EGFR tyrosine kinase inhibitors and uses thereof

Abstract

The present invention provides a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and use thereof in preparing a drug for preventing or treating related diseases caused by EGFR mutation.

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine, and specifically relates to an EGFR tyrosine kinase inhibitor and a use thereof. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which NSCLC includes squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma. Compared with SCLC, the cancer cells of NSCLC grow and divide more slowly, and spread and metastasize relatively late. NSCLC accounts for about 80% of all lung cancers and has a poor prognosis. About 75% of patients are already in the middle and late stages when they are discovered. Overexpression and mutation of epidermal growth factor receptor (EGFR) have been clearly proven to lead to uncontrollable cell growth and are related to the progression of most cancer diseases, especially NSCLC (Oncotarget. 2016 Jul 5; 7 (27): 41691-41702).

[0003] EGFR is a member of the epidermal growth factor receptor (HER) family, which consists of EGFR (Erb-Bl), Erb-B2 (HER-2 / neu), Erb-B3 and Erb-B4. EGFR is a glycoprotein and a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. It is a tyrosine kinase receptor that penetrates the cell membrane and is located on the cell membrane surface (Clin Cancer Res; 21 (3) February 1, 2015, 526-533).

[0004] In NSCLC patients, EGFR is not only overexpressed, but also has abnormal activation mutations in its tyrosine kinase structural region that are independent of ligand binding. The most common kinase activity mutations are EGFR (Exon 19delE746-A750) and EGFR (Exon 21L858R). The first-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) gefitinib and erlotinib have been approved for the treatment of these two mutations, but the first-generation EGFR-TKIs develop resistance during treatment, and the development of resistance is related to the secondary mutation of threonine at position 790 of EGFR replaced by methionine (T790M). The second generation of irreversible covalent inhibitors, such as afatinib, is more effective in treating EGFR (delE746-A750) and EGFR (L858R), but is not as effective against the drug-resistant T790M mutant. The reason is that it is more active against wild-type EGFR than against EGFR (delE746-A750 / T790M) and EGFR (L858R / T790M) drug-resistant mutants, thus showing dose-dependent toxicity (Biologics, 2014, 8: 183-192), and it is impossible to obtain an effective treatment window. Therefore, the third generation of EGFR-TKIs, such as AZD9291, CO-1686 and HM61713, have been developed. The third generation of EGFR-TKIs are tyrosine kinase inhibitors with specific selectivity. Compared with the first and second generation EGFR-TKIs, the third generation EGFR-TKIs reduce the inhibition of wild-type EGFR, reduce clinical toxicity and side effects, and can use higher clinical doses to achieve better therapeutic effects (J Clin Oncol 2014; 32:abstr 8009; J Clin Oncol 2014; 32:abstr 8010).

[0005] Another abnormally activated EGFR mutation that induces cancer is the exon 20 insertion mutation (EGFRexon20ins). Previous studies have shown that this type of mutation accounts for 4%-10% of all EGFR-mutated lung cancers (PLoS ONE 201510(7):e0133859). Currently, there is still no drug available for treatment in the clinic (Mol Cancer Ther. 2013,12,220). Patients with this type of mutation are not sensitive to the currently available EGFR inhibitors. The current standard treatment is cytotoxic chemotherapy, which has poor prognosis and strong side effects. Currently, there is no targeted drug available. Poziotinib and Ref-1, candidate drugs targeting EGFR exon20 insertion mutations, have entered clinical research, but they have very strong inhibitory ability against wild-type EGFR, and therefore can cause significant side effects, such as skin toxicity. This severely limits the increase in clinical doses and clinical efficacy. In addition, there are compounds CLN-081 and DZ9008 that have just entered the clinic. Although their in vitro activity shows that they reduce the toxicity to the wild type, their clinical effects have not been further confirmed. In summary, compounds with higher activity and lower toxicity are still to be developed. Summary of the invention

[0006] The invention discloses a class of compounds that can be used as EGFR protein kinase inhibitors and uses thereof in preparing drugs for preventing or treating EGFR-related diseases.

[0007] In one aspect, the present invention provides a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, Z1 is -NH-, -N(CH3)-, -O- or -S-;

[0010] Z2, Z3, Z4, and Z5 are each independently selected from C or N;

[0011] Each R1 is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxyl, nitro, amino, cyano or alkoxy; two adjacent R1s may form a five- to six-membered saturated or unsaturated aromatic ring or aromatic heterocyclic ring together with the atoms to which they are connected;

[0012] Each R2 is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;

[0013] R3 is selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;

[0014] R4 is selected from hydrogen, C1-C6 alkyl or -NR5R6;

[0015] R5 and R6 are each independently selected from hydrogen, C1-C6 alkyl, -R7N(R8)(R9);

[0016] R7, R8, and R9 are each independently selected from hydrogen or C1-C6 alkyl;

[0017] m and n are independently selected from 1, 2 or 3;

[0018] Dashed lines are single or double bonds.

