CN111732597B - Preparation and application of 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy - Google Patents
Preparation and application of 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy Download PDFInfo
- Publication number
- CN111732597B CN111732597B CN202010568748.7A CN202010568748A CN111732597B CN 111732597 B CN111732597 B CN 111732597B CN 202010568748 A CN202010568748 A CN 202010568748A CN 111732597 B CN111732597 B CN 111732597B
- Authority
- CN
- China
- Prior art keywords
- amino
- pyrimidin
- oxy
- phenyl
- thiopyran
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
Abstract
The invention relates to preparation and application of 2-aminopyrimidine heterocyclic compounds containing 4-amidophenoxy and shown in general formulas I and II, and pharmaceutically acceptable salts, hydrates, solvates and prodrugs thereof, wherein substituents X and R1And R2Have the meanings given in the description. The compounds of the general formulas I and II have strong EGFR and EGFR inhibitionT790M/L858R、EGFRT790MThe function of kinase, and has very low inhibitory activity to normal human hepatocyte LO2, and also relates to the application of the compounds and the pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof in preparing medicines for treating diseases caused by abnormal activation and high expression of EGFR kinase and positive T790M and L858R mutationIn particular for the preparation of a medicament for the treatment and/or prevention of cancer.
Description
Technical Field
The invention relates to a preparation method of a novel 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy and pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof, a pharmaceutical composition containing the compound, and application of the compound in preparing a medicament for treating and/or preventing cancers.
Technical Field
Cancer, i.e., malignant tumor, is a disease that can cause abnormal division by losing control of the proliferation and differentiation of normal cells, and has many biological pathological characteristics such as infiltrative and metastatic properties, which are serious hazards to human health. According to statistics of cancer data of 2018 by the international cancer research institution, 1810 ten thousand cancer cases exist all the year around the world, and the mortality rate is 52.9%; worldwide cancer cases are expected to rise dramatically to 2950 million by 2040 years.
EGFR has been found to be involved in the inhibition of a range of vital activities of tumor cells, such as proliferation, invasion, angiogenesis, tumor metastasis and apoptosis. EGFR family members have been the dominant target for cancer therapy, especiallyIt is a treatment for NSCLC. The growth of tumors can be effectively inhibited by inhibiting the kinase activity of EGFR tyrosine and blocking the signal pathway conduction. A large number of third-generation EGFR small-molecule inhibitors (represented by the following structural formula) have been reported, among which WZ4002 is reported to have a high effect on biological cell activity and antitumor activity in the literature (Novel tissue-selective EGFR kinase inhibitors obtained from EGFR T790M. 2009; 462-1070-1074), and besides, many EGFR small-molecule inhibitors have excellent antitumor activity, such as Osteinib (Structure-and reactivity-based modification of tumor inhibition for TOF MS of epidermal growth factor receptor (EGFR J Medium Chem.2013;56 7025-7048), CO 6 (Discovery of tissue-receptor mutation) and EGFR kinase inhibitors obtained from Streptococcus kinase (ATCC 6174, 31-LB) and the like. An in vitro proliferation study of human NSCLC cell line shows that the EGFR is expresseddel19HCC827 cells of (1), expressing EGFRL858R/T790MH1975 cells and expression of EGFRWTThe concentration of the cells under half maximal growth inhibition is respectively 9.2nmol/L, 10 nmol/L and 2225nmol/L, which shows that the cells have stronger selectivity to EGFR mutation positive.
In order to develop a novel efficient antitumor drug, the inventor of the invention has conducted extensive research on amino heterocyclic pyrimidine compounds, maintains the U-shaped structural scaffold of the third-generation EGFR inhibitor compound on the basis of retaining active groups such as aminopyrimidine and michael acceptor, introduces active groups such as small molecule alkyl side chains and halogens on pyrimidine rings and acrylamide side chains to adjust the reactivity of the compound, changes the parent nucleus structure through molecular docking results, and designs and synthesizes a series of 2-amino pyrimidine heterocyclic compounds containing 4-amidophenoxy group with novel structures. The invention mainly inspects the antitumor activity of the 4-amidophenoxy-containing 2-aminopyrimidine heterocyclic compounds with different parent nucleus and different substituents so as to screen antitumor drugs with better activity and selectivity.
Disclosure of Invention
The invention relates to 2-aminopyrimidine heterocyclic compounds containing 4-amidophenoxy and shown in general formulas I and II, and pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof.
Compounds of general formulae i and ii and pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof.
Wherein:
the fused ring on which the X ring is located is selected from:
R1selected from:
z is N, C;
-R2(CH)2R3selected from:
wherein R is6Selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydroxy, amino, mercapto, carboxyl, trifluoromethoxy, methyl, ethyl, propyl, butyl, cyclopropane, ethylene, propylene, acetylene, propyne, methoxy, ethoxy, propoxy, isopropoxy, butoxy, or azido;
R7selected from halogen, hydroxy, trifluoromethyl, trifluoromethoxy, amino, azido, cyano, mercapto, C1～C4Alkyl radical, C3～C6Cycloalkyl, C1～C4Alkenyl radical, C1～C4Alkynyl, C1～C4An alkoxy group.
Very particularly preferred derivatives of the formulae I and II according to the invention, including the racemates or optical isomers thereof, and the pharmaceutically acceptable salts and/or hydrates thereof, are not meant to limit the invention in any way:
n- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) but-2-enamide
N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) pent-2-enamide
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) hex-2-enamide
2-fluoro-N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) but-2-enamide
N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) pent-2-enamide
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) hex-2-enamide
N- (3- ((2- ((3-cyanophenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
(E) -N- (3- ((2- ((3-cyanophenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) but-2-enamide
N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide
(E) -N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide
(E) -N- (3- ((2- ((3-cyanophenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) hex-2-amide
N- (3- ((2- ((3-methoxyphenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
2-fluoro-N- (3- ((2- ((3-methoxyphenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
(E) -4- (dimethylamino) -N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -but-2-enamide
(E) -N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -4- (piperidin-1-yl) -but-2-enamide
N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6, 7-dihydrothiophene [3,2-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
(E) -N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6, 7-dihydrothieno [3,2-d ] pyrimidin-4-yl) oxy) phenyl) -pent-2-enamide
N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
2-fluoro-N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
(E) -4- (dimethylamino) -N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -but-2-enamide
(E) -N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -4- (piperidin-1-yl) -but-2-enamide
The invention also provides a medicinal composition which contains the 2-aminopyrimidine heterocyclic compounds containing 4-amidophenoxy in the general formulas I and II, geometrical isomers thereof, pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof as active ingredients and pharmaceutically acceptable excipients.
