CN111973602B

CN111973602B - Application of morpholinyl acetamido quinazoline compound as EGFR (epidermal growth factor receptor) inhibitor

Info

Publication number: CN111973602B
Application number: CN201910426415.8A
Authority: CN
Inventors: 薛松涛; 饶国武; 陈明浩; 任欣悦; 靳浩
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2022-02-11
Anticipated expiration: 2039-05-21
Also published as: CN111973602A

Abstract

The invention discloses an application of a morpholinyl acetamido quinazoline compound shown in a formula (I) as an EGFR inhibitor in preparation of a medicine for treating or preventing EGFR-mediated diseases or a medicine for inhibiting EGFR. The compounds provided by the invention have excellent inhibitory activity on wild-type or mutant EGFR or EGFR-mutated cancer cells.

Description

Application of morpholinyl acetamido quinazoline compound as EGFR (epidermal growth factor receptor) inhibitor

(I) technical field

The invention relates to a quinazoline compound and application thereof, in particular to application of a morpholinyl acetamido quinazoline compound as an EGFR inhibitor in preparation of a medicament for treating or preventing EGFR-mediated diseases or a medicament for inhibiting EGFR.

(II) background of the invention

The quinazoline compounds have a plurality of good biological activities and are widely applied in the field of medicine, particularly, some quinazoline derivatives with special structures have obvious antiviral activity, antibacterial activity, antitumor activity and the like, and the quinazoline compounds are marketed as antitumor drugs. For example, Gefitinib (Gefitinib), Erlotinib (Erlotinib), and Lapatinib (Lapatinib), which are marketed for the treatment of cancer, all belong to the quinazoline class of compounds. Novel quinazoline compounds and their biological activities are also commonly reported in the literature (see y. -y. ke, h. -y. shiao, y. c. hsu, c. -y. chu, w. -c. wang, y. -c. lee, w. -h. lin, c. -h. chen, j. t. a. hsu, c. -w. chang, c. -w. lin, t. -k. yeh, y. -s. chao, m.s. coumar, h. -p. hsieh, chemed chem 2013,8, 136-148; a.garofalo, a.farce, s.ravez, a.lemoine, p.six, p.vachatte, l.gos, p.depenux, j.chem. 1204, d. chem. 1189). Of course most quinazoline compounds do not have anti-tumor activity.

EGFR is epidermal growth factor receptor on the surface of cell membrane, and can effectively inhibit the growth and proliferation of tumor by inhibiting the activity expression of protein tyrosine kinase. EGFR is a constitutively expressed component of many normal epithelial tissues (e.g., skin and hair follicles), while in most solid tumors, EGFR is either overexpressed or highly expressed. For example, the expression rate of EGFR in lung cancer reaches 40-80%. Therefore, the aim of treating the lung cancer can be achieved by selectively inhibiting the EGFR and interfering the signal transduction pathway mediated by the EGFR. The early results of EGFR inhibitors are encouraging, and the majority of patients with activation-mutated non-small cell lung cancer respond well early. Patients develop resistance to treatment with first generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) within a few months. The most common mechanism for acquired resistance is the secondary T790M (threonine instead of methionine) target point mutation in exon 20, which accounts for 60% of all mutations. The generation of drug resistance not only reduces the sensitivity of the patient to the drug, but also greatly reduces the life quality of the tumor patient.

To overcome the resistance caused by the T790M mutation, several second and third generation irreversible EGFR-TKIs have been developed, but they are less selective. Therefore, the research and development of a novel EGFR inhibitor for selectively inhibiting T790M mutation and overcoming clinical drug resistance has important clinical significance and application prospect.

Disclosure of the invention

The invention aims to provide application of a novel quinazoline compound, namely a morpholinyl acetamido quinazoline compound as an EGFR inhibitor in preparation of medicines for treating or preventing EGFR-mediated diseases or medicines for inhibiting EGFR.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of morpholinyl acetamido quinazoline compounds shown in a formula (I) or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing EGFR (epidermal growth factor receptor) -mediated diseases or medicines for inhibiting EGFR (epidermal growth factor receptor),

examples of pharmaceutically acceptable salts of the morpholinoacetamidoquinazoline compounds of the present invention include, but are not limited to, inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and inorganic and organic base salts with bases such as sodium hydroxy, TRIS (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methylglucamine.

The EGFR of the present invention, including but not limited to: EGFR^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK and EGFR^T790M-TK kinase. The morpholinyl acetamido quinazoline compound (I) of the invention is used for treating EGFR^wt-TK，EGFR^L858R-TK and EGFR^T790MThe TK has remarkable inhibitory effect, especially on EGFR^T790MIC of-TK₅₀Is obviously superior to gefitinib.

In a specific embodiment of the invention, the EGFR-mediated disease is cancer. Such cancers include, but are not limited to: non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma and solid tumors. In a preferred embodiment, the cancer is EGFR-mediated non-small cell lung cancer or breast cancer, and particularly has an inhibitory activity against HCC827, NCI-H1975, and MDA-MB-231 cancer cells that is superior to that of existing drugs.

