologically adverse effects. Such as acid (including organic and inorganic) addition salts or base addition salts (including organic and inorganic bases).
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.
The term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or pharmaceutical agent that is non-toxic but achieves the desired effect.
The term "pharmaceutically acceptable carrier" refers to those carriers which do not significantly irritate the body and which do not impair the biological activity and performance of the active compound. Including but not limited to any diluents, disintegrants, binders, glidants, wetting agents approved by the national food and drug administration for use in humans or animals.
The abbreviations used in the claims and the specification have the following meanings:
M:mol/L；
mM：mmol/L；
μM：μMol/L；
nM：nmol/L。
drawings
FIG. 1 results of Transwell experiment
FIG. 2 Effect of Compounds on apoptosis of K562 cells
FIG. 3 Effect of Compounds on apoptosis-related protein expression
Detailed Description
The following more specifically describes the preparation of the compounds of the present invention, but these specific preparation methods do not set any limit to the scope of the present invention. In addition, reaction conditions such as reactants, solvents, bases, amounts of compounds used, reaction temperature, reaction time and the like are not limited to the following examples.
The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art.
1 H-NMR and 13 C-NMR was recorded on a 500MHz NMR spectrometer. Significant peaks are tabulated in order of split number (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br.s, broad singlet) and proton number.
In the following examples, the abbreviations given below are used: TLC (thin layer chromatography); NMR (nuclear magnetic resonance); HR-MS (high resolution mass spectrometry); min (minutes); h (hours); m.p (melting point); mL (milliliters); l (liter); mg (mg).
Example 1- ((4-ethylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), N-ethylpiperazine (154 μ L,0.990 mmol) and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0ml of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material point disappeared (dichloromethane: methanol = 10. The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the target product, 4- ((4-ethylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (140 mg), was obtained by flash column chromatography as a white solid in a yield of 57.26%. m.p.68.3-68.7 ℃;
HR-MS m/z:472.2322[M+H] + ； 1 H NMR(500MHz,Chloroform-d)δ9.04(s,1H),8.24(s,1H),7.90(d,J＝8.1Hz,2H),7.83(s,1H),7.79(s,1H),7.46(d,J＝8.0Hz,2H),7.34(s,1H),7.08(s,1H),3.58(s,2H),2.59–2.50(m,4H),2.46(q,J＝7.3Hz,3H),2.27(s,3H),1.11(t,J＝7.2Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ166.19,143.35,140.65,140.11,138.41,134.50,132.67,129.38,127.35,124.30,122.13,115.42,115.05,114.53,112.76,62.43,52.92,52.69,52.29,13.57,11.82.
example 1a 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round-bottomed flask were added 3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) aniline (0.121g, 0.5 mmol) and cesium carbonate (0.179g, 0.55mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.113g, 0.6 mmol) was also dissolved in dichloromethane, the reaction solution was slowly dropped at room temperature, and after 4 hours of reaction under sealed stirring, the starting material point was monitored to disappear (developing agent: PE: EA = 1. The reaction solution was extracted with saturated brine and dichloromethane, and white crystals were naturally precipitated from the organic phase to obtain 0.191g of the objective product with a yield of 97.0%. HR-MS m/z of 394.0933[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ9.09(s,1H),8.18(s,1H),7.93(d,J＝7.8Hz,2H),7.83(s,1H),7.76(s,1H),7.50(d,J＝7.8Hz,2H),7.34(s,1H),7.06(s,1H),4.63(s,2H),2.24(s,3H). 13 C NMR(126MHz,Chloroform-d)δ166.01,142.06,140.68,140.25,138.56,134.67,134.05,129.13,128.02,124.46,122.29,115.67,115.36,114.78,113.19,45.39,13.70.
Example 2- ((4-Isopropylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), 1-isopropylpiperazine (141 μ L,0.990 mmol), and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0mL N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored by disappearance of the starting point (dichloromethane: methanol = 10. Reaction solutionExtraction was performed with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the target product, 4- ((4-isopropylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (85 mg), was obtained by flash column chromatography as a white solid in 31.47% yield. m.p.72.2-73.6 ℃; HR-MS m/z 486.2486[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ8.80(s,1H),8.23(s,1H),7.88(d,J＝8.0Hz,2H),7.80(d,J＝4.5Hz,2H),7.45(d,J＝7.9Hz,2H),7.34(s,1H),7.08(s,1H),3.57(s,2H),2.69(dt,J＝12.8,6.4Hz,1H),2.55(d,J＝32.5Hz,8H),2.27(s,3H),1.07(d,J＝6.5Hz,6H). 13 C NMR(126MHz,Chloroform-d)δ166.23,143.30,140.67,140.06,138.39,134.48,132.67,129.45,127.34,115.41,115.04,114.53,112.75,62.50,54.54,53.31,48.59,18.58,13.55.
Example 3- ((4-Acetylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), 1-acetylpiperazine (127 μ L,0.990 mmol), and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0ml of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material point disappeared (dichloromethane: methanol = 10. The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the target product, 4- ((4-acetylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (192 mg), was obtained by flash column chromatography as a white solid in a yield of 76.38%. m.p.195.1-196.3 ℃; HR-MS m/z 486.2126[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ9.57(s,1H),8.32(s,1H),8.04–7.84(m,3H),7.79(s,1H),7.42(d,J＝7.5Hz,2H),7.33(s,1H),7.08(s,1H),3.56(s,2H),3.49(d,J＝34.1Hz,4H),2.40(d,J＝60.3Hz,4H),2.26(s,3H),2.07(s,3H). 13 C NMR(126MHz,Chloroform-d)δ169.14,166.27,142.52,141.03,140.06,138.35,134.51,133.18,129.06,127.71,124.40,122.23,115.47,115.14,114.52,112.54,62.28,53.26,52.51,46.34,41.51,21.33,13.61.
Example 4N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (morpholinomethyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), morpholine (86 μ L,0.990 mmol), and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0ml N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 10. The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the objective product N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (morpholinomethyl) benzamide (194 mg) was obtained as a white solid in 84.34% yield by flash column chromatography. m.p.81.6-82.5 ℃; HR-MS m/z 445.1848[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ9.40(s,1H),8.20(s,1H),7.90(d,J＝8.0Hz,2H),7.84(s,1H),7.74(s,1H),7.44(d,J＝8.0Hz,2H),7.30(s,1H),7.04(s,1H),3.77–3.63(m,4H),3.54(s,2H),2.43(s,4H),2.22(s,3H). 13 C NMR(126MHz,Chloroform-d)δ166.54,142.99,140.98,140.09,138.43,134.60,133.02,129.51,127.66,124.46,122.29,115.65,115.40,114.76,112.84,67.07,63.00,53.75,13.62.
Example 5N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (2-methylmorpholinyl) methyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), 2-methylmorpholine (112. Mu.L, 0.990 mmol) and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0mL N, N-bisAfter stirring the reaction at room temperature for 4h in methyl formamide, TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 10. The reaction mixture was extracted with saturated brine and ethyl acetate, and excess solvent was removed by evaporation under reduced pressure, followed by flash column chromatography to give the objective product N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (2-methylmorpholinyl) methyl) benzamide (183 mg) as a white solid in 76.93% yield. m.p.69.4-71.6 deg.c; HR-MS m/z, 459.2004[ M + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.28(d,J＝16.7Hz,1H),8.21(s,1H),7.90(d,J＝7.9Hz,2H),7.82(s,1H),7.75(s,1H),7.45(d,J＝8.0Hz,2H),7.31(s,1H),7.05(s,1H),3.82(d,J＝11.3Hz,1H),3.64(dt,J＝17.1,8.7Hz,2H),3.53(s,2H),2.64(dd,J＝29.0,11.2Hz,2H),2.22(s,3H),2.16(td,J＝11.3,2.9Hz,1H),1.85(t,J＝10.6Hz,1H),1.11(d,J＝6.3Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ166.31,142.97,140.76,140.01,140.01,138.33,134.47,132.83,129.36,127.47,124.30,122.13,115.49,115.17,114.60,112.72,71.80,66.67,62.64,60.03,52.93,19.07,13.51.
