CN111704603B - Anti-tumor compound and application thereof - Google Patents
Anti-tumor compound and application thereof Download PDFInfo
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- CN111704603B CN111704603B CN202010537921.7A CN202010537921A CN111704603B CN 111704603 B CN111704603 B CN 111704603B CN 202010537921 A CN202010537921 A CN 202010537921A CN 111704603 B CN111704603 B CN 111704603B
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
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- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
Abstract
The invention relates to a compound which has a structure shown in a formula I, wherein a substituent R is C6-C8 naphthenic base, aromatic base, n R1Substituted aromatic radical, n R1Substituted C6-C8 cycloalkyl; wherein n is 1-4. R1Independently selected from the following: halogen atom, C1-C6 alkyl, C2-C4 alkenyl, C2-C3 alkynyl, fluorine substituted C1-C4 alkyl, phenyl, substituted phenyl, nitro, pyridyl, pyrrolyl, pyrazinyl, piperazinyl, furyl; r2 is C1-C3 alkyl; r3 is C1-C4 alkyl; r4 is O-aminophenyl, m-aminophenyl, p-aminophenyl, pyrrolyl, pyridyl, piperidyl, piperazinyl, pyrazinyl, pyrimidinyl or quinolyl.
Description
Technical Field
The invention relates to a small molecular compound, in particular to a small molecular compound with antitumor activity.
Background
Lung cancer is one of the most common causes of high mortality in many cancers, with about 160 million people dying from lung cancer every year worldwide, the most common primary malignancy. In 2007, by systematically exploring and searching for new oncogenes involved in non-small cell lung cancer pathogenesis, it was eventually discovered that ALK-activated rearrangement in non-small cell lung cancer. To date, a variety of small molecule compounds have been demonstrated to possess some degree of ALK tyrosine kinase inhibitory activity and have been supported by in vivo and in vitro studies. In recent years, the FDA approved a part of ALK inhibitors for clinical treatment of non-small cell lung cancer, and brought new eosin for treatment of ALK-positive non-small cell lung cancer patients. These drug atmosphere first, second and third generation inhibitors may be administered according to their chronological order of development of resistance
The first generation of ALK inhibitors included cribzotinib (PF-02341066, Pfizer), to which most patients developed resistance within one to two years. The second generation ALK inhibitor comprises Ceritinib as a representative, and has good clinical curative effect on drug-resistant mutants (comprising L1196M, G1269A, I1171T and S1206Y) generated by Crizotinib treatment. The third-generation ALK tyrosine kinase inhibitor contains a large ring in the chemical structure and shows extremely strong activity, and comprises Lorlatinib (Pfizer) and the like.
Although the ALK kinase inhibitor on the market can relieve the treatment problem of ALK specific lung cancer and solve the drug resistance problem to a certain extent within a certain time, the problems of low activity and secondary drug resistance still exist. The continuous development of resistance is the largest reef used by ALK inhibitors in clinical applications. Therefore, there is a need for more highly effective, highly selective ALK inhibitors in clinical therapy to overcome these problems.
Disclosure of Invention
The invention aims to provide a brand-new micromolecular compound aiming at the problem that the mutation of drug resistance genes of several existing ALK micromolecular inhibitors causes the continuous occurrence of drug resistance. The small molecular compound has good antitumor activity, and the in vitro antitumor proliferation activity is superior to that of the existing antitumor compounds of the same type.
In order to achieve the purpose, the invention adopts the technical scheme that:
a compound having the structure of formula I:
wherein the substituent R is C6-C8 cycloalkyl, aryl, n R1Substituted aromatic radical, n R1Substituted C6-C8 cycloalkyl; wherein n is 1-4.
R1Independently selected from the following: halogen atom, C1-C6 alkyl, C2-C4 alkenyl, C2-C3 alkynyl, fluorine substituted C1-C4 alkyl, phenyl, substituted phenyl, nitro, pyridyl, pyrrolyl, pyrazinyl, piperazinyl, furyl.
R2 is C1-C3 alkyl.
R3 is C1-C4 alkyl.
R4 is O-aminophenyl, m-aminophenyl, p-aminophenyl, pyrrolyl, pyridyl, piperidyl, piperazinyl, pyrazinyl, pyrimidinyl or quinolyl.
The compound has good antitumor activity, can inhibit the proliferation of non-small cell lung cancer cells, and has good antitumor activity.
The alkyl of C1-C3 refers to methyl, ethyl, propyl and isopropyl.
The alkyl of C1-C4 refers to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl.
C1-C6 alkyl means methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl.
C2-C4 alkenyl means ethenyl, propenyl, butenyl.
C2-C3 alkynyl means ethynyl, propynyl.
Fluorine substituted C1-C4 alkyl refers to C1-C4 alkyl substituted with at least one fluorine atom. It may be an alkyl group substituted with a plurality of fluorine atoms, or a perfluoro-substituted alkyl group.
As a preferred embodiment of the present invention, the compound has the structure of formula II:
wherein the substituent R is C6-C8 cycloalkyl, aryl, n R1Substituted aromatic radical, n R1Substituted C6-C8 cycloalkyl; wherein n is 1-4.
R1Independently selected from the following: halogen atom, C1-C6 alkyl, C2-C4 alkenyl, C2-C3 alkynyl, fluorine substituted C1-C4 alkyl, phenyl, substituted phenyl, nitro, pyridyl, pyrrolyl, pyrazinyl, piperazinyl, furyl.
If there are more than 1 substituent R1Each substituent R1May be the same or different.