[0019] In some embodiments, Formula (I) is Formula (Ia):

[0020]

[0021] Z1 is -NH-, -N(CH3)-, -O- or -S-;

[0022] Z2, Z3, Z4, and Z5 are each independently selected from C or N;

[0023] Each R1 is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxyl, nitro, amino, cyano or alkoxy; two adjacent R1s may form a five- to six-membered saturated or unsaturated aromatic ring or aromatic heterocyclic ring together with the atoms to which they are connected;

[0024] Each R2 is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;

[0025] m and n are independently selected from 1, 2 or 3;

[0026] Dashed lines are single or double bonds.

[0027] In other embodiments, Z1 is -NH-, -N(CH3)-, or -O-;

[0028] Z2, Z3, Z4, and Z5 are each independently selected from C or N, and Z3 and Z4 are not N at the same time;

[0029] Each R1 is independently selected from hydrogen, C1-C3 alkyl or halogen; two adjacent R1s may form a benzene ring together with the atoms to which they are attached;

[0030] Each R2 is independently selected from hydrogen, C1-C3 alkyl, halogenated C1-C3 alkyl, halogen, cyano or methoxy;

[0031] m and n are independently selected from 1 or 2;

[0032] Dashed lines are single or double bonds.

[0033] In other embodiments, Selected from:

[0034]

[0035] In other embodiments, Selected from:

[0036]

[0037] The aforementioned aryl group is an all-carbon monocyclic or condensed polycyclic aromatic group having a conjugated π electron system. The aryl group may have 6 to 10 carbon atoms in one or more rings. Most commonly, the aryl group has 6 carbon atoms in the ring. For example, a C6-10 aryl group is an aromatic group containing 6 to 10 carbon atoms, such as a phenyl or naphthyl group.

[0038] The aforementioned heteroaryl, i.e., a monocyclic or condensed polycyclic aromatic heterocyclic group having one or more heteroatom ring members (ring atoms) in at least one ring, wherein the heteroatom ring members are independently selected from O, S and N. The heteroaryl group has 5 to 14 ring atoms, including 1 to 13 carbon atoms and 1 to 8 heteroatoms selected from O, S and N. In some embodiments, the heteroaryl group has 5 to 10 ring atoms, including one to four heteroatoms. The heteroaryl group may also contain one to three oxo or thiocarbonyl (i.e., =S) groups. In some embodiments, the heteroaryl group has 5 to 8 ring atoms, including one, two or three heteroatoms. For example, a 5-membered heteroaryl group is a monocyclic heteroaryl group as defined above, having 5 ring-forming atoms in the monocyclic heteroaryl ring; a 6-membered heteroaryl group is a monocyclic heteroaryl group as defined above, having 6 ring-forming atoms in the monocyclic heteroaryl ring; and a 5- to 10-membered heteroaryl group is a monocyclic or bicyclic heteroaryl group as defined above, having 5, 6, 7, 8, 9 or 10 ring-forming atoms in the monocyclic or bicyclic heteroaryl ring.

[0039] In other embodiments, the compound is selected from the following compounds:

[0040]

[0041]

[0042] The aforementioned compounds can be used to prepare drugs for preventing or treating receptor tyrosine kinase mutations, especially EGFR mutation-related diseases; the EGFR mutation-related diseases are cancers, especially cancers related to mutations in the exon 20 domain of EGFR, such as non-small cell lung cancer, breast cancer, brain cancer, ovarian cancer, pancreatic cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal tissue cancer, esophageal cancer, thyroid cancer, leukemia, lymphoma and multiple myeloma, etc.

[0043] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or isomer thereof; a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient may comprise any conventional pharmaceutical carrier or excipient. Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents such as hydrates and solvents. If necessary, the pharmaceutical composition may contain additional ingredients such as flavoring agents, binders, excipients, etc.

[0044] The aforementioned pharmaceutical composition may further comprise one or more other anticancer drugs, wherein the anticancer drugs are small molecule drugs, monoclonal antibodies, or fusion protein drugs.

[0045] The compounds with EGFR tyrosine kinase inhibitory activity provided by the present application have strong inhibitory ability to the kinase activity of EGFR (D770_N771insNPG) insertion mutation. The inhibition of the proliferation ability of wild-type EGFR high-expressing mouse proto-B cells (BaF3) is much lower than Poziotinib and Ref-1, which are currently in the clinical research stage. At the same time, it can effectively inhibit the proliferation of BaF3 cells with high expression of EGFR exon20 insertion mutation. Compared with the single mutant EGFR high-expressing tumor cell proliferation inhibitory activity, the selectivity of mutant cells relative to wild cells is a more important and critical indicator as a clinical treatment window. Therefore, compared with the current clinical compounds, the compounds provided by the present application can be used in higher doses in clinical applications without causing obvious toxicity, greatly improving the efficacy and obtaining better prognosis. And it has a good inhibitory effect on the abnormal proliferation of common EGFR mutations, EGFR (delE746-A750), and Her2 insertion mutation high-expressing tumor cells. DETAILED DESCRIPTION

[0046] Example 1 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-(4-methoxyphenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0047]

[0048] Step 1:

[0049]

[0050] Compound 1.1 (15.000 g, 54.57 mmol, 1 eq.) was dissolved in DMSO (100 mL), and compound 1.2 (6.67 g, 60.03 mmol, 1.1 eq.) and K2CO3 (15.08 g, 109.14 mmol, 2 eq.) were added, and the mixture was heated to 80°C and reacted for 15 hours. The mixture was poured into water (200 mL), allowed to stand for 30 min, filtered, and the filter cake was dried to obtain a brown solid (7.350 g, yield 38.53%).