Example 6N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (pyrrolidin-1-ylmethyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), tetrahydropyrrole (81 μ L,0.990 mmol), and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0ml of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material point disappeared (dichloromethane: methanol = 10. The reaction mixture was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the desired product, N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (pyrrolidin-1-ylmethyl) benzamide (82 mg), was obtained as a white solid in a yield of 36.83% by flash column chromatography. m.p.58.3-59.8 ℃; HR-MS m/z, 429.1910[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ8.86(s,1H),8.24(s,1H),7.89(d,J＝8.1Hz,2H),7.82(d,J＝12.6Hz,2H),7.49(d,J＝8.1Hz,2H),7.36(s,1H),7.09(s,1H),3.71(s,2H),2.55(s,4H),2.28(s,3H),1.82(s,4H). 13 C NMR(126MHz,Chloroform-d)δ166.12,143.98,140.59,140.15,138.47,134.52,132.65,129.26,127.37,115.36,114.95,114.50,112.79,60.16,54.22,23.49,13.60.
Example 7- ((4-Butylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), 1-butylpiperazine (157 μ L,0.990 mmol) and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0ml of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material point disappeared (dichloromethane: methanol = 10. The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the objective product, 4- ((4-butylpiperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (158 mg), was obtained as a white solid in a yield of 61.31% by flash column chromatography. m.p.57.7-59.9 ℃; HR-MS m/z 500.2636[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.03(s,1H),8.23(s,1H),7.89(d,J＝8.1Hz,2H),7.83(s,1H),7.78(s,1H),7.46(d,J＝8.0Hz,2H),7.34(s,1H),7.08(s,1H),3.57(s,2H),2.50(s,8H),2.37–2.32(m,2H),2.27(s,3H),1.48(p,J＝7.6Hz,2H),1.33(h,J＝7.3Hz,2H),0.92(t,J＝7.3Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ166.23,143.40,140.67,140.08,138.40,134.48,132.66,129.40,127.33,124.30,122.13,115.41,115.06,114.53,112.73,62.49,58.48,53.18,53.08,28.99,20.79,14.04,13.56.
Example 8 (S) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (3-methylmorpholinyl) methyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), (S) -3-methylmorpholine (112 μ L,0.990 mmol) and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0mL N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 10. The reaction mixture was extracted with saturated brine and ethyl acetate, and excess solvent was removed by evaporation under reduced pressure, followed by flash column chromatography to give the desired product (S) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (3-methylmorpholinyl) methyl) benzamide (106 mg) as a white solid in 44.55% yield. m.p.81.3-82.7 ℃; HR-MS m/z:459.2014[ M + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.14(s,1H),8.21(s,1H),7.89(d,J＝8.0Hz,2H),7.81(s,1H),7.77(s,1H),7.46(d,J＝7.9Hz,2H),7.32(s,1H),7.06(s,1H),4.08(d,J＝13.8Hz,1H),3.71(dd,J＝11.4,3.5Hz,2H),3.58(td,J＝10.6,2.4Hz,1H),3.31(dd,J＝11.2,9.0Hz,1H),3.21(d,J＝13.8Hz,1H),2.58–2.46(m,2H),2.24(s,3H),2.23–2.17(m,1H),1.06(d,J＝6.3Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ166.27,143.97,140.73,140.05,138.37,134.47,132.64,129.27,127.39,124.30,122.13,115.44,115.08,114.58,112.72,72.90,67.33,57.96,55.61,51.45,14.44,13.53.
Example 9- (2, 6-Dimethylmorpholinyl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), 2, 6-dimethylmorpholine (122 μ L,0.990 mmol), and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0mL N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored by disappearance of the starting point (dichloromethane: methanol = 10. The reaction mixture was extracted with saturated brine and ethyl acetate, the excess solvent was removed by evaporation under reduced pressure,flash column chromatography gave the desired product, 4- ((2, 6-dimethylmorpholine) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (112 mg) as a white solid in 45.71% yield. m.p.65.7-66.2 ℃; HR-MS m/z 473.2165[ M + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ8.89(s,1H),8.22(s,1H),7.90(d,J＝8.1Hz,2H),7.79(s,2H),7.47(d,J＝8.0Hz,2H),7.34(s,1H),7.07(s,1H),3.70(dq,J＝12.3,6.3Hz,2H),3.54(s,2H),2.67(d,J＝10.9Hz,2H),2.25(s,3H),1.79(t,J＝10.7Hz,2H),1.13(d,J＝6.3Hz,6H). 13 C NMR(126MHz,Chloroform-d)δ166.03,143.09,140.53,140.18,138.49,134.51,132.78,129.51,129.49,127.34,115.37,114.91,114.53,112.87,71.66,62.44,59.42,19.11,13.60.
Example 10- ((4- (tert-butyl) piperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), N-tert-butylpiperazine (118 μ L,0.743 mmol) and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0mL of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material point disappeared (dichloromethane: methanol = 10. The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the desired product, 4- ((4- (tert-butyl) piperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (134 mg), was obtained by flash column chromatography as a white solid in 51.78% yield. m.p.80.5-81.2 ℃; HR-MS m/z 500.2633[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ8.91(s,1H),8.23(s,1H),7.88(d,J＝8.1Hz,2H),7.83(s,1H),7.78(s,1H),7.44(d,J＝8.0Hz,2H),7.33(s,1H),7.07(s,1H),3.56(s,2H),2.58(d,J＝61.9Hz,8H),2.27(s,3H),1.10(s,9H). 13 C NMR(126MHz,Chloroform-d)δ166.15,143.25,140.62,140.11,138.43,134.51,132.67,129.49,127.32,124.31,122.14,115.39,115.00,114.51,112.74,62.42,53.50,45.62,29.71,25.75,13.59.
Example 11- ((4- (Cyclopropanecarbonyl) piperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200g, 0.508mmol), 1-cyclopropylformylpiperazine (141 μ L,0.990 mmol), and potassium carbonate (0.075g, 0.545mmol) dissolved in 4.0mL of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4H, TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 10. The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the target product, 4- ((4- (cyclopropanecarbonyl) piperazin-1-yl) methyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (185 mg), was obtained by flash column chromatography as a white solid in a yield of 69.72%. m.p.82.3-84.0 ℃; HR-MS m/z 512.2266[ m + H ])] + ； 1 H NMR(500MHz,Chloroform-d)δ9.54(s,1H),8.22(s,1H),7.86(d,J＝7.9Hz,2H),7.82(s,1H),7.73(s,1H),7.37(d,J＝7.9Hz,2H),7.27(s,1H),7.02(s,1H),3.65(s,2H),3.51(s,2H),3.50(s,2H),2.45(s,2H),2.28(s,2H),2.21(s,3H),1.68(tt,J＝8.0,4.8Hz,1H),0.93–0.85(m,2H),0.73–0.67(m,2H). 13 C NMR(126MHz,Chloroform-d)δ172.16,166.32,142.55,141.02,140.00,138.30,134.50,133.11,129.11,127.68,124.38,115.56,115.28,114.53,112.57,62.32,53.46,52.59,45.54,42.20,13.57,11.02,7.45.