The compound parent nucleus and the branched chain structure are optimally designed, have good binding property with ALK kinase protein, have good anti-tumor activity for various tumor cell lines, have half inhibition concentration as low as 0.16 mu M, have good inhibition rate in a low concentration range, and block the cell cycle at the G1/S phase. Has the potential of being used for treating anaplastic large cell lymphoma.
In a preferred embodiment of the present invention, the substituent R is phenyl, n R1Substituted phenyl, naphthyl, n R1Substituted naphthyl. For example, n ═ 2, phenyl substituted with two substituents; n-1, having one substituentA phenyl group.
As a preferred embodiment of the present invention, the C1-C6 alkyl group includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, hexyl.
As a preferred embodiment of the present invention, the substituent R1The substitution is in the ortho, meta or para position.
As a preferred embodiment of the present invention, the compound is one of the following compounds:
(1) 5-chloro-N2- (4-fluorophenyl) -N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(2) 5-chloro-N2 (3-chlorophenyl) -N4 (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(3) N2- (3, 5-bis (trifluoromethyl) phenyl) -5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(4) N2- ([1,1' -biphenyl ] -3-yl) -5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(5) 5-chloro-N2 (3, 5-dichloropyridin-4-yl) -N4 (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(6) 5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) -N2- (3-nitrophenyl) pyrimidine-2, 4-diamine.
(7) 5-chloro-N2 (4-chlorophenyl) -N4 (2-isopropoxy-5-methyl-4 piperidine-phenyl) pyrimidine-2, 4-diamine.
(8) 5-chloro-N2- (4-chloro-3- (trifluoromethyl) phenyl) -N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(9) 5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) -N2- (2-methyl-5-nitrophenyl) pyrimidine-2, 4-diamine.
(10) 5-chloro-N2 (α -naphthyl) -N4 (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(11) 5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) -N2- (4-nitrophenyl) pyrimidine-2, 4-diamine.
(12) 5-chloro-N2, N4-bis (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine.
(13) 2-phenylamino-3- [ (3-methyl-4-piperazinyl-6-isopropyloxy-phenyl) -amino ] -4-chloro-pyrimidine.
As a preferred embodiment of the present invention, the compound is one of the following:
the invention also aims to provide an active ingredient for preparing the medicine and application of the active ingredient in preparing the medicine for treating tumors.
In particular to the application of the compound in preparing a medicine for treating tumor.
Preferably, the compounds act by binding to ALK kinase proteins.
Preferably, the tumor treatment drug is a tumor treatment drug for non-small cell lung cancer.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the synthesized small molecular compound has good anti-tumor activity, has the same anti-tumor activity as the existing chemical component Ceritinib, has good binding force of ALK kinase, and can realize the anti-tumor inhibition effect at lower concentration.
2. The compound synthesized by the invention has high blocking rate on the tumor cells in the G1/S phase.
Description of the drawings:
FIG. 1 is a plot of inhibition versus concentration curve fit for compounds CR-B1 and CR-B5.
FIG. 2 is the in vitro antiproliferative activity (IC) of compounds CR-B1, CR-B5 and Ceritinib against Karpas-299 cells within 24-96h50) Graph is shown.
FIG. 3 shows the inhibition of Karpas-299 cells by the compounds CR-B1, CR-B5 and Ceritinib in the concentration range of 0.1-100. mu.M.
FIG. 4 is a graph of the effect of compounds CR-B1, CR-B5 and Ceritinib (0.5. mu.M) on the cell cycle of Karpas-299.
FIG. 5 is the in vitro antiproliferative activity (IC) of compounds CR-B1, CR-B5 and Ceritinib in 9-strain cells50：μM)。
FIG. 6 shows the number of compounds in the 12 CR-B series which showed superior activity to Ceritinib on 9 tumor cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The main reagent source information applied in the following examples is as follows:
para-fluoroaniline, 3-chloroaniline, 3, 5-bis (trifluoromethyl) aniline, 3-aminodiamine, 3, 5-dichloro-4-aminopyridine, 3-nitroaniline, 4-chloroaniline, 4-chloro-3-trifluoromethylaniline, 2-methyl-5-nitroaniline, 1-naphthylamine, 4-nitroaniline, 2-isopropoxy-5-methyl-4 piperidine-aniline, 2,4, 5-trichloropyrimidine, BOC anhydride, triethylamine, trifluoroacetic acid were purchased from Anan Ji-Ji chemistry. Methylene chloride, methanol, isopropanol, N-diisopropylethylamine were purchased from a chemical reagent plant of the metropolis family. Silica gel, 200 meshes and 300 meshes, purchased from Qingdao ocean factories.
The synthetic route of the CR-B series target compound is designed as follows:
reagents and conditions: (i) et3N, DCM, RT;
(ii)IPA，DIPEA，80℃；
(iii)IPA，HCl，80℃；
(iv)TFA，RT。
example 1
Synthesis of tert-butyl 4- (4-amino-5-isopropoxy-2-methyl-phenyl) piperidine-1-carboxylate (Compound a)
1mmol (284.20mg) of 2-isopropoxy-5-methyl-4- (4-piperidine) aniline hydrochloride was weighed into a 25ml round bottom reaction flask, and appropriate dichloromethane was added as a solvent, after shaking to dissolve it, 3mmol (383.54. mu.l) of triethylamine was added, and finally 4 equivalents of BOC anhydride were added to dissolve it completely as a colorless liquid, and stirred at room temperature for 4 hours, and the progress of the reaction was monitored by Thin Layer Chromatography (TLC). After the reaction is completed, directly spin-drying the reaction solution to obtain yellow oily matter, filling silica gel column with 200-plus-300-mesh silica gel powder, and performing dichloromethane: and (4) performing column separation with methanol of 2:1, detecting a product by TLC, and performing spin drying to obtain a colorless oily intermediate a. The yield was 95.7%.