Compared with the prior art, the invention has the beneficial effects that: the compound (I) provided by the invention has excellent inhibitory activity on wild type or mutant EGFR or cancer cells mutated by EGFR.

(IV) detailed description of the preferred embodiments

The invention is further illustrated by reference to specific examples, which are intended to illustrate the invention, but not to limit it in any way.

For comparison, the present invention produced compound (II). Preparation of Compounds (III) and (IV) according to the method of reference (CN 108014114A).

Example 1: preparation of morpholinyl acetamido quinazoline (I)

Adding 3.25 g (5.73mmol) of chloroacetamidoquinazoline (IV) and 0.598 g (6.86mmol) of morpholine, 3.626 g (45.84mmol) of pyridine and 32.5 ml of methanol into a 50 ml reaction bottle in sequence, heating to 40 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirring for 10 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 10 ml of ethyl acetate into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 1.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of the dried concentrate and the silica gel, loading the mixture into a column, and then performing column chromatography by using a volume ratio of 1: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating a collected solution, and drying at 50 ℃ to obtain an off-white solid product shown in the formula (I), wherein the yield is 65%, and the melting point is 122-125 ℃.¹H NMR(500MHz,CDCl₃)δ:2.65-2.72(m,4H),3.19(s,2H),3.30(m,1H),3.54(d,J＝15.3Hz,1H),3.74(s,3H),3.81-3.84(m,11H),3.99-4.01(m,2H),4.64(dd,J＝8.2.14.2Hz,1H),5.27(t,J＝8.6Hz,1H),6.67(s,1H),6.87(d,J＝8.6Hz,2H),7.07(d,J＝8.6Hz,2H),7.40(dd,J＝2.0,8.9Hz,1H),7.81(d,J＝8.9Hz,1H),8.58(s,1H),8.84(s,1H),9.29(s,1H)。HRMS-ESI m/z:618.2477[M+H]⁺。

Example 2: preparation of cinnamyl amido quinazoline (II)

0.27 g (0.55mmol) of aminoquinazoline (III), 0.13 g (1.64mmol) of pyridine and 20 ml of tetrahydrofuran are added into a reaction bottle in sequence, 0.733 g (4.40mmol) of cinnamoyl chloride is added dropwise under the stirring condition at-10 ℃, and TLC tracking detection is carried out after dropwise addition (a developing solvent is ethyl acetate/petroleum ether is 1: 1) at-10 DEG CAnd (2) filtering for 12 hours, evaporating the solvent from the filtrate, adding 10 ml of ethyl acetate into the concentrate to dissolve the concentrate to obtain a dissolved solution, adding 0.60 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of a dried concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 1: eluting by using a petroleum ether/ethyl acetate mixed solution of 10 as an eluent, tracking and detecting by TLC (a developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (II) (the Rf value is 0.5) according to TLC detection, concentrating a collected solution, and drying at 50 ℃ to obtain the cinnamoylaminoquinazoline shown in the formula (II) as an off-white solid with the yield of 68% and the melting point of 222-224 ℃.¹H NMR(500MHz,CDCl₃)δ:3.28-3.34(m,1H),3.54(dt,J＝3.5,15.0Hz,1H),3.75(s,3H),3.79-3.82(m,7H),4.00-4.12(m,2H),4.70(dd,J＝8.0,14.2Hz,1H),5.31(t,J＝8.6Hz,1H),6.65(d,J＝15.5Hz,1H),6.71(s,1H),6.90(d,J＝8.7Hz,2H),7.10(d,J＝8.6Hz,2H),7.42-7.44(m,3H),7.56-7.59(m,3H),7.80-7.84(m,3H),8.59(s,1H),8.87(br,1H)；HRMS-ESI m/z:621.2270[M+H]⁺。

Example 3: in vitro kinase Activity assay

Kinase-Lumi^TMA kinase activity detection kit by a chemiluminescence method is a kit for quantitatively detecting the activity of kinase by measuring the residual amount of ATP in a solution after the kinase reaction by the chemiluminescence method. Kinase-Lumi is adopted in the experiment^TMChemiluminescence method kinase activity detection kit (Beyotime) for testing compounds (I) and (II) respectively on EGFR under room temperature condition^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK and EGFR^T790M-inhibitory activity of TK kinase.

The following is a 96-well plate recommended detection system.

(1) Preparation of ATP Standard Curve

The reaction buffer was prepared with 1mM manganese dichloride, 5mM magnesium dichloride, 1mM Dithiothreitol (DTT).

Setting 0, 0.03, 0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 μ MATP standard sample hole (all above ATP concentration are the final concentration of the substance when the total volume in the standard sample hole reaches 100 μ L). For preparation, 50. mu.L of ATP was first diluted with reaction buffer. Then 50 mu L of Kinase-Lumi is added^TMAnd (3) mixing the chemiluminescence kinase detection reagent and the mixture. After reaction at room temperature (about 25 ℃) for 10 minutes, chemiluminescence detection was carried out using a multifunctional microplate reader, and an ATP standard curve was prepared.

(2) Sample detection

Sample wells were configured to contain 0.1. mu.g/mL polyglutamic acid and tyrosine (4:1) kinase substrates, 5. mu.MATP and 10. mu.g/L kinase (EGFR) at a total volume of 100. mu.L per well^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK or EGFR^T790MTK), compound (I) or compound (II) or gefitinib or lapatinib in different concentrations (100, 200, 400, 800, 1000 nM). When preparing, polyglutamic acid and tyrosine (4:1) kinase substrate, ATP, kinase (EGFR) are added into each hole^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK or EGFR^T790MTK) and compound (I) or compound (II) or gefitinib or lapatinib, adding reaction buffer and diluting to total volume of 50 μ L; then 50 mu L of Kinase-Lumi is added^TMAnd (3) mixing the chemiluminescence kinase detection reagent and the mixture. The reaction was carried out at room temperature (about 25 ℃ C.) for 10 minutes. Then using a multifunctional microplate reader to perform chemiluminescence detection. The amount of ATP remaining in the sample wells was calculated from the standard curve, and then the enzyme activity was calculated according to the definition of enzyme activity. Finally calculate the IC₅₀The value is obtained.

The results of the in vitro kinase activity experiments are shown in Table 1, and Table 1 shows that compounds (I) and (II) and positive drugs gefitinib and lapatinib are shown to act on EGFR^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK and EGFR^T790M-activity data of TK kinase. From the data in the table, it can be seen that compounds (I) and (II) are active against EGFR^wt-TK activity is inhibited to various degrees, wherein the compound (I) inhibits EGFR^wtThe TK inhibition is strongest, and the IC thereof₅₀Is 25.8nM and is superior to lapatinib which is a drug on the market. In addition, the compound (I) is directed against EGFR^L858R/T790MNo significant inhibition of-TK, but no EGFR^T790MThe activity of TK, the IC of which is strongly inhibited₅₀Is 36.2nMIs superior to gefitinib which is a medicament on the market. Thus, in vitro kinase activity assays indicate that Compound (I) is active against EGFR^wt-TK，EGFR^L858R-TK and EGFR^T790MThe TK has remarkable inhibitory effect, especially on EGFR^T790MIC of-TK₅₀Is obviously superior to gefitinib.

TABLE 1 inhibitory Activity of Compounds (I) and (II) on EGFR

Example 4: in vitro test for anti-cancer Activity

The prepared compounds (I) and (II) are respectively subjected to the biological activity tests of HCC827, NCI-H1975 and MDA-MB-231 cancer cell lines.

The test method comprises the following steps: tetrazolium salt reduction (MTT process).

Cell lines: HCC827, NCI-H1975, MDA-MB-231. The tumor cell strain is purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.

The experimental procedure was as follows:

(1) preparation of samples: for soluble samples, each 1mg was dissolved in 40. mu.L DMSO, 2. mu.L was diluted with 1000. mu.L of medium to a concentration of 100. mu.g/mL, and then serially diluted with medium to the use concentration.

(2) Culture of cells

Preparation of culture medium, each 1000mL of DMEM culture medium (Gibco) contains 80 ten thousand units of penicillin, 1.0g of streptomycin and 10% inactivated fetal bovine serum.

② culturing cells: inoculating tumor cells into culture medium, standing at 37 deg.C and 5% CO₂Culturing in an incubator, and carrying out passage for 3-5 days.

Measuring the inhibition of the sample on the growth of tumor cells

The 5 th generation cells were digested with EDTA-pancreatin and diluted to 1X 10 with medium⁶Perml, 100. mu.L/well in 96-well cell culture plates, 37 ℃ 5% CO₂Culturing in an incubator. After 24 hours of inoculation, 30. mu.M, 15. mu.M, 7.5. mu.M, 4.0. mu.M, diluted with the culture medium, were added,mu.M or 1. mu.M samples, 100. mu.L per well, 3 wells per concentration, at 37 ℃ with 5% CO₂Incubate in the incubator, after 36h add 20. mu.L of 5mg/mL MTT solution to the cell culture wells, incubate at 37 ℃ for 3h, add DMSO, 150. mu.L per well, shake with a shaker, completely solubilize the formazan, and color at 490nm using a microplate reader. Using cells cultured in the same DMSO concentration medium without sample under the same conditions as a control, the IC of the sample on tumor cell growth was calculated₅₀。

The results of the tests are shown in table 2:

TABLE 2 in vitro antitumor Activity of Compounds (I) and (II) against cancer cell lines

Claims

1. An application of morpholinyl acetamido quinazoline compounds shown in a formula (I) or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing EGFR mediated diseases,

the EGFR-mediated disease is cancer, and the cancer cells of the cancer are HCC827, NCI-H1975 or MDA-MB-231 cells.