[0051] Step 2:

[0052]

[0053] Compound 1.3 (2.500 g, 7.15 mmol, 1 eq.) was dissolved in dioxane (100 mL), and compound 1.4 (1.30 g, 8.58 mmol, 1.2 eq.), Pd(dppf)Cl2 (523.35 mg, 715.25 umol, 0.1 eq.), K3PO4 (1.52 g, 7.15 mmol, 1 eq.), and H2O (10 mL) were added. The mixture was replaced with nitrogen three times, and the mixture was protected with nitrogen. The mixture was heated to 60°C and reacted for 1.5 hours. The mixture was poured into water (150 mL), extracted with EtOAC (300 mL), and the organic phase was separated. The organic phase was washed once with a saturated aqueous solution of NaCl, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and subjected to column chromatography (n-heptane: EtOAC = 15:1) to give a white solid (2.100 g, yield 89.04%).

[0054] Step 3:

[0055]

[0056] Compound 1.5 (2.100 g, 6.37 mmol, 1 eq.) was dissolved in dioxane (100 mL), and compound 1.6 (1.19 g, 6.37 mmol, 1 eq.), Pd2(dba)3 (583.16 mg, 636.84 umol, 0.1 eq.), x-phos (607.18 mg, 1.27 mmol, 0.2 eq.), Cs2CO3 (4.15 g, 12.74 mmol, 2 eq.) were added, and nitrogen was replaced three times, and nitrogen protection was used. The mixture was heated to 100°C and reacted for 4 hours. The system was cooled, filtered, and the filtrate was directly used for the next step.

[0057] Step 4:

[0058]

[0059] The filtrate from the previous step was added with N,N,N'-trimethylethylenediamine (3.25 g, 31.81 mmol, 5 eq.), heated to 80°C, reacted for 15 hours, poured into water (120 mL), extracted with EtOAC (250 mL), separated, the organic phase was washed once with a saturated aqueous NaCl solution, separated, the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatography (DCM: MeOH = 30:1) was performed to obtain a red oil (2.940 g, yield: 82.29%).

[0060] Step 5:

[0061]

[0062] Compound 1.8 (2.940 g, 5.23 mmol, 1 eq.) was dissolved in EtOH (80 mL), and Fe (1.46 g, 26.17 mmol, 5 eq.), NH4Cl (1.29 g, 26.17 mmol, 5 eq.), and H2O (20 mL) were added, and the mixture was heated to 80°C and reacted for 5 hours. The system was filtered and washed with EtOAC (200 mL), and water (100 mL) was added to the filtrate, and the liquids were separated. The organic phase was washed once with a saturated aqueous NaCl solution, and the liquids were separated. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a brown solid (2.140 g, yield: 76.89%).

[0063] Step 6:

[0064]

[0065] Compound 1.9 (2.140 g, 4.03 mmol, 1 eq.) was dissolved in DCM (20 mL), the temperature was lowered to -20°C in a dry ice ethanol bath, triethylamine (814.66 mg, 8.05 mmol, 2 eq.) and compound 1.10 (364.33 mg, 4.03 mmol, 1 eq.) were added, and the reaction was continued for 10 min. Water (20 mL) was added to the system, and the mixture was extracted with DCM (100 mL). The mixture was separated, and the organic phase was washed once with a saturated aqueous NaCl solution. The organic phase was separated, dried over anhydrous magnesium sulfate, filtered, concentrated, and chromatographed (DCM: MeOH = 30: 1). The mixture was then slurried with EtOAC to give an off-white solid (0.800 g, 33.93%). 1H NMR(400MHz,DMSO-d6)(ppm):10.07(s,1H),8.53(s,1H),7.96-8.00(t,1H),7.90(m,2H),7.61(s,1H),7.41-7.44(d,2H),6.97-7.17(m,6H),6 .36-6.43(dd,1H),6.17-6.22(dd,1H),5.72-5.75(dd,1H),3.79-3.81( d,6H),2.84-2.87(t,2H),2.70(s,3H),2.28-2.31(t,2H),2.20(s,6H). LC-MS(m / z):587.00[M+H] + .

[0066] Example 2 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-(p-tolylamino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0067]

[0068] Using a similar method to Example 1, replace compound 1.2 in step 1 with p-toluidine, and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):10.13(s,1H),8.60(s,1H),7.96(s,1H),7.86(s, 1H),7.84(s,1H),7.47-7.50(m,2H),7.42-7.45(d,2H),7.28-7.32(t,2H),6.9 4-6.98(m,3H),6.40-6.44(m,1H),6.18-6.22(dd,1H),5.73-5.76(dd,1H),3.8 0(s,3H),2.85-2.88(t,2H),2.71(s,3H),2.23-2.31(t,2H),2.18-2.22(m,9H). LC-MS (m / z): 569.69 [M+H] + .

[0069] Example 3 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-((2-methylpyrimidin-5-yl)amino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0070]

[0071] Using a similar method to Example 1, replace compound 1.2 in step 1 with 2-methylpyrimidin-5-amine, and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):10.08(brs,1H),8.84(s,2H),8.55(brs,1H),8.37(s,1H),8.12(s,1H),7.93(s,1H),7.50-7.54(m,2H),7.29-7 .34(t,2H),6.97(s,1H),6.34(s,1H),6.14-6.18(d,1H),5.70-5.73(m ,1H),3.79(s,3H),2.91-2.93(m,2H),2.71(s,3H),2.28-2.50(m,9H). LC-MS(m / z):571.66[M+H] + .

[0072] Example 4 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-((5-methylpyrazin-2-yl)amino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0073]

[0074] Using a similar method to Example 1, replace compound 1.2 in step 1 with 5-methylpyrazine-2-amine, and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):10.17(s,1H),9.24(s,1H),8.57(s,1H),8.4 1(s,1H),8.09(s,1H),8.03(s,1H),7.88(s,1H),7.51-7.54(m,2H),7.32-7 .37(m,2H),7.02(s,1H),6.32-6.39(m,1H),6.13-6.17(dd,1H),5.70-5.7 2(dd,1H),3.80(s,3H),2.92(m,2H),2.74(m,3H),2.35(m,5H),2.24(m5H). LC-MS(m / z):571.28[M+H] + .

[0075] Example 5 N-(5-((5-(3-chloro-4-fluorophenyl)-4-(p-tolylamino)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide

[0076]

[0077] Using a similar method to Example 1, replace compound 1.2 in step 1 with p-toluidine, and replace compound 1.4 in step 2 with (3-chloro-4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):10.10(s,1H),8.57(s,1H),8.11(s,1H),7.88(s,1H),7.86(s,1H),7.63-7.66(dd,1H),7.41-7.51(m,4H),6.94- 7.0(m,3H),6.38-6.44(m,1H),6.18-6.23(dd,1H),5.73-5.74(d,1H), 3.80(s,3H),2.88(m,2H),2.71(s,3H),2.31(m,2H),2.19-2.22(m,9H). LC-MS(m / z):603.25[M+H] + .

[0078] Example 6 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-(methyl(p-tolyl)amino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0079]

[0080] Using a similar method to Example 1, replace compound 1.2 in step 1 with N,4-dimethylaniline and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1H NMR(400MHz,DMSO-d6)(ppm):10.07(s,1H),9.06(s,1H),7.86(s,1H),7.78(s,1H),7.01-7.03(m,3H),6.71-6.83(m,6H),6.35-6.41(dd ,1H),6.18-6.23(dd,1H),5.70-5.73(dd,1H),3.89(s,3H),3.44(s,3H),2.87-2.88(m,2H),2.70(s,3H),2.20-2.30(m,8H),2.08(s,3H). LC-MS(m / z):583.77[M+H] + .

[0081] Example 7 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-(p-tolyloxy)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0082]

[0083] Using a similar method to Example 1, replace compound 1.2 in step 1 with p-cresol and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz, DMSO-d6)(ppm):10.02(s,1H),8.41(s,1H),8.37(s,1H),8.16(s,1H),7.67-7.72(m,2H),7.10-7.15(m,4H),6.91( s,1H),6.39-6.43(dd,1H),6.22-6.27(dd,1H),5.75-5.78(dd,1H),3.76(s,3H),2.85(m,2H),2.67(s,3H),2.23-2.34(m,10H). LC-MS(m / z):570.75[M+H] + .

[0084] Example 8 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-(naphthalen-1-yloxy)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0085]

[0086] Using a similar method to Example 1, replace compound 1.2 in step 1 with naphthalene-1-ol, and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound.1 H NMR(400MHz,DMSO-d6)(ppm):10.0(brs,1H),8.45(s,1H),8.28(brs,1H),8.09(s,1H),7.96-7.98(m,1H),7.77-7.85(m,4H),7.43-7.57(m,4H) ,7.30-7.35(m,2H),6.83(s,1H),6.40(m,1H),6.22-6.26(d,1H),5.76- 5.79(d,1H),3.66(s,3H),2.82(m,2H),2.64(s,3H),2.23-2.34(m,8H). LC-MS(m / z):606.71[M+H] + .

[0087] Example 9 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluorophenyl)-4-((2-fluorophenyl)amino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0088]

[0089] Using a similar method to Example 1, replacing compound 1.4 in step 2 with (4-fluorophenyl)boronic acid, the title compound was obtained. 1 H NMR(400MHz,DMSO-d6)(ppm):10.07(s,1H),8.50(s,1H),7.93(s,1H),7.78 -7.88(m,3H),7.52-7.56(m,2H),7.31-7.36(t,2H),7.12-7.14(m,1H),6.95 -7.05(m,3H),6.36-6.40(dd,1H),6.18-6.22(dd,1H),5.72-5.75(dd,1H),3 .78(s,3H),2.83-2.86(t,3H),2.70(s,2H),2.27-2.30(t,2H),2.20(s,6H). LC-MS (m / z): 574.95 [M+H] + .

[0090] Example 10 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluoro-5-methylphenyl)amino)-5-(4-fluorophenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0091]

[0092] Using a similar method to Example 1, replace compound 1.2 in step 1 with 2-fluoro-5-methylaniline and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):9.92(brs,1H),8.45(brs,1H),8.11(s,1H),7.92(m,2H),7.50-7.58(m,3H),7.36-7.30(m,2H),7.0-7.04 (m,1H),6.94(s,1H),6.85-6.86(m,1H),6.19-6.23(d,1H)5.73-5.76(d,1H),3.81(s,3H),2.93(m,2H),2.66(s,3H),2.18-2.37(m,9H). LC-MS(m / z):589.04[M+H] + .

[0093] Example 11 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluoro-4-methylphenyl)amino)-5-(4-fluorophenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0094]

[0095] Using a similar method to Example 1, replace compound 1.2 in step 1 with 2-fluoro-4-methylaniline and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):10.04(s,1H),8.57(s,1H),7.91(s,1H),7.78(s,1H),7.65-7.70(t,3H),7.51-7.55(m,2H),7.31-7.36(m,2H) ,6.96-6.99(m,2H),6.37-6.43(m,1H),6.18-6.23(dd,1H),5.73-5.76(dd,1H),3.79(s,3H),2.86(m,2H),2.70(s,3H),2.10-2.40(m,11H). LC-MS(m / z):589.01[M+H] + .

[0096] Example 12 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluoro-3-methylphenyl)amino)-5-(4-fluorophenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0097]

[0098] Using a similar method to Example 1, replace compound 1.2 in step 1 with 2-fluoro-3-methylaniline and replace compound 1.4 in step 2 with (4-fluorophenyl)boronic acid to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)(ppm):10.04(s,1H),8.52(s,1H),7.94(s,2H),7.7 6-7.80(m,1H),7.60(s,1H),7.52-7.57(m,2H),7.32-7.36(m,2H),6.97(s, 1H),6.85-6.90(m,2H),6.36-6.42(dd,1H),6.17-6.21(dd,1H),5.72-5.75 (dd,1H),3.79(s,3H),2.85-2.88(t,2H),2.70(s,3H),2.17-2.40(m,11H). LC-MS(m / z):588.50[M+H] + .

[0099] Example 13 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-(4-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0100]

[0101] Using a similar method to Example 1, replacing compound 1.4 in step 2 with (4-(trifluoromethyl)phenyl)boronic acid, the title compound was obtained. 1 H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.46(s,1H),8.10(s,1H),8.01(s ,1H),7.89(s,1H),7.82-7.84(d,2H),7.73-7.75(d,2H),7.65-7.69(m,1H),7 .00-7.16(m,3H),6.94(s,1H),6.36-6.43(dd,1H),6.18-6.23(dd,1H),5.73- 5.76(dd,1H),3.78(s,3H),2.83-2.86(t,2H),2.69(s,3H),2.21-2.30(m,8H). LC-MS (m / z): 625.16 [M+H] + .

[0102] Example 14 N-(5-((5-(4-cyanophenyl)-4-((2-fluorophenyl)amino)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide

[0103]

[0104] Using a similar method to Example 1, replacing compound 1.4 in step 2 with (4-cyanophenyl)boronic acid, the title compound was obtained. 1 H NMR(400MHz, DMSO-d6)(ppm):10.04(s,1H),8.46(s,1H),8.15(s,1H),8.02(s,1H),7.92-7.95(m,3H),7.73(d,2H),7.63-7.67(m,1H),7.0-7. 16(m,3H),6.94(s,1H),6.37-6.43(dd,1H),6.19-6.23(dd,1H),5.73-5 .76(dd,1H),3.78(s,3H),2.85(m,2H),2.68(s,3H),2.21-2.40(m,8H). LC-MS(m / z):582.10[M+H] + .

[0105] Example 15 N-(5-((5-(4,4-difluorocyclohex-1-en-1-yl)-4-((2-fluorophenyl)amino)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide

[0106]

[0107] A similar method to Example 1 was used to obtain the title compound by replacing compound 1.4 in step 2 with (4,4-difluorocyclohex-1-en-1-yl)boronic acid. 1 H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.49(s,1H),7.94(s,2H),7.71-7.85(m,2H),7.16-7.21(m.3H),6.93(s,1H),6.3 5-6.42(m,1H),6.17-6.22(m,1H),5.72-5.75(dd,1H),3.78(s,3H),2.82-2.85(t,2H),2.68-2.71(m,5H),2.20-2.28(m,9H). LC-MS(m / z):596.48[M+H] + .

[0108] Example 16 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-(4-propylphenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0109]

[0110] A similar method to Example 1 was used to obtain the title compound by replacing compound 1.4 in step 2 with (4-propylphenyl)boronic acid. 1 H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.51(s,1H),7.93-7.98(m,3H),7.66(s, 1H),7.41-7.43(d,2H),7.33-7.35(d,2H),7.11-7.16(m,1H),6.97-7.02(m,3H),6.37 -6.43(dd,1H),6.18-6.22(dd,1H),5.72-5.75(dd,1H),3.79(s,3H),2.87(m,2H),2.7 0(s,3H),2.60-2.64(t,2H),2.22-2.31(m,8H),1.62-1.67(m,2H),0.92-0.96(t,3H). LC-MS(m / z):599.27[M+H] + .

[0111] Example 17 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluoro-2-methylphenyl)-4-((2-fluorophenyl)amino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0112]

[0113] A similar method to Example 1 was used to obtain the title compound by replacing compound 1.4 in step 2 with (4-fluoro-2-methylphenyl)boronic acid. 1H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.52(s,1H),7.77(s,1H),7.7 2-7.75(m,2H),7.41(s,1H),7.29-7.32(m,1H),7.21-7.24(m,1H),7.0-7.1 7(m,4H),6.95(s,1H),6.36-6.43(dd,1H),6.18-6.22(dd,1H),5.73-5.76 (dd,1H),3.80(s,3H),2.83-2.86(t,3H),2.69(s,3H),2.21-2.30(m,11H). L C-MS(m / z):589.04[M+H] + .

[0114] Example 18 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((5-(4-fluoro-3-methylphenyl)-4-((2-fluorophenyl)amino)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0115]

[0116] A similar method to Example 1 was used to obtain the title compound by replacing compound 1.4 in step 2 with (4-fluoro-3-methylphenyl)boronic acid. 1 H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.52(s,1H),7.93(s,1H),7.86-7.93(m ,2H),7.73(s,1H),7.41-7.43(m,1H)7.33-7.34(m,1H),7.24-7.28(t,1H),7.14(m,1 H),7.0-7.04(m,2H),6.96(s,1H),6.36-6.38(m,1H),6.18-6.22(d,1H),5.72-5.75 (d,1H)3.79(s,3H),2.84-2.87(t,2H),2.70(s,3H),2.28-2.30(m,5H),2.21(s,6H). LC-MS(m / z):589.04[M+H] + .

[0117] Example 19 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-(pyridin-4-yl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0118]

[0119] A similar method to Example 1 was used to obtain the title compound by replacing compound 1.4 in step 2 with pyridin-4-ylboronic acid. 1 H NMR(400MHz,DMSO-d6)(ppm):10.03(s,1H),8.63-8.64(d,2H),8.45(s,1H) ,8.18(s,1H),8.06(s,1H),7.97(s,1H),7.67-7.70(t,1H),7.55-7.56(d,2H ),7.01-7.17(m,3H),6.94(s,1H),6.37-6.43(dd,1H),6.19-6.23(d,1H),5 .73-5.76(d,1H),3.78(s,3H),2.85(m,2H),2.69(s,3H),2.21-2.30(m,8H). LC-MS (m / z): 557.78 [M+H] + .

[0120] Example 20 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-(2-methoxyphenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0121]

[0122] A similar method to Example 1 was used to obtain the title compound by replacing compound 1.4 in step 2 with (2-methoxyphenyl)boronic acid. 1 H NMR(400MHz,DMSO-d6)(ppm):10.06(s,1H),8.53(s,1H),8.05(m,1H),7.95(s, 1H),7.90(s,1H),7.43-7.45(m,1H),7.30-7.32(m.2H),7.09-7.19(m,3H),6.96 -6.99(m,3H),6.36-6.39(dd,1H),6.17-6.22(dd,1H),5.72-5.75(dd,1H),3.8 0-3.83(d,6H),2.85-2.88(m,2H),2.71(s,3H),2.29-2.31(m,2H),2.21(s,6H). LC-MS (m / z): 587.18 [M+H] + .

[0123] Example 21 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-(3-methoxyphenyl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide

[0124]

[0125] Using a similar method to Example 1, replacing compound 1.4 in step 2 with (3-methoxyphenyl)boronic acid, the title compound was obtained. 1 H NMR(400MHz, DMSO-d6)(ppm):10.06(s,1H),8.51(s,1H),7.96-8.0(m,3H),7.75(s,1H),7.41-7.45(m,1H),6.97-7.18(m,7H),6. 46-6.43(dd,1H),6.17-6.22(dd,1H),5.72-5.75(dd,1H),3.80-3.85(d,6H),2.85-2,88(t,2H),2.70(s,3H),2.20-2.40(m,8H). LC-MS(m / z):587.07[M+H] + .

[0126] Experimental Example 1 EGFR D770_N771insNPG kinase activity inhibition assay

[0127] EGFR D770_N771insNPG kinase expressed in a baculovirus expression system was purchased from Shanghai UnivBiological Inc. The TK Kinase HTRF Detection Kit (#62TK0PEC) containing biotin-labeled peptide substrate TK Substrate-biotin, Eu-labeled specific phosphorylated peptide antibody TK Antibody-Cryptate, HTRF fluorescent receptor reagent Streptavidin-XL665, 5× kinase reaction buffer solution, and detection buffer was purchased from Cisbio Bioassays (Codolet, France). Dithiothreitol (DTT), magnesium chloride, manganese chloride, adenosine triphosphate (ATP), dimethyl sulfoxide (DMSO), and HEPES buffer were obtained from Sigma at the highest purity level available.

[0128] General method for EGFR kinase activity inhibition assay: The phosphorylation reaction buffer consists of 1M HEPES (pH 7.0), 5mMMgCl2, and 1mM MnCl2. 1mM DTT is added to the buffer immediately before the experiment. Prepare a DMSO storage stock solution of the test compound, and use DMSO to perform a three-fold concentration gradient dilution according to the experimental requirements. The resulting gradient dilution solution is further diluted with the phosphorylation reaction buffer solution to obtain a working solution of the test compound dissolved in a reaction buffer containing 5% DMSO. Add 2μL of the compound working solution and 4μL of the EGFR kinase solution diluted in the reaction buffer to a white low-volume 384-well microtiter plate, and then add 4μL of a mixed solution of ATP and biotin-labeled peptide substrate dissolved in the reaction buffer to start the phosphorylation reaction. In the WT EGFR experiment, the final concentrations of WT kinase, peptide substrate, ATP and DMSO were 0.01ng / ul, 500nM, 4μM and 1%, respectively. In the EGFR D770_N771insNPG experiment, the final concentrations of kinase, peptide substrate, ATP and DMSO were 0.01ng / ul, 200nM, 4μM and 1%, respectively. The reaction was performed at room temperature for 60 minutes under light-proof conditions. 5μL of TK Antibody-Cryptate antibody and Streptavidin-XL665 diluted in detection buffer were added respectively and incubated at room temperature for 60 minutes. The final concentration of Streptavidin-XL665 in the WT EGFR kinase reaction system was 15.61nM, and the final concentration of Streptavidin-XL665 in the EGFR D770_N771insNPG kinase reaction system was 31.25nM. The antibodies were diluted according to the final concentration provided by the supplier. Tecan ( The plate was read using a multifunctional microplate reader Spark in Switzerland to detect the homogeneous time-resolved fluorescence intensity of the two groups, with an excitation wavelength of 320 nm and emission wavelengths of 665 nm and 620 nm, respectively. Prism 7 (La Jolla, 15CA) was used to plot the 665 nm / 620 nm fluorescence intensity ratio against the inhibitor concentration, and the S-shaped dose-dependent curve model was used to record the obtained inhibition curve and obtain the IC value of the inhibitor. 50 IC values ​​of EGFRD770_N771insNPG kinase inhibitory activity of some compounds prepared according to the above examples 50 The measurement results are shown in Table 1.

[0129] Table 1

[0130] Compound <![CDATA[IC 50 (nM)]]> Example 1 0.85 Example 2 0.83 Example 9 2.00 Example 10 5.67 Embodiment 11 4.93 Example 12 8.94

[0131] The experimental results show that the compounds described in the present application can effectively inhibit the kinase activity of EGFR D770_N771insNPG, wherein the IC 50 All less than 1nM.

[0132] Experimental Example 2 Cell proliferation inhibition experiment

[0133] H358, HCC827, NCI-H1781, and Calu-3 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai), BaF3 EGFRWT, BaF3 EGFR D770_N771insSVD, BaF3 EGFR V769_D770insASV, and BaF3 ERBB2A775_G776insYVMA cells were purchased from Beijing Kangyuan Bochuang, and MDA-MB-231, SK-BR-3, and BT474 were purchased from Nanjing Kebai Biotechnology Co., Ltd. MEM medium, RPMI1640 medium, penicillin-streptomycin double antibody, 0.5% trypsin (10X), and epidermal growth factor EGF were purchased from ThermoFisher (Waltham, MA, USA). Certified fetal bovine serum (FBS) was purchased from Biological Industries (Israel). Corning 96- and 384-well cell culture plates were purchased from CORNING (USA). Cell-Titer Purchased from Promega Corporation (Madison, WI, USA).

[0134] To evaluate the inhibitory ability of the synthesized compounds on the proliferation of lung cancer cells H358, HCC827, NCI-H1781, Calu-3 and BaF3EGFRWT, BaF3 EGFR D770_N771insSVD, BaF3 EGFR V769_D770insASV, BaF3 ERBB2 A775_G776insYVMA, the exponentially growing cells of Calu-3 were seeded in MEM medium containing 10% bovine serum and 1% penicillin-streptomycin, the exponentially growing cells of H358, HCC827, NCI-H1781, BaF3 EGFR D770_N771insSVD, BaF3 EGFR V769_D770insASV, BaF3 ERBB2 A775_G776insYVMA were seeded in RPMI1640 medium containing 10% bovine serum and 1% penicillin-streptomycin, and the Exponentially growing cells of EGFR WT were seeded in RPMI1640 medium containing 10% bovine serum, 1% penicillin-streptomycin and 50ng / mL EGF, HCC827, NCI-H1781, Calu-3, BaF3 ERBB2 A775_G776insYVMA at a density of 75,000 cells / mL, H358 at a density of 100,000 cells / mL, BaF3 EGFR D770_N771insSVD, BaF3 V769_D770insASV at a density of 50,000 cells / mL, and BaF3 EGFR WT at a density of 125,000 cells / mL in 384-well plates with 20 μL per well and placed in an incubator at 37°C, 5% CO2 overnight. Compounds were diluted in DMSO to a 12-point, 3-fold serial dilution starting from 2mM. Add 1 μL of DMSO solution from the compound stock plate to 99 μL of cell culture medium (the final maximum concentration of the compound in the assay is 10 μM, and the final concentration of DMSO is 0.5%). Add 20 μL of compound solution in the culture medium to each well of the 384-well plate with cells according to a gradient. After adding the compound solution, the 384-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 3 days. Cell viability was determined by quantifying the ATP present in the cell culture using the CellTiter-Glo detection kit from Promega (Madison, WI, USA). After 20 minutes of incubation, the readings were taken using the TECAN SPARK multi-function microplate reader under the chemiluminescence program. The S-shaped dose-response model (variable slope, four parameters) was used in Prism 7 (LaJolla, CA) to determine the concentration (IC) of the compound that inhibits cell viability by 50%. 50 value).

[0135] The drug control substances Poziotinib, Osimertinib, and Pyrotinib used in the experiment were all commercially available. Ref-1 was prepared according to the preparation method described in WO2015195228A1, and its structure is as follows:

[0136]

[0137] Table 2 shows the results of the inhibition of proliferation of BaF3 EGFR WT, BaF3 EGFR D770_N771insSVD, and BaF3 EGFR V769_D770insASV cells by some of the compounds prepared according to the aforementioned examples.

[0138] Table 2

[0139]

[0140]

[0141] The results show that the activity of the compounds of the present invention on the inhibition of WT EGFR high-expression BaF3 cell proliferation is much lower than the two candidate drugs currently in the clinical research stage, indicating that the toxicity brought about by this will be greatly reduced. For the selectivity obtained by comparing the inhibitory activity of the two most widely expressed mutations D770_N771insSVD and V769_D770insASV in WT EGFR and EGFR Exon20 insertion, the two clinical compounds are currently 0.18 / 0.33 and 0.25 / 0.18, respectively, indicating that the clinical efficacy dose is much higher than the toxic dose, so that clinical application will be greatly limited. The selectivity of most of the compounds of the present invention is significantly higher than that of clinical compounds, such as Examples 9 and 13, the selectivity is 5.5 / 5.0 and 1.3 / 3.3, respectively, which are 30 / 15, 7.2 / 10 and 22 / 28, 5.2 / 18 times of Poziotininb and Ref-1, respectively, indicating that the clinical treatment space is greatly improved and the efficacy is more obvious.

[0142] The results of the inhibition of HCC 827 and H358 WT cell proliferation by some of the compounds prepared according to the aforementioned examples are shown in Table 3.

[0143] Table 3

[0144]

[0145]

[0146] The results showed that the inhibition of the proliferation of H358 NSCLC cells expressing wild-type EGFR by the compounds of the present invention was much lower than that of Poziotinib, and was close to or even lower than that of Osimertinib, a third-generation EGFR inhibitor with fully verified clinical safety.

[0147] Table 4 shows the results of the inhibition of proliferation of Calu-3, NCI-H1781 and BaF3 ERBB2A775_G776insYVMA cells by some of the compounds prepared according to the aforementioned examples.

[0148] Table 4

[0149]

[0150] The results showed that although Example 2 had a lower inhibitory effect on the proliferation of BaF3 ERBB2 A775_G776insYVMA and NCI-H1781 cells than Pyrotinib, which is currently in the clinical research stage for the treatment of non-small cell lung cancer with high expression of ERBB2 A775_G776insYVMA and ERBB2 G776_V777insV, its inhibition on the proliferation of WT ERBB2 high-expressing Calu-3 was significantly lower than Pyrotinib, showing better selectivity and clinical treatment space.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: Z1 is -NH-, -N(CH3)-, -O- or -S-; Z2, Z3, Z4, and Z5 are each independently selected from C or N; Each R1 is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxyl, nitro, amino, cyano or alkoxy; two adjacent R1s may form a five- to six-membered saturated or unsaturated aromatic ring or aromatic heterocyclic ring together with the atoms to which they are connected; Each R2 is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy; m and n are independently selected from 1, 2 or 3; Dashed lines are single or double bonds.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: Z1 is -NH-, -N(CH3)- or -O-; Z2, Z3, Z4, and Z5 are each independently selected from C or N, and Z3 and Z4 are not N at the same time; Each R1 is independently selected from hydrogen, C1-C3 alkyl or halogen; two adjacent R1s may form a benzene ring together with the atoms to which they are attached; Each R2 is independently selected from hydrogen, C1-C3 alkyl, halogenated C1-C3 alkyl, halogen, cyano or methoxy; m and n are independently selected from 1 or 2; Dashed lines are single or double bonds.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Selected from:

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Selected from:

5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the following compounds:

6. Use of the compound according to any one of claims 1 to 5 for preparing a drug for preventing or treating diseases related to receptor tyrosine kinase EGFR mutation.

7. The use according to claim 6, wherein the related disease caused by the EGFR mutation is cancer.

8. The method of claim 7, wherein the cancer is non-small cell lung cancer, breast cancer, brain cancer, ovarian cancer, pancreatic cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal tissue cancer, esophageal cancer, thyroid cancer, leukemia, lymphoma or multiple myeloma.

9. The use according to claim 6, wherein the disease related to the EGFR mutation is a cancer related to a mutation in the exon 20 domain of EGFR.

10. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient.

11. The pharmaceutical composition according to claim 10, further comprising one or more other anticancer drugs, wherein the anticancer drugs are small molecule drugs, monoclonal antibodies or fusion protein drugs.

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