Example 2
Synthesis of tert-butyl 4- (4- (2, 5-dichloro-4-pyrimidine) amino-5-isopropoxy-2-methylphenyl) piperidine-1-carboxylate (Compound b)
Weighing 1mmol (350.20mg) of compound a, placing the compound a in a 25ml round-bottom reaction flask, adding a proper amount of isopropanol as a solvent, and then adding 3mmol (523.98 mu l) of N, N-diisopropylethylamine; 1.5mmol (275.88mg) of 2,4, 5-trichloropyrimidine is weighed out and dissolved in 1ml of isopropanol, added dropwise into the reaction system, reacted for about 5h, and the progress of the reaction is monitored by TLC. After the reaction is completed, adding about 15ml of water for dispersion and extracting with dichloromethane for a few times, combining all dichloromethane organic layers after the extraction is completed, adding a proper amount of saturated sodium chloride solution for washing for 2-3 times, adding anhydrous magnesium sulfate for drying for about 30 minutes, and concentrating and drying under reduced pressure to obtain a solid intermediate b. The yield was 84.9%.
Example 3
(1) Method for synthesizing 5-chloro-N2- (4-fluorophenyl) -N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B1):
1mmol (495.20mg) of intermediate b and 1.5mmol (166.68mg) of para-fluoroaniline were weighed into a 25ml round bottom flask, appropriate isopropanol as solvent was added, a catalytic amount (ca.1%) of concentrated HCl was added and the reaction was continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring for 1 hour at room temperature, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B1. The yield was 71.3%.
1H NMR(400MHz,DMSO)δ10.18(s,1H),9.10(s,2H),8.30(s,1H),7.64(s,1H),7.51(s,2H),7.05(t,J＝8.8Hz,2H),6.89(s,1H),3.36(d,J＝11.9Hz,2H),3.06(s,3H),2.23(s,3H),1.90(dd,J＝54.5,12.8Hz,4H),1.24(d,J＝5.9Hz,6H)ppm.HRMS(ESI)m/z:(M+H)+calcd for:C25H29ClFN5O:469.2045,found:470.2122.
Example 4
(2) 5-chloro-N2 (3-chlorophenyl) -N4 (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-bis
Amine (CR-B2) Synthesis:
1mmol (495.20mg) of intermediate b and 1.5mmol (191.35mg) of 3-chloroaniline were weighed into a 25ml round bottom flask, an appropriate amount of isopropanol was added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid was added, and the reaction was continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B2. The yield was 69.6%.
1H NMR(400MHz,DMSO)δ10.02(s,1H),9.04(s,1H),8.90(s,1H),8.63(s,1H),8.28(s,1H),7.68(s,2H),7.45(s,1H),7.21(t,J＝8.1Hz,1H),6.99(d,J＝7.9Hz,1H),6.88(s,1H),4.53(s,1H),3.36(d,J＝12.1Hz,2H),3.02(s,3H),2.26(s,3H),1.96(s,2H),1.85(s,2H),1.23(s,6H)ppm.HRMS(ESI)m/z:(M+H)+calcd for:C25H29Cl2N5O:485.1749,found:486.1828.
Example 5
(3) Method for synthesizing N2- (3, 5-bis (trifluoromethyl) phenyl) -5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B3):
weigh 1mmol (495.20mg) of intermediate b and 1.5mmol (343.69mg) of 3, 5-bis (trifluoromethyl) aniline into a 25ml round bottom flask, add the appropriate amount of isopropanol as solvent, add a catalytic amount (about 1%) of concentrated hydrochloric acid, and continue the reaction at 80 ℃ overnight. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B3. The yield was 59.6%.
1H NMR(400MHz,DMSO)δ9.98(s,1H),8.66(s,2H),8.35(s,2H),8.29(s,1H),8.23(s,1H),7.80(s,1H),7.52(s,1H),6.84(s,1H),4.52(s,1H),3.41–3.27(m,6H),3.04(s,3H),2.26(s,3H),1.84(s,3H),1.23(s,5H)ppm.HRMS(ESI)m/z:(M+H)+calcd for Chemical Formula:C27H28ClF6N5O:587.1887,found:588.1959.
Example 6
(4) Method for synthesizing N2- (1,1' -biphenyl) -5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B4):
1mmol (495.20mg) of intermediate b and 1.5mmol (253.84mg) of 3-aminodiamine are weighed into a 25ml round-bottomed flask, an appropriate amount of isopropanol is added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid is added, and the reaction is continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B4. The yield was 60.4%.
1H NMR(400MHz,DMSO)δ10.21(s,1H),9.06(d,J＝30.9Hz,2H),8.83(s,1H),8.33(s,1H),7.61(d,J＝7.8Hz,1H),7.56(d,J＝7.3Hz,2H),7.43(t,J＝7.4Hz,2H),7.33(dt,J＝15.5,7.5Hz,4H),6.86(s,1H),3.35(d,J＝12.0Hz,2H),3.00(s,4H),2.13(s,3H),1.93(d,J＝12.1Hz,3H),1.77(d,J＝13.2Hz,2H),1.33(s,1H),1.25(d,J＝6.0Hz,7H),1.02(d,J＝6.2Hz,1H),0.83(t,J＝7.4Hz,2H)ppm.HRMS(ESI)m/z:(M+H)+calcd for Chemical Formula:C31H34ClN5O:527.2452,found:528.2523.
Example 7
(5) Method for synthesizing 5-chloro-N2 (3, 5-dichloropyridin-4-yl) -N4 (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B5):
1mmol (495.20mg) of intermediate b and 1.5mmol (244.50mg) of 3, 5-dichloro-4-aminopyridine were weighed into a 25ml round-bottomed flask, and a suitable amount of isopropanol was added as solvent, followed by a catalytic amount (ca.1%) of concentrated hydrochloric acid and the reaction was continued at 80 ℃ overnight. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B5. The yield was 72.9%.
1H NMR(400MHz,DMSO)δ9.51(s,1H),9.26(s,1H),9.11(s,2H),8.66(s,1H),7.56(s,1H),6.94(s,1H),4.53(s,1H),3.07(d,J＝9.8Hz,2H),2.34(s,1H),1.98(s,1H),1.86(s,1H),1.22(d,J＝6.0Hz,3H)ppm.HRMS(ESI)m/z:(M+H)+calcd for Chemical Formula:C24H27Cl3N6O:520.1312,found:521.1388.
Example 8
(6) Method for synthesizing 5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) -N2- (3-nitrophenyl) pyrimidine-2, 4-diamine (CR-B6):
1mmol (495.20mg) of intermediate b and 1.5mmol (207.19mg) of 3-nitroaniline were weighed into a 25ml round bottom flask, an appropriate amount of isopropanol was added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid was added, and the reaction was continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B6. The yield was 55.6%.
1H NMR(400MHz,DMSO)δ9.87(s,1H),8.79(s,1H),8.52(s,1H),8.25(s,1H),8.20(s,1H),8.07(d,J＝14.3Hz,1H),7.85(s,1H),7.76(d,J＝9.7Hz,1H),7.46(t,J＝8.2Hz,1H),6.85(s,1H),4.51(s,1H),3.37(d,J＝12.6Hz,1H),3.05(t,J＝11.6Hz,1H),2.22(s,1H),1.86(s,1H),1.25(s,1H)ppm.HRMS(ESI)m/z:(M+H)+calcd for Chemical Formula:C25H29ClN6O3:496.1990,found:497.2063.
Example 9
(7) Method for synthesizing 5-chloro-N2 (4-chlorophenyl) -N4 (2-isopropoxy-5-methyl-4 piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B7):
1mmol (495.20mg) of intermediate b and 1.5mmol (191.36mg) of 4-chloroaniline were weighed into a 25ml round bottom flask, an appropriate amount of isopropanol was added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid was added, and the reaction was continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B7. The yield was 70.4%.
1H NMR(400MHz,DMSO)δ9.95(s,1H),9.00(s,1H),8.86(s,1H),8.60(s,1H),8.26(s,1H),7.73(s,1H),7.55(d,J＝8.7Hz,1H),7.23(d,J＝8.8Hz,1H),6.87–6.82(m,1H),4.52(s,1H),3.35(s,1H),3.06(s,1H),2.25(s,1H),1.89(d,J＝31.8Hz,1H),1.82(s,1H),1.24(d,J＝6.0Hz,1H)ppm.HRMS(ESI)m/z:(M+H)+calcd for Chemical Formula C25H29Cl2N5O:485.1749,found:486.1826.
Example 10
(8) Method for synthesizing 5-chloro-N2- (4-chloro-3- (trifluoromethyl) phenyl) -N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B8):
weigh 1mmol (495.20mg) of intermediate b and 1.5mmol (293.35mg) of 4-chloro-3- (trifluoromethyl) aniline into a 25ml round-bottomed flask, add the appropriate amount of isopropanol as solvent, add a catalytic amount (about 1%) of concentrated hydrochloric acid, and continue the reaction at 80 ℃ overnight. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B8. The yield was 48.6%.
1H NMR(400MHz,DMSO)δ9.91(s,1H),8.97(s,1H),8.78(s,1H),8.29(d,J＝30.1Hz,1H),8.05(s,1H),7.96(d,J＝10.4Hz,1H),7.78(s,1H),7.47(d,J＝8.8Hz,1H),6.86(s,1H),3.50(s,1H),3.36(s,1H),3.06(s,1H),2.26(s,1H),1.85(s,2H),1.24(s,2H),1.03(s,1H),0.83(s,1H)ppm.HRMS(ESI)m/z:(M+H)+calcd for Chemical Formula:C HCl F N O:553.1623,found:554.1699.
Example 11
(9) Method for synthesizing 5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) -N2- (2-methyl-5-nitrophenyl) pyrimidine-2, 4-diamine (CR-B9):
1mmol (495.20mg) of intermediate b and 1.5mmol (228.23mg) of 2-methyl-5-nitroaniline were weighed into a 25ml round bottom flask, an appropriate amount of isopropanol was added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid was added, and the reaction was continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B9. The yield was 58.8%.
Example 12
(10) Method for synthesizing 5-chloro-N2 (3-chlorophenyl) -N4 (2-isopropoxy-5-methyl-4 piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B10):
1mmol (495.20mg) of intermediate b and 1.5mmol (214.78mg) of 1-naphthylamine were weighed into a 25ml round-bottomed flask, an appropriate amount of isopropanol was added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid was added, and the reaction was continued at 80 ℃ overnight. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B10. The yield was 52.8%.
Example 13
(11) Method for synthesizing 5-chloro-N4- (2-isopropoxy-5-methyl-4-piperidine-phenyl) -N2- (4-nitrophenyl) pyrimidine-2, 4-diamine (CR-B11):
1mmol (495.20mg) of intermediate b and 1.5mmol (207.19mg) of 4-nitroaniline were weighed into a 25ml round bottom flask, an appropriate amount of isopropanol was added as solvent, a catalytic amount (about 1%) of concentrated hydrochloric acid was added, and the reaction was continued overnight at 80 ℃. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B11. The yield was 71.2%.
Example 14
(12) Method for synthesizing 5-chloro-N2, N4-bis (2-isopropoxy-5-methyl-4-piperidine-phenyl) pyrimidine-2, 4-diamine (CR-B12):
weigh 1mmol (495.20mg) of intermediate b and 1.5mmol (372.56mg) of 2-isopropoxy-5-methyl-4-piperidine-aniline into a 25ml round bottom flask, add the appropriate amount of isopropanol as solvent, add a catalytic amount (ca.1%) of concentrated hydrochloric acid, and continue the reaction at 80 ℃ overnight. TLC monitored the progress of the reaction. And after the reaction is completed, cooling the reaction liquid to room temperature, adding a small amount (about 5 drops) of trifluoroacetic acid, stirring at room temperature for 1h, separating out corresponding solid, filtering to remove the solvent, collecting the solid, washing with water for multiple times, and drying in vacuum to obtain the target product CR-B12. The yield was 60.3%.
Example 15
MTT method: MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide) is yellow powder, is easy to decompose by visible light, and therefore needs to be protected from light in the using process. MTT solution acts on living cells to form water-insoluble blue-purple crystalline Formazan (Formazan), but does not act on apoptotic or necrotic cells. Formazan is solubilized by Dimethylsulfoxide (DMSO) or 20% SDS (sodium dodecyl sulfate), and the absorbance (OD value) is measured at a wavelength of 490nm or 570nm by an enzyme-linked immunosorbent detector. Within a certain cell number range, the OD value is in direct proportion to the number of living cells, and can reflect the capacity of the medicine for inhibiting or killing tumor cells. In the research, the MTT method is mainly used for detecting the in-vitro anti-tumor activity of the compound on different tumor cells.
Preparation of RPMI 1640 and DMEM complete medium
The double antibody (Penicillin/Streptomycin Solution) and fetal calf serum frozen at-20 deg.C are thawed by placing in advance at 4 deg.C or room temperature, and RPMI 1640 and DMEM basal medium are placed in a water bath at room temperature or 37 deg.C. Respectively adding 10% fetal calf serum and 1% double antibody into RPMI 1640 and DMEM basal medium in a super clean operating platform, slightly inverting or shaking to mix them uniformly, subpackaging into 50ml centrifuge tubes, and storing in a refrigerator at 4 deg.C.
Preparation of cell freezing medium
Fetal bovine serum frozen at-20 ℃ was thawed by placing it at 4 ℃ or room temperature in advance, and the amount of fetal bovine serum: and (3) preparing DMSO at a ratio of 9:1, fully and uniformly mixing, subpackaging into 15ml centrifuge tubes, and storing in a refrigerator at-20 ℃. The fetal calf serum plays an important role in maintaining the activity of cryopreserved cells, the higher the serum content is, the better the cell protection is, and the highest content can reach 90%. DMSO plays a role of a permeability protective agent in frozen stock solution, and can rapidly permeate into cells, so that the permeability of cell membranes to water is improved, the freezing point is lowered, water in the cells is permeated to the outside of the cells before freezing in the slow freezing storage process, small ice crystals are formed outside the cells, the formation of intracellular ice crystals is reduced or avoided, and the damage of the ice crystals to the cells is reduced. DMSO is used at a concentration of 5% to 10%, typically 10%.
MTT (250mg) powder stored at-20 ℃ was allowed to stand at room temperature in advance. And (3) taking 50ml of PBS buffer solution into a 50ml centrifuge tube wrapped by tinfoil paper (processed in a dark place), taking MTT powder out of the centrifuge tube, washing the tube wall by PBS for multiple times, and ensuring that all MTT in the tube is completely taken out. Putting the mixture into an ultrasonic instrument, and heating and carrying out ultrasonic treatment for about 1h to fully dissolve the MTT. After the dissolution was complete, the mixture was filtered through a 0.22 μm microfiltration membrane. The final concentration was 5 mg/ml. Storing at 4 deg.C in dark or storing at-20 deg.C in dark for a long time.
Preparation of 20% SDS solution
40g was weighed into a 250ml beaker, 200ml RO water (reverse osmosis water) was added and placed in a sonicator for at least 1h to dissolve completely. After complete dissolution, one thousandth of concentrated HCl, namely 200ul, is added and stored at normal temperature.
The target compound CR-B1-12 synthesized in the above example and the positive control drug Ceritinib were stored in an ambient temperature desiccator. In preparation, the target compounds CR-B1-12 and Ceritinib were precisely weighed into a 1.5ml EP tube, and the volume of DMSO required for compound preparation was calculated according to the concentration calculation formula C ═ M/M · V, and C ═ 10mM (see table 1). Adding DMSO with corresponding volume into a super clean bench to fully dissolve the compound, and storing at 4 ℃ or-20 ℃ for a long time.
TABLE 1 preparation of mother liquor of positive control Ceritinib and CR-B series compounds
Cell cycle experiments:
the cell cycle refers to the whole time cycle that a cell undergoes division once, and can be divided into 4 stages, wherein the DNA content of each stage is different, and the stages comprise (1) G0/G1: the gap time from completion of cell mitosis to before DNA replication, the DNA content being diploid (2N); (2) and (4) S period: in the DNA replication period, the DNA content is between that of the diploid and the tetraploid; (3) stage G2: the time until completion of DNA replication to the onset of mitosis; (4) the DNA content in G2/M phase was tetraploid (4N) at the time from the beginning to the end of cell division in M phase. Propidium Iodide (PI) is a dye that binds to intracellular DNA and RNA when used in the dark. After a proper amount of RNase is added in an experiment to degrade RNA, PI is only combined with DNA, the fluorescence intensity of PI is detected by a flow cytometer to reflect the DNA content in cells, and then the DNA percentage in each period is calculated by specific software, so that the period of the cells is judged. This method is used to determine the effect of candidate compounds on the cell cycle.
Cell resuscitation
The water bath kettle is opened in advance and set to 37 ℃, a 15ml centrifuge tube is taken out from a clean bench, the cell types are marked, and 9ml of complete culture medium required by cell growth is taken out and placed in the centrifuge tube for standby. Taking out cells from a cell freezing liquid nitrogen tank or a refrigerator at minus 80 ℃, rapidly putting the cells into a water bath to rapidly melt the cells, transferring the cells into a marked centrifugal tube after the cells are completely dissolved, centrifuging the cells at 1200rpm/min for 5min after uniform mixing, removing clear liquid, taking 2ml of fresh culture medium to suspend the cells, adding the cell suspension into 8ml of corresponding complete culture medium, fully and uniformly shaking the cells, and putting the cells into a carbon dioxide incubator at 37 ℃ and 5 percent for culture. Wherein MGC803, HGC27, HCT116, H1975, PC-9-C797S and Karpas-299 are completely cultured in RPMI 1640; HepG2, Eca109, a549 and 293T cells were in DMEM complete medium, the same below.
Cell exchange liquid
The growth state of the cells was observed every day, and the cell culture medium was changed when the cell growth morphology was intact, the cell number was less than 80-90% coverage, but the medium was yellow in color or more floating cells were observed. The specific operation is as follows: 1. adherent cells (MGC803, HGC27, HCT116, H1975, HepG2, Eca109, A549, 293T cells, the same below) and semi-suspension cells (PC-9-C797S cells, the same below): discard the supernatant, add 2ml PBS along the wall, gently shake the dish for washing, discard PBS, add 10ml corresponding complete medium along the wall, place in a 5% carbon dioxide incubator at 37 deg.C for culturing. 2. Suspension of cells: karpas-299 cells. Collecting cell sap by a 15ml centrifuge tube, centrifuging at 1200rpm/min for 5min after resuspending 3ml PBS, discarding PBS, taking 2ml fresh culture medium to suspend cells, adding the cells into a culture dish containing 8ml corresponding culture medium, and placing the culture dish in a 5% carbon dioxide incubator at 37 ℃ for culture.
Cell passage
The growth state of the cells was observed every day, and when the growth morphology of the cells was observed to be good and the number of cells reached 85-90% of the culture dish, the cells were passaged. The specific operation is as follows: 1. adherent cells: discarding supernatant, adding 2-3ml PBS, washing for 1-2 times, adding 1ml 0.25% trypsin, digesting for 1-3min (different digestion time according to cell types), and after digestion, according to pancreatin: complete medium ═ 1: 2-1: 4, adding more than 2ml of prepared complete culture medium to stop digestion, blowing off cells into a single state, collecting cell sap, centrifuging at 1200rpm/min for 5min, removing supernatant, suspending the cells by 2-3ml of fresh complete culture medium, and performing cell suspension according to the ratio of 1:2 or 1:3, adding the mixture into a culture dish containing 8ml of corresponding culture medium, and culturing in a 5% carbon dioxide incubator at 37 ℃.2. Suspension of cells: directly collecting the suspension cell liquid to a 15ml centrifuge tube, centrifuging at 1200rpm/min for 5min, centrifuging at 1200rpm/min for 4min after resuspending 3ml PBS, discarding PBS, taking 2-3ml fresh complete culture medium to suspend cells, adding the cells into a culture dish containing 8ml of corresponding culture medium according to the proportion of 1:2 or 1:3, and placing the cells into a carbon dioxide incubator for culture. 3. Semi-suspension cell: collecting supernatant into 15ml centrifuge tube, washing with 2-3ml PBS for 1-2 times, adding 1ml 0.25% trypsin for digestion for 1-3min, adding 2-4ml complete culture medium to stop digestion, collecting cell liquid into 15ml centrifuge tube containing supernatant, centrifuging at 1200rpm/min for 5min, pouring off supernatant, suspending cells with 2-3ml fresh complete culture medium, adding into culture dish containing 8ml corresponding culture medium according to proportion of 1:2 or 1:3, and culturing in carbon dioxide incubator.
Cell seeding
Tumor cells and 293T cells which have good growth state under microscopic observation and cell number coverage of about 85-90% are selected, cell suspensions are prepared by passaging cells under-4.2.2.3 |, and the cell concentration is adjusted to 2X 104-1X 105/ml (different cell concentrations are prepared according to cell types and growth rates). The adjusted cell suspension is evenly inoculated into the middle 60 holes of a 96-hole plate, and the same amount of physiological saline is added into 100 mul/hole and 36 holes at the edge to avoid the edge effect. The well plates were incubated at 37 ℃ in a 5% carbon dioxide incubator and the next day of administration.
Administration of CR-B series of compounds and Ceritinib
The prepared CR-B series compound and Ceritinib stock solution (10mM) were diluted to the concentration required for the experiment with complete medium adapted to cell culture, and the concentration gradient of this experiment was 40, 20, 10, 5, 2.5, 1.25. mu.M, and 100. mu.l/well was added to the well plate, and 3 wells were repeated for each concentration. A solvent control (DMSO) and a blank control (medium) were set simultaneously. The well plate is placed in a 5% carbon dioxide incubator at 37 ℃ for culture, and MTT is added for detection after 48 hours.
MTT assay
After the compound acted on the cells for 48 hours, MTT (20. mu.l) with 10% of the liquid amount in the wells was added under the condition of keeping out of the light, and the cells were further cultured in a 5% carbon dioxide incubator at 37 ℃ for 2-4 hours. For adherent cells, the supernatant was gently spun off while 150. mu.l/well of DMSO was added. Shaking the shaking table for 10min, and setting lambda at 490nm for detecting OD value by ELISA detector after the crystal is completely dissolved. For semi-adherent cells and suspension cells, 20% SDS solution is directly added, 50. mu.l/well is cultured in a 5% carbon dioxide incubator at 37 ℃ for at least 8h, and after crystals are completely dissolved, an enzyme linked immunosorbent assay detector sets lambda as 570nm to detect OD value. The OD value of each repeated drug concentration is taken as the average value, and the formula is calculated according to the relative inhibition rate: the inhibition rate (blank OD value-experimental OD value)/blank OD value was calculated, the relative inhibition rate of the compound at each concentration gradient was calculated, and the half maximal Inhibitory Concentration (IC) of the compound was calculated using the data analysis software GraphPad Prism 650) The value is obtained.
Results of in vitro antiproliferative Activity of CR-B series Compounds
MTT method for detecting the in vitro anti-proliferation activity of CR-B series compounds on MGC803, HGC27, HCT116, H1975, PC-9-C797S, Karpas-299, HepG2, Eca109, A549 tumor cells and 293T cells is shown in Table 2 and Table 3. The results show that all 12 compounds have better anti-tumor activity, and the in vitro anti-proliferation activity of a plurality of compounds on the 9 tumor cell lines is better than that of a positive control drug Ceritinib. Wherein 5 compounds (CR-B1, CR-B4, CR-B5, CR-B6 and CR-B12) have better activity on non-small cell lung cancer cell A549Ceritinib; the anti-proliferative activity of 9 compounds on human lung adenocarcinoma H1975 cells is stronger than that of Ceritinib; the activity of 12 new synthesized compounds on the lung cancer cell PC-9-C797S with three-gene mutation is better than that of Ceritinib; except CR-B3 and CR-B8, the rest compounds have obviously better antiproliferative activity on NPM-ALK fused mature cell Karpas-299 than Ceritinib, and the compounds CR-B1 and CR-B5 have the most obvious activity and IC500.56. mu.M and 0.16. mu.M, respectively, and inhibition-concentration curves were fitted as shown in FIG. 1.
TABLE 2 in vitro antiproliferative Activity of CR-B series of Compounds
TABLE 3 in vitro antiproliferative Activity of CR-B series of Compounds
Example 16
Time dependency examination of CR-B1 and CR-B5
And (3) selecting Karpas-299 cells with good growth state to inoculate the cells in a 96-well plate. The following day compounds CR-B1, CR-B5 were administered at concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125. mu.M, 3 wells per drug concentration. And (5) respectively culturing in a carbon dioxide incubator for 24h, 48h, 72h and 96h, adding MTT, and detecting.
The results are shown in table 4 and fig. 2. The experimental results show that the compounds CR-B1 and CR-B5 have the in vitro inhibitory activity (IC) on Karpas-299 cells within 24-96h50) No significant change, and CR-B1 and CR-B5 were both more active than Ceritinib, with CR-B5 being the best.
TABLE 4 time-dependent tolerance examination of the compounds CR-B1 CR-B5 and Ceritinib (IC 50: μ M)
Example 17
Concentration dependency examination of CR-B1 and CR-B5
The cells of Karpas-299 which grew well were selected and seeded in a 96-well plate according to the cell seeding protocol of item-4.3.1.1 |. The following day compounds CR-B1, CR-B5 were administered at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,4, 8, 16, 20, 40, 80, 100. mu.M, 3 wells per drug concentration. The culture was continued at 37 ℃ in a 5% carbon dioxide incubator for 48h, and MTT was added and the assay was carried out as described for-4.3.1.3 |.
The results are shown in table 5 and fig. 3. From the results of the experiments it can be seen that the compounds CR-B1 and CR-B5 were concentration-dependent on the antiproliferative activity of Karpas-299 cells in vitro in the concentration range of 0.5-16. mu.M, and reached the maximum inhibition at 16. mu.M, and did not increase with increasing concentration. In addition, the inhibition rate of CR-B1 and CR-B5 in the concentration range of 0.5-0.9 mu M is obviously higher than that of Ceritinib.
TABLE 5 concentration (. mu.M) -inhibition (%) -of the compounds CR-B1, CR-B5 and Ceritinib
Example 18
Cell cycle experiments with CR-B1 and CR-B5
Selecting Karpas-299 cells with good growth state, collecting cells according to the cell passage operation method of item-4.2.2.3 |, resuspending into cell suspension, counting with cell counting plate, and adjusting cell concentration to 2 × 105One/ml, seeded in 6 well cell culture plates, 2ml per well. The following day, compounds CR-B1 and CR-B5 were administered at concentrations of 0.1, 0.5, 1 μ M, and were incubated at 37 deg.C in a 5% carbon dioxide incubator for an additional 48 h. Collecting cells by a flow cytometry tube, centrifuging for 5min at 1200rpm/min, removing a supernatant culture medium, adding 1ml PBS to gently blow off the cells, centrifuging for 5min at 1200rpm/min, and repeatedly washing for 3 times. The supernatant was decanted and the flow tube was flicked gently with the fingers to disperse the cells sufficiently. Adding 70% glacial ethanol 500 μ l into each tube, adding dropwise while shaking gently to avoid cell aggregation, mixing, and placing in 4 deg.C refrigeratorOvernight. The next day, centrifuging at 800rpm/min for 5min, discarding ethanol, adding 1ml PBS, washing repeatedly for 3 times, adding 100 μ l RNase in DNA content detection kit, and placing in water bath at 37 deg.C for 30 min. Centrifuge at 800rpm/min for 5min, discard the supernatant and wash 2 times with PBS. Adding PI in the kit, staining for 20min, filtering with 200 mesh (75 μm) cell filter screen before machine, and detecting the filtrate on machine.
Results of the experiments are shown in Table 6 and FIG. 4, and the cell cycle results show that Karpas-299 cells treated with a newly synthesized ALK inhibitor for 48h were trapped at the G1/S stage. Administration of 0.5 μ M CR-B1 induced 83.3% of the cells to be captured at G1/S, which was slightly greater than 81.1% of the cells treated with Ceritinib; while the 0.5 μ M treatment with CR-B5 induced 90.3% of the cells to be trapped at G1/S, which was significantly larger than the group with Ceritinib; CR-B1 and CR-B5 reduced G2/M cells from 24.6% to 15.8% and 8.3%, respectively; these data indicate that CR-B5 was the most effective at capturing G1/S cells and inducing cell cycle arrest in these 3 compounds. Cell cycle results were analyzed using Flow Jo software.
TABLE 6 cell cycle analysis data for compounds CR-B1, CR-B5, and Ceritinib
Example 19
The 12 new synthesized compounds of the invention all have obvious in vitro anti-tumor activity. Wherein the activity of the compounds CR-B1 and CR-B5 on 9 different tumor cells is better than that of Ceritinib. In the 9 different tumor cells, the antiproliferative activity of the compounds CR-B1 and CR-B5 on NPM-ALK fused mature cell Karpas-299 is obviously better than that of other cells, as shown in figure 5. In addition, the 12 CR-B series compounds also have antiproliferative activity superior to Ceritinib on gastric cancer cells MGC-823(7/12) and HGC-27(10/12), colorectal cancer cells H1975(9/12), liver cancer cells HepG-2(8/12) and PC-9-C797S (12/12), and as shown in FIG. 6, the 12 newly synthesized compounds have broad-spectrum antitumor effect.
It is apparent from the activity data that different anti-proliferative activities of tumors can be caused when R is different substituent groups. Firstly, we substituted 2-isopropoxy-5-methyl-4-piperidine-aniline on the right side of the Ceritinib structure for 2,4, 5-trichloropyrimidine at the same time to obtain compound CR-12, and the activity data shows that the substitution is meaningful, and CR-B12 has an anti-proliferative activity superior to Ceritinib on 6 of 9 tumor cells. The activity data supports our design of compounds. Because the subject mainly aims at the structural optimization of Ceritinib, the research on the in vitro anti-tumor activity of the CR-B series compounds on NPM-ALK fused mature cells Karpas-299 is mainly discussed in the structure-activity relationship.
When R is both-NO2When substituted (CR-B6 and CR-B11), the substitution position of-NO 2 on the benzene ring has little influence on the activity of the compound, and the introduction of a methyl group (CR-B9) at the neck position can slightly increase the antitumor activity of the compound, but the activity of the 3 compounds is better than that of Ceritinib. When R is both substituted by-Cl (CR-B2 and CR-B7), the substitution position of-Cl on the benzene ring also has no significant effect on the activity of the compound; when-Cl is para-substituted and-CF 3(CR-B8) is introduced at its collar, the antitumor activity is significantly lower than that of CR-B7 and weaker than that of Ceritinib. In addition, the addition of a substitution of-CF 3 (CR-B3) also did not improve the antitumor activity, and was still weaker than Ceritinib. When R is substituted by benzene ring (CR-B4 and CR-B10), the antitumor activity is obviously improved compared with Ceritinib, and the phenyl substitution (CR-B4) is superior to the biphenyl (CR-B10). When the substitution positions are para-substitution and the R group is strongly electron-withdrawing substitution (-NO2, -F, -Cl), the activity of CR-B1, CR-B7 and CR-B11 is all greater than that of Ceritinib, so that the activity of fluorine substitution (CR-B1) is optimal. When the benzene ring of the R-based parent nucleus is changed into a pyridine ring, the tumor antiproliferative activity of CR-B5 is obviously stronger than that of Ceritinib, and is stronger than that of CR-B1 with the R-based parent nucleus as the benzene ring.
In vitro antiproliferative activity research obtains compounds CR-B1 and CR-B5 with obvious tumor inhibition effect and better than positive control Ceritinib. The 2 compounds showed no significant difference in time of action, were concentration-dependent in the concentration range of 0.5-16. mu.M and reached maximum inhibition at 16. mu.M, which was significantly higher than Ceritinib in the concentration range of 0.5-0.9. mu.M. Cell cycle experiments show that the compounds CR-B1 and CR-B5 can block the cell cycle in the G1/S phase, and the CR-B5 blocking rate is higher. Particularly, the compound has the best effect of inhibiting the NPM-ALK fused mature cell strain Karpas-299.
The descriptions of each patent, patent application, and publication cited in this application are incorporated herein by reference in their entirety. Citation of any reference shall not be construed as an admission that such reference is available as "prior art" to the present application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. A compound having the structure of formula II:
Wherein the substituent R is phenyl, n R1Substituted phenyl, naphthyl; wherein n = 1-4;
R1independently selected from the following: halogen atom, C1-C6 alkyl, fluorine substituted C1-C4 alkyl, phenyl and nitro.
2. The compound of claim 1, wherein said C1-C6 alkyl group comprises methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, hexyl.
3. A compound according to claim 1, wherein R is a substituent1The substitution is in the ortho, meta or para position.
4. The compound of claim 1, wherein the compound is one of the following compounds:
5. use of a compound according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of tumours.
6. The use of claim 5, wherein the compound acts by binding to an ALK kinase protein.
7. The use of claim 5, wherein the tumor treatment drug is a tumor treatment drug for non-small cell lung cancer.
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