The following synthetic schemes illustrate the preparation of the general formulae I and II of the present invention, all starting materials being prepared by the means described in the synthetic schemes, by methods well known to those of ordinary skill in the art of organic chemistry or commercially available. All of the final 4-amidophenoxy-containing 2-aminopyrimidine heterocycles of the invention are prepared by the methods described in the synthetic schemes or by methods analogous thereto, which are well known to those of ordinary skill in the art of organic chemistry. All variables used in the synthetic routes are as defined below or as defined herein.
Taking N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide as an example, the synthetic method is as follows, and all the raw materials are in commercial analytical purity.
Taking N- (3- ((2- ((3-cyanophenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide as an example, the synthesis method is shown below, and all the raw materials are in commercial analytical purity.
The method comprises the steps of firstly synthesizing an intermediate VII, and then carrying out butt joint or other modes with different side chain amines and different substituted micromolecule acyl chlorides to obtain a target compound.
The quinazoline compounds of the general formulae I and II described above in the present invention may be formed into pharmaceutically acceptable salts with acids according to methods common in the art to which the present invention pertains. Pharmaceutically acceptable addition salts include inorganic and organic acid addition salts, with the following being particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, and the like.
In addition, the present invention also includes prodrugs of the derivatives of the present invention. Prodrugs of the derivatives of the invention are those of formula I above which may themselves be less active or even inactive, but which, upon administration, are converted to the corresponding biologically active form under physiological conditions (e.g., by metabolism, solvolysis, or otherwise).
The invention can contain the 2-aminopyrimidine heterocyclic compounds containing 4-amidophenoxy in the general formulas I and II, and pharmaceutically acceptable salts, hydrates or solvates thereof as active ingredients, and the active ingredients are mixed with pharmaceutically acceptable carriers or excipients to prepare a composition and prepare a clinically acceptable dosage form, wherein the pharmaceutically acceptable excipients refer to any diluents, auxiliary agents and/or carriers which can be used in the pharmaceutical field. The derivatives of the present invention may be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions.
Clinical dosages of 4-amidophenoxy-containing 2-aminopyrimidine heterocycles of the invention of the above general formulae I and II in patients can be based on: the therapeutic efficacy and bioavailability of the active ingredients in vivo, their metabolism and excretion rates, and the age, sex, and disease stage of the patient are suitably adjusted, although the daily dose for an adult should generally be 10 to 500mg, preferably 50 to 300mg. These formulations may be administered in several doses (preferably one to six times) at regular intervals, according to the guidance of a doctor or pharmacist.
The pharmaceutical composition of the present invention can be formulated into several dosage forms containing some excipients commonly used in the pharmaceutical field. The above-mentioned several dosage forms can adopt the dosage forms of injection, tablet, capsule, aerosol, suppository, membrane, dripping pill, external liniment and ointment, etc.
The carriers for the pharmaceutical compositions of the present invention are of the usual type available in the pharmaceutical field, including: binder, lubricant, disintegrating agent, cosolvent, diluent, stabilizer, suspending agent, pigment-free, correctant, antiseptic, solubilizer, matrix, etc. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if certain drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.
The invention also discloses application of the 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy in preparing medicaments for treating and/or preventing proliferative diseases. The active compounds of the present invention or their pharmaceutically acceptable salts and solvates thereof may be used alone as the sole anti-proliferative agent or in combination with anti-proliferative agents now on the market for the treatment and/or prevention of proliferative diseases such as psoriasis, benign prostatic hypertrophy, atherosclerosis and restenosis.
The invention also discloses application of the 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy in preparing a medicament for treating and/or preventing cancer. The compound of the present invention has an activity of inhibiting the growth of tumor cells in vitro, and therefore, it can be used for the preparation of a medicament for the treatment and/or prevention of cancers, such as cancers of breast, lung, liver, kidney, colon, rectum, stomach, prostate, bladder, uterus, pancreas, bone marrow, testis, ovary, lymph, soft tissues, head and neck, thyroid, esophagus, leukemia, neuroblastoma, and the like.
The invention also discloses application of the 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy in preparing medicines for treating and/or preventing prostatic cancer, lung cancer, cervical cancer and breast cancer.
Through in vitro inhibition lung cancer cells A549, A431 and H1975 (T790M/L858R double mutation), human breast cancer cells MCF-7, cervical cancer cells Hela and human normal liver cells LO2 activity tests, the compound has a remarkable inhibition effect on lung cancer cells, breast cancer cells and cervical cancer cells, has very low inhibition activity on human normal liver cells, and indicates low toxic and side effects of the compound.
The EGFR kinase activity test shows that the compound has obvious EGFR kinase activity inhibition effect, has strong inhibition effect on EGFR high-expression lung cancer cells, and is particularly used for preparing medicaments for treating and/or preventing lung cancer.
The active compound or the medicinal salt and the solvate thereof can be used alone as a unique antitumor medicament or can be used together with the antitumor medicaments (such as platinum medicament cisplatin, camptothecin medicament irinotecan, vinca base medicament novinova, deoxycytidine medicament gemcitabine, etoposide, taxol and the like) on the market at present. Combination therapy is achieved by administering the individual therapeutic components simultaneously, sequentially or separately.
The results of screening the anti-tumor activity of various EGFR inhibitor high-expression cell strains in vitro show that the EGFR inhibitor high-expression cell strains have stronger anti-tumor activity and selectivity, activity tests on human normal cells LO2 show that most compounds have extremely low activity on the cells, and experiments show that some compounds have high-efficiency anti-tumor activity and lower side effects.
Detailed Description
In order to better explain the invention, the following embodiments are further detailed, but they do not limit the invention.
The examples are intended to illustrate, but not to limit, the scope of the invention. NMR spectra of the derivatives were taken from Bruker ARX-400 and mass spectra were taken from Agilent 1100LC/TOF MSD; all reagents used were analytically or chemically pure.
4-amidophenoxy-containing 2-aminopyrimidine heterocycles of the general formulae I and II:
the structural formulas of examples 1 to 28 of the present invention are shown in table 1 below.