Example 12N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask was added 4- (chloromethyl) -N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) benzamide (0.200 g,0.508 mmol), N-methylpiperazine (157 μ L,0.990 mmol) and potassium carbonate (0.075g, 0.545mmol) were dissolved in 4.0ml of N, N-dimethylformamide, and after stirring the reaction at room temperature for 4 hours, TLC monitored until the starting material point disappeared (dichloromethane: methanol =10: 1). The reaction solution was extracted with saturated brine and ethyl acetate, excess solvent was removed by evaporation under reduced pressure, and the target product N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide (186 mg) was obtained as a white solid by flash column chromatography (yield 80.38%). m.p.76.3-77.9 ℃; HR-MS m/z of 458.2176[ M + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.17(s,1H),8.21(s,1H),7.88(d,J＝8.0Hz,2H),7.81(s,1H),7.75(s,1H),7.43(d,J＝8.0Hz,2H),7.31(s,1H),7.05(s,1H),3.54(s,2H),2.75–2.30(m,8H),2.29(s,3H),2.23(s,3H). 13 C NMR(126MHz,Chloroform-d)δ166.28,143.39,140.70,140.06,138.36,134.48,132.68,129.33,127.38,124.30,122.13,115.46,115.11,114.56,112.74,62.40,55.04,53.01,45.94,13.54.
Example 13N- (3- (2, 4-dimethyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask was added 3- (2, 5-dimethyl-1H-imidazol-1-yl) -5- (trifluoromethyl) aniline (0.085g, 0.333mmol) and potassium carbonate (0.051g, 0.366mmol) dissolved in 4.0mL dichloromethane, p-chlorobenzoyl chloride (0.9g, 0.366mmol) also dissolved in dichloromethane, the reaction was slowly added dropwise at room temperature, and after 4H of reaction with stirring in a sealed environment, the reaction was monitored until the starting material point disappeared (developing solvent: DCM: meOH = 10. The solvent was evaporated under reduced pressure to give a light yellow solid, dissolved in N, N-dimethylformamide, added with potassium carbonate (0.023g, 0.167mmol), methylpiperazine (56 μ L,0.500 mmol), stirred overnight at room temperature, monitored by TLC until the starting material spot disappeared (dichloromethane: methanol = 5. Removing excessive solvent by evaporation under reduced pressure, and separating by flash column chromatography to obtain target product N- (3- (2, 5-dimethyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazine-1-Yl) methyl) benzamide (97 mg) as a pale yellow solid in 61.78% yield. m.p.75.5-76.7 ℃; HR-MS m/z 472.2327[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.20(s,1H),7.97(s,1H),7.94(s,1H),7.89(d,J＝8.0Hz,2H),7.43(d,J＝7.9Hz,2H),7.25(s,1H),6.75(s,1H),3.55(s,2H),2.57–2.34(m,8H),2.31(s,3H),2.28(s,3H),2.17(s,3H). 13 C NMR(126MHz,Chloroform-d)δ166.41,143.98,143.60,140.52,139.24,137.25,132.85,129.52,127.58,124.48,122.31,120.16,117.32,117.06,116.21,62.63,55.25,53.26,46.16,13.94,13.46.
Example 13a 3- (2, 4-dimethyl-1H-imidazol-1-yl) -5- (trifluoromethyl) aniline
To a 25mL sealed tube was added 2, 4-dimethylimidazole (0.345g, 3.60mmol), 3-amino-5-bromo-trifluorotoluene (423. Mu.L, 3.000 mmol), cuprous iodide (0.174g, 1.200mmol), 8-hydroxyquinoline (0.087g, 0.600mmol) and potassium carbonate (0.684g, 4.950mmol) dissolved in 10mL of dimethyl sulfoxide, and after three nitrogen replacements, the reaction was stirred in an oil bath at 120 ℃ for 24h and TLC monitored until the starting point disappeared (dichloromethane: methanol = 20. The reaction solution was filtered to remove the solid, washed with aqueous ammonia and ethyl acetate, the filtrate was extracted with water and ethyl acetate 2 times, washed with saturated brine once, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove excess solvent, and subjected to flash column chromatography to give the desired product 3- (2, 5-dimethyl-1H-imidazol-1-yl) -5- (trifluoromethyl) aniline 100mg as a yellow solid in 13.06% yield. HR-MS m/z 256.1063[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ6.88(s,1H),6.83(s,1H),6.67(d,J＝22.7Hz,2H),4.23(s,2H),2.26(d,J＝57.7Hz,6H). 13 C NMR(126MHz,Chloroform-d)δ148.18,132.69,124.57,122.40,114.16,111.48,110.63,29.69.
Example 14N- (3- (2, 4-dimethyl-1H-imidazol-1-yl) phenyl) -4- ((4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask was added 3- (2, 5-dimethyl-1H-imidazol-1-yl) aniline (0.231g, 1.233mmol) and potassium carbonate (0.188g, 1.357mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.257g, 1.357mmol) also dissolved in dichloromethane, the reaction was slowly dropped at room temperature, and after 4H of reaction stirring under sealed conditions, the starting material point was monitored to disappear (developing agent: DCM: meOH = 10. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in N, N-dimethylformamide, potassium carbonate (0.199g, 1.437 mmol), methylpiperazine (0.144g, 1.437 mmol) were added, stirred at room temperature overnight, and monitored by TLC until the starting material spot disappeared (dichloromethane: methanol = 5. Excess solvent was removed by evaporation under reduced pressure and the title product, N- (3- (2, 5-dimethyl-1H-imidazol-1-yl) phenyl) -4- ((4-methylpiperazin-1-yl) methyl) benzamide (115 mg), was obtained by flash column chromatography as a pale yellow solid in 23.11% yield. m.p.70.4-71.9 ℃; HR-MS m/z 404.245[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ8.98(s,1H),7.87(d,J＝8.2Hz,2H),7.69(q,J＝2.6,2.0Hz,1H),7.66–7.59(m,1H),7.39(t,J＝8.6Hz,3H),6.99(dd,J＝7.6,1.5Hz,1H),6.73–6.68(m,1H),3.53(s,2H),2.47(s,8H),2.28(d,J＝4.3Hz,6H),2.17–2.14(m,3H). 13 C NMR(126MHz,Chloroform-d)δ166.20,143.85,142.97,139.57,138.59,136.50,133.37,129.86,129.34,127.42,120.85,119.59,117.39,117.08,62.57,55.15,53.16,46.07,13.81,13.41.
Example 14a 3- (2, 4-dimethyl-1H-imidazol-1-yl) aniline
To a 25mL sealed tube was added 2, 4-dimethylimidazole (0.300g, 3.121mmol), m-bromoaniline (288. Mu.L, 2.601 mmol), cuprous iodide (0.149g, 0.780mmol), 8-hydroxyquinoline (0.076 g, 0.520mmol) and potassium carbonate (0.593g, 4.292mmol) dissolved in 10mL of dimethyl sulfoxide, and after three nitrogen replacements, the reaction was stirred in an oil bath at 120 ℃ for 24h and TLC monitored until the starting material point disappeared (dichloromethane: methanol = 20. The reaction solution was filtered to remove the solid,washing with ammonia water and ethyl acetate, extracting the filtrate with water and ethyl acetate for 2 times, washing with saturated brine once, drying with anhydrous sodium sulfate, evaporating under reduced pressure to remove excess solvent, and separating by flash column chromatography to obtain the desired product 3- (2, 5-dimethyl-1H-imidazol-1-yl) aniline 231mg as a yellow solid with a yield of 47.43%. HR-MS m/z of 188.1193[ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ7.19(t,J＝7.9Hz,1H),6.72(d,J＝2.1Hz,1H),6.67(ddd,J＝8.1,2.3,0.9Hz,1H),6.61(dt,J＝7.8,1.3Hz,1H),6.53(t,J＝2.1Hz,1H),3.85(s,2H),2.31(s,3H),2.21(s,3H). 13 C NMR(126MHz,Chloroform-d)δ147.44,130.11,115.27,114.33,111.71,13.80,13.48.
Example 15N- (5- (2, 4-dimethyl-1H-imidazol-1-yl) -2-methoxyphenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask were added 5- (2, 4-dimethyl-1H-imidazol-1-yl) -2-methoxyaniline (0.100g, 0.460mmol) and potassium carbonate (0.70g, 0.506mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.095g, 0.673mmol) was also dissolved in dichloromethane, the reaction was slowly dropped at room temperature, and after 4H of reaction with stirring under sealing, the starting material point was monitored to disappear (developing agent: DCM: meOH = 10. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in N, N-dimethylformamide, potassium carbonate (0.064g, 0.461mmol), methylpiperazine (133 μ L,1.197 mmol) were added, stirred at room temperature overnight, and monitored by TLC until the starting material spot disappeared (dichloromethane: methanol = 5. Excess solvent was removed by evaporation under reduced pressure and the title product, N- (5- (2, 4-dimethyl-1H-imidazol-1-yl) -2-methoxyphenyl) -4- ((4-methylpiperazin-1-yl) methyl) benzamide (66 mg), was obtained by flash column chromatography as a pale yellow solid in 16.54% yield. m.p.69.4-69.9 deg.c; HR-MS m/z 434.2560[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ8.58(s,1H),8.55(dd,J＝2.0,0.9Hz,1H),7.82(d,J＝8.3Hz,2H),7.46(d,J＝8.1Hz,2H),6.95(d,J＝1.9Hz,2H),6.76–6.69(m,1H),3.97(s,3H),3.57(s,2H),2.50(s,8H),2.34(s,3H),2.31(s,3H),2.22(d,J＝1.1Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ165.23,147.35,142.89,136.11,133.53,131.44,129.41,128.36,127.06,120.30,117.14,110.00,62.43,56.21,55.04,52.92,45.88,13.73,13.40.
Example 15a 5- (2, 4-dimethyl-1H-imidazol-1-yl) -2-methoxyaniline
To a 25mL sealed tube was added 2, 4-dimethylimidazole (0.200g, 2.081mmol), 5-bromo-2-methoxyaniline (216. Mu.L, 1.734 mmol), cuprous iodide (0.099g, 0.520mmol), 8-hydroxyquinoline (0.050g, 0.347mmol) and potassium carbonate (0.395g, 2.861mmol) dissolved in 10mL of dimethyl sulfoxide, and after three nitrogen replacements, the reaction was stirred in an oil bath at 120 ℃ for 24h and TLC was monitored until the starting point disappeared (dichloromethane: methanol = 20. The reaction solution was filtered to remove the solid, washed with aqueous ammonia and ethyl acetate, the filtrate was extracted with water and ethyl acetate 2 times, washed with saturated brine once, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove excess solvent, and subjected to flash column chromatography to give the desired product 5- (2, 4-dimethyl-1H-imidazol-1-yl) -2-methoxyaniline in 331mg as a yellow solid with a yield of 87.86%. HR-MS m/z 218.1299[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ6.93–6.66(m,2H),6.57(d,J＝7.4Hz,2H),3.96(s,2H),3.88(s,3H),2.25(s,3H),2.20(s,3H). 13 C NMR(126MHz,Chloroform-d)δ146.70,136.93,115.08,111.82,110.24,55.73,13.81.
Example 16N- (3- (4-cyano-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask was added N- (3- (4-cyano-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide (0.200g, 0.793 mmol) and potassium carbonate (0.132g, 0.952mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.180g, 0.952mmol) also dissolved in dichloromethane, the reaction solution was slowly dropped at room temperature, and after 4 hours of reaction under sealed stirring, the mixture was monitored until the starting point disappeared (developing solvent: DCM: meOH = 10.
The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in 4ml of n, n-dimethylformamide, potassium carbonate (0.055g, 0.400mmol), methylpiperazine (132 μ L,1.190 mmol) were added, stirred at room temperature overnight, and TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 10. Excess solvent was removed by evaporation under reduced pressure and the desired product, N- (3- (4-cyano-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide (156 mg) was obtained as a white solid in 41.99% yield by flash column chromatography. m.p.86.7-88.4 ℃; HR-MS m/z 469.1963[ 2 ] M + H] + ； 1 H NMR(500MHz,Chloroform-d)δ8.99(s,1H),8.43(t,J＝2.0Hz,1H),8.02(s,1H),7.97–7.89(m,4H),7.44(d,J＝7.9Hz,2H),7.37(s,1H),3.60(s,2H),2.64(d,J＝38.3Hz,8H),2.44(s,3H). 13 C NMR(126MHz,Chloroform-d)δ166.04,142.82,141.16,137.09,136.68,132.53,129.45,127.61,126.54,124.02,121.85,116.82,116.46,116.13,114.13,113.52,62.08,54.66,51.86,45.18,29.71.
Example 16941- (3-amino-5- (trifluoromethyl) phenyl) -1H-imidazole-4-carbonitri-nitrile
To a 25mL sealed tube was added 1H-imidazole-4-carbonitrile (0.279g, 3.000mmol), 3-amino-5-bromo-trifluorotoluene (354 μ L,2.500 mmol), cuprous iodide (0.095g, 0.750mmol), 8-hydroxyquinoline (0.073g, 0.500mmol) and potassium carbonate (0.570g, 4.125mmol) dissolved in 10mL dimethyl sulfoxide, and after three nitrogen replacements, the reaction was stirred in an oil bath at 120 ℃ for 24h and TLC was monitored until the starting point disappeared (dichloromethane: methanol = 20. Filtering the reaction solution to remove solid, washing with ammonia water and ethyl acetate, extracting the filtrate with water and ethyl acetate for 2 times, washing with saturated saline solution, drying with anhydrous sodium sulfate, evaporating under reduced pressure to remove excessive solvent, and separating by flash column chromatography to obtain the target product N- (3- (4-cyano-1H-imidazole)Oxazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide 225mg as a yellow solid in 35.69% yield. 1 H NMR(500MHz,DMSO-d 6 )δ8.71(s,1H),8.47(s,1H),7.10(d,J＝1.7Hz,1H),6.99(d,J＝2.0Hz,1H),6.94(d,J＝1.7Hz,1H),6.02(s,2H).
Example 17N- (3- (1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask were added 3- (1H-imidazol-1-yl) -5- (trifluoromethyl) aniline (0.150g, 0.660 mmol) and potassium carbonate (0.109g, 0.792mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.150g, 0.792mmol) also dissolved in dichloromethane, the reaction was slowly dropped at room temperature, and after stirring for 4H under sealing, the reaction was monitored until the starting material point disappeared (developing agent: DCM: meOH = 10.
The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in 4ml of n, n-dimethylformamide, potassium carbonate (0.046 g, 0.330mmol), methylpiperazine (110 μ L,0.990 mmol) were added, stirred at room temperature overnight, and TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 5. Excess solvent was removed by evaporation under reduced pressure and the desired product, N- (3- (1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-methylpiperazin-1-yl) methyl) benzamide (167 mg) was obtained by flash column chromatography as a white solid in 57.06% yield. m.p.78.2-78.7 deg.c; HR-MS m/z of 444.2014[ M + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.21(s,1H),8.30(t,J＝2.0Hz,1H),7.92(d,J＝7.9Hz,4H),7.46(d,J＝8.0Hz,2H),7.38(s,2H),7.23(s,1H),3.58(s,2H),2.52(s,8H),2.34(s,3H). 13 C NMR(126MHz,Chloroform-d)δ166.25,143.24,140.84,138.30,132.73,130.86,129.35,127.45,115.99,115.54,113.19,62.33,54.95,52.77,45.78.
Example 17a 3- (1H-imidazol-1-yl) -5- (trifluoromethyl) aniline
To a 25mL sealed tube, imidazole (0.600g, 2.500mmol), 3-amino-5-bromo-trifluorotoluene (354. Mu.L, 3.00 mmol), cuprous iodide (0.095g, 0.750mmol), 8-hydroxyquinoline (0.073g, 0.500mmol) and potassium carbonate (0.570g, 4.125mmol) were added and dissolved in 10mL of dimethyl sulfoxide, and after nitrogen substitution three times, the reaction was stirred in an oil bath at 120 ℃ for 24h, and TLC was monitored until the starting material point disappeared (dichloromethane: methanol = 20. The reaction solution was filtered to remove the solid, washed with aqueous ammonia and ethyl acetate, the filtrate was extracted with water and ethyl acetate 2 times, washed with saturated brine once, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove excess solvent, and subjected to flash column chromatography to give the desired product 3- (1H-imidazol-1-yl) -5- (trifluoromethyl) aniline 500mg as a white solid in 88.08% yield. HR-MS m/z 228.0753 2 [ m ] +H] + ； 1 H NMR(500MHz,Chloroform-d)δ7.88(s,1H),7.24(d,J＝30.1Hz,2H),6.93(s,1H),6.85(s,1H),6.81(s,1H),4.34(s,2H). 13 C NMR(126MHz,Chloroform-d)δ148.63,138.70,135.47,133.13,130.38,124.54,122.37,118.21,110.04,107.44.
Example 18N- (3- (1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-butylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask was added 3- (1H-imidazol-1-yl) -5- (trifluoromethyl) aniline (0.150g, 0.660 mmol) and potassium carbonate (0.109g, 0.792mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.150g, 0.792mmol) also dissolved in dichloromethane, the reaction was slowly dropped at room temperature, and after 4H of sealed stirring reaction, the mixture was monitored until the starting point disappeared (developing agent: DCM: meOH = 10.
The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in 4ml of n, n-dimethylformamide, and potassium carbonate (0.046 g, 0.330mmol), 1-n-butylpiperazine (156 μ L,0.990 mmol) were added, stirred at room temperature overnight, and monitored by TLC until the starting material spot disappeared (dichloromethane: methanol = 5. Evaporating under reduced pressure to remove excessive solventThe title product, N- (3- (1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -4- (4-butylpiperazin-1-yl) methyl) benzamide (206 mg) was obtained by flash column chromatography as a pale yellow solid in 64.28% yield. m.p.175.1-177.2 ℃; HR-MS m/z 486.2478[ 2 ], [ M + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.35(s,1H),8.26(s,1H),7.89(d,J＝8.3Hz,3H),7.85(s,1H),7.42(d,J＝8.2Hz,2H),7.33(s,2H),7.18(s,1H),3.54(s,2H),2.74–2.37(m,8H),2.37–2.31(m,2H),1.50–1.43(m,2H),1.29(h,J＝7.4Hz,2H),0.89(t,J＝7.3Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ166.34,143.26,140.91,138.23,132.70,130.74,129.34,127.46,118.36,116.01,115.60,113.14,62.41,58.40,53.11,52.85,28.78,20.74,14.00.
Example 19N- (4-methyl-3- (4-methyl-1H-imidazol-1-yl) phenyl) -4- ((4-methylpiperazin-1-yl) methyl) benzamide
To a 100mL round bottom flask were added 4-methyl-3- (4-methyl-1H-imidazol-1-yl) aniline (0.150g, 0.801mmol) and potassium carbonate (0.133g, 0.961mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.182g, 0.961mmol) also dissolved in dichloromethane, the reaction was slowly dropped at room temperature, and after stirring the reaction for 4H under sealing, the starting material point was monitored to disappear (developing agent: DCM: meOH = 10. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in 4ml of n, n-dimethylformamide, potassium carbonate (0.055g, 0.401mmol), methylpiperazine (133 μ L,1.201 mmol) were added, stirred at room temperature overnight, and TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 5. Excess solvent was removed by evaporation under reduced pressure and the desired product, N- (4-methyl-3- (4-methyl-1H-imidazol-1-yl) phenyl) -4- ((4-methylpiperazin-1-yl) methyl) benzamide (158 mg) was obtained by flash column chromatography as a white solid in 48.88% yield. m.p.75.1-78.2 ℃; HR-MS m/z 404.2454[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ8.78(s,1H),7.88(d,J＝8.2Hz,2H),7.65(dd,J＝8.3,2.2Hz,1H),7.57(d,J＝2.1Hz,1H),7.44–7.38(m,3H),7.26(d,J＝8.4Hz,1H),6.76(s,1H),3.55(s,2H),2.53(s,8H),2.35(s,3H),2.23(s,3H),2.15(s,3H). 13 C NMR(126MHz,Chloroform-d)δ165.87,142.42,138.30,137.11,136.90,136.55,133.46,131.56,129.25,129.15,127.39,120.47,118.40,117.06,62.29,54.85,52.46,45.58,17.31,13.57.
EXAMPLE 19a 4-methyl-3- (4-methyl-1H-imidazol-1-yl) phenylamine
To a 25mL sealed tube was added 4-methylimidazole (0.250g, 3.046 mmol), 3-bromo-4-methylaniline (0.472g, 2.538mmol), cuprous iodide (0.145g, 0.761 mmol), 8-hydroxyquinoline (0.075g, 0.508mmol) and potassium carbonate (0.579g, 4.188mmol) dissolved in 10mL dimethyl sulfoxide, and after three nitrogen replacements, the reaction was stirred at 120 ℃ in an oil bath for 24h and monitored by TLC until the starting point disappeared (dichloromethane: methanol = 20. The reaction solution was filtered to remove the solid, washed with ammonia water and ethyl acetate, the filtrate was extracted with water and ethyl acetate 2 times, washed with saturated brine once, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove excess solvent, and subjected to flash column chromatography to give the desired product 4-methyl-3- (4-methyl-1H-imidazol-1-yl) aniline 350mg as a white solid in 73.70% yield. HR-MS m/z:188.1191[ 2 ] M + H] + ； 1 H NMR(500MHz,Chloroform-d)δ7.82–7.33(m,1H),7.04(d,J＝8.1Hz,2H),6.69–6.59(m,1H),6.55–6.40(m,1H),3.76(s,2H),2.25(s,3H),2.03(s,3H).
Example 20- ((4-Butylpiperazin-1-yl) methyl) -N- (4-methyl-3- (4-methyl-1H-imidazol-1-yl) phenyl) benzamide
A100 mL round-bottomed flask was charged with 4-methyl-3- (4-methyl-1H-imidazol-1-yl) aniline (0.150g, 0.801mmol) and potassium carbonate (0.133g, 0.961mmol) dissolved in 4.0mL dichloromethane, p-chloromethylbenzoyl chloride (0.182g, 0.961mmol) was also dissolved in dichloromethane, the reaction mixture was slowly dropped at room temperature, and the mixture was stirred in a sealed state for 4 hours and then reactedMonitoring should be done until the starting point disappears (developing solvent: DCM: meOH = 10. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was dissolved in 4ml of n, n-dimethylformamide, potassium carbonate (0.055g, 0.401mmol), 1-n-butylpiperazine (190 μ L,1.201 mmol) was added, stirred at room temperature overnight, and TLC monitored until the starting material spot disappeared (dichloromethane: methanol = 5. Excess solvent was removed by evaporation under reduced pressure and the desired product, 4- ((4-butylpiperazin-1-yl) methyl) -N- (4-methyl-3- (4-methyl-1H-imidazol-1-yl) phenyl) benzamide (99 mg), was obtained by flash column chromatography as a white solid in 27.74% yield. m.p.61.1-63.5 ℃; HR-MS m/z 446.2930[ m + H ]] + ； 1 H NMR(500MHz,Chloroform-d)δ9.09(s,1H),7.88(d,J＝8.0Hz,2H),7.70–7.62(m,1H),7.58(s,1H),7.38(t,J＝13.2Hz,3H),7.24(d,J＝8.3Hz,1H),6.91–6.64(m,1H),3.53(s,2H),2.53(s,8H),2.44–2.39(m,2H),2.20(s,3H),2.13(s,3H),1.50(td,J＝11.7,9.8,6.1Hz,2H),1.33–1.22(m,2H),0.89(t,J＝7.3Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ166.01,142.21,137.25,136.84,133.48,131.50,129.20,129.02,127.51,120.63,118.47,62.29,58.20,52.90,52.25,28.25,20.64,17.30,13.93,13.56.
Biological activity assay
Cell culture
Human chronic myelogenous leukemia cell K562 and human acute promyelocytic leukemia cell HL-60 were purchased from the cell bank of Chinese academy of sciences (Shanghai, china). K562 cells were cultured in IMEM medium (KeyGEN BioTECH, jiangsu, china) containing 10% of total bone Serum Standard (FBS, NEWZERUM, christchurch, NZL); HL-60 cells were cultured in IMEM medium (KeygEN BioTECH, jiangsu, china) containing 20% of Total bone Serum Standard; MCF-7 cells were cultured in DMEM medium (KeygEN BioTECH, jiangsu, china) containing 10% of Fetal bone Serum Standard (FBS, NEWZERUM, christchurch, NZL); all cells were at 37 ℃ and contained 5% CO 2 Culturing in the environment.
Transwell migration test
The upper cell suspension is IMEM medium containing 0.2% bovine serum albumin (BSA, VICMED, jiangsu, china), and the lower cell suspension is IMEM medium containing 10% FBS, so as to form certain chemotaxis and make tumor fineCells mimic migration in vitro. K562 cells were pretreated for 24h with different concentrations of the compound of example 7 and example 10 (0,2.5. Mu.M, 5. Mu.M and 10. Mu.M). Resuspending the cells in IMEM medium (containing 0.2% BSA) and adjusting the cell density to 5X 10 cells/ml 5 And (4) cells. 200 μ L of cell suspension was taken and carefully added to the upper chamber of the Transwell chamber. The lower chamber was filled with 600. Mu.L of medium (10% FBS). Put at 37 ℃ with 5% CO 2 Incubate in incubator for 24h. The cell was removed and centrifuged in a plate centrifuge (5 min, 2000rpm). The number of migrated cells was measured in accordance with the MTT method. Cell mobility was plotted using GraphPad Prism 9.
K562 cells are currently one of the most commonly used cells for leukemia studies. It is necessary to further investigate the effect of compounds on cell invasion in vitro. The results of Transwell experiments showed that as the dose was increased, the number of migrated cells was significantly decreased, the results of the compound of example 7 for inhibiting migration (DMSO: 100%; 2.5. Mu.M: 83.05. + -. 3.38%; 5. Mu.M: 48.79. + -. 6.10%; 10. Mu.M: 34.45. + -. 2.87%; Y-10: 33.88. + -. 2.84%), the results of the compound of example 10 for inhibiting migration (DMSO: 100%; 2.5. Mu.M: 72.68. + -. 11.70%; 5. Mu.M: 39.62. + -. 6.28%; 10. Mu.M: 31.55. + -. 9.98%; Y-10: 36.14. + -. 7.86%), confirming that the compounds of examples 7 and 10 had significant anti-migration ability for K562 cells, and that examples 7 and 10 had comparable anti-migration ability to Imatinib (Y-10), while that of example 10 was superior to Imatinib (FIG. 1).
Detection of apoptosis by flow cytometry
Induction of apoptosis in tumor cells is considered an effective strategy for tumor chemotherapy. In general, drug-induced apoptosis is one of the major mechanisms of action for treating tumors. Annexin V and PI double staining, which is a classical method for flow detection of apoptosis, is based on the principle that Phosphatidylserine (PS) on the cell membrane turns from the inside of the cell membrane to the surface of the cell membrane in the early stage of apoptosis. Annexin V, when used in combination with PI, was used to identify normal, apoptotic, and dead cells. Cells were cultured in six-well plates, treated with different concentrations of compound of example 10 (0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M) at 48h, blanked with DMSO,the cells were collected by trypsinization (without EDTA) and the number of cells was adjusted to 1X 10 per ml 6 And (4) cells. After two washes with PBS, it was resuspended in a 1 × bubbling buffer. PI and Annexin V-FITC are added to stain the cells in a dark place according to the instructions of an Annexin V-FITC apoptosis detection kit (KeygEN BioTECH, jiangsu, china) and the cells are incubated for 5-15min at room temperature. The assay was completed in 1h on a guava easyCyte 8HT microfluidic cytoanalyzer. Apoptosis data analysis FlowJo v10 software was used.
Flow cytometry was used to further determine whether the inhibitory effect of the compound on cellular activity was associated with the induction of apoptosis. After 24h of treatment, the effect on apoptosis of K562 cells was examined. As shown in FIG. 2, the proportion of apoptotic cells (Q2 + Q3) increased from 5.72% (DMSO) to 37.58% (example 10, 10. Mu.M). The results indicate that the compound is a potential apoptosis inducer for K562 cells and has stronger ability to induce apoptosis than imatinib (Y-10, 10. Mu.M, 23.10%).
Western blot experiment for detecting expression of apoptosis-related protein
Antibodies ABL1, CRKL and p-CRKL were purchased from Beyotime (Shanghai, china), and β -Actin (Santa Cruz, USA) was used as an internal control. The cells were harvested 4h after treatment of K562 cells with varying concentrations of the compounds of example 7 and example 10 (0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M). Adding an appropriate amount of 1 xSDS loading buffer according to the cell amount, boiling in water bath at 99 ℃ for 5min, and freezing for 5min, and repeating for three times. Protein quantification was performed according to grey value analysis. Proteins were separated in 1 xSDS-PAGE running buffer and then transferred to PVDF membrane. After the transfer printing is finished, 5% of skimmed milk powder is used for 60r/min, and the shaking table is closed for 45min. At the end of blocking, the membrane was washed three times with PBST for 5min each. The corresponding primary antibody dilution was then added and incubated overnight at 4 ℃ in a shaker. Washing the membrane three times with PBST 5min each time the next day, adding the second antibody diluent, incubating for 45min in a shaker at room temperature, and washing the membrane three times with PBST 5min each time. HRP-ECL imaging (Tanon, shanghai, china), beta-actin as an internal control, detecting the expression level of the target protein.
In order to investigate the effect of the compounds on leukemia-associated proteins, the expression of the genes of the compounds at the protein level was investigated by Western blot assay, and the results showed that examples 7 and 10 had a dose-dependent inhibition of ABL1 protein production, and that the inhibition of ABL1 at a maximum concentration of 10. Mu.M was superior to that of the positive drug imatinib. The compound also has a certain inhibition effect on CRKL protein, but has no dose dependence. The compound can also affect the phosphorylation levels of ABL1 and CRKL, reduce the phosphorylation levels and inhibit the phosphorylation signals.
The Caspase family plays a very important role in mediating the process of apoptosis, wherein Caspase-3 is a key executive molecule that functions in many pathways of apoptosis signaling. Caspase-3 is a cysteine-aspartic protease, plays a key role in the process of apoptosis, and is widely used as a biomarker of apoptosis. WB results show that the series of compounds can cause obvious apoptosis of K562 cells, accumulation of Caspase-3 protein and activated Caspase-3 protein, and cause the apoptosis effect of K562 cells to be better than that of positive drug imatinib (Y-10) at the maximum concentration of 10 mu M, as shown in figure 3.
In vitro inhibition data
MTT method for detecting cytotoxicity
Thiazole blue (MTT) was used for the determination of cell activity. The hemacytometer plate method was used for cell counting, and cell viability was greater than 95% in all experiments. K562, HL-60 and MCF-7 cells at 1X 10 per ml 4 Individual cells were seeded in 96-well plates. Each well was added 100. Mu.L of medium (containing 1% FBS) dissolved with different concentrations of the compound of the example (0-50. Mu.M), and incubated for 24h, respectively. Using a plate centrifuge (5min, 2000rpm), the supernatant was discarded, 10. Mu.L of MTT (5 mg/mL) solution was added to each well, incubated at 37 ℃ for 4h, the plate was centrifuged to remove the supernatant, 100. Mu.L of DMSO was added to each well, and the mixture was shaken on a shaker for 10min to fully dissolve the Formazan crystals. The absorbance was measured at a wavelength of 570nm using a microplate reader (BioTek, USA). Cytotoxicity assays were performed compared to control (DMSO). The concentration of drug-induced 50% inhibition of cell growth (IC) was determined by non-linear regression using a curve fitting algorithm for GraphPad Prism 9 (GraphPad software, la Jolla, calif., USA) 50 )。
To investigate the effect of compounds on the activity of tumor cells, MThe TT method detects cytotoxicity. As shown in Table 1, the effect of the compounds on the activity of different tumor cells, the half inhibitory concentration IC of the drug on each cell line was calculated 50 (24h) In that respect The results show that the compound has obvious inhibition effect on K562, HL60 and MCF-7 tumor cells.
TABLE 1 inhibitory Activity of the Compounds on different tumor cells
NA indicates no activity
BCR-ABL1 kinase inhibition assay
1X assay buffer preparation: enzymatic buffer 5 ×, mgCl 2 (1M)，DTT(0.2M)，MnCl 2 (0.2M), SEB (2500 nM); the enzyme stock solution, the substrate and the ATP stock solution are melted on ice, and the above reagents need to be always placed on ice in the experimental process; taking 4 μ L of compound working solution per well, adding to 384 microwell plates according to the experimental arrangement, adding 4 μ L of 1X assay buffer per well containing 2.5% DMSO to the positive control (Imatinib), adding 1X assay buffer per well containing 2.5% DMSO to the Blank control (Blank); preparing an enzyme working solution, taking 0.5 mu L of BCR-ABL1 enzyme stock solution (200 ng/. Mu.L), and diluting the enzyme to 0.25 ng/. Mu.L by using 1X assay buffer. Adding 2 muL/hole into a white microplate, adding 2 muL/hole 1X assay buffer into a blank control hole, and performing the step on ice; after the addition, the microporous plate is centrifuged for 1 minute on a centrifuge at 1000 revolutions; preparing a Substrate TK Substrate-biotin/ATP mixed solution: 0.6 mu L of TK Substrate-biotin is added with 0.6 mu L of ATP (100 mM) and 238.8 mu L of 1X assay buffer to dilute the Substrate, wherein the concentration of the Substrate is 1.25 mu M, the concentration of the ATP is 25 mu M, and the mixed solution of the Substrate and the ATP is placed on ice for later use; taking 4 mu L/hole substrate/ATP mixed solution into a white micropore plate, wherein the concentration of the substrate is 0.5 mu M, the concentration of the ATP is 10 mu M, and centrifuging the micropore plate for 1 minute after the substrate is added by 1000 revolutions; attaching a film to the microporous plate, pressing the film, and incubating for 1h at 25 ℃; get5 μ L/well of the mixture (62.5 nM Streptavidin-XL665, detection Buffer) was added to the microplate and centrifuged for 1 min at 1000 rpm; adding 5 mu L/hole mixed solution (1 xTK Antibody-Cryptate, detection Buffer) into a microplate, and centrifuging for 1 min at 1000 rpm; the microporous plate is placed at 25 ℃ after being sealed and incubated for 1 hour; after the incubation was finished, TR-FRET detection was performed using Nivo, reading fluorescence values (Excitation at 320nm;
calculation of enzyme inhibition:
Ratio＝665/615
Inhibition％＝(Ratio(Sample)-Ratio(Pos.Ctrl)))/(Ratio(Blank)-Ratio Pos.Ctrl))×100％
| examples | BCR-ABL1 kinase inhibition% |
| Example 7 | 65.03 |
| Example 10 | 68.48 |
| Example 12 | 56.37 |
Examples 7, 10 and 12 have inhibitory activities on BCR-ABL1 kinase of 65.03%, 68.48% and 56.37%, and have significant target inhibitory activities.
Claims (10)
1. A compound represented by formula I or a pharmaceutically acceptable salt thereof:
wherein R is 6 Is composed of
X is selected from CH 2 R 7 、NR 8 Or O, n is an integer of 0 to 4, and R is 6 Optionally substituted by one or more R 9 Substitution; said R is 9 Selected from the group consisting of hydrogen, C1-C6 alkyl, halogen, cyano, -NR 10 R 11 、NO 2 (C1-C6 alkyl) -O-),
Acetyl or C1-C6 alkyl substituted with 1-3 halogens; n is an integer of 0 to 1;
R 1 、R 2 、R 3 、R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, halogen, cyano, -NR 10 R 11 、NO 2 (C1-C6 alkyl) -O-),
Acetyl, 1-3 halo-substituted C1-C6 alkyl;
R 8 and R 9 Each independently selected from hydrogen, C1-C6 alkyl,
Or an acetyl group;
R 10 and R 11 Each independently selected from H or C1-C6 alkyl.
2. The compound or pharmaceutically acceptable salt of claim 1, wherein the compound of formula I has a structure as shown in formula II,
wherein the hydrogen atom on the piperazine ring A may be substituted by one or more R 9 Substituted, R 1 、R 2 、R 3 、R 4 、R 5 、R 8 And R 9 As defined for compounds of formula I;
R 1 selected from hydrogen, C1-C6 alkyl or cyano, preferably, R 1 Selected from hydrogen, methyl or cyano, more preferably, R 1 Is selected from methyl;
R 2 selected from hydrogen or C1-C6 alkyl, preferably, R 2 Selected from hydrogen or methyl, more preferably, R 2 Selected from hydrogen;
R 3 selected from hydrogen or C1-C6 alkyl, preferably, R 3 Selected from hydrogen or methyl, more preferably, R 3 Selected from hydrogen;
R 4 selected from hydrogen or C1-C6 alkyl substituted by 1-3 halogens, preferably R 3 Selected from hydrogen or trifluoromethyl, more preferably, R 3 Is selected from trifluoromethyl;
R 5 selected from hydrogen or (C1-C6 alkyl) -O-, preferably, R 5 Selected from hydrogen or methoxy, more preferably, R 5 Selected from hydrogen;
R 8 selected from hydrogen,
Acetyl, n-butyl, methyl, tert-butyl, ethyl or isopropyl, preferably R 8 Selected from n-butyl, methyl or tert-butyl;
R 9 selected from hydrogen.
3. The compound or pharmaceutically acceptable salt of claim 1, wherein the compound of formula I has a structure as shown in formula III,
wherein the hydrogen atoms of the morpholine ring B may be substituted by one or more R 9 Substituted, R 1 、R 2 、R 3 、R 4 、R 5 And R 9 As defined for compounds of formula I;
R 1 、R 2 、R 3 、R 4 and R 5 Selected from hydrogen, C1-C6 alkyl or C1-C6 alkyl substituted by 1-3 halogens, preferably R 1 、R 2 、R 3 、R 4 And R 5 Selected from hydrogen, methyl or trifluoromethyl, more preferably, R 1 Is methyl, R 4 Is trifluoromethyl, R 2 、R 3 And R 5 Are all hydrogen;
R 9 is hydrogen or C1-C6 alkyl, preferably, R 9 Is hydrogen or methyl.
4. The compound or pharmaceutically acceptable salt of claim 1, wherein the compound of formula I has a structure as shown in formula IV,
wherein the hydrogen atoms on the pyrrolidine C may be substituted by one or more R 9 Substituted, R 1 、R 2 、R 3 、R 4 、R 5 And R 9 As defined for compounds of formula I;
R 1 is methyl, R 4 Is trifluoromethyl, R 2 、R 3 And R 5 Are all hydrogen;
R 9 is hydrogen.
5. The following compounds or pharmaceutically acceptable salts thereof:
6. the following compounds or pharmaceutically acceptable salts thereof:
7. a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, II, III, or VI, or a pharmaceutically acceptable salt thereof; preferably, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula I, II, III or VI or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
8. Use of a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 7 for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease which responds to an inhibition of protein kinase activity.
9. Use of a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 7 for the manufacture of a medicament for the prevention and/or treatment of a neoplastic disease; preferably, the neoplastic disorder is selected from chronic myelogenous leukemia or acute lymphocytic leukemia.
10. A process for preparing a compound of formula I, comprising the following synthetic scheme:
wherein R is 1 、R 2 、R 3 、R 4 、R 5 、R 9 And X is as defined for compounds of formula I, P and M are independently selected from Cl, br or I;
carrying out coupling reaction on the compound of the formula 1 and the compound of the formula 2 in the presence of a catalyst to prepare a compound of a formula 3; carrying out amide forming reaction on the compound of the formula 3 and the compound of the formula 4 to obtain a compound of a formula 5; reacting the compound shown in the formula 5 with the compound shown in the formula 6 under alkaline conditions to obtain the compound shown in the formula 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211372805.XA CN115925632A (en) | 2022-11-02 | 2022-11-02 | Imidazole phenyl aminobenzamide compound with antitumor activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211372805.XA CN115925632A (en) | 2022-11-02 | 2022-11-02 | Imidazole phenyl aminobenzamide compound with antitumor activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115925632A true CN115925632A (en) | 2023-04-07 |
Family
ID=86653483
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211372805.XA Pending CN115925632A (en) | 2022-11-02 | 2022-11-02 | Imidazole phenyl aminobenzamide compound with antitumor activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115925632A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016033304A1 (en) * | 2014-08-28 | 2016-03-03 | Codexis, Inc. | Imidazoyl anilide derivatives and methods of use |
| CN110869017A (en) * | 2017-05-12 | 2020-03-06 | 英安塔制药有限公司 | Apoptosis signal-regulating kinase 1 inhibitors and methods of use thereof |
| CN111606887A (en) * | 2019-02-26 | 2020-09-01 | 安徽中科拓苒药物科学研究有限公司 | Novel kinase inhibitor |
-
2022
- 2022-11-02 CN CN202211372805.XA patent/CN115925632A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016033304A1 (en) * | 2014-08-28 | 2016-03-03 | Codexis, Inc. | Imidazoyl anilide derivatives and methods of use |
| CN110869017A (en) * | 2017-05-12 | 2020-03-06 | 英安塔制药有限公司 | Apoptosis signal-regulating kinase 1 inhibitors and methods of use thereof |
| CN111606887A (en) * | 2019-02-26 | 2020-09-01 | 安徽中科拓苒药物科学研究有限公司 | Novel kinase inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| ELENA KALINICHENKO等: "Synthesis, biological activities and docking studies of novel 4-(arylaminomethyl)benzamide derivatives as potential tyrosine kinase inhibitors", 《MOLECULES》, vol. 24, no. 19, pages 3543 * |
| 王秀军等: "伊马替尼哌嗪环的结构优化及抗肿瘤活性研究", 《沈阳药科大学学报》, vol. 39, no. 5, pages 529 - 535 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2518063B1 (en) | Pyridazinones and furan-containing compounds | |
| CN102977014B (en) | New quinoline compounds and uses thereof | |
| EA018163B1 (en) | Imidazopyridazinecarbonitriles useful as kinase inhibitors | |
| US20050009849A1 (en) | Pyridopyrimidine kinase inhibitors | |
| CN114555586A (en) | Novel KRASG12C protein inhibitor and preparation method and application thereof | |
| JP2015524448A (en) | Alkynyl heteroaromatic compounds and their applications | |
| TW201236684A (en) | Pharmaceutically acceptable salts of (E)-N-[4-[[3-chloro-4-(2-pyridylmethoxy)phenyl]amino]-3-cyano-7-ethoxy-6-quinolyl]-3-[(2R)-1-methylpyrrolidin-2-yl]prop-2-enamide, preparation process and pharmaceutical use there of | |
| CN112771053A (en) | Biomarker-based therapeutic compositions | |
| CN107266461B (en) | A kind of alkoxy dibenzazepines class compound, preparation method and medical usage | |
| JP6917448B2 (en) | Crystal form, salt form and production method of tyrosine kinase inhibitor | |
| US20220033381A1 (en) | 3-azabicyclo[3,1,1]heptane derivative and pharmaceutical composition including same | |
| WO2013044596A1 (en) | Kidney-type glutaminase inhibitor and preparation method and use thereof | |
| KR102214225B1 (en) | 5-membered heterocyclic amide type WNT pathway inhibitor | |
| JP2009516658A (en) | 3,6-dihydro-2-oxo-6H-1,3,4-thiadiazine derivative | |
| CN107383016A (en) | The preparation and application of the Pyrrolopyrimidine compounds of the structure containing heteroaryl amide | |
| CN107151233B (en) | Hydrazone-containing pyrimidine derivative and application thereof | |
| CA2722197A1 (en) | Multiple acting anti-angiogenic and cytotoxic compounds and methods for using the same | |
| CN115925632A (en) | Imidazole phenyl aminobenzamide compound with antitumor activity | |
| JP4365586B2 (en) | Phthalazine derivatives with angiogenesis inhibitory activity | |
| CN115197167B (en) | 1,2, 4-thiadiazolidine-3, 5-dione compound, and preparation method and application thereof | |
| WO2019170088A1 (en) | Oxazino-quinazoline and oxazino-quinazoline type compound, preparation method therefor, and uses thereof | |
| CN105646448B (en) | Pyridine compounds and their and application thereof | |
| CN114763360A (en) | Chiral macrocyclic compounds as protein kinase inhibitors and uses thereof | |
| CN102216280B (en) | Bisarylurea derivatives and their use | |
| CN104211682A (en) | pyridine compounds and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |