CN109053594B - 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidine-4-yl) urea compound and preparation and application thereof - Google Patents
1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidine-4-yl) urea compound and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a 1- (3, 5-dimethoxyphenyl) -3- (substitutedThe structure of the compound is shown as a formula (I), research results show that the compound has no toxic effect on proliferation of BEAS-2B cells (lung normal cells), and has certain inhibition effect on five selected non-small cell lung cancer cell lines including A549 cells (WT), PC-9 cells (EGFR del E746-A75), H520 cells (FGFR amplification), H1581 cells (amplification FGFR) and H226 cells (FGFR amplification/over-expressed EGFR), and the compound shows certain antitumor activity.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea EGFR/FGFR dual small molecule inhibitor and a preparation method and application thereof.
Background
Receptor Tyrosine Kinases (RTKs) are the largest class of enzyme-linked receptors in the human body, and there are 58 known RTKs in the human genome. RTKs play a crucial role in activities such as cell growth, proliferation, migration and differentiation, and signals from many RTKs, such as Epidermal Growth Factor Receptors (EGFRs), Fibroblast Growth Factor Receptors (FGFRs), Hepatocyte Growth Factor Receptors (HGFRs), and platelet-derived growth factor receptors (PDGFR), have been pharmacologically shown to be crucial for the survival of cancers expressing relevant protein mutations. Tyrosine Kinase Inhibitors (TKIs), which target these deregulated RTKs, may provide significant clinical benefit to cancer patients. Both EGFR and FGFR in RTKs have been well characterized as important oncogenic drivers of NSCLC. In the past decade, many EGFR-TKIs have been directly approved for clinical treatment of specific NSCLC patients, such as the first generation EGFR-TKIs Gefitinib (Gefitinib) and Erlotinib (Erlotinib); second generation EGFR-TKIs Afatinib (Afatinib); and third generation EGFR-TKIs oxitinib (Osimetinib). In addition, FGFR serving as another important drug action target for resisting NSCLC has no corresponding drug on the market, but in recent years, a large number of in vitro studies and independent experiments show that FGFR1 gene amplification and FGFR2 overexpression exist in NSCLC. And in a quantitative RT-PCR experiment on 59 NSCLC patients, the transcriptional activity of the tumor tissue FGFR1 of the NSCLC patients is more than two times that of the paracancerous tissue. Therefore, more and more FGFR-TKIs are also being reported in research studies for treating NSCLC.
However, although various chemical types of small molecule inhibitors have been extensively studied as EGFR-TKIs and FGFR-TKIs against NSCLC, so far, no related report exists that can be really used as a dual small molecule inhibitor for two kinase receptors. It has been proved that after clinical treatment with EGFR-TKIs such as gefitinib or afatinib, the amplification of FGFR1 of NSCLC cell strains is increased in a negative feedback manner to generate drug resistance (the principle is shown in figure 1), and the FGFR1 is knocked out in a gene silencing manner or is treated by combining with the FGFR-TKIs, so that the formation of drug-resistant strains can be effectively inhibited. Therefore, the activation of FGFR negative feedback caused by EGFR targeted inhibition, namely bypass activation, is also one of the important reasons for the drug resistance of EGFR-TKIs; furthermore, as the ability to perform single cell sequencing has improved, tumors have been reported that carry both oncogenic alleles of mutant EGFR and FGFR. In consideration of the fact that different types of medicines are used together, the patients often have inconvenience in medication, drug interaction or incompatibility, and some toxic and side effects or adverse reactions appear as a result. Therefore, the development of the dual inhibitor of EGFR and FGFR has urgent clinical needs and development prospects, particularly for NSCLC and drug-resistant treatment thereof.
Disclosure of Invention
The invention provides a 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidine-4-yl) urea compound, and a preparation method and an application thereof, wherein the compound has better antitumor activity and can prevent drug resistance to a certain extent.
The invention adopts the following technical scheme:
the chemical structure of the 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea EGFR/FGFR dual small molecule inhibitor is as follows:
tables 1 to 1: structure of Y series compound
Preferably, the compound 1- (3, 5-dimethoxyphenyl) -3- (6- ((5-iodo-2-methoxyphenyl) amino) pyrimidin-4-yl) urea (Y6) has the following chemical structure:
preferably, the compound 1- (3, 5-dimethoxyphenyl) -3- (6- ((2-methoxy-5- (trifluoromethyl) phenyl) amino) pyrimidin-4-yl) urea (Y13) has the following chemical structure:
the invention also provides a method for preparing 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea EGFR/FGFR dual small molecule inhibitor, which comprises
First step synthesis of intermediate product:
the method comprises the following steps: a dry three-mouth reaction flask is taken and put into a magnet. 4-amino-6-chloropyrimidine (1eq), KI (0.5eq) was added and dissolved in anhydrous ethanol (35 mL). After heating with stirring for 10min on a magnetic stirrer, trifluoroacetic acid (200mL) was added. And (4) activating. After about 1h, anhydrous ethanol (15mL) dissolved substituted aniline (0.8eq) was added for reaction. It is noted that the reaction solution is added in a dropwise manner to achieve the effect of long-term excess reaction, and the dropwise adding time is controlled to be about 1 h.
Step two: the progress and effect of the reaction were checked by TLC. Generally, after 36 hours of reaction, the reaction was almost complete. After the reaction is completed, the solvent absolute ethyl alcohol is firstly dried in a spinning mode, and then a certain amount of ethyl acetate is added for dissolution. Removing acid with appropriate amount of 20% sodium bicarbonate solution, adding appropriate amount of 50% sodium chloride solution, extracting, collecting organic layer, spin drying to obtain residual amount, adding appropriate amount of anhydrous sodium sulfate, and removing water overnight.
Step three: and (3) carrying out suction filtration and sand making, weighing column layer silica gel powder which is 15 to 20 times of the total amount of the raw materials, filling the column layer silica gel powder into a column, and passing the column to collect product points. The first point (aniline point) is generally crossed by the petroleum ether and ethyl acetate (2: 1), the second point (pyrimidine point) is crossed by the petroleum ether and ethyl acetate (1: 1), and the product point is flushed by ethyl acetate or methanol. And (4) collecting the spin-dried product points, drying in an oven, performing mass spectrometry and nuclear magnetism, and verifying.
The invention also provides a method for preparing the 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea EGFR/FGFR dual small molecule inhibitor, which comprises the following steps: and (3) synthesizing an intermediate product in the second step:
the method comprises the following steps: a dry three-mouth reaction flask is taken and added with a magnet. Triphosgene (0.5eq) was weighed out and dissolved in dichloromethane (20mL) and sonicated to completion. 3, 5-Dimethoxyaniline (1eq) dissolved in dichloromethane (10mL) was slowly added dropwise at 0 ℃ one drop per minute over a period of about 0.5 h. After dropping, 2-3 drops of triethylamine are added. Heating and refluxing for 3-6 h, and the reaction is generally complete. Concentrating under reduced pressure to dryness, dissolving the residue with ethyl acetate, sequentially removing alkali with 10% potassium hydrogen sulfate solution, removing acid with 20% sodium bicarbonate solution and washing with 50% sodium chloride solution to remove inorganic phase, collecting organic phase, spin drying, dewatering with anhydrous sodium sulfate, drying overnight, filtering, and making into sand.
Step two: preparing sand, filling the sand into a column, and separating and purifying isocyanate after 3, 5-dimethoxyaniline is obtained. And sending to mass spectrum and nuclear magnetism for verification.
The invention also provides a method for preparing the 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea EGFR/FGFR dual small molecule inhibitor, which is characterized by comprising the following steps: and thirdly, synthesizing a target product:
the method comprises the following steps: a dry three-mouth reaction flask is taken and added with a magnet. Weighing pyrimidine aniline (1eq) and isocyanate (1.2eq), dissolving in toluene (10mL), heating and refluxing at 70-80 ℃ for 8-10 h, and performing TLC detection to obtain the pyrimidine aniline (1eq) and isocyanate (1.2 eq). And cooling to room temperature, washing the filter cake for 2-3 times by using toluene, dissolving the filter cake by using ethyl acetate after scraping, washing by using a 20% sodium bicarbonate solution to remove acid, and adding a 50% sodium chloride solution for extraction and layering. The organic layer was collected and spin dried.
Step two: and (5) counting plates to detect purity, sending to mass spectrometry and nuclear magnetism, and verifying.
The 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea derivative can be used for treating tumors.
The 1- (3, 5-dimethoxyphenyl) -3- (6- (substituted anilino) pyrimidine-4-yl) urea derivative shows a certain antitumor activity. According to the result of an antitumor activity test, the compounds Y6 and Y13 show certain inhibitory activity on five non-small cell lung cancer cell lines; among them, the compound Y13 is relatively preferable. IC of Compound Y13 on 5 cancer cells50The values were 15.06. + -. 1.75. mu.M, 6.22. + -. 0.91. mu.M, 4.45. + -. 0.63. mu.M, 7.35. + -. 1.01. mu.M, and 14.57. + -. 1.65. mu.M, respectively. The results show that compound Y13 is a relatively potent EGFR/FGFR inhibitor.
Drawings
FIG. 1 is a schematic diagram of the principle of FGFR1 activation mediated EGFR-TKIs acquired drug resistance generation.
FIG. 2 shows the results of the antitumor cell test obtained in example 2.
Detailed Description
The following examples are further detailed descriptions of the present invention.
Synthesis of the Compound of example 1
1.1A specific synthetic route for the compounds is shown below:
1.2 synthetic procedure
a. First step synthesis of intermediate product:
the method comprises the following steps: a dry three-mouth reaction flask is taken and put into a magnet. 4-amino-6-chloropyrimidine (1eq), KI (0.5eq) was added and dissolved in anhydrous ethanol (35 mL). After heating with stirring for 10min on a magnetic stirrer, trifluoroacetic acid (200mL) was added. And (4) activating. After about 1h, anhydrous ethanol (15mL) dissolved substituted aniline (0.8eq) was added for reaction. It is noted that the reaction solution is added in a dropwise manner to achieve the effect of long-term excess reaction, and the dropwise adding time is controlled to be about 1 h.
Step two: the progress and effect of the reaction were checked by TLC. Generally, after 36 hours of reaction, the reaction was almost complete. After the reaction is completed, the solvent absolute ethyl alcohol is firstly dried in a spinning mode, and then a certain amount of ethyl acetate is added for dissolution. Removing acid with appropriate amount of 20% sodium bicarbonate solution, adding appropriate amount of 50% sodium chloride solution, extracting, collecting organic layer, spin drying to obtain residual amount, adding appropriate amount of anhydrous sodium sulfate, and removing water overnight.
Step three: and (3) carrying out suction filtration and sand making, weighing column layer silica gel powder which is 15 to 20 times of the total amount of the raw materials, filling the column layer silica gel powder into a column, and passing the column to collect product points. The first point (aniline point) is generally crossed by the petroleum ether and ethyl acetate (2: 1), the second point (pyrimidine point) is crossed by the petroleum ether and ethyl acetate (1: 1), and the product point is flushed by ethyl acetate or methanol. And (4) collecting the spin-dried product points, drying in an oven, performing mass spectrometry and nuclear magnetism, and verifying.
b. And (3) synthesizing an intermediate product in the second step:
the method comprises the following steps: a dry three-mouth reaction flask is taken and added with a magnet. Triphosgene (0.5eq) was weighed out and dissolved in dichloromethane (20mL) and sonicated to completion. 3, 5-Dimethoxyaniline (1eq) dissolved in dichloromethane (10mL) was slowly added dropwise at 0 ℃ one drop per minute over a period of about 0.5 h. After dropping, 2-3 drops of triethylamine are added. Heating and refluxing for 3-6 h, and the reaction is generally complete. Concentrating under reduced pressure to dryness, dissolving the residue with ethyl acetate, sequentially removing alkali with 10% potassium hydrogen sulfate solution, removing acid with 20% sodium bicarbonate solution and washing with 50% sodium chloride solution to remove inorganic phase, collecting organic phase, spin drying, dewatering with anhydrous sodium sulfate, drying overnight, filtering, and making into sand.
Step two: preparing sand, filling the sand into a column, and separating and purifying isocyanate after 3, 5-dimethoxyaniline is obtained. And sending to mass spectrum and nuclear magnetism for verification.
c. And thirdly, synthesizing a target product:
the method comprises the following steps: a dry three-mouth reaction flask is taken and added with a magnet. Weighing pyrimidine aniline (1eq) and isocyanate (1.2eq), dissolving in toluene (10mL), heating and refluxing at 70-80 ℃ for 8-10 h, and performing TLC detection to obtain the pyrimidine aniline (1eq) and isocyanate (1.2 eq). And cooling to room temperature, washing the filter cake for 2-3 times by using toluene, dissolving the filter cake by using ethyl acetate after scraping, washing by using a 20% sodium bicarbonate solution to remove acid, and adding a 50% sodium chloride solution for extraction and layering. The organic layer was collected and spin dried.
Step two: and (5) counting plates to detect purity, sending to mass spectrometry and nuclear magnetism, and verifying.
1.3 results of the experiment
All the target compound structures synthesized are shown in the table 1-1 above; MS of a part of a target compound including an active compound synthesized,1H NMR and13the physicochemical data such as C NMR are as follows:
1- (3, 5-dimethoxy) -3- (6- ((3, 5-dimethoxy) amino) pyrimidin-4-yl) urea (Y1).
White powder, yield 58.3%; Mp/DEG C of 192.6 to 194.1; ESI-MS [ M + Na ]]+:425.92；1H NMR(600MHz,CDCL3-d6)(ppm):8.360(s,1H,-NH-),7.049(s,1H, 2-pyrimidine-H),6.757(s,1H,Ar-H),6.597(m,3H,Ar-H),6.240(s,2H, Ar-H),6.222(s,1H,5-pyrimidine-H),3.809(s,6H,-OCH3),3.788(s,6H, -OCH3).13C NMR(150MHz,DMSO-d6)(ppm):162.528,161.995,161.788, 159.417,158.654,155.302,154.253,144.014,143.067,112.977,111.051, 109.206,91.311,56.700,55.987.
1- (6- ((5-bromo-2-methoxy) amino) pyrimidin-4-yl) -3- (3, 5-dimethoxyphenyl) urea (Y3).
Yellow powder, yield 45.2%; Mp/DEG C of 203.8 to 205.7; ESI-MS [ M + Na ]]+:474.13；1H NMR(600MHz,DMSO-d6)(ppm):9.857(s,1H,-NH-),9.202(s,1H, -NH-),8.856(s,1H,-NH-),8.319(s,1H,2-pyrimidine-H),8.124(s,1H,Ar-H), 7.172(s,1H,Ar-H),7.118(s,1H,Ar-H),6.978(d,J＝6Hz,1H,Ar-H),6.963(d, J＝6Hz,1H,Ar-H),6.665(s,1H,Ar-H),6.160(s,1H,5-pyrimidine-H),3.782 (s,3H,-OCH3),3.686(s,6H,-OCH3).13C NMR(150MHz,DMSO-d6) (ppm):161.245,160.681,156.998,151.588,140.516,130.022,125.806, 124.627,113.239,1111.424,96.963,94.778,91.146,55.957,55.093.
1- (3, 5-dimethoxyphenyl) -3- (6- ((5-iodo-2-methoxyphenyl) amino) pyrimidin-4-yl) urea (Y6).
White powder, yield 38.4%; 198.3 to 199.4 Mp/DEG C; ESI-MS [ M + Na ]]+:522.14；1H NMR(600MHz,CDCL3-d6)(ppm):8.451(s,1H,-NH-),8.362(s,1H, 2-pyrimidine-H),7.449(d,J＝7.8Hz,1H,Ar-H),7.436(d,J＝7.8Hz,1H, Ar-H),6.769(d,J＝1.8Hz,1H,Ar-H),6.766(d,J＝1.8Hz,1H,Ar-H),6.714(s, 1H,Ar-H),6.519(s,1H,Ar-H),6.242(s,1H,5-pyrimidine-H),3.888(s,3H, -OCH3),3.791(s,6H,-OCH3).13C NMR(150MHz,DMSO-d6)(ppm): 161.033,160.663,157.295,151.683,140.501,130.582,111.586,96.935, 94.741,85.607,56.569,55.088,55.032.
1- (3, 5-dimethoxy) -3- (6- ((3-fluoro-5-methoxyphenyl) amino) pyrimidin-4-yl) urea (Y10).
White powder with a yield of 50.4%; Mp/DEG C of 225.6 to 226.2; ESI-MS [ M + Na ]]+:397.88；1H NMR(600MHz,CDCL3-d6)(ppm):8.188(s,1H,-NH-),7.177(s,2H, 2-pyrimidine-H+Ar-H),7.083(s,1H,Ar-H),7.014(s,1H,Ar-H),6.929(s,1H, Ar-H),6.594(s,1H,Ar-H),6.231(s,1H,Ar-H),5.830(s,1H,5-pyrimidine-H), 3.776(s,6H,-OCH3),2.276(s,3H,-CH3).13C NMR(150MHz,DMSO-d6) (ppm):160.933,160.668,157.365,157.259,151.650,140.463,115.521, 96.900,94.801,91.090,55.074,55.023,21.137.
1- (3, 5-dimethoxy) -3- (6- ((2-methoxy-5- (trifluoromethyl) phenyl) amino) pyrimidin-4-yl) urea (Y13).
White powder, yield 39.3%; 231.7 to 232.6 Mp/DEG C; ESI-MS [ M + Na ]]+:464.20；1H NMR(600MHz,CDCL3-d6)(ppm):8.502(s,1H,-NH-),7.373(s,1H, 2-pyrimidine-H),7.224(s,1H,Ar-H),7.005(d,J＝8.4Hz,1H,Ar-H),6.991(d, J＝8.4Hz,1H,Ar-H),6.857(s,1H,Ar-H),6.793(s,1H,Ar-H),6.584(s,1H, Ar-H),6.248(s,1H,5-pyrimidine-H),3.975(s,3H,-OCH3),3.860(s,6H, -OCH3).13C NMR(150MHz,DMSO-d6)(ppm):160.854,160.670,160.605, 159.993,157.317,151.645,142.364,141.283,140.436,108.320,96.904, 96.560,94.818,93.982,91.401,55.500,55.066,55.018.
1- (3, 5-dimethoxy) -3- (6- ((3-ethylphenyl) amino) pyrimidin-4-yl) urea (Y15).
White powder, yield 51.0%; Mp/DEG C of 209.2 to 209.9; ESI-MS [ M + Na ]]+: 393.79；1HNMR(600MHz,CDCL3-d6)(ppm):8.383(s,1H,-NH-),7.937(s, 1H,2-pyrimidine-H),7.855(d,J＝2.4Hz,1H,Ar-H),7.851(d,J＝2.4Hz,1H, Ar-H),6.818(m,3H,Ar-H),6.557(m,3H,5-pyrimidine-H+Ar-H),3.845(s, 6H,-OCH3),2.671(m,2H,-CH2CH3),1.254(t,J＝7.2Hz,3H,-CH3).13C NMR(150MHz,DMSO-d6)(ppm):161.205,160.674,157.325,157.233, 151.684,144.261,140.503,139.953,128.582,121.843,119.373,117.534, 96.948,96.593,94.785,90.083,55.077,55.030,28.262,15.473.
1- (6- ((4- (tert-butyl) phenyl) amino) pyrimidin-4-yl) -3- (3, 5-dimethoxyphenyl) urea (Y18).
White powder,yield:50.7％；Mp/℃:212.3～213.1；ESI-MS[M+Na]+: 422.21；1H NMR(600MHz,CDCL3-d6)(ppm):8.335(s,1H,-NH-),7.364(s, 1H,2-pyrimidine-H),7.311(s,1H,Ar-H),7.185(s,1H,Ar-H),7.006(s,1H, Ar-H),6.764(d,J＝1.8Hz,1H,Ar-H),6.761(d,J＝1.8Hz,1H,Ar-H),6.604(d, J＝1.8Hz,1H,Ar-H),6.601(d,J＝1.8Hz,1H,Ar-H),6.231(s,1H, 5-pyrimidine-H),3.771(s,6H,-OCH3),1.351(s,9H,-CH3).13C NMR (150MHz,DMSO-d6)(ppm):160.615,152.238,141.290,96.602,94.948, 94.001,56.252,55.026,31.206.
The properties and solubility of the target compound synthesized by the present invention are as follows:
the yield of the target compound is generally higher. The compounds Y1-2, Y6-7, Y9-11, Y13-15 and Y17-19 are all white solids; compound Y3 is a yellow solid; compounds Y4, Y8 and Y16, as a beige solid; compounds Y5, Y12 and Y20 are black solids. The compound is easily dissolved in ethyl acetate, acetonitrile, dichloromethane, DMSO and DMF; slightly soluble in petroleum ether, methanol and ethanol; insoluble in toluene.
The target compounds synthesized by the invention all show [ M +1 ] in MS spectrogram]+Peaks, and strong signals, and partial compounds have isotopic peaks.1The H-NMR spectrum result shows that hydrogen signals of all target compounds and chemical shifts of the target compounds can be clearly seen on the spectrum. With DMSO-d6When the solvent is adopted, the nuclear magnetic hydrogen spectrum data shows completely, namely the theoretical number of the compound hydrogen is matched with the number of hydrogen on a nuclear magnetic hydrogen spectrum diagram; and with CDCL3-d6In the case of solvent, the nuclear magnetic hydrogen spectrum data of most target compounds shows incomplete, and the nuclear magnetic hydrogen spectrum usually has no two hydrogens on the carbamide amine.13The C-NMR spectrum results show that the carbon peak shifts and the number of the target compound basically accord with theoretical data.
EXAMPLE 2 antitumor cell Activity of Compounds
2.1 testing the antitumor Activity of Compounds by the MTT method
The MTT method was used in this experiment. The selected normal lung cells are BEAS-2B cells; the five selected non-small cell lung cancer cells include lung adenocarcinoma cell A549(WT EGFR), lung adenocarcinoma cell PC-9(EGFR d746-750), lung squamous carcinoma cell H520(FGFR amplification), large cell lung cancer H1581(FGFR amplification), and lung squamous carcinoma cell H226(FGFR amplification/overexpressed EGFR). SelectingThe cells were taken for logarithmic growth, digested, collected and counted using a cell counting plate. Subsequently, the counted cells were diluted to an appropriate concentration (5X 10^ 4/mL-8X 10^ 4/mL), the diluted cell suspension was added to a 96-well plate by a discharging gun per 100. mu.L of each well, and culture was performed, and a blank control well containing only a culture medium was placed on the same well plate; after plating overnight culture, replacing with fresh culture medium, adding a series of tested target compounds diluted by concentration gradient and positive drugs BGJ398 and G into each hole, and detecting the survival rate of cells after the drugs act for 72 hours; after 20. mu.L of MTT assay solution was added to each well of the 96-well plate, the 96-well plate was placed in an incubator at 37 ℃ and incubated for four hours. The supernatant was removed and 150 μ L of DMSO was added, solubilizing the MTT formazan precipitate. Finally, detecting the light absorption value of each hole with ultraviolet absorption wavelength of 490nm by a microplate reader, and calculating the corresponding cell survival rate, inhibition rate or IC50Values, etc. The experiment needs to be repeated at least three times, so that the experiment error is reduced.
2.2 results of the experiment
The obtained test results are shown in fig. 2, and as shown in a in fig. 2, at a concentration of 10 μ M, the cell survival rate of all tested target compounds was 95% or more; the positive control group with the two drugs combined has a low cell survival rate of 75%. B in FIG. 2 shows the inhibitory effect of the test compounds on A549 cells, wherein the compounds with an inhibition rate of more than 50% are Y3-Y11, Y13-Y14, Y16-Y17 and Y19; FIG. 2C shows the inhibitory effect of the test compounds on PC-9 cells, wherein compounds Y1-Y4, Y6-Y20 with an inhibitory rate of more than 50%; the inhibitory effect of the test compounds on H520 cells, as shown in D in fig. 2, the inhibitory rate of both compounds Y6 and Y13 was over 50%; the inhibitory effect of the test compound on H1581 cells is shown in E in FIG. 2, the inhibitory rate of the compounds Y6-Y7, Y13-Y14 and Y19 is more than 50%; on the other hand, as shown by F in fig. 2, the inhibitory effect on H226 cells was more than 50% for all of compounds Y6, Y13, Y14, Y16 and Y19.
EXAMPLE 3 IC of Compounds Y6 and Y13 on five tumor cells50Experiment of
3.1 MTT method IC of test Compound50Value of
Based on the results of the toxic effect of all the compounds of interest on normal lung cells and the inhibitory effect on five NSCLC cell lines, we obtained compounds Y6 and Y13 which have inhibitory effects on all five NSCLC cell lines. We selected these two compounds for further IC50And (5) carrying out experiments. Six concentrations (20. mu.M, 10. mu.M, 1.0. mu.M, 0.50. mu.M, 0.10. mu.M and 0.01. mu.M) were initially set for both compounds. Then, five non-small cell lung cancer cell lines, including a549 cells, PC-9 cells, H520 cells, H1581 cells, and H226 cells, were treated with the two compounds at different concentrations, respectively. Optical Density (OD) values were obtained, inhibition calculated, and IC calculated for different compounds by GraphPad Prism 5 software50The value is obtained.
3.2 results of the experiment
IC for five cancer cells, A549 cell, PC-9 cell, H520 cell, H1581 cell and H226 cell50The values determined for compound Y6 are respectively>20 mu M, 3.54 +/-0.85 mu M, 9.87 +/-1.26 mu M, 8.86 +/-1.14 mu M and 19.43 +/-2.23 mu M; compound Y13 showed 15.06. + -. 1.75. mu.M, 6.22. + -. 0.91. mu.M, 4.45. + -. 0.63. mu.M, 7.35. + -. 1.01. mu.M and 14.57. + -. 1.65. mu.M, respectively. Y13 is the most active compound in this series.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidin-4-yl) urea compound is characterized in that the compound is 1- (3, 5-dimethoxyphenyl) -3- (6- ((2-methoxy-5- (trifluoromethyl) phenyl) amino) pyrimidin-4-yl) urea (Y13), and has the following chemical structure:
2. a process for the preparation of a 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidin-4-yl) urea compound as claimed in claim 1, comprising
(1) 4-amino-6-chloropyrimidine reacts with substituted aniline to obtain a pyrimidine aniline intermediate product;
(2) triphosgene and 3, 5-dimethoxyaniline react to obtain an isocyanate intermediate product;
(3) and (3) reacting the pyrimidine aniline intermediate product in the step (1) with the isocyanate intermediate product in the step (2) to obtain the 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidine-4-yl) urea compound.
3. The use of a 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidin-4-yl) urea compound as claimed in claim 1, wherein said 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidin-4-yl) urea compound is used for the preparation of an anti-tumor medicament.
4. The use of 1- (3, 5-dimethoxyphenyl) -3- (substituted pyrimidin-4-yl) urea as claimed in claim 3 wherein said antineoplastic agent is an EGFR/FGFR dual small molecule inhibitor.
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