Table 1 structural formulae of examples 1 to 28:
example 15N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
Step A preparation method of methyl 4-oxytetrahydro-2H-thiopyran-3-carboxylate (III 1) dimethyl 3,3' -thiodipropionate (II) (1130.0 g,630.3 mmol) and NaH (60%, 22.7g,945.5 mmol) were charged into a flask containing 500mL of tetrahydrofuran. The mixture was stirred at room temperature for 4h. After completion of the reaction, the mixture was added to 1000mL of water, and extracted with methylene chloride several times. The organic phase was dried over anhydrous sodium sulfate and methylene chloride was recovered under reduced pressure to give III 1 as a yellow liquid (104.3 g) in 95.0% yield.
Step B preparation of 7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidine-2, 4-diol (IV 1)
Compound III 1 (50.0g, 287.0 mmol) and urea (103.5g, 1723.3mmol) were dissolved in 500mL of a sodium methoxide solution. After stirring the reaction at 80 ℃ for about 5h, the reaction was complete. 500mL of water is slowly added into the reaction solution, and in the mixing process, visible fine particles are separated out, and after decompression, suction filtration and drying, the compound IV 1 is obtained, wherein the yield is 76.2%, and the light yellow solid is 40.29 g.
Process for the preparation of step C2, 4-dichloro-7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidine (V1)
Compound IV 1 (20.0 g,108.5 mmol) is dissolved in 80mL of phosphorus oxychloride solution and stirred for 3h at 120 ℃, after the reaction is finished, the reaction mixture is cooled to room temperature, then 500mL of ice water is slowly added and stirred vigorously, and a solid precipitates. After suction filtration under reduced pressure, the filter cake was washed with distilled water. After drying the filter cake 19.8g of a light grey solid are obtained in 82.6% yield.
Step D preparation method of (22-chloro-4- (3-nitrophenoxy) -7, 8-dihydro-5H-thiopyran [4,3-D ] pyrimidine (VI 1)
Compound V1 (19.0g, 85.9mmol), m-nitrophenol (12.0g, 86.3 mmol) and cesium carbonate (31.0g, 95.1mmol) were put in a flask containing 120mL of 4-dioxane, and stirred at room temperature for 6 hours, so that a large amount of solid was precipitated during the reaction. After the reaction was completed, the reaction mixture was poured into 250mL of water, stirred for 30min, filtered and dried to obtain 27.0g of a white solid with a yield of 97.1%.
Process for the preparation of step E N- (3-methoxyphenyl) -4- (3-nitrophenoxy) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-2-amine (VII 1 a)
Compound VI 1 (5 g,15.4 mmol) was dissolved in 60mL of acetonitrile, and then p-toluenesulfonic acid (5.6 g,32.5 mmol) and amino side chain a (16.5 mmol) were added thereto in this order. After stirring at 100 ℃ for 3h, the reaction was complete. After cooling to room temperature, the mixture was concentrated under reduced pressure, and acetonitrile was concentrated to about 50%. After the addition of twice the amount of water, a large amount of solid precipitated. The solid was suction filtered under reduced pressure and dried to obtain the key intermediate vii 1a.
Preparation method of step F4- (3-aminophenoxy) -N- (3-methoxyphenyl) -7, 8-dihydro-5H-thiopyranolo [4,3-d ] pyrimidin-2-amine (VIII 1 a)
Compound VII 1a (13.0 mmol) was dissolved in 60mL of ethanol. Ferric trichloride hexahydrate (15.6 mmol) and activated carbon (91.0 mmol) were added successively. After heating to 80 ℃, hydrazine hydrate (130.0 mmol) was mixed with 10mL ethanol and added to the above solution. The mixture was stirred under reflux for 4h, and after the reaction was complete, the solid was removed from the reaction system by filtration. The filter cake was then washed with 10mL of absolute ethanol, the filtrate was collected, and the solvent was recovered by vacuum distillation. The residue was added to 60mL of saturated aqueous sodium bicarbonate and the solid was precipitated by vigorous stirring. The solid was suction filtered under reduced pressure and dried to obtain the key intermediate viii 1a.
Step G preparation method of N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide (IX 1 a)
Compound VIII 1a (1.5 mmol) was dissolved in 30mL of dichloromethane and DIPEA (3.0 mmol) was added. The solution was stirred in an ice bath for 5 minutes, and then an amide (3.0 mmol) diluted with an equal amount of dichloromethane was slowly dropped into the above solution. The reaction was completed within 2h, and after completion of the reaction, the reaction mixture was filtered, and the solvent was recovered by distillation under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane/methanol = 70.
Example 1N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
m.p.:225.9–227.3℃；TOF MS ES+(m/z):(M+H)+:435.14；1H NMR(400MHz,DMSO-d6)δ10.43(s,1H),9.40(s,1H),7.67 (s,1H),7.54(d,J＝6.8Hz,1H),7.39(t,J＝7.5Hz,1H),7.19(s, 1H),7.04(d,J＝6.0Hz,1H),6.93(s,2H),6.51–6.41(m,1H), 6.38(d,J＝6.8Hz,1H),6.25(d,J＝16.9Hz,1H),5.76(d,J＝9.7 Hz,1H),3.77(s,2H),3.54(s,3H),2.97(s,4H).
The synthesis was carried out according to example 1, starting from intermediate VIII 1a with different substituents R2The reaction yielded the compounds of examples 2 to 7.
Example 2 (E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) but-2-enamide
m.p.:198.2–202.7℃；TOF MS ES+(m/z):(M+H)+:449.16；1H NMR(400MHz,DMSO-d6)δ10.53(s,1H),9.37(s,1H),7.70 (s,1H),7.57(d,J＝6.5Hz,1H),7.40–7.31(m,1H),7.19(s, 1H),7.03(s,1H),6.96–6.85(m,2H),6.76(s,1H),6.38(d, J＝5.0Hz,1H),6.25(d,J＝14.4Hz,1H),3.76(s,2H),3.53(s, 3H),2.96(s,4H),1.85(d,J＝5.8Hz,3H).
Example 3N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide
m.p.:199.7–201.6℃；TOF MS ES+(m/z):(M+H)+:463.18；1H NMR(400MHz,DMSO-d6)δ9.35(s,1H),7.27(s,1H),7.13 (d,J＝8.0Hz,1H),7.05(t,J＝8.0Hz,1H),6.96(t,J＝8.1Hz,1H), 6.45(d,J＝8.1Hz,1H),6.40(d,J＝8.0Hz,1H),6.35(s,1H), 6.30(s,1H),5.25(s,2H),3.72(s,2H),3.55(s,3H),2.95(s, 4H),2.18(s,3H),1,84(s,3H).
Example 4 (E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) pent-2-enamide
m.p.:200.7–202.4℃；TOF MS ES+(m/z):(M+H)+:463.18；1H NMR(400MHz,DMSO-d6)δ10.31(s,1H),9.37(s,1H),7.63 (s,1H),7.48(d,J＝8.6Hz,1H),7.35(t,J＝8.1Hz,1H),7.18(d, J＝9.2Hz,1H),7.02(d,J＝7.3Hz,1H),6.89(t,J＝8.1Hz,2H), 6.36(d,J＝7.2Hz,1H),6.10(d,J＝15.3Hz,1H),5.95(d,J＝15.5 Hz,1H),3.75(s,2H),3.51(s,3H),2.95(s,4H),2.06–1.89(m, 2H),1.22(d,J＝10.0Hz,3H).
Example 5 (E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide
m.p.:230.5–234.5℃；TOF MS ES+(m/z):(M+H)+:477.19；1H NMR(400MHz,DMSO-d6)δ10.41(s,1H),9.34(s,1H),7.27 (s,1H),7.12(d,J＝8.1Hz,1H),7.04(t,J＝7.9Hz,1H),6.96(t, J＝8.1Hz,1H),6.45(d,J＝7.8Hz,1H),6.42–6.34(m,2H),6.30 (d,J＝7.6Hz,1H),5.27(s,2H),3.72(s,2H),3.55(s,3H), 2.95(s,4H),1.98(s,1H),1.23(s,6H).
Example 6 (E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) hex-2-enamide
m.p.:226.7–229.4℃；TOF MS ES+(m/z):(M+H)+:477.19；1H NMR(400MHz,DMSO-d6)δ10.18(s,1H),9.20(s,1H),7.64 (s,1H),7.51(s,1H),7.39(t,J＝8.0Hz,1H),7.31(d,J＝8.4 Hz,2H),6.89(d,J＝7.7Hz,1H),6.78(dd,J＝14.8,7.4Hz,1H), 6.59(d,J＝7.6Hz,2H),6.11(d,J＝15.2Hz,1H),3.76(s,2H), 3.63(s,3H),2.99–2.89(m,4H),2.17(q,J＝6.7Hz,2H),1.44 (dt,J＝14.4,7.3Hz,2H),0.94–0.89(m,3H).
Example 7-fluoro-N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
m.p.:215.2–217.4℃；TOF MS ES+(m/z):(M+H)+:453.14；1H NMR(400MHz,DMSO-d6)δ10.18(s,1H),9.36(s,1H),7.27 (s,1H),7.12(d,J＝8.0Hz,1H),7.05(t,J＝7.9Hz,1H),6.96(t, J＝8.1Hz,1H),6.45(d,J＝7.9Hz,1H),6.40(d,J＝7.9Hz,1H), 6.35(s,1H),6.31(d,J＝7.9Hz,1H),5.26(s,2H),3.72(s,2H), 3.55(s,3H),2.95(s,4H).
VI 1 is synthesized according to the method of example 1, reacted with an amino side chain b to produce VII 1b, reduced to give VIII 1b, and the intermediate VIII 1b is used with different substituents R2The reaction yielded the compounds of examples 8 to 13.
Example 8N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
m.p.:189.2–190.7℃；TOF MS ES+(m/z):(M+H)+:436.14；1H NMR(400MHz,DMSO-d6)δ10.24(s,1H),9.32(s,1H),8.21 (s,1H),7.78(d,J＝8.7Hz,1H),7.65(s,1H),7.48(d,J＝9.1 Hz,1H),7.37(t,J＝8.1Hz,1H),6.89(dd,J＝8.0,2.2Hz,1H),6.78 (dt,J＝14.3,7.0Hz,1H),6.48(d,J＝8.2Hz,1H),6.12(d,J＝15.3 Hz,1H),5.53(d,J＝14.3Hz,1H),3.76(s,2H),3.74(s,3H), 2.95(s,4H)
Example 9 (E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) but-2-enamide
m.p.:187.6–190.1℃；TOF MS ES+(m/z):(M+H)+:450.16；1H NMR(400MHz,DMSO-d6)δ10.14(s,1H),9.27(s,1H),8.17 (s,1H),7.75(d,J＝8.6Hz,1H),7.45(d,J＝7.4Hz,1H),7.38(d, J＝9.1Hz,1H),6.89(d,J＝7.3Hz,1H),6.83–6.73(m,2H),6.45 (s,1H),6.11(d,J＝14.8Hz,1H),3.74(s,2H),3.72(s,3H), 2.95–2.91(m,4H),1.85(d,J＝6.7Hz,3H).
Example 10N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide
m.p.:221.1–224.1℃；TOF MS ES+(m/z):(M+H)+:450.16；1H NMR(400MHz,DMSO-d6)δ10.08(s,1H),9.83(s,1H),8.28 (d,J＝5.4Hz,1H),8.05(s,1H),7.92(d,J＝8.4Hz,1H),7.39(s, 1H),7.37–7.32(m,2H),7.29(d,J＝6.5Hz,1H),6.53(d,J＝ 8.1Hz,1H),3.75–3.70(m,5H),2.96(d,J＝21.6Hz,4H),2.10 (s,3H),1.84(s,3H).
Example 11 (E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) pent-2-enamide
m.p.:214.3–216.4℃；TOF MS ES+(m/z):(M+H)+:464.17；1H NMR(400MHz,DMSO-d6)δ10.14(s,1H),9.31(s,1H),8.21 (s,1H),7.79(s,1H),7.65(s,1H),7.47(d,J＝7.6Hz,1H), 7.39(d,J＝7.6Hz,1H),6.93–6.81(m,2H),6.49(s,1H),6.09 (d,J＝15.4Hz,1H),3.76(s,3H),3.74(s,2H),2.95(s,4H), 2.27–2.16(m,2H),1.03(t,J＝7.1Hz,3H).
Example 12 (E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4, 3-d)]Pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide m.p. 226.8-229.2 ℃; TOF MS ES + (M/z): M + H) +:478.19;1H NMR(400MHz,DMSO-d6)δ10.15(s,1H),9.30(s,1H),8.19 (s,1H),7.76(d,J＝8.4Hz,1H),7.63(s,1H),7.45(d,J＝8.0 Hz,1H),7.37(d,J＝8.2Hz,1H),6.88(d,J＝7.8Hz,1H),6.81– 6.73(m,1H),6.46(d,J＝6.5Hz,1H),6.04(d,J＝15.4Hz,1H), 3.74(s,2H),3.72(s,3H),2.94(d,J＝10.9Hz,4H),2.44(dd, J＝13.0,6.5Hz,1H),1.02(d,J＝6.7Hz,6H).
example 13 (E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) hex-2-enamide
m.p.:230.1–231.7℃；TOF MS ES+(m/z):(M+H) +:478.19；1H NMR(400MHz,DMSO-d6)δ10.24(s,1H),9.33(s, 1H),8.21(s,1H),7.77(d,J＝6.9Hz,1H),7.65(s,1H),7.48 (d,J＝8.2Hz,1H),7.37(t,J＝7.5Hz,1H),6.89(d,J＝7.9Hz, 1H),6.78(dt,J＝14.6,6.9Hz,1H),6.48(d,J＝8.3Hz,1H),6.12 (d,J＝15.3Hz,1H),3.75(s,2H),3.73(s,3H),2.95(s,4H), 2.24–2.12(m,2H),1.49–1.42(m,2H),0.90(t,J＝7.0Hz,3H).
Example 14N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
Step A preparation method of 2, 4-dichloro-6- (thiophene-2-yl) pyrimidine (V2)
A mixed liquid of 1, 2-dimethoxyethane and water 5 was used as a solvent, 2,4, 6-trichloropyrimidine (70.0 g,381.6 mmol), bis (triphenylphosphine) palladium dichloride and thiophen-2-ylboronic acid (25.2 g,196.8 mmol) were charged into a 500mL flask, and the reaction was completed after the compound was left to react at 90 ℃ for about 1.5 hours with stirring by using a one-pot method, and the system was cloudy in black color. The solvent in the system was evaporated to dryness, and the residue was purified by silica gel column chromatography using ethyl acetate/diethyl ether = 70.
Step B, preparation method of 2-chloro-4- (3-nitrophenoxy) -6- (thiophene-2-yl) pyrimidine (VI 2)
Compound V2 (19.0g, 82.3mmol) is dissolved in 120mL of 1, 4-dioxane, m-nitrophenol (12.0g, 86.3mmol) and cesium carbonate (32.0 g, 99.5 mmol) are sequentially added into a solvent, and stirring is carried out at room temperature for 4 hours, so that a large amount of solid is separated out in the reaction process. After the reaction was completed, the reaction solution was poured into 250mL of water and stirred for 10 minutes, and the solid was filtered off by suction under reduced pressure and dried to obtain 24.0g of a yellow solid, with a yield of 87.4%.
Step D preparation method of 3- ((4- (3-nitrophenoxy) -6- (thiophene-2-yl) pyrimidine-2-yl) amino) benzonitrile (VII 2 c)
Compound VI 2 (5.3g, 16.0 mmol) was dissolved in 60mL of acetonitrile, and p-toluenesulfonic acid (5.6 g,32.5 mmol) and amino side chain c (16.0 mmol) were added to the above solution in this order. Stirring at 100 ℃, completing the reaction for 4-5h, cooling to room temperature, and concentrating the reaction system by using a rotary evaporator. After the acetonitrile was concentrated to about 50%, twice the amount of water was added to precipitate a large amount of solid. And (4) filtering out a solid under reduced pressure, and drying to obtain a key intermediate VII 2c.
Step E preparation method of 3- ((4- (3-aminophenoxy) -6- (thiophene-2-yl) pyrimidine-2-yl) amino) benzonitrile (VIII 2 c)
Compound VII 2c (13.0 mmol) was dissolved in 60mL of ethanol. Ferric chloride hexahydrate (15.6 mmol) and activated carbon (91.0 mmol) were added sequentially. After heating to 80 ℃, hydrazine hydrate (130.0 mmol) was mixed with 10mL ethanol and added to the above solution. The mixture was stirred under reflux for 4h, and after the reaction was complete, the solid in the reaction system was removed by filtration. The filter cake was then washed with 10mL of absolute ethanol and the filtrate was collected. Most of the solvent was removed by vacuum distillation. 60mL of saturated aqueous sodium bicarbonate solution was added and the solid was precipitated by vigorous stirring. Compound viii 2c was obtained by filtration and drying of the filter cake.
Step F preparation method of N- (3- ((2- ((3-cyanophenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide (IX 2 c)
Compound VIII 2c (1.5 mmol) was dissolved in 30mL of dichloromethane and 3.0mmol of N, N-Diisopropylethylamine (DIPEA) was added. The solution was stirred in an ice bath for 5min, and then an amide (3.0 mmol) diluted with an equal amount of dichloromethane was slowly dropped into the above solution. The reaction is completed within 2h, after the reaction is completed, the solid in the reaction system is removed by vacuum filtration, and the filtrate is recovered by vacuum distillation. The residue was purified by silica gel column chromatography using dichloromethane/methanol = 70.
Example 14N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
m.p.:207.2–208.4℃；TOF MS ES+(m/z):(M+H)+:440.11；1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),9.95(s,1H),8.08–8.05(m, 1H),7.84(d,J＝5.2Hz,2H),7.71(d,J＝6.3Hz,1H),7.52(d,J＝8.3 Hz,1H),7.44(t,J＝8.1Hz,2H),7.33(s,2H),7.28–7.24(m,1H),7.16 (s,1H),7.03–6.99(m,1H),6.43(dd,J＝16.9,10.1Hz,1H),6.25(dd,J ＝16.9,2.0Hz,1H),5.79–5.74(m,1H).
Following the procedure of example 14, intermediates VIII 2c were substituted with various substituents R2The reaction yielded the compounds of examples 15 to 18.
Example 15 (E) -N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) but-2-enamide
m.p.:209.9–211.1℃；TOF MS ES+(m/z):(M+H)+:454.13；1H NMR(400MHz,DMSO-d6)δ10.13(s,1H),9.95(s,1H),8.07(d,J＝ 5.8Hz,2H),7.84(d,J＝5.1Hz,2H),7.69(d,J＝10.9Hz,1H),7.49(d,J ＝8.2Hz,1H),7.43(d,J＝7.9Hz,1H),7.34(s,2H),7.27–7.24(m,1H), 7.16(s,1H),6.98(d,J＝8.1Hz,1H),6.79(dd,J＝15.3,7.6Hz,1H), 6.11(d,J＝15.3Hz,1H),1.85(d,J＝7.0Hz,3H).
Example 16N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide
m.p.:197.2–199.2℃；TOF MS ES+(m/z):(M+H)+:468.15；1H NMR(400MHz,DMSO-d6)δ10.01(s,1H),9.95(s,1H),8.06(d,J ＝3.8Hz,1H),7.84(d,J＝4.9Hz,2H),7.67(s,1H),7.42(dt,J＝16.2, 8.2Hz,3H),7.34(s,2H),7.27–7.24(m,1H),7.15(s,1H),6.95(d,J＝ 8.1Hz,1H),5.86(s,1H),2.12(s,3H),1.85(s,3H).
Example 17 (E) -N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide
m.p.:210.5.2–211.9℃；TOF MS ES+(m/z):(M+H)+:482.16；1H NMR(400MHz,DMSO-d6)δ10.19(s,1H),9.94(s,1H),8.06(d,J＝ 3.8Hz,1H),7.84(d,J＝5.2Hz,2H),7.69(d,J＝4.5Hz,1H),7.49(d,J ＝8.3Hz,1H),7.42(t,J＝8.1Hz,1H),7.33(s,2H),7.25(t,J＝4.4Hz, 1H),7.15(s,1H),6.98(d,J＝8.0Hz,1H),6.81(d,J＝6.3Hz,1H),6.79 –6.74(m,1H),6.06(d,J＝15.4Hz,1H),2.43(dd,J＝13.1,6.6Hz,1H), 1.03(d,J＝6.5Hz,6H).
Example 18 (E) -N- (3- ((2- ((3-cyanophenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) hexan-2-amide
m.p.:222.3–223.5℃；TOF MS ES+(m/z):(M+H)+:482.16；1H NMR(400MHz,DMSO-d6)δ10.15(s,1H),9.95(s,1H),8.07(d,J＝ 3.7Hz,1H),7.85(d,J＝5.0Hz,2H),7.68(s,1H),7.49(d,J＝8.4Hz, 1H),7.42(t,J＝8.0Hz,2H),7.33(s,2H),7.26(dd,J＝5.0,3.8Hz,1H), 7.16(s,1H),6.98(d,J＝7.9Hz,1H),6.82–6.76(m,1H),6.10(d,J＝ 15.4Hz,1H),2.18(d,J＝7.0Hz,2H),1.45(d,J＝7.3Hz,2H),0.90(d,J ＝3.5Hz,3H).
Example 19N- (3- ((2- ((3-methoxyphenyl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
TOF MS ES+(m/z):(M+H)+:445.13.
Example 20-fluoro-N- (3- ((2- ((3-methoxyphenyl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
TOF MS ES+(m/z):(M+H)+:463.12.
Example 21 (E) -4- (dimethylamino) -N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -but-2-enamide
TOF MS ES+(m/z):(M+H)+:476.18.
Example 22 (E) -N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -4- (piperidin-1-yl) -but-2-enamide TOF MS ES + (M/z) (M + H)+:516.21.
Example 23N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6, 7-dihydrothieno [3,2-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide
TOF MS ES+(m/z):(M+H)+:395.12.
Example 24 (E) -N- (3- ((2- ((1-methyl-1H-pyrazol-3-yl) amino) -6, 7-dihydrothieno [3,2-d ] pyrimidin-4-yl) oxy) phenyl) -pent-2-enamide
TOF MS ES+(m/z):(M+H)+:423.16.
Example 25N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thien-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
TOF MS ES+(m/z):(M+H)+:448.09.
Example 26-fluoro-N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) acrylamide
TOF MS ES+(m/z):(M+H)+:466.08.
Example 27 (E) -4- (dimethylamino) -N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -but-2-enamide TOF MS ES + (M/z) (M + H)+:505.14.
Example 28 (E) -N- (3- ((2- ((2-mercaptopyridin-4-yl) amino) -6- (thiophen-2-yl) pyrimidin-4-yl) oxy) phenyl) -4- (piperidin-1-yl) -but-2-enamide
TOF MS ES+(m/z):(M+H)+:545.17.
Pharmacological study of the products of the invention
In vitro cytotoxic Activity
The quinoline derivatives containing pyrimidine and pyridine of the general formulas I and II are subjected to in vitro activity screening for inhibiting lung cancer cells A549, breast cancer cells MCF-7, cervical cancer cells Hela and human normal cells LO2, and the reference substance is the temotinib.
(1) After cells were thawed and passaged for 2-3 stabilities, they were digested from the bottom of the flask with trypsin solution (0.25%). After pouring the cell digest into the centrifuge tube, the culture medium is added to stop the digestion. Centrifuging the centrifuge tube at 800r/min for 10min, discarding supernatant, adding 5mL culture solution, blowing and beating the mixed cells, sucking 10 μ L cell suspension, adding into cell counting plate, counting, and adjusting cell concentration to 104One for each well. 100. Mu.L of the cell suspension was added to the 96-well plate except that the A1 well was blank. The 96-well plate was placed in an incubator for 24h.
(2) The test sample was dissolved in 50. Mu.L of dimethyl sulfoxide, and then an appropriate amount of culture solution was added to dissolve the sample into a 2mg/mL liquid medicine, and then the sample was diluted to 20,4,0.8,0.16, 0.032. Mu.g/mL in a 24-well plate.
3 wells were added for each concentration, two columns of cells surrounding each, which were greatly affected by the environment, and only used as blank wells. The 96-well plate was placed in an incubator for 72h.
(3) The drug-containing culture solution in the 96-well plate is discarded, the cells are washed twice by using Phosphate Buffer Solution (PBS), 100 mu L of MTT (tetrazole) (0.5 mg/mL) is added into each well and put into an incubator for 4h, the MTT solution is discarded, and 100 mu L of dimethyl sulfoxide is added. Vibrating on a magnetic oscillator to fully dissolve the viable cells and the MTT reaction product formazan, and putting the formazan into an enzyme labeling instrument to measure the result. Determination of drug IC by Bliss method50The value is obtained.
The results of the compound on inhibiting the activity of lung cancer cells A549, breast cancer cells MCF-7, cervical cancer cells Hela and human normal cells LO2 (see table II) are shown.
EGFR kinase Activity assay
Testing IC of newly synthesized compound for inhibiting various cancer cells by using the HTRF technology with the imatinib as the positive control50Values, and test for inhibition of EGFR by some compounds.
The specific method comprises the following steps: preparing a working solution of ATP, TK Substrate-biotin (TK-Substrate biotin) and Kinase buffer with required concentrations, and uniformly mixing the ATP, TK Substrate-biotin and Kinase buffer according to a volume ratio of 2; diluting the medicine with a Kinase buffer to prepare the required concentration; and preparing an EGFR enzyme working solution. In a white 384-well plate, 6. Mu.L of the homogenate, 2. Mu.L of the drug and 2. Mu.L of the kinase were added to each well, mixed, and reacted at 37 ℃ for 30min. Then 5. Mu.L of streptokinase labeled XL-665 and 5. Mu.L of Eu3+ bound cryptate antibody are added and mixed. Standing at room temperature for 30min, exciting at 314nm with enzyme labeling instrument, detecting fluorescence at 665 and 620nm wavelength, calculating kinase inhibition rate, and calculating IC of each medicine by Bliss method according to absorbance50The value is obtained.
Inhibition rate (%) = (Ratio 665/620 control wells-Ratio 665/620 dosing wells)/Ratio 665/620 control wells × 100%.
The experimental data are expressed by mean values +/-standard deviation, the inter-group significance test of the measured data adopts one-factor variance analysis, two-two comparison adopts t test, the significance difference is when p is less than 0.05, and the significance difference is when p is less than 0.01.
TABLE 2 in vitro antitumor Activity of some of the target Compounds
Table 3 partial target Compound enzymatic Activity
From the test results, it is clear that the compounds of the general formulas I and II to be protected by the invention have good in vitro anti-cell proliferation activity and anti-EGFR kinase activity, the toxicity of all target compounds on human normal cells LO2 is lower than that of the temotinib, and the inhibition activity of part of the compounds on L858R/T790M double mutant cell lung cancer cells H1975 is better than that of the temotinib. As can be seen from the data in the table above, the exemplified compounds have a certain selectivity for EGFR double mutant kinase over anti-EGFR wild type kinase activity, which also demonstrates that the exemplified compounds target well and are highly selective, as in examples 1 and 6. It can be concluded that the compounds of formulae I and II of the present invention are potential EGFR inhibitors.
While the invention has been described with reference to specific embodiments, modifications and equivalent arrangements will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
Application example 1: capsule preparation
10g of the compound in example 9 is mixed with 20g of auxiliary materials according to the requirement of a pharmaceutical capsule, and then the mixture is filled into hollow capsules, wherein each capsule weighs 300mg.
Application example 2: tablet formulation
10g of the compound obtained in example 3 was mixed with 20g of an adjuvant by a usual tableting method in pharmacy, and the mixture was compressed into 100 tablets each weighing 300mg.
Application example 3 ointment
Prepared by grinding 10g of the compound of example 4, and then uniformly grinding the ground compound with 500g of oil-based substances such as vaseline and the like.
While the invention has been described with reference to specific embodiments, modifications and equivalent arrangements will be apparent to those skilled in the art and are intended to be included within the scope of the invention.
Application example 4: aerosol formulation
Dissolving 10g of the compound in example 9 in a proper amount of propylene glycol, adding distilled water and other additives, and preparing into 500mL of clear solution.
Application example 5 drop pill
10g of the compound obtained in example 12 and 50g of a base such as gelatin are heated, melted and mixed uniformly, and then the mixture is dropped into low-temperature liquid paraffin to obtain 1000 pills.
Application example 6 Liniment for external use
10g of the compound of example 16 was mixed with 2.5g of an emulsifier and other adjuvants according to a conventional pharmaceutical method, and the mixture was ground, and then distilled water was added thereto to make 200 mL.
Application example 7 film agent
10g of the compound obtained in example 19 was swelled with polyvinyl alcohol, medicinal glycerin, water, etc. under stirring, dissolved by heating, filtered through a 80-mesh screen, and dissolved by adding the compound obtained in example 14 to the filtrate under stirring, and 100 films were formed on a film-coating machine.
Application example 8 suppository
10g of the compound of example 24 was ground and added with an appropriate amount of glycerin, and after grinding to uniformity, the mixture was added with molten glycerin gelatin, ground to uniformity, and poured into a mold coated with a lubricant to prepare 10 suppositories.
Claims (3)
1. A2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy is characterized in that the structure is shown as the following general formula I:
wherein the fused ring on which the X ring is located is selected from:
R1selected from benzene ring or pyridine, the benzene ring or pyridine contains 1 methoxy substituent;
R2selected from hydrogen or F;
R3selected from methyl, ethyl, isopropyl, hydrogen or propyl.
2. A 4-amidophenoxy-containing 2-aminopyrimidine heterocyclic compound according to claim 1 characterized by: the compound of the general formula I is one selected from the following compounds:
n- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide;
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) but-2-enamide;
n- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide;
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) pent-2-enamide;
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide;
(E) -N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) hex-2-enamide;
2-fluoro-N- (3- ((2- ((3-methoxyphenyl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide;
n- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide;
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) but-2-enamide;
n- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -3-methyl-2-enamide;
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) pent-2-enamide;
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) -4-methylpent-2-enamide;
(E) -N- (3- ((2- ((6-methoxypyridin-3-yl) amino) -7, 8-dihydro-5H-thiopyran [4,3-d ] pyrimidin-4-yl) oxy) phenyl) hex-2-enamide.
3. Use of a 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy group according to any of claims 1-2 for the preparation of a medicament for the treatment and/or prevention of breast cancer, lung cancer or cervical cancer.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211474427.6A CN116239603A (en) | 2020-06-20 | 2020-06-20 | 2-aminopyrimidine heterocyclic compound and application thereof |
| CN202010568748.7A CN111732597B (en) | 2020-06-20 | 2020-06-20 | Preparation and application of 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010568748.7A CN111732597B (en) | 2020-06-20 | 2020-06-20 | Preparation and application of 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211474427.6A Division CN116239603A (en) | 2020-06-20 | 2020-06-20 | 2-aminopyrimidine heterocyclic compound and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111732597A CN111732597A (en) | 2020-10-02 |
| CN111732597B true CN111732597B (en) | 2022-11-01 |
Family
ID=72651879
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211474427.6A Pending CN116239603A (en) | 2020-06-20 | 2020-06-20 | 2-aminopyrimidine heterocyclic compound and application thereof |
| CN202010568748.7A Active CN111732597B (en) | 2020-06-20 | 2020-06-20 | Preparation and application of 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211474427.6A Pending CN116239603A (en) | 2020-06-20 | 2020-06-20 | 2-aminopyrimidine heterocyclic compound and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN116239603A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113880859B (en) * | 2021-11-16 | 2022-06-21 | 江西科技师范大学 | 2-aryl-4-arylmethylamino pyrimidine compound and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102947316A (en) * | 2010-06-23 | 2013-02-27 | 韩美科学株式会社 | Novel fused pyrimidine derivatives for inhibition of tyrosine kinase activity |
| WO2018139883A1 (en) * | 2017-01-26 | 2018-08-02 | 부광약품 주식회사 | Fused pyrimidine derivative as multi-target kinase inhibitor |
| CN109280048A (en) * | 2018-11-16 | 2019-01-29 | 江西科技师范大学 | A kind of pyrimidines of the structure of acrylamide containing substituted-phenyl and its application |
| CN110078732A (en) * | 2018-01-26 | 2019-08-02 | 沈阳药科大学 | Purine compound and application thereof |
| WO2020060268A1 (en) * | 2018-09-20 | 2020-03-26 | 한미약품 주식회사 | Novel sulfonamide derivative with fused pyrimidine skeleton, having epidermal growth factor receptor mutation inhibitory effect |
-
2020
- 2020-06-20 CN CN202211474427.6A patent/CN116239603A/en active Pending
- 2020-06-20 CN CN202010568748.7A patent/CN111732597B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102947316A (en) * | 2010-06-23 | 2013-02-27 | 韩美科学株式会社 | Novel fused pyrimidine derivatives for inhibition of tyrosine kinase activity |
| WO2018139883A1 (en) * | 2017-01-26 | 2018-08-02 | 부광약품 주식회사 | Fused pyrimidine derivative as multi-target kinase inhibitor |
| CN110078732A (en) * | 2018-01-26 | 2019-08-02 | 沈阳药科大学 | Purine compound and application thereof |
| WO2020060268A1 (en) * | 2018-09-20 | 2020-03-26 | 한미약품 주식회사 | Novel sulfonamide derivative with fused pyrimidine skeleton, having epidermal growth factor receptor mutation inhibitory effect |
| CN109280048A (en) * | 2018-11-16 | 2019-01-29 | 江西科技师范大学 | A kind of pyrimidines of the structure of acrylamide containing substituted-phenyl and its application |
Non-Patent Citations (5)
| Title |
|---|
| Xiaohan Hu等.Design, Synthesis, and Antitumor Activity of Olmutinib Derivatives Containing Acrylamide Moiety.《Molecules》.2021,第26卷第3041页. * |
| Zhen Xiao等.Design, synthesis and antitumor activity of novel thiophene-pyrimidine derivatives as EGFR inhibitors overcoming T790M and L858R/T790M mutations.《European Journal of Medicinal Chemistry》.2020,第203卷第112511页. * |
| Zhen Xiao等.Discovery of thiapyran-pyrimidine derivatives as potential EGFR inhibitors.《Bioorganic & Medicinal Chemistry》.2020,第28卷第115669页. * |
| 沈益飞.EGFR抑制剂N-[3-(2-嘧啶氨基)苯基]丙烯酰胺类化合物的设计、合成及活性研究.《中国优秀硕士学位论文全文数据库 工程科技I辑》.2016,(第06期),第E079-21页. * |
| 肖珍.奥莫替尼衍生物的设计、合成及抗肿瘤活性研究.《中国优秀硕士学位论文全文数据库 工程科技I辑》.2021,(第04期),第B016-464页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111732597A (en) | 2020-10-02 |
| CN116239603A (en) | 2023-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6047184B2 (en) | Triazines, pyrimidines, and pyridine analogs, and their use as therapeutics and diagnostic probes | |
| CN101479274B (en) | Pharmaceutical compounds | |
| US9809572B2 (en) | Deuterated diaminopyrimidine compounds and pharmaceutical compositions comprising such compounds | |
| EP3936502A1 (en) | Pyrazine derivative and application thereof in inhibiting shp2 | |
| JP5719770B2 (en) | Icotinib hydrochloride, compound, crystallographic form, concomitant drug and its use | |
| CN112300153B (en) | Heterocyclic compound, pharmaceutical composition and application | |
| CN114656482A (en) | Macrocyclic heterocyclic compound as EGFR inhibitor and application thereof | |
| CN104230952B (en) | Compound containing pyrimidine skeleton, and preparation method and use of compound | |
| CN105732616A (en) | Pyrrolopyridine compounds containing biaryl amide structure, preparation method and applications thereof | |
| CN102108078B (en) | 1,4-substituted phthalazine compound and preparation method and applications thereof | |
| CN104557913B (en) | Pyridopyrimidine compounds as well as preparation method and application thereof | |
| CN111732597B (en) | Preparation and application of 2-aminopyrimidine heterocyclic compound containing 4-amidophenoxy | |
| CN104086562B (en) | The preparation of the heterocycle miazines compound containing virtue hydrazone structure and application | |
| CN116322697A (en) | Quinazoline compound and pharmaceutical composition thereof | |
| CN110467616B (en) | Preparation and application of triazolopyrazine compound containing heteroaryl substituted pyridazinone structure | |
| CN108456214B (en) | Quinazoline compound containing oxazole or imidazole structure and application thereof | |
| CN106866642B (en) | Quinazoline compound containing aryl acylhydrazone structure and application thereof | |
| CN113880859B (en) | 2-aryl-4-arylmethylamino pyrimidine compound and application thereof | |
| CN106892907B (en) | Quinazoline compound containing acylhydrazone structure and application thereof | |
| CN111836819A (en) | Arylamine-substituted pyrrolopyrimidine compound, and preparation method and application thereof | |
| TWI535724B (en) | New polymorphic forms of icotinib phosphate and uses thereof | |
| US8563743B2 (en) | Benzofuranone compound and pharmaceutical composition containing same | |
| CN109942562B (en) | Five-membered heterocyclic diazine compound containing aryl structure and preparation method and application thereof | |
| WO2023025164A1 (en) | Crystal forms, preparation method and application of aryl phosphine oxide compound | |
| CN114031561B (en) | 4-phenoxy-containing quinazoline compound and